Role of Allopurinol on Oxidative Stress and Mitochondrial Alterations in Skeletal Muscle of Diabetic Patients

Study Overview
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Brief Summary
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Our recent data in mice have demonstrated a key role of xanthine oxidase in hyperglycemia-induced by Reactive oxygen species production, and a preventive role of allopurinol (inhibitor of xanthine oxidase) on the keeping of mitochondria number and structure, in skeletal muscle of diabetic mice. The investigators want to initiate a clinical trial in order to evaluate the efficacy of allopurinol on the improvement of mitochondrial alterations, oxidative capacities and insulin sensitivity, in skeletal muscle of type 2 diabetic patients.

Official Title
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Involvement of Reactive Oxygen Species Produced by the Xanthine Oxidase in Mitochondrial Alterations in Skeletal Muscle of Type 2 Diabetic Patients

Conditions
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Type 2 Diabetes Mellitus

Intervention / Treatment
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* Drug: 2 capsules of allopurinol 150 mg daily for 3 month
* Drug: 2 capsules of lactose daily for 3 month


Participation Criteria
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Eligibility Criteria
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Inclusion Criteria: BMI from 25 to 40 kg/m² Type 2 diabetes known for over one year but less than 10 years, treated with Oral anti-diabetic drugs or a Glucagon-like peptide-1 (GLP1-analog) well controlled hypertension (untreated or currently treated) with a systolic blood pressure of 95 to 140 mmHg and diastolic blood pressure of 45 to 90 mmHg and heart frequency of 40 to 100 per minute Recent HbA1c < 9 % Uricemia > 300 µmol/l For women : Menopausal or contraception Renal function as defined by glomerular filtration rate (GFR) ≥ 80 mL/min/1.73 m2 Exclusion Criteria: Tobacco ( more than 5 cigarettes) Excessive drinking Known pathology Hypersensitivity to allopurinol Treatment by anticoagulants, allopurinol, regular steroids or Nonsteroidal anti-inflammatory drug (NSAID), fibrate or insulin

Ages Eligible for Study
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Minimum Age: 30 Years
Maximum Age: 60 Years

Sexes Eligible for Study
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All

Accepts Healthy Volunteers
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No

Study Plan
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How is the study designed?
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Design Details

Primary Purpose: Basic Science
Allocation: Randomized
Intervention Model: Parallel Assignment
Masking: Double


Arms and Interventions

| Participant Group/Arm | Intervention/Treatment |
| --- | --- |
| Experimental: Allopurinol<br>Allopurinol, experimental arms, type 2 diabetes subjects receive 2 capsules of allopurinol 150 mg daily for 3 month | Drug: 2 capsules of allopurinol 150 mg daily for 3 month<br>* 2 capsules of allopurinol 150 mg daily for 3 month<br>|
| Placebo Comparator: Placebo<br>Placebo, type 2 diabetes subjects receive 2 capsules of lactose (placebo) daily for 3 month | Drug: 2 capsules of lactose daily for 3 month<br>* 2 capsules of lactose daily for 3 month<br>|

What is the study measuring?
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Primary Outcome Measures

| Outcome Measure | Measure Description | Time Frame |
| --- | --- | --- |
| muscle oxidative stress in diabetic patients | muscular protein carbonylation level (unit: reactive carbonyl derivates, arbitrary unit) by Western blot (oxyblot kit from Chemicon) and pro et antioxidant genes expression (unit: mRNA levels normalized by housekeeping gene, arbitrary ratio) by real time polymerase chain reaction (RT-PCR) | At 3 months of treatment | 

Secondary Outcome Measures

| Outcome Measure | Measure Description | Time Frame |
| --- | --- | --- |
| - plasmatic oxidative stress by dosing plasmaticmarkers: | Dosing plasmatic malondialdéhyde, plasmatic H2O2, protein carbonylation of plasmatic protein and urinary isoprostans, and finally antioxidants (vitamins C and E, glutathione (unit: from µM to M) | At 3 months of treatment | 
| alterations in mitochondrial structure of skeletal muscle with transmission electron microscopy | Analysis of mitochondria area (in µm2) and density (in %) | At 3 months of treatment | 
| mitochondrial density by measuring the ratio mitochondrial Deoxyribonucleic acid (mtDNA)/nuclear DNA by real-time polymerase chain reaction (PCR) in skeletal muscle |  | At 3 months of treatment | 
| mitochondrial function | Expression of genes implicated in mitochondrial action (messenger Ribonucleic acid (mRNA) levels by Reverse transcription polymerase chain reaction (RT-PCR) and proteins levels by Western Blot)(unit: arbitrary ratio relative to housekeeping gene/protein). | At 3 months of treatment | 
| quantification of intramuscular lipids by histology (biopsy analysis) | Staining Oil Red O evaluate the intramuscular lipid accumulation using the software ImageJ(unit: % of labelling by field). | At 3 months of treatment | 
| sensitivity to insulin using a hyperinsulinemic euglycemic clamp | sensitivity to insulin will be expressed as the glucose infusion rate (GIR)/insulinemia ratio. | At 3 months of treatment | 
| uricemia and xanthine oxidase activity in sera and muscles(unit: mg/l for uricemia and mU/ml for XO activity) | Plasma concentrations of uric acid will be measured before and after treatment to assess patient compliance . The reduction of xanthine oxidase activity in serum and muscle protein lysates will be measured using the kit  Amplex Red xanthine / xanthine oxidase assay kit from Molecular Probes | At 3 months of treatment | 
| Tolerance of the treatment measured by any adverse events during treatment and between each visit. | Any adverse events during treatment and between each visit. | during the 3 months of treatment | 

 Terms related to the study
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Keywords Provided by Centre Hospitalier Valida
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Type 2 diabetes mellitus, oxidative stress, insulin sensitivity, insulin resistance, mitochondrial dysfunction, skeletal muscle, xanthine oxidase, allopurinol