Long-term rates of mitochondrial protein synthesis are increased in mouse skeletal muscle with high-fat feeding regardless of insulin-sensitizing treatment

Abstract

Skeletal muscle mitochondrial protein synthesis is regulated in part by insulin. The development of insulin resistance with diet-induced obesity may therefore contribute to impairments to protein synthesis and decreased mitochondrial respiration. Yet the impact of diet-induced obesity and insulin resistance on mitochondrial energetics is controversial, with reports varying from decreases to increases in mitochondrial respiration. We investigated the impact of changes in insulin sensitivity on long-term rates of mitochondrial protein synthesis as a mechanism for changes to mitochondrial respiration in skeletal muscle. Insulin resistance was induced in C57BL/6J mice using 4 wk of a high-fat compared with a low-fat diet. For 8 additional weeks, diets were enriched with pioglitazone to restore insulin sensitivity compared with nonenriched control low-fat or high-fat diets. Skeletal muscle mitochondrial protein synthesis was measured using deuterium oxide labeling during High-resolution respirometry was performed using palmitoyl-l-carnitine, glutamate+malate, and glutamate+malate+succinate as substrates for mitochondria isolated from quadriceps. Mitochondrial protein synthesis and palmitoyl- l-carnitine oxidation were increased in mice consuming a high-fat diet, regardless of differences in insulin sensitivity with pioglitazone treatment. There was no effect of diet or pioglitazone treatment on ADP-stimulated respiration or HO emission using glutamate+malate or glutamate+malate+succinate. The results demonstrate no impairments to mitochondrial protein synthesis or respiration following induction of insulin resistance. Instead, mitochondrial protein synthesis was increased with a high-fat diet and may contribute to remodeling of the mitochondria to increase lipid oxidation capacity. Mitochondrial adaptations with a high-fat diet appear driven by nutrient availability, not intrinsic defects that contribute to insulin resistance.

Mitochondrial adaptations to exercise do not require Bcl2‐mediated autophagy but occur with BNIP3/Parkin activation

“…Additional stress on protein turnover may reveal a more significant role of exercise‐induced autophagy through the Bcl2 pathway for maintaining mitochondrial respiratory function. High‐fat feeding is a model that increases reliance on whole body and mitochondrial lipid respiration, which we and others have shown increases mitochondrial abundance and protein turnover and possibly restricts adaptations to exercise . Compensatory changes in protein turnover may occur differently during high‐fat feeding, when autophagy may be compromised.…”

“…Glucose tolerance tests (GTT) and insulin tolerance tests (ITT) were performed in unrestrained mice at baseline and repeated at 4 weeks and 12 weeks. Mice were fasted and placed in individual cages with free access to water for 6 hours prior to the GTT and 4 hours prior to the ITT as previously described . Body weight was recorded after the fasting period to calculate injection volumes.…”

“…High‐resolution respirometry was performed using Oxygraph‐2k units (Oroboros Instruments, Innsbruck, Austria) with MiR05 respiration buffer (0.5 mM EGTA, 3 mM MgCl 2 ‐6H 2 O, 60 mM lactobionic acid, 20 mM taurine, 10 mM KH 2 PO 4 , 20 mM HEPES, 110 mM sucrose, and 1 g/L bovine serum albumin). Two separate protocols were performed to measure oxygen consumption and hydrogen peroxide (H 2 O 2 ) emission (10 μM Amplex red, 5 U/ml superoxide dismutase, and 1 U/mL horseradish peroxidase) as previously described and with a few variations . Each protocol was performed in duplicate chambers (A and B) on one machine and the rate of oxygen consumed (JO 2 pmol O 2 /sec/mL) at each point was calculated as the average values from the two chambers.…”

“…Mice were fasted and placed in individual cages with free access to water for 6 hours prior to the GTT and 4 hours prior to the ITT as previously described. 22 Body weight was recorded after the fasting period to calculate injection volumes. Blood glucose concentrations were measured by a handheld glucometer (Alphatrak2, Zoetis, Parsippany, NJ, USA) from a tail vein.…”

“…In week 10, mice received an intraperitoneal injection of D 2 O saline solution (~99%; Cambridge Isotope Laboratories, Tewksbury, MA, USA) at 30 μL/g body wt to raise body water enrichment to ~5% (assuming 60% water weight) followed by continuous consumption of D 2 O enriched at 8% in the drinking water. 22 Plasma samples collected at the time of euthanasia were analyzed for D 2 O enrichment to calculate precursor enrichment. Plasma was placed in the inner well of an o-ring screw on cap and placed inverted on a heating block overnight at 80°C.…”