Deep Proteome Profiling of White Adipose Tissue Reveals Marked Conservation and Distinct Features Between Different Anatomical Depots.

White adipose tissue is deposited mainly as subcutaneous adipose tissue (SAT), often associated with metabolic protection, and abdominal/visceral adipose tissue, which contributes to metabolic disease. To investigate the molecular underpinnings of these differences, we conducted comprehensive proteomics profiling of whole tissue and isolated adipocytes from these two depots across two diets from C57Bl/6J mice. The adipocyte proteomes from lean mice were highly conserved between depots, with the major depot-specific differences encoded by just 3% of the proteome. Adipocytes from SAT (SAdi) were enriched in pathways related to mitochondrial complex I and beiging, whereas visceral adipocytes (VAdi) were enriched in structural proteins and positive regulators of mTOR presumably to promote nutrient storage and cellular expansion. This indicates that SAdi are geared toward higher catabolic activity, while VAdi are more suited for lipid storage. By comparing adipocytes from mice fed chow or Western diet (WD), we define a core adaptive proteomics signature consisting of increased extracellular matrix proteins and decreased fatty acid metabolism and mitochondrial Coenzyme Q biosynthesis. Relative to SAdi, VAdi displayed greater changes with WD including a pronounced decrease in mitochondrial proteins concomitant with upregulation of apoptotic signaling and decreased mitophagy, indicating pervasive mitochondrial stress. Furthermore, WD caused a reduction in lipid handling and glucose uptake pathways particularly in VAdi, consistent with adipocyte de-differentiation. By overlaying the proteomics changes with diet in whole adipose tissue and isolated adipocytes, we uncovered concordance between adipocytes and tissue only in the visceral adipose tissue, indicating a unique tissue-specific adaptation to sustained WD in SAT. Finally, an in-depth comparison of isolated adipocytes and 3T3-L1 proteomes revealed a high degree of overlap, supporting the utility of the 3T3-L1 adipocyte model. These deep proteomes provide an invaluable resource highlighting differences between white adipose depots that may fine-tune their unique functions and adaptation to an obesogenic environment.

Mitochondrial oxidants, but not respiration, are sensitive to glucose in adipocytes.

Insulin action in adipose tissue is crucial for whole-body glucose homeostasis, with insulin resistance being a major risk factor for metabolic diseases such as type 2 diabetes. Recent studies have proposed mitochondrial oxidants as a unifying driver of adipose insulin resistance, serving as a signal of nutrient excess. However, neither the substrates for nor sites of oxidant production are known. Because insulin stimulates glucose utilization, we hypothesized that glucose oxidation would fuel respiration, in turn generating mitochondrial oxidants. This would impair insulin action, limiting further glucose uptake in a negative feedback loop of "glucose-dependent" insulin resistance. Using primary rat adipocytes and cultured 3T3-L1 adipocytes, we observed that insulin increased respiration, but notably this occurred independently of glucose supply. In contrast, glucose was required for insulin to increase mitochondrial oxidants. Despite rising to similar levels as when treated with other agents that cause insulin resistance, glucose-dependent mitochondrial oxidants failed to cause insulin resistance. Subsequent studies revealed a temporal relationship whereby mitochondrial oxidants needed to increase before the insulin stimulus to induce insulin resistance. Together, these data reveal that (a) adipocyte respiration is principally fueled from nonglucose sources; (b) there is a disconnect between respiration and oxidative stress, whereby mitochondrial oxidant levels do not rise with increased respiration unless glucose is present; and (c) mitochondrial oxidative stress must precede the insulin stimulus to cause insulin resistance, explaining why short-term, insulin-dependent glucose utilization does not promote insulin resistance. These data provide additional clues to mechanistically link nutrient excess to adipose insulin resistance.

Lactate production is a prioritized feature of adipocyte metabolism.

Adipose tissue is essential for whole-body glucose homeostasis, with a primary role in lipid storage. It has been previously observed that lactate production is also an important metabolic feature of adipocytes, but its relationship to adipose and whole-body glucose disposal remains unclear. Therefore, using a combination of metabolic labeling techniques, here we closely examined lactate production of cultured and primary mammalian adipocytes. Insulin treatment increased glucose uptake and conversion to lactate, with the latter responding more to insulin than did other metabolic fates of glucose. However, lactate production did not just serve as a mechanism to dispose of excess glucose, because we also observed that lactate production in adipocytes did not solely depend on glucose availability and even occurred independently of glucose metabolism. This suggests that lactate production is prioritized in adipocytes. Furthermore, knocking down lactate dehydrogenase specifically in the fat body of Drosophila flies lowered circulating lactate and improved whole-body glucose disposal. These results emphasize that lactate production is an additional metabolic role of adipose tissue beyond lipid storage and release.

Insulin signaling requires glucose to promote lipid anabolism in adipocytes.

Adipose tissue is essential for metabolic homeostasis, balancing lipid storage and mobilization based on nutritional status. This is coordinated by insulin, which triggers kinase signaling cascades to modulate numerous metabolic proteins, leading to increased glucose uptake and anabolic processes like lipogenesis. Given recent evidence that glucose is dispensable for adipocyte respiration, we sought to test whether glucose is necessary for insulin-stimulated anabolism. Examining lipogenesis in cultured adipocytes, glucose was essential for insulin to stimulate the synthesis of fatty acids and glyceride-glycerol. Importantly, glucose was dispensable for lipogenesis in the absence of insulin, suggesting that distinct carbon sources are used with or without insulin. Metabolic tracing studies revealed that glucose was required for insulin to stimulate pathways providing carbon substrate, NADPH, and glycerol 3-phosphate for lipid synthesis and storage. Glucose also displaced leucine as a lipogenic substrate and was necessary to suppress fatty acid oxidation. Together, glucose provided substrates and metabolic control for insulin to promote lipogenesis in adipocytes. This contrasted with the suppression of lipolysis by insulin signaling, which occurred independently of glucose. Given previous observations that signal transduction acts primarily before glucose uptake in adipocytes, these data are consistent with a model whereby insulin initially utilizes protein phosphorylation to stimulate lipid anabolism, which is sustained by subsequent glucose metabolism. Consequently, lipid abundance was sensitive to glucose availability, both during adipogenesis and in Drosophila flies in vivo Together, these data highlight the importance of glucose metabolism to support insulin action, providing a complementary regulatory mechanism to signal transduction to stimulate adipose anabolism.

Skeletal muscle excitation-metabolism coupling.

Mitochondria represent the main source of ATP in skeletal muscle and mitochondria activity increases after muscle fiber depolarization. The regulation of mitochondrial function during contraction in skeletal muscle, however, is poorly understood. Skeletal muscle has a particular distribution of mitochondria where three distinct populations can be recognized. The most studied populations are the ones positioned deep into the myofibers between the myofibrils (intermyofibrillar mitochondria), and that located immediately beneath sarcolemma (subsarcolemmal mitochondria); a less studied population locates covering the myonuclei, as a continuation of the subsarcolemmal population. All mitochondria populations undergo fusion and fission events and intermyofibrillar mitochondria are interconnected; mitochondrial communication is necessary to maintain not only the energetic homeostasis of the muscle but its contractile function, as well. The mechanism supporting communication between subsarcolemmal and intermyofibrillar mitochondria is unknown. The recently described MCU complex of proteins has provided a new insight into the role of calcium as a regulator of mitochondrial function. Whether the different mitochondria populations have different calcium handling capacity and whether mitochondria Ca2+ has a role in energy transmission along the mitochondria network are intriguing issues that emerge when studying the link between electrical stimulation of the muscle fiber and the mitochondria metabolic output.

GDF-11 prevents cardiomyocyte hypertrophy by maintaining the sarcoplasmic reticulum-mitochondria communication.

Growth differentiation factor 11 (GDF11) is a novel factor with controversial effects on cardiac hypertrophy both in vivo and in vitro. Although recent evidence has corroborated that GDF11 prevents the development of cardiac hypertrophy, its molecular mechanism remains unclear. In our previous work, we showed that norepinephrine (NE), a physiological pro-hypertrophic agent, increases cytoplasmic Ca2+ levels accompanied by a loss of physical and functional communication between sarcoplasmic reticulum (SR) and mitochondria, with a subsequent reduction in the mitochondrial Ca2+ uptake and mitochondrial metabolism. In order to study the anti-hypertrophic mechanism of GDF11, our aim was to investigate whether GDF11 prevents the loss of SR-mitochondria communication triggered by NE. Our results show that: a) GDF11 prevents hypertrophy in cultured neonatal rat ventricular myocytes treated with NE. b) GDF11 attenuates the NE-induced loss of contact sites between both organelles. c) GDF11 increases oxidative mitochondrial metabolism by stimulating mitochondrial Ca2+ uptake. In conclusion, the GDF11-dependent maintenance of physical and functional communication between SR and mitochondria is critical to allow Ca2+ transfer between both organelles and energy metabolism in the cardiomyocyte and to avoid the activation of Ca2+-dependent pro-hypertrophic signaling pathways.

Deletion of miPEP in adipocytes protects against obesity and insulin resistance by boosting muscle metabolism.

Mitochondria facilitate thousands of biochemical reactions, covering a broad spectrum of anabolic and catabolic processes. Here we demonstrate that the adipocyte mitochondrial proteome is markedly altered across multiple models of insulin resistance and reveal a consistent decrease in the level of the mitochondrial processing peptidase miPEP. To experimentally test this observation, we generated adipocyte-specific miPEP knockout mice to interrogate its role in the aetiology of insulin resistance. We observed a strong phenotype characterised by enhanced insulin sensitivity and reduced adiposity, despite normal food intake and physical activity. Strikingly, these phenotypes vanished when mice were housed at thermoneutrality, suggesting that metabolic protection conferred by miPEP deletion hinges upon a thermoregulatory process. Tissue specific analysis of miPEP deficient mice revealed an increment in muscle metabolism, and upregulation of the protein FBP2 that is involved in ATP hydrolysis in the gluconeogenic pathway. These findings suggest that miPEP deletion initiates a compensatory increase in skeletal muscle metabolism acting as a protective mechanism against diet-induced obesity and insulin resistance.

Erratum: Proteomic pathways to metabolic disease and type 2 diabetes in the pancreatic islet.

[This corrects the article DOI: 10.1016/j.isci.2021.103099.].

Insulin-dependent H2O2 production is higher in muscle fibers of mice fed with a high-fat diet.

Insulin resistance is defined as a reduced ability of insulin to stimulate glucose utilization. C57BL/6 mice fed with a high-fat diet (HFD) are a model of insulin resistance. In skeletal muscle, hydrogen peroxide (H2O2) produced by NADPH oxidase 2 (NOX2) is involved in signaling pathways triggered by insulin. We evaluated oxidative status in skeletal muscle fibers from insulin-resistant and control mice by determining H2O2 generation (HyPer probe), reduced-to-oxidized glutathione ratio and NOX2 expression. After eight weeks of HFD, insulin-dependent glucose uptake was impaired in skeletal muscle fibers when compared with control muscle fibers. Insulin-resistant mice showed increased insulin-stimulated H2O2 release and decreased reduced-to-oxidized glutathione ratio (GSH/GSSG). In addition, p47phox and gp91phox (NOX2 subunits) mRNA levels were also high (~3-fold in HFD mice compared to controls), while protein levels were 6.8- and 1.6-fold higher, respectively. Using apocynin (NOX2 inhibitor) during the HFD feeding period, the oxidative intracellular environment was diminished and skeletal muscle insulin-dependent glucose uptake restored. Our results indicate that insulin-resistant mice have increased H2O2 release upon insulin stimulation when compared with control animals, which appears to be mediated by an increase in NOX2 expression.

A high-content endogenous GLUT4 trafficking assay reveals new aspects of adipocyte biology.

Insulin-induced GLUT4 translocation to the plasma membrane in muscle and adipocytes is crucial for whole-body glucose homeostasis. Currently, GLUT4 trafficking assays rely on overexpression of tagged GLUT4. Here we describe a high-content imaging platform for studying endogenous GLUT4 translocation in intact adipocytes. This method enables high fidelity analysis of GLUT4 responses to specific perturbations, multiplexing of other trafficking proteins and other features including lipid droplet morphology. Using this multiplexed approach we showed that Vps45 and Rab14 are selective regulators of GLUT4, but Trarg1, Stx6, Stx16, Tbc1d4 and Rab10 knockdown affected both GLUT4 and TfR translocation. Thus, GLUT4 and TfR translocation machinery likely have some overlap upon insulin-stimulation. In addition, we identified Kif13A, a Rab10 binding molecular motor, as a novel regulator of GLUT4 traffic. Finally, comparison of endogenous to overexpressed GLUT4 highlights that the endogenous GLUT4 methodology has an enhanced sensitivity to genetic perturbations and emphasises the advantage of studying endogenous protein trafficking for drug discovery and genetic analysis of insulin action in relevant cell types.

Phosphoproteomics reveals rewiring of the insulin signaling network and multi-nodal defects in insulin resistance.

The failure of metabolic tissues to appropriately respond to insulin ("insulin resistance") is an early marker in the pathogenesis of type 2 diabetes. Protein phosphorylation is central to the adipocyte insulin response, but how adipocyte signaling networks are dysregulated upon insulin resistance is unknown. Here we employ phosphoproteomics to delineate insulin signal transduction in adipocyte cells and adipose tissue. Across a range of insults causing insulin resistance, we observe a marked rewiring of the insulin signaling network. This includes both attenuated insulin-responsive phosphorylation, and the emergence of phosphorylation uniquely insulin-regulated in insulin resistance. Identifying dysregulated phosphosites common to multiple insults reveals subnetworks containing non-canonical regulators of insulin action, such as MARK2/3, and causal drivers of insulin resistance. The presence of several bona fide GSK3 substrates among these phosphosites led us to establish a pipeline for identifying context-specific kinase substrates, revealing widespread dysregulation of GSK3 signaling. Pharmacological inhibition of GSK3 partially reverses insulin resistance in cells and tissue explants. These data highlight that insulin resistance is a multi-nodal signaling defect that includes dysregulated MARK2/3 and GSK3 activity.

Mitochondrial electron transport chain, ceramide and Coenzyme Q are linked in a pathway that drives insulin resistance in skeletal muscle.

Insulin resistance (IR) is a complex metabolic disorder that underlies several human diseases, including type 2 diabetes and cardiovascular disease. Despite extensive research, the precise mechanisms underlying IR development remain poorly understood. Here, we provide new insights into the mechanistic connections between cellular alterations associated with IR, including increased ceramides, deficiency of coenzyme Q (CoQ), mitochondrial dysfunction, and oxidative stress. We demonstrate that elevated levels of ceramide in the mitochondria of skeletal muscle cells results in CoQ depletion and loss of mitochondrial respiratory chain components, leading to mitochondrial dysfunction and IR. Further, decreasing mitochondrial ceramide levels in vitro and in animal models (under chow and high fat diet) increased CoQ levels and was protective against IR. CoQ supplementation also rescued ceramide-associated IR. Examination of the mitochondrial proteome from human muscle biopsies revealed a strong correlation between the respirasome system and mitochondrial ceramide as key determinants of insulin sensitivity. Our findings highlight the mitochondrial Ceramide-CoQ-respiratory chain nexus as a potential foundation of an IR pathway that may also play a critical role in other conditions associated with ceramide accumulation and mitochondrial dysfunction, such as heart failure, cancer, and aging. These insights may have important clinical implications for the development of novel therapeutic strategies for the treatment of IR and related metabolic disorders.

Mitochondrial electron transport chain, ceramide, and coenzyme Q are linked in a pathway that drives insulin resistance in skeletal muscle.

Insulin resistance (IR) is a complex metabolic disorder that underlies several human diseases, including type 2 diabetes and cardiovascular disease. Despite extensive research, the precise mechanisms underlying IR development remain poorly understood. Previously we showed that deficiency of coenzyme Q (CoQ) is necessary and sufficient for IR in adipocytes and skeletal muscle (Fazakerley et al., 2018). Here, we provide new insights into the mechanistic connections between cellular alterations associated with IR, including increased ceramides, CoQ deficiency, mitochondrial dysfunction, and oxidative stress. We demonstrate that elevated levels of ceramide in the mitochondria of skeletal muscle cells result in CoQ depletion and loss of mitochondrial respiratory chain components, leading to mitochondrial dysfunction and IR. Further, decreasing mitochondrial ceramide levels in vitro and in animal models (mice, C57BL/6J) (under chow and high-fat diet) increased CoQ levels and was protective against IR. CoQ supplementation also rescued ceramide-associated IR. Examination of the mitochondrial proteome from human muscle biopsies revealed a strong correlation between the respirasome system and mitochondrial ceramide as key determinants of insulin sensitivity. Our findings highlight the mitochondrial ceramide-CoQ-respiratory chain nexus as a potential foundation of an IR pathway that may also play a critical role in other conditions associated with ceramide accumulation and mitochondrial dysfunction, such as heart failure, cancer, and aging. These insights may have important clinical implications for the development of novel therapeutic strategies for the treatment of IR and related metabolic disorders.

Endoplasmic reticulum-mitochondria coupling increases during doxycycline-induced mitochondrial stress in HeLa cells.

Subcellular organelles communicate with each other to regulate function and coordinate responses to changing cellular conditions. The physical-functional coupling of the endoplasmic reticulum (ER) with mitochondria allows for the direct transfer of Ca2+ between organelles and is an important avenue for rapidly increasing mitochondrial metabolic activity. As such, increasing ER-mitochondrial coupling can boost the generation of ATP that is needed to restore homeostasis in the face of cellular stress. The mitochondrial unfolded protein response (mtUPR) is activated by the accumulation of unfolded proteins in mitochondria. Retrograde signaling from mitochondria to the nucleus promotes mtUPR transcriptional responses aimed at restoring protein homeostasis. It is currently unknown whether the changes in mitochondrial-ER coupling also play a role during mtUPR stress. We hypothesized that mitochondrial stress favors an expansion of functional contacts between mitochondria and ER, thereby increasing mitochondrial metabolism as part of a protective response. Hela cells were treated with doxycycline, an antibiotic that inhibits the translation of mitochondrial-encoded proteins to create protein disequilibrium. Treatment with doxycycline decreased the abundance of mitochondrial encoded proteins while increasing expression of CHOP, C/EBPß, ClpP, and mtHsp60, markers of the mtUPR. There was no change in either mitophagic activity or cell viability. Furthermore, ER UPR was not activated, suggesting focused activation of the mtUPR. Within 2 h of doxycycline treatment, there was a significant increase in physical contacts between mitochondria and ER that was distributed throughout the cell, along with an increase in the kinetics of mitochondrial Ca2+ uptake. This was followed by the rise in the rate of oxygen consumption at 4 h, indicating a boost in mitochondrial metabolic activity. In conclusion, an early phase of the response to doxycycline-induced mitochondrial stress is an increase in mitochondrial-ER coupling that potentiates mitochondrial metabolic activity as a means to support subsequent steps in the mtUPR pathway and sustain cellular adaptation.

Dissecting the biology of mTORC1 beyond rapamycin.

Rapamycin extends maximal life span and increases resistance to starvation in many organisms. The beneficial effects of rapamycin are thought to be mediated by its inhibitory effects on the mechanistic target of rapamycin complex 1 (mTORC1), although it only partially inhibits the kinase activity of mTORC1. Other mTOR kinase inhibitors have been developed, such as Torin-1, but these readily cross-react with mTORC2. Here, we report the distinct characteristics of a third-generation mTOR inhibitor called RapaLink1. We found that low doses of RapaLink1 inhibited the phosphorylation of all mTORC1 substrates tested, including those whose phosphorylation is sensitive or resistant to inhibition by rapamycin, without affecting mTORC2 activity even after prolonged treatment. Compared with rapamycin, RapaLink1 showed better efficacy for inhibiting mTORC1 and potently blocked cell proliferation and induced autophagy. Moreover, using RapaLink1, we demonstrated that mTORC1 and mTORC2 exerted differential effects on cell glycolysis and glucose uptake. Last, we found that RapaLink1 and rapamycin had opposing effects on starvation resistance in Drosophila. Consistent with the effects of RapaLink1, genetic blockade of mTORC1 activity made flies more sensitive to starvation, reflecting the complexity of the mTORC1 network that extends beyond effects that can be inhibited by rapamycin. These findings extend our understanding of mTOR biology and provide insights into some of the beneficial effects of rapamycin.

Proteomic pathways to metabolic disease and type 2 diabetes in the pancreatic islet.

Pancreatic islets are essential for maintaining physiological blood glucose levels, and declining islet function is a hallmark of type 2 diabetes. We employ mass spectrometry-based proteomics to systematically analyze islets from 9 genetic or diet-induced mouse models representing a broad cross-section of metabolic health. Quantifying the islet proteome to a depth of >11,500 proteins, this study represents the most detailed analysis of mouse islet proteins to date. Our data highlight that the majority of islet proteins are expressed in all strains and diets, but more than half of the proteins vary in expression levels, principally due to genetics. Associating these varied protein expression levels on an individual animal basis with individual phenotypic measures reveals islet mitochondrial function as a major positive indicator of metabolic health regardless of strain. This compendium of strain-specific and dietary changes to mouse islet proteomes represents a comprehensive resource for basic and translational islet cell biology.

Changes in Gene Expression of the MCU Complex Are Induced by Electrical Stimulation in Adult Skeletal Muscle.

The slow calcium transient triggered by low-frequency electrical stimulation (ES) in adult muscle fibers and regulated by the extracellular ATP/IP3/IP3R pathway has been related to muscle plasticity. A regulation of muscular tropism associated with the MCU has also been described. However, the role of transient cytosolic calcium signals and signaling pathways related to muscle plasticity over the regulation of gene expression of the MCU complex (MCU, MICU1, MICU2, and EMRE) in adult skeletal muscle is completely unknown. In the present work, we show that 270 0.3-ms-long pulses at 20-Hz ES (and not at 90 Hz) transiently decreased the mRNA levels of the MCU complex in mice flexor digitorum brevis isolated muscle fibers. Importantly, when ATP released after 20-Hz ES is hydrolyzed by the enzyme apyrase, the repressor effect of 20 Hz on mRNA levels of the MCU complex is lost. Accordingly, the exposure of muscle fibers to 30 µM exogenous ATP produces the same effect as 20-Hz ES. Moreover, the use of apyrase in resting conditions (without ES) increased mRNA levels of MCU, pointing out the importance of extracellular ATP concentration over MCU mRNA levels. The use of xestospongin B (inhibitor of IP3 receptors) also prevented the decrease of mRNA levels of MCU, MICU1, MICU2, and EMRE mediated by a low-frequency ES. Our results show that the MCU complex can be regulated by electrical stimuli in a frequency-dependent manner. The changes observed in mRNA levels may be related to changes in the mitochondria, associated with the phenotypic transition from a fast- to a slow-type muscle, according to the described effect of this stimulation frequency on muscle phenotype. The decrease in mRNA levels of the MCU complex by exogenous ATP and the increase in MCU levels when basal ATP is reduced with the enzyme apyrase indicate that extracellular ATP may be a regulator of the MCU complex. Moreover, our results suggest that this regulation is part of the axes linking low-frequency stimulation with ATP/IP3/IP3R.

Is Mitochondrial Dysfunction a Common Root of Noncommunicable Chronic Diseases?

Mitochondrial damage is implicated as a major contributing factor for a number of noncommunicable chronic diseases such as cardiovascular diseases, cancer, obesity, and insulin resistance/type 2 diabetes. Here, we discuss the role of mitochondria in maintaining cellular and whole-organism homeostasis, the mechanisms that promote mitochondrial dysfunction, and the role of this phenomenon in noncommunicable chronic diseases. We also review the state of the art regarding the preclinical evidence associated with the regulation of mitochondrial function and the development of current mitochondria-targeted therapeutics to treat noncommunicable chronic diseases. Finally, we give an integrated vision of how mitochondrial damage is implicated in these metabolic diseases.

Mitochondrial Calcium Increase Induced by RyR1 and IP3R Channel Activation After Membrane Depolarization Regulates Skeletal Muscle Metabolism.

Aim: We hypothesize that both type-1 ryanodine receptor (RyR1) and IP3-receptor (IP3R) calcium channels are necessary for the mitochondrial Ca2+ increase caused by membrane depolarization induced by potassium (or by electrical stimulation) of single skeletal muscle fibers; this calcium increase would couple muscle fiber excitation to an increase in metabolic output from mitochondria (excitation-metabolism coupling). Methods: Mitochondria matrix and cytoplasmic Ca2+ levels were evaluated in fibers isolated from flexor digitorium brevis muscle using plasmids for the expression of a mitochondrial Ca2+ sensor (CEPIA3mt) or a cytoplasmic Ca2+ sensor (RCaMP). The role of intracellular Ca2+ channels was evaluated using both specific pharmacological inhibitors (xestospongin B for IP3R and Dantrolene for RyR1) and a genetic approach (shIP3R1-RFP). O2 consumption was detected using Seahorse Extracellular Flux Analyzer. Results: In isolated muscle fibers cell membrane depolarization increased both cytoplasmic and mitochondrial Ca2+ levels. Mitochondrial Ca2+ uptake required functional inositol IP3R and RyR1 channels. Inhibition of either channel decreased basal O2 consumption rate but only RyR1 inhibition decreased ATP-linked O2 consumption. Cell membrane depolarization-induced Ca2+ signals in sub-sarcolemmal mitochondria were accompanied by a reduction in mitochondrial membrane potential; Ca2+ signals propagated toward intermyofibrillar mitochondria, which displayed increased membrane potential. These results are compatible with slow, Ca2+-dependent propagation of mitochondrial membrane potential from the surface toward the center of the fiber. Conclusion: Ca2+-dependent changes in mitochondrial membrane potential have different kinetics in the surface vs. the center of the fiber; these differences are likely to play a critical role in the control of mitochondrial metabolism, both at rest and after membrane depolarization as part of an "excitation-metabolism" coupling process in skeletal muscle fibers.

IP3 receptor blockade restores autophagy and mitochondrial function in skeletal muscle fibers of dystrophic mice.

Duchenne muscular dystrophy (DMD) is characterized by a severe and progressive destruction of muscle fibers associated with altered Ca2+ homeostasis. We have previously shown that the IP3 receptor (IP3R) plays a role in elevating basal cytoplasmic Ca2+ and that pharmacological blockade of IP3R restores muscle function. Moreover, we have shown that the IP3R pathway negatively regulates autophagy by controlling mitochondrial Ca2+ levels. Nevertheless, it remains unclear whether IP3R is involved in abnormal mitochondrial Ca2+ levels, mitochondrial dynamics, or autophagy and mitophagy observed in adult DMD skeletal muscle. Here, we show that the elevated basal autophagy and autophagic flux levels were normalized when IP3R was downregulated in mdx fibers. Pharmacological blockade of IP3R in mdx fibers restored both increased mitochondrial Ca2+ levels and mitochondrial membrane potential under resting conditions. Interestingly, mdx mitochondria changed from a fission to an elongated state after IP3R knockdown, and the elevated mitophagy levels in mdx fibers were normalized. To our knowledge, this is the first study associating IP3R1 activity with changes in autophagy, mitochondrial Ca2+ levels, mitochondrial membrane potential, mitochondrial dynamics, and mitophagy in adult mouse skeletal muscle. Moreover, these results suggest that increased IP3R activity in mdx fibers plays an important role in the pathophysiology of DMD. Overall, these results lead us to propose the use of specific IP3R blockers as a new pharmacological treatment for DMD, given their ability to restore both autophagy/mitophagy and mitochondrial function.