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.

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.

Tirzepatide induces a thermogenic-like amino acid signature in brown adipose tissue.

OBJECTIVES: Tirzepatide, a dual GIP and GLP-1 receptor agonist, delivered superior glycemic control and weight loss compared to selective GLP-1 receptor (GLP-1R) agonism in patients with type 2 diabetes (T2D). These results have fueled mechanistic studies focused on understanding how tirzepatide achieves its therapeutic efficacy. Recently, we found that treatment with tirzepatide improves insulin sensitivity in humans with T2D and obese mice in concert with a reduction in circulating levels of branched-chain amino (BCAAs) and keto (BCKAs) acids, metabolites associated with development of systemic insulin resistance (IR) and T2D. Importantly, these systemic effects were found to be coupled to increased expression of BCAA catabolic genes in thermogenic brown adipose tissue (BAT) in mice. These findings led us to hypothesize that tirzepatide may lower circulating BCAAs/BCKAs by promoting their catabolism in BAT. METHODS: To address this question, we utilized a murine model of diet-induced obesity and employed stable-isotope tracer studies in combination with metabolomic analyses in BAT and other tissues. RESULTS: Treatment with tirzepatide stimulated catabolism of BCAAs/BCKAs in BAT, as demonstrated by increased labeling of BCKA-derived metabolites, and increases in levels of byproducts of BCAA breakdown, including glutamate, alanine, and 3-hydroxyisobutyric acid (3-HIB). Further, chronic administration of tirzepatide increased levels of multiple amino acids in BAT that have previously been shown to be elevated in response to cold exposure. Finally, chronic treatment with tirzepatide led to a substantial increase in several TCA cycle intermediates (α-ketoglutarate, fumarate, and malate) in BAT. CONCLUSIONS: These findings suggest that tirzepatide induces a thermogenic-like amino acid profile in BAT, an effect that may account for reduced systemic levels of BCAAs in obese IR mice.

Modified UCN2 peptide treatment improves skeletal muscle mass and function in mouse models of obesity-induced insulin resistance.

BACKGROUND: Type 2 diabetes and obesity are often seen concurrently with skeletal muscle wasting, leading to further derangements in function and metabolism. Muscle wasting remains an unmet need for metabolic disease, and new approaches are warranted. The neuropeptide urocortin 2 (UCN2) and its receptor corticotropin releasing factor receptor 2 (CRHR2) are highly expressed in skeletal muscle and play a role in regulating energy balance, glucose metabolism, and muscle mass. The aim of this study was to investigate the effects of modified UCN2 peptides as a pharmaceutical therapy to counteract the loss of skeletal muscle mass associated with obesity and casting immobilization. METHODS: High-fat-fed mice (C57Bl/6J; 26 weeks old) and ob/ob mice (11 weeks old) were injected daily with a PEGylated (Compound A) and non-PEGylated (Compound B) modified human UCN2 at 0.3 mg/kg subcutaneously for 14 days. A separate group of chow-fed C57Bl/6J mice (12 weeks old) was subjected to hindlimb cast immobilization and, after 1 week, received daily injections with Compound A. In vivo functional tests were performed to measure protein synthesis rates and skeletal muscle function. Ex vivo functional and molecular tests were performed to measure contractile force and signal transduction of catabolic and anabolic pathways in skeletal muscle. RESULTS: Skeletal muscles (extensor digitorum longus, soleus, and tibialis anterior) from high-fat-fed mice treated with Compound A were ~14% heavier than muscles from vehicle-treated mice. Chronic treatment with modified UCN2 peptides altered the expression of structural genes and transcription factors in skeletal muscle in high-fat diet-induced obesity including down-regulation of Trim63 and up-regulation of Nr4a2 and Igf1 (P < 0.05 vs. vehicle). Signal transduction via both catabolic and anabolic pathways was increased in tibialis anterior muscle, with increased phosphorylation of ribosomal protein S6 at Ser235/236 , FOXO1 at Ser256 , and ULK1 at Ser317 , suggesting that UCN2 treatment modulates protein synthesis and degradation pathways (P < 0.05 vs. vehicle). Acutely, a single injection of Compound A in drug-naïve mice had no effect on the rate of protein synthesis in skeletal muscle, as measured via the surface sensing of translation method, while the expression of Nr4a3 and Ppargc1a4 was increased (P < 0.05 vs. vehicle). Compound A treatment prevented the loss of force production from disuse due to casting. Compound B treatment increased time to fatigue during ex vivo contractions of fast-twitch extensor digitorum longus muscle. Compound A and B treatment increased lean mass and rates of skeletal muscle protein synthesis in ob/ob mice. CONCLUSIONS: Modified human UCN2 is a pharmacological candidate for the prevention of the loss of skeletal muscle mass associated with obesity and immobilization.

Muscle-Liver Trafficking of BCAA-Derived Nitrogen Underlies Obesity-Related Glycine Depletion.

Glycine levels are inversely associated with branched-chain amino acids (BCAAs) and cardiometabolic disease phenotypes, but biochemical mechanisms that explain these relationships remain uncharted. Metabolites and genes related to BCAA metabolism and nitrogen handling were strongly associated with glycine in correlation analyses. Stable isotope labeling in Zucker fatty rats (ZFRs) shows that glycine acts as a carbon donor for the pyruvate-alanine cycle in a BCAA-regulated manner. Inhibition of the BCAA transaminase (BCAT) enzymes depletes plasma pools of alanine and raises glycine levels. In high-fat-fed ZFRs, dietary glycine supplementation raises urinary acyl-glycine content and lowers circulating triglycerides but also results in accumulation of long-chain acyl-coenzyme As (acyl-CoAs), lower 5' adenosine monophosphate-activated protein kinase (AMPK) phosphorylation in muscle, and no improvement in glucose tolerance. Collectively, these studies frame a mechanism for explaining obesity-related glycine depletion and also provide insight into the impact of glycine supplementation on systemic glucose, lipid, and amino acid metabolism.

Nucleotides released from palmitate-activated murine macrophages attract neutrophils.

Obesity and elevation of circulating free fatty acids are associated with an accumulation and proinflammatory polarization of macrophages within metabolically active tissues, such as adipose tissue, muscle, liver, and pancreas. Beyond macrophages, neutrophils also accumulate in adipose and muscle tissues during high-fat diets and contribute to a state of local inflammation and insulin resistance. However, the mechanisms by which neutrophils are recruited to these tissues are largely unknown. Here we used a cell culture system as proof of concept to show that, upon exposure to a saturated fatty acid, palmitate, macrophages release nucleotides that attract neutrophils. Moreover, we found that palmitate up-regulates pannexin-1 channels in macrophages that mediate the attraction of neutrophils, shown previously to allow transfer of nucleotides across membranes. These findings suggest that proinflammatory macrophages release nucleotides through pannexin-1, a process that may facilitate neutrophil recruitment into metabolic tissues during obesity.

Imidazopyridine and Pyrazolopiperidine Derivatives as Novel Inhibitors of Serine Palmitoyl Transferase.

To develop novel treatments for type 2 diabetes and dyslipidemia, we pursued inhibitors of serine palmitoyl transferase (SPT). To this end compounds 1 and 2 were developed as potent SPT inhibitors in vitro. 1 and 2 reduce plasma ceramides in rodents, have a slight trend toward enhanced insulin sensitization in DIO mice, and reduce triglycerides and raise HDL in cholesterol/cholic acid fed rats. Unfortunately these molecules cause a gastric enteropathy after chronic dosing in rats.

Biomarkers of Ectopic Fat Deposition: The Next Frontier in Serum Lipidomics.

CONTEXT: Strong evidence suggests that ectopic fat rather than fat mass per se drives risk for type 2 diabetes. Nonetheless, biomarkers of ectopic fat have gone unexplored. OBJECTIVE: To determine the utility of serum lipidomics to predict ectopic lipid deposition. DESIGN: Cross-sectional. SETTING: The Clinical Translational Research Center at the University of Colorado Anschutz Medical Campus. PARTICIPANTS: Endurance-trained athletes (n = 15, 41 ± 0.9 y old; body mass index 24 ± 0.6 kg/m(2)) and obese people with or without type 2 diabetes (n = 29, 42 ± 1.4 y old; body mass index 32 ± 2.5 kg/m(2)). INTERVENTION: Blood sampling and skeletal muscle biopsy. MAIN OUTCOME MEASURES: Multivariable models determined the ability of serum lipids to predict intramuscular (im) lipid accumulation of triacylglycerol (TAG), diacylglycerol (DAG), and ceramide (liquid chromatography tandem mass spectroscopy). RESULTS: Among people with obesity, serum ganglioside C22:0 and lactosylceramide C14:0 predicted muscle TAG (overall model R(2) = 0.48), whereas serum DAG C36:1 and free fatty acid (FFA) C18:4 were strong predictors of muscle DAG (overall model R(2) = 0.77), as were serum TAG C58:5, FFA C14:2 and C14:3, phosphotidylcholine C38:1, and cholesterol ester C24:1 to predict muscle ceramide (overall model R(2) = 0.85). Among endurance-trained athletes, serum FFA C14:1 and sphingosine were significant predictors of muscle TAG (overall model R(2) = 0.81), whereas no models could predict intramuscular DAG or ceramide in this group. CONCLUSIONS: Different serum lipids predict intramuscular TAG accumulation in obese people vs athletes. The ability of serum lipidomics to predict intramuscular DAG and ceramide in insulin-resistant humans may prove a new biomarker to determine risk for diabetes.

Nucleotides released from palmitate-challenged muscle cells through pannexin-3 attract monocytes.

Obesity-associated low-grade inflammation in metabolically relevant tissues contributes to insulin resistance. We recently reported monocyte/macrophage infiltration in mouse and human skeletal muscles. However, the molecular triggers of this infiltration are unknown, and the role of muscle cells in this context is poorly understood. Animal studies are not amenable to the specific investigation of this vectorial cellular communication. Using cell cultures, we investigated the crosstalk between myotubes and monocytes exposed to physiological levels of saturated and unsaturated fatty acids. Media from L6 myotubes treated with palmitate-but not palmitoleate-induced THP1 monocyte migration across transwells. Palmitate activated the Toll-like receptor 4 (TLR4)/nuclear factor-κB (NF-κB) pathway in myotubes and elevated cytokine expression, but the monocyte chemoattracting agent was not a polypeptide. Instead, nucleotide degradation eliminated the chemoattracting properties of the myotube-conditioned media. Moreover, palmitate-induced expression and activity of pannexin-3 channels in myotubes were mediated by TLR4-NF-κB, and TLR4-NF-κB inhibition or pannexin-3 knockdown prevented monocyte chemoattraction. In mice, the expression of pannexin channels increased in adipose tissue and skeletal muscle in response to high-fat feeding. These findings identify pannexins as new targets of saturated fatty acid-induced inflammation in myotubes, and point to nucleotides as possible mediators of immune cell chemoattraction toward muscle in the context of obesity.

Inhibition of ceramide synthesis ameliorates glucocorticoid-, saturated-fat-, and obesity-induced insulin resistance.

Insulin resistance occurs in 20%-25% of the human population, and the condition is a chief component of type 2 diabetes mellitus and a risk factor for cardiovascular disease and certain forms of cancer. Herein, we demonstrate that the sphingolipid ceramide is a common molecular intermediate linking several different pathological metabolic stresses (i.e., glucocorticoids and saturated fats, but not unsaturated fats) to the induction of insulin resistance. Moreover, inhibition of ceramide synthesis markedly improves glucose tolerance and prevents the onset of frank diabetes in obese rodents. Collectively, these data have two important implications. First, they indicate that different fatty acids induce insulin resistance by distinct mechanisms discerned by their reliance on sphingolipid synthesis. Second, they identify enzymes required for ceramide synthesis as therapeutic targets for combating insulin resistance caused by nutrient excess or glucocorticoid therapy.

Disruption of cortical actin in skeletal muscle demonstrates an essential role of the cytoskeleton in glucose transporter 4 translocation in insulin-sensitive tissues.

Cell culture work suggests that signaling to polymerize cortical filamentous actin (F-actin) represents a required pathway for the optimal redistribution of the insulin-responsive glucose transporter, GLUT4, to the plasma membrane. Recent in vitro study further suggests that the actin-regulatory neural Wiskott-Aldrich syndrome protein (N-WASP) mediates the effect of insulin on the actin filament network. Here we tested whether similar cytoskeletal mechanics are essential for insulin-regulated glucose transport in isolated rat epitrochlearis skeletal muscle. Microscopic analysis revealed that cortical F-actin is markedly diminished in muscle exposed to latrunculin B. Depolymerization of cortical F-actin with latrunculin B caused a time- and concentration-dependent decline in 2-deoxyglucose transport. The loss of cortical F-actin and glucose transport was paralleled by a decline in insulin-stimulated GLUT4 translocation, as assessed by photolabeling of cell surface GLUT4 with Bio-LC-ATB-BMPA. Although latrunculin B impaired insulin-stimulated GLUT4 translocation and glucose transport, activation of phosphatidylinositol 3-kinase and Akt by insulin was not rendered ineffective. In contrast, the ability of insulin to elicit the cortical F-actin localization of N-WASP was abrogated. These data provide the first evidence that actin cytoskeletal mechanics are an essential feature of the glucose transport process in intact skeletal muscle. Furthermore, these findings support a distal actin-based role for N-WASP in insulin action in vivo.

Defective signaling through Akt-2 and -3 but not Akt-1 in insulin-resistant human skeletal muscle: potential role in insulin resistance.

Recent evidence has shown that activation of phosphatidyinositol-3-kinase (PI3K) and Akt, necessary for insulin stimulation of glucose transport, is impaired in insulin resistance. It is unknown, however, which Akt isoform shows impaired activation in insulin resistance. Additionally, related growth factors (epidermal or platelet-derived vascular) also stimulate PI3K, but it is unknown whether production of 3,4,5 phosphatidyinositol is sufficient to stimulate glucose transport in insulin-resistant muscle. Moreover, these studies were performed in rodents, and little data exists from humans. Hence, we investigated the stimulation of PI3K and Akt-1, -2, and -3 by insulin and epidermal growth factors (EGFs) in skeletal muscles from lean and obese insulin-resistant humans. Insulin activated all Akt isoforms in lean muscles, whereas only Akt-1 was activated in obese muscles. Insulin receptor substrate (IRS)-1 was associated with PI3K activity, which is necessary for Akt activation by insulin, and was reduced in obese muscles, and this was accompanied by decreased IRS-1 expression. In contrast, insulin- or EGF-stimulated phosphotyrosine-associated PI3K activity was not different between lean and obese muscles. These results show that a defect in the ability of insulin to activate Akt-2 and -3 may explain the impaired insulin-stimulated glucose transport in insulin resistance. Additionally, these data also show that different upstream or downstream signals may regulate the activity of the various Akt isoforms.