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.

Bilirubin deficiency renders mice susceptible to hepatic steatosis in the absence of insulin resistance.

BACKGROUND & AIMS: Plasma concentrations of bilirubin, a product of heme catabolism formed by biliverdin reductase A (BVRA), inversely associate with the risk of metabolic diseases including hepatic steatosis and diabetes mellitus in humans. Bilirubin has antioxidant and anti-inflammatory activities and may also regulate insulin signaling and peroxisome proliferator-activated receptor alpha (PPARα) activity. However, a causal link between bilirubin and metabolic diseases remains to be established. Here, we used the global Bvra gene knockout (Bvra-/-) mouse as a model of deficiency in bilirubin to assess its role in metabolic diseases. APPROACH & RESULTS: We fed mice fat-rich diets to induce hepatic steatosis and insulin resistance. Bile pigments were measured by LC-MS/MS, and hepatic lipids by LC-MS/MS (non-targeted lipidomics), HPLC-UV and Oil-Red-O staining. Oxidative stress was evaluated measuring F2-isoprostanes by GC-MS. Glucose metabolism and insulin sensitivity were verified by glucose and insulin tolerance tests, ex vivo and in vivo glucose uptake, and Western blotting for insulin signaling. Compared with wild type littermates, Bvra-/- mice contained negligible bilirubin in plasma and liver, and they had comparable glucose metabolism and insulin sensitivity. However, Bvra-/- mice exhibited an inflamed and fatty liver phenotype, accompanied by hepatic accumulation of oxidized triacylglycerols and F2-isoprostanes, in association with depletion of α-tocopherol. α-Tocopherol supplementation reversed the hepatic phenotype and observed biochemical changes in Bvra-/- mice. CONCLUSIONS: Our data suggests that BVRA deficiency renders mice susceptible to oxidative stress-induced hepatic steatosis in the absence of insulin resistance.

Genome-wide analysis in Drosophila reveals diet-by-gene interactions and uncovers diet-responsive genes.

Genetic and environmental factors play a major role in metabolic health. However, they do not act in isolation, as a change in an environmental factor such as diet may exert different effects based on an individual's genotype. Here, we sought to understand how such gene-diet interactions influenced nutrient storage and utilization, a major determinant of metabolic disease. We subjected 178 inbred strains from the Drosophila genetic reference panel (DGRP) to diets varying in sugar, fat, and protein. We assessed starvation resistance, a holistic phenotype of nutrient storage and utilization that can be robustly measured. Diet influenced the starvation resistance of most strains, but the effect varied markedly between strains such that some displayed better survival on a high carbohydrate diet (HCD) compared to a high-fat diet while others had opposing responses, illustrating a considerable gene × diet interaction. This demonstrates that genetics plays a major role in diet responses. Furthermore, heritability analysis revealed that the greatest genetic variability arose from diets either high in sugar or high in protein. To uncover the genetic variants that contribute to the heterogeneity in starvation resistance, we mapped 566 diet-responsive SNPs in 293 genes, 174 of which have human orthologs. Using whole-body knockdown, we identified two genes that were required for glucose tolerance, storage, and utilization. Strikingly, flies in which the expression of one of these genes, CG4607 a putative homolog of a mammalian glucose transporter, was reduced at the whole-body level, displayed lethality on a HCD. This study provides evidence that there is a strong interplay between diet and genetics in governing survival in response to starvation, a surrogate measure of nutrient storage efficiency and obesity. It is likely that a similar principle applies to higher organisms thus supporting the case for nutrigenomics as an important health strategy.

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.

Phosphoproteomics of Acute Cell Stressors Targeting Exercise Signaling Networks Reveal Drug Interactions Regulating Protein Secretion.

Exercise engages signaling networks to control the release of circulating factors beneficial to health. However, the nature of these networks remains undefined. Using high-throughput phosphoproteomics, we quantify 20,249 phosphorylation sites in skeletal muscle-like myotube cells and monitor their responses to a panel of cell stressors targeting aspects of exercise signaling in vivo. Integrating these in-depth phosphoproteomes with the phosphoproteome of acute aerobic exercise in human skeletal muscle suggests that co-administration of ß-adrenergic and calcium agonists would activate complementary signaling relevant to this exercise context. The phosphoproteome of cells treated with this combination reveals a surprising divergence in signaling from the individual treatments. Remarkably, only the combination treatment promotes multisite phosphorylation of SERBP1, a regulator of Serpine1 mRNA stability, a pro-fibrotic secreted protein. Secretome analysis reveals that the combined treatments decrease secretion of SERPINE1 and other deleterious factors. This study provides a framework for dissecting phosphorylation-based signaling relevant to acute exercise.

Drug Screening in Human PSC-Cardiac Organoids Identifies Pro-proliferative Compounds Acting via the Mevalonate Pathway.

We have previously developed a high-throughput bioengineered human cardiac organoid (hCO) platform, which provides functional contractile tissue with biological properties similar to native heart tissue, including mature, cell-cycle-arrested cardiomyocytes. In this study, we perform functional screening of 105 small molecules with pro-regenerative potential. Our findings reveal surprising discordance between our hCO system and traditional 2D assays. In addition, functional analyses uncovered detrimental effects of many hit compounds. Two pro-proliferative small molecules without detrimental impacts on cardiac function were identified. High-throughput proteomics in hCO revealed synergistic activation of the mevalonate pathway and a cell-cycle network by the pro-proliferative compounds. Cell-cycle reentry in hCO and in vivo required the mevalonate pathway as inhibition of the mevalonate pathway with a statin attenuated pro-proliferative effects. This study highlights the utility of human cardiac organoids for pro-regenerative drug development, including identification of underlying biological mechanisms and minimization of adverse side effects.

Mitochondrial CoQ deficiency is a common driver of mitochondrial oxidants and insulin resistance.

Insulin resistance in muscle, adipocytes and liver is a gateway to a number of metabolic diseases. Here, we show a selective deficiency in mitochondrial coenzyme Q (CoQ) in insulin-resistant adipose and muscle tissue. This defect was observed in a range of in vitro insulin resistance models and adipose tissue from insulin-resistant humans and was concomitant with lower expression of mevalonate/CoQ biosynthesis pathway proteins in most models. Pharmacologic or genetic manipulations that decreased mitochondrial CoQ triggered mitochondrial oxidants and insulin resistance while CoQ supplementation in either insulin-resistant cell models or mice restored normal insulin sensitivity. Specifically, lowering of mitochondrial CoQ caused insulin resistance in adipocytes as a result of increased superoxide/hydrogen peroxide production via complex II. These data suggest that mitochondrial CoQ is a proximal driver of mitochondrial oxidants and insulin resistance, and that mechanisms that restore mitochondrial CoQ may be effective therapeutic targets for treating insulin resistance.

Carcinogenic Chromium(VI) Compounds Formed by Intracellular Oxidation of Chromium(III) Dietary Supplements by Adipocytes.

Chromium(III) nutritional supplements are widely consumed for their purported antidiabetic activities. X-ray fluorescence microscopy (XFM) and X-ray absorption near-edge structure (XANES) studies have now shown that non-toxic doses of [Cr3 O(OCOEt)6 (OH2 )3 ](+) (A), a prospective antidiabetic drug that undergoes similar H2 O2 induced oxidation reactions in the blood as other Cr supplements, was also oxidized to carcinogenic Cr(VI) and Cr(V) in living cells. Single adipocytes treated with A had approximately 1 µm large Cr hotspots containing Cr(III) , Cr(V) , and Cr(VI) (primarily Cr(VI) thiolates) species. These results strongly support the hypothesis that the antidiabetic activity of Cr(III) and the carcinogenicity of Cr(VI) compounds arise from similar mechanisms involving highly reactive Cr(VI) and Cr(V) intermediates, and highlight concerns over the safety of Cr(III) nutritional supplements.

Next-generation Akt inhibitors provide greater specificity: effects on glucose metabolism in adipocytes.

Many human tumours exhibit activation of the PI3K (phosphoinositide 3-kinase)/Akt pathway, and inhibition of this pathway slows tumour growth. This led to the development of specific Akt inhibitors for in vivo use. However, activation of Akt is also necessary for processes including glucose metabolism. Therefore a potential complication of such anticancer drugs is insulin resistance and/or diabetes. In the process of characterizing the metabolic effects of early-phase Akt inhibitors, we discovered an off-target inhibitory effect on mammalian facilitative glucose transporters. In view of the crucial role of glucose transport for all mammalian cells, such an off-target effect would have major implications for further development of this family of compounds. In the present study, we have characterized a next-generation Akt inhibitor, MK-2206. MK-2206 is an orally active allosteric Akt inhibitor under development for treating solid tumours. We report that MK-2206 potently inhibits Thr308Akt and Ser473Akt phosphorylation in 3T3-L1 adipocytes (IC50 0.11 and 0.18 µM respectively) as well as downstream effects of insulin on GLUT4 (glucose transporter 4) translocation (IC50 0.47 µM) and glucose transport (IC50 0.14 µM). Notably, the potency of MK-2206 is approximately 1 log higher than previous inhibitors and its specificity is significantly improved with modest inhibitory effects on glucose transport in GLUT4-expressing adipocytes and GLUT1-rich human erythrocytes, independently of Akt. Nevertheless, MK-2206 clearly has potent effects on Akt2, the principal isoform involved in peripheral insulin action, in which case insulin resistance will probably be a major complication following in vivo administration. We conclude that MK-2206 provides an optimal tool for studying the effects of Akt in vitro.

CaMKII-mediated phosphorylation of the myosin motor Myo1c is required for insulin-stimulated GLUT4 translocation in adipocytes.

The unconventional myosin Myo1c has been implicated in insulin-regulated GLUT4 translocation to the plasma membrane in adipocytes. We show that Myo1c undergoes insulin-dependent phosphorylation at S701. Phosphorylation was accompanied by enhanced 14-3-3 binding and reduced calmodulin binding. Recombinant CaMKII phosphorylated Myo1c in vitro and siRNA knockdown of CaMKIIdelta abolished insulin-dependent Myo1c phosphorylation in vivo. CaMKII activity was increased upon insulin treatment and the CaMKII inhibitors CN21 and KN-62 or the Ca(2+) chelator BAPTA-AM blocked insulin-dependent Myo1c phosphorylation and insulin-stimulated glucose transport in adipocytes. Myo1c ATPase activity was increased after CaMKII phosphorylation in vitro and after insulin stimulation of CHO/IR/IRS-1 cells. Expression of wild-type Myo1c, but not S701A or ATPase dead mutant K111A, rescued the inhibition of GLUT4 translocation by siRNA-mediated Myo1c knockdown. These data suggest that insulin regulates Myo1c function via CaMKII-dependent phosphorylation, and these events play a role in insulin-regulated GLUT4 trafficking in adipocytes likely involving Myo1c motor activity.

Berberine, a natural plant product, activates AMP-activated protein kinase with beneficial metabolic effects in diabetic and insulin-resistant states.

Berberine has been shown to have antidiabetic properties, although its mode of action is not known. Here, we have investigated the metabolic effects of berberine in two animal models of insulin resistance and in insulin-responsive cell lines. Berberine reduced body weight and caused a significant improvement in glucose tolerance without altering food intake in db/db mice. Similarly, berberine reduced body weight and plasma triglycerides and improved insulin action in high-fat-fed Wistar rats. Berberine downregulated the expression of genes involved in lipogenesis and upregulated those involved in energy expenditure in adipose tissue and muscle. Berberine treatment resulted in increased AMP-activated protein kinase (AMPK) activity in 3T3-L1 adipocytes and L6 myotubes, increased GLUT4 translocation in L6 cells in a phosphatidylinositol 3' kinase-independent manner, and reduced lipid accumulation in 3T3-L1 adipocytes. These findings suggest that berberine displays beneficial effects in the treatment of diabetes and obesity at least in part via stimulation of AMPK activity.