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Mild uncoupling of oxidative phosphorylation is an intrinsic property of all mitochondria, allowing for adjustments in cellular energy metabolism to maintain metabolic homeostasis. Small molecule uncouplers have been extensively studied for their potential to increase metabolic rate, and recent research has focused on developing safe and effective mitochondrial uncoupling agents for the treatment of obesity and cardiometabolic syndrome (CMS). Here, we provide a brief overview of CMS and cover the recent mechanisms by which chemical uncouplers regulate CMS-associated risk-factors and comorbidities, including dyslipidemia, insulin resistance, steatotic liver disease, type 2 diabetes, and atherosclerosis. Additionally, we review the current landscape of uncoupling agents, focusing on repurposed FDA-approved drugs and compounds in advanced preclinical or early-stage clinical development. Lastly, we discuss recent molecular insights by which chemical uncouplers enhance cellular energy expenditure, highlighting their potential as a new addition to the current CMS drug landscape, and outline several limitations that need to be addressed before these agents can successfully be introduced into clinical practice. [ABSTRACT FROM AUTHOR]

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 diabetes.sup.1-3, 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. A role and mechanism of action are identified for INSP3R1 in the stimulation of hepatic gluconeogenesis and mitochondrial oxidation by glucagon, suggesting that INSP3R1 may be a target for ameliorating dysregulation of hepatic glucose metabolism., Author(s): Rachel J. Perry [sup.1] [sup.2] , Dongyan Zhang [sup.1] , Mateus T. Guerra [sup.1] , Allison L. Brill [sup.2] , Leigh Goedeke [sup.1] , Ali R. Nasiri [sup.1] , [...]

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 PKC[epsilon]-mediated IRK T1150 phosphorylation, which resulted in impaired activation of hepatic insulin signaling and reduced hepatic glycogen storage. These findings provide insights into the mechanisms that underlie Dyrk1binduced hepatic lipogenesis and hepatic insulin resistance and identify Dyrk1b as a therapeutic target for NASH and insulin resistance in the liver., Introduction Nonalcoholic fatty liver disease (NAFLD) is a rapidly growing disorder affecting nearly 25% of the adult population worldwide and is a major risk factor for nonalcoholic steatohepatitis (NASH), atherosclerosis, [...]

Genome-wide association studies (GWASs) have linked genes to various pathological traits. However, the potential contribution of regulatory noncoding RNAs, such as microRNAs (miRNAs), to a genetic predisposition to pathological conditions has remained unclear. We leveraged GWAS meta-analysis data from [greater than] 188,000 individuals to identify 69 miRNAs in physical proximity to single-nucleotide polymorphisms (SNPs) associated with abnormal levels of circulating lipids. Several of these miRNAs (miR-128-1, miR-148a, miR-130b, and miR-301b) control the expression of key proteins involved in cholesterol-lipoprotein trafficking, such as the low-density lipoprotein (LDL) receptor (LDLR) and the ATP-binding cassette A1 (ABCA1) cholesterol transporter. Consistent with human liver expression data and genetic links to abnormal blood lipid levels, overexpression and antisense targeting of miR-128-1 or miR-148a in high-fat diet-fed C57BL/6J and Apoe-null mice resulted in altered hepatic expression of proteins involved in lipid trafficking and metabolism, and in modulated levels of circulating lipoprotein-cholesterol and triglycerides. Taken together, these findings support the notion that altered expression of miRNAs may contribute to abnormal blood lipid levels, predisposing individuals to human cardiometabolic disorders., Author(s): Alexandre Wagschal [1, 2]; S Hani Najafi-Shoushtari [1, 2]; Lifeng Wang [1, 2]; Leigh Goedeke [3]; Sumita Sinha [4]; Andrew S deLemos [5]; Josh C Black [1, 6]; Cristina [...]

The hepatic low-density lipoprotein receptor (LDLR) pathway is essential for clearing circulating LDL cholesterol (LDL-C). Whereas the transcriptional regulation of LDLR is well characterized, the post-transcriptional mechanisms that govern LDLR expression are just beginning to emerge. Here we develop a high-throughput genome-wide screening assay to systematically identify microRNAs (miRNAs) that regulate LDLR activity in human hepatic cells. From this screen we identified and characterized miR-148a as a negative regulator of LDLR expression and activity and defined a sterol regulatory element-binding protein 1 (SREBP1)-mediated pathway through which miR-148a regulates LDL-C uptake. In mice, inhibition of miR-148a increased hepatic LDLR expression and decreased plasma LDL-C. Moreover, we found that miR-148a regulates hepatic expression of ATP-binding cassette, subfamily A, member 1 (ABCA1) and circulating high-density lipoprotein cholesterol (HDL-C) levels in vivo. These studies uncover a role for miR-148a as a key regulator of hepatic LDL-C clearance through direct modulation of LDLR expression and demonstrate the therapeutic potential of inhibiting miR-148a to ameliorate an elevated LDL-C/HDL-C ratio, a prominent risk factor for cardiovascular disease., Author(s): Leigh Goedeke [1, 2, 3, 4]; Noemi Rotllan [1, 2]; Alberto Canfrán-Duque [1, 2]; Juan F Aranda [1, 2, 3]; Cristina M Ramírez [1, 2]; Elisa Araldi [1, 2, [...]

Cardiovascular disease remains the leading cause of mortality in westernized countries, despite optimum medical therapy to reduce the levels of low-density lipoprotein (LDL)-associated cholesterol. The pursuit of novel therapies to target the residual risk has focused on raising the levels of high-density lipoprotein (HDL)-associated cholesterol in order to exploit its atheroprotective effects (1). MicroRNAs (miRNAs) have emerged as important post-transcriptional regulators of lipid metabolism and are thus a new class of target for therapeutic intervention (2). MicroRNA-33a and microRNA-33b (miR-33a/b) are intronic miRNAs whose encoding regions are embedded in the sterol-response-element-binding protein genes SREBF2 and SREBF1 (refs 3-5), respectively. These miRNAs repress expression of the cholesterol transporter ABCA1, which is a key regulator of HDL biogenesis. Recent studies in mice suggest that antagonizing miR-33a may be an effective strategy for raising plasma HDL levels (3-5) and providing protection against atherosclerosis (6); however, extrapolating these findings to humans is complicated by the fact that mice lack miR-33b, which is present only in the SREBF1 gene of medium and large mammals. Here we show in African green monkeys that systemic delivery of an anti-miRNA oligonucleotide that targets both miR-33a and miR-33b increased hepatic expression of ABCA1 and induced a sustained increase in plasma HDL levels over 12 weeks. Notably, miR-33 antagonism in this non-human primate model also increased the expression of miR-33 target genes involved in fatty acid oxidation (CROT, CPT1A, HADHB and PRKAA1) and reduced the expression of genes involved in fatty acid synthesis (SREBF1, FASN, ACLY and ACACA), resulting in a marked suppression of the plasma levels of very-low-density lipoprotein (VLDL)-associated triglycerides, a finding that has not previously been observed in mice. These data establish, in a model that is highly relevant to humans, that pharmacological inhibition of miR-33a and miR33b is a promising therapeutic strategy to raise plasma HDL and lower VLDL triglyceride levels for the treatment of dyslipidaemias that increase cardiovascular disease risk., Recent advances in the understanding of lipid metabolism have revealed that the genetic loci encoding the transcription factors SREBP1 and SREBP2 (known as SREBF1 and SREBF2) also encode the miRNAs [...]

Metformin exerts its plasma glucose-lowering therapeutic effect primarily through inhibition of hepatic gluconeogenesis. However, the precise molecular mechanism by which metformin inhibits hepatic gluconeogenesis is still unclear. Although inhibition of mitochondrial complex I is frequently invoked as metformin’s primary mechanism of action, the metabolic effects of complex I inhibition have not been thoroughly evaluated in vivo. Here, we show that acute portal infusion of piericidin A, a potent and specific complex I inhibitor, does not reduce hepatic gluconeogenesis in vivo. In contrast, we show that metformin, phenformin, and galegine selectively inhibit hepatic gluconeogenesis from glycerol. Specifically, we show that guanides/biguanides interact with complex IV to reduce its enzymatic activity, leading to indirect inhibition of glycerol-3-phosphate (G3P) dehydrogenase (GPD2), increased cytosolic redox, and reduced glycerol-derived gluconeo-genesis. We report that inhibition of complex IV with potassium cyanide replicates the effects of the guanides/biguanides in vitro by selectively reducing glycerol-derived gluconeogenesis via increased cytosolic redox. Finally, we show that complex IV inhibition is sufficient to inhibit G3P-mediated respiration and gluconeo-genesis from glycerol. Taken together, we propose a mechanism of metformin action in which complex IV–mediated inhibition of GPD2 reduces glycerol-derived hepatic gluconeogenesis. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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 PKCe 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 fulllength 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. [ABSTRACT FROM AUTHOR]

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 Aβ in neuronal cells and impaired the clearance of extracellular Aβ 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. [ABSTRACT FROM AUTHOR]

Uncoupling mitochondria from liver disease: The mitochondrial uncoupler 2,4-dinitrophenol showed potential in treating nonalcoholic fatty liver disease (NAFLD) but was beset by toxicity issues. Here, Goedeke et al. show that a modified, liver-specific mitochondrial uncoupler, previously shown to be effective in rodent models metabolic disease, improved metabolic symptoms in two diet-induced nonhuman primate models of NAFLD. Their controlled-release mitochondrial protonophore (CRMP) improved insulin resistance, dyslipidemia, and hepatic steatosis in nonhuman primates treated over the course of 6 weeks, without increases in oxidative stress, liver enzymes, or adverse events. This preclinical study supports further work to translate CRMP for the treatment of metabolic diseases in humans. 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 β-hydroxybutyrate (β-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. [ABSTRACT FROM AUTHOR]

Type 2 diabetes (T2D) is characterized by persistent hyperglycemia despite hyperinsulinemia, affects more than 400 million people worldwide, and is a major cause of morbidity and mortality. Insulin resistance, of which ectopic lipid accumulation in the liver [nonalcoholic fatty liver disease (NAFLD)] and skeletal muscle is the root cause, plays a major role in the development of T2D. Although lifestyle interventions and weight loss are highly effective at reversing NAFLD and T2D, weight loss is difficult to sustain, and newer approaches aimed at treating the root cause of T2D are urgently needed. In this review, we highlight emerging pharmacological strategies aimed at improving insulin sensitivity and T2D by altering hepatic energy balance or inhibiting key enzymes involved in hepatic lipid synthesis. We also summarize recent research suggesting that liver-targeted mitochondrial uncoupling may be an attractive therapeutic approach to treat NAFLD, nonalcoholic steatohepatitis, and T2D. [ABSTRACT FROM AUTHOR]

Aims/hypothesis: Targeting regulators of adipose tissue lipoprotein lipase could enhance adipose lipid clearance, prevent ectopic lipid accumulation and consequently ameliorate insulin resistance and type 2 diabetes. Angiopoietin-like 8 (ANGPTL8) is an insulin-regulated lipoprotein lipase inhibitor strongly expressed in murine adipose tissue. However, Angptl8 knockout mice do not have improved insulin resistance. We hypothesised that pharmacological inhibition, using a second-generation antisense oligonucleotide (ASO) against Angptl8 in adult high-fat-fed rodents, would prevent ectopic lipid accumulation and insulin resistance by promoting adipose lipid uptake.Methods: ANGPTL8 expression was assessed by quantitative PCR in omental adipose tissue of bariatric surgery patients. High-fat-fed Sprague Dawley rats and C57BL/6 mice were treated with ASO against Angptl8 and insulin sensitivity was assessed by hyperinsulinaemic-euglycaemic clamps in rats and glucose tolerance tests in mice. Factors mediating lipid-induced hepatic insulin resistance were assessed, including lipid content, protein kinase Cε (PKCε) activation and insulin-stimulated Akt phosphorylation. Rat adipose lipid uptake was assessed by mixed meal tolerance tests. Murine energy balance was assessed by indirect calorimetry.Results: Omental fat ANGPTL8 mRNA expression is higher in obese individuals with fatty liver and insulin resistance compared with BMI-matched insulin-sensitive individuals. Angptl8 ASO prevented hepatic steatosis, PKCε activation and hepatic insulin resistance in high-fat-fed rats. Postprandial triacylglycerol uptake in white adipose tissue was increased in Angptl8 ASO-treated rats. Angptl8 ASO protected high-fat-fed mice from glucose intolerance. Although there was no change in net energy balance, Angptl8 ASO increased fat mass in high-fat-fed mice.Conclusions/interpretation: Disinhibition of adipose tissue lipoprotein lipase is a novel therapeutic modality to enhance adipose lipid uptake and treat non-alcoholic fatty liver disease and insulin resistance. In line with this, adipose ANGPTL8 is a candidate therapeutic target for these conditions. [ABSTRACT FROM AUTHOR]

MicroRNA-122 (miR-122) is abundant in the liver and involved in lipid homeostasis, but its relevance to the long-term risk of developing metabolic disorders is unknown. We therefore measured circulating miR-122 in the prospective population-based Bruneck Study (n = 810; survey year 1995). Circulating miR-122 was associated with prevalent insulin resistance, obesity, metabolic syndrome, type 2 diabetes, and an adverse lipid profile. Among 92 plasma proteins and 135 lipid subspecies quantified with mass spectrometry, it correlated inversely with zinc-α-2-glycoprotein and positively with afamin, complement factor H, VLDL-associated apolipoproteins, and lipid subspecies containing monounsaturated and saturated fatty acids. Proteomics analysis of livers from antagomiR-122-treated mice revealed novel regulators of hepatic lipid metabolism that are responsive to miR-122 inhibition. In the Anglo-Scandinavian Cardiac Outcomes Trial (ASCOT, n = 155), 12-month atorvastatin reduced circulating miR-122. A similar response to atorvastatin was observed in mice and cultured murine hepatocytes. Over up to 15 years of follow-up in the Bruneck Study, multivariable adjusted risk ratios per one-SD higher log miR-122 were 1.60 (95% CI 1.30-1.96; P < 0.001) for metabolic syndrome and 1.37 (1.03-1.82; P = 0.021) for type 2 diabetes. In conclusion, circulating miR-122 is strongly associated with the risk of developing metabolic syndrome and type 2 diabetes in the general population. [ABSTRACT FROM AUTHOR]

Small non-coding RNAs (microRNAs) are important regulators of gene expression that modulate many physiological processes; however, their role in regulating intracellular transport remains largely unknown. Intriguingly, we found that the dynamin (DNM) genes, a GTPase family of proteins responsible for endocytosis in eukaryotic cells, encode the conserved miR-199a and miR-199b family of miRNAs within their intronic sequences. Here, we demonstrate that miR-199a and miR-199b regulate endocytic transport by controlling the expression of important mediators of endocytosis such as clathrin heavy chain (CLTC), Rab5A, low-density lipoprotein receptor (LDLR) and caveolin-1 (Cav-1). Importantly, miR-199a-5p and miR-199b-5p overexpression markedly inhibits CLTC, Rab5A, LDLR and Cav-1 expression, thus preventing receptor-mediated endocytosis in human cell lines (Huh7 and HeLa). Of note, miR-199a-5p inhibition increases target gene expression and receptor-mediated endocytosis. Taken together, our work identifies a new mechanism by which microRNAs regulate intracellular trafficking. In particular, we demonstrate that the DNM, miR-199a-5p and miR-199b-5p genes act as a bifunctional locus that regulates endocytosis, thus adding an unexpected layer of complexity in the regulation of intracellular trafficking. [ABSTRACT FROM AUTHOR]

Atherosclerosis is the major cause of death and disability in diabetic and obese subjects with insulin resistance. Akt2, a phosphoinositide-dependent serine-threonine protein kinase, is highly express in insulin-responsive tissues; however, its role during the progression of atherosclerosis remains unknown. Thus, we aimed to investigate the contribution of Akt2 during the progression of atherosclerosis. We found that germ-line Akt2-deficient mice develop similar atherosclerotic plaques as wild-type mice despite higher plasma lipids and glucose levels. It is noteworthy that transplantation of bone marrow cells isolated from Akt2-/- mice to Ldlr-/- mice results in marked reduction of the progression of atherosclerosis compared with Ldlr-/- mice transplanted with wild-type bone marrow cells. In vitro studies indicate that Akt2 is required for macrophage migration in response to proathero- genic cytokines (monocyte chemotactic protein-1 and macrophage colony-stimulating factor). Moreover, Akt2-/- macrophages accumulate less cholesterol and have an alternative activated or M2-type phenotype when stimulated with proinflammatory cytokines. Together, these results provide evidence that macrophage Akt2 regulates migration, the inflammatory response and cholesterol metabolism and suggest that targeting Akt2 in macrophages might be beneficial for treating atherosclerosis. [ABSTRACT FROM AUTHOR]

Metabolic diseases are characterized by the failure of regulatory genes or proteins to effectively orchestrate specific pathways involved in the control of many biological processes. In addition to the classical regulators, recent discoveries have shown the remarkable role of small noncoding RNAs (microRNAs [miRNAs]) in the posttranscriptional regulation of gene expression. In this regard, we have recently demonstrated that miR-33a and miR33b, intronic miRNAs located within the sterol regulatory element-binding protein (SREBP) genes, regulate lipid metabolism in concert with their host genes. Here, we show that miR-33b also cooperates with SREBP1 in regulating glucose metabolism by targeting phosphoenolpyruvate carboxykinase (PCK1) and glucose-6-phosphatase (G6PC), key regulatory enzymes of hepatic gluconeogenesis. Overexpression of miR-33b in human hepatic cells inhibits PCK1 and G6PC expression, leading to a significant reduction of glucose production. Importantly, hepatic SREBP1c/miR-33b levels correlate inversely with the expression of PCK1 and G6PC upon glucose infusion in rhesus monkeys. Taken together, these results suggest that miR-33b works in concert with its host gene to ensure a fine-tuned regulation of lipid and glucose homeostasis, highlighting the clinical potential of miR-33a/b as novel therapeutic targets for a range of metabolic diseases. [ABSTRACT FROM AUTHOR]

hsa-miR-33a and hsa-miR-33b, intronic microRNAs (miRNAs) located within the sterol regulatory element-binding protein 2 and 1 genes (Srebp-2 and -1), respectively, have recently been shown to regulate lipid homeostasis in concert with their host genes. Although the functional role of miR-33a and -b has been highly investigated, the role of their passenger strands, miR-33a* and -b*, remains unclear. Here, we demonstrate that miR-33a* and -b* accumulate to steady-state levels in human, mouse, and nonhuman primate tissues and share a similar lipid metabolism target gene network as their sister strands. Analogous to miR-33, miR-33* represses key enzymes involved in cholesterol efflux (ABCA1 and NPC1), fatty acid metabolism (CROT and CPT1a), and insulin signaling (IRS2). Moreover, miR-33* also targets key transcriptional regulators of lipid metabolism, including SRC1, SRC3, NFYC, and RIP140. Importantly, inhibition of either miR-33 or miR-33* rescues target gene expression in cells overex- pressing pre-miR-33. Consistent with this, overexpression of miR-33* reduces fatty acid oxidation in human hepatic cells. Altogether, these data support a regulatory role for the miRNA* species and suggest that miR-33 regulates lipid metabolism through both arms of the miR-33/miR-33* duplex. [ABSTRACT FROM AUTHOR]

Cholesterol homeostasis is among the most intensely regulated processes in biology. Since its isolation from gallstones at the time of the French Revolution, cholesterol has been extensively studied. Insufficient or excessive cellular cholesterol results in pathological processes including atherosclerosis and metabolic syndrome. Mammalian cells obtain cholesterol from the circulation in the form of plasma lipoproteins or intracellularly, through the synthesis of cholesterol from acetyl coenzyme A (acetyl-CoA). This process is tightly regulated at multiple levels. In this review, we provide an overview of the multiple mechanisms by which cellular cholesterol metabolism is regulated. We also discuss the recent advances in the post-transcriptional regulation of cholesterol homeostasis, including the role of small non-coding RNAs (microRNAs). These novel findings may open new avenues for the treatment of dyslipidemias and cardiovascular diseases. [ABSTRACT FROM AUTHOR]

The hepatic low-density lipoprotein receptor (LDLR) pathway is essential for clearing circulating LDL-cholesterol (LDL-C). While the transcriptional regulation of LDLR is well-characterized, the post-transcriptional mechanisms which govern LDLR expression are just beginning to emerge. Here, we developed a high-throughput genome-wide screening assay to systematically identify microRNAs (miRNAs) that regulate LDLR activity in human hepatic cells. From this screen, we characterize miR-148a as a negative regulator of LDLR expression and activity, and define a novel SREBP1-mediated pathway by which miR-148a regulates LDL-C uptake. Importantly, inhibition of miR-148a increases hepatic LDLR expression and decreases plasma LDL-C in vivo. We also provide evidence that miR-148a regulates hepatic ABCA1 expression and circulating HDL-C levels. Collectively, these studies uncover miR-148a as an important regulator of hepatic LDL-C clearance through direct regulation of LDLR expression, and demonstrate the therapeutic potential of inhibiting miR-148a to ameliorate the elevated LDL-C/HDL-C ratio, a prominent risk factor for cardiovascular disease.

Angiopoietin-like 4 (ANGPTL4), a key protein involved in lipoprotein metabolism, has diverse effects. There is an association between Angptl4 and diabetic kidney disease; however, this association has not been well investigated. We show that both podocyte-and tubule-specific ANGPTL4 are crucial fibrogenic molecules in diabetes. Diabetes accelerates the fibrogenic phenotype in control mice but not in ANGPTL4 mutant mice. The protective effect observed in ANGPTL4 mutant mice is correlated with a reduction in stimulator of interferon genes pathway activation, expression of pro-inflammatory cytokines, reduced epithelial-to-mesenchymal transition and endothelial-to-mesenchymal transition, lessened mitochondrial damage, and increased fatty acid oxidation. Mechanistically, we demonstrate that podocyte-or tubule-secreted Angptl4 interacts with Integrin β1 and influences the association between dipeptidyl-4 with Integrin β1. We demonstrate the utility of a targeted pharmacologic therapy that specifically inhibits Angptl4 gene expression in the kidneys and protects diabetic kidneys from proteinuria and fibrosis. Together, these data demonstrate that podocyte-and tubule-derived Angptl4 is fibrogenic in diabetic kidneys. [ABSTRACT FROM AUTHOR]

Recent clinical outcome studies demonstrate that SGLT2 inhibitors (i) significantly reduce major adverse cardiovascular events and heart failure in patients with and without type 2 diabetes; however, the mechanisms by which SGLT2i exert their cardiovascular benefits remain unclear. Here, we aimed to elucidate the acute effects of SGLT2i (dapagliflozin, dapa) on cardiac mitochondrial substrate oxidation. Using LC-MS/MS methodology combined with an infusion of [13C6]glucose and [13C4]βOHB, we assessed the ratio of cardiac mitochondrial pyruvate (VPDH) and βOHB (VβOHB) oxidation rates to rates of mitochondrial citrate synthase flux (VCS) in awake male Sprague Dawley (SD) rats. Metabolic studies were performed 4-6 h after oral dapa (1.5 mg/kg) or vehicle treatment in control rats and after induction of heart failure induced by permanent ligation of the left anterior descending coronary artery. Acute dapa treatment led to marked glycosuria with a 15% reduction in fasting plasma glucose concentrations and 50% increase in whole-body βOHB turnover and plasma βOHB concentrations (all P<0.05 vs. VEH). Dapa caused a 40-60% decrease in myocardial [14C]deoxyglucose uptake and pyruvate oxidation (VPDH/VCS) and a 60% increase in myocardial ketone oxidation (VβOHB/VCS) (all P<0.05 vs. VEH). Similar effects were observed in heart failure rats. Dapa treatment caused a 15% decrease in fasting plasma glucose concentrations, 50% increase in plasma βOHB levels and 60% increase in left ventricular VβOHB/VCS (all P<0.05 vs. VEH). Conclusion: Collectively, these studies demonstrate that dapa shifts both the normal and failing heart to increased mitochondrial ketone oxidation, which may be mediated by increased hepatic ketogenesis and βOHB availability. Disclosure: L. Goedeke: None. X. Zhang: None. R. J. Perry: None. G. Cline: None. L. H. Young: None. G. I. Shulman: Consultant; Self; 89bio, Inc., BridgeBio, Ionis Pharmaceuticals, Maze Therapeutics, Novo Nordisk, Other Relationship; Self; AstraZeneca, Esperion Therapeutics, Inc, Generian Pharmaceuticals, Inc., Gilead Sciences, Inc., iMetabolic Biopharma Corporation, Janssen Research & Development, LLC, Merck & Co., Inc., The Liver Company. J. Lee: None. Y. Ma: None. X. Hu: None. J. Zhang: n/a. J. Dong: None. K. D. Galsgaard: None. N. Guerrera: None. S. Haedersdal: None. Funding: National Institutes of Health (R01DK113984, R01DK045735, K99HL150234); AstraZeneca [ABSTRACT FROM AUTHOR]