Testing the Efficacy of β‐hydroxy‐β‐methylbutyrate (HMB) to Preserve Mitochondrial Function in Aging Drosophila Melanogaster
Authors: Callahan Damien M. , Mead Andrew F. , Maughan David W. 
Published: November 2023

Introduction

Mitochondrial function (capacity for oxygen consumption and ATP production) declines with advancing age across most animal species. In striated muscle, mitochondrial dysfunction may play a role in age‐related loss of muscle mass (sarcopenia) and contractile dysfunction. β‐hydroxy‐β‐methylbutyrate (HMB), a metabolite of L‐leucine, has been shown to enhance the effects of resistance exercise and limit muscle atrophy with disuse and aging. Some data suggest an effect of HMB on enhancing mitochondrial function, a mechanism by which atrophy and contractile dysfunction might be attenuated.

Objective

Drosophila melanogaster were used as a model organism to test the efficacy of HMB to limit age‐related declines in mitochondrial function. We tested the hypothesis that HMB supplementation would attenuate age‐related mitochondrial dysfunction in Drosophila compared with standard diet. A third group of 1‐week old controls raised on standard food served as a reference for the primary effects of age.

Methods

Flies were reared in vials containing standard cornmeal fly food (Control), or standard cornmeal fly food containing calcium HMB monohydrate (10 mg/ml; HMB) and collected upon eclosion. Twenty flies per group were tested at 1‐week (Young) and 5‐weeks (Old). Flight ability was assessed the day prior to testing. Older flies were matched for flight ability in HMB and Control groups. Mitochondrial function (peak O2 consumption rate, OCR, and respiratory control ratio, RCR) was assessed in mitochondria isolated from IFM. Fly thoraces were dissected and homogenized in isolation buffer at 4°C to liberate mitochondria from the IFM. Following isolation by differential centrifugation, mitochondria were loaded by protein content (8 μg) and respiration was measured using an XF96e analyzer (Seahorse Biosciences/Agilent Technologies, Santa Clara CA). Respiration was measured under basal (pyruvate/malate) state 3 (ADP stimulated) and state 4 (oligomycin) conditions. OCR and RCR (state 3 OCR/state 4) was assessed in each group. Statistical measures were not applied to these preliminary data because a single value was obtained for each group of flies.

Results

Young controls had higher flight scores than old flies in each group. Age‐related declines in OCR were noted in both HMB and Control (basal and state 3 OCR). Young flies had the highest OCR, followed by Old Controls and Old HMB (59.6, 22.4 and 21.1pmol/ug/min, respectively). Similarly, RCR was highest in Young (8.0) followed by Old Control (5.9) and Old HMB (4.1).

Conclusion

We find a ~60% reduction in OCR with age regardless of group, suggesting HMB was not sufficient to attenuate age‐related reduction in mitochondrial function. Because measurements were made on similar quantities of mitochondria in each group, these reductions reflect qualitative reductions in mitochondrial function, an interpretation buttressed by reductions in RCR.

Support or Funding Information

Supported by The UVM Foundation.