Metformin and Molecular Aging in Prediabetes

Overview

The incidence and prevalence of type 2 diabetes mellitus (T2DM) are increasing globally. The global prevalence of diabetes has nearly doubled since 1980, rising from 4.7% to 8.5% in the adult population. Asian Indians have one of the highest incidence rates of pre-diabetes (10.3%) and type 2 diabetes mellitus (T2DM) (8.8%) among all major ethnic groups, and the conversion from pre-diabetes to diabetes occurs more rapidly in this population. Metformin has been shown to effectively prevent the progression of prediabetes to overt diabetes. Furthermore, metformin improves lifespan in animal models through an anti-ageing pathway driven by mTOR. Metformin has also been shown to protect endothelial cells from hyperglycaemic damage by directly stimulating the expression of Sirtuin-1 (SIRT1), a deacetylase involved in metabolism and longevity by modulating SIRT1 downstream targets FoxO1 and p53/p21. It is important to note thatSIRT1, andmammalian target of rapamycin (mTOR) form a network that connects cellular metabolism and longevity programmes. Only one study is available which has explored the relationship of metformin with longevity. Previous study conducted a single-blind randomized placebo-controlled trial in prediabetic subjects in Italy (n, 38) who received metformin 1500mg/day (n, 19) or placebo (n, 19) for 2 months. They demonstrated that metformin use significantly increased insulin sensitivity and metabolic parameters, SIRT1 gene/protein expression, and SIRT1 promoter chromatin accessibility. They also demonstrated that metformin use increased mTOR gene expression with a concurrent decrease in p70S6K phosphorylation and altered the plasma N-glycan profile. These authors concluded that in individuals with prediabetes, metformin ameliorated effector pathways that have been shown to regulate longevity in animal models. The investigators recently did a study on 797 prediabetic women from north India (492 of whom were obese). In this study the investigators reported that age, obesity, and subcutaneous adiposity (predominantly truncal) are the main causes of leukocyte telomere shortening. It is yet unknown how metformin impacts aging-related genes and surrogate markers of ageing in the Asian Indian population. This clinical trial aims to evaluate the effects of metformin treatment on surrogate markers of ageing (leukocyte telomere length and telomerase activity), in the setting of pre-diabetes. We intend to compare treatment with metformin for six months, versus placebo in pre-diabetic subjects. We will assess the surrogate markers of ageing (leukocyte telomere length and telomerase activity) and the expression of longevity genes SIRT1, p66Shc, p53 and mTOR in peripheral blood mononuclear cells (PBMCs) before and after 6 months of metformin treatment.

Research methodology and outcomes measures: 1. Clinical History and Examination: Clinical history and blood pressure. 2. Body mass Index: BMI will be calculated by using formula weight (Kg)/height (m2). 3. Anthropometric Assessment: All circumferences (waist, hip, mid-thigh, mid arm and neck) and skinfolds (biceps, triceps, anterior axillary, subscapular, suprailiac, lateral thoracic and thigh) measurements will be taken. All the measurements will be repeated three times at same position and conditions. 4. Body Composition: Body composition will be measured through multi-frequency bioelectrical impedance (MF-BIA; InBody 770, Cerritos, CA, USA) Visit-2 (Day 90) Visit-3 (Day 180) Compliance check Enrolment of prediabetes subjects (BMI\>25kg/m2) (n=112) Investigations: Fasting blood glucose, IGT (2h post-oral glucose load (75g) and HBA1c Group-I: Metformin Group (n=56) Group-II: Placebo Group (n=56) Final Analysis Measurements same as in visit 1 Baseline investigations: Clinical and dietary profiles, blood pressure, anthropometric assessments \[body mass index, circumferences (waist, hip, mid-thigh, mid-arm and neck) and skinfolds (biceps, triceps, sub scapular, suprailiac, thigh, lateral thoracic and calf)\], body fat, handgrip muscle strength, glycemic and lipid profile other metabolic parameters, fasting serum insulin, C-peptide and HOMA-IR,leukocyte telomerase length and telomerase activity and gene expression of SIRT1, mTOR, p53, p66Shc genes. Randomization: Visit 1 (Day 0) Screening Diet and exercise (run in two weeks) 5 5. Handgrip Muscle Strength: Grip strength will be measured using a Jamar Analogue Hand Dynamometer with participants seated, their elbow by their side and flexed to right angles, and a neutral wrist position, the dynamometer handle position II and provision of support underneath the dynamometer. 6. Biochemical measurements: The blood sample shall be drawn after 12 hours overnight fast, subject having taken normal diet in the previous three days. A 75-g OGTT will be performed. Blood samples shall be analyzed for following; blood glucose, A1C, lipids, fasting insulin, C-peptide, Homeostasis Model Assessment (HOMA)- Insulin Resistance (IR), and HOMA- Beta (ß%) and serum glucagon levels. 7. DNA Isolation and Quantification: DNA will be separated from peripheral blood mononuclear cells using the QIAamp DNA extraction kit (Qiagen, Hilden, Germany) and will be stored at -20oC for future experiments. After DNA isolation, the DNA samples will be quantified and diluted to 50 ng/μL. The concentration and quality of DNA will be both measured by using a nanodrop (Nanodrop Technologies, Wilmington, NC, USA) and samples will be included for analysis all will have an optical density ratio A 260/A280\> 1.8. h) Measurement of Leukocyte Telomere Length: LTL will be analysed with a quantitative polymerase chain reaction (qPCR) based technique that compares telomere repeat sequence copy number (T) to a reference single copy-gene copy number (S). The telomere length for each sample will be estimated using the telomere to single copy gene ratio (T/S ratio) with the calculation of ΔCt \[Ct (telomere)/Ct(single gene)\]. T/S ratio for each sample (x) will be normalized to the mean T/S ratio of the reference sample \[2-(ΔCtx-ΔCtr) = 2-ΔΔCt\], which will be used for the standard curve, both as a reference sample and as a validation sample. i) Gene expression by real-time PCR: Total RNA will be extracted using aRNeasy Mini Kit, (QIAGEN) cDNA and synthesized with an iScript cDNA synthesis kit (Bio-Rad, USA). Q-PCR assay will be performed in a Thermal Cycler (iCycler iQ5, Bio-Rad, Hercules, CA). Primers for Sirt1, p66Shc, p53, and mTORwill be designed from sequences derived from the GenBank database using Primer 3 (Whitehead Institute, Massachusetts, USA) and Operon's Oligo software (Operon, California, USA), purchased from Eurofins MWG (Ebersberg, Germany). The comparative threshold cycle method (ΔΔCq), which compares differences in the threshold cycle values between groups, will be used to obtain the relative fold change of gene expression.