David E. Lee

Diet-induced obesity alters anabolic signalling in mice at the onset of skeletal muscle regeneration

Obesity is classified as a metabolic disorder that is associated with delayed muscle regeneration following damage. For optimal skeletal muscle regeneration, inflammation along with extracellular matrix remodelling and muscle growth must be tightly regulated. Moreover, the regenerative process is dependent on the activation of myogenic regulatory factors (MRFs) for myoblast proliferation and differentiation. The purpose of this study was to determine how obesity alters inflammatory and protein synthetic signalling and MRF expression at the onset of muscle regeneration in mice.

Mitochondrial quality control, promoted by PGC‐1α, is dysregulated by Western diet‐induced obesity and partially restored by moderate physical activity in mice

Skeletal muscle mitochondrial degeneration is a hallmark of insulin resistance/obesity marked by lost function, enhanced ROS emission, and altered morphology which may be ameliorated by physical activity (PA). However, no prior report has examined mitochondrial quality control regulation throughout biogenesis, fusion/fission dynamics, autophagy, and mitochondrial permeability transition pore (MPTP) in obesity. Therefore, we determined how each process is impacted by Western diet (WD)‐induced obesity and whether voluntary PA may alleviate derangements in mitochondrial quality control mechanisms. Despite greater mitochondrial content following WD (COX‐IV and Cytochrome C), induction of biogenesis controllers appears impaired (failed induction of PGC‐1α). Mitochondrial fusion seems diminished (reduced MFN2, Opa1 proteins), with no significant changes in fission, suggesting a shift in balance of dynamics regulation favoring fission. Autophagy flux was promoted in WD (reduced p62, increased LC3II:I ratio); however, mitophagy marker BNIP3 is reduced in WD which may indicate reduced mitophagy despite enhanced total autophagy flux. MPTP regulator Ant mRNA is reduced by WD. Few processes were impacted by physical activity. Finally, mitochondrial quality control processes are partially promoted by PGC‐1α, as PGC‐1α transgenic mice display elevated mitochondrial biogenesis and autophagy flux. Additionally, these mice exhibit elevated Mfn1 and Opa1 mRNA, with no change in protein content suggesting these factors are transcriptionally promoted by PGC‐1α overexpression. These data demonstrate dysfunctions across mitochondrial quality control in obesity and that PGC‐1α is sufficient to promote multiple, but not necessarily all, aspects of mitochondrial quality control. Mitochondrial quality control may therefore be an opportune target to therapeutically treat metabolic disease.

microRNA‐16 Is Downregulated During Insulin Resistance and Controls Skeletal Muscle Protein Accretion

Insulin resistant diabetes, currently at epidemic levels in developed countries, begins in the skeletal muscle and is linked to altered protein turnover. microRNAs downregulate targeted mRNA translation decreasing the amount of translated protein, thereby regulating many cellular processes. Regulation of miRNAs and their function in skeletal muscle insulin resistance is largely unexplored. The purpose of this study was to identify the effects of insulin resistance on contents of skeletal muscle miRNAs with potential functions in protein turnover. We examined miRs ‐1, ‐16, ‐23, ‐27, ‐133a, ‐133b, and ‐206 in muscles of Zucker rats. miR‐1 was 5‐ to 10‐fold greater in obesity, whereas miRs‐16 and ‐133b were repressed ∼50% in obese compared to lean rats, with no other alterations in miRNA contents. miR‐16 correlated to protein synthesis in lean, but not obese rats. miR‐16 reduction by lipid overload was verified in‐vivo by diet‐induced obesity and in‐vitro using a diacylglycerol analog. A role for miR‐16 in protein turnover of skeletal myocytes was established using transient overexpression and anti‐miR inhibition. miR‐16 overexpression resulted in lower protein synthesis (puromycin incorporation, ∼25–50%), mTOR (∼25%), and p70S6K1 (∼40%) in starved and insulin stimulated myoblasts. Conversely, anti‐miR‐16 increased basal protein synthesis (puromycin incorporation, ∼75%), mTOR (∼100%), and p70S6K1 (∼100%). Autophagy was enhanced by miR‐16 overexpression (∼50% less BCL‐2, ∼100% greater LC3II/I, ∼50% less p62) and impaired with miR‐16 inhibition (∼45% greater BCL‐2, ∼25% less total LC3, ∼50% greater p62). This study demonstrates reduced miR‐16 during insulin resistance and establishes miR‐16 control of protein accretion in skeletal muscle. J. Cell. Biochem. 117: 1775–1787, 2016.

Translational machinery of mitochondrial mRNA is promoted by physical activity in Western diet-induced obese mice.

Mitochondria‐encoded proteins are necessary for oxidative phosphorylation; however, no report has examined how physical activity (PA) and obesity affect mitochondrial mRNA translation machinery. Our purpose was to determine whether Western diet (WD)‐induced obesity and voluntary wheel running (VWR) impact mitochondrial mRNA translation machinery and whether expression of this machinery is dictated by oxidative phenotype.

Impaired exercise-induced mitochondrial biogenesis in the obese Zucker rat, despite PGC-1α induction, is due to compromised mitochondrial translation elongation.

Previously, we demonstrated that high-volume resistance exercise stimulates mitochondrial protein synthesis (a measure of mitochondrial biogenesis) in lean but not obese Zucker rats. Here, we examined factors involved in regulating mitochondrial biogenesis in the same animals. PGC-1α was 45% higher following exercise in obese but not lean animals compared with sedentary counterparts. Interestingly, exercised animals demonstrated greater PPARδ protein in both lean (47%) and obese (>200%) animals. AMPK phosphorylation (300%) and CPT-I protein (30%) were elevated by exercise in lean animals only, indicating improved substrate availability/flux. These findings suggest that, despite PGC-1α induction, obese animals were resistant to exercise-induced synthesis of new mitochondrial and oxidative protein. Previously, we reported that most anabolic processes are upregulated in these same obese animals regardless of exercise, so the purpose of this study was to assess specific factors associated with the mitochondrial genome as possible culprits for impaired mitochondrial biogenesis. Exercise resulted in higher mRNA contents of mitochondrial transcription factor A (∼50% in each phenotype) and mitochondrial translation initiation factor 2 (31 and 47% in lean and obese, respectively). However, mitochondrial translation elongation factor-Tu mRNA was higher following exercise in lean animals only (40%), suggesting aberrant regulation of mitochondrial translation elongation as a possible culprit in impaired mitochondrial biogenesis following exercise with obesity.

Moderate physical activity promotes basal hepatic autophagy in diet-induced obese mice

Obesity is a known risk factor for the development of hepatic disease; obesity-induced fatty liver can lead to inflammation, steatosis, and cirrhosis and is associated with degeneration of the mitochondria. Lifestyle interventions such as physical activity may ameliorate this condition. The purpose of this study was to investigate regulation of mitochondrial and autophagy quality control in liver following Western diet-induced obesity and voluntary physical activity. Eight-week-old C57BL/6J mice were fed a Western diet (WD) or normal chow (NC, control) for 4 weeks; afterwards, groups were divided into voluntary wheel running (VWR) or sedentary (SED) conditions for an additional 4 weeks. WD-SED animals had a median histology score of 2, whereas WD-VWR was not different from NC groups (median score 1). There was no difference in mRNA of inflammatory markers Il6 and Tnfa in WD animals. WD animals had 50% lower mitochondrial content (COX IV and Cytochrome C proteins), 50% lower Pgc1a mRNA content, and reduced content of mitochondrial fusion and fission markers. Markers of autophagy were increased in VWR animals, regardless of obesity, as measured by 50% greater LC3-II/I ratio and 40% lower p62 protein content. BNIP3 protein content was 30% less in WD animals compared with NC animals, regardless of physical activity. Diet-induced obesity results in derangements in mitochondrial quality control that appear to occur prior to the onset of hepatic inflammation. Moderate physical activity appears to enhance basal autophagy in the liver; increased autophagy may provide protection from hepatic fat accumulation.

Mitochondrial degeneration precedes the development of muscle atrophy in progression of cancer cachexia in tumour-bearing mice

Cancer cachexia is largely irreversible, at least via nutritional means, and responsible for 20–40% of cancer‐related deaths. Therefore, preventive measures are of primary importance; however, little is known about muscle perturbations prior to onset of cachexia. Cancer cachexia is associated with mitochondrial degeneration; yet, it remains to be determined if mitochondrial degeneration precedes muscle wasting in cancer cachexia. Therefore, our purpose was to determine if mitochondrial degeneration precedes cancer‐induced muscle wasting in tumour‐bearing mice.

Cancer cachexia-induced muscle atrophy: evidence for alterations in microRNAs important for muscle size

Muscle atrophy is a hallmark of cancer cachexia resulting in impaired function and quality of life and cachexia is the immediate cause of death for 20-40% of cancer patients. Multiple microRNAs (miRNAs) have been identified as being involved in muscle development and atrophy; however, less is known specifically on miRNAs in cancer cachexia. The purpose of this investigation was to examine the miRNA profile of skeletal muscle atrophy induced by cancer cachexia to uncover potential miRNAs involved with this catabolic condition. Phosphate-buffered saline (PBS) or Lewis lung carcinoma cells (LLC) were injected into C57BL/6J mice at 8 wk of age. LLC animals were allowed to develop tumors for 4 wk to induce cachexia. Tibialis anterior muscles were extracted and processed to isolate small RNAs, which were used for miRNA sequencing. Sequencing results were assembled with mature miRNAs, and functions of miRNAs were analyzed by Ingenuity Pathway Analysis. LLC animals developed tumors that contributed to significantly smaller tibialis anterior muscles (18.5%) and muscle cross-sectional area (40%) compared with PBS. We found 371 miRNAs to be present in the muscle above background levels. Of these, nine miRNAs were found to be differentially expressed. Significantly altered groups of miRNAs were categorized into primary functionalities including cancer, cell-to-cell signaling, and cellular development among others. Gene network analysis predicted specific alterations of factors contributing to muscle size including Akt, FOXO3, and others. These results create a foundation for future research into the sufficiency of targeting these genes to attenuate muscle loss in cancer cachexia.

Differential effects of leucine supplementation in young and aged mice at the onset of skeletal muscle regeneration

Aging decreases the ability of skeletal muscle to respond to injury. Leucine has been demonstrated to target protein synthetic pathways in skeletal muscle thereby enhancing this response. However, the effect of aging on leucine-induced alterations in protein synthesis at the onset of skeletal muscle regeneration has not been fully elucidated. The purpose of this study was to determine if aging alters skeletal muscle regeneration and leucine-induced alterations in markers of protein synthesis. The tibialis anterior of young (3 months) and aged (24 months) female C57BL/6J mice were injected with either bupivacaine or PBS, and the mice were given ad libitum access to leucine-supplemented or normal drinking water. Protein and gene expression of markers of protein synthesis and degradation, respectively, were analyzed at three days post-injection. Following injury in young mice, leucine supplementation was observed to elevate only p-p70S6K. In aged mice, leucine was shown to elicit higher p-mTOR content with and without injury, and p-4EBP-1 content post-injury. Additionally in aged mice, leucine was shown to elicit higher content of relative p70S6K post-injury. Our study shows that leucine supplementation affects markers of protein synthesis at the onset of skeletal muscle regeneration differentially in young and aged mice.

PGC‐1α4 gene expression is suppressed by the IL‐6—MEK—ERK 1/2 MAPK signalling axis and altered by resistance exercise, obesity and muscle injury

PGC‐1α4 is a novel regulator of muscle hypertrophy; however, there is limited understanding of the regulation of its expression and role in many (patho)physiological conditions. Therefore, our purpose was to elicit signalling mechanisms regulating gene expression of Pgc1α4 and examine its response to (patho)physiological stimuli associated with altered muscle mass.