Intrinsic Mitochondrial Function Impacts the Outcomes of Metformin Treatment on Skeletal Muscle Mitochondrial Morphology in Aged Rat

Abstract

Aging is the main risk factor for many costly, irreversible, and comorbid chronic diseases. The anti‐diabetic drug Metformin is being considered as an anti‐aging treatment although little is known about its effects in healthy individuals. Previous data from our lab suggest that metformin is beneficial to individuals that are relatively insulin resistant and may be detrimental to those who are insulin sensitive. We aimed to understand how intrinsic function of mitochondria affects metformin treatment outcomes in skeletal muscle. We hypothesized that the effects of metformin on skeletal muscle mitochondrial morphology and remodeling will differ based on intrinsic mitochondrial function. To test our hypothesis, we used the High‐Capacity Runner/Low‐Capacity Runner (HCR/LCR) rat model system which comprises two selectively bred lines of genetically heterogeneous rats that diverge for exercise capacity, cardiovascular risk factors, lifespan, and healthspan. In drinking water, we treated six HCR and four LCR male rats (age 18 months) with a dose of 100 mg/kg/day metformin for a week, followed by three weeks of 200 mg/kg/day. We included HCR and LCR control groups (n=5) for a total of four groups. A week prior to sacrifice, we initiated deuterium oxide (D2O) labeling to assess protein turnover and electroporated DNA constructs that encode for mitochondrial matrix‐targeted YFP and tdTomato‐tagged TOM20, a protein located on the mitochondrial outer membrane. We analyzed protein turnover in hindlimb muscles using GC‐MS, and prepared histological sections of the Tibialis Anterior (TA) muscle for further analysis of mitochondrial morphology. Normalized to tibial length, the mass of the gastrocnemius (‐16.6%) and plantaris (‐20.8%) muscles were lower in metformin treated HCR rats compared to untreated, while the normalized mass of the TA muscle was lower in both HCR (‐15%) and LCR (‐20.1%) metformin treated rats compared to control. There were no significant differences in mean fiber cross sectional area (CSA) of the TA muscle. We detected no significant differences in bulk protein synthesis of both the myofibrillar and mitochondrial fractions of the TA, gastrocnemius, and soleus muscles. The number of mitochondrial branches observed in a cross section of TA fibers from metformin treated LCR rats were 30.5% lower compared to control LCR rats. However, mean CSA of each mitochondrial branch in metformin treated LCR rats increased by 82.8% compared to control LCR rats, which, together with the decrease in branch number, resulted in maintenance of the cumulative mitochondrial CSA in each fiber. Analysis based on mitochondrial sub‐populations showed that the subsarcolemmal mitochondrial population was more impacted than intermyofibrillar mitochondria. There were no significant differences in mitochondrial morphology in control vs metformin treated HCR rats. Our data supports our hypothesis that metformin has differential effects in skeletal muscle based on intrinsic mitochondrial function prior to treatment. Additional studies are needed to determine what impact these disparities have on the aging process.