Metformin Reduces the Progression of Atherogenesis by Regulating the Sestrin2-mTOR Pathway in Obese and Diabetic Rats.

Objective: In previous research, we found that Sestrin2 has a strong association with plasma atherogenicity and combats the progression of atherogenesis by regulating the AMPK-mTOR pathway. Metformin, an activator of AMPK, is widely used as a first-line therapy for diabetes, but its role in preventing atherosclerosis and cardiac outcomes is unclear. Hence, we aimed to assess the effect of metformin on preventing atherosclerosis and its regulatory role in the Sestrin2-AMPK -mTOR pathway in obese/diabetic rats. Methods: Animals were fed a high-fat diet to induce obesity, administered streptozotocin to induce diabetes, and then treated with metformin (150 mg/kg body weight) for 14 weeks. Aorta and heart tissues were analyzed for Sestrin2 status by western blotting and immunohistochemistry, AMPK and mTOR activities were investigated using western blotting, and atherogenicity-related events were evaluated using reverse transcription quantitative polymerase chain reaction and histology. Results: Obese and diabetic rats showed significant decrease in Sestrin2 levels and AMPK activity, accompanied by increased mTOR activity in the heart and aorta tissues. Metformin treatment significantly restored Sestrin2 and AMPK levels, reduced mTOR activity, and restored the altered expression of inflammatory markers and adhesion molecules in obese and diabetic rats to normal levels. A histological analysis of samples from obese and diabetic rats showed atherosclerotic lesions both in aorta and heart tissues. The metformin-treated rats showed a decrease in atherosclerotic lesions, cardiac hypertrophy, and cardiomyocyte degeneration. Conclusion: This study presents further insights into the beneficial effects of metformin and its protective role against atherosclerosis through regulation of the Sestrin2-AMPK-mTOR pathway.

Augmentation of RBP4/STRA6 signaling leads to insulin resistance and inflammation and the plausible therapeutic role of vildagliptin and metformin.

A role of Retinol Binding Protein-4 (RBP4) in insulin resistance is widely studied. However, there is paucity of information on its receptor viz., Stimulated by Retinoic Acid-6 (STRA6) with insulin resistance. To address this, we investigated the regulation of RBP4/STRA6 expression in 3T3-L1 adipocytes exposed to glucolipotoxicity (GLT) and in visceral adipose tissue (VAT) from high fat diet (HFD) fed insulin-resistant rats. 3T3-L1 adipocytes were subjected to GLT and other experimental maneuvers with and without vildagliptin or metformin. Real-time PCR and western-blot experiments were performed to analyze RBP4, STRA6, PPARγ gene and protein expression. Adipored staining and glucose uptake assay were performed to evaluate lipid and glucose metabolism. Oral glucose tolerance test (OGTT) and Insulin Tolerance Test (ITT) were performed to determine the extent of insulin resistance in HFD fed male Wistar rats. Total serum RBP4 was measured by quantitative sandwich enzyme-linked immunosorbent assay kit. Adipocytes under GLT exhibited significantly increased RBP4/STRA6 expressions and decreased insulin sensitivity/glucose uptake. Vildagliptin and metformin not only restored the above but also decreased the expression of IL-6, NFκB, SOCS-3 along with lipid accumulation. Furthermore, HFD fed rats exhibited significantly increased serum levels of RBP4 along with VAT expression of RBP4, STRA6, PPARγ, IL-6. These molecules were significantly altered by the vildagliptin/ metformin treatment. We conclude that RBP4/STRA6 pathway is primarily involved in mediating inflammation and insulin resistance in adipocytes and visceral adipose tissues under glucolipotoxicity and in insulin resistant rats.

Plausible diagnostic value of urinary isomeric dimethylarginine ratio for diabetic nephropathy.

Altered circulatory asymmetric and symmetric dimethylarginines have been independently reported in patients with end-stage renal failure suggesting their potential role as mediators and early biomarkers of nephropathy. These alterations can also be reflected in urine. Herein, we aimed to evaluate urinary asymmetric to symmetric dimethylarginine ratio (ASR) for early prediction of diabetic nephropathy (DN). In this cross-sectional study, individuals with impaired glucose tolerance (IGT), newly diagnosed diabetes (NDD), diabetic microalbuminuria (MIC), macroalbuminuria (MAC), and normal glucose tolerance (NGT) were recruited from Dr. Mohans' Diabetes Specialties centre, India. Urinary ASR was measured using a validated high-throughput MALDI-MS/MS method. Significantly lower ASR was observed in MIC (0.909) and MAC (0.741) in comparison to the NGT and NDD groups. On regression models, ASR was associated with MIC [OR: 0.256; 95% CI: 0.158-0.491] and MAC [OR 0.146; 95% CI: 0.071-0.292] controlled for all the available confounding factors. ROC analysis revealed ASR cut-point of 0.95 had C-statistic of 0.691 (95% CI: 0.627-0.755) to discriminate MIC from NDD with 72% sensitivity. Whereas, an ASR cut-point of 0.82 had C-statistic of 0.846 (95% CI: 0.800 - 0.893) had 91% sensitivity for identifying MAC. Our results suggest ASR as a potential early diagnostic biomarker for DN among the Asian Indians.

High molecular weight adiponectin reduces glucolipotoxicity-induced inflammation and improves lipid metabolism and insulin sensitivity via APPL1-AMPK-GLUT4 regulation in 3T3-L1 adipocytes.

BACKGROUND AND AIMS: Although the importance of adipokines in modulating the disease process of type 2 diabetes is well recognized, there is dearth of data on the specific role of high molecular weight adiponectin (HMW Ad) on insulin resistance and obesity. Therefore, we tested the effects of HMW Ad on glucolipotoxcity-induced inflammation and insulin resistance in 3T3-L1 adipocytes. METHODS: 3T3-L1 adipocytes were subject to glucolipotoxicity with and without HMW Ad treatment. Real-time PCR and Western-blot experiments were performed to analyse gene and protein expressions, respectively. Lipolysis, adipored staining, and glucose uptake assay were performed to evaluate alterations in lipid and glucose metabolism. RESULTS: Adipocytes subject to glucolipotoxicity showed significantly (p < 0.05) decreased mRNA expression of adiponectin, AdipoR2, GLUT4, and increased inflammation, lipid accumulation as well as lipolysis. Treatment with HMW Ad beneficially modulated lipid metabolism, reduced inflammation and improved glucose uptake in adipocytes. HMW Ad also beneficially regulated APPL1 and AMPK signaling in adipocytes. Silencing of APPL1 gene in adipocytes significantly reduced the effects of HMW Ad on pAMPK protein expression, indicating that HMW Ad plays an important role in regulating AMPK phosphorylation via APPL1 in 3T3-L1 adipocytes. CONCLUSIONS: HMW Ad treatment improved glucose homeostasis and resulted in reduced lipolysis, inflammation and insulin resistance in adipocytes subject to glucolipotoxicity. The beneficial modulation and regulation of APPL1 and AMPK signals by HMW Ad observed in this study represent a novel mechanism. Raising endogenous HMW Ad levels either by pharmacological or lifestyle modification could have a therapeutic value.

Effect of lifestyle improvement program on the biomarkers of adiposity, inflammation and gut hormones in overweight/obese Asian Indians with prediabetes.

AIMS: While lifestyle modification is known to offer several metabolic benefits, there is paucity of comprehensive data on changes in biomarkers of adiposity, inflammation as well as gut hormones. We investigated these biomarkers in overweight/obese individuals with prediabetes randomized to either 4 months of a lifestyle improvement program or standard care and followed them up for a year. METHODS: Participants [standard care and intervention arm (n = 75 each)] were randomly selected from the Diabetes Community Lifestyle Improvement Program trial. Glycemic and lipid control and anthropometric measurements were assessed by standard protocols. Adipokines, inflammatory markers and gut hormones were measured using multiplex and standard ELISA kits. RESULTS: Along with modest benefits in primary outcomes (glycemic and lipid control and weight reduction), participants in the intervention group showed significant reductions (p < 0.001) in plasma levels of leptin (17.6%), TNF-α (35%), IL-6 (33.3%), MCP-1 (22.3%) and PYY (28.3%) and increased levels of adiponectin (33.1%) and ghrelin (23.6%) at the end of 4 months of lifestyle intervention. The changes were independent of weight and persisted even at 1 year of follow-up. In contrast, participants from the standard care arm did not show any statistically significant improvements on the above parameters. CONCLUSIONS: Participants who underwent an intensive lifestyle improvement program showed metabolic benefits as well as favorable beneficial changes in systemic levels of adipokines, cytokines and gut hormones, not only during the intervention period, but also during 12-month follow-up period.

RELATIONSHIP OF ADIPOKINES AND PROINFLAMMATORY CYTOKINES AMONG ASIAN INDIANS WITH OBESITY AND YOUTH ONSET TYPE 2 DIABETES.

OBJECTIVE: It is well known that inflammation is associated with diabetes, but it is unclear whether obesity mediates this association in individuals with youth-onset type 2 diabetes mellitus (T2DM-Y). METHODS: We recruited individuals with T2DM-Y (age at onset <25 years) and age-matched normal glucose tolerance (NGT) subjects. Participants were further classified using Asia-Pacific body mass index cut-points for obesity and categorized as: nonobese NGT (n = 100), Obese NGT (n = 50), nonobese T2DM-Y (n = 50), and obese T2DM-Y (n = 50). We compared adipokines (adiponectin and leptin) and proinflammatory cytokines (tumor necrosis factor alpha [TNF-α] and monocyte chemotactic protein-1 [MCP-1]) across groups. RESULTS: Compared to nonobese NGT, the other 3 groups (obese NGT, nonobese T2DM-Y, and obese T2DM-Y) were found to have lower adiponectin (7.7 vs. 5.7, 4.2, 3.8 µg/mL, P<.01), and higher leptin (3.6 vs. 5.4, 5.7, 7.9 µg/mL, P<.001) and MCP 1 (186 vs. 272, 340, 473 pg/mL, P<.001) respectively. However, TNF-α levels were higher only among nonobese T2DM-Y (112 pg/mL) and obese T2DM-Y (141 pg/mL, P<.01 for each). After adjusting for age, sex, waist, hypertension, homeostatic model assessment of insulin resistance (HOMA-IR), serum cholesterol, triglycerides, and family history of diabetes, adiponectin was associated with 33% and 41% lower odds of being nonobese T2DM and obese T2DM, respectively. However, adjusted for same factors, leptin, TNF-α, and MCP-1 were associated with markedly higher odds (5- to 14-fold) of nonobese and obese T2DM. CONCLUSION: In young Asian Indians, leptin and proinflammatory cytokines are positively, and adiponectin negatively, associated with both nonobese and obese T2DM-Y compared to nonobese NGT individuals.

ALTERED CIRCULATING LEVELS OF RETINOL BINDING PROTEIN 4 AND TRANSTHYRETIN IN RELATION TO INSULIN RESISTANCE, OBESITY, AND GLUCOSE INTOLERANCE IN ASIAN INDIANS.

OBJECTIVE: Retinol binding protein 4 (RBP4) has been implicated in metabolic disorders including type 2 diabetes mellitus (T2DM), but few studies have looked at transthyretin (TTR) with which RBP4 is normally bound to in the circulation. We report on the systemic levels of RBP4 and TTR and their associations with insulin resistance, obesity, prediabetes, and T2DM in Asian Indians. METHODS: Age-matched individuals with normal glucose tolerance (NGT, n = 90), impaired glucose tolerance (IGT, n = 70) and T2DM (n = 90) were recruited from the Chennai Urban Rural Epidemiology Study (CURES). Insulin resistance was estimated using the homeostasis model assessment of insulin resistance (HOMA-IR). RBP4 and TTR levels were measured by enzyme-linked immunosorbent assay (ELISA). RESULTS: Circulatory RBP4 and TTR levels (in µg/mL) were highest in T2DM (RBP4: 13 ± 3.9, TTR: 832 ± 310) followed by IGT (RBP4: 10.5 ± 3.2; TTR: 720 ± 214) compared to NGT (RBP4: 8.7 ± 2.5; TTR: 551 ± 185; P<.001). Compared to nonobese NGT individuals, obese NGT, nonobese T2DM, and obese T2DM had higher RBP4 (8.1 vs. 10.6, 12.1, and 13.2 µg/mL, P<.01) and TTR levels (478 vs. 737, 777, and 900 µg/mL, P<.01). RBP4 but not TTR was significantly (P<.001) correlated with insulin resistance even among NGT subjects. In regression analysis, RBP4 and TTR showed significant associations with T2DM after adjusting for confounders (RBP4 odds ratio [OR]: 1.107, 95% confidence interval [CI]: 1.008-1.216; TTR OR: 1.342, 95% CI: 1.165-1.547). CONCLUSION: Circulatory levels of RBP4 and TTR showed a significant associations with glucose intolerance, obesity, T2DM and RBP4 additionally, with insulin resistance.

Relationship of betatrophin with youth onset type 2 diabetes among Asian Indians.

BACKGROUND AND AIMS: Betatrophin is emerging as a marker for compensatory beta cell proliferation. While betatrophin has been mainly investigated in adults, there is a lack of data on betatrophin levels in youth-onset type 2 diabetes mellitus (T2DM-Y). The aim of this study was to determine levels of betatrophin and its association with T2DM-Y in Asian Indian participants. METHODS: We recruited 100 individuals with normal glucose tolerance (NGT; n=50) and newly-diagnosed cases (within 18 months of first diagnosis) of T2DM-Y (n=50) with onset between 12 and 24 years of age from a large tertiary diabetes center in Chennai in southern India. Insulin resistance was measured by homeostatic model (HOMA-IR) and insulin secretion by oral disposition index (DIO). Betatrophin levels were measured by enzyme-linked immunosorbent assay. RESULTS: Betatrophin levels were significantly lower in the T2DM-Y group compared with the NGT group (803 vs 1104 pg/ml, p<0.001). Betatrophin showed a significant inverse correlation with waist circumference (p=0.035), HOMA-IR (p<0.001), fasting and 2 h postprandial glucose (p<0.01), glycated hemoglobin (p=0.019) and a positive correlation with fasting C-peptide (p<0.001) and DIO (p=0.012). In regression analysis, betatrophin was independently associated with T2DM-Y even after adjustment for age, gender, and waist circumference (OR per standard deviation: 0.562, 95% CI: 0.342-0.899, p=0.019). However, the association was lost when HOMA-IR was included in the model (OR: 1.141, 95% CI: 0.574-2.249; p=0.646). CONCLUSION: Betatrophin levels are lower in T2DM-Y and this association is likely mediated through insulin resistance.

Association of Soluble (Pro) Renin Receptor with Gestational Diabetes Mellitus.

OBJECTIVE: There is a need to identify biomarkers for gestational diabetes mellitus (GDM). Recently the soluble pro-renin receptor (s[Pro]RR) has been shown to be associated with GDM. We investigated the association of s(Pro) RR levels in Asian Indians with GDM. METHODS: We recruited 222 pregnant females, 147 without GDM (non-GDM) and 75 with GDM visiting antenatal clinics in Tamilnadu in South India. We included singleton pregnancy and excluded those with pre-existing diabetes mellitus or hypertension. Oral glucose tolerance tests (OGTTs) were performed, and GDM was diagnosed using the International Association of Diabetes and Pregnancy Study Group criteria. s(Pro)RR was measured by enzyme-linked immunosorbent assay (ELISA). Receiver operating characteristic (ROC) curves were used to identify s(Pro) RR cut-off points to identify GDM. RESULTS: The mean levels of the s(Pro)RR were significantly higher in subjects with GDM (34.0 ± 12 ng/mL, P<.001) compared to non-GDM (21.4 ± 6.5 ng/mL). The proportions of subjects with GDM were 11 (15%) in the first tertile of s(Pro)RR (<19.61 ng/mL), 20 (27%) in the second (19.62-26.8 ng/mL), and 44 (59%) in the third tertile (>26.8 ng/mL). In multiple logistic regression analysis, s(Pro)RR showed a significant association with GDM (odds ratio [OR]: 1.201, 95% confidence interval [CI]: 1.065-1.355, P = .003) after adjusting for potential confounders. A s(Pro)RR cut-off of 23.3 ng/mL had a C statistic of 0.828 (95% CI: 0.738-0.918, P<.001), sensitivity of 68%, and specificity of 70% to identify GDM. CONCLUSIONS: s(Pro)RR levels are higher in females with GDM, and this could be used as a potential biomarker.