Macrophage immunometabolism in diabetes-associated atherosclerosis.

Macrophages play fundamental roles in atherosclerotic plaque formation, growth, and regression. These cells are extremely plastic and perform different immune functions depending on the stimuli they receive. Initial in vitro studies have identified specific metabolic pathways that are crucial for the proper function of pro-inflammatory and pro-resolving macrophages. However, the plaque microenvironment, especially in the context of insulin resistance and type 2 diabetes, constantly challenges macrophages with several simultaneous inflammatory and metabolic stimuli, which may explain why atherosclerosis is accelerated in diabetic patients. In this mini review, we discuss how macrophage mitochondrial function and metabolism of carbohydrates, lipids, and amino acids may be affected by this complex plaque microenvironment and how risk factors associated with type 2 diabetes alter the metabolic rewiring of macrophages and disease progression. We also briefly discuss current challenges in assessing macrophage metabolism and identify future tools and possible strategies to alter macrophage metabolism to improve treatment options for diabetes-associated atherosclerosis.

Mammary duct luminal epithelium controls adipocyte thermogenic programme.

Sympathetic activation during cold exposure increases adipocyte thermogenesis via the expression of mitochondrial protein uncoupling protein 1 (UCP1)1. The propensity of adipocytes to express UCP1 is under a critical influence of the adipose microenvironment and varies between sexes and among various fat depots2-7. Here we report that mammary gland ductal epithelial cells in the adipose niche regulate cold-induced adipocyte UCP1 expression in female mouse subcutaneous white adipose tissue (scWAT). Single-cell RNA sequencing shows that glandular luminal epithelium subtypes express transcripts that encode secretory factors controlling adipocyte UCP1 expression under cold conditions. We term these luminal epithelium secretory factors 'mammokines'. Using 3D visualization of whole-tissue immunofluorescence, we reveal sympathetic nerve-ductal contact points. We show that mammary ducts activated by sympathetic nerves limit adipocyte UCP1 expression via the mammokine lipocalin 2. In vivo and ex vivo ablation of mammary duct epithelium enhance the cold-induced adipocyte thermogenic gene programme in scWAT. Since the mammary duct network extends throughout most of the scWAT in female mice, females show markedly less scWAT UCP1 expression, fat oxidation, energy expenditure and subcutaneous fat mass loss compared with male mice, implicating sex-specific roles of mammokines in adipose thermogenesis. These results reveal a role of sympathetic nerve-activated glandular epithelium in adipocyte UCP1 expression and suggest that mammary duct luminal epithelium has an important role in controlling glandular adiposity.

Hepatocyte-specific miR-33 deletion attenuates NAFLD-NASH-HCC progression.

The complexity of the multiple mechanisms underlying non-alcoholic fatty liver disease (NAFLD) progression remains a significant challenge for the development of effective therapeutics. miRNAs have shown great promise as regulators of biological processes and as therapeutic targets for complex diseases. Here, we study the role of hepatic miR-33, an important regulator of lipid metabolism, during the progression of NAFLD. We report that miR-33 is overexpressed in hepatocytes isolated from mice with NAFLD and demonstrate that its specific suppression in hepatocytes (miR-33 HKO ) improves multiple aspects of the disease, including insulin resistance, steatosis, and inflammation and limits the progression to non-alcoholic steatohepatitis (NASH), fibrosis and hepatocellular carcinoma (HCC). Mechanistically, we find that hepatic miR-33 deficiency reduces lipid biosynthesis and promotes mitochondrial fatty acid oxidation to reduce lipid burden in hepatocytes. Additionally, miR-33 deficiency improves mitochondrial function, reducing oxidative stress. In miR-33 deficient hepatocytes, we found an increase in AMPKα activation, which regulates several pathways resulting in the attenuation of liver disease. The reduction in lipid accumulation and liver injury resulted in decreased transcriptional activity of the YAP/TAZ pathway, which may be involved in the reduced progression to HCC in the HKO livers. Together, these results suggest suppressing hepatic miR-33 may be an effective therapeutic approach at different stages of NAFLD/NASH/HCC disease progression.

Q-Flux: A method to assess hepatic mitochondrial succinate dehydrogenase, methylmalonyl-CoA mutase, and glutaminase fluxes in vivo.

The mammalian succinate dehydrogenase (SDH) complex has recently been shown as capable of operating bidirectionally. Here, we develop a method (Q-Flux) capable of measuring absolute rates of both forward (VSDH(F)) and reverse (VSDH(R)) flux through SDH in vivo while also deconvoluting the amount of glucose derived from four discreet carbon sources in the liver. In validation studies, a mitochondrial uncoupler increased net SDH flux by >100% in awake rodents but also increased SDH cycling. During hyperglucagonemia, attenuated pyruvate cycling enhances phosphoenolpyruvate carboxykinase efficiency to drive increased gluconeogenesis, which is complemented by increased glutaminase (GLS) flux, methylmalonyl-CoA mutase (MUT) flux, and glycerol conversion to glucose. During hyperinsulinemic-euglycemic clamp, both pyruvate carboxylase and GLS are suppressed, while VSDH(R) is increased. Unstimulated MUT is a minor anaplerotic reaction but is readily induced by small amounts of propionate, which elicits glucagon-like metabolic rewiring. Taken together, Q-Flux yields a comprehensive picture of hepatic mitochondrial metabolism and should be broadly useful to researchers.

Antagonism of miR-148a attenuates atherosclerosis progression in APOBTGApobec-/-Ldlr+/- mice: A brief report.

OBJECTIVE: miR-148a-3p (miR-148a) is a hepatic and immune-enriched microRNA (miRNA) that regulates macrophage-related lipoprotein metabolism, cholesterol homeostasis, and inflammation. The contribution of miR-148a-3p to the progression of atherosclerosis is unknown. In this study, we determined whether miR-148a silencing mitigated atherogenesis in APOBTGApobec-/-Ldlr+/- mice. METHODS: APOBTGApobec-/-Ldlr+/- mice were fed a typical Western-style diet for 22 weeks and injected with a nontargeting locked nucleic acid (LNA; LNA control) or miR-148a LNA (LNA 148a) for the last 10 weeks. At the end of the treatment, the mice were sacrificed, and circulating lipids, hepatic gene expression, and atherosclerotic lesions were analyzed. RESULTS: Examination of atherosclerotic lesions revealed a significant reduction in plaque size, with marked remodeling of the lesions toward a more stable phenotype. Mechanistically, miR-148a levels influenced macrophage cholesterol efflux and the inflammatory response. Suppression of miR-148a in murine primary macrophages decreased mRNA levels of proinflammatory M1-like markers (Nos2, Il6, Cox2, and Tnf) and increased the expression of anti-inflammatory genes (Arg1, Retlna, and Mrc1). CONCLUSIONS: Therapeutic silencing of miR148a mitigated the progression of atherosclerosis and promoted plaque stability. The antiatherogenic effect of miR-148a antisense therapy is likely mediated by the anti-inflammatory effects observed in macrophages treated with miR-148 LNA and independent of significant changes in circulating low-density lipoprotein cholesterol (LDL-C) and high-density lipoprotein cholesterol (HDL-C).

Insulin increases placental triglyceride as a potential mechanism for fetal adiposity in maternal obesity.

OBJECTIVE: Maternal obesity increases the incidence of excess adiposity in newborns, resulting in lifelong diabetes risk. Elevated intrauterine fetal adiposity has been attributed to maternal hyperglycemia; however, this hypothesis does not account for the increased adiposity seen in newborns of mothers with obesity who have euglycemia. We aimed to explore the placental response to maternal hyperinsulinemia and the effect of insulin-like growth factor 2 (IGF-2) in promoting fetal adiposity by increasing storage and availability of nutrients to the fetus. METHODS: We used placental villous explants and isolated trophoblasts from normal weight and obese women to assess the effect of insulin and IGF-2 on triglyceride content and insulin receptor signaling. Stable isotope tracer methods were used ex vivo to determine effect of hormone treatment on de novo lipogenesis (DNL), fatty acid uptake, fatty acid oxidation, and esterification in the placenta. RESULTS: Here we show that placentae from euglycemic women with normal weight and obesity both have abundant insulin receptor. Placental depth and triglyceride were greater in women with obesity compared with normal weight women. In syncytialized placental trophoblasts and villous explants, insulin and IGF-2 activate insulin receptor, induce expression of lipogenic transcription factor SREBP-1 (sterol regulatory element-binding protein 1), and stimulate triglyceride accumulation. We demonstrate elevated triglyceride is attributable to increased esterification of fatty acids, without contribution from DNL and without an acceleration of fatty acid uptake. CONCLUSIONS: Our work reveals that obesity-driven aberrations in maternal metabolism, such as hyperinsulinemia, alter placental metabolism in euglycemic conditions, and may explain the higher prevalence of excess adiposity in the newborns of obese women.

Metformin, phenformin, and galegine inhibit complex IV activity and reduce glycerol-derived gluconeogenesis.

SignificanceMetformin is the most commonly prescribed drug for the treatment of type 2 diabetes mellitus, yet the mechanism by which it lowers plasma glucose concentrations has remained elusive. Most studies to date have attributed metformin's glucose-lowering effects to inhibition of complex I activity. Contrary to this hypothesis, we show that inhibition of complex I activity in vitro and in vivo does not reduce plasma glucose concentrations or inhibit hepatic gluconeogenesis. We go on to show that metformin, and the related guanides/biguanides, phenformin and galegine, inhibit complex IV activity at clinically relevant concentrations, which, in turn, results in inhibition of glycerol-3-phosphate dehydrogenase activity, increased cytosolic redox, and selective inhibition of glycerol-derived hepatic gluconeogenesis both in vitro and in vivo.

Sex- and strain-specific effects of mitochondrial uncoupling on age-related metabolic diseases in high-fat diet-fed mice.

Mild uncoupling of oxidative phosphorylation is an intrinsic property of all mitochondria and may have evolved to protect cells against the production of damaging reactive oxygen species. Therefore, compounds that enhance mitochondrial uncoupling are potentially attractive anti-aging therapies; however, chronic ingestion is associated with a number of unwanted side effects. We have previously developed a controlled-release mitochondrial protonophore (CRMP) that is functionally liver-directed and promotes oxidation of hepatic triglycerides by causing a subtle sustained increase in hepatic mitochondrial inefficiency. Here, we sought to leverage the higher therapeutic index of CRMP to test whether mild mitochondrial uncoupling in a liver-directed fashion could reduce oxidative damage and improve age-related metabolic disease and lifespan in diet-induced obese mice. Oral administration of CRMP (20 mg/[kg-day] × 4 weeks) reduced hepatic lipid content, protein kinase C epsilon activation, and hepatic insulin resistance in aged (74-week-old) high-fat diet (HFD)-fed C57BL/6J male mice, independently of changes in body weight, whole-body energy expenditure, food intake, or markers of hepatic mitochondrial biogenesis. CRMP treatment was also associated with a significant reduction in hepatic lipid peroxidation, protein carbonylation, and inflammation. Importantly, long-term (49 weeks) hepatic mitochondrial uncoupling initiated late in life (94-104 weeks), in conjugation with HFD feeding, protected mice against neoplastic disorders, including hepatocellular carcinoma (HCC), in a strain and sex-specific manner. Taken together, these studies illustrate the complex variation of aging and provide important proof-of-concept data to support further studies investigating the use of liver-directed mitochondrial uncouplers to promote healthy aging in humans.

Dyrk1b promotes hepatic lipogenesis by bypassing canonical insulin signaling and directly activating mTORC2 in mice.

Mutations in Dyrk1b are associated with metabolic syndrome and nonalcoholic fatty liver disease in humans. Our investigations showed that DYRK1B levels are increased in the liver of patients with nonalcoholic steatohepatitis (NASH) and in mice fed with a high-fat, high-sucrose diet. Increasing Dyrk1b levels in the mouse liver enhanced de novo lipogenesis (DNL), fatty acid uptake, and triacylglycerol secretion and caused NASH and hyperlipidemia. Conversely, knockdown of Dyrk1b was protective against high-calorie-induced hepatic steatosis and fibrosis and hyperlipidemia. Mechanistically, Dyrk1b increased DNL by activating mTORC2 in a kinase-independent fashion. Accordingly, the Dyrk1b-induced NASH was fully rescued when mTORC2 was genetically disrupted. The elevated DNL was associated with increased plasma membrane sn-1,2-diacylglyerol levels and increased PKCε-mediated IRKT1150 phosphorylation, which resulted in impaired activation of hepatic insulin signaling and reduced hepatic glycogen storage. These findings provide insights into the mechanisms that underlie Dyrk1b-induced hepatic lipogenesis and hepatic insulin resistance and identify Dyrk1b as a therapeutic target for NASH and insulin resistance in the liver.

MMAB promotes negative feedback control of cholesterol homeostasis.

Intricate regulatory networks govern the net balance of cholesterol biosynthesis, uptake and efflux; however, the mechanisms surrounding cholesterol homeostasis remain incompletely understood. Here, we develop an integrative genomic strategy to detect regulators of LDLR activity and identify 250 genes whose knockdown affects LDL-cholesterol uptake and whose expression is modulated by intracellular cholesterol levels in human hepatic cells. From these hits, we focus on MMAB, an enzyme which catalyzes the conversion of vitamin B12 to adenosylcobalamin, and whose expression has previously been linked with altered levels of circulating cholesterol in humans. We demonstrate that hepatic levels of MMAB are modulated by dietary and cellular cholesterol levels through SREBP2, the master transcriptional regulator of cholesterol homeostasis. Knockdown of MMAB decreases intracellular cholesterol levels and augments SREBP2-mediated gene expression and LDL-cholesterol uptake in human and mouse hepatic cell lines. Reductions in total sterol content were attributed to increased intracellular levels of propionic and methylmalonic acid and subsequent inhibition of HMGCR activity and cholesterol biosynthesis. Moreover, mice treated with antisense inhibitors of MMAB display a significant reduction in hepatic HMGCR activity, hepatic sterol content and increased expression of SREBP2-mediated genes. Collectively, these findings reveal an unexpected role for the adenosylcobalamin pathway in regulating LDLR expression and identify MMAB as an additional control point by which cholesterol biosynthesis is regulated by its end product.

Increased exosome secretion in neurons aging in vitro by NPC1-mediated endosomal cholesterol buildup.

As neurons age, they show a decrease in their ability to degrade proteins and membranes. Because undegraded material is a source of toxic products, defects in degradation are associated with reduced cell function and survival. However, there are very few dead neurons in the aging brain, suggesting the action of compensatory mechanisms. We show in this work that ageing neurons in culture show large multivesicular bodies (MVBs) filled with intralumenal vesicles (ILVs) and secrete more small extracellular vesicles than younger neurons. We also show that the high number of ILVs is the consequence of the accumulation of cholesterol in MVBs, which in turn is due to decreased levels of the cholesterol extruding protein NPC1. NPC1 down-regulation is the consequence of a combination of upregulation of the NPC1 repressor microRNA 33, and increased degradation, due to Akt-mTOR targeting of NPC1 to the phagosome. Although releasing more exosomes can be beneficial to old neurons, other cells, neighbouring and distant, can be negatively affected by the waste material they contain.

miR-33 in cardiometabolic diseases: lessons learned from novel animal models and approaches.

miRNAs have emerged as critical regulators of nearly all biologic processes and important therapeutic targets for numerous diseases. However, despite the tremendous progress that has been made in this field, many misconceptions remain among much of the broader scientific community about the manner in which miRNAs function. In this review, we focus on miR-33, one of the most extensively studied miRNAs, as an example, to highlight many of the advances that have been made in the miRNA field and the hurdles that must be cleared to promote the development of miRNA-based therapies. We discuss how the generation of novel animal models and newly developed experimental techniques helped to elucidate the specialized roles of miR-33 within different tissues and begin to define the specific mechanisms by which miR-33 contributes to cardiometabolic diseases including obesity and atherosclerosis. This review will summarize what is known about miR-33 and highlight common obstacles in the miRNA field and then describe recent advances and approaches that have allowed researchers to provide a more complete picture of the specific functions of this miRNA.

Insulin-stimulated endoproteolytic TUG cleavage links energy expenditure with glucose uptake.

TUG tethering proteins bind and sequester GLUT4 glucose transporters intracellularly, and insulin stimulates TUG cleavage to translocate GLUT4 to the cell surface and increase glucose uptake. This effect of insulin is independent of phosphatidylinositol 3-kinase, and its physiological relevance remains uncertain. Here we show that this TUG cleavage pathway regulates both insulin-stimulated glucose uptake in muscle and organism-level energy expenditure. Using mice with muscle-specific Tug (Aspscr1)-knockout and muscle-specific constitutive TUG cleavage, we show that, after GLUT4 release, the TUG C-terminal cleavage product enters the nucleus, binds peroxisome proliferator-activated receptor (PPAR)γ and its coactivator PGC-1α and regulates gene expression to promote lipid oxidation and thermogenesis. This pathway acts in muscle and adipose cells to upregulate sarcolipin and uncoupling protein 1 (UCP1), respectively. The PPARγ2 Pro12Ala polymorphism, which reduces diabetes risk, enhances TUG binding. The ATE1 arginyltransferase, which mediates a specific protein degradation pathway and controls thermogenesis, regulates the stability of the TUG product. We conclude that insulin-stimulated TUG cleavage coordinates whole-body energy expenditure with glucose uptake, that this mechanism might contribute to the thermic effect of food and that its attenuation could promote obesity.

Therapeutic potential of mitochondrial uncouplers for the treatment of metabolic associated fatty liver disease and NASH.

BACKGROUND: Mitochondrial uncouplers shuttle protons across the inner mitochondrial membrane via a pathway that is independent of adenosine triphosphate (ATP) synthase, thereby uncoupling nutrient oxidation from ATP production and dissipating the proton gradient as heat. While initial toxicity concerns hindered their therapeutic development in the early 1930s, there has been increased interest in exploring the therapeutic potential of mitochondrial uncouplers for the treatment of metabolic diseases. SCOPE OF REVIEW: In this review, we cover recent advances in the mechanisms by which mitochondrial uncouplers regulate biological processes and disease, with a particular focus on metabolic associated fatty liver disease (MAFLD), nonalcoholic hepatosteatosis (NASH), insulin resistance, and type 2 diabetes (T2D). We also discuss the challenges that remain to be addressed before synthetic and natural mitochondrial uncouplers can successfully enter the clinic. MAJOR CONCLUSIONS: Rodent and non-human primate studies suggest that a myriad of small molecule mitochondrial uncouplers can safely reverse MAFLD/NASH with a wide therapeutic index. Despite this, further characterization of the tissue- and cell-specific effects of mitochondrial uncouplers is needed. We propose targeting the dosing of mitochondrial uncouplers to specific tissues such as the liver and/or developing molecules with self-limiting properties to induce a subtle and sustained increase in mitochondrial inefficiency, thereby avoiding systemic toxicity concerns.

Loss of hepatic miR-33 improves metabolic homeostasis and liver function without altering body weight or atherosclerosis.

miR-33 is an intronic microRNA within the gene encoding the SREBP2 transcription factor. Like its host gene, miR-33 has been shown to be an important regulator of lipid metabolism. Inhibition of miR-33 has been shown to promote cholesterol efflux in macrophages by targeting the cholesterol transporter ABCA1, thus reducing atherosclerotic plaque burden. Inhibition of miR-33 has also been shown to improve high-density lipoprotein (HDL) biogenesis in the liver and increase circulating HDL-C levels in both rodents and nonhuman primates. However, evaluating the extent to which these changes in HDL metabolism contribute to atherogenesis has been hindered by the obesity and metabolic dysfunction observed in whole-body miR-33-knockout mice. To determine the impact of hepatic miR-33 deficiency on obesity, metabolic function, and atherosclerosis, we have generated a conditional knockout mouse model that lacks miR-33 only in the liver. Characterization of this model demonstrates that loss of miR-33 in the liver does not lead to increased body weight or adiposity. Hepatic miR-33 deficiency actually improves regulation of glucose homeostasis and impedes the development of fibrosis and inflammation. We further demonstrate that hepatic miR-33 deficiency increases circulating HDL-C levels and reverse cholesterol transport capacity in mice fed a chow diet, but these changes are not sufficient to reduce atherosclerotic plaque size under hyperlipidemic conditions. By elucidating the role of miR-33 in the liver and the impact of hepatic miR-33 deficiency on obesity and atherosclerosis, this work will help inform ongoing efforts to develop novel targeted therapies against cardiometabolic diseases.

Mechanisms by which adiponectin reverses high fat diet-induced insulin resistance in mice.

Adiponectin has emerged as a potential therapy for type 2 diabetes mellitus, but the molecular mechanism by which adiponectin reverses insulin resistance remains unclear. Two weeks of globular adiponectin (gAcrp30) treatment reduced fasting plasma glucose, triglyceride (TAG), and insulin concentrations and reversed whole-body insulin resistance, which could be attributed to both improved insulin-mediated suppression of endogenous glucose production and increased insulin-stimulated glucose uptake in muscle and adipose tissues. These improvements in liver and muscle sensitivity were associated with ∼50% reductions in liver and muscle TAG and plasma membrane (PM)-associated diacylglycerol (DAG) content and occurred independent of reductions in total ceramide content. Reductions of PM DAG content in liver and skeletal muscle were associated with reduced PKCε translocation in liver and reduced PKCθ and PKCε translocation in skeletal muscle resulting in increased insulin-stimulated insulin receptor tyrosine1162 phosphorylation, IRS-1/IRS-2-associated PI3-kinase activity, and Akt-serine phosphorylation. Both gAcrp30 and full-length adiponectin (Acrp30) treatment increased eNOS/AMPK activation in muscle and muscle fatty acid oxidation. gAcrp30 and Acrp30 infusions also increased TAG uptake in epididymal white adipose tissue (eWAT), which could be attributed to increased lipoprotein lipase (LPL) activity. These data suggest that adiponectin and adiponectin-related molecules reverse lipid-induced liver and muscle insulin resistance by reducing ectopic lipid storage in these organs, resulting in decreased plasma membrane sn-1,2-DAG-induced nPKC activity and increased insulin signaling. Adiponectin mediates these effects by both promoting the storage of TAG in eWAT likely through stimulation of LPL as well as by stimulation of AMPK in muscle resulting in increased muscle fat oxidation.

miR-27b Modulates Insulin Signaling in Hepatocytes by Regulating Insulin Receptor Expression.

Insulin resistance (IR) is one of the key contributing factors in the development of type 2 diabetes mellitus (T2DM). However, the molecular mechanisms leading to IR are still unclear. The implication of microRNAs (miRNAs) in the pathophysiology of multiple cardiometabolic pathologies, including obesity, atherosclerotic heart failure and IR, has emerged as a major focus of interest in recent years. Indeed, upregulation of several miRNAs has been associated with obesity and IR. Among them, miR-27b is overexpressed in the liver in patients with obesity, but its role in IR has not yet been thoroughly explored. In this study, we investigated the role of miR-27b in regulating insulin signaling in hepatocytes, both in vitro and in vivo. Therefore, assessment of the impact of miR-27b on insulin resistance through the hepatic tissue is of special importance due to the high expression of miR-27b in the liver together with its known role in regulating lipid metabolism. Notably, we found that miR-27b controls post-transcriptional expression of numerous components of the insulin signaling pathway including the insulin receptor (INSR) and insulin receptor substrate 1 (IRS1) in human hepatoma cells. These results were further confirmed in vivo showing that overexpression and inhibition of hepatic miR-27 enhances and suppresses hepatic INSR expression and insulin sensitivity, respectively. This study identified a novel role for miR-27 in regulating insulin signaling, and this finding suggests that elevated miR-27 levels may contribute to early development of hepatic insulin resistance.

Glucagon stimulates gluconeogenesis by INSP3R1-mediated hepatic lipolysis.

Although it is well-established that reductions in the ratio of insulin to glucagon in the portal vein have a major role in the dysregulation of hepatic glucose metabolism in type-2 diabetes1-3, the mechanisms by which glucagon affects hepatic glucose production and mitochondrial oxidation are poorly understood. Here we show that glucagon stimulates hepatic gluconeogenesis by increasing the activity of hepatic adipose triglyceride lipase, intrahepatic lipolysis, hepatic acetyl-CoA content and pyruvate carboxylase flux, while also increasing mitochondrial fat oxidation-all of which are mediated by stimulation of the inositol triphosphate receptor 1 (INSP3R1). In rats and mice, chronic physiological increases in plasma glucagon concentrations increased mitochondrial oxidation of fat in the liver and reversed diet-induced hepatic steatosis and insulin resistance. However, these effects of chronic glucagon treatment-reversing hepatic steatosis and glucose intolerance-were abrogated in Insp3r1 (also known as Itpr1)-knockout mice. These results provide insights into glucagon biology and suggest that INSP3R1 may represent a target for therapies that aim to reverse nonalcoholic fatty liver disease and type-2 diabetes.

Controlled-release mitochondrial protonophore (CRMP) reverses dyslipidemia and hepatic steatosis in dysmetabolic nonhuman primates.

Nonalcoholic fatty liver disease (NAFLD) is estimated to affect up to one-third of the general population, and new therapies are urgently required. Our laboratory previously developed a controlled-release mitochondrial protonophore (CRMP) that is functionally liver-targeted and promotes oxidation of hepatic triglycerides. Although we previously demonstrated that CRMP safely reverses hypertriglyceridemia, fatty liver, hepatic inflammation, and fibrosis in diet-induced rodent models of obesity, there remains a critical need to assess its safety and efficacy in a model highly relevant to humans. Here, we evaluated the impact of longer-term CRMP treatment on hepatic mitochondrial oxidation and on the reversal of hypertriglyceridemia, NAFLD, and insulin resistance in high-fat, fructose-fed cynomolgus macaques (n = 6) and spontaneously obese dysmetabolic rhesus macaques (n = 12). Using positional isotopomer nuclear magnetic resonance tracer analysis (PINTA), we demonstrated that acute CRMP treatment (single dose, 5 mg/kg) increased rates of hepatic mitochondrial fat oxidation by 40%. Six weeks of CRMP treatment reduced hepatic triglycerides in both nonhuman primate models independently of changes in body weight, food intake, body temperature, or adverse reactions. CRMP treatment was also associated with a 20 to 30% reduction in fasting plasma triglycerides and low-density lipoprotein (LDL)-cholesterol in dysmetabolic nonhuman primates. Oral administration of CRMP reduced endogenous glucose production by 18%, attributable to a 20% reduction in hepatic acetyl-coenzyme A (CoA) content [as assessed by whole-body ß-hydroxybutyrate (ß-OHB) turnover] and pyruvate carboxylase flux. Collectively, these studies provide proof-of-concept data to support the development of liver-targeted mitochondrial uncouplers for the treatment of metabolic syndrome in humans.

MicroRNA 7 Impairs Insulin Signaling and Regulates Aß Levels through Posttranscriptional Regulation of the Insulin Receptor Substrate 2, Insulin Receptor, Insulin-Degrading Enzyme, and Liver X Receptor Pathway.

Brain insulin resistance is a key pathological feature contributing to obesity, diabetes, and neurodegenerative disorders, including Alzheimer's disease (AD). Besides the classic transcriptional mechanism mediated by hormones, posttranscriptional regulation has recently been shown to regulate a number of signaling pathways that could lead to metabolic diseases. Here, we show that microRNA 7 (miR-7), an abundant microRNA in the brain, targets insulin receptor (INSR), insulin receptor substrate 2 (IRS-2), and insulin-degrading enzyme (IDE), key regulators of insulin homeostatic functions in the central nervous system (CNS) and the pathology of AD. In this study, we found that insulin and liver X receptor (LXR) activators promote the expression of the intronic miR-7-1 in vitro and in vivo, along with its host heterogeneous nuclear ribonucleoprotein K (HNRNPK) gene, encoding an RNA binding protein (RBP) that is involved in insulin action at the posttranscriptional level. Our data show that miR-7 expression is altered in the brains of diet-induced obese mice. Moreover, we found that the levels of miR-7 are also elevated in brains of AD patients; this inversely correlates with the expression of its target genes IRS-2 and IDE. Furthermore, overexpression of miR-7 increased the levels of extracellular Aß in neuronal cells and impaired the clearance of extracellular Aß by microglial cells. Taken together, these results represent a novel branch of insulin action through the HNRNPK-miR-7 axis and highlight the possible implication of these posttranscriptional regulators in a range of diseases underlying metabolic dysregulation in the brain, from diabetes to Alzheimer's disease.