Advanced glycation end products are direct modulators of β-cell function.
Impact factor: 7.72
Level of evidence: II
Sample Size: In the first study, groups of 10 male Sprague-Dawley rats were given AGE-modified rat serum albumin (AGE-RSA) or saline (sham) for 4 months. Other groups of rodents were given AGE-RSA and treated with alagebrium. In the second study, groups 20 male Sprague-Dawley rats were randomized to be fed a low AGE diet or a high AGE diet. Rats were randomized again to receive sham or alagebrium. In the third study, groups of 20 female non-obese mice were analyzed for prediabetes at age 75 days with or without alagebrium chloride being administered.
Protocol: The effects of AGEs on insulin secretion by MIN6N8 cells and mouse islet cells in-vitro were measured. Also, three separated in-vivo rodent studies were done: Both Sprague-Dawley and nonobese diabetic rats were injected with AGE or fed high AGE diets and rodents were also treated with alagebrium, an AGE-lowering agent.
Statistical Analysis: Studies done with more than two rodent groups were assessed via ANOVA followed by Tukey’s post hoc. Student t-test and Mann-Whitney U tests were preformed when appropriate. Mantel-Coz was used to find the incidence of diabetes in non-obese mice. P-value: 0.05.
Study Type: Randomized control
Results: Beta cells that were exposed to AGEs showed insulin secretory defects, defects in mitochondria, excess superoxide generation, a decrease in ATP, a loss of SOD activity, reduced calcium flux, and increased uptake of glucose. All of these features were improved with the administration of alagebrium or with SOD overexpression. Isolated islet cells exposed to AGEs also showed insulin secretory defects, as well as an increase if superoxide production and a decrease in ATP. These features were also improved with alagebrium or the SOD mimetic, MnTBAP. Non-obese mice specifically showed increased circulating AGEs, as well as an increase in islet superoxide formation which was prevented by alagebrium.
Conclusion: AGEs cause defects in the secretion of insulin, most likely by inhibiting mitochondrial function. This may contribute to the development of Type I Diabetes.
Coughlan MT, Yap FY, Tong DC, Andrikopoulos S, Gasser A, Thallas-Bonke V, Webster DE, Miyazaki J, Kay TW, Slattery RM, Kaye DM, Drew BG, Kingwell BA, Fourlanos S, Groop PH, Harrison LC, Knip M, Forbes JM. Advanced glycation end products are direct modulators of β-cell function. Diabetes. 2011 Oct;60(10):2523-32. doi: 10.2337/db10-1033. Epub 2011 Sep 12. PMID: 21911745; PMCID: PMC3178291.
Advanced glycation end products in children with type 1 diabetes: family matters?
Study Type: Cross-sectional.
Sample size: 52 type 1 diabetics, age 12 (55% boys); 28 nondiabetic, age 11 (48% boys). Both groups were balanced in terms of BMI, waist circumference, and blood pressure.
Protocol: Skin autofluorescence (SAF) was measured using an AGE reader in three consecutive rounds. GHb of the diabetic children were also noted.
Statistical Analysis: Mann-Whitney U P-value: 0.001. For factors associated with SAF, multivariant regression model with age and sex as confounding variables. P-value: 0.05.
Results: The level of SAF was significantly higher in children with diabetes compared to the siblings group. SAF was also associated with GHb levels.
Conclusion: There may be factors outside of hyperglycemia that are involved in the skin AGE levels in diabetic children. The sample size is a limitation of this study.
Barat P, Cammas B, Lacoste A, Harambat J, Vautier V, Nacka F, Corcuff JB. Advanced glycation end products in children with type 1 diabetes: family matters? Diabetes Care. 2012 Jan;35(1):e1. doi: 10.2337/dc11-1398. PMID: 22187444; PMCID: PMC3241329.
Perinatal exposure to high dietary advanced glycation end products in transgenic NOD8.3 mice leads to pancreatic beta cell dysfunction.
Sample size: 10 offspring/high or low AGE diet group, 5 litters
Protocol: A strain of mice susceptible to type 1 diabetes were used. Pregnant female mice were maintained on either a high AGE diet or low AGE diet. Islet cells were isolated and hormone concentration was measured using an insulin ELISA kit.
Statistical Analysis: Mann-Whitney U P-value: 0.05; Chi-squared for trends in insulitis frequency
Study Type: Rat study
Results: Rat offspring who were exposed to low AGE diets during pregnancy and early life had lower circulating AGE concentrations compared to those exposed to high AGE diets. Higher insulin, proinsulin, and glucagon levels were also observed in isolated islet cells from the low AGE diet group. The lower AGE diet group also showed higher expression of Ins2 (encoding preproinsulin) and lower glucagon, AGEs and AGE receptors in isolated islet cells, as well as lower immune cell infiltration.
Conclusion: In the diabetic susceptible mice strain, reduced AGE diet during pregnancy, lactation, and early offspring life may enhance islet secretions and immune infiltration.
Borg DJ, Yap FYT, Keshvari S, Simmons DG, Gallo LA, Fotheringham AK, Zhuang A, Slattery RM, Hasnain SZ, Coughlan MT, Kantharidis P, Forbes JM. Perinatal exposure to high dietary advanced glycation end products in transgenic NOD8.3 mice leads to pancreatic beta cell dysfunction. Islets. 2018 Jan 2;10(1):10-24. doi: 10.1080/19382014.2017.1405189. Epub 2017 Dec 22. PMID: 29157116; PMCID: PMC5796486.
Oral advanced glycation end products (AGEs) promote insulin resistance and diabetes by depleting the antioxidant defenses AGE receptor-1 and sirtuin 1.
Impact factor: 9.412
Level of evidence: VII
Sample size: 18-20 mice per generation (F0, F1-3)
Important methods: 8-week-old C57BL/6 mice in the F0 generation were fed a standard diet without heat exposure (low-AGE). The offspring F1-F3 were put into two pair-fed groups, with one group eating low-AGE (MG- diet) and the other eating the same formula with added AGEs (MG+ diet). AGEs in the serum, cells, and tissues were measured by competitive ELISA for CML and for MG-derivatives. AGE lipids were determined by direct ELISA. Glucose tolerance was assessed by doing an IGTT every 6 months after the age of 1 year. Adipocytes from fat pads and the thigh of 18 month old were used for insulin-stimulated glucose uptake. RNA isolation and RT-PCR, Western Analysis, Immuno-precipitation, and ROS Determination.
Statistical Analysis: Student’s t test was used to determine the significance between groups and one-way ANOVA with Bonferroni correction analysis for comparisons among the three groups. A value of p < 0.05 was considered significant.
Study Type: Multigenerational mouse studies, In vivo
Results: MG+ diet promotes weight gain, adiposity, and metabolic changes in wild-type mice. These results look like replicated metabolic dysfunctions that exist in humans. MG+ diet altered insulin receptor (IRS-1) and Akt levels in pathways that require insulin.
Conclusion: It showed that having a high amount of AGEs in the diet can cause early depletion of certain defense mechanisms that will allow the elevation of oxidative species. A link has been identified between exogenous AGEs and metabolic dysfunction and is supported by the activation of inflammation in macrophages and adipocytes. AGEs present in human food can cause long-term disease outcomes by depleting AGEr1 and SIRT1. Reducing/restricting AGEs in the diet acan help regain gene function and lower risk of T2D or seriousness of T2D and other diseases linked to high oxidative species.
Cai W, Ramdas M, Zhu L, Chen X, Striker GE, Vlassara H. Oral advanced glycation end products (AGEs) promote insulin resistance and diabetes by depleting the antioxidant defenses AGE receptor-1 and sirtuin 1. Proc Natl Acad Sci U S A. 2012 Sep 25;109(39):15888-93. doi: 10.1073/pnas.1205847109. Epub 2012 Aug 20. PMID: 22908267; PMCID: PMC3465382.
Increased skin autofluorescence of children and adolescents with type 1 diabetes despite a well-controlled HbA1c: results from a cohort study.
Sample Size: 77 type 1 diabetic patients recruited from an outpatient clinic, 118 healthy patients recruited from a school; separated into age groups (11-12, 13-14, 15-16, 17-19).
Protocol: Skin autofluorescence (SAF) were measured three times via a Diabeter and the mean measurement was used. HbA1c, age, and diabetes duration were evaluated via retrospective cohort study.
Statistical Analysis: SAF- Mann-Whitney U, P-value: 0.009; HbAc1, age, and SAF- multivariant analysis.
Study Type: Cross-sectional
Results: Children and adolescents with type 1 diabetes had significantly higher SAF than healthy patients in all age groups; however, not all age groups showed a correlation between SAF and HbA1c levels.
Conclusion: This study was consistent with previous studies that show elevated SAF levels in type 1 diabetic patients. Finding were also consistent with studies that show accelerated SAF levels in older patients. The results showed only a weak association between SAF and HbA1c levels that disappeared when adjusted according to age and diabetes duration, which conflicts with some previous studies.
Van der Heyden JC, Birnie E, Mul D, Bovenberg S, Veeze HJ, Aanstoot HJ. Increased skin autofluorescence of children and adolescents with type 1 diabetes despite a well-controlled HbA1c: results from a cohort study. BMC Endocr Disord. 2016 Sep 9;16(1):49. doi: 10.1186/s12902-016-0129-3. PMID: 27613110; PMCID: PMC5017065.
Restriction of advanced glycation end products improves insulin resistance in human type 2 diabetes: potential role of AGER1 and SIRT1.
Impact Factor: 16.019
Level of Evidence: II
Sample size: 36 subjects, 18 type 2 diabetics (age 61± 4 years, 18 healthy (age 67 ± 1. 4 years).
Protocol: Subjects were randomly chosen assigned to a standard diet of >20 AGE equivalents/day, (n=6) or an isocaloric AGE-restricted diet of <10 AGE Eq/day, (n=12) for 4 months. All subjects were still receiving standard care. The AGE-restricted groups were told to boil, poach, stew, or steam food and to avoid frying, baking or grilling. Nutritional intake was closely monitored in both groups. CML and MG were measured in the serum. Insulin resistence was estimated by the HOMA index.
Statistical Analysis: Kolmogorov-Smirnov goodness-of-fit to test for normal distribution, those that were not were adjusted. Differences of means between groups were analyzed by Student t test or ANOVA followed by the Bonferroni correction for multiple comparisons. Correlation analyses were also used by the Peason correlation coefficient. Significance defined as p<0.05 and are based on two-sided tests.
Study Type: Randomized control trial; in vitro
Results: Type 2 diabetics had significantly higher fasting blood glucose, plasma insulin, BMI , CML, sMG, and leptin. They also had significantly higher levels of AGEs. Receptors for AGEs were lower in those with type 2 diabetes than in healthy subjuects. After AGE-restricted diet, levels of mRNA for receptor for AGEs in type 2 diabetics was increased by 50% (almost normal range.) In diabetic patients who ate the standard diet, the receptors were still low.
Conclusion: AGE restriction can improve insulin resistance in type 2 diabetes. Improvements in plasma insulin, markers of inflammation, and oxidative stress were found in diabetic patients on AGE-restrictive diet. Plasma insulin was near normal in type 2 diabetics when on AGE-restricted diet, showing that AGEs in the diet can have an effect in the metabolic state. This study shows that chronic exposure to exogenous AGEs in the diet can impair native antioxidant defenses and insulin action. Reducing dietary intake of AGEs can reduce that.
Uribarri J, Cai W, Ramdas M, Goodman S, Pyzik R, Chen X, Zhu L, Striker GE, Vlassara H. Restriction of advanced glycation end products improves insulin resistance in human type 2 diabetes: potential role of AGER1 and SIRT1. Diabetes Care. 2011 Jul;34(7):1610-6. doi: 10.2337/dc11-0091. PMID: 21709297; PMCID: PMC3120204.
Maternally transmitted and food-derived glycotoxins: a factor preconditioning the young to diabetes?
Impact factor: 16.019
Level of evidence: II
Sample Size: n=60 mothers in labor, their infants, and infant foods.
Protocol: AGEs were tested in the sera of healthy mothers in labor, their infants, and infant foods. Fasting blood samples were obtained from the mothers at the time of the delivery. A full clinical assessment of the babies was done and done again at 6 and 12 months. At the 6 and 12 month assessment, questions included ones about feeding patterns, illness, and a sample of blood was taken from the baby. Glucose and insulin were measured, as well as CML (AGE) and other methylglyoxal derivatives using ELISA. Plasma 8-isoprostane, adiponectin, leptin, and insulin levels were also tested using ELISA kits. Insulin resistance was calculated using HOMA index as fasting insulin x fasting glucose (mmol/L)/22.5.
Statistical Analysis: Differences of mean values between groups by unpaired Student t-test or ANOVA followed by Bonferroni correction depending on the number of groups. Correlation analysis by Pearson correlation coefficient. Significance considered p < 0.05 and based on two-sided tests.
Study Type: Observational study where the relationship of maternal blood and food AGEs to circulating glycoxidants, inflammatory markers, and insulin levels in infants up to 1 year of age.
Results: Correlations were found between newborn and maternal serum CML (r=0.734, p=0.001), serum methyglycoxal (r=0.59, p=0.001), and 8-isoprostanes (r=0.6.44, p=0.001). Infant adiponectin at 12 months negatively correlated with maternal sCML (r= -0.467, p=0.001). High maternal sMGs predicted higher insulin in infants (p=0.027). Infant soluble AGEs significantly increased while starting processed food intake, raising daily AGE intake to around 7.5 fold in 1 year.
Conclusion: Maternal blood and AGEs from the diet raise AGEs in children to the levels seen in a normal adult. This preconditions the children to high oxidant stress and inflammation. There is a possibly that this can cause an early onset of diabetes. An excess in maternal AGEs by consuming AGE-rich foods while pregnant could affect fetal development, infancy, or adult health. AGEs can impair insulin production and function. Lower adiponectin was seen in infants whose mothers had high AGE levels. This burden of taking on higher glycoxidants from the mother at infancy can contribute to increased incidence of diabetes in the young. Limitations to this study were small sample size, low access to infant blood samples, and lack of detailed food intake assessment, but these results have been repeated in rat studies.
Mericq V, Piccardo C, Cai W, Chen X, Zhu L, Striker GE, Vlassara H, Uribarri J. Maternally transmitted and food-derived glycotoxins: a factor preconditioning the young to diabetes? Diabetes Care. 2010 Oct;33(10):2232-7. doi: 10.2337/dc10-1058. Epub 2010 Jul 13. PMID: 20628088; PMCID: PMC2945165.
Fetal or neonatal low-glycotoxin environment prevents autoimmune diabetes in NOD mice.
Impact factor: 7.72
Level of evidence: IV
Sample Size: 34 female mice and 12 brother-sister mating pairs aged 6 weeks.
Protocol: NOD mice were exposed to a high-AGE diet (H-AGE) and a different group was fed a diet similar in nutritional value, but with AGE levels fivefold-lower (L-AGE). The H-AGE diet was prepared by exposing to heat two times while the L-AGE was only exposed one time and contained fivefold less CML-like AGEs. Body weights and urine samples were gathered and the mice were randomly assigned H-AGE and L-AGE diets and were followed up to 56 weeks of age. In the F0 generation, 22 females and 8 mating pairs were placed on the L-AGE diet, and 12 females and 4 mating pairs were place on the H-AGE diet at 6 weeks of age. After weaning, the F1 (n=55) and F2 (n=51) generations’ offspring were assigned to diets: 21 female mice from F1 and 30 females from F2 NOD mice continued on the L-AGE diets, while 18 females from F1 and 21 females from F2 NOD mice continued on the H-AGE diets. Also, and additional 16 females of F1 offspring from L-AGE fed mothers were switched to the H-AGE diet after weaning. Body weight and urinary glucose were measured once per week until onset of glucosuria and by tail vein blood after that. The mice were considered to have diabetes if they had persistent hyperglycemia (>250 mg/dl) for over a week. Diabetic mice were treated with insulin twice dialy to maintain blood glucose levels at 150-200 mg/dl for up to 56 weeks. Twelve hour blood fasting levels and 24 hour urine samples were collected at 1-2 month intervals. In group F1, some mice were sacrificed at 9, 12 and 16 weeks for collection of blood and tissue specimens. At 16 weeks of age, an intraperitoneal glucose tolerence test (IGTT) in groups of 12 mice from 4 different litters after an overnight fast (13-15h). BG and insulin levels were measured before and at half-hour intervals up to 120 minutes. ELISA was used to measure insulin levels. Serum and using AGE levels were determined by ELISA-1. Albumin in the urine was measured by an anti-mouse albumin ELISA kit, and creatine in the urine was assessed by a colorimetric method. Renal function was assessed as the albumin-to-creatinine ratio. Immunohistology and flow cytometry was used to examine the tissue for lymphocyte isolation.
Statistical Analysis: Two-tailed unpaired t test (Mann-Whitney) or Student’s t test was used to evaluate differences between the means of grouped data from mice fed with H-AGE and L-AGE diets. A p value of <0.05 was considered significant.
Study Type: Multigenerational mouse study
Results: Food and water intake were similar between groups during they study period. There were no significant differences in body weights between the H-AGE and L-AGE diet groups. Weight was only lost in mice with Type 1 diabetic mice. H-AGE fed mice ingested fivefold more AGE than the L-AGE mice determined by fasting serum AGE. Reduction in the cumulative incidence of type 1 diabetes was evident in the AGE-restricted groups compared to those on the H-AGE diet. Of the 16 F0 mice, 15 developed diabetes by 25 weeks, and only 6 of the 30 L-AGE fed mice had diabetes or with a delay of 15 weeks. In the first generation group 11 of 18 H-AGE fed mice we diabetic by 25 weeks, as compared to only 3 of 21 of L-AGE fed mice that didn’t develop diabetes until about 40 weeks or with a 15 week lag. Similar results were found in the F2 subgroup; 13 of 21 (62%) of H-AGE fed mice were diabetic at 25 weeks, while only 4 of 30 (13.3%) L-AGE fed mice by 40 weeks. F1 H-AGE mice that were derived from L-AGE fed mothers (56%) also became diabetic by 24 weeks (no lag). Overall, 76% of mice fed the L-AGE diet lived up to 56 weeks, where none of the H-AGE fed mice lived after 44 weeks even when treated with insulin.
When comparing dietary AGE and Beta-cell function at about 16 weeks of age, L-AGE fed mice that were prediabetic showed a lower fasting blood glucose and higher insulin levels compared with mice that were the same age but fed the H-AGE diet. L-AGE fed mice also showed a lower glycemic response to IGTT, and greater insulin responses to glucose.
Upon histological examination of the pancreas, >50% of islets showed inflammatory infiltration interrupting normal function of these cells, in H-AGE fed mice at 9 weeks. At 48 weeks, the islets from diabetic mice given the H-AGE diet showed that all of them had lymphocytic infiltration and were seriously affected (100% > grade III). Only 20% of the islets from mice that were diabetic L-AGE fed mice were affected as seriously. The other 80% showed mild insulitis. Of the mice that were non-diabetic, but given the H-AGE diet, 50% showed severe insulitis (>grade III), and only 10% of L-AGE fed mice with the same amount of damage. The rest showed little to no unsulitis.
Conclusion: A diet low in glycotoxins lead to blockage of type 1 diabetes in the NOD mouse that is genetically susceptible. The incidence of type 1 diabetes was decreased the earlier in life the restriction of AGEs in the diet was applied. Islet cell infiltration by cytotoxic T cells was suppressed. A marked increase in overall survival was seen, and if the disease did occur in the L-AGE fed mice, it was delayed in onset and reduced in severity. The L-AGE diet had shorter heat exposure during processing and therefore had less glycotoxin content. Around 10% of AGEs in the diet are absorbed orally and some are able to cause tissue damage. It is possible that AGEs from food contribute to autoimmune diabetes. Low-AGE diets may be an effective strategy for diabetes prevention. There has been an increase in type 1 diabetes with the rise in the Western-type AGE-enriched diets, and this supports what is found in this study.
Peppa M, He C, Hattori M, McEvoy R, Zheng F, Vlassara H. Fetal or neonatal low-glycotoxin environment prevents autoimmune diabetes in NOD mice. Diabetes. 2003 Jun;52(6):1441-8. doi: 10.2337/diabetes.52.6.1441. PMID: 12765955.
Methylglyoxal Metabolism and Aging-Related Disease: Moving from Correlation toward Causation.
Impact factor: 11.641
Level of evidence: Level V
Type of study and any information related to it: Review study
Discussion/Conclusion: This review article highlights current research on methylglyoxal (MG), its metabolism, and advanced glycation end-products (AGEs) and the causal role they play in various diseases. To start, MG nonenzymatically reacts with biopolymers to form AGEs in the body. Finding a consistent and reliable method to accurately measure MGs has proven difficult as MGs can be lost from samples through detoxification, degradation, or auto-oxidation if samples are not handled quickly and properly stored. Additionally, there have been reports of glassware being contaminated with MG, which can lead to inaccurate measurements. The current method to measure MG is to stabilize it through chemical derivatization and use liquid chromatography-mass spectrometry (LC-MS) based quantification. Human correlation studies have demonstrated that elevated levels of MG and AGEs in the plasma are associated with cardiovascular disease and types 1 and 2 diabetes. Increased MG is associated with degenerative changes in the kidney including loss of function in type 2 diabetes, diabetic neuropathy, and end-stage kidney disease in patients with chronic kidney disease. It has even been observed that increased AGEs and MG can be associated with diabetic complications independently of hemoglobin A1c. This evidence suggests that MG and AGEs may be main contributors to diabetic complications. The glyoxalase (GLO1 and GLO2) enzyme system is our body's main tool for ridding itself of MG. Glutathione must first form a hemithioacetal with MG before the glyoxalase system can convert MG into D-lactate. Researchers have created cell and animal models with knockdown (KD) or overexpression of the glyoxalase system to elucidate the role MG and AGEs play in diseases. In nondiabetic mice with KD of GLO1 there was an increase in AGEs, oxidative stress, and degenerative changes in the kidneys synonymous to those caused by diabetes. Conversely, diabetic mice with an overexpression of GLO1 showed lower levels of MG and AGEs and subsequently less oxidative and renal damage. Additional human data demonstrated that individuals protected from DN express higher levels of GLO1 and aldose reductase (AR) compared to individuals susceptible to DN. Interestingly, GLO1 knockout (KO) systems did not result in an increase in MG concentration. The KO of GLO1 in cell and animal models demonstrated that in an undisturbed system MG levels were within normal limits, however, the lethal concentration of MG had been significantly reduced. Evidence from the KO model suggest that there are secondary mechanisms for MG removal that can compensate for the loss of the glyoxalase system. The human correlation studies along with the KD, KO, and overexpression studies suggest a causal relationship between GLO1, MG, and AGEs and diabetes. Aldo-keto reductases (AKRs), aldehyde dehydrogenases (ALDHs), Parkinson associated protein DJ-1, ketone bodies, and acetoacetate represent some of the additional pathways beyond the glyoxalase system for the detoxification of MG. The expression of AKR levels was shown to be elevated in GLO1 KO mice and to have partially contributed to MG detoxification. DJ-1 has been shown to possess glyoxalase activity with the ability to convert MG into L-lactate. Ketone bodies and acetoacetate can non enzymatically scavenge MG through an aldol reaction. These ancillary processes allow the body to handle excessive amounts of oxidative damage from MG. Several studies have suggested that exposure to low doses of MG may increase MG tolerance through an induction method. Specifically, treating GLO1 KO models with MG has been shown to increase the median and maximum life span in organisms such as Drosophila and C. elegans. Pharmacologic treatments are being developed that target the KEAP1-Nrf2-ATE pathway which is responsible for the regulation of GLO1 expression and MG metabolism. Sulforaphane (SFN) is a small compound found in broccoli sprout and it is an Nrf2 inducer which has been shown to increase GLO1 activity and glutathione levels in human cell models. The phenolic compounds 6-gingerol and phloretin can also activate Nrf2 leading to increased GLO1 and reduced AGEs. GLO1 inhibitors can potentially be used as antitumor agents as cancerous cells have high MG levels and require the glyoxalase system to survive.
Kold-Christensen R, Johannsen M. Methylglyoxal Metabolism and Aging-Related Disease: Moving from Correlation toward Causation. Trends Endocrinol Metab. 2020;31(2):81-92. doi:10.1016/j.tem.2019.10.003
Simple non-invasive assessment of advanced glycation end product accumulation.
Impact factor: 7.518
Level of evidence: Level VI
Type of study and any information related to it: This was a cross-sectional study seeking to validate the use of the Autofluorescence Reader (AFR) as a non-invasive tool to measure the accumulation of advanced glycation end products (AGEs) in the skin.
Sample size: 92 patients were selected, 46 of which had type 1 and 2 diabetes while the other 46 were control subjects. A majority of the subjects were Caucasian. Diabetic patients were randomly recruited from the diabetes outpatient clinic at University Hospitz Groningen in the Netherlands.
Important methods: The AFR was used to measure skin autofluorescence, from the subject’s forearm, with light intensities ranging from 300 to 600 nm. Full-thickness skin biopsies from the subject’s forearm were collected from the same area the AFR reading was taken and then analyzed for collagen-linked fluorescence (CFL) and specific AGEs pentosidine, Nε-(carboxymethyl)-lysine (CML) and Nε-(carboxyethyl)lysine (CEL).
Statistical tests ran: The Student’s t test was used to test the differences between skin autofluorescence in the diabetic and control groups. Multivariable stepwise regression analysis was used to observe the independent effects of autofluorescence. A two-tailed p-value of less than 0.05 was considered statistically significant.
Results: This study demonstrated that AFR was a valid measure of skin AGEs. There was a significant correlation between skin autofluorescence and CLF (Type 1 r=0.64, Type 2 r= 0.58; p= 0.01), pentosidine (r= 0.55, -<0.001), CML (r= 0.55, p<0.001) and CEL (r= 0.47, p= 0.002). The study also found that diabetic patients had a mean skin autofluorescence 25% higher than the control group. Skin autofluorescence was higher in patients with Type 2 diabetes than in patients with Type 1 diabetes. Additionally, skin autofluorescence was significantly correlated with age, serum creatinine, and hemoglobin A1c.
Discussion/Conclusion: This study demonstrated that the AFR can be a valid measure of AGE accumulation in skin. While the overlap in skin autofluorescence between the control group and diabetic group limit AFRs use as a screening tool for diabetes, it may have clinical use in assessing risk of AGE associated chronic complications of diabetes such as renal failure (serum creatinine) or glycemic control (hemoglobin A1c).
Limitations: This study is limited by the fact that not all AGEs exhibit fluorescent properties, so only a subset of the total AGEs in the body are being measured by the AFR. It is also impossible to rule out the possibility of other fluorochromes being present in the skin and leading to inaccurate AFR readings. The subjects in the study were primarily Caucasian, so differences between different skin pigmentation could not be established. Finally, the sample size in this study was small making it difficult to establish a causal relationship between AFR and assessing chronic diabetic complications.
Wang CC, Shen MY, Chang KC, Wang GJ, Liu SH, Chang CT. Skin autofluorescence is associated with rapid renal function decline in subjects at increased risk of coronary artery disease. PLoS One. 2019;14(5):e0217203. Published 2019 May 22. doi:10.1371/journal.pone.0217203
Influence of physical activity intervention on circulating soluble receptor for advanced glycation end products in elderly subjects.
Level of evidence: Level VI
Type of study and any information related to it: This was an interventional study investigating the influence that physical activity has on the serum levels of the soluble receptor for advanced glycation end products (sRAGE) in elderly subjects. The association between sRAGE and paraoxonasel (PON1), an antioxidative enzyme, activity was also studied.
Sample size: 30 community-dwelling Japanese volunteers (15 men, 15 women) with an average age of 65 were selected for the study. Each subject was in good health and had not been diagnosed with diabetes mellitus or kidney, liver, nutritional or collagen diseases.
Important methods: The subjects were enrolled in a 6-month interventional program aimed at increasing the physical activity of the volunteers. The program had the patients walk at a pace 60% of their maximal heart rate for ≥ 30 minutes. The researchers measured the volunteers BMI, serum PON1 activity and sRAGE levels before and after the intervention. PON1 activity was measured kinetically using a Versamax Microplate Reader and sRAGE levels were measured using an enzyme-linked immunosorbent assay.
Statistical tests ran: Paired t test was used to compare the pre- and post-phase values of the measured variables. Single linear regression analysis and stepwise multiple linear regression were used to determine any correlation between variables in the pre-phase and change between the pre- and post- phase values. A p-value of less than 0.05 was considered statistically significant.
Results: There was a statistically significant reduction in sRAGE levels (Pre= 1103 ± 496 ng/L, Post= 1030 ± 437 ng/L; p= 0.019) after the 6-month interventional program. Additionally, the change in sRAGE was significantly and inversely related to PON1 activity (β= -0.511, p<0.01).
Discussion/Conclusion: This study suggests that circulating sRAGE levels can be significantly reduced by increasing physical activity over a 6-month period. Additionally, changes in sRAGE are inversely correlated with the activity of PON1. The findings from this research provide evidence that physical activity intervention may be a useful tool for medical professionals to help individuals lower their sRAGE levels and increase the activity of antioxidative enzymes, such as PON1.
Limitations: Firstly, the small sample size and limited intervention period (6-months) merit future research to confirm the findings of this study and help establish a causal relationship. Secondly, future studies could include more genetically and ethnically diverse patients to make the result of the study more applicable to different populations. Lastly, future research could measure the level of advanced glycation end products (AGEs) and after a physical activity interventional program.
Kotani K, Caccavello R, Sakane N, Yamada T, Taniguchi N, Gugliucci A. Influence of Physical Activity Intervention on Circulating Soluble Receptor for Advanced Glycation end Products in Elderly Subjects. J Clin Med Res. 2011;3(5):252-257. doi:10.4021/jocmr704w
The role of glycation in the pathogenesis of aging and its prevention through herbal products and physical exercise.
Level of evidence: Level V
Type of study and any information related to it: This is a review article that describes the role AGEs play in the aging process and what the potential inhibitory activity of exercise and herbal products are on non-enzymatic glycation related organ dysfunction.
Discussion/Conclusion: AGEs are produced in a nonenzymatic reaction between sugars and proteins from foods; this is called the Maillard reaction. Glycation occurs spontaneously over time which has been shown in experiments in D. melanogaster flies with older flies having a higher concentration of AGEs compared to younger flies. Glycation can also occur in pathological processes (e.g. cardiovascular disease, diabetes, renal disease, Alzheimer’s) and is thought to contribute to cellular dysfunction in these diseases.
AGEs contribute to aging through three separate processes: the accumulation of AGEs in extracellular matrix (ECM) and subsequent cross linking with the surrounding tissue, glycation of intracellular proteins leading to cellular dysfunction, and inflammation, ROS generation, and apoptosis resulting from the interaction of AGEs with RAGE. The crosslinking of AGEs with collagen and elastin decreases the flexibility of that tissue and will have negative effects on vascular structure and functions. There is an association between N-(carboxymethyl)-lysine (CML) and vascular stiffness in older individuals. Intracellular glycation of proteins can disturb cellular gene expression and decrease the activity of antioxidant enzymes. Glycation of intracellular enzymes that play a role in proteasome mediated proteolysis can lead to higher levels of glycated proteins inside the cell as well.
There is much interest in discovering pharmacologic therapies that can break AGE-mediated cross links and prevent the formation of AGEs. Aminoguanidine was formerly used as an AGE inhibitor because of its ability to react with carbonyl species; however, due to safety concerns aminoguanidine is not used clinically. Various AGE breakers, which disrupt AGE-mediated cross linking, have been studied. So far, none of the therapies have been successful for various reasons and there are no AGE breakers in clinical use.
Herbal products are considered a promising therapy because they are safe for human consumption and can have strong anti-glycation properties. Flavonoids, the major class of polyphenols, have been shown to inhibit the AGE formation in vitro. One flavonoid, epicathechin has been shown to have AGE breaking properties in vivo.
Physical exercise has been shown to reduce AGE accumulation in animal models and in human subjects. In one study, a 12-week exercise regimen reduced AGEs and RAGE levels in the aortic vessels of rats. Another study discovered that AGE levels were lower in the patellar tendon of life-long athletes when compared to untrained persons. Regular physical activity is thought to decrease AGE formation by decreasing ROS generation and improving the function of antioxidant enzymes. Exercise can further decrease AGE formation through improved glycemic control and higher energy demands that decrease available reactive intermediates for glycation.
Kim, C. S., Park, S., & Kim, J. (2017). The role of glycation in the pathogenesis of aging and its prevention through herbal products and physical exercise. Journal of exercise nutrition & biochemistry, 21(3), 55.
Plasma Proteins Modified by Advanced Glycation End Products (AGEs) Reveal site-specific susceptibilities to glycemic control in patients with type 2 diabetes.
Type of study: Case-Cohort study with median follow up of 10 years
Sample size: 134 cardiovascular case subjects, 218 random individuals (includes 65 cardiovascular cases) all with T2DM Important methods: Plasma levels of protein- bound N«-(carboxymethyl)lysine, N«-(carboxyethyl)lysine, and pentosidine were measured
Statistical tests ran: AGEs were transformed into a Z-score, association with cardiovascular events was measured with cox proportional hazard regression
Results: Higher plasma AGE z-scores were associated with increases cardiovascular events in individuals with and without prior events
Discussion/Conclusion: In individuals with T2DM, AGEs are associated with an increased risk of cardiovascular incidence
Greifenhagen U, Frolov A, Blüher M, Hoffmann R. Plasma Proteins Modified by Advanced Glycation End Products (AGEs) Reveal Site-specific Susceptibilities to Glycemic Control in Patients with Type 2 Diabetes. J Biol Chem. 2016 Apr 29;291(18):9610-6. doi: 10.1074/jbc.M115.702860. Epub 2016 Mar 1. PMID: 26933035; PMCID: PMC4850298.
Advanced glycation end-products regulate extracellular matrix-adipocyte metabolic crosstalk in diabetes.
Impact factor: 3.998
Level of Evidence: III
Sample Size: A total of 165 subjects were used. Diabetic patients were considered to have an HbA1c > 6.5%. 90 nondiabetic adipose tissue samples and 75 diabetic adipose tissue samples.
Protocol: Visceral adipose tissue (VAT) was collected from the greater omentum, subcutaneous adipose tissue was collected from the abdominal wall during bariatric surgery. AGE levels were measured from subcutaneous (SAT) and visceral (VAT) adipose tissues from obese diabetic patients (DM) and nondiabetic pateints (NDM) using fluorescence microscopy. In a 3D-ECM-adipocyte culture system, the effects of AGE-modified ECM on adipocyte metabolism was studied. Human adipose tissue was treated with high levels of glucose for 72 hours to maximize AGE-induction with a medium containing 17mM, 50mM, or 100 mM glucose, or 100 mM mannitol for negative control before isolation of ECM. ECM was isolated from tissues and treated with a deglycosylating enzyme PNGase for the last 24 hours of the 72-hour glucose treatment. Glucose uptake was evaluated in AGE-modified 3D-ECM-adipocyte cultures. Basal and insulin-stimulated glucose uptake were compared between DM and NDM cultures. ECM was isolated from treated visceral adipose tissue (VAT) and combined with VAT pradipocytes. NDM ECM and NDM preadipocytes were combined, while DM ECM was combined with DM preadipocytes. This was referred to as disease-matched ECM. Fluorescence microscopy analysis was used to analyze data.
Statistical Analysis: A p of 0.05 was considered significant. Linear mixed model was used to compare AGE-immunofluorescence in tissues by diabetes status. A linear mixed model was also used to compare the relationship between AGE-immunofluorescence and HbA1c in VAT and SAT separatley. The linear mixed model was used again to detect differences in glucose uptake by treatment and diabetes status in the 2D-cultures. A two-way ANOVA mixed model was used to compare values from RT-PCR data. For the 3D-cultures, linear mixed models were used to observe the effect of different levels of ECM glycation and diabetes status as well as addditional factors of RAGE antibody, and SMIFH2 on glucose uptake. Random intercept was used for all models to account for within-subject correlation, and controlled for age, sex, and BMI of the adipocyte and the ECM patient source. Post-hoc pairwise comparisons were used to find differences in outcome variables at different levels of independent variables using Vonferroni’s correction for multiple testing. Standard errors of the mean were shown.
Study Type: In vivo 2D and 3D-ECM culture system.
Results: The 100 mM glucose treatmented induced AGE on ECM to levels near those observed in native DM VAT, while PNGase decreased AGE levels. AGE are increased in adipose tissue of obese diabetic patients. AGE-modified ECM impairs adipocyte glucose uptake in 3D culture. DM ECM-adipocyte cultures showed decreased basal and insulin-stimulated uptake of glucose compared to NDM cultures. High glucose AGE-modified ECM showed a great decrease in insulin-stimulated glucose uptake compared to ECM conditioned in low glucose environments in both DM and NDM ECM-adipocyte cultures, but did not affect the basal uptake of glucose. This effect was more procounceed in DM ECM-adipocyte cultures.
Conclusion: AGE are increased in the kidneys, muscles, skin, and liver in humans what are obese with diabetes (DM). Using the 3D-adipocyte-ECM cultures, and inhibitory effect of AGE-modified ECM on the metabolism of glucose by adipocytes was reversed using PNGase treatment, showing that glycated ECM contributes to adipocute insulin resistance associated with DM. Complete removal of ECM glycosylation in vitro improves adipocyte glucose uptake.
Strieder-Barboza C, Baker NA, Flesher CG, Karmakar M, Neeley CK, Polsinelli D, Dimick JB, Finks JF, Ghaferi AA, Varban OA, Lumeng CN, O'Rourke RW. Advanced glycation end-products regulate extracellular matrix-adipocyte metabolic crosstalk in diabetes. Sci Rep. 2019 Dec 24;9(1):19748. doi: 10.1038/s41598-019-56242-z. PMID: 31875018; PMCID: PMC6930305.
The relationship between circulating irisin levels and tissues AGE accumulation in type 2 diabetes patients.
Impact factor: 2.942
Level of evidence: II
Sample size: 362 Chinese Type 2 diabetic patients (179 men and 183 women aged 37-64 years) and 100 age- and sex-matched healthy controls.
Protocol: Patients were recruited from Tianjin Metabolic Diseases Hospital in China. They were diagnosed with diabetes per WHO criteria from 1999.Patients with T1DM were excluded. T2DM was considered a patient with no signs of ketosis, a C-peptide assay stating that sufficient beta cells were present. A questionnaire was completed by each patient to gather information about age, sex, weight, height, BMI, age of onset of DM, fasting glucose levels, physical exercise as well as blood pressure. Patients who were obese with a BMI >30 kg/m2 and those with malignant neoplasms, renal or liver diseases or endocrinological diseases other than diabetes were excluded. None of the patients were taking any antihypertensive drugs, diabetes medications, estrogen supplements, or diuretics. Blood samples were taken after an overnight fast. Concentrations of triglyceride (TG), total cholesterol (TC), LDL-C, HDL-C, and glucose were obtained. Serum fasting insulin concentration was measured by immunoassay. Insulin resistance was measured (HOMA-IR). Serum irisin concentrations were measured using ELISA. HbA1c was measured through high-performance chromatography and CRP was measured. Skin AF was measured using and AGE Reader.
Statistical Analysis: Unpaired Student’s t test was applied to examine differences in variables between the groups. Chi-square analysis was used to compare categorical and nominal variables. ANOVA and Mann-Whitney U test with Tukey’s post hoc analysis. A multiple stepwise regression analysis was applied to determine independent association between AF and other independent variables. A p value of < 0.05 was considered significant.
Study Type: Cross-sectional
Results: Patients with T2DM were found to have higher skin AF, or AGEs, than those that were healthy. The patients with highest levels of skin AF had higher HbA1C, TC, TG, LDL-C and CRP and lower levels of irisin (cardiovascular protectant) and HDL-C. There was a negative correlation between skin AF and circulating irisin (r=0.227, P<0.001).
Conclusion: Irisin is cardiovascular protective and were found to be lower in patients with T2DM compared to healthy controls. Lower levels of irisin were associated with higher levels of AGEs in the skin indicating increased AGE accumulation. AGE accumulation based on skin AF levels in patients with T2DM was higher than that in healthy controls. HbA1c levels also increased with increasing skin AF values. Limitations include sample size, and although the correlation between plasma irisin and AGE accumulation provides evidence in the role of metabolic disorders, it does not address the pathology of angiopathy in T2DM.
Zhu Li, Gang Wang, Yan-juan Zhu, Chen-guang Li, Yun-zhao Tang, Zhen-huan Jiang, Min Yang, Chang-Lin Ni, Li-ming Chen, Wen-yan Niu; The relationship between circulating irisin levels and tissues AGE accumulation in type 2 diabetes patients. Biosci Rep 30 June 2017; 37 (3): BSR20170213. doi: https://doi.org/10.1042/BSR20170213
The advanced glycation end-product Nϵ -carboxymethyllysine promotes progression of pancreatic cancer: implications for diabetes-associated risk and its prevention.
Impact Factor: 6.021
Level of Evidence: VII
Type of Study: in vitro, in vivo, genetically engineered mouse model
Sample Size: 39; 11 Ctr treatment, 11 CML treatment, 10 Ctr+RAP treatment, 7 CML+RAP treatment
Introduction: The investigators seek to determine the role of AGEs in pancreatic cancer (PaC). The focus was an AGE found in vivo, Nϵ -carboxymethyllysine (CML), and its tumor-promoting role in a genetically-engineered mouse model of human PaC and in pancreatic ductal adenocarcinoma (PDA) cell lines.
Methods: An in vitro study investigated the tumor-promoting effects of CML on PDA cell lines by assessing cell growth, the tumorigenic pathways involved in PaC promotion, and whether the effects were RAGE-mediated. An in vivo study investigated how CML affects the progression of early murine pancreatic neoplasia. KC mice (Pdx1-Cre;LSL-KrasG12D/+) were interbred with MITO-Luc (mitosis luciferase) reporter mice to obtain KC-Mito (KCM) mice. Pancreatic tissue samples were analyzed by IHC for distribution of RAGE and CD166/ALCAM. Immunofluorescence was used to analyze co-localization of RAGE and CML. Western blot was used to analyze pancreatic protein levels of p-STAT3, RAGE, and CD166/ALCAM. ELISA kits were used to analyze serum levels of CML, sRAGE, and IL-6.
Statistical analysis: All data was expressed as mean values ± standard deviation and/or a percentage. A Student’s t-test or one-way ANOCE followed by the student-Newman-Keuls test was used to assess the differences between cell types/treatments or animal groups. A Fisher’s exact test was used to assess differences between animal groups in PaC prevalence at 11-weeks-old. A P-value of <0.05 indicated statistical significance.
Results: CML was shown to promote PDA cell proliferation as well as induce RAGE up-regulation. Further, the results showed that CML favored development and progression of early murine PaC. In the KCM mice, CML increased RAGE protein levels in pancreatic intraepithelial neoplasia's (PanINs) and well-differentiated regions of invasive PaC. The AGE/RAGE axis was also shown to modulate tumorigenic pathways involved in PaC. RAP was shown to fail the prevention of CML-inducted PaC promotion and progression but did delay PanIN lesion development in Ctr-KCM mice. Western blot and IHC showed an increase of the RAGE homolog CD166/ALCAM protein level in mice treated with CML+RAP compared with mice treated with CML alone, but not in those treated with RAP alone. Finally, it was revealed that RAP+CML treatment decreased serum levels of sRAGE and increased the CML:sRAGE ratio.
Discussion/Conclusions: The AGE/RAGE axis modulates PaC development in a genetically-engineered mouse model of the disease, which may explain the higher risk of PaC caused by diabetes mellitus and other conditions related to AGE accumulation. An AGE reduction therapy might be more beneficial than blocking RAGE, which was shown in the study to be ineffective in stopping CML-induced promotion of tumorigenesis. AGE reduction may decrease PaC risk associated with diabetes mellitus, obesity, western diet, and tobacco smoking. Limitations: Experiments could only be performed on mice, and the sample size for each experimental group was small.
Menini S, Iacobini C, de Latouliere L, Manni I, Ionta V, Blasetti Fantauzzi C, Pesce C, Cappello P, Novelli F, Piaggio G, Pugliese G. The advanced glycation end-product Nϵ -carboxymethyllysine promotes progression of pancreatic cancer: implications for diabetes-associated risk and its prevention. J Pathol. 2018 Jun;245(2):197-208. doi: 10.1002/path.5072. Epub 2018 Apr 4. PMID: 29533466.
Extracellular matrix glycation and receptor for advanced glycation end-products activation: a missing piece in the puzzle of the association between diabetes and cancer.
Impact Factor: 4.603
Level of Evidence: V
Type of Study: Review article
Introduction: This article addresses the link between cancer and diabetes, while emphasizing the lack of research on the actual effects of hyperglycemia on cancer. Advanced glycation end-products (AGEs) are long-term markers of hyperglycemia.
Summary: AGE-mediated crosslinking of collagen IV and laminin promotes the stiffening of basal lamina matrix, favoring the invasiveness of tumor cells and cancer progression. Further, since tumors rely on anaerobic metabolism, showing a higher rate of glucose uptake and glycolysis, AGE formation in cancer cells is favored. Collagens, elastins, laminins and fibronectin are highly glycated in diabetes/hyperglycemia, and may be a source of AGEs-modified proteins, activating RAGE-dependent mechanisms. AGE-mediated activation of RAGE evokes the production of pro-inflammatory cytokines, such as nuclear factor-kappa B (NK-kB), signal transducer activator of transcription 3 (STAT3) and hypoxia inducible factor 1 (HIF-1). The increased activation of these transcription factors eventually leads to recruitment and activation of inflammatory cells in the tumor microenvironment. Due to the proinflammatory nature of RAGE activation as well as reports showing how some miRNAs change expression profiles after RAGE activation, there is a possibility that either the repression of tumor suppressor miRNAs or the activation of onco miRNAs in a RAGE-mediated inflammation-dependent manner.
Conclusions: Chronic hyperglycemia results in the irreversible glycation of proteins and lipids, resulting in AGEs formation. While AGEs are linked to promoting a tumor microenvironment through modification of ECM components, the role of RAGE-mediation mechanisms is less known.
Rojas A, Añazco C, González I, Araya P. Extracellular matrix glycation and receptor for advanced glycation end-products activation: a missing piece in the puzzle of the association between diabetes and cancer. Carcinogenesis. 2018 Apr 5;39(4):515-521. doi: 10.1093/carcin/bgy012. PMID: 29373651.
High Serum Advanced Glycation End Products Are Associated with Decreased Insulin Secretion in Patients with Type 2 Diabetes: A Brief Report
Impact Factor: 2.965
Summary: A study was done with 15 patients that had Type 2 diabetes (T2DM) and 20 healthy controls. The study aimed to figure out if advanced end glycation products (AGE’s) were related to insulin secretion defects. They measured the levels of carboxymethyllysine (CML, an AGE) and carboxyethyllysine (CEL, an AGE). Levels of CML and CEL were significantly higher in T2DM. The study also showed a negative correlation between insulin secretion and the level of AGE’s. This suggested AGE’s involvement in insulin secretory defects in T2DM.
Okura T, Ueta E, Nakamura R, Fujioka Y, Sumi K, Matsumoto K, Shoji K, Matsuzawa K, Izawa S, Nomi Y, Mihara H, Otsuka Y, Kato M, Taniguchi SI, Yamamoto K. High Serum Advanced Glycation End Products Are Associated with Decreased Insulin Secretion in Patients with Type 2 Diabetes: A Brief Report. J Diabetes Res. 2017;2017:5139750. doi: 10.1155/2017/5139750. Epub 2017 Jun 12. PMID: 28695132; PMCID: PMC5485485.
Perinatal exposure to high dietary advanced glycation end products in transgenic NOD8.3 mice leads to pancreatic beta cell dysfunction
Summary: A mouse study was done on a population that was susceptible to type one diabetes and were randomly selected to partake in a low AGE or high AGE diet. In this study they measured AGE circulating levels and how it affected the pancreatic islets and contributed to beta cell dysfunction. The study showed that insulin secretion was higher in the mouse offspring with a low AGE diet and mice with a high AGE diet had lower levels of insulin that was secreted. This was found to be statistically significant at a p value of 0.002 which was determined by a Mann Whitney U test. They also were able to show that low AGE diets decreased insulitis in the population but did not affect the number of islet cells. This was statistically significant with a p value of 0.0008 and was shown with a box and whisker plot. At the end of the study they concluded that only circulating AGE levels differed between the populations of low age diet and high age diet and that plasma insulin, proinsulin and blood glucose was no different between the groups.
Borg DJ, Yap FYT, Keshvari S, Simmons DG, Gallo LA, Fotheringham AK, Zhuang A, Slattery RM, Hasnain SZ, Coughlan MT, Kantharidis P, Forbes JM. Perinatal exposure to high dietary advanced glycation end products in transgenic NOD8.3 mice leads to pancreatic beta cell dysfunction. Islets. 2018 Jan 2;10(1):10-24. doi: 10.1080/19382014.2017.1405189. Epub 2017 Dec 22. PMID: 29157116; PMCID: PMC5796486.
Long term dietary restriction of advanced glycation end-products (AGEs) in older adults with type 2 diabetes Is feasible and efficacious-results from a pilot RCT.
Impact factor: 4.546
Level of Evidence: V
Sample Size: 75 participants with T2D and subjective memory complaints over the age of 65 y/o. 35 were in the AGEs Lowering arm and 40 were in the Standard of Care (SOC) arm.
Protocol: 40 participants were assigned to the SOC group and were instructed to follow a diet for 6 months that was designed for achieving good glycemic control. The other 35 participants were assigned the same diet with additional instruction on reducing dietary AGE intake. The guidelines for good glycemic control were intake of carbohydrates, and information on serving sizes, counting, and quality. It was recommended to get carbohydrate intake from vegetables, fruits, whole grains, legumes, and low-fat dairy procuts while reducing consumption of refined sugars. They were also told to eat mono and poly-unsaturated fatty acids in place of saturated and trans fat, and to eat lean meat, low sodium and low processed food items. Participants were asked to not make any lifestyle changes or diet changes for the next 6 months. The AGE Lowering group of 35 were given the same guidance for glycemic control, but also given instructions on how to reduce dietary AGEs intake by cooking differently and reducing cooking time and temperature. These modifications were to boil, poach, stew, or steam foods and avoid frying, baking, or grilling. All participants in both arms were called once a week for encouragement to keep following the guidelines. A change in serum AGEs at the end of the 6 months was looked for. Other considerations were adherence to diet and its association with serum AGEs. Imaging of the brain and cognitive function were also studied but not discussed further.
Statistical Analysis: The primary effect of the intervention used intent-to-treat (ITT) methods. The secondary analyses used pre-protocol (PP) methods. All of the participants in the ITT analysis were randomized and finished 6 months of follow-up. In the PP analysis, participants were considered only 80% compliant to their assigned diet. Chi square analysis was used to compare the 2 arms for categorical outcomes, t-test for continuous outcomes, and Mann-Whitney for non-normal distributions. Changes in AGEs concentration in the serum from the beginning to 6 months were analyzed by paired t-test or Wilcoxin tests for within-arm changes and by 2 sided t-test for between-arms analysis. A p value of 0.05 was considered significant for all analyses.
Study Type: Pilot trial
Results: There was a significant change over time in many serum AGEs compared to the SOC guidelines. Of the participants in the very high adherence (above 80%) to the AGE-lowering diet, there was a greater reduction in the serum AGEs levels. There were no significant differences between the two arms in analysis of any other metabolic markers.
Discussion/Conclusion: Dietary restriction of AGEs long-term is feasible in adults with T2D, especially if it is followed. The intervention was based off of many earlier randomized controlled trials that lowered AGEs in the diet by altering cooking methods in healthy individuals, those with metabolic syndrome, diabetes, and renal failure. In this study, a reduction of 8-10% was seen of serum AGEs, while the earlier studies showed 23-35% reduction. It is thought this occurred because of the older age of the participants in this study. It was also taken into consideration that AGEs concentrations increase with age and diabetes, and renal function is also decreased. The SOC arm showed overall increases in serum AGEs over the 6 months.
Limitations: The nature of the design because neither the investigators nor the patients were blind to the intervention. The sample size may also not have been big enough to know the actual effecto of AGEs reduction on metabolic markers in older adults with T2D.
Lotan R, Ganmore I, Shelly S, Zacharia M, Uribarri J, Beisswenger P, Cai W, Troen AM, Schnaider Beeri M. Long Term Dietary Restriction of Advanced Glycation End-Products (AGEs) in Older Adults with Type 2 Diabetes Is Feasible and Efficacious-Results from a Pilot RCT. Nutrients. 2020 Oct 15;12(10):3143. doi: 10.3390/nu12103143. PMID: 33076217; PMCID: PMC7602440.
The receptor for advanced glycation endproducts drives T cell survival and inflammation in type 1 diabetes mellitus.
Impact Factor: 4.886
Summary: RAGE expression has been shown to be increased in patients and at-risk relatives of type one diabetes mellitus. Detectable levels of the RAGE have been found in T1D. T!D showed a difference in the signaling pathway that is associated with the upregulation of cell activation and survival. In addition, AGE products can trigger cell activation like the RAGE’s. Memory cells were analyzed in these patients and they had an increased level of inflammatory function, due to increased levels of RAGE+ CD8+ cells. These effector cells showed the same inflammatory markers in at risk relatives. This suggests that the increased expression of RAGE predates the onset of Type 1 Diabetes mellitus. This suggests that RAGE directly impacts the inflammatory process of t-cells and contributes to a possible relation to the chronic autoimmune response (due to increased cell injury and killing by CD8+ RAGE cells) in T1D patients. This was performed via an in-vivo case control study. The data was analyzed by Mann-U Whitney after they collected the data from immunohistochemistry. The data they collected ranged from a P value of .01-.05 based on the specific control-case comparison. This indicates the data described within the study is statistically relevant and significant.
Durning SP, Preston-Hurlburt P, Clark PR, Xu D, Herold KC; Type 1 Diabetes TrialNet Study Group. The Receptor for Advanced Glycation Endproducts Drives T Cell Survival and Inflammation in Type 1 Diabetes Mellitus. J Immunol. 2016 Oct 15;197(8):3076-3085. doi: 10.4049/jimmunol.1600197. Epub 2016 Sep 21. PMID: 27655844; PMCID: PMC5101164.
The relationship between advanced glycation end products and gestational diabetes: a systematic review and meta analysis.
Impact factor: 2.74
Level of evidence: 1
Summary: A meta analysis was done on 16 studies that compared the level of advanced end glycation products to healthy pregnant controls and women with gestational diabetes. The analysis concluded that women with gestational diabetes had significantly higher levels of AGE’s ( p value of 0.00001). However the study also found that women with gestational diabetes had higher BMIs as well which can contribute to AGE formation.
Sisay M, Edessa D, Ali T, Mekuria AN, Gebrie A. The relationship between advanced glycation end products and gestational diabetes: A systematic review and meta-analysis. PLoS One. 2020 Oct 21;15(10):e0240382. doi: 10.1371/journal.pone.0240382. PMID: 33085688; PMCID: PMC7577486.
Vitamin D status is associated with skin autofluorescence in patients with type 2 diabetes mellitus: a preliminary report.
Impact Factor: 6.036
Level of Evidence: IV
Summary: A randomized control trial was done of 245 patients with T2DM treated with metformin. The goal of the study was to determine the relation of vitamin D status and advanced end glycation products. Each patient was given vitamin D (50,000IU) or a placebo. They determined the AGE content by skin autofluorescence. They were able to determine that the skin autofluorescence was significantly higher in those with SAF <50 nmol/l (P value 0.001). However, the study saw no difference after 6 months of vitamin D compared to the placebo group. They were able to conclude that vitamin d status is independent of SAF in well controlled T2DM.
Krul-Poel YH, Agca R, Lips P, van Wijland H, Stam F, Simsek S. Vitamin D status is associated with skin autofluorescence in patients with type 2 diabetes mellitus: a preliminary report. Cardiovasc Diabetol. 2015 Jul 16;14:89. doi: 10.1186/s12933-015-0250-z. PMID: 26173772; PMCID: PMC4503297.
Evaluation of anti fluorescent property of hemoglobin advanced glycation end product as long term glycemic index in diabetes.
Impact Factor: 3.01
Level of Evidence: V
Summary: This article is about the relationship between HbA(1c) levels and advanced glycation products that can be used as a therapeutic mechanism. The article describes that to monitor glucose levels that occur in the blood, the Hb-AGE can be used as a measurement molecule to help provide more information about people who suffer from diabetes so they are better equipped to fight the implications that come with having the disease. Hemoglobin was taken and inoculated in male Wistar rats to induce the formation of the fluorophore with the rat. Phenyl Borate Chromatography was used with an enzyme linked immunosorbent assay.
Statistical Analysis: This experiment was done with both in vitro and in vivo studies. The first study that was done was the one that was in vitro. The in vitro reaction was taking hemoglobin with glucose and then identifying the fluorophores that were formed due to nonenzymatic glycosylation of the hemoglobin. On the other hand, there was also an in vivo study that showed that there was a significant increase in fluorophores after 2 months of the hyperglycemia. Both of these studies suggest that the Hb-AGE biomarker can be advanced therapy of Diabetes.
Study Type: Rat study
Gopalkrishnapillai B, Nadanathangam V, Karmakar N, Anand S, Misra A. Evaluation of autofluorescent property of hemoglobin-advanced glycation end product as a long-term glycemic index of diabetes. Diabetes. 2003 Apr;52(4):1041-6. doi: 10.2337/diabetes.52.4.1041. PMID: 12663478.
Dietary advanced glycation end products restriction diminishes inflammation markers and oxidative stress in patients with type 2 diabetes mellitus.
Impact factor: 2.405
Level of evidence: IV
Sample Size: 26 subjects with T2DM (13 with a standard diet, 13 with a low dAGEs diet)
Protocol: Anthropometric, dietary history, lifestyle, etc. information was collected on all the subjects. The low dAGE group received specific instructions on what to eat and how to cook it, while the standard diet group had regular meals prepared according to the American Diabetes Association. AGEs content of each food item was measured according to the database of 250 foods reported by Goldbert et al. Serum glucose and lipids were measured by enzymatic methods. HbA1c was measured via ion exchange chromatography. Urine samples were obtained from all subjects. Serum fluorescent AGEs, C-reactive protein, TNF-α by ELISA, and malondialdehyde (MDA) were measured by extraction-spectrophotometric procedure.
Statistical Analysis: Compared groups of treatment with the t test for independent samples or with the Mann-Whitney U test. Statistical significance was defined as p < 0.05.
Study Type: Cross sectional
Results: Baseline dAGEs was similar in both groups. After the 6 week study, it was unchanged in the standard diet group but significantly decreased in the low dAGEs group by 44%. No changes were found for insulin levels or HOMA-IR. The dAGEs restriction significantly decreased TNF-α and malondialdehyde levels.
Conclusion: Restricting dAGE resulted in decreased TNF-α and malondialdehyde levels compared to the standard diet group. This study offers the first prospective evidence on the relationship between dAGEs and oxidative stress markers (MDA) in T2DM patients. AGEs induce reactive oxygen species, which increase oxidative stress that stimulates the synthesis and release of proinflammatory cytokines. In this study, 6 weeks of dAGEs restriction resulted in reduced inflammation markers in T2DM patients. Further studies on low dAGEs meals on diabetes control and on the risk of complications is necessary in order to bring about formal food preparation recommendations to delay and/or prevent diabetes and its complications.
Luévano-Contreras C, Garay-Sevilla ME, Wrobel K, Malacara JM, Wrobel K. Dietary advanced glycation end products restriction diminishes inflammation markers and oxidative stress in patients with type 2 diabetes mellitus. J Clin Biochem Nutr. 2013 Jan;52(1):22-6. doi: 10.3164/jcbn.12-40. Epub 2012 Dec 6. PMID: 23341693; PMCID: PMC3541414.