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.
