Inhibition of mitochondrial complex 1 by the S6K1 inhibitor PF-4708671 partly contributes to its glucose metabolic effects in muscle and liver cells.

mTOR complex 1 (mTORC1) and p70 S6 kinase (S6K1) are both involved in the development of obesity-linked insulin resistance. Recently, we showed that the S6K1 inhibitor PF-4708671 (PF) increases insulin sensitivity. However, we also reported that PF can increase glucose metabolism even in the absence of insulin in muscle and hepatic cells. Here we further explored the potential mechanisms by which PF increases glucose metabolism in muscle and liver cells independent of insulin. Time course experiments revealed that PF induces AMP-activated protein kinase (AMPK) activation before inhibiting S6K1. However, PF-induced glucose uptake was not prevented in primary muscle cells from AMPK α1/2 double KO (dKO) mice. Moreover, PF-mediated suppression of hepatic glucose production was maintained in hepatocytes derived from AMPK α1/2-dKO mice. Remarkably, PF could still reduce glucose production and activate AMPK in hepatocytes from S6K1/2 dKO mice. Mechanistically, bioenergetics experiments revealed that PF reduces mitochondrial complex I activity in both muscle and hepatic cells. The stimulatory effect of PF on glucose uptake was partially reduced by expression of the Saccharomyces cerevisiae NADH:ubiquinone oxidoreductase in L6 cells. These results indicate that PF-mediated S6K1 inhibition is not required for its effect on insulin-independent glucose metabolism and AMPK activation. We conclude that, although PF rapidly activates AMPK, its ability to acutely increase glucose uptake and suppress glucose production does not require AMPK activation. Unexpectedly, PF rapidly inhibits mitochondrial complex I activity, a mechanism that partially underlies PF's effect on glucose metabolism.

AMPK in skeletal muscle function and metabolism.

Skeletal muscle possesses a remarkable ability to adapt to various physiologic conditions. AMPK is a sensor of intracellular energy status that maintains energy stores by fine-tuning anabolic and catabolic pathways. AMPK's role as an energy sensor is particularly critical in tissues displaying highly changeable energy turnover. Due to the drastic changes in energy demand that occur between the resting and exercising state, skeletal muscle is one such tissue. Here, we review the complex regulation of AMPK in skeletal muscle and its consequences on metabolism ( e.g., substrate uptake, oxidation, and storage as well as mitochondrial function of skeletal muscle fibers). We focus on the role of AMPK in skeletal muscle during exercise and in exercise recovery. We also address adaptations to exercise training, including skeletal muscle plasticity, highlighting novel concepts and future perspectives that need to be investigated. Furthermore, we discuss the possible role of AMPK as a therapeutic target as well as different AMPK activators and their potential for future drug development.-Kjøbsted, R., Hingst, J. R., Fentz, J., Foretz, M., Sanz, M.-N., Pehmøller, C., Shum, M., Marette, A., Mounier, R., Treebak, J. T., Wojtaszewski, J. F. P., Viollet, B., Lantier, L. AMPK in skeletal muscle function and metabolism.

Pharmacological inhibition of S6K1 increases glucose metabolism and Akt signalling in vitro and in diet-induced obese mice.

AIMS/HYPOTHESIS: The mammalian target of rapamycin complex 1 (mTORC1)/p70 ribosomal S6 kinase (S6K)1 pathway is overactivated in obesity, leading to inhibition of phosphoinositide 3-kinase (PI3K)/Akt signalling and insulin resistance. However, chronic mTORC1 inhibition by rapamycin impairs glucose homeostasis because of robust induction of liver gluconeogenesis. Here, we compared the effect of rapamycin with that of the selective S6K1 inhibitor, PF-4708671, on glucose metabolism in vitro and in vivo. METHODS: We used L6 myocytes and FAO hepatocytes to explore the effect of PF-4708671 on the regulation of glucose uptake, glucose production and insulin signalling. We also treated high-fat (HF)-fed obese mice for 7 days with PF-4708671 in comparison with rapamycin to assess glucose tolerance, insulin resistance and insulin signalling in vivo. RESULTS: Chronic rapamycin treatment induced insulin resistance and impaired glucose metabolism in hepatic and muscle cells. Conversely, chronic S6K1 inhibition with PF-4708671 reduced glucose production in hepatocytes and enhanced glucose uptake in myocytes. Whereas rapamycin treatment inhibited Akt phosphorylation, PF-4708671 increased Akt phosphorylation in both cell lines. These opposite effects of the mTORC1 and S6K1 inhibitors were also observed in vivo. Indeed, while rapamycin treatment induced glucose intolerance and failed to improve Akt phosphorylation in liver and muscle of HF-fed mice, PF-4708671 treatment improved glucose tolerance and increased Akt phosphorylation in metabolic tissues of these obese mice. CONCLUSIONS/INTERPRETATION: Chronic S6K1 inhibition by PF-4708671 improves glucose homeostasis in obese mice through enhanced Akt activation in liver and muscle. Our results suggest that specific S6K1 blockade is a valid pharmacological approach to improve glucose disposal in obese diabetic individuals.

Hepatocyte-specific Ptpn6 deletion promotes hepatic lipid accretion, but reduces NAFLD in diet-induced obesity: potential role of PPARγ.

UNLABELLED: Hepatocyte-specific Shp1 knockout mice (Ptpn6(H-KO)) are protected from hepatic insulin resistance evoked by high-fat diet (HFD) feeding for 8 weeks. Unexpectedly, we report herein that Ptpn6(H-KO) mice fed an HFD for up to 16 weeks are still protected from insulin resistance, but are more prone to hepatic steatosis, as compared with their HFD-fed Ptpn6(f/f) counterparts. The livers from HFD-fed Ptpn6(H-KO) mice displayed 1) augmented lipogenesis, marked by increased expression of several hepatic genes involved in fatty acid biosynthesis, 2) elevated postprandial fatty acid uptake, and 3) significantly reduced lipid export with enhanced degradation of apolipoprotein B (ApoB). Despite more extensive hepatic steatosis, the inflammatory profile of the HFD-fed Ptpn6(H-KO) liver was similar (8 weeks) or even improved (16 weeks) as compared to their HFD-fed Ptpn6(f/f) littermates, along with reduced hepatocellular damage as revealed by serum levels of hepatic enzymes. Interestingly, comparative microarray analysis revealed a significant up-regulation of peroxisome proliferator-activated receptor gamma (PPARγ) gene expression, confirmed by quantitative polymerase chain reaction. Elevated PPARγ nuclear activity also was observed and found to be directly regulated by Shp1 in a cell-autonomous manner. CONCLUSION: These findings highlight a novel role for hepatocyte Shp1 in the regulation of PPARγ and hepatic lipid metabolism. Shp1 deficiency prevents the development of severe hepatic inflammation and hepatocellular damage in steatotic livers, presenting hepatocyte Shp1 as a potential novel mediator of nonalcoholic fatty liver diseases in obesity.

Insulin activates RSK (p90 ribosomal S6 kinase) to trigger a new negative feedback loop that regulates insulin signaling for glucose metabolism.

We previously demonstrated that the mTORC1/S6K1 pathway is activated by insulin and nutrient overload (e.g. amino acids (AA)), which leads to the inhibition of the PI3K/Akt pathway via the inhibitory serine phosphorylation of IRS-1, notably on serine 1101 (Ser-1101). However, even in the absence of AA, insulin can still promote IRS-1 Ser-1101 phosphorylation by other kinases that remain to be fully characterized. Here, we describe a new negative regulator of IRS-1, the p90 ribosomal S6 kinase (RSK). Computational analyses revealed that Ser-1101 within IRS-1 falls into the consensus motif of RSK. Moreover, recombinant RSK phosphorylated IRS-1 C-terminal fragment on Ser-1101, which was prevented by mutations of this site or when a kinase-inactive mutant of RSK was used. Using antibodies directed toward the phosphorylation sites located in the activation segment of RSK (Ser-221 or Ser-380), we found that insulin activates RSK in L6 myocytes in the absence of AA overload. Inhibition of RSK using either the pharmacological inhibitor BI-D1870 or after adenoviral expression of a dominant negative RSK1 mutant (RSK1-DN) showed that RSK selectively phosphorylates IRS-1 on Ser-1101. Accordingly, expression of the RSK1-DN mutant in L6 myocytes and FAO hepatic cells improved insulin action on glucose uptake and glucose production, respectively. Furthermore, RSK1 inhibition prevented insulin resistance in L6 myocytes chronically exposed to high glucose and high insulin. These results show that RSK is a novel regulator of insulin signaling and glucose metabolism and a potential mediator of insulin resistance, notably through the negative phosphorylation of IRS-1 on Ser-1101.

Management of excessive movable tissue: a modified impression technique.

Excessive movable tissue is a challenge in complete denture prosthetics. A modified impression technique is presented with polyvinyl siloxane impression material and a custom tray with relief areas and perforations in the area of the excessive movable tissue.

The mitochondrial biliverdin exporter ABCB10 in hepatocytes mitigates neutrophilic inflammation in alcoholic hepatitis.

Acute liver failure caused by alcoholic hepatitis (AH) is only effectively treated with liver transplantation. Livers of patients with AH show a unique molecular signature characterized by defective hepatocellular redox metabolism, concurrent to hepatic infiltration of neutrophils that express myeloperoxidase (MPO) and form neutrophil extracellular traps (NETs). Exacerbated NET formation and MPO activity contribute to liver damage in mice with AH and predicts poor prognosis in AH patients. The identification of pathways that maladaptively exacerbate neutrophilic activity in liver could inform of novel therapeutic approaches to treat AH. Whether the redox defects of hepatocytes in AH directly exacerbate neutrophilic inflammation and NET formation is unclear. Here we identify that the protein content of the mitochondrial biliverdin exporter ABCB10, which increases hepatocyte-autonomous synthesis of the ROS-scavenger bilirubin, is decreased in livers from humans and mice with AH. Increasing ABCB10 expression selectively in hepatocytes of mice with AH is sufficient to decrease MPO gene expression and histone H3 citrullination, a specific marker of NET formation. These anti-inflammatory effects can be explained by ABCB10 function reducing ROS-mediated actions in liver. Accordingly, ABCB10 gain-of-function selectively increased the mitochondrial GSH/GSSG ratio and decreased hepatic 4-HNE protein adducts, without elevating mitochondrial fat expenditure capacity, nor mitigating steatosis and hepatocyte death. Thus, our study supports that ABCB10 function regulating ROS-mediated actions within surviving hepatocytes mitigates the maladaptive activation of infiltrated neutrophils in AH. Consequently, ABCB10 gain-of-function in human hepatocytes could potentially decrease acute liver failure by decreasing the inflammatory flare caused by excessive neutrophil activity.

HSDL2 links nutritional cues to bile acid and cholesterol homeostasis.

In response to energy and nutrient shortage, the liver triggers several catabolic processes to promote survival. Despite recent progress, the precise molecular mechanisms regulating the hepatic adaptation to fasting remain incompletely characterized. Here, we report the identification of hydroxysteroid dehydrogenase-like 2 (HSDL2) as a mitochondrial protein highly induced by fasting. We show that the activation of PGC1α-PPARα and the inhibition of the PI3K-mTORC1 axis stimulate HSDL2 expression in hepatocytes. We found that HSDL2 depletion decreases cholesterol conversion to bile acids (BAs) and impairs FXR activity. HSDL2 knockdown also reduces mitochondrial respiration, fatty acid oxidation, and TCA cycle activity. Bioinformatics analyses revealed that hepatic Hsdl2 expression positively associates with the postprandial excursion of various BA species in mice. We show that liver-specific HSDL2 depletion affects BA metabolism and decreases circulating cholesterol levels upon refeeding. Overall, our report identifies HSDL2 as a fasting-induced mitochondrial protein that links nutritional signals to BAs and cholesterol homeostasis.

Angiotensin II type 2 receptor promotes adipocyte differentiation and restores adipocyte size in high-fat/high-fructose diet-induced insulin resistance in rats.

This study was aimed at establishing whether specific activation of angiotensin II (ANG II) type 2 receptor (AT2R) modulates adipocyte differentiation and function. In primary cultures of subcutaneous (SC) and retroperitoneal (RET) preadipocytes, both AT2R and AT1R were expressed at the mRNA and protein level. Cells were stimulated with ANG II or the AT2R agonist C21/M24, alone or in the presence of the AT1R antagonist losartan or the AT2R antagonist PD123,319. During differentiation, C21/M24 increased PPARγ expression in both RET and SC preadipocytes while the number of small lipid droplets and lipid accumulation solely increased in SC preadipocytes. In mature adipocytes, C21/M24 decreased the mean size of large lipid droplets. Upon abolishment of AT2R expression using AT2R-targeted shRNAs, expressions of AT2R, aP2, and PPARγ remained very low, and cells were unable to differentiate. In Wistar rats fed a 6-wk high-fat/high-fructose (HFHF) diet, a significant shift toward larger adipocytes was observed in RET and SC adipose tissue depots. C21/M24 treatments for 6 wk restored normal adipocyte size distribution in both these tissue depots. Moreover, C21/M24 and losartan decreased hyperinsulinemia and improved insulin sensitivity impaired by HFHF diet. A strong correlation between adipocyte size area and glucose infusion rate during euglycemic-hyperinsulinemic clamp was observed. These results indicate that AT2R is involved in early adipocyte differentiation, while in mature adipocytes and in a model of insulin resistance AT2R activation restores normal adipocyte morphology and improves insulin sensitivity.

Quantifying mitochondrial redox and bilirubin content in intact primary hepatocytes of obese mice using fluorescent reporters.

Assessing the physiological role of H2O2 requires sensitive techniques to quantify H2O2 and antioxidants in live cells. Here, we present a protocol to assess the mitochondrial redox state and unconjugated bilirubin levels in intact live primary hepatocytes from obese mice. We described steps to quantify H2O2, GSSG/GSH, and bilirubin content in the mitochondrial matrix and the cytosol using the fluorescent reporters roGFP2-ORP1, GRX1-roGFP2, and UnaG, respectively. We detail hepatocyte isolation, plating, and transduction and live-cell imaging using a high-content imaging reader. For complete details on the use and execution of this protocol, please refer to Shum et al.1.

Restoration of lysosomal acidification rescues autophagy and metabolic dysfunction in non-alcoholic fatty liver disease.

Non-alcoholic fatty liver disease (NAFLD) is the most common liver disease in the world. High levels of free fatty acids in the liver impair hepatic lysosomal acidification and reduce autophagic flux. We investigate whether restoration of lysosomal function in NAFLD recovers autophagic flux, mitochondrial function, and insulin sensitivity. Here, we report the synthesis of novel biodegradable acid-activated acidifying nanoparticles (acNPs) as a lysosome targeting treatment to restore lysosomal acidity and autophagy. The acNPs, composed of fluorinated polyesters, remain inactive at plasma pH, and only become activated in lysosomes after endocytosis. Specifically, they degrade at pH of ~6 characteristic of dysfunctional lysosomes, to further acidify and enhance the function of lysosomes. In established in vivo high fat diet mouse models of NAFLD, re-acidification of lysosomes via acNP treatment restores autophagy and mitochondria function to lean, healthy levels. This restoration, concurrent with reversal of fasting hyperglycemia and hepatic steatosis, indicates the potential use of acNPs as a first-in-kind therapeutic for NAFLD.

Isocitrate dehydrogenase 1 sustains a hybrid cytoplasmic-mitochondrial tricarboxylic acid cycle that can be targeted for therapeutic purposes in prostate cancer.

The androgen receptor (AR) is an established orchestrator of cell metabolism in prostate cancer (PCa), notably by inducing an oxidative mitochondrial program. Intriguingly, AR regulates cytoplasmic isocitrate dehydrogenase 1 (IDH1), but not its mitochondrial counterparts IDH2 and IDH3. Here, we aimed to understand the functional role of IDH1 in PCa. Mouse models, in vitro human PCa cell lines, and human patient-derived organoids (PDOs) were used to study the expression and activity of IDH enzymes in the normal prostate and PCa. Genetic and pharmacological inhibition of IDH1 was then combined with extracellular flux analyses and gas chromatography-mass spectrometry for metabolomic analyses and cancer cell proliferation in vitro and in vivo. In PCa cells, more than 90% of the total IDH activity is mediated through IDH1 rather than its mitochondrial counterparts. This profile seems to originate from the specialized prostate metabolic program, as observed using mouse prostate and PDOs. Pharmacological and genetic inhibition of IDH1 impaired mitochondrial respiration, suggesting that this cytoplasmic enzyme contributes to the mitochondrial tricarboxylic acid cycle (TCA) in PCa. Mass spectrometry-based metabolomics confirmed this hypothesis, showing that inhibition of IDH1 impairs carbon flux into the TCA cycle. Consequently, inhibition of IDH1 decreased PCa cell proliferation in vitro and in vivo. These results demonstrate that PCa cells have a hybrid cytoplasmic-mitochondrial TCA cycle that depends on IDH1. This metabolic enzyme represents a metabolic vulnerability of PCa cells and a potential new therapeutic target.

Comparative Expedited Regulatory Programs of U.S Food & Drug Administration and Project Orbis Partners.

Project Orbis was initiated in May 2019 by the Oncology Center of Excellence to facilitate faster patient access to innovative cancer therapies by providing a framework for concurrent submissions and review of oncology products among international partners. Since its inception, Australia's Therapeutic Goods Administration (TGA), Canada's Health Canada (HC), Singapore's Health Sciences Authority (HSA), Switzerland's Swissmedic (SMC), Brazil's National Health Surveillance Agency (ANVISA), United Kingdom's Medicines and Healthcare Products Regulatory Agency (MHRA), and most recently Israel's Ministry of Health (IMoH) Medical Technologies, Health Information, Innovation and Research (MTIIR) Directorate, have joined Project Orbis. While each country has its own expedited review pathways to bring promising therapies to patients, there are some similarities and differences in pathways and timelines. FDA's fast-track designation and MHRA's marketing authorization under exceptional circumstances (MAEC) allow non-clinical and limited clinical evidence to support approval under these programs. HC's Extraordinary Use New Drug (EUND) pathway allows granting exceptional use authorization with limited clinical evidence. ANVISA, HSA, MTIIR, and TGA do not have standard pathways that allow non-clinical evidence and limited clinical evidence. While there is no definite regulatory pathway for HSA, the current framework for approval does allow flexibility in the type of data (non-clinical or clinical) required to demonstrate the benefit-risk profile of a product. HSA may register a product if the agency is satisfied that the overall benefit outweighs the risk. All Project Orbis Partner (POP) countries have similar programs to the FDA accelerated approval program except ANVISA. Although HSA and MTIIR do not have defined pathways for accelerated approval programs, there are opportunities to request accelerated approval per these agencies. All POP countries have pathways like the FDA priority review except MHRA. Priority review timelines for new drugs range from 120 to 264 calendar days (cd). Standard review timelines for new drugs range from 180 to 365 cd.

γ-Actin regulates cell migration and modulates the ROCK signaling pathway.

Cell migration plays a crucial role in numerous cellular functions, and alterations in the regulation of cell migration are required for invasive transformation of a tumor cell. While the mechanistic process of actin-based migration has been well documented, little is known as to the specific function of the nonmuscle actin isoforms in mammalian cells. Here, we present a comprehensive examination of γ-actin's role in cell migration using an RNAi approach. The partial suppression of γ-actin expression in SH-EP neuroblastoma cells resulted in a significant decrease in wound healing and transwell migration. Similarly, the knockdown of γ-actin significantly reduced speed of motility and severely affected the cell's ability to explore, which was, in part, due to a loss of cell polarity. Moreover, there was a significant increase in the size and number of paxillin-containing focal adhesions, coupled with a significant decrease in phosphorylated paxillin in γ-actin-knockdown cells. In addition, there was a significant increase in the phosphorylation of cofilin and myosin regulatory light chain, suggesting an overactivated Rho-associated kinase (ROCK) signaling pathway in γ-actin-knockdown cells. The alterations in the phosphorylation of paxillin and myosin regulatory light chain were unique to γ-actin and not ß-actin knockdown. Inhibition of the ROCK pathway with the inhibitor Y-27632 restored the ability of γ-actin-knockdown cells to migrate. This study demonstrates γ-actin as a potential upstream regulator of ROCK mediated cell migration.

Deletion of ABCB10 in beta-cells protects from high-fat diet induced insulin resistance.

OBJECTIVE: The contribution of beta-cell dysfunction to type 2 diabetes (T2D) is not restricted to insulinopenia in the late stages of the disease. Elevated fasting insulinemia in normoglycemic humans is a major factor predicting the onset of insulin resistance and T2D, demonstrating an early alteration of beta-cell function in T2D. Moreover, an early and chronic increase in fasting insulinemia contributes to insulin resistance in high-fat diet (HFD)-fed mice. However, whether there are genetic factors that promote beta-cell-initiated insulin resistance remains undefined. Human variants of the mitochondrial transporter ABCB10, which regulates redox by increasing bilirubin synthesis, have been associated with an elevated risk of T2D. The effects of T2D ABCB10 variants on ABCB10 expression and the actions of ABCB10 in beta-cells are unknown. METHODS: The expression of beta-cell ABCB10 was analyzed in published transcriptome datasets from human beta-cells carrying the T2D-risk ABCB10 variant. Insulin sensitivity, beta-cell proliferation, and secretory function were measured in beta-cell-specific ABCB10 KO mice (Ins1Cre-Abcb10flox/flox). The short-term role of beta-cell ABCB10 activity on glucose-stimulated insulin secretion (GSIS) was determined in isolated islets. RESULTS: Carrying the T2Drisk allele G of ABCB10 rs348330 variant was associated with increased ABCB10 expression in human beta-cells. Constitutive deletion of Abcb10 in beta-cells protected mice from hyperinsulinemia and insulin resistance by limiting HFD-induced beta-cell expansion. An early limitation in GSIS and H2O2-mediated signaling caused by elevated ABCB10 activity can initiate an over-compensatory expansion of beta-cell mass in response to HFD. Accordingly, increasing ABCB10 expression was sufficient to limit GSIS capacity. In health, ABCB10 protein was decreased during islet maturation, with maturation restricting beta-cell proliferation and elevating GSIS. Finally, ex-vivo and short-term deletion of ABCB10 in islets isolated from HFD-fed mice increased H2O2 and GSIS, which was reversed by bilirubin treatments. CONCLUSIONS: Beta-cell ABCB10 is required for HFD to induce insulin resistance in mice by amplifying beta-cell mass expansion to maladaptive levels that cause fasting hyperinsulinemia.

Determining Basal Energy Expenditure and the Capacity of Thermogenic Adipocytes to Expend Energy in Obese Mice.

Energy expenditure measurements are necessary to understand how changes in metabolism can lead to obesity. Basal energy expenditure can be determined in mice by measuring whole-body oxygen consumption, CO2 production, and physical activity using metabolic cages. Thermogenic brown/beige adipocytes (BA) contribute significantly to rodent energy expenditure, particularly at low ambient temperatures. Here, measurements of basal energy expenditure and total BA capacity to expend energy in obese mice are described in two detailed protocols: the first explaining how to set up the assay to measure basal energy expenditure using analysis of covariance (ANCOVA), a necessary analysis given that energy expenditure co-varies with body mass. The second protocol describes how to measure BA energy expenditure capacity in vivo in mice. This procedure involves anesthesia, needed to limit expenditure caused by physical activity, followed by the injection of beta3-adrenergic agonist, CL-316,243, which activates energy expenditure in BA. These two protocols and their limitations are described in sufficient detail to allow a successful first experiment.

Mitochondrial oxidative function in NAFLD: Friend or foe?

BACKGROUND: Mitochondrial oxidative function plays a key role in the development of non-alcoholic fatty liver disease (NAFLD) and insulin resistance (IR). Recent studies reported that fatty liver might not be a result of decreased mitochondrial fat oxidation caused by mitochondrial damage. Rather, NAFLD and IR induce an elevation in mitochondrial function that covers the increased demand for carbon intermediates and ATP caused by elevated lipogenesis and gluconeogenesis. Furthermore, mitochondria play a role in regulating hepatic insulin sensitivity and lipogenesis by modulating redox-sensitive signaling pathways. SCOPE OF REVIEW: We review the contradictory studies indicating that NAFLD and hyperglycemia can either increase or decrease mitochondrial oxidative capacity in the liver. We summarize mechanisms regulating mitochondrial heterogeneity inside the same cell and discuss how these mechanisms may determine the role of mitochondria in NAFLD. We further discuss the role of endogenous antioxidants in controlling mitochondrial H2O2 release and redox-mediated signaling. We describe the emerging concept that the subcellular location of cellular antioxidants is a key determinant of their effects on NAFLD. MAJOR CONCLUSIONS: The balance of fat oxidation versus accumulation depends on mitochondrial fuel preference rather than ATP-synthesizing respiration. As such, therapies targeting fuel preference might be more suitable for treating NAFLD. Similarly, suppressing maladaptive antioxidants, rather than interfering with physiological mitochondrial H2O2-mediated signaling, may allow the maintenance of intact hepatic insulin signaling in NAFLD. Exploration of the subcellular compartmentalization of different antioxidant systems and the unique functions of specific mitochondrial subpopulations may offer new intervention points to treat NAFLD.

Isolation and functional analysis of peridroplet mitochondria from murine brown adipose tissue.

Mitochondria play a central role in lipid metabolism and can bind to lipid droplets. However, the role and functional specialization of the population of peridroplet mitochondria (PDMs) remain unclear, as methods to isolate functional PDMs were not developed until recently. Here, we describe an approach to isolate intact PDMs from murine brown adipose tissue based on their adherence to lipid droplets. PDMs isolated using our approach can be used to study their specialized function by respirometry. For complete information on the use and execution of this protocol, please refer to Benador et al. (2018).

Sex-specific genetic regulation of adipose mitochondria and metabolic syndrome by Ndufv2.

We have previously suggested a central role for mitochondria in the observed sex differences in metabolic traits. However, the mechanisms by which sex differences affect adipose mitochondrial function and metabolic syndrome are unclear. Here we show that in both mice and humans, adipose mitochondrial functions are elevated in females and are strongly associated with adiposity, insulin resistance and plasma lipids. Using a panel of diverse inbred strains of mice, we identify a genetic locus on mouse chromosome 17 that controls mitochondrial mass and function in adipose tissue in a sex- and tissue-specific manner. This locus contains Ndufv2 and regulates the expression of at least 89 mitochondrial genes in females, including oxidative phosphorylation genes and those related to mitochondrial DNA content. Overexpression studies indicate that Ndufv2 mediates these effects by regulating supercomplex assembly and elevating mitochondrial reactive oxygen species production, which generates a signal that increases mitochondrial biogenesis.

ABCB10 exports mitochondrial biliverdin, driving metabolic maladaptation in obesity.

Although the role of hydrophilic antioxidants in the development of hepatic insulin resistance and nonalcoholic fatty liver disease has been well studied, the role of lipophilic antioxidants remains poorly characterized. A known lipophilic hydrogen peroxide scavenger is bilirubin, which can be oxidized to biliverdin and then reduced back to bilirubin by cytosolic biliverdin reductase. Oxidation of bilirubin to biliverdin inside mitochondria must be followed by the export of biliverdin to the cytosol, where biliverdin is reduced back to bilirubin. Thus, the putative mitochondrial exporter of biliverdin is expected to be a major determinant of bilirubin regeneration and intracellular hydrogen peroxide scavenging. Here, we identified ABCB10 as a mitochondrial biliverdin exporter. ABCB10 reconstituted into liposomes transported biliverdin, and ABCB10 deletion caused accumulation of biliverdin inside mitochondria. Obesity with insulin resistance up-regulated hepatic ABCB10 expression in mice and elevated cytosolic and mitochondrial bilirubin content in an ABCB10-dependent manner. Revealing a maladaptive role of ABCB10-driven bilirubin synthesis, hepatic ABCB10 deletion protected diet-induced obese mice from steatosis and hyperglycemia, improving insulin-mediated suppression of glucose production and decreasing lipogenic SREBP-1c expression. Protection was concurrent with enhanced mitochondrial function and increased inactivation of PTP1B, a phosphatase disrupting insulin signaling and elevating SREBP-1c expression. Restoration of cellular bilirubin content in ABCB10 KO hepatocytes reversed the improvements in mitochondrial function and PTP1B inactivation, demonstrating that bilirubin was the maladaptive effector linked to ABCB10 function. Thus, we identified a fundamental transport process that amplifies intracellular bilirubin redox actions, which can exacerbate insulin resistance and steatosis in obesity.