Scientific support for and against a diet higher in protein than the RDA

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Protein is made up of multiple building blocks and not all protein sources are alike, so to say more protein is better or worse under-appreciates the intricacies involved. The challenge is in hitting the nutritional sweet spots as individually required since protein needs vary by age, athletic demands, health conditions such as pregnancy, illness, post-surgical recovery, if you’re healing your gut, recovering from toxic exposures, etc.

Amino Acids - the Building Blocks of Protein

The protein we eat is comprised of 20 amino acids. Your body breaks the protein down into amino acids, which are then absorbed into your bloodstream. The body uses these amino acids to create around 50,000 different proteins, each with a specific structure and function based on how the amino acids are arranged.

From the Cleveland clinic “You can think of amino acids like the letters of the alphabet. When you combine letters in various ways, you make different words. The same goes for amino acids — when you combine them in various ways, you make different proteins.” Source: Amino Acids

Your body uses amino acids to make proteins. The different amino acids and the way they’re put together determine the function of each protein. Amino acids are involved in many important roles in your body including making hormones and neurotransmitters, maintaining a healthy gut, boosting your immune system, building muscle when combined with exercise, and more.

Source: MedlinePlus website, https://medlineplus.gov/ency/article/002222.htm with a review date of 1/19/2023

There are essential amino acids and non-essential amino acids. While the label “essential” seems to imply those amino acids are more important for the body, this isn’t true, read through the next sections addressing each of these amino acids to see the important functions they all provide.

Essential Amino Acids.

Nine Amino Acids that we consume are essential, you must get them from the food you eat, but more of each essential amino acids doesn’t necessarily mean it’s better (e.g.s methionine and the branched chain amino acids: leucine, isoleucine, and valine, as you will read below.) The nine essential amino acids are:
Histidine: Histidine helps make a brain chemical (neurotransmitter) called histamine. Histamine plays an important role in your body’s immune function, digestion, sleep and sexual function.
Isoleucine: A Branched-chain Amino Acid, isoleucine is involved with your body’s muscle metabolism and immune function. It also helps your body make hemoglobin and regulate energy.
Leucine: A Branched-chain Amino Acid, leucine helps your body make protein and growth hormones. It also helps grow and repair muscle tissue, heal wounds and regulate blood sugar levels.
Lysine: Lysine is involved in the production of hormones and energy. It’s also important for calcium and immune function.
Methionine: Methionine helps with your body’s tissue growth, metabolism and detoxification. Methionine also helps with the absorption of essential minerals, including zinc and selenium.
Phenylalanine: Phenylalanine is needed for the production of your brain’s chemical messengers, including dopamine, epinephrine and norepinephrine. It’s also important for the production of other amino acids.
Threonine: Threonine plays an important role in collagen and elastin. These proteins provide structure to your skin and connective tissue. They also help with forming blood clots, which help prevent bleeding. Threonine plays an important role in fat metabolism and your immune function, too.
Tryptophan: Tryptophan helps maintain your body’s correct nitrogen balance. It also helps make a brain chemical (neurotransmitter) called serotonin. Serotonin regulates your mood, appetite and sleep.
Valine: A Branched-chain Amino Acid, valine is involved in muscle growth, tissue regeneration and making energy.

Conditionally Non-Essential Amino Acids

Some nonessential amino acids are classified as conditional. This means they’re considered essential when you’re pregnant, ill, recovering, stressed, etc. Different references cite slightly different lists of these conditionally essential amino acids. Using Medline Plus - Amino Acids with a review date of 1/19/2023, (MedlinePlus is a service of the National Library of Medicine (NLM), the world's largest medical library, which is part of the National Institutes of Health (NIH)) conditionally essential amino acids include:
Arginine: Increases production of the vasodilator nitric oxide, which improves blood flow and reduces blood pressure.
Cysteine: A sulfur-containing amino acid that helps give proteins their structure. Cysteine also plays an important role in detoxification, neurotransmitter production, and collagen formation.
Glutamine: important for a variety of functions, including protein synthesis, energy production, ammonia detoxification, digestive tract health, glucose regulation, and immune system function.
Glycine: The most abundant amino acid in collagen, accounting for one third of the amino acids present. Glycine helps the body make glutathione and acts as an immunomodulator. Glycine also functions as a neurotransmitter, helps calm the central nervous system, and participates in the processing of motor and sensory information that permits movement, vision, and hearing. Glycine helps break down ingested fats by regulating the secretion of bile acids from the gallbladder into the small intestine.
Proline: Proline plays an important role in cellular regeneration and tissue repair. Proline is also a component of collagen and helps support healthy blood pressure and arterial elasticity, thus decreasing the risk of arteriosclerosis.
Serine: A keyplayer in metabolism for other amino acids and folate. Cell membranes rely on serine as well, as it’s incorporated into the phospholipids that surround and protect the cells. In addition, serine is essential for appropriate physical and mental functioning, it’s especially important for proper functioning of the brain and central nervous system.
Tyrosine: Tyrosine plays a role in protein synthesis and is involved in regulation of blood pressure as well as production of thyroid hormones, melanin, and a number of brain chemicals that affect mood, focus, and other aspects of cognitive function. Some of these important neurotransmitters include dopamine, epinephrine, and norepinephrine.

Nonessential Amino Acids

Your body produces the rest of the 11 amino acids you need. These are called nonessential amino acids. The nonessential amino acids are:

Alanine: Alanine breaks down tryptophan and vitamin B-6. It is a source of energy for muscles and the central nervous system. It strengthens the immune system and helps the body use sugars.
Asparagine Asparagine is widely used for the production of other nutrients such as glucose, proteins, lipids, and nucleotides. Asparagine is important in the metabolism of toxic ammonia in the body.
Aspartic acid: Aspartic acid helps every cell in the body work. It plays a role in hormone production and release, as well as normal nervous system function.
Glutamic acid: Glutamic acid turns into glutamate in the body. This is a chemical (neurotransmitter) that helps nerve cells in the brain send and receive information from other cells. It may be involved in learning and memory.
• Arginine*
• Cysteine*
• Glutamine*
• Glycine*
• Proline*
• Serine*
• Tyrosin*
Those marked with * are conditionally non-essential and addressed in the above discussion "Conditionally Non-Essential Amino Acids".

Branched-chain amino acids (BCAAs)

Three of the essential amino acids: leucine, isoleucine, and valine, are branched-chain amino acids (BCAAs). They stimulate the building of protein in muscle and possibly reduce muscle breakdown. Sacropenia, a condition where muscle mass, strength, and performance are significantly compromised with age is certainly a condition to be cognizant of as there are papers that have associated sarcopenia with cognitive decline, thus appearing to make BCAAs critical to ApoE4s. But BCAAs are a double edged sword, read more on cautions regarding BCAAs in the section below, "Issues for ApoE4s to consider regarding protein sources".


While loved by body-builders, Branched-chain Amino Acids (leucine, isoleucine, valine) have been correlated with severity of insulin resistance and possibly mitochondrial dysfunction, issues ApoE4s should be very mindful of. Read more on BCAAs in the "Issues for ApoE4s to consider regarding protein sources" below. Source of graphic: https://www.bodybuilding.com/content/ask-the-macro-manager-what-are-the-best-bcaa-food-sources.html

Aromatic amino acids

The word "aromatic" here refers to a specific class of organic compounds in chemistry, not to molecules with scents. Aromatic amino acids have a particular chemical structure, an aromatic ring. Among the 20 standard amino acids, histidine, phenylalanine, and tryptophan (essential amino acids) plus tyrosine (a non-essential/conditional amino acid) are classified as aromatic. In the MedicalXpress article, Research shows protein-rich diets may influence gut microbiome and body composition (American Society for Microbiology, 15 June 2024) the mice consuming aromatic amino-acid-rich proteins experienced the greatest weight and fat mass loss compared to those on standard protein and branched-chain amino-acid-rich protein diets.

Complete protein

A complete protein contains at least some of all nine essential amino acids. Most complete proteins come from animal sources (meat, fish, eggs, and dairy), but some plants, such as hemp and soy-derived foods, are also complete.

Incomplete protein

An incomplete protein is missing at least one of those nine essential amino acids. Most plant proteins are likely to be incomplete.

Don’t assume incomplete proteins are less nutritious than complete proteins. Many incomplete proteins happen to be great sources of fiber, vitamins, minerals, and antioxidants.

If you eat predominantly incomplete protein sources (such as if you’re a vegetarian or vegan), you do have to be more calculated about your intake. But with consuming a variety of these incomplete protein sources, adequate amounts of all nine essential amino acids can be obtained.

Glucogenic and Ketogenic Amino Acids

Amino acids can also be classified as glucogenic, ketogenic, or both according to how they are processed in the Krebs cycle, also known as the TCA (tricarboxylic acid cycle) cycle or the citric acid cycle.

Glucogenic amino acids – Glucogenic amino acids are amino acids that can be used for gluconeogenesis. Gluconeogenesis is a process whereby glucose is produced from non-carbohydrate sources. Amino acids that form pyruvate or intermediates of the Krebs/TCA cycle are glucogenic.

Ketogenic amino acids – Ketogenic amino acids are broken down into acetyl-CoA directly without passing through pyruvate, i.e. they form ketone bodies.

According to Ketogenic Amino Acid and Glucogenic Amino Acid

Glucogenic amino acids Both glucogenic and ketogenic Ketogenic amino acids
• alanine • isoleucine • leucine
• arginine • threonine • lysine
• cysteine • phenylalanine
• glycine • tyrosine
• glutamate • tryptophan
• histidine
• hydroxyproline
• proline
• methionine
• serine
• threonine
• valine

Why the need for more protein when older and why best to consume protein in a large dose vs sprinkled throughout the day

Among the many benefits that protein offers, protein consumption can help maintain muscle mass and strength.

Sarcopenia is a condition where muscle mass, strength, and performance become significantly compromised with age. Studies indicate that loss of muscle mass is correlated with loss of cognition, a concern of ApoE4s.

According to Sarcopenia in older adults (Jeremy D. Walston, 23 Jun 2014) causes of sarcopenia are multifactorial and interventions emphasize exercise and nutritional interventions.

One of these nutritional interventions is to consume protein in boluses (a single large dose) to overcome anabolic resistance.

According to Defining anabolic resistance: implications for delivery of clinical care nutrition (Robert W. Morton et al, April 2018):

Anabolic resistance is responsible, in part, for skeletal muscle atrophy with aging, muscle disuse, and during disease states. Anabolic resistance describes the reduced stimulation of muscle protein synthesis to a given dose of protein/amino acids and contributes to declines in skeletal muscle mass.

Also according to this paper,

Older individuals should perform exercise using both heavy and light loads three times per week, ingest at least 1.2 g of protein/kg/day, evenly distribute their meals into protein boluses of 0.40 g/kg, and consume protein within 2 h of retiring for sleep.

Consuming protein within 2 hours of retiring for sleep conflicts with brain health advice of overnight nightly fasting of a minimum of 12 hours for non-ApoE4 carriers and 14-16 hours for ApoE4 carriers with no food/caloric drink consumption at least 3 before bed. The rationale for this is this allows insulin to stay low and not suppress production of melatonin in the evening thus aiding a good night sleep which is critical for brain health. This abstinence before bed also allows the body to concentrate on other processes besides digestion while sleeping, such as brain waste cleaning. So weigh the pros and cons of this according to your individual situation.


Issues for ApoE4s to consider regarding protein sources

The three macronutrients. Source: Know Your Macros—Why Macronutrients Are Key to Healthy Eating https://www.cedars-sinai.org/blog/what-are-macronutrients.html

Protein is one of three macronutrients. Protein sources are not just strictly the macronutrient protein. For example, an egg has protein, fat, and even a little carbohydrate. So the source of the protein is an important consideration in light of the fact that in comparison to carriers of the other alleles, ApoE4s are at greater risk for dementia and cardiovascular disease, are poorer agers, pro-inflammatory, and need to pay particular attention to insulin sensitivity.

The below list is daunting and at first blush seems to offer no viable sources for protein consumption, but it can be done. The key may be in not over consuming one particular source, but in consuming a variety of clean, properly cooked, natural sources, and eating ancestrally since ApoE4 is the ancestral gene.

Despite the below cautions regarding certain plant sources, plants do offer valuable qualities to ApoE4s, including neuroprotective antioxidant, anti-inflammatory, anti-amyloid, and anticholinesterase properties. To read more, see the ApoE4.info wiki: Beneficial (and some negative) Plant/Natural Chemicals.

ApoE4s need to keep an eye on Saturated Fat and lipopolysaccharides (LPS)

It is well documented that ApoE4s hyper-absorb dietary fat and often see higher total cholesterol and LDL-C (low density lipoprotein cholesterol) levels with saturated fat intake, so protein sources high in saturated fat (meat and dairy) should especially be viewed with caution. Cholesterol imbalance can lead to cognitive concerns (source: Cholesterol imbalance and neurotransmission defects in neurodegeneration(Kyung Chul Shin et al, 1 Aug 2024) and saturated fat intake on Alzheimer's risk may be most evident in (or even limited to) carriers of the APOE ε4 allele (source: Saturated and trans fats and dementia: a systematic review(Neal D. Barnard et al, Sept 2014). For more info on ApoE4 and saturated fat, see the "Saturated Fat Sensitivity" section in ApoE ε4 and health conditions besides (or maybe contributing to) Alzheimer’s.

A high saturated fat diet comes with an additional concern not specific to ApoE4s, lipopolysaccharides (LPS). LPS are bacterial toxins in the body (an endotoxin) that, under normal circumstances are safely contained, but when introduced to the bloodstream, LPS are a significant cause of inflammation. Humans are greatly more sensitive to LPS than other animals (sources: Sepsis and the evolution of human increased sensitivity to lipopolysaccharide(Jessica F. Brinkworth & Negin Valizadegan, 10 Mar 2021) and Resilience to Bacterial Infection: Difference between Species Could Be Due to Proteins in Serum (H. Shaw Warren et al, 15 Jan 2010).

Saturated fat can increase the intestinal absorption of LPS. (High–Saturated Fat Diet Increases Endotoxemia4 High-Saturated Fat Diet increases Endotoxemia) (Judy Fulop ND, FABNO, 21 Jun 2018).

Here are some ways that saturated fat can increase LPS levels and inflammation:

•Saturated fat can promote the production of chylomicrons (particles that transport dietary lipids) in the gut, which can then transport LPS to other parts of the body. (Dietary fat induced chylomicron-mediated LPS translocation in a bicameral Caco-2cell model(Monic M. M. Tomassen et al, 12 Jan 2023)
•Saturated fat can increase gut permeability to LPS (Saturated Fat Ingestion Promotes Lipopolysaccharide-Mediated Inflammation and Insulin Resistance in Polycystic Ovary Syndrome(Frank González et al, Mar 2019)
•Saturated fat can promote changes in the gut microbiome that are similar to those observed in obesity, which can increase fatty acid absorption. (Saturated Fat Ingestion Promotes Lipopolysaccharide-Mediated Inflammation and Insulin Resistance in Polycystic Ovary Syndrome(Frank González et al, Mar 2019) and High–Saturated Fat Diet Increases Endotoxemia(Judy Fulop ND, FABNO, 21 Jun 2018).

Researchers have found that high blood levels of lipopolysaccharides (LPS) are associated with large amyloid deposits in the brain (Short-Chain Fatty Acids and Lipopolysaccharide as Mediators Between Gut Dysbiosis and Amyloid Pathology in Alzheimer’s Disease(Moira Marizzoni, et al, 10 Nov 2020).

This just briefly touches on this subject, to read more, see these two ApoE4.info wikis: Gut-Brain Connection: Leaky Gut/Leaky Brain, Microbiome (gut bugs) and Inflammation & LPS.

Use cooking methods to minimize harmful compounds

Meat, poultry, fish, etc. should be cooked by steaming, boiling, stewing, slow cooking or other methods to reduce production of Advanced Glycation End-products (AGEs), heterocyclic amines (HCAs), polycyclic aromatic hydrocarbons (PAHs).

AGEs are a group of proteins and lipids that are formed when they are exposed to sugars and undergo nonenzymatic glycation. They accumulate in the body and damage cells and tissues, thus producing ailments associated with aging. High levels of AGEs have been linked to oxidative stress, inflammation and multiple chronic diseases, including Alzheimer’s, heart disease, diabetes and more.

Cooking methods matter. Dry heat promotes new dietary AGE formation by >10 to 100-fold above the uncooked state (source: Advanced Glycation End Products in Foods and a Practical Guide to Their Reduction in the Diet (Jaime Uribarri et al, 8Jul 2013).

Several animal studies have shown that eating a low-AGE diet results in a lower risk of heart and kidney disease, increased insulin sensitivity, and lower levels of AGEs in blood and tissues by up to 53%. Similar results were observed in human studies. To read more on the health effects of AGEs, see the write-up under the subheading Negative health issues associated with Advanced Glycation End-Products (AGEs) in the "A Deeper Dive into the Science" section below.

HCAs and PAHs have been connected with certain cancers. HCAs are chemical compounds that can form when meat, poultry, or fish are cooked at high temperatures, like grilling, broiling, or pan-frying. HCAs are created when amino acids, creatine or creatinine, and sugars react together. PAHs are a class of organic compounds that are made up of multiple aromatic rings fused together. PAHs generally have a high degree of acute toxicity in humans.

Foods high in Advanced Glycation End-products (AGEs) (Based on data from Uribarri, et.al., 2010) Source of figure from The Nutrition Master Class article on AGEs https://www.nutritionmasterclass.com.ph/articles/ages-matter

Wild caught or Grass-fed and Grass Finished preferred

Wild caught fish or grass fed/finished meat sources are preferred over farmed sources. What an animal eats affects the quality and nutritional value of the product. Sources of meat, poultry, and fish generally aren’t fed well under farmed conditions. For example, cattle are natural grass eaters, but are typically given a diet rich in unnatural sources such as grain, corn, and soy, to fatten them up fast and cheap. Farmed beef is generally higher in fat content, and lower in nutrients including omega-3s. Omega-3s are especially important for ApoE4s.

Mind the Methionine

Methionine is an essential amino acid, the body needs it for important functions and as an essential amino acid, methionine must be obtained from food. However, more isn't necessarily better. Methionine elevates homocysteine so consumption should be moderated. (to read more see ApoE4.info wiki: Homocysteine: the brain, and vascular/cerebrovascular concerns. There is a well-established link between high levels of homocysteine in the blood and Alzheimer's disease (20 year literature review: Homocysteine and Dementia: An International Consensus Statement(A. David Smith et al, 20 Feb 2018). Sources high in methionine mostly come from animal sources of protein, but some plant sources can contain methionine too, see graphic below.

The western diet is high in the amino acid methionine. A link between high methionine intake and hyperhomocysteinemia and oxidative stress, mitochondrial dysfunction, inflammation, and accelerated epigenetic aging (all issues of concern for ApoE4s) has been found. Source: USDA Nutrient Database Release 28


Methionine glycine balance2.jpg

Methionine should also be consumed in balance with glycine. Glycine is a non-essential amino acid, meaning the body can make glycine on its own, but it is also consumed in the diet. Each gram of methionine increases the body's need for glycine by 0.5 to 1 gram. Glycine is used throughout the body and provides many benefits. (See this Medicine Net article: Top 9 Benefits and Uses of Glycine)

Methionine, glycine, and arginine are the three amino acids along with two enzymes that the body needs to make creatine. Creatine increases the levels of ATP (adenosine triphosphate) the primary energy source for cells. (see ApoE4.info wiki Mitochondria).

Creatine is well known for its muscle benefit: strength, lean muscle mass and muscle recovery, but it also promotes brain health. Studies have shown that creatine can improve cognitive performance in individuals with mild cognitive impairment, Alzheimer's disease, and Parkinson's disease. Sources:

Creatine2.jpg

To read more on the importance of the need to balance methionine and glycine see:






Neu5gc and inflammation

Meat from cattle, pigs and sheep contain Neu5gc which humans cannot synthesize, thus is a source of inflammation and consumption is best minimized. N-glycolylneuraminic acid, aka Neu5Gc, is a sialic acid molecule found in most non-human mammals. You may feel fine after consumption because inflammation is often silent and undetectable until health issues start to manifest as a result of chronic exposure. One of the health issues from chronic inflammation is Alzheimer's Disease (one source: Association between an inflammatory biomarker score and future dementia diagnosis in the population-based UK Biobank cohort of 500,000 people (Krisztina Mekli et al, 19 Jul 2023) also see Bredesen Protocol).

Be mindful of iron consumption

Iron overload is directly proportional to cognitive decline in Alzheimer's disease (AD). It may be that Alzheimer's disease itself causes an increase in iron levels in the brain, but maybe not. From this paper ApoE4 disrupts intracellular trafficking and iron homeostasis in an improved iPSC-based model of human brain endothelial cells (Luisa Bell et al, 3 Sept 2024) "Extensive evidence documents the impact of ApoE4 on BBB permeability and function 16-21. In this manuscript, we demonstrate for the first time how ApoE4 expression in brain endothelial cells leads to both disruption of sorting tubule formation and altered iron homeostasis." And from this paper, Autocrine Effects of Brain Endothelial Cell-Produced Human Apolipoprotein E on Metabolism and Inflammation in vitro (Felecia M. Marottoli et al, 9 Jun 2021), "Mitochondrial activity generates reactive oxygen species, and, with APOE4, there were higher mitochondrial superoxide levels, lower levels of antioxidants related to heme and glutathione and higher markers/outcomes of oxidative damage to proteins and lipids." Read more on heme iron next.

Heme Iron and insulin resistance connection

Heme iron from red meat and other animal products has been found to be associated with developing type 2 diabetes (T2D) and there is a significant coneection between insulin resistance and Alzheimer's, perhaps you've heard Alzheimer's sometimes nickname, Type 3 Diabetes. (For more info see ApoE4 wikis: Insulin Resistance, Insulin Resistance in the brain). This paper, Red meat intake and risk of type 2 diabetes in a prospective cohort study of United States females and males (Xiao Gu et al, 19 Oct 2023) tracked the eating habits of more than 200,000 people enrolled in long-term health studies for up to 36 years and found that those who regularly consumed a lot of red meat — more than a serving per day — had a significantly higher risk of developing Type 2 diabetes. And this paper Integration of epidemiological and blood biomarker analysis links haem iron intake to increased type 2 diabetes risk (Fenglei Wang et al, 13 Aug 2024) more clearly established and explained the connection between a higher intake of heme iron and higher risk of developing Type 2 diabetes. The paper is behind a paywall, however, you can read more about the paper from this MedicalXpress article, Significant link found between heme iron, found in red meat and other animal products, and type 2 diabetes risk. There have been other previous studies also identifying this association.

Higher intake of heme iron has been associated with a higher risk of developing insulin resistance and Type 2 diabetes. Source of graphic https://snacks-with-jax.com/2020/02/01/infant-iron-needs/

Branched-chain Amino Acids and insulin resistance connection

Branched-chain Amino Acids in excess may be troublemakers for ApoE4s as they’ve been found to be correlated with insulin resistance severity which can result in mitochondrial dysfunction. [Source: Excess Branched-Chain Amino Acids Suppress Mitochondrial Function and Biogenic Signaling but Not Mitochondrial Dynamics in a Myotube Model of Skeletal Muscle Insulin Resistance(Lindsey R. VanDerStad et al, 17 July 2024)]. Proper mitochondrial function is very important in ApoE4s, for more info see the ApoE4.info wiki: Mitochondria.

Insulin resistance and insulin resistance in the brain are also problematic concerns of ApoE4s (see wikis Insulin Resistance and Insulin Resistance in the brain).

Some studies regarding a connection beyween branched-chain amino acids and insulin resistance:

Insulin Resistance and Impaired Branched-Chain Amino Acid Metabolism in Alzheimer’s Disease (Liu, Rui et al, 30 May 2023)
Mounting evidence supports the concept that AD is an age-related metabolic neurodegenerative disease mediated in part by brain insulin resistance, and sharing similar metabolic dysfunctions and brain pathological characteristics that occur in type 2 diabetes mellitus (T2DM) and other insulin resistance disorders. Brain insulin signal pathway is a major regulator of branched-chain amino acid (BCAA) metabolism. In the past several years, impaired BCAA metabolism has been described in several insulin resistant states such as obesity, T2DM and cardiovascular disease. Disrupted BCAA metabolism leading to elevation in circulating BCAAs and related metabolites is an early metabolic phenotype of insulin resistance and correlated with future onset of T2DM. Brain is a major site for BCAA metabolism. BCAAs play pivotal roles in normal brain function, especially in signal transduction, nitrogen homeostasis, and neurotransmitter cycling. Evidence from animal models and patients support the involvement of BCAA dysmetabolism in neurodegenerative diseases including Huntington’s disease, Parkinson’s disease, and maple syrup urine disease.
Insulin action, type 2 diabetes, and branched-chain amino acids: A two-way street (Phillip J. White et al, Oct 2021)
In human cohorts, BCAA and related metabolites are now well established as among the strongest biomarkers of obesity, insulin resistance, T2D, and cardiovascular diseases. Lowering of BCAA and branched-chain ketoacid (BCKA) levels by feeding BCAA-restricted diet or by the activation of the rate-limiting enzyme in BCAA catabolism, branched-chain ketoacid dehydrogenase (BCKDH), in rodent models of obesity have clear salutary effects on glucose and lipid homeostasis, but BCAA restriction has more modest effects in short-term studies in human T2D subjects.
The Emerging Role of Branched-Chain Amino Acids in Insulin Resistance and Metabolism (Mee-Sup Yoon, 1 July 2016)
Insulin is required for maintenance of glucose homeostasis. Despite the importance of insulin sensitivity to metabolic health, the mechanisms that induce insulin resistance remain unclear. Branched-chain amino acids (BCAAs) belong to the essential amino acids, which are both direct and indirect nutrient signals. Even though BCAAs have been reported to improve metabolic health, an increased BCAA plasma level is associated with a high risk of metabolic disorder and future insulin resistance, or type 2 diabetes mellitus (T2DM). The activation of mammalian target of rapamycin complex 1 (mTORC1) by BCAAs has been suggested to cause insulin resistance. In addition, defective BCAA oxidative metabolism might occur in obesity, leading to a further accumulation of BCAAs and toxic intermediates. This review provides the current understanding of the mechanism of BCAA-induced mTORC1 activation, as well as the effect of mTOR activation on metabolic health in terms of insulin sensitivity. Furthermore, the effects of impaired BCAA metabolism will be discussed in detail.
Brain Insulin Lowers Circulating BCAA Levels by Inducing Hepatic BCAA Catabolism (Andrew C Shin, et al, 9 Oct 2014)
• Insulin, but not glucose, is a major regulator of circulating BCAAs
• Hypothalamic insulin lowers plasma BCAAs and induces hepatic BCAA catabolism
• Acute overfeeding impairs the ability of brain insulin to lower circulating BCAAs
• High-fat feeding in primates or obese/diabetic humans decreases hepatic BCKDH (branched-chain α-keto acid dehydrogenase)
Branched-chain amino acids in metabolic signalling and insulin resistance ((Christopher J. Lynch & Sean H. Adams, 7 Oct 2014)
Frequently, BCAAs have been reported to mediate antiobesity effects, especially in rodent models. However, circulating levels of BCAAs tend to be increased in individuals with obesity and are associated with worse metabolic health and future insulin resistance or type 2 diabetes mellitus (T2DM). A hypothesized mechanism linking increased levels of BCAAs and T2DM involves leucine-mediated activation of the mammalian target of rapamycin complex 1 (mTORC1), which results in uncoupling of insulin signalling at an early stage. A BCAA dysmetabolism model proposes that the accumulation of mitotoxic metabolites (and not BCAAs per se) promotes β-cell mitochondrial dysfunction, stress signalling and apoptosis associated with T2DM. Alternatively, insulin resistance might promote aminoacidaemia by increasing the protein degradation that insulin normally suppresses, and/or by eliciting an impairment of efficient BCAA oxidative metabolism in some tissues. Whether and how impaired BCAA metabolism might occur in obesity is discussed in this Review.

Eggs - pasture-raised or Omega-3 are preferred.

• As discussed above, farm fed sources of meat/fish/poultry generally aren’t fed well under farmed conditions, the effect extends to the eggs the chickens produce. With pasture-raised eggs, each hen gets a minimum of 108 square feet of pasture for themselves versus free-range eggs where the hens only receive a minimum of 2 square feet of pasture for themselves. Pastured hens roam free through rotated pastures. They get plenty of fresh air and sunshine with access to their natural nutrition sources: grass, worms, and bugs. Pasture-raised eggs may have more vitamin A, vitamin D, omega-3 fats, and carotenoid antioxidants.

• Omega-3s are critical for normal brain function and especially important for ApoE4s (see the ApoE4.info wiki: Fats, Omega -3(ω-3) & -6(ω-6), DHA and More under the subsection “Why Omega-3s are important for ApoE4s”). Chickens that produce Omega-3 eggs are fed a diet containing flaxseed, a plant that is a rich source of Omega-3. The eggs are higher in Omega-3s than regular eggs, but still not nearly as high as what can be obtained through fish.

Natural vs processed sources of protein preferred

• Natural sources of protein are preferred over processed sources which include protein powder, protein drinks, and protein bars.
• Clean, natural sources of meat are preferred over processed meats (bacon, ham, spam, hot dogs/sausages, deli meats, dried meat/jerky, cured meat, smoked meat, corned beef,etc.). As partially supported by papers in the Deeper Dive section below, processed meats are associated with numerous health concerns. Processed meats contain various chemical compounds that are not present in fresh meat. Studies consistently find strong links between processed meat consumption and various chronic diseases. An easy to read, introduction on processed meats can be found in this Healthline article, Why Processed Meat is Bad For You

Milk/dairy and Inflammation

Most people are unknowingly sensitive to diary. The sensitivity lies either in the sugar (lactose) or the protein (A-1 casein), thus raising inflammation. Of note, dairy can also raise homocysteine. If you must have some dairy, try A-2 cow milk. Or even better, try goat milk or sheep milk, both have the less inflammatory A-2 casein protein and contain Medium Chain Triglycerides (MCTs) which more easily convert to beneficial ketones.

Cheese

• Cheese is a dairy product, and as just discussed may also be a source of inflammation depending on sensitivity and milk source. If cheese is a non-negotiable in your diet, it may be best to stick with fermented cheese that contain probiotics, if the milk source of the fermented cheese is A-2, all the better. Fermented cheeses are hard cheeses that have been aged longer. They include varieties such as Cheddar, Swiss, and Parmesan.
• Do note some cheeses are not really cheese. The U.S. Food and Drug Administration (FDA) sets the rules for what qualifies as cheese and what doesn't. For instance, a product must contain at least 51% real cheese just to be labeled as a "pasteurized process cheese food." Anything that falls below that barely half-cheese standard may be considered a "cheese product". Popular products that are "cheese adjacent" include Velveeta, Kraft singles, Kraft Parmesan, Cheese Whiz, and in a spray can Easy Cheese. These often contain other ingredients which make it more a processed food than a natural, clean food source.

Fish - best sources and especially beneficial for ApoE4s

• Mercury is a neurotoxin that can cause cognitive decline. Since they are at the top of the food chain, large fish tend to have the highest levels of mercury and should therefore be avoided. The grouping of fish called SMASH fish (Salmon, Mackerel, Anchovy, Sardine, Herring) and shellfish (shrimp [preferably wild-caught], crab [preferably wild-caught], mussels, clams, oysters) are preferable to large fish sources.
• Dr. Hussein Yassine is an expert in brain lipid metabolism. His lab at the University of Southern California (USC), the Yassine Lab conducts research with a focus on Alzheimer’s disease as it relates to APOE. Dr Yassine recommends ApoE4s consume fish AT LEAST once a week.
• From Table 2 of Diet’s Role in Modifying Risk of Alzheimer’s Disease: History and Present Understanding (William B. Grant and Steven M. Blake 28 Oct 2023), while meat supply held the highest correlation with Alzheimer's Disease, fish held a positive (inverse) association.
• High fish intake is associated with a lower risk of mortality and slowed cognitive decline among APOE4 carriers, suggesting a protective effect.

Interaction between APOE ε4 and dietary protein intake on cognitive decline: A longitudinal cohort study (Yun Zhang et al, 29 April 2021) found that frequent fish intake is linked to slower cognitive decline in APOE4 carriers, with a more pronounced benefit observed in women. It also found that diversified protein intake, including fish, may mitigate the negative cognitive effects associated with the APOE4 allele.
Apolipoprotein E Genotype, Meat, Fish, and Egg Intake in Relation to Mortality Among Older Adults: A Longitudinal Analysis in China (Xurui Jin et al, 20 Jul 2021) observed an inverse association between fish intake and mortality which was only significant among APOE ε4 carriers.

Fish Roe (fish eggs) come with a bonus for ApoE4s

The Omega-3 fatty acids EPA and DHA are critical for normal brain function (see the ApoE4.info wiki: Fats, Omega -3(ω-3) & -6(ω-6), DHA and More under the subsection “Why Omega-3s are important for ApoE4s”). Fish Roe (fish eggs) can be a nice source of protein with the extra added bonus of providing a source of DHA that is thought to be especially beneficial for ApoE4s. Fish and particularly fish roe provides DHA in phospholipid form. This form of DHA is more efficiently absorbed by the brain in individuals with the APOE4 gene, as ApoE4s have impaired transport of the more common nonesterified form of DHA often found in fish oil supplements.

Organic plant protein preferred over non-organic

Organic sources are better than those sprayed with herbicides, pesticides or are genetically modified.
Apoe4s may be even more sensitive to non-organic foods. This paper Elevated Serum Pesticide Levels and Risk for Alzheimer Diseasepaper found, "Elevated serum DDE levels are associated with an increased risk for AD and carriers of an APOE4 ε4 allele may be more susceptible to the effects of DDE. Both DDT and DDE increase amyloid precursor protein levels, providing mechanistic plausibility for the association of DDE exposure with AD."

Legumes and inflammatory lectins

Legumes (peas, peanuts, beans, lentils) are high in lectins which are inflammatory, however if the legumes are pressure cooked, the lectins can be inactivated.

Grains/gluten and leaky gut

Hyman grains.jpg

•Grains (wheat, rice, oats, cornmeal, barley) and grain products (bread, pasta, breakfast cereals, grits, and tortillas) can be sources of protein, however, they contain gluten. Gluten activates the protein zonulin in the body. Zonulin opens of the tight junctions between cells of the wall of the digestive tract leading to leaky gut which in turn can lead to a leaky brain barrier thus introduing neuroinflammation. This occurs even if someone doesn't have celiac disease. (see Gut-Brain Connection: Leaky Gut/Leaky Brain, Microbiome (gut bugs)).
•In the introduction of the section we mentioned about eating ancestrally. ApoE4 is the ancestral gene having existed since the dawn of man and for the vast majority of human history everyone was an APOEε4/4 which we share with evolutionary primate anscestors. Our human hunter-gatherer ancestors ate a variety of food, but no grains. Grains were introduced to the human diet very recently in evolutionary terms. Dr Dale Bredesen, neurologist and author of books, The End of Alzheimer's and The End of Alzheimer's Program as well as Dr David Perlmutter, neurologist and author of The Grain Brain along with other books, both recommend abstaining from grains as a measure to reduce the risk of Alzheimer's.

Whole grains and Wheat Germ Agglutinin

Whole grains, but not refined grains that have been milled to remove the bran and germ, have the additional concern of Wheat Germ Agglutinin (WGA). Papers on this subject were found to be very science-heavy so for a simple explanation, per this source Wheat Germ Agglutinin: The Evil Relative of Gluten wheat germ agglutinin is a lectin that:

• Acts like insulin, causing weight gain and insulin resistance by pumping sugar into fat cells, where that sugar turns into fat.
• Blocks sugar from nourishing muscles, instead turning that sugar into fat.
• Impedes digestion of protein.

Watch the glycemic load of plant protein sources

The carbohydrate content of plant protein sources are best moderated to maintain proper blood glucose and insulin levels.

Soy and phytoestrogens

Soy is probably best if not overconsumed as they contain phytoestrogens which are similar in function to human estrogen although with much weaker effects; soy isoflavones can bind to estrogen receptors in the body and cause either weak estrogenic or anti-estrogenic activity.

A Deeper Dive into the Science

The below references pay particular attention to ApoE4 concerns of dementia, cardiovascular disease, inflammation, insulin resistance, mitochondrial health, and longevity.


Research in favor of higher protein or certain amino acids

This paper, Proportions of macronutrients, including specific dietary fats, in prospective anti-Alzheimer’s diet(Marcin Studnicki et al, 2019) states that protein consumption should vary based on the phase of a person's life with early and late stages needing the most protein. From the conclusions section of this paper:

We predict that the proportions of macronutrients in the diet in four periods of life (youth, early middle age, late middle age, late age) should differ, especially in regard to protein intake. Specifically, we postulate that protein intake in early middle age and late middle age should be reduced by half by comparison to historical consumption in the USA. Conversely, in the late age, the protein intake should be increased above the historical consumption. We postulate that applying these changes in the consumption of protein throughout the whole life of an individual will act prophylactically against development of AD and possibly will extend health-span and the duration of life.


This article, Research shows protein-rich diets may influence gut microbiome and body composition(American Society for Microbiology, 15 June 2024) discusses a mouse study (which don’t always translate to humans) presented at the annual meeting of the American Society for Microbiology, held June 13–17, 2024, in Atlanta, Georgia, (unknown if there is a peer-reviewed publication of that study) where a switch to a protein-rich diet resulted in significant weight loss, reduced body fat and induced immediate changes to the gut microbiome. Greater results in weight loss and reduced fat came from the mice consuming aromatic amino-acid-rich proteins compared to those on standard protein and branched-chain amino-acid-rich protein diets. The presentation also explored how undigested protein in the colon can be fermented to produce beneficial metabolites, such as short-chain fatty acids (SCFAs), or lead to the production of harmful metabolites like ammonia and sulfides, which are linked to gastrointestinal disorders and other health issues.


Research against high protein or certain amino acids

Excess Branched-Chain Amino Acids Suppress Mitochondrial Function and Biogenic Signaling but Not Mitochondrial Dynamics in a Myotube Model of Skeletal Muscle Insulin Resistance (Lindsey R. VanDerStad et al, 17 July 2024)

Abstract
Branched-chain amino acids (BCAA) are correlated with severity of insulin resistance, which may partially result from mitochondrial dysfunction. Mitochondrial dysfunction is also common during insulin resistance and is regulated in part by altered mitochondrial fusion and fission (mitochondrial dynamics). To assess the effect of BCAA on mitochondrial dynamics during insulin resistance, the present study examined the effect of BCAA on mitochondrial function and indicators of mitochondrial dynamics in a myotube model of insulin resistance. C2C12 myotubes were treated with stock DMEM or DMEM with additional BCAA at 0.2 mM, 2 mM, or 20 mM to achieve a continuum of concentrations ranging from physiologically attainable to supraphysiological (BCAA overload) both with and without hyperinsulinemia-mediated insulin resistance. qRT-PCR and Western blot were used to measure gene and protein expression of targets associated with mitochondrial dynamics. Mitochondrial function was assessed by oxygen consumption, and mitochondrial content was measured using mitochondrial-specific staining. Insulin resistance reduced mitochondrial function, peroxisome proliferator-activated receptor gamma coactivator 1-alpha mRNA, and citrate synthase expression mRNA, but not protein expression. Excess BCAA at 20 mM also independently reduced mitochondrial function in insulin-sensitive cells. BCAA did not alter indicators of mitochondrial dynamics at the mRNA or protein level, while insulin resistance reduced mitochondrial fission protein 1 mRNA, but not protein expression. Collectively, BCAA at excessively high levels or coupled with insulin resistances reduce mitochondrial function and content but do not appear to alter mitochondrial dynamics under the tested conditions.


From Metabolomic and Proteomic Analysis of ApoE4-Carrying H4 Neuroglioma Cells in Alzheimer’s Disease Using OrbiSIMS and LC-MS/MS(Li Lu et al, 11 July 2024)

Our results have elucidated the metabolic alterations in H4 neuroglioma cells in the presence of ApoE4, supporting the hypothesis on the low ability of ApoE4 in transporting lipids and discovering amino acid pathways that may be involved in AD.
In addition, the remaining nine secondary ions assigned by comparison with the HMDB are enriched in the ApoE4 KI cells compared to control, mostly in alanine, aspartate, and glutamate metabolism (Figure 2d,e), suggesting the metabolic disorders of these three amino acids.
Levels of alanine, aspartate, and glutamate all increase in the ApoE4 KI group, which partially contrasts with other metabolomics studies showing their low concentrations in AD patients. (27)”
Combining both methods, we identified that the presence of ApoE4 in H4 cells significantly affects the lipid metabolism (glycerophospholipids and sphingolipids) and amino acid metabolism, including alanine, aspartate, and glutamate metabolism; aminoacyl-tRNA biosynthesis; glutamine metabolism; and taurine and hypotaurine metabolism.


Protein restriction slows the development and progression of pathology in a mouse model of Alzheimer’s disease(Reji Babygirija et al, 18 June 2024). Caution advised as this is a mouse model which don't always translate to humans, but from the paper:

Dietary protein is a critical regulator of metabolic health and aging. Low protein diets are associated with healthy aging in humans, and dietary protein restriction extends the lifespan and healthspan of mice. In this study, we examined the effect of protein restriction (PR) on metabolic health and the development and progression of Alzheimer’s disease (AD) in the 3xTg mouse model of AD. Here, we show that PR promotes leanness and glycemic control in 3xTg mice, specifically rescuing the glucose intolerance of 3xTg females. PR induces sex-specific alterations in circulating and brain metabolites, downregulating sphingolipid subclasses in 3xTg females. PR also reduces AD pathology and mTORC1 activity, increases autophagy, and improves the cognition of 3xTg mice. Finally, PR improves the survival of 3xTg mice. Our results suggest that PR or pharmaceutical interventions that mimic the effects of this diet may hold promise as a treatment for AD.


BCAAs acutely drive glucose dysregulation and insulin resistance: role of AgRP neurons(Harsh Shah et al, 6 June 2024)

These findings suggest that BCAAs per se can acutely impair glucose homeostasis and insulin sensitivity, thus offering an explanation for how they may disrupt glucose metabolism in the long-term as observed in obesity and diabetes. Our findings also reveal that AgRP neuronal regulation of blood glucose is mediated through BCAAs, further elucidating a novel mechanism by which brain controls glucose homeostasis.


Dietary restriction of isoleucine increases healthspan and lifespan of genetically heterogeneous mice (Cara L. Green et al, 7 Nov 2023)

Highlights
• Isoleucine restriction (IleR) improves metabolic health in both sexes
• IleR reprograms hepatic metabolism in a sex- and age-dependent manner
• IleR reduces frailty and increases lifespan, with stronger effects on male lifespan
• Amino acid restriction begun at 6 months extends healthspan but not lifespan
Summary
Low-protein diets promote health and longevity in diverse species. Restriction of the branched-chain amino acids (BCAAs) leucine, isoleucine, and valine recapitulates many of these benefits in young C57BL/6J mice. Restriction of dietary isoleucine (IleR) is sufficient to promote metabolic health and is required for many benefits of a low-protein diet in C57BL/6J males. Here, we test the hypothesis that IleR will promote healthy aging in genetically heterogeneous adult UM-HET3 mice. We find that IleR improves metabolic health in young and old HET3 mice, promoting leanness and glycemic control in both sexes, and reprograms hepatic metabolism in a sex-specific manner. IleR reduces frailty and extends the lifespan of male and female mice, but to a greater degree in males. Our results demonstrate that IleR increases healthspan and longevity in genetically diverse mice and suggests that IleR, or pharmaceuticals that mimic this effect, may have potential as a geroprotective intervention.


The Role of Methionine-Rich Diet in Unhealthy Cerebrovascular and Brain Aging: Mechanisms and Implications for Cognitive Impairment(Anna Ungvari et al, 3 Nov 2023)

As aging societies in the western world face a growing prevalence of vascular cognitive impairment and Alzheimer’s disease (AD), understanding their underlying causes and associated risk factors becomes increasingly critical. A salient concern in the western dietary context is the high consumption of methionine-rich foods such as red meat. The present review delves into the impact of this methionine-heavy diet and the resultant hyperhomocysteinemia on accelerated cerebrovascular and brain aging, emphasizing their potential roles in cognitive impairment. Through a comprehensive exploration of existing evidence, a link between high methionine intake and hyperhomocysteinemia and oxidative stress, mitochondrial dysfunction, inflammation, and accelerated epigenetic aging is drawn. Moreover, the microvascular determinants of cognitive deterioration, including endothelial dysfunction, reduced cerebral blood flow, microvascular rarefaction, impaired neurovascular coupling, and blood–brain barrier (BBB) disruption, are explored. The mechanisms by which excessive methionine consumption and hyperhomocysteinemia might drive cerebromicrovascular and brain aging processes are elucidated. By presenting an intricate understanding of the relationships among methionine-rich diets, hyperhomocysteinemia, cerebrovascular and brain aging, and cognitive impairment, avenues for future research and potential therapeutic interventions are suggested.


High-protein diets increase cardiovascular risk by activating macrophage mTOR to suppress mitophagy (X. Zhang et al, 23 Jan 2020) From Abstract:

Here, we show that dietary protein drives atherosclerosis and lesion complexity. Protein ingestion acutely elevates amino acid levels in blood and atherosclerotic plaques, stimulating macrophage mTOR signaling. This is causal in plaque progression as the effects of dietary protein are abrogated in macrophage-specific Raptor-null mice. Mechanistically, we find amino acids exacerbate macrophage apoptosis induced by atherogenic lipids, a process that involves mTORC1-dependent inhibition of mitophagy, accumulation of dysfunctional mitochondria, and mitochondrial apoptosis. Using macrophage-specific mTORC1- and autophagy-deficient mice we confirm this amino acid-mTORC1-autophagy signaling axis in vivo. Our data provide the first insights into the deleterious impact of excessive protein ingestion on macrophages and atherosclerotic progression. Incorporation of these concepts in clinical studies will be important to define the vascular effects of protein-based weight loss regimens.


Protein Quantity and Source, Fasting-Mimicking Diets, and Longevity (Sebastian Brandhorst and Valter D Longo, 15 Nov 2019)

ABSTRACT
Dietary modifications, including caloric restriction, dietary restriction, various intervals of fasting, and even limiting the time when food is consumed can have a pronounced impact on longevity. In addition, dietary modifications are powerful interventions to delay, prevent, or treat many aging-related diseases such as cancer and diabetes. Restricting amino acid and protein intake generally decreases aging-related comorbidities and thereby increases health and longevity. However, chronic dietary interventions are likely not feasible for most people due to low adherence to dietary protocols or resistance to drastic changes to lifestyle, and might even cause detrimental effects, possibly by negatively affecting the immune system and wound healing. The periodic use of low-protein, low-calorie fasting-mimicking diets (FMDs) has the potential to promote health benefits, while minimizing the burden of chronic restriction. Protein restriction and FMDs together have the potential to play an important complementary role in medicine by promoting disease prevention and treatment, and by delaying the aging process at least in part by stimulating stem cell–based regeneration in periods of normal food intake after periodic FMD cycles. The aim of this narrative review is to summarize research on the impact of protein restriction on health and longevity in model organisms and to discuss the implementation of an FMD in mice and in human clinical trials and its effects on biomarkers of healthy aging. Taking into account the importance of sex on aging and diet, we include this information in all discussed studies. Whereas for some model organisms of aging, such as rodents, many studies are available, results are more limited for primates and/or humans.


Low Protein Intake is Associated with a Major Reduction in IGF-1, Cancer, and Overall Mortality in the 65 and Younger but Not Older Population (Morgan E. Levine et al, 4 Mar 2014)

Mice and humans with Growth Hormone Receptor/IGF-1 deficiencies display major reductions in age-related diseases. Because protein restriction reduces GHR-IGF-1 activity, we examined links between protein intake and mortality. Respondents (n=6,381) aged 50–65 reporting high protein intake had a 75% increase in overall mortality and a 4-fold increase in cancer and diabetes mortality during an 18 year follow up period. These associations were either abolished or attenuated if the source of proteins was plant-based. Conversely, in respondents over age 65, high protein intake was associated with reduced cancer and overall mortality. Mouse studies confirmed the effect of high protein intake and the GHR-IGF-1 axis on the incidence and progression of breast and melanoma tumors, and also the detrimental effects of a low protein diet in the very old. These results suggest that low protein intake during middle age followed by moderate protein consumption in old subjects may optimize healthspan and longevity.


Chapter Eleven - The Impact of Dietary Methionine Restriction on Biomarkers of Metabolic Health (Manda L. Orgeron et al, 26 Dec 2013)

Over the last 20 years, dietary methionine restriction (MR) has emerged as a promising DR [dietary restriction] mimetic because it produces a comparable extension in life span, but surprisingly, does not require food restriction. Dietary MR also reduces adiposity but does so through a paradoxical increase in both energy intake and expenditure. The increase in energy expenditure fully compensates for increased energy intake and effectively limits fat deposition. Perhaps more importantly, the diet increases metabolic flexibility and overall insulin sensitivity and improves lipid metabolism while decreasing systemic inflammation.


Adverse Effects Associated with Protein Intake above the Recommended Dietary Allowance for Adults(Ioannis Delimaris, 18 July 2013)

The adverse effects associated with long-term high protein/high meat intake in humans were (a) disorders of bone and calcium homeostasis, (b) disorders of renal function, (c) increased cancer risk, (d) disorders of liver function, and (e) precipitated progression of coronary artery disease. Conclusions. The findings of the present study suggest that there is currently no reasonable scientific basis in the literature to recommend protein consumption above the current RDA (high protein diet) for healthy adults due to its potential disease risks.


Low carbohydrate–high protein diet and mortality in a cohort of Swedish women(P. Lagiou et al, 22 March 2007). This study consisted of 237 women, 30–49 years old at baseline.

Conclusions. A diet characterized by low carbohydrate and high protein intake was associated with increased total and particularly cardiovascular mortality amongst women. Vigilance with respect to long-term adherence to such weight control regimes is advisable.


The atherogenic effect of excess methionine intake ((Aron M. Troen et al, 1 Dec 2003). Atherogenic refers to the tendency to promote the formation of fatty plaques in the arteries. Caution, mouse study.

Methionine is the precursor of homocysteine, a sulfur amino acid intermediate in the methylation and transsulfuration pathways. Elevated plasma homocysteine (hyperhomocysteinemia) is associated with occlusive vascular disease. Whether homocysteine per se or a coincident metabolic abnormality causes vascular disease is still an open question. Animals with genetic hyperhomocysteinemia have so far not displayed atheromatous lesions. However, when methionine-rich diets are used to induce hyperhomocysteinemia, vascular pathology is often observed. Such studies have not distinguished the effects of excess dietary methionine from those of hyperhomocysteinemia. We fed apolipoprotein E-deficient mice with experimental diets designed to achieve three conditions: (i) high methionine intake with normal blood homocysteine; (ii) high methionine intake with B vitamin deficiency and hyperhomocysteinemia; and (iii) normal methionine intake with B vitamin deficiency and hyperhomocysteinemia. Mice fed methionine-rich diets had significant atheromatous pathology in the aortic arch even with normal plasma homocysteine levels, whereas mice fed B vitamin-deficient diets developed severe hyperhomocysteinemia without any increase in vascular pathology. Our findings suggest that moderate increases in methionine intake are atherogenic in susceptible mice. Although homocysteine may contribute to the effect of methionine, high plasma homocysteine was not independently atherogenic in this model. Some product of excess methionine metabolism rather than high plasma homocysteine per se may underlie the association of homocysteine with vascular disease.

Animal vs Plant Sources of protein

From Diet’s Role in Modifying Risk of Alzheimer’s Disease: History and Present Understanding (William B. Grant and Steven M. Blake 28 Oct 2023):

As Table 2 shows, meat supply became the food group with the highest correlation with AD, followed by fat, fish (inverse), and total energy.


The Isocaloric Substitution of Plant-Based and Animal-Based Protein in Relation to Aging-Related Health Outcomes: A Systematic Review (Jiali Zheng et al, 9 Jan 2022). Isocaloric means having the equal caloric value.

Plant-based and animal-based protein intake have differential effects on various aging-related health outcomes, but less is known about the health effect of isocaloric substitution of plant-based and animal-based protein. This systematic review summarized current evidence of the isocaloric substitutional effect of plant-based and animal-based protein on aging-related health outcomes. PubMed and Embase databases were searched for epidemiologic observational studies published in English up to 15 March 2021. Studies that included adults ≥18 years old; use of a nutritional substitution model to define isocaloric substitution of plant protein and animal protein; health outcomes covering mortality, aging-related diseases or indices; and reported association estimates with corresponding 95% confidence intervals were included. Nine cohort studies and 3 cross-sectional studies were identified, with a total of 1,450,178 subjects included in this review. Consistent and significant inverse association of substituting plant protein for various animal proteins on all-cause mortality was observed among 4 out of 5 studies with relative risks (RRs) from 0.54 to 0.95 and on cardiovascular disease (CVD) mortality among all 4 studies with RRs from 0.58 to 0.91. Among specific animal proteins, the strongest inverse association on all-cause and CVD mortality was identified when substituting plant protein for red and/or processed meat protein, with the effect mainly limited to bread, cereal, and pasta protein when replacing red meat protein. Isocaloric substitution of plant-based protein for animal-based protein might prevent all-cause and CVD-specific mortality. More studies are needed on this topic, particularly for cancer incidence and other specific aging-related diseases.
5. Conclusions
In summary, findings from this review suggested isocaloric substitution of plant-based protein for animal-based protein was inversely associated with risk of all-cause and CVD mortality, with the protective effect primarily contributed by substituting bread, cereal, and pasta protein for red meat protein. Given the limited number of studies on each outcome of this review, more studies with different aging-related health outcomes and diverse study populations are needed to accumulate more evidence and confirm our findings. These preliminary findings may provide important public health implications as well as recommendations of introducing plant protein-rich sources to replace animal proteins to prevent aging-related diseases, and promote longevity and healthy aging.


Protein foods from animal sources, incident cardiovascular disease and all-cause mortality: a substitution analysis(Victor W Zhong et al, 7 Jan 2021). This was a pooled analysis of six prospective cohort studies of 29 682 US participants. Data were collected in 1985–2016.

Conclusions
Nuts, whole grains, legumes and fish appeared to be healthier protein sources than eggs, processed meat, unprocessed red meat and poultry for preventing incident CVD and premature death. The magnitude of lower risk for incident CVD and all-cause mortality was driven by amount and number of animal protein foods substituted.
Key Messages
• Nuts, whole grains, legumes and fish appeared to be healthier sources of protein than eggs, processed meat, unprocessed red meat and poultry for preventing cardiovascular disease and premature death.
• Substituting two or more healthier protein foods for unhealthier protein foods, even for a small amount such as one serving per week, was associated with appreciably lower risks of cardiovascular disease and premature death.
• Considerably lower risks of cardiovascular disease and premature death were seen in smaller subsets of participants who were consuming more animal protein foods (e.g. one serving per day or more) and thus had greater room for reducing intake.
• Effect sizes resulting from substituting different protein sources wisely were comparable to effect sizes associated with improving other lifestyle factors, in relation to risks of cardiovascular disease and premature death, such as alcohol intake, exercise, smoking and body weight.


Association Between Plant and Animal Protein Intake and Overall and Cause-Specific Mortality (Jiaqi Huang PhD et al, 13 Jul 2020) In this cohort of 237,036 men and 179,068 women with 16 years of observation and nearly 78,000 deaths, greater intake of plant protein was significantly associated with lower overall mortality and cardiovascular disease mortality independent of several other risk factors.

Conclusions and Relevance
In this large prospective cohort, higher plant protein intake was associated with small reductions in risk of overall and cardiovascular disease mortality. Our findings provide evidence that dietary modification in choice of protein sources may influence health and longevity.


Dietary protein intake and all-cause and cause-specific mortality: results from the Rotterdam Study and a meta-analysis of prospective cohort studies(Zhangling Chen et al, 19 Feb 2020)

Abstract
Evidence for associations between long-term protein intake with mortality is not consistent. We aimed to examine associations of dietary protein from different sources with all-cause and cause-specific mortality. We followed 7786 participants from three sub-cohorts of the Rotterdam Study, a population-based cohort in the Netherlands. Dietary data were collected using food-frequency questionnaires at baseline (1989–1993, 2000–2001, 2006–2008). Deaths were followed until 2018. Associations were examined using Cox regression. Additionally, we performed a highest versus lowest meta-analysis and a dose–response meta-analysis to summarize results from the Rotterdam Study and previous prospective cohorts. During a median follow-up of 13.0 years, 3589 deaths were documented in the Rotterdam Study. In this cohort, after multivariable adjustment, higher total protein intake was associated with higher all-cause mortality [e.g. highest versus lowest quartile of total protein intake as percentage of energy (Q4 versus Q1), HR = 1.12 (1.01, 1.25)]; mainly explained by higher animal protein intake and CVD mortality [Q4 versus Q1, CVD mortality: 1.28 (1.03, 1.60)]. The association of animal protein intake and CVD was mainly contributed to by protein from meat and dairy. Total plant protein intake was not associated with all-cause or cause-specific mortality, mainly explained by null associations for protein from grains and potatoes; but higher intake of protein from legumes, nuts, vegetables, and fruits was associated with lower risk of all-cause and cause-specific mortality. Findings for total and animal protein intake were corroborated in a meta-analysis of eleven prospective cohort studies including the Rotterdam Study (total 64,306 deaths among 350,452 participants): higher total protein intake was associated with higher all-cause mortality [pooled RR for highest versus lowest quantile 1.05 (1.01, 1.10)]; and for dose–response per 5 energy percent (E%) increment, 1.02 (1.004, 1.04); again mainly driven by an association between animal protein and CVD mortality [highest versus lowest, 1.09 (1.01, 1.18); per 5 E% increment, 1.05 (1.02, 1.09)]. Furthermore, in the meta-analysis a higher plant protein intake was associated with lower all-cause and CVD mortality [e.g. for all-cause mortality, highest versus lowest, 0.93 (0.87, 0.99); per 5 E% increment, 0.87 (0.78, 0.98), for CVD mortality, highest versus lowest 0.86 (0.73, 1.00)]. Evidence from prospective cohort studies to date suggests that total protein intake is positively associated with all-cause mortality, mainly driven by a harmful association of animal protein with CVD mortality. Plant protein intake is inversely associated with all-cause and CVD mortality. Our findings support current dietary recommendations to increase intake of plant protein in place of animal protein.
In conclusion, our study provides evidence that higher total protein intake is associated with higher all-cause mortality, primarily driven by a positive association between animal protein intake and CVD mortality. In contrast, higher plant protein intake is associated with lower all-cause and CVD mortality. Food source and level of protein may play a substantial role as we observed harmful associations of total and animal protein mainly in North American and European populations and beneficial associations of plant protein mainly in North American and Japanese populations. Further studies in other populations with different amounts and food sources of protein intakes or with different protein requirements are needed to improve global dietary recommendations and to define optimal ranges and sources of protein intake for different populations.


The Role of Meat Protein in Generation of Oxidative Stress and Pathophysiology of Metabolic Syndromes(Muhammad Ijaz Ahmad et al, Jan 2020)

Various processing methods have a great impact on the physiochemical and nutritional properties of meat that are of health concern. Hence, the postmortem processing of meat by different methods is likely to intensify the potential effects on protein oxidation. The influence of meat protein oxidation on the modulation of the systemic redox status and underlying mechanism is well known. However, the effects of processed meat proteins isolated from different sources on gut microbiota, oxidative stress biomarkers, and metabolomic markers associated with metabolic syndromes are of growing interest. The application of advanced methodological approaches based on OMICS, and mass spectrometric technologies has enabled to better understand the molecular basis of the effect of processed meat oxidation on human health and the aging process. Animal studies indicate the involvement of dietary proteins isolated from different sources on health disorders, which emphasizes the impact of processed meat protein on the richness of bacterial taxa such as (Mucispirillum, Oscillibacter), accompanied by increased expression of lipogenic genes. This review explores the most recent evidences on meat processing techniques, meat protein oxidation, underlying mechanisms, and their potential effects on nutritional value, gut microbiota composition and possible implications on human health.
Conclusion
There is a growing interest in processed meat proteins and impact of processing methods on nutrition and health. A full understanding of the chemistry behind the meat protein oxidation is paramount in terms of food quality and consumer health. Oxidative and nitrosative modification of proteins in muscle is a topic to be further explored. The identification of chemistry fundamentals of protein oxidation products in processed meat is required to assess their potential toxicity. Although the effects of isolated dietary protein from different sources by our group open a new chapter to better understand the underlying mechanism and potential pathways on gut health and different axis in the field of nutrigenomics. Future studies are needed to better understand the effects of processing methods on the dietary protein composition, nutritional value and their consequences. Furthermore, meta-proteomic, metabolomic, and meta-transcriptomic studies are needed to identify the changes in the gut microbiota as a result of dietary challenge.


Association of changes in red meat consumption with total and cause specific mortality among US women and men: two prospective cohort studies (Yan Zheng et al, 12 June 2019)

Participants: 53,553 women and 27,916 men without cardiovascular disease or cancer at baseline.
Conclusion: Increases in red meat consumption, especially processed meat, were associated with higher overall mortality rates.


Dietary proteins and protein sources and risk of death: the Kuopio Ischaemic Heart Disease Risk Factor Study(Virtanen Heli EK et al, May 2019). The study population consisted of 2,641 Finnish men, aged 42–60 y at baseline in 1984–1989.

Conclusions
Higher ratio of animal to plant protein in diet and higher meat intake were associated with increased mortality risk. Higher total protein intake appeared to be associated with mortality mainly among those with a predisposing disease. This trial was registered at clinicaltrials.gov as NCT03221127.


Patterns of plant and animal protein intake are strongly associated with cardiovascular mortality: the Adventist Health Study-2 cohort(Marion Tharrey et al, 2 Apr 2018). This study selected 81,337 men and women from the Adventist Health Study-2.

Conclusions
Associations between the ‘Meat’ and ‘Nuts & Seeds’ protein factors and cardiovascular outcomes were strong and could not be ascribed to other associated nutrients considered to be important for cardiovascular health. Healthy diets can be advocated based on protein sources, preferring low contributions of protein from meat and higher intakes of plant protein from nuts and seeds.


Health Risks Associated with Meat Consumption: A Review of Epidemiological Studies(Evelyne Battaglia Richi et al, 18 Jan 2016)

Abstract. Recent evidence from large prospective US and European cohort studies and from meta-analyses of epidemiological studies indicates that the long-term consumption of increasing amounts of red meat and particularly of processed meat is associated with an increased risk of total mortality, cardiovascular disease, colorectal cancer and type 2 diabetes, in both men and women. The association persists after inclusion of known confounding factors, such as age, race, BMI, history, smoking, blood pressure, lipids, physical activity and multiple nutritional parameters in multivariate analysis. The association has not always been noted with red meat, and it has been absent with white meat. There is evidence of several mechanisms for the observed adverse effects that might be involved, however, their individual role is not defined at present. It is concluded that recommendations for the consumption of unprocessed red meat and particularly of processed red meat should be more restrictive than existing recommendations. Restrictive recommendations should not be applied to subjects above about 70 years of age, as the studies quoted herein did not examine this age group, and the inclusion of sufficient protein supply (e. g. in the form of meat) is particularly important in the elderly.
Conclusions. Meat is a valuable source of macro- and micro-nutrients, particularly of proteins, vitamins A, B 1 ,B 12 , niacin, iron, and zinc. Not consuming meat carries certain risks. These are especially present if no animal-based foods at all are consumed(vegan diet).Evidence from cohort studies leads to the conclusion that long-term consumption of increasing amounts of red meat and particularly of processed meat may result in a certain increase in the risk of mortality, cardiovascular disease, certain forms of cancer such as colon cancer and type 2 diabetes. There is evidence that several mechanisms might be involved, such as curing salt, however, their significance is not yet clearly known. It is concluded that recommendations for consumption of unprocessed red meat should be more restrictive than existing recommendations in Switzer-land [44]. The recommendation for the consumption of processed red meat should be even more restrictive. The Harvard School of Public Health [45] and the World Cancer Research Fund [46] both went further and recommended avoiding processed meat altogether. The present recommendations apply to adults aged about 35 – 70 years, as the studies quoted in this report examined these age groups. Restrictive recommendations are not warranted for the elderly, as the consumption of sufficient amounts of dietary proteins (e. g. in the form of meat) is particularly important for them [42, 43, 47]


Association of Animal and Plant Protein Intake With All-Cause and Cause-Specific Mortality(Mingyang Song et al, Oct 2016)

Conclusions and Relevance. High animal protein intake was positively associated with cardiovascular mortality and high plant protein intake was inversely associated with all-cause and cardiovascular mortality, especially among individuals with at least 1 lifestyle risk factor. Substitution of plant protein for animal protein, especially that from processed red meat, was associated with lower mortality, suggesting the importance of protein source.


Potential health hazards of eating red meat(A. Wolk, 6 Sep 2016)

Abstract
Red meat (beef, veal, pork, lamb and mutton) consumption contributes several important nutrients to the diet, for example essential amino acids, vitamins (including B12) and minerals (including iron and zinc). Processed red meat (ham, sausages, bacon, frankfurters, salami, etc.) undergoes treatment (curing, smoking, salting or the use of chemical preservatives and additives) to improve its shelf life and/or taste. During recent decades, consumption of red meat has been increasing globally, especially in developing countries. At the same time, there has been growing evidence that high consumption of red meat, especially of processed meat, may be associated with an increased risk of several major chronic diseases. Here, a comprehensive summary is provided of the accumulated evidence based on prospective cohort studies regarding the potential adverse health effects of red meat consumption on major chronic diseases, such as diabetes, coronary heart disease, heart failure, stroke and cancer at several sites, and mortality. Risk estimates from pooled analyses and meta-analyses are presented together with recently published findings. Based on at least six cohorts, summary results for the consumption of unprocessed red meat of 100 g day−1 varied from nonsignificant to statistically significantly increased risk (11% for stroke and for breast cancer, 15% for cardiovascular mortality, 17% for colorectal and 19% for advanced prostate cancer); for the consumption of 50 g day−1 processed meat, the risks were statistically significantly increased for most of the studied diseases (4% for total prostate cancer, 8% for cancer mortality, 9% for breast, 18% for colorectal and 19% for pancreatic cancer, 13% for stroke, 22% for total and 24% for cardiovascular mortality and 32% for diabetes). Potential biological mechanisms underlying the observed risks and the environmental impact of red meat production are also discussed. The evidence-based integrated message is that it is plausible to conclude that high consumption of red meat, and especially processed meat, is associated with an increased risk of several major chronic diseases and preterm mortality. Production of red meat involves an environmental burden. Therefore, some European countries have already integrated these two issues, human health and the ‘health of the planet’, into new dietary guidelines and recommended limiting consumption of red meat.


Adverse Effects Associated with Protein Intake above the Recommended Dietary Allowance for Adults(Ioannis Delimaris, 18 July 2013)

The adverse effects associated with long-term high protein/high meat intake in humans were (a) disorders of bone and calcium homeostasis, (b) disorders of renal function, (c) increased cancer risk, (d) disorders of liver function, and (e) precipitated progression of coronary artery disease. Conclusions. The findings of the present study suggest that there is currently no reasonable scientific basis in the literature to recommend protein consumption above the current RDA (high protein diet) for healthy adults due to its potential disease risks.


Negative health issues associated with Advanced Glycation End-Products (AGEs)

Toxicity of advanced glycation end products (Review) (Aleksandra Kuzan, 18 Mar 2021)

Advanced glycation end products (AGEs) are proteins or lipids glycated nonenzymatically by glucose, or other reducing sugars and their derivatives, such as glyceraldehyde, glycolaldehyde, methyloglyoxal and acetaldehyde. There are three different means of AGE formation: i) Maillard reactions, the polyol pathway and lipid peroxidation. AGEs participate in the pathological mechanisms underlying the development of several diseases, such as diabetes and its complications, retinopathy or neuropathy, neurological disorders (for example, Parkinson's disease and Alzheimer's disease), atherosclerosis, hypertension and several types of cancer. AGE levels are increased in patients with hyperglycaemia, and is likely the result of the high concentration of glycation substrates circulating in the blood. The present review summarises the formation and nomenclature of advanced glycation end products, with an emphasis on the role of AGEs in the development of diabetes, neurological disorders, as well as in cancer and other pathologies. A particular focus is placed on the functions of toxic AGEs. Additionally, studies which have shown the cytotoxicity of glycated albumin and other AGEs are also discussed. Finally, the diagnostic relevance of AGEs as well as for targeting in therapeutic strategies are highlighted.


Impact of advanced glycation end products (AGEs) signaling in coronary artery disease (Marinos Kosmopoulos et al, 1 Mar 2019)

Coronary artery disease remains the leading cause of mortality in adult diabetic population with however, a high predominance also in non-diabetic subjects. In search of common molecular mechanisms and metabolic by-products with potential pathogenic role, increased advanced glycation end products (AGEs) present a critical biomarker for CAD development in both cases. Interaction of AGEs with their transmembrane cell receptor, RAGE in endothelial and smooth muscle cells as well as in platelets, activates intracellular signaling that leads to endothelial injury, modulation of vascular smooth muscle cell function and altered platelet activity. Furthermore, tissue accumulation of AGEs affects current treatment approaches being involved in stent restenosis. The present review provides an update of AGE-induced molecular mechanisms involved in CAD pathophysiology while it discusses emerging therapeutic interventions targeting AGE reduction and AGE-RAGE signaling with beneficial clinical outcome.


Perspective of Advanced Glycation End Products on Human Health (Jer-An Lin et al, 8 Feb 2019)

Source: Perspective of Advanced Glycation End Products on Human Health https://pubs.acs.org/doi/abs/10.1021/acs.jafc.7b05943
In the last 20 years, the effects of advanced glycation end products (AGEs) on health have received increasing attention. High AGE levels in the body correlate with the progression of many diseases, such as diabetes, cardiovascular disease, and some cancers. However, whether AGEs are a cause of these diseases or represent accompanying symptoms of these diseases still needs to be elucidated by more comprehensive research. Recently, many researchers have begun to investigate the effects of AGE intake-induced variations of gut microbiota on disease progression, which will further explain the impact of AGEs on health and open a new chapter in AGE research.


Oral AGE restriction ameliorates insulin resistance in obese individuals with the metabolic syndrome: a randomised controlled trial (Helen Vlassara et al, 29 Jul 2016) This 1-year study investigated the effects of a low-AGE diet in 138 people with obesity. It noted increased insulin sensitivity, a modest decrease in body weight, and lower levels of AGE, oxidative stress, and inflammation.

Aims/hypothesis
We previously reported that obese individuals with the metabolic syndrome (at risk), compared with obese individuals without the metabolic syndrome (healthy obese), have elevated serum AGEs that strongly correlate with insulin resistance, oxidative stress and inflammation. We hypothesised that a diet low in AGEs (L-AGE) would improve components of the metabolic syndrome in obese individuals, confirming high AGEs as a new risk factor for the metabolic syndrome.
Methods
A randomised 1 year trial was conducted in obese individuals with the metabolic syndrome in two parallel groups: L-AGE diet vs a regular diet, habitually high in AGEs (Reg-AGE). Participants were allocated to each group by randomisation using random permuted blocks. At baseline and at the end of the trial, we obtained anthropometric variables, blood and urine samples, and performed OGTTs and MRI measurements of visceral and subcutaneous abdominal tissue and carotid artery. Only investigators involved in laboratory determinations were blinded to dietary assignment. Effects on insulin resistance (HOMA-IR) were the primary outcome.
Results
Sixty-one individuals were randomised to a Reg-AGE diet and 77 to an L-AGE diet; the data of 49 and 51, respectively, were analysed at the study end in 2014. The L-AGE diet markedly improved insulin resistance; modestly decreased body weight; lowered AGEs, oxidative stress and inflammation; and enhanced the protective factors sirtuin 1, AGE receptor 1 and glyoxalase I. The Reg-AGE diet raised AGEs and markers of insulin resistance, oxidative stress and inflammation. There were no effects on MRI-assessed measurements; no side effects from the intervention were identified. HOMA-IR came down from 3.1±1.8 to 1.9±1.3 (p<0.001) in the L-AGE group, while it increased from 2.9±1.2 to 3.6±1.7 (p<0.002) in the Reg-AGE group.
Conclusions/interpretation
L-AGE ameliorates insulin resistance in obese people with the metabolic syndrome, and may reduce the risk of type 2 diabetes, without necessitating a major reduction in adiposity. Elevated serum AGEs may be used to diagnose and treat ‘at-risk’ obesity.


Dietary Advanced Glycation End Products and Risk Factors for Chronic Disease: A Systematic Review of Randomised Controlled Trials (Rachal E Clarke et al, 1 Mar 2016)

Dietary advanced glycation end-products (AGEs) form during heating and processing of food products and are widely prevalent in the modern Western diet. Recent systematic reviews indicate that consumption of dietary AGEs may promote inflammation, oxidative stress and insulin resistance. Experimental evidence indicates that dietary AGEs may also induce renal damage, however, this outcome has not been considered in previous systematic reviews. The purpose of this review was to examine the effect of consumption of a high AGE diet on biomarkers of chronic disease, including chronic kidney disease (CKD), in human randomized controlled trials (RCTs). Six databases (SCOPUS, CINHAL, EMBASE, Medline, Biological abstracts and Web of Science) were searched for randomised controlled dietary trials that compared high AGE intake to low AGE intake in adults with and without obesity, diabetes or CKD. Twelve dietary AGE interventions were identified with a total of 293 participants. A high AGE diet increased circulating tumour necrosis factor-alpha and AGEs in all populations. A high AGE diet increased 8-isoprostanes in healthy adults, and vascular cell adhesion molecule-1 (VCAM-1) in patients with diabetes. Markers of CKD were not widely assessed. The evidence presented indicates that a high AGE diet may contribute to risk factors associated with chronic disease, such as inflammation and oxidative stress, however, due to a lack of high quality randomised trials, more research is required.


Advanced Glycation End Products and Oxidative Stress in Type 2 Diabetes Mellitus (Kerstin Nowotny et al, 16 Mar 2015)

Type 2 diabetes mellitus (T2DM) is a very complex and multifactorial metabolic disease characterized by insulin resistance and β cell failure leading to elevated blood glucose levels. Hyperglycemia is suggested to be the main cause of diabetic complications, which not only decrease life quality and expectancy, but are also becoming a problem regarding the financial burden for health care systems. Therefore, and to counteract the continually increasing prevalence of diabetes, understanding the pathogenesis, the main risk factors, and the underlying molecular mechanisms may establish a basis for prevention and therapy. In this regard, research was performed revealing further evidence that oxidative stress has an important role in hyperglycemia-induced tissue injury as well as in early events relevant for the development of T2DM. The formation of advanced glycation end products (AGEs), a group of modified proteins and/or lipids with damaging potential, is one contributing factor. On the one hand it has been reported that AGEs increase reactive oxygen species formation and impair antioxidant systems, on the other hand the formation of some AGEs is induced per se under oxidative conditions. Thus, AGEs contribute at least partly to chronic stress conditions in diabetes. As AGEs are not only formed endogenously, but also derive from exogenous sources, i.e., food, they have been assumed as risk factors for T2DM. However, the role of AGEs in the pathogenesis of T2DM and diabetic complications—if they are causal or simply an effect—is only partly understood. This review will highlight the involvement of AGEs in the development and progression of T2DM and their role in diabetic complications.

Elevated Serum Advanced Glycation Endproducts in Obese Indicate Risk for the Metabolic Syndrome: A Link Between Healthy and Unhealthy Obesity? (Jaime Uribarri et al, 2015 Feb 19)

Context:
Although obesity can predispose to the metabolic syndrome (MS), diabetes, and cardiovascular disease, not all obese subjects develop MS, hence the need for new indicators of risk for this syndrome. Advanced glycation end products (AGEs) correlate with factors involved in the MS, including inflammation and insulin resistance (IR). Because AGEs can be derived from food and are modifiable, it is important to determine whether they are a risk factor for MS.
Objective:
The objective of this study was to assess the association of endogenous and exogenous AGEs with MS criteria.
Design:
The following data were collected in a cross-sectional study of subjects with and without the MS: serum AGEs (sAGEs) and mononuclear cell AGEs, metabolites, pro- and antiinflammatory markers, body fat mass measures, including abdominal magnetic resonance imaging, and caloric and dietary AGE (dAGE) consumption.
Setting:
The study was conducted in the general community.
Participants:
Participants included 130 MS and 139 non-MS subjects of both sexes, older than 50 years.
Results:
sAGEs (ϵN-carboxymethyllysine, methylglyoxal) were markedly elevated in obese persons with more than one other MS criteria but not in obese without MS criteria. sAGEs directly correlated with markers of IR (HOMA) and inflammation (leptin, TNFα, RAGE) and inversely with innate defenses (SIRT1, AGE receptor 1 [AGER1], glyoxalase-I, adiponectin). sAGEs correlated with dAGEs but not with calories, nutrient consumption, or fat mass measures. Consumption of dAGE, but not of calories, was markedly higher in MS than in non-MS.
Conclusion:
High sAGEs, a modifiable risk factor for IR, may indicate risk for the MS, type 2 diabetes, and cardiovascular disease. High dietary AGE consumption and serum AGE levels may link healthy obesity to at-risk obesity.


Effect of advanced glycation end product intake on inflammation and aging: a systematic review (Katrien Van Puyvelde, et al, 1 Oct 2014)

Aging is associated with a chronic low-grade inflammatory status that contributes to chronic diseases such as age-related muscle wasting, kidney disease, and diabetes mellitus. Since advanced glycation end products (AGEs) are known to be proinflammatory, this systematic review examined the relation between the dietary intake of AGEs and inflammatory processes. The PubMed and Web of Science databases were screened systematically. Seventeen relevant studies in humans or animals were included. The intervention studies in humans showed mainly a decrease in inflammation in subjects on a low-AGE diet, while an increase in inflammation in subjects on a high-AGE diet was less apparent. About half of the observational studies found a relationship between inflammatory processes and AGEs in food. When the results are considered together, the dietary intake of AGEs appears to be related to inflammatory status and the level of circulating AGEs. Moreover, limiting AGE intake may lead to a decrease in inflammation and chronic diseases related to inflammatory status. Most of the trials were conducted in patients with chronic kidney disease or diabetes, and thus additional studies in healthy individuals are needed. Further investigation is needed to elucidate the effects of lifetime exposure of dietary AGEs on aging and health.


Vascular effects of advanced glycation endproducts: Clinical effects and molecular mechanisms(Alin Stirban et al, Apr 2014)

The enhanced generation and accumulation of advanced glycation endproducts (AGEs) have been linked to increased risk for macrovascular and microvascular complications associated with diabetes mellitus. AGEs result from the nonenzymatic reaction of reducing sugars with proteins, lipids, and nucleic acids, potentially altering their function by disrupting molecular conformation, promoting cross-linking, altering enzyme activity, reducing their clearance, and impairing receptor recognition. AGEs may also activate specific receptors, like the receptor for AGEs (RAGE), which is present on the surface of all cells relevant to atherosclerotic processes, triggering oxidative stress, inflammation and apoptosis. Understanding the pathogenic mechanisms of AGEs is paramount to develop strategies against diabetic and cardiovascular complications.


Advanced glycation end products in neurodegeneration: More than early markers of oxidative stress? (G. Münch PhD et al, 21 Feb 2014)

Oxidative stress is believed to play a decisive role in the pathogenesis of Parkinson's disease (PD). In addition, Lewy bodies, densely crosslinked intracellular protein deposits formed from cytoskeletal components, accumulate in presymptomatic stages of the disease. Recent findings indicate that “advanced glycation end products” (AGEs) are the major structural crosslinkers that cause the transformation of soluble neurofilament proteins to insoluble Lewy bodies. AGE formation is increased under conditions of oxidative stress, such as early GSH depletion, that are evident in the substantia nigra of PD patients, and is inhibited by radical scavengers and thiol antioxidants. Because AGEs not only are markers of oxidative stress but are also active participants in cell signaling by activation of glial cells to produce superoxide and nitric oxide, they can be considered part of a vicious cycle, which finally leads to neuronal cell death in the substantia nigra in PD.


Role of advanced glycation end products in cellular signaling(Christiane Ott et al, 2014)

Improvements in health care and lifestyle have led to an elevated lifespan and increased focus on age-associated diseases, such as neurodegeneration, cardiovascular disease, frailty and arteriosclerosis. In all these chronic diseases protein, lipid or nucleic acid modifications are involved, including cross-linked and non-degradable aggregates, such as advanced glycation end products (AGEs). Formation of endogenous or uptake of dietary AGEs can lead to further protein modifications and activation of several inflammatory signaling pathways. This review will give an overview of the most prominent AGE-mediated signaling cascades, AGE receptor interactions, prevention of AGE formation and the impact of AGEs during pathophysiological processes.


Advanced glycation endproducts in food and their effects on health (Malene W. Poulsen et al, October 2013)

Advanced glycation endproducts (AGEs) form by Maillard-reactions after initial binding of aldehydes with amines or amides in heated foods or in living organisms. The mechanisms of formation may include ionic as well as oxidative and radical pathways. The reactions may proceed within proteins to form high-molecular weight (HMW) AGEs or among small molecules to form low-molecular weight (LMW) AGEs. All free amino acids form AGEs, but lysine or arginine side chains dominate AGE formation within proteins. The analysis of AGEs in foods and body fluids is most often performed by ELISA or LC-MS; however, none of the methodologies cover all HMW and LMW AGEs. Most research is, therefore, carried out using ’representative’ AGE compounds, most often Nε-carboxymethyl-lysine (CML). Only LMW AGEs, including peptide-bound forms, and carbonyls may be absorbed from the gut and contribute to the body burden of AGEs. Some AGEs interact with specific pro- or anti-inflammatory receptors. Most studies on the biological effects of AGEs have been carried out by administering heated foods. The pro-inflammatory and deteriorating biological effects of AGEs in these studies, therefore, need further confirmation. The current review points out several research needs in order to address important questions on AGEs in foods and health.


Advanced Glycation End Products in Foods and a Practical Guide to Their Reduction in the Diet (Jaime Uribarri et al, 8 Jul 2013)

Modern diets are largely heat-processed and as a result contain high levels of advanced glycation end products (AGEs). Dietary advanced glycation end products (dAGEs) are known to contribute to increased oxidant stress and inflammation, which are linked to the recent epidemics of diabetes and cardiovascular disease. This report significantly expands the available dAGE database, validates the dAGE testing methodology, compares cooking procedures and inhibitory agents on new dAGE formation, and introduces practical approaches for reducing dAGE consumption in daily life. Based on the findings, dry heat promotes new dAGE formation by >10- to 100-fold above the uncooked state across food categories. Animal-derived foods that are high in fat and protein are generally AGE-rich and prone to new AGE formation during cooking. In contrast, carbohydrate-rich foods such as vegetables, fruits, whole grains, and milk contain relatively few AGEs, even after cooking. The formation of new dAGEs during cooking was prevented by the AGE inhibitory compound aminoguanidine and significantly reduced by cooking with moist heat, using shorter cooking times, cooking at lower temperatures, and by use of acidic ingredients such as lemon juice or vinegar. The new dAGE database provides a valuable instrument for estimating dAGE intake and for guiding food choices to reduce dAGE intake.


Molecular effects of advanced glycation end products on cell signalling pathways, ageing and pathophysiology (O. Nedić et al, 21 May 2013)

Advanced glycation end-products (AGEs) are a heterogeneous group of compounds formed by the Maillard chemical process of non- enzymatic glycation of free amino groups of proteins, lipids and nucleic acids. This chemical modification of biomolecules is triggered by endogeneous hyperglycaemic or oxidative stress-related processes. Additionally, AGEs can derive from exogenous, mostly diet-related, sources. Considering that AGE accumulation in tissues correlates with ageing and is a hallmark in several age-related diseases it is not surprising that the role of AGEs in ageing and pathology has become increasingly evident. The receptor for AGEs (RAGE) is a single transmembrane protein being expressed in a wide variety of human cells. RAGE binds a broad repertoire of extracellular ligands and mediates responses to stress conditions by activating multiple signal transduction pathways being mostly responsible for acute and/or chronic inflammation. RAGE activation has been implicated in ageing as well as in a number of age-related diseases, including atherosclerosis, neurodegeneration, arthritis, stoke, diabetes and cancer. Here we present a synopsis of findings that relate to AGEs-reported implication in cell signalling pathways and ageing, as well as in pathology. Potential implications and opportunities for translational research and the development of new therapies are also discussed.


Pinocembrin protects against β-amyloid-induced toxicity in neurons through inhibiting receptor for advanced glycation end products (RAGE)-independent signaling pathways and regulating mitochondrion-mediated apoptosis (Rui Liu et al, 18 Sep 2012)

Background
It is known that amyloid-β peptide (Aβ) plays a pivotal role in the pathogenesis of Alzheimer's disease (AD). Interaction between Aβ and the receptor for advanced glycation end products (RAGE) has been implicated in neuronal degeneration associated with this disease. Pinocembrin, a flavonoid abundant in propolis, has been reported to possess numerous biological activities beneficial to health. Our previous studies have demonstrated that pinocembrin has neuroprotective effects on ischemic and vascular dementia in animal models. It has been approved by the State Food and Drug Administration of China for clinical use in stroke patients. Against this background, we investigated the effects of pinocembrin on cognitive function and neuronal protection against Aβ-induced toxicity and explored its potential mechanism.
Methods
Mice received an intracerebroventricular fusion of Aβ25-35. Pinocembrin was administrated orally at 20 mg/kg/day and 40 mg/kg/day for 8 days. Behavioral performance, cerebral cortex neuropil ultrastructure, neuronal degeneration and RAGE expression were assessed. Further, a RAGE-overexpressing cell model and an AD cell model were used for investigating the mechanisms of pinocembrin. The mechanisms underlying the efficacy of pinocembrin were conducted on target action, mitochondrial function and potential signal transduction using fluorescence-based multiparametric technologies on a high-content analysis platform.
Results
Our results showed that oral administration of pinocembrin improved cognitive function, preserved the ultrastructural neuropil and decreased neurodegeneration of the cerebral cortex in Aβ25-35-treated mice. Pinocembrin did not have a significant effect on inhibiting Aβ1-42 production and scavenging intracellular reactive oxygen species (ROS). However, pinocembrin significantly inhibited the upregulation of RAGE transcripts and protein expression both in vivo and in vitro, and also markedly depressed the activation of p38 mitogen-activated protein kinase (MAPK)-MAPKAP kinase-2 (MK2)-heat shock protein 27 (HSP27) and stress-activated protein kinase (SAPK)/c-Jun N-terminal kinase (JNK)-c-Jun pathways and the downstream nuclear factor κB (NFκB) inflammatory response subsequent to Aβ-RAGE interaction. In addition, pinocembrin significantly alleviated mitochondrial dysfunction through improving mitochondrial membrane potential and inhibiting mitochondrial oxidative stress, and regulated mitochondrion-mediated apoptosis by restoration of B cell lymphoma 2 (Bcl-2) and cytochrome c and inactivation of caspase 3 and caspase 9.
Conclusions
Pinocembrin was shown to infer cognitive improvement and neuronal protection in AD models. The mechanisms of action of the compound were illustrated on RAGE-dependent transduction inhibition and mitochondrion protection. It appears to be a promising candidate for the prevention and therapy of AD.

Role of advanced glycation end products (AGEs) and oxidative stress in vascular complications in diabetes (Sho-ichi Yamagish et al, May 2012)

Abstract
Background
A non-enzymatic reaction between reducing sugars and amino groups of proteins, lipids and nucleic acids contributes to the aging of macromolecules, whose process has been known to progress at an accelerated rate under hyperglycemic and/or oxidative stress conditions. Over a course of days to weeks, early glycation products undergo further reactions such as rearrangements and dehydration to become irreversibly cross-linked, fluorescent protein derivatives termed advanced glycation end products (AGEs).
Scope of review
In this paper, we review the role of AGE–oxidative stress axis and its therapeutic interventions in vascular complications in diabetes.
Major conclusions
AGEs elicit oxidative stress generation and subsequently cause inflammatory and thrombogenic reactions in various types of cells via interaction with a receptor for AGEs (RAGE), thereby being involved in vascular complications in diabetes. In addition, mitochondrial superoxide generation has been shown to play an important role in the formation and accumulation of AGEs under diabetic conditions. Further, we have recently found that a pathophysiological crosstalk between AGE–RAGE axis and renin–angiotensin system (RAS) could contribute to the progression of vascular damage in diabetes.
General significance
These observations suggest that inhibition of AGE–RAGE–oxidative stress axis or blockade of its interaction with RAS is a novel therapeutic strategy for preventing vascular complications in diabetes.


Role of advanced glycation end products in cardiovascular disease (Z Hegab et al, 26 Apr 2012)

Advanced glycation end products (AGEs) are produced through the non enzymatic glycation and oxidation of proteins, lipids and nucleic acids. Enhanced formation of AGEs occurs particularly in conditions associated with hyperglycaemia such as diabetes mellitus (DM). AGEs are believed to have a key role in the development and progression of cardiovascular disease in patients with DM through the modification of the structure, function and mechanical properties of tissues through crosslinking intracellular as well as extracellular matrix proteins and through modulating cellular processes through binding to cell surface receptors [receptor for AGEs (RAGE)]. A number of studies have shown a correlation between serum AGE levels and the development and severity of heart failure (HF). Moreover, some studies have suggested that therapies targeted against AGEs may have therapeutic potential in patients with HF. The purpose of this review is to discuss the role of AGEs in cardiovascular disease and in particular in heart failure, focussing on both cellular mechanisms of action as well as highlighting how targeting AGEs may represent a novel therapeutic strategy in the treatment of HF.


Advanced glycation endproducts and their receptor RAGE in Alzheimer's disease (Velandai Srikanth et al, May 2011)

Alzheimer's disease (AD) is the most common dementing disorder of late life. Although there might be various different triggering events in the early stages of the disease, they seem to converge on a few characteristic final pathways in the late stages, characterized by inflammation and neurodegeneration. In this review, we revisit the hypothesis that advanced glycation endproducts (AGEs) and their receptor RAGE may play an important role in disease pathogenesis. Accumulation of AGEs in cells and tissues is a normal feature of aging, but is accelerated in AD. In AD, AGEs can be detected in pathological deposits such as amyloid plaques and neurofibrillary tangles. AGEs explain many of the neuropathological and biochemical features of AD such as extensive protein crosslinking, glial induction of oxidative stress and neuronal cell death. Oxidative stress and AGEs initiate a positive feedback loop, where normal age-related changes develop into a pathophysiological cascade. RAGE and its decoy receptor soluble RAGE, may contribute to or protect against AD pathogenesis by influencing transport of β-amyloid into the brain or by manipulating inflammatory mechanisms. Targeted pharmacological interventions using AGE-inhibitors, RAGE-antagonists, RAGE-antibodies, soluble RAGE or RAGE signalling inhibitors such as membrane-permeable antioxidants may be promising therapeutic strategies to slow down the progression of AD.

Dietary Advanced Glycation End Products and Aging (Claudia Luevano-Contreras & Karen Chapman-Novakofski, 13 Dec 2010)

Advanced glycation end products (AGEs) are a heterogeneous, complex group of compounds that are formed when reducing sugar reacts in a non-enzymatic way with amino acids in proteins and other macromolecules. This occurs both exogenously (in food) and endogenously (in humans) with greater concentrations found in older adults. While higher AGEs occur in both healthy older adults and those with chronic diseases, research is progressing to both quantify AGEs in food and in people, and to identify mechanisms that would explain why some human tissues are damaged, and others are not. In the last twenty years, there has been increased evidence that AGEs could be implicated in the development of chronic degenerative diseases of aging, such as cardiovascular disease, Alzheimer’s disease and with complications of diabetes mellitus. Results of several studies in animal models and humans show that the restriction of dietary AGEs has positive effects on wound healing, insulin resistance and cardiovascular diseases. Recently, the effect of restriction in AGEs intake has been reported to increase the lifespan in animal models. This paper will summarize the work that has been published for both food AGEs and in vivo AGEs and their relation with aging, as well as provide suggestions for future research.


Advanced Glycation End Products in Foods and a Practical Guide to Their Reduction in the Diet(Jaime Uribarri et al, June 2010)

Modern diets are largely heat-processed and as a result contain high levels of advanced glycation end products (AGEs). Dietary advanced glycation end products (dAGEs) are known to contribute to increased oxidant stress and inflammation, which are linked to the recent epidemics of diabetes and cardiovascular disease. This report significantly expands the available dAGE database, validates the dAGE testing methodology, compares cooking procedures and inhibitory agents on new dAGE formation, and introduces practical approaches for reducing dAGE consumption in daily life. Based on the findings, dry heat promotes new dAGE formation by >10- to 100-fold above the uncooked state across food categories. Animal-derived foods that are high in fat and protein are generally AGE-rich and prone to new AGE formation during cooking. In contrast, carbohydrate-rich foods such as vegetables, fruits, whole grains, and milk contain relatively few AGEs, even after cooking. The formation of new dAGEs during cooking was prevented by the AGE inhibitory compound aminoguanidine and significantly reduced by cooking with moist heat, using shorter cooking times, cooking at lower temperatures, and by use of acidic ingredients such as lemon juice or vinegar. The new dAGE database provides a valuable instrument for estimating dAGE intake and for guiding food choices to reduce dAGE intake.


Advanced glycation end products and their circulating receptors predict cardiovascular disease mortality in older community-dwelling women (Richard D Semba et al, 1 Apr 2010)

Aims: To characterize the relationship between advanced glycation end products (AGEs) and circulating receptors for AGEs (RAGE) with cardiovascular disease mortality. Methods: The relationships between serum AGEs, total RAGE (sRAGE), and endogenous secretory RAGE (esRAGE), and mortality were characterized in 559 community-dwelling women, ‡ 65 years, in Baltimore, Maryland. Results: During 4.5 years of follow-up, 123 (22%) women died, of whom 54 died with cardiovascular disease. The measure of serum AGEs was carboxymethyl-lysine (CML), a dominant AGE. Serum CML predicted cardiovascular disease mortality (Hazards Ratio [HR] for highest vs lower three quartiles, 1.94, 95% Confidence Interval [CI] 1.08–3.48, p=0.026), after adjusting for age, race, body mass index, and renal insufficiency. Serum sRAGE (ng/mL) and esRAGE (ng/mL) predicted cardiovascular disease mortality (HR per 1 Standard Deviation [SD] 1.27, 95% CI 0.98–1.65, p=0.07; HR 1.28, 95% CI 1.02–1.63, p=0.03), after adjusting for the same covariates. Among non-diabetic women, serum CML, sRAGE, and esRAGE, respectively, predicted cardiovascular disease mortality (HR for highest vs lower three quartiles, 2.29, 95% CI 1.21–4.34, p=0.01; HR per 1 SD, 1.24, 95% CI0.92–1.65, p=0.16; HR per 1 SD 1.45, 95% CI 1.08–1.93, p=0.01), after adjusting for the same covariates. Conclusions: High circulating AGEs and RAGE predict cardiovascular disease mortality among older community-dwelling women. AGEs are a potential target for interventions, as serum AGEs can be lowered by change in dietary pattern and pharmacological treatment.


The role of advanced glycation end products in retinal ageing and disease (Josephine V. Glenn & Alan W. Stitt, 0ct 2009)

The retina is exposed to a lifetime of potentially damaging environmental and physiological factors that make the component cells exquisitely sensitive to age-related processes. Retinal ageing is complex and a raft of abnormalities can accumulate in all layers of the retina. Some of this pathology serves as a sinister preamble to serious conditions such as age-related macular degeneration (AMD) which remains the leading cause of irreversible blindness in the Western world.

The formation of advanced glycation end products (AGEs) is a natural function of ageing but accumulation of these adducts also represents a key pathophysiological event in a range of important human diseases. AGEs act as mediators of neurodegeneration, induce irreversible changes in the extracellular matrix, vascular dysfunction and pro-inflammatory signalling. Since many cells and tissues of the eye are profoundly influenced by such processes, it is fitting that advanced glycation is now receiving considerable attention as a possible pathogenic factor in visual disorders. This review presents the current evidence for a pathogenic role for AGEs and activation of the receptor for AGEs (RAGE) in initiation and progression of retinal disease. It draws upon the clinical and experimental literature and highlights the opportunities for further research that would definitively establish these adducts as important instigators of retinal disease. The therapeutic potential for novel agents that can ameliorate AGE formation of attenuate RAGE signalling in the retina is also discussed.


Protection against Loss of Innate Defenses in Adulthood by Low Advanced Glycation End Products (AGE) Intake: Role of the Antiinflammatory AGE Receptor (Helen Vlassara et al, 9 Oct 2009)

Context: Increased oxidant stress and inflammation (OS/infl) are linked to both aging-related diseases and advanced glycation end products (AGEs). Whereas AGE receptor-1 (AGER1) reduces OS/infl in animals, this has not been assessed in normal humans.
Objective: The objectives of the study were to determine whether AGER1 correlates with AGEs and OS/infl and a reduction of dietary AGEs (dAGEs) lowers OS/infl in healthy adults and chronic kidney disease (CKD-3) patients.
Design: This study was cross-sectional with 2-yr follow-up studies of healthy adults and CKD-3 patients, a subset of which received a reduced AGE or regular diet.
Setting: The study was conducted at general community and renal clinics.
Participants: Participants included 325 healthy adults (18–45 and >60 yr old) and 66 CKD-3 patients.
Intervention: An isocaloric low-AGE (30–50% reduction) or regular diet was given to 40 healthy subjects for 4 months and to nine CKD-3 patients for 4 wk.
Main Outcome: Relationships between age, dAGEs, serum AGEs, peripheral mononuclear cell AGE-receptors, and OS/Infl before and after reduction of dAGE intake were measured.
Results: AGEs, oxidant stress, receptor for AGE, and TNFα were reduced in normal and CKD-3 patients after the low-AGE diet, independently of age. AGER1 levels in CKD-3 patients on the low-AGE diet resembled 18- to 45-yr-old normal subjects. Dietary, serum, and urine AGEs correlated positively with peripheral mononuclear cell AGER1 levels in healthy participants. AGER1 was suppressed in CKD-3 subjects, whereas receptor for AGE and TNFα were increased.
Conclusions: Reduction of AGEs in normal diets may lower oxidant stress/inflammation and restore levels of AGER1, an antioxidant, in healthy and aging subjects and CKD-3 patients. AGE intake has implications for health outcomes and costs and warrants further testing.


Advanced Glycation End-products and Atherosclerosis (H Vlassara 8 Jul 2009)

The late rearrangements of the covalent nonenzymatic modification of proteins by glucose, called advanced glycation end-products (AGEs), have been shown to accumulate in diabetic and ageing tissues. AGEs elicit a wide range of cell-mediated responses leading to vascular dysfunction, matrix expansion and athero- and glomerulosclerosis. Cellular responses are thought to be largely induced through an AGE-specific cell-surface receptor complex (AGEr). Interaction of AGE-modified proteins with these cells may serve diverse purposes, including disposal of senescent AGE-modified molecules and initiation of tissue repair and protein turnover. In humans, the normal renal clearance rate for the AGE-degradation products found in serum, AGE peptides (AGEp), correlates inversely with renal creatinine clearance rate. Of note, circulating AGEp include reactive intermediates which readily attach covalently to either insoluble matrix collagen or serum proteins, e.g. low-density lipoproteins (LDL), to form AGEp collagen and AGEp-LDL. Consistent with this, diabetic and nondiabetic patients with renal failure (a group highly susceptible to accelerated atherosclerosis) exhibit markedly elevated AGE-modified serum LDL. In summary, in addition to glucose-derived AGEs, the endo-genously produced degradation products, AGE peptides, can amplify tissue damage and thus account as distinct toxins. The effects may particularly accelerate glucose toxicity in certain individuals that are genetically susceptible to diabetic renal and extrarenal disease.


Circulating Glycotoxins and Dietary Advanced Glycation Endproducts: Two Links to Inflammatory Response, Oxidative Stress, and Aging (Jaime Uribarri et al, 20 Feb 2009)

Background
Oxidative stress (OS) and inflammatory mediators increase with aging. The levels of advanced glycation endproducts (AGEs), prooxidant factors linked to chronic diseases such as diabetes, cardiovascular disease, and renal disease, also increase with aging. AGEs are readily derived from heat-treated foods. We propose that the excess consumption of certain AGEs via the diet enhances OS and inflammatory responses in healthy adults, especially in elderly persons.
Methods
We examined 172 young (<45 years old) and older (>60 years old) healthy individuals to determine whether the concentration of specific serum AGEs (Nε-carboxymethyl-lysine [CML] or methylglyoxal [MG] derivatives) were higher in older compared to younger persons and whether, independent of age, they correlated with the intake of dietary AGEs, as well as with circulating markers of OS and inflammation.
Results
Body weight, body mass index (BMI), and serum AGE, CML, and MG derivatives were higher in older participants, independent of gender. Serum CML correlated with levels of 8-isoprostanes (r =0.448, p =.0001) as well as with Homeostasis Model Assessment index (HOMA), an index of insulin resistance (r = 0.247, p = .044). The consumption of dietary AGEs, but not of calories, correlated independently with circulating AGEs (CML: r =0.415, p = .0001 and MG: r = 0.282, p = .002) as well as with high sensitivity C-reactive protein (hsCRP) (r = 0.200, p = .042).
Conclusions
Circulating indicators of AGEs (CML and MG derivatives), although elevated in older participants, correlate with indicators of inflammation and OS across all ages. Indicators of both AGEs and OS are directly influenced by the intake of dietary AGEs, independent of age or energy intake. Thus, reduced consumption of these oxidants may prove a safe economic policy to prevent age-related diseases, especially in an aging population.


Glycation Endproducts, Soluble Receptor for Advanced Glycation Endproducts and Cytokines in Diabetic and Non-diabetic Pregnancies (Magdalena Pertyńska-Marczewska et al, 11 January 2009)

Problem Cytokines, advanced glycation end products (AGEs), and their receptor RAGE have been recently suggested to play a role in human pregnancy. In this study, we sought to determine the alterations of plasma AGEs, soluble RAGE (sRAGE), and proinflammatory cytokines in normal pregnancies and those complicated with type 1 diabetes mellitus.
Method of study These parameters were measured in samples from healthy non-pregnant (C), diabetic non-pregnant (D), healthy pregnant (HP), and pregnant diabetic (DP) women.
Results In the first trimester, DP showed lower sRAGE and higher AGEs compared to HP. In the DP group, significant negative correlations were seen between TNF-α and lipopolysaccharide (LPS)-stimulated ΙL-6 in the first trimester and sRAGE in the third trimester. LPS-stimulated IL-12 was positively correlated with levels of AGEs in the third trimester.
Conclusion We detected several differences in the levels of AGEs, sRAGE, and proinflammatory cytokines between euglycemic and diabetic pregnancies.


Advanced glycation end products induce in vitro cross-linking of α-synuclein and accelerate the process of intracellular inclusion body formation (Shamim Shaikh, Louise F.B. Nicholson 11 March 2008)

Cross-linking of α-synuclein and Lewy body formation have been implicated in the dopaminergic neuronal cell death observed in Parkinson's disease (PD); the mechanisms responsible, however, are not clear. Reactive oxygen species and advanced glycation end products (AGEs) have been found in the intracellular, α-synuclein-positive Lewy bodies in the brains of both PD as well as incidental Lewy body disease patients, suggesting a role for AGEs in α-synuclein cross-linking and Lewy body formation. The aims of the present study were to determine 1) whether AGEs can induce cross-linking of α-synuclein peptides, 2) the progressive and time-dependent intracellular accumulation of AGEs and inclusion body formation, and 3) the effects of extracellular or exogenous AGEs on intracellular inclusion formation. We first investigated the time-dependent cross-linking of recombinant human α-synuclein in the presence of AGEs in vitro, then used a cell culture model based on chronic rotenone treatment of human dopaminergic neuroblastoma cells (SH-SY5Y) over a period of 1–4 weeks, in the presence of different doses of AGEs. Cells (grown on coverslips) and cell lysates, collected at the end of every week, were analyzed for the presence of intracellular reactive oxygen species, AGEs, α-synuclein proteins, and intracellular α-synuclein- and AGE-positive inclusion bodies by using immunocytochemical, biochemical, and Western blot techniques. Our results show that AGEs promote in vitro cross-linking of α-synuclein, that intracellular accumulation of AGEs precedes α-synuclein-positive inclusion body formation, and that extracellular AGEs accelerate the process of intracellular α-synuclein-positive inclusion body formation.


Effects of advanced glycation end products on renal fibrosis and oxidative stress in cultured NRK-49F cells (YAN, Hai-dong et al, May 2007)

Background
Advanced glycation end products (AGEs) play a critical role in the development of diabetic nephropathy. Reactive oxygen species (ROS) may play a critical role in AGEs induced growth factor expression. In this study, the effects of AGEs on transforming growth factor β1 (TGF-β1), connective tissue growth factor (CTGF) and fibronectin (Fn) mRNA expression and oxidative stress in cultured NRK-49F cells were examined.
Methods
NRK-49F cells were incubated with medium containing different doses of AGEs (50, 100 or 200 μg/ml) for 24 hours, or with AGEs 100 μg/ml for different times (0, 12, 24 or 48 hours). Cells in the serum-free medium or medium containing 25 mmol/L glucose were controls. Cells were treated with 25 mmol/L glucose and 100 μg/ml AGEs for 24 hours to determine the effects between AGEs and glucose. We clarified the role of antioxidant by pretreating cells with N-acetylcysteine (10 mmol/L), ginkgo biloba extract (50 or 100 mg/L) for 24 hours and with 100 μg/ml AGEs for further 24 hours. Alamarblue dye assay was used to analyze cell growth; intracellular ROS generation was measured by flow cytometry; intracellular glutathione by fluorescence spectrophotometry; expressions of TGF-β1, CTGF and Fn mRNA by semiquantitative RT-PCR.
Results
AGEs significantly increased the expressions of TGF-β1, CTGF, Fn mRNA and intracellular ROS generation, and decreased the glutathion level in NRK-49F cells in dose- and time-dependent manners. High glucose and AGEs together significantly increased the expression of TGF-β1, CTGF and Fn mRNA, compared with AGEs and high glucose separately. Preincubation with N-acetylcysteine or ginkgo biloba extract increased GSH level, suppressed AGEs-induced oxidative stress and TGF-β1, CTGF and Fn mRNA overexpression.
Conclusions
AGEs can significantly increase expression of TGF-β1, CTGF, Fn mRNA in NRK-49F cells through enhancement of oxidative stress. The accumulation of AGEs may play a pivotal role in the pathogenesis of tubulointerstitial fibrosis in diabetic nephropathy. Suppression of AGEs induced TGF-β1, CTGF and Fn mRNA overexpression in renal fibroblasts through inhibition of oxidative stress may be a mechanism underlying effect of ginkgo biloba extract in diabetic nephropathy. In addition, antioxidant therapy may help prevent AGEs accumulation and its induced damage.


Circulating Glycotoxins and Dietary Advanced Glycation Endproducts: Two Links to Inflammatory Response, Oxidative Stress, and Aging(Jaime Uribarri et al, 1 Apr 2007)

Background. Oxidative stress (OS) and inflammatory mediators increase with aging. The levels of advanced glycation endproducts (AGEs), prooxidant factors linked to chronic diseases such as diabetes, cardiovascular disease, and renal disease, also increase with aging. AGEs are readily derived from heat-treated foods. We propose that the excess consumption of certain AGEs via the diet enhances OS and inflammatory responses in healthy adults, especially in elderly persons.
Methods. We examined 172 young (<45 years old) and older (>60 years old) healthy individuals to determine whether the concentration of specific serum AGEs (Nϵ-carboxymethyl-lysine [CML] or methylglyoxal [MG] derivatives) were higher in older compared to younger persons and whether, independent of age, they correlated with the intake of dietary AGEs, as well as with circulating markers of OS and inflammation.
Results. Body weight, body mass index (BMI), and serum AGE, CML, and MG derivatives were higher in older participants, independent of gender. Serum CML correlated with levels of 8-isoprostanes (r = 0.448, p =.0001) as well as with Homeostasis Model Assessment index (HOMA), an index of insulin resistance (r = 0.247, p =.044). The consumption of dietary AGEs, but not of calories, correlated independently with circulating AGEs (CML: r = 0.415, p =.0001 and MG: r = 0.282, p =.002) as well as with high sensitivity C-reactive protein (hsCRP) (r = 0.200, p =.042).
Conclusions. Circulating indicators of AGEs (CML and MG derivatives), although elevated in older participants, correlate with indicators of inflammation and OS across all ages. Indicators of both AGEs and OS are directly influenced by the intake of dietary AGEs, independent of age or energy intake. Thus, reduced consumption of these oxidants may prove a safe economic policy to prevent age-related diseases, especially in an aging population.


Insulin-Resistance-and-Type-2-Diabetes-in-High-Fat Insulin Resistance and Type 2 Diabetes in High-Fat–Fed Mice Are Linked to High Glycotoxin Intake (Oana Sandu et al, August 01 2005)

Dietary advanced glycosylation end products (AGEs) have been linked to insulin resistance in db/db(++) mice. To test whether dietary AGEs play a role in the progression of insulin resistance in normal mice fed high-fat diets, normal C57/BL6 mice were randomly assigned to high-fat diets (35% g fat), either high (HAGE-HF group; 995.4 units/mg AGE) or low (by 2.4-fold LAGE-HF group; 329.6 units/mg AGE) in AGE content for 6 months. Age-matched C57/BL6 and db/db(++) mice fed regular diet (5% g fat, 117.4 units/mg AGE) served as controls. After 6 months, 75% of HAGE-HF mice were diabetic and exhibited higher body weight (P < 0.001), fasting glucose (P < 0.001), insulin (P < 0.001), and serum AGEs (P < 0.01) than control mice, while none of the LAGE-HF mice were diabetic despite a similar rise in body weight and plasma lipids. The HAGE-HF group displayed markedly impaired glucose and insulin responses during glucose tolerance tests and euglycemic and hyperglycemic clamps and altered pancreatic islet structure and function compared with those of LAGE-HF mice, in which findings resembled those of control mice. The HAGE-HF group had more visceral fat (by two- and fourfold) and more AGE-modified fat (by two- and fivefold) than LAGE-HF and control mice, respectively. In the HAGE-HF group, plasma 8-isoprostane was higher (P < 0.01) and adiponectin lower (P < 0.001) than control mice, while in the LAGE-HF group, these were more modestly affected (P < 0.05). These results demonstrate that the development of insulin resistance and type 2 diabetes during prolonged high-fat feeding are linked to the excess AGEs/advanced lipoxidation end products inherent in fatty diets.


Increased levels of serum advanced glycation end-products in women with polycystic ovary syndrome (Evanthia Diamanti-Kandarakis, et al, 29 November 2004)

objective Women with polycystic ovary syndrome (PCOS) carry a number of cardiovascular risk factors and are considered to be at increased risk for atherosclerosis. Elevated concentrations of advanced glycation end-products (AGE), which exert their effects through interaction with specific receptors (RAGE), have been implicated in the cellular and tissue damage during atherosclerotic processes.
design/patients We investigated serum AGE levels in 29 young women with PCOS as well as the expression of their receptor, RAGE, in circulating monocytes and compared them levels with 22 healthy control women.
measurements/results Women with PCOS had higher levels of serum AGE proteins compared to healthy individuals (9·81 ± 0·16 vs. 5·11 ± 0·16, P < 0·0001), and increased RAGE expression was observed in monocytes of PCOS women compared to controls (30·91 ± 10·11 vs. 7·97 ± 2·61, P < 0·02). A positive correlation was observed between AGE proteins and testosterone (T) levels (r = 0·73, P < 0·0001). The correlation between AGE proteins and T levels remained high (partial correlation coefficient = 0·61, P = 0·0001) after controlling for body mass index (BMI), insulin levels and the area under the curve for glucose (AUCGLU) during an oral glucose tolerance test (OGTT). A positive correlation was also observed between AGE proteins and the free androgen index (FAI) (r = 0·58, P < 0·0001), waist-to-hip ratio (WHR) (r = 0·31, P < 0·02), insulin (r = 0·46, P < 0·001), homeostasis model assessment (HOMA) (r = 0·47, P < 0·0001), AUCGLU (r = 0·52, P < 0·002) and RAGE (r = 0·59, P < 0·01). A negative correlation was observed between AGE proteins and glucose/insulin ratio (GLU/INS) (r = −0·35, P < 0·01), and the quantitative insulin sensitivity check index (QUICKI) (r =−0·50, P < 0·01). In multiple regression analysis T was the only independent predictor of AGE levels (P < 0·0001, b = 0·044) between BMI, insulin, SHBG and AUCGLU (adjusted R2 = 0·59, F = 44·41, P < 0·0001).
conclusion These data clearly demonstrate, for the first time, that PCOS women without overt hyperglycaemia have increased AGE levels and elevated RAGE expression when compared with controls.


[ Advanced glycation end products, oxidant stress and vascular lesions (O. Chappey et al, 29 Oct 2003)

The formation of advanced glycation end products (AGEs) is observed in conditions such as diabetes mellitus and ageing, both associated with vascular disorders. AGEs form by the interaction of an aldose with NH2 of proteins, and the subsequent Amadori rearrangement leads to complex molecules. The heterogeneous class of AGE molecules is found in plasma, cells and tissues and accumulates in the vessel wall and the kidney. AGE reactions can generate reactive oxygen intermediates (ROIs), which can act as signal mediators and can be deleterious for molecules or cells. The AGEs and ROI-induced cellular dysfunctions can interfere with the gene expression of peptides and cytokines regulating cell proliferation and vascular functions. The interaction of AGEs with the AGE receptor (RAGE) is followed by a series of intracellular modifications that may be involved in the development of atherosclerosis. An attempt to minimize AGE formation and to limit ROI production by an appropriate therapy may result in the reduction or slowing of vascular disease in patients with diabetes mellitus.


Fetal-or-Neonatal-Low-Glycotoxin-Environment Fetal or Neonatal Low-Glycotoxin Environment Prevents Autoimmune Diabetes in NOD Mice (Melpomeni Peppa et al, June 01 2003)

Advanced glycation end products (AGEs) are implicated in β-cell oxidant stress. Diet-derived AGE (dAGE) are shown to contribute to end-organ toxicity attributed to diabetes. To assess the role of dAGE on type 1 diabetes, NOD mice were exposed to a high-AGE diet (H-AGE) and to a nutritionally similar diet with approximate fivefold-lower levels of Nε-carboxymethyllysine (CML) and methylglyoxal-derivatives (MG) (L-AGE). Suppression of serum CML and MG in L-AGE-fed mice was marked by suppression of diabetes (H-AGE mice >94% vs. L-AGE mice 33% in founder [F]0, 14% in F1, and 13% in F2 offspring, P < 0.006) and by a delay in disease onset (4-month lag). Survival for L-AGE mice was 76 vs. 0% after 44 weeks of H-AGE mice. Reduced insulitis in L-AGE versus H-AGE mice (P < 0.01) was marked by GAD- and insulin-unresponsive pancreatic interleukin (IL)-4-positive CD4+ cells compared with the GAD- and insulin-responsive interferon (IFN)-γ-positive T-cells from H-AGE mice (P < 0.005). Splenocytes from L-AGE mice consisted of GAD- and insulin-responsive IL-10-positive CD4+ cells compared with the IFN-γ-positive T-cells from H-AGE mice (P < 0.005). Therefore, high AGE intake may provide excess antigenic stimulus for T-cell-mediated diabetes or direct β-cell injury in NOD mice; both processes are ameliorated by maternal or neonatal exposure to L-AGE nutrition.


https://journals.lww.com/jasn/fulltext/2003/03000/restriction_of_dietary_glycotoxins_reduces.21.aspx Restriction of Dietary Glycotoxins Reduces Excessive Advanced Glycation End Products in Renal Failure Patients] (Uribarri, Jaime et al, March 2003)

Advanced glycation endproduct (AGE) levels are elevated in renal failure patients and may contribute to the excessive cardiovascular disease in this population. Diet-derived AGE are major contributors to the total body AGE pool. It was postulated that a reduction in dietary AGE intake might impact on the high circulating AGE levels in renal failure patients. Twenty-six nondiabetic renal failure patients on maintenance peritoneal dialysis were randomized to either a high or a low AGE diet for 4 wk. Three-day dietary records, fasting blood, 24-h urine, and dialysis fluid collections were obtained at baseline and end of study. AGE levels were determined by ELISA for Nε-carboxymethyl-lysine (CML) and methylglyoxal-derivatives (MG). Eighteen patients completed the study. Low dietary AGE intake decreased serum CML (34%; P < 0.002), serum MG (35%; P < 0.008), CML-LDL (28%; P < 0.011), CML-apoB (25%; P < 0.028), dialysate CML (39%; P < 0.03), and dialysate MG output (40%; P < 0.04). High dietary AGE intake increased serum CML (29%; P < 0.028), serum MG (26%; P < 0.09), CML-LDL (50%; P < 0.011), CML-apoB (67%; P < 0.028), and dialysate CML output (27%; P < 0.01). Serum AGE correlated with BUN (r = 0.6, P < 0.002 for CML; r = 0.4, P < 0.05 for MG), serum creatinine (r = 0.76, P < 0.05 for CML; r = 0.55, P < 0.004 for MG), total protein (r = 0.4, P < 0.05 for CML; r = 0.4, P < 0.05 for MG), albumin (r = 0.4, P < 0.02 for CML; r = 0.4, P < 0.05 for MG), and phosphorus (r = 0.5, P < 0.006 for CML; r = 0.5, P < 0.01 for MG). It is concluded that dietary glycotoxins contribute significantly to the elevated AGE levels in renal failure patients. Moreover, dietary restriction of AGE is an effective and feasible method to reduce excess toxic AGE and possibly cardiovascular associated mortality.


Inflammatory mediators are induced by dietary glycotoxins, a major risk factor for diabetic angiopathy (Helen Vlassara et al, 2002 Nov 12)

Diet is a major environmental source of proinflammatory AGEs (heat-generated advanced glycation end products); its impact in humans remains unclear. We explored the effects of two equivalent diets, one regular (high AGE, H-AGE) and the other with 5-fold lower AGE (L-AGE) content on inflammatory mediators of 24 diabetic subjects: 11 in a 2-week crossover and 13 in a 6-week study. After 2 weeks on H-AGE, serum AGEs increased by 64.5% (P = 0.02) and on L-AGE decreased by 30% (P = 0.02). The mononuclear cell tumor necrosis factor-α/β-actin mRNA ratio was 1.4 ± 0.5 on H-AGE and 0.9 ± 0.5 on L-AGE (P = 0.05), whereas serum vascular adhesion molecule-1 was 1,108 ± 429 and 698 ± 347 ng/ml (P = 0.01) on L- and H-AGE, respectively. After 6 weeks, peripheral blood mononuclear cell tumor necrosis factor-α rose by 86.3% (P = 0.006) and declined by 20% (P, not significant) on H- or L-AGE diet, respectively; C-reactive protein increased by 35% on H-AGE and decreased by 20% on L-AGE (P = 0.014), and vascular adhesion molecule-1 declined by 20% on L-AGE (P < 0.01) and increased by 4% on H-AGE. Serum AGEs were increased by 28.2% on H-AGE (P = 0.06) and reduced by 40% on L-AGE (P = 0.02), whereas AGE low density lipoprotein was increased by 32% on H-AGE and reduced by 33% on L-AGE diet (P < 0.05). Thus in diabetes, environmental (dietary) AGEs promote inflammatory mediators, leading to tissue injury. Restriction of dietary AGEs suppresses these effects.


Oxidative stress-inducing carbonyl compounds from common foods: novel mediators of cellular dysfunction (Weijing Cai et al, Jul 2002)

BACKGROUND: The general increase in reactive oxygen species generated from glucose-derived advanced glycation endproducts (AGEs) is among the key mechanisms implicated in tissue injury due to diabetes. AGE-rich foods could exacerbate diabetic injury, at least by raising the endogenous AGE. MATERIALS AND METHODS: Herein, we tested whether, prior to ingestion, diet-derived AGEs contain species with cell activating (TNFalpha), chemical (cross-linking) or cell oxidative properties, similar to native AGEs. Glutathione (GSH) and GSH peroxidase (GPx) were assessed after exposure of human umbilical vein endothelial cell (HUVECs) to affinity-purified food-AGE extracts, each exposed to 250 degrees C, for 10 min, along with synthetic AGEs. RESULTS: Animal product-derived AGE, like synthetic methylglyoxal-bovine serum albumin (MG-BSA), AGE-BSA, and AGE-low density lipoprotein (AGE-LDL), induced a dose- and time-dependent depletion of GSH (()60-75%, p, 0.01) and an increase in GPx activity (()500-600%, p < 0.01), consistent with marked TNFalpha and cross-link formation (p < 0.05); this contrasted with the low bioreactivity of starch/vegetable AGE-extracts, which was similar to that of control BSA and CML- BSA and BSA (p:NS). Anti-AGE-R1,2,3 and -RAGE IgG each inhibited cell-associated (125) I-dAGE by approximately 30-55%; GSH/GPx were effectively blocked by N-acetyl-cysteine (NAC, 800 uM, p < 0.01) and aminoguanidine-HCl (AG, 100 uM, p < 0.01). CONCLUSION: Thus, food-derived AGE, prior to absorption, contain potent carbonyl species, that can induce oxidative stress and promote inflammatory signals.


Prevention of diabetic nephropathy in mice by a diet low in glycoxidation products (Feng Zheng et al, 16 May 2002)

Background
Reactive advanced glycation end products (AGEs), known to promote diabetic tissue damage, occur endogenously as well as in heated foods and are orally absorbed. The relative contribution of diet-derived AGEs to diabetic nephropathy (DN) remains unclear.
Methods
We tested a standard mouse food (AIN-93G) found to be rich in AGEs (H-AGE diet) in parallel with a similar diet that contained six-fold lower AGE content (L-AGE), but equal calories, macronutrients, and micronutrients. Non-obese diabetic mice (NOD) with type 1 diabetes (T1D) and db/db mice with type 2 diabetes (T2D) were randomly assigned to each formula for either 4 or 11 months, during which time renal parameters and AGE levels were assessed.
Results
Compared to the progressive DN and short survival seen in NOD mice exposed to long-term H-AGE feeding, L-AGE-fed NOD mice developed minimal glomerular pathology and a modest increase in urinary albumin:creatinine ratio (p<0.005), and a significantly extended survival (p<0.0001), consistent with lower serum (p<0.025) and kidney AGEs (p<0.01). Also, in the 4-month study, and in contrast to the H-AGE-fed mice, L-AGE-fed NOD and db/db mice exhibited low levels of renal cortex TGFβ-1 (p<0.05), laminin B1 mRNA (p<0.01) and α1 IV collagen mRNA (p<0.05) and protein, in concert with reduced serum and kidney AGEs (p<0.05, respectively).
Conclusion
Intake of high-level, food-derived AGEs is a major contributor to DN in T1D and T2D mice. Avoidance of dietary AGEs provides sustained protection against DN in mice; providing the rationale for similar studies in human diabetic patients.


Improved Insulin Sensitivity Is Associated With Restricted Intake of Dietary Glycoxidation Products in the db/db Mouse (Susanna M. Hofmann et al, 2002)

Advanced glycation end products (AGEs), known promoters of diabetic complications, form abundantly in heated foods and are ingested in bioreactive forms. To test whether dietary AGEs play a role in the progression of insulin resistance, C57/BL/KsJ db/db mice were randomly placed for 20 weeks on a diet with either a low AGE content (LAD) or a 3.4-fold higher content of AGE (high AGE diet [HAD]), including εN-carboxymethyllysine (CML) and methylglyoxal (MG). LAD-fed mice showed lower fasting plasma insulin levels throughout the study (P = 0.01). Body weight was reduced by ∼13% compared with HAD-fed mice (P = 0.04) despite equal food intake. LAD-fed mice exhibited significantly improved responses to both glucose (at 40 min, P = 0.003) and insulin (at 60 min, P = 0.007) tolerance tests, which correlated with a twofold higher glucose uptake by adipose tissue (P = 0.02). Compared with the severe hypertrophy and morphological disorganization of islets from HAD-fed mice, LAD-fed mice presented a better-preserved structure of the islets. LAD-fed mice demonstrated significantly increased plasma HDL concentrations (P < 0.0001). Consistent with these observations, LAD-fed mice exhibited twofold lower serum CML and MG concentrations compared with HAD-fed mice (P = 0.02). These results demonstrate that reduced AGE intake leads to lower levels of circulating AGE and to improved insulin sensitivity in db/db mice.


Advanced glycation end-products: a review(R Singh etal, 2001)

Advanced glycation end-products are a complex and heterogeneous group of compounds that have been implicated in diabetes related complications. At present it is not known if they are the cause or the consequence of the complications observed. We discuss the chemistry of advanced glycated end-product formation and their patho-biochemistry particularly in relation to the diabetic microvascular complications of retinopathy, neuropathy and nephropathy as well as their role in the accelerated vasculopathy observed in diabetes. The concept of carbonyl stress as a cause for advanced glycated end-product toxicity is mentioned. We discuss alterations in the concentrations of advanced glycated end-products in the body, particularly in relation to changes occuring with age, diabetes and its complications such as nephropathy. Problems relating to current methods of advanced glycated end-product detection and measurement are highlighted including the lack of a universally established method of detection or unit of measurement. Agents used for the treatment of advanced glycated end-product accumulation are reviewed, with an emphasis on the results of the recent phase III trials using aminoguanidine and diabetes related complications.


Protein glycation, diabetes, and aging (P Ulrich & A Cerami, 2001)

Biological amines react with reducing sugars to form a complex family of rearranged and dehydrated covalent adducts that are often yellow-brown and/or fluorescent and include many cross-linked structures. Food chemists have long studied this process as a source of flavor, color, and texture changes in cooked, processed, and stored foods. During the 1970s and 1980s, it was realized that this process, called the Maillard reaction or advanced glycation, also occurs slowly in vivo. Advanced glycation endproducts (AGEs) that form are implicated, causing the complications of diabetes and aging, primarily via adventitious and crosslinking of proteins. Long-lived proteins such as structural collagen and lens crystallins particularly are implicated as pathogenic targets of AGE processes. AGE formation in vascular wall collagen appears to be an especially deleterious event, causing crosslinking of collagen molecules to each other and to circulating proteins. This leads to plaque formation, basement membrane thickening, and loss of vascular elasticity. The chemistry of these later-stage, glycation-derived crosslinks is still incompletely understood but, based on the hypothesis that AGE formation involves reactive carbonyl groups, the authors introduced the carbonyl reagent aminoguanidine hydrochloride as an inhibitor of AGE formation in vivo in the mid 1980s. Subsequent studies by many researchers have shown the effectiveness of aminoguanidine in slowing or preventing a wide range of complications of diabetes and aging in animals and, recently, in humans. Since, the authors have developed a new class of agents, exemplified by 4,5-dimethyl-3-phenacylthiazolium chloride (DPTC), which can chemically break already-formed AGE protein-protein crosslinks. These agents are based on a new theory of AGE crosslinking that postulates that alpha-dicarbonyl structures are present in AGE protein-protein crosslinks. In studies in aged animals, DPTC has been shown to be capable of reverting indices of vascular compliance to levels seen in younger animals. Human clinical trials are underway.


Advanced glycation end products: A nephrologist's perspective (Dominic S.C. Raj MD et al, March 2000)

Advanced glycation end products (AGEs) are a heterogeneous group of molecules that accumulate in plasma and tissues with advancing age, diabetes, and renal failure. There is emerging evidence that AGEs are potential uremic toxins and may have a role in the pathogenesis of vascular and renal complications associated with diabetes and aging. AGEs are formed when a carbonyl of a reducing sugar condenses with a reactive amino group in target protein. These toxic molecules interact with specific receptors and elicit pleiotropic responses. AGEs accelerate atherosclerosis through cross-linking of proteins, modification of matrix components, platelet aggregation, defective vascular relaxation, and abnormal lipoprotein metabolism. In vivo and in vitro studies indicate that AGEs have a vital role in the pathogenesis of diabetic nephropathy and the progression of renal failure. The complications of normal aging, such as loss of renal function, Alzheimer's disease, skin changes, and cataracts, may also be mediated by progressive glycation of long-lived proteins. AGEs accumulate in renal failure as a result of decreased excretion and increased generation resulting from oxidative and carbonyl stress of uremia. AGE-modified β2 -microglobulin is the principal pathogenic component of dialysis-related amyloidosis in patients undergoing dialysis. Available dialytic modalities are not capable of normalizing AGE levels in patients with end-stage renal disease. A number of reports indicated that restoration of euglycemia with islet-cell transplantation normalized and prevented further glycosylation of proteins. Aminoguanidine (AGN), a nucleophilic compound, not only decreases the formation of AGEs but also inhibits their action. A number of studies have shown that treatment with AGN improves neuropathy and delays the onset of retinopathy and nephropathy. N -Phenacylthiazolium bromide is a prototype AGE cross-link breaker that reacts with and can cleave covalent AGE-derived protein cross-links. Thus, there is an exciting possibility that the complications of diabetes, uremia, and aging may be prevented with these novel agents.


Mechanism of autoxidative glycosylation: identification of glyoxal and arabinose as intermediates in the autoxidative modification of proteins by glucose (K J Wells-Knecht et al, 21 Mar 1995)

Glycation and oxidation reactions contribute to protein modification in aging and diabetes. Formation of dicarbonyl sugars during autoxidation of glucose is the hypothetical first step in the autoxidative glycosylation and subsequent browning of proteins by glucose [Wolff, S. P., & Dean, R. T. (1987) Biochem. J. 245, 243-250]. In order to identify the dicarbonyl sugar(s) formed during autoxidation of glucose under physiological conditions, glucose was incubated in phosphate buffer (pH 7.4) at 37 degrees C under air (oxidative conditions) or nitrogen with transition metal chelators (antioxidative conditions). Dicarbonyl compounds were analyzed spectrophotometrically and by HPLC after reaction with Girard-T reagent. Carbohydrates were analyzed by gas chromatography-mass spectrometry. Both dicarbonyl sugar and arabinose concentrations increased with time and glucose concentration in incubations conducted under oxidative conditions; only trace amounts of these products were detected in glucose incubated under antioxidative conditions. HPLC analysis of adducts formed with Girard-T reagent indicated that glyoxal was the only alpha-dicarbonyl sugar formed on autoxidation of glucose. Glyoxal and arabinose accounted for > or = 50% of the glucose lost during a 21 day incubation. Neither glucosone nor its degradation product, ribulose, was detectable. Reaction of glyoxal with RNase yielded the glycoxidation product, N epsilon-(carboxymethyl)lysine, while arabinose is a source of pentosidine. Our results implicate glyoxal and arabinose as intermediates in the browning and crosslinking of proteins by glucose under oxidative conditions. They also provide a mechanism by which antioxidants and dicarbonyl trapping reagents, such as aminoguanidine, limit glycoxidation reactions and support further evaluation of these types of compounds for inhibition of chemical modification and crosslinking of proteins during aging and diabetes.


Cellular receptors for advanced glycation end products. Implications for induction of oxidant stress and cellular dysfunction in the pathogenesis of vascular lesions (Ann Marie Schmidt et al, Oct 1994)

Advanced glycation end products (AGEs) form bythe interaction of aldoses with proteins and the subsequent molecular rearrangements of the covalently linked sugars, eventuating in a diverse group of fluorescent compounds of yellow-brown color. This heterogeneous class of nonenzymatically glycated proteins or lipids is found in the plasma and accumulates in the vessel wall and tissues even in normal aging. As a consequence of hyperglycemia, AGE formation and deposition are much enhanced in diabetes, in which their presence has been linked to secondary complications, especially microvascular disease. This review summarizes the cellular interactions of AGEs and describes the central role of a novel receptor for AGE (RAGE). RAGE, an immunoglobulin superfamily member, mediates the binding of AGEs to endothelial cells and mononuclear phagocytes, interacts with a lactoferrin-like polypeptide that also binds AGEs, and appears to activate intracellular signal transduction mechanisms consequent to its interaction with the glycated ligand. RAGE is expressed by ECs, mononuclear phagocytes, smooth muscle cells, mesangial cells, and neurons, indicating a potential role in the regulation of their properties in homeostasis and/or their dysfunction in the development of diabetic complications. Since AGEs have been shown to generate reactive oxygen intermediates, tethering of AGEs to the cell surface bytheir receptors focuses oxidant stress on cellular targets, resulting in changes in gene expression and the cellular phenotype. The discovery of RAGE and development of reagents to block its interaction with AGEs should provide insights into the role of this ligand-receptor interaction in the pathogenesis of diabetic complications and, potentially, atherosclerosis.