CBL is rapidly phosphorylated upon insulin receptor activation. Mice whole body CBL depletion improved insulin sensitivity and glucose clearance; however, the precise mechanisms remain unknown. We depleted either CBL or its associated protein SORBS1/CAP independently in myocytes and assessed mitochondrial function and metabolism compared to control cells. CBL- and CAP-depleted cells showed increased mitochondrial mass with greater proton leak. Mitochondrial respiratory complex I activity and assembly into respirasomes were reduced. Proteome profiling revealed alterations in proteins involved in glycolysis and fatty acid degradation. Our findings demonstrate CBL/CAP pathway couples insulin signaling to efficient mitochondrial respiratory function and metabolism in muscle.

FATP1 mediates skeletal muscle cell fatty acid import, yet its intracellular localization and metabolic control role are not completely defined. Here, we examine FATP1 localization and metabolic effects of its overexpression in mouse skeletal muscle. The FATP1 protein was detected in mitochondrial and plasma membrane fractions, obtained by differential centrifugation, of mouse gastrocnemius muscle. FATP1 was most abundant in purified mitochondria, and in the outer membrane and soluble intermembrane, but not in the inner membrane plus matrix, enriched subfractions of purified mitochondria. Immunogold electron microscopy localized FATP1-GFP in mitochondria of transfected C2C12 myotubes. FATP1 was overexpressed in gastrocnemius mouse muscle, by adenovirus-mediated delivery of the gene into hindlimb muscles of newborn mice, fed after weaning a chow or high-fat diet. Compared to GFP delivery, FATP1 did not alter body weight, serum fed glucose, insulin and triglyceride levels, and whole-body glucose tolerance, in either diet. However, fatty acid levels were lower and β-hydroxybutyrate levels were higher in FATP1- than GFP-mice, irrespective of diet. Moreover, intramuscular triglyceride content was lower in FATP1- versus GFP-mice regardless of diet, and β-hydroxybutyrate content was unchanged in high-fat-fed mice. Electroporation-mediated FATP1 overexpression enhanced palmitate oxidation to CO2, but not to acid-soluble intermediate metabolites, while CO2 production from β-hydroxybutyrate was inhibited and that from glucose unchanged, in isolated mouse gastrocnemius strips. In summary, FATP1 was localized in mitochondria, in the outer membrane and intermembrane parts, of mouse skeletal muscle, what may be crucial for its metabolic effects. Overexpressed FATP1 enhanced disposal of both systemic fatty acids and intramuscular triglycerides. Consistently, it did not contribute to the high-fat diet-induced metabolic dysregulation. However, FATP1 lead to hyperketonemia, likely secondary to the sparing of ketone body oxidation by the enhanced oxidation of fatty acids.

Sarcopenia is one of the main factors contributing to the disability of aged people. Among the possible molecular determinants of sarcopenia, increasing evidences suggest that chronic inflammation contributes to its development. However, a key unresolved question is the nature of the factors that drive inflammation during aging and that participate in the development of sarcopenia. In this regard, mitochondrial dysfunction and alterations in mitophagy induce inflammatory responses in a wide range of cells and tissues. However, whether accumulation of damaged mitochondria (MIT) in muscle could trigger inflammation in the context of aging is still unknown. Here, we demonstrate that BCL2 interacting protein 3 (BNIP3) plays a key role in the control of mitochondrial and lysosomal homeostasis, and mitigates muscle inflammation and atrophy during aging. We show that muscle BNIP3 expression increases during aging in mice and in some humans. BNIP3 deficiency alters mitochondrial function, decreases mitophagic flux and, surprisingly, induces lysosomal dysfunction, leading to an upregulation of Toll-like receptor 9 (TLR9)-dependent inflammation and activation of the NLRP3 (nucleotide-binding oligomerization domain (NOD)-, leucine-rich repeat (LRR)-, and pyrin domain-containing protein 3) inflammasome in muscle cells and mouse muscle. Importantly, downregulation of muscle BNIP3 in aged mice exacerbates inflammation and muscle atrophy, and high BNIP3 expression in aged human subjects associates with a low inflammatory profile, suggesting a protective role for BNIP3 against age-induced muscle inflammation in mice and humans. Taken together, our data allow us to propose a new adaptive mechanism involving the mitophagy protein BNIP3, which links mitochondrial and lysosomal homeostasis with inflammation and is key to maintaining muscle health during aging.

Some forms of mitochondrial dysfunction induce sterile inflammation through mitochondrial DNA recognition by intracellular DNA sensors. However, the involvement of mitochondrial dynamics in mitigating such processes and their impact on muscle fitness remain unaddressed. Here we report that opposite mitochondrial morphologies induce distinct inflammatory signatures, caused by differential activation of DNA sensors TLR9 or cGAS. In the context of mitochondrial fragmentation, we demonstrate that mitochondria-endosome contacts mediated by the endosomal protein Rab5C are required in TLR9 activation in cells. Skeletal muscle mitochondrial fragmentation promotes TLR9-dependent inflammation, muscle atrophy, reduced physical performance and enhanced IL6 response to exercise, which improved upon chronic anti-inflammatory treatment. Taken together, our data demonstrate that mitochondrial dynamics is key in preventing sterile inflammatory responses, which precede the development of muscle atrophy and impaired physical performance. Thus, we propose the targeting of mitochondrial dynamics as an approach to treating disorders characterized by chronic inflammation and mitochondrial dysfunction.

Circadian rhythms are generated by an autoregulatory feedback loop of transcriptional activators and repressors. Circadian rhythm disruption contributes to type 2 diabetes (T2D) pathogenesis. We elucidated whether altered circadian rhythmicity of clock genes is associated with metabolic dysfunction in T2D. Transcriptional cycling of core-clock genes BMAL1, CLOCK, and PER3 was altered in skeletal muscle from individuals with T2D, and this was coupled with reduced number and amplitude of cycling genes and disturbed circadian oxygen consumption. Inner mitochondria–associated genes were enriched for rhythmic peaks in normal glucose tolerance, but not T2D, and positively correlated with insulin sensitivity. Chromatin immunoprecipitation sequencing identified CLOCK and BMAL1 binding to inner-mitochondrial genes associated with insulin sensitivity, implicating regulation by the core clock. Inner-mitochondria disruption altered core-clock gene expression and free-radical production, phenomena that were restored by resveratrol treatment. We identify bidirectional communication between mitochondrial function and rhythmic gene expression, processes that are disturbed in diabetes.

Type 2 diabetes mellitus (T2D) affects millions of people worldwide and is one of the leading causes of morbidity and mortality. The skeletal muscle (SKM) is one of the most important tissues involved in maintaining glucose homeostasis and substrate oxidation, and it undergoes insulin resistance in T2D. In this study, we identify the existence of alterations in the expression of mitochondrial aminoacyl-tRNA synthetases (mt-aaRSs) in skeletal muscle from two different forms of T2D: early-onset type 2 diabetes (YT2) (onset of the disease before 30 years of age) and the classical form of the disease (OT2). GSEA analysis from microarray studies revealed the repression of mitochondrial mt-aaRSs independently of age, which was validated by real-time PCR assays. In agreement with this, a reduced expression of several encoding mt-aaRSs was also detected in skeletal muscle from diabetic (db/db) mice but not in obese ob/ob mice. In addition, the expression of the mt-aaRSs proteins most relevant in the synthesis of mitochondrial proteins, threonyl-tRNA, and leucyl-tRNA synthetases (TARS2 and LARS2) were also repressed in muscle from db/db mice. It is likely that these alterations participate in the reduced expression of proteins synthesized in the mitochondria detected in db/db mice. We also document an increased iNOS abundance in mitochondrial-enriched muscle fractions from diabetic mice that may inhibit aminoacylation of TARS2 and LARS2 by nitrosative stress. Our results indicate a reduced expression of mt-aaRSs in skeletal muscle from T2D patients, which may participate in the reduced expression of proteins synthesized in mitochondria. An enhanced mitochondrial iNOS could play a regulatory role in diabetes.

Mitochondrial fusion and fission events, collectively known as mitochondrial dynamics, act as quality control mechanisms to ensure mitochondrial function and fine-tune cellular bioenergetics. Defective mitofusin 2 (Mfn2) expression and enhanced mitochondrial fission in skeletal muscle are hallmarks of insulin-resistant states. Interestingly, Mfn2 is highly expressed in brown adipose tissue (BAT), yet its role remains unexplored. Using adipose-specific Mfn2 knockout (Mfn2-adKO) mice, we demonstrate that Mfn2, but not Mfn1, deficiency in BAT leads to a profound BAT dysfunction, associated with impaired respiratory capacity and a blunted response to adrenergic stimuli. Importantly, Mfn2 directly interacts with perilipin 1, facilitating the interaction between the mitochondria and the lipid droplet in response to adrenergic stimulation. Surprisingly, Mfn2-adKO mice were protected from high-fat diet-induced insulin resistance and hepatic steatosis. Altogether, these results demonstrate that Mfn2 is a mediator of mitochondria to lipid droplet interactions, influencing lipolytic processes and whole-body energy homeostasis.

Type 2 Diabetes (T2D) is a highly prevalent chronic metabolic disease with strong co-morbidity with obesity and cardiovascular diseases. There is growing evidence supporting the notion that a crosstalk between mitochondria and the insulin signaling cascade could be involved in the etiology of T2D and insulin resistance. In this study we investigated the molecular basis of this crosstalk by using systems biology approaches. We combined, filtered, and interrogated different types of functional interaction data, such as direct protein-protein interactions, co-expression analyses, and metabolic and signaling dependencies. As a result, we constructed the mitochondria-insulin (MITIN) network, which highlights 286 genes as candidate functional linkers between these two systems. The results of internal gene expression analysis of three independent experimental models of mitochondria and insulin signaling perturbations further support the connecting roles of these genes. In addition, we further assessed whether these genes are involved in the etiology of T2D using the genome-wide association study meta-analysis from the DIAGRAM consortium, involving 8,130 T2D cases and 38,987 controls. We found modest enrichment of genes associated with T2D amongst our linker genes (p = 0.0549), including three already validated T2D SNPs and 15 additional SNPs, which, when combined, were collectively associated to increased fasting glucose levels according to MAGIC genome wide meta-analysis (p = 8.12×10(-5)). This study highlights the potential of combining systems biology, experimental, and genome-wide association data mining for identifying novel genes and related variants that increase vulnerability to complex diseases.