Hepatocyte mARC1 promotes fatty liver disease.

Background & Aims: Non-alcoholic fatty liver disease (NAFLD) has a prevalence of ∼25% worldwide, with significant public health consequences yet few effective treatments. Human genetics can help elucidate novel biology and identify targets for new therapeutics. Genetic variants in mitochondrial amidoxime-reducing component 1 (MTARC1) have been associated with NAFLD and liver-related mortality; however, its pathophysiological role and the cell type(s) mediating these effects remain unclear. We aimed to investigate how MTARC1 exerts its effects on NAFLD by integrating human genetics with in vitro and in vivo studies of mARC1 knockdown. Methods: Analyses including multi-trait colocalisation and Mendelian randomisation were used to assess the genetic associations of MTARC1. In addition, we established an in vitro long-term primary human hepatocyte model with metabolic readouts and used the Gubra Amylin NASH (GAN)-diet non-alcoholic steatohepatitis mouse model treated with hepatocyte-specific N-acetylgalactosamine (GalNAc)-siRNA to understand the in vivo impacts of MTARC1. Results: We showed that genetic variants within the MTARC1 locus are associated with liver enzymes, liver fat, plasma lipids, and body composition, and these associations are attributable to the same causal variant (p.A165T, rs2642438 G>A), suggesting a shared mechanism. We demonstrated that increased MTARC1 mRNA had an adverse effect on these traits using Mendelian randomisation, implying therapeutic inhibition of mARC1 could be beneficial. In vitro mARC1 knockdown decreased lipid accumulation and increased triglyceride secretion, and in vivo GalNAc-siRNA-mediated knockdown of mARC1 lowered hepatic but increased plasma triglycerides. We found alterations in pathways regulating lipid metabolism and decreased secretion of 3-hydroxybutyrate upon mARC1 knockdown in vitro and in vivo. Conclusions: Collectively, our findings from human genetics, and in vitro and in vivo hepatocyte-specific mARC1 knockdown support the potential efficacy of hepatocyte-specific targeting of mARC1 for treatment of NAFLD. Impact and implications: We report that genetically predicted increases in MTARC1 mRNA associate with poor liver health. Furthermore, knockdown of mARC1 reduces hepatic steatosis in primary human hepatocytes and a murine NASH model. Together, these findings further underscore the therapeutic potential of targeting hepatocyte MTARC1 for NAFLD.

MLX plays a key role in lipid and glucose metabolism in humans: Evidence from in vitro and in vivo studies.

BACKGROUND AND AIM: Enhanced hepatic de novo lipogenesis (DNL) has been proposed as an underlying mechanism for the development of NAFLD and insulin resistance. Max-like protein factor X (MLX) acts as a heterodimer binding partner for glucose sensing transcription factors and inhibition of MLX or downstream targets has been shown to alleviate intrahepatic triglyceride (IHTG) accumulation in mice. However, its effect on insulin sensitivity remains unclear. As human data is lacking, the aim of the present work was to investigate the role of MLX in regulating lipid and glucose metabolism in primary human hepatocytes (PHH) and in healthy participants with and without MLX polymorphisms. METHODS: PHH were transfected with non-targeting or MLX siRNA to assess the effect of MLX knockdown on lipid and glucose metabolism, insulin signalling and the hepatocellular transcriptome. A targeted association analysis on imputed genotype data for MLX on healthy individuals was undertaken to assess associations between specific MLX SNPs (rs665268, rs632758 and rs1474040), plasma biochemistry, IHTG content, DNL and gluconeogenesis. RESULTS: MLX knockdown in PHH altered lipid metabolism (decreased DNL (p < 0.05), increased fatty acid oxidation and ketogenesis (p < 0.05), and reduced lipid accumulation (p < 0.001)). Additionally, MLX knockdown increased glycolysis, lactate secretion and glucose production (p < 0.001) and insulin-stimulated pAKT levels (p < 0.01) as assessed by transcriptomic, steady-state and dynamic measurements. Consistent with the in vitro data, individuals with the rs1474040-A and rs632758-C variants had lower fasting plasma insulin (p < 0.05 and p < 0.01, respectively) and TG (p < 0.05 and p < 0.01, respectively). Although there was no difference in IHTG or gluconeogenesis, individuals with rs632758 SNP had notably lower hepatic DNL (p < 0.01). CONCLUSION: We have demonstrated using human in vitro and in vivo models that MLX inhibition favored lipid catabolism over anabolism and increased glucose production, despite increased glycolysis and phosphorylation of Akt, suggesting a metabolic mechanism that involves futile cycling.

Large-Scale Functional Genomics Screen to Identify Modulators of Human ß-Cell Insulin Secretion.

Type 2 diabetes (T2D) is a chronic metabolic disorder affecting almost half a billion people worldwide. Impaired function of pancreatic ß-cells is both a hallmark of T2D and an underlying factor in the pathophysiology of the disease. Understanding the cellular mechanisms regulating appropriate insulin secretion has been of long-standing interest in the scientific and clinical communities. To identify novel genes regulating insulin secretion we developed a robust arrayed siRNA screen measuring basal, glucose-stimulated, and augmented insulin secretion by EndoC-ßH1 cells, a human ß-cell line, in a 384-well plate format. We screened 521 candidate genes selected by text mining for relevance to T2D biology and identified 23 positive and 68 negative regulators of insulin secretion. Among these, we validated ghrelin receptor (GHSR), and two genes implicated in endoplasmic reticulum stress, ATF4 and HSPA5. Thus, we have demonstrated the feasibility of using EndoC-ßH1 cells for large-scale siRNA screening to identify candidate genes regulating ß-cell insulin secretion as potential novel drug targets. Furthermore, this screening format can be adapted to other disease-relevant functional endpoints to enable large-scale screening for targets regulating cellular mechanisms contributing to the progressive loss of functional ß-cell mass occurring in T2D.

Retained differentiation capacity of human skeletal muscle satellite cells from spinal cord-injured individuals.

Despite the well-known role of satellite cells in skeletal muscle plasticity, the effect of spinal cord injury on their function in humans remains unknown. We determined whether spinal cord injury affects the intrinsic ability of satellite cells to differentiate and produce metabolically healthy myotubes. We obtained vastus lateralis biopsies from eight spinal cord-injured and six able-bodied individuals. Satellite cells were isolated, grown and differentiated in vitro. Gene expression was measured by quantitative PCR. Abundance of differentiation markers and regulatory proteins was determined by Western blotting. Protein synthesis and fatty acid oxidation were measured by radioactive tracer-based assays. Activated satellite cells (myoblasts) and differentiated myotubes derived from skeletal muscle of able-bodied and spinal cord-injured individuals expressed similar (P > 0.05) mRNA levels of myogenic regulatory factors. Myogenic differentiation factor 1 expression was higher in myoblasts from spinal cord-injured individuals. Desmin and myogenin protein content was increased upon differentiation in both groups, while myotubes from spinal cord-injured individuals contained more type I and II myosin heavy chain. Phosphorylated and total protein levels of Akt-mechanistic target of rapamycin and forkhead box protein O signalling axes and protein synthesis rate in myotubes were similar (P > 0.05) between groups. Additionally, fatty acid oxidation of myotubes from spinal cord-injured individuals was unchanged (P > 0.05) compared to able-bodied controls. Our results indicate that the intrinsic differentiation capacity of satellite cells and metabolic characteristics of myotubes are preserved following spinal cord injury. This may inform potential interventions targeting satellite cell activation to alleviate skeletal muscle atrophy.

Protein kinase N2 regulates AMP kinase signaling and insulin responsiveness of glucose metabolism in skeletal muscle.

Insulin resistance is central to the development of type 2 diabetes and related metabolic disorders. Because skeletal muscle is responsible for the majority of whole body insulin-stimulated glucose uptake, regulation of glucose metabolism in this tissue is of particular importance. Although Rho GTPases and many of their affecters influence skeletal muscle metabolism, there is a paucity of information on the protein kinase N (PKN) family of serine/threonine protein kinases. We investigated the impact of PKN2 on insulin signaling and glucose metabolism in primary human skeletal muscle cells in vitro and mouse tibialis anterior muscle in vivo. PKN2 knockdown in vitro decreased insulin-stimulated glucose uptake, incorporation into glycogen, and oxidation. PKN2 siRNA increased 5'-adenosine monophosphate-activated protein kinase (AMPK) signaling while stimulating fatty acid oxidation and incorporation into triglycerides and decreasing protein synthesis. At the transcriptional level, PKN2 knockdown increased expression of PGC-1α and SREBP-1c and their target genes. In mature skeletal muscle, in vivo PKN2 knockdown decreased glucose uptake and increased AMPK phosphorylation. Thus, PKN2 alters key signaling pathways and transcriptional networks to regulate glucose and lipid metabolism. Identification of PKN2 as a novel regulator of insulin and AMPK signaling may provide an avenue for manipulation of skeletal muscle metabolism.

Human Carboxylesterase 2 Reverses Obesity-Induced Diacylglycerol Accumulation and Glucose Intolerance.

Serine hydrolases are a large family of multifunctional enzymes known to influence obesity. Here, we performed activity-based protein profiling to assess the functional level of serine hydrolases in liver biopsies from lean and obese humans in order to gain mechanistic insight into the pathophysiology of metabolic disease. We identified reduced hepatic activity of carboxylesterase 2 (CES2) and arylacetamide deacetylase (AADAC) in human obesity. In primary human hepatocytes, CES2 knockdown impaired glucose storage and lipid oxidation. In mice, obesity reduced CES2, whereas adenoviral delivery of human CES2 reversed hepatic steatosis, improved glucose tolerance, and decreased inflammation. Lipidomic analysis identified a network of CES2-regulated lipids altered in human and mouse obesity. CES2 possesses triglyceride and diacylglycerol lipase activities and displayed an inverse correlation with HOMA-IR and hepatic diacylglycerol concentrations in humans. Thus, decreased CES2 is a conserved feature of obesity and plays a causative role in the pathogenesis of obesity-related metabolic disturbances.

Altered DNA methylation of glycolytic and lipogenic genes in liver from obese and type 2 diabetic patients.

OBJECTIVE: Epigenetic modifications contribute to the etiology of type 2 diabetes. METHOD: We performed genome-wide methylome and transcriptome analysis in liver from severely obese men with or without type 2 diabetes and non-obese men to discover aberrant pathways underlying the development of insulin resistance. Results were validated by pyrosequencing. RESULT: We identified hypomethylation of genes involved in hepatic glycolysis and insulin resistance, concomitant with increased mRNA expression and protein levels. Pyrosequencing revealed the CpG-site within ATF-motifs was hypomethylated in four of these genes in liver of severely obese non-diabetic and type 2 diabetic patients, suggesting epigenetic regulation of transcription by altered ATF-DNA binding. CONCLUSION: Severely obese non-diabetic and type 2 diabetic patients have distinct alterations in the hepatic methylome and transcriptome, with hypomethylation of several genes controlling glucose metabolism within the ATF-motif regulatory site. Obesity appears to shift the epigenetic program of the liver towards increased glycolysis and lipogenesis, which may exacerbate the development of insulin resistance.

Acute overactive endocannabinoid signaling induces glucose intolerance, hepatic steatosis, and novel cannabinoid receptor 1 responsive genes.

Endocannabinoids regulate energy balance and lipid metabolism by stimulating the cannabinoid receptor type 1 (CB1). Genetic deletion and pharmacological antagonism have shown that CB1 signaling is necessary for the development of obesity and related metabolic disturbances. However, the sufficiency of endogenously produced endocannabinoids to cause hepatic lipid accumulation and insulin resistance, independent of food intake, has not been demonstrated. Here, we show that a single administration of isopropyl dodecylfluorophosphonate (IDFP), perhaps the most potent pharmacological inhibitor of endocannabinoid degradation, increases hepatic triglycerides (TG) and induces insulin resistance in mice. These effects involve increased CB1 signaling, as they are mitigated by pre-administration of a CB1 antagonist (AM251) and in CB1 knockout mice. Despite the strong physiological effects of CB1 on hepatic lipid and glucose metabolism, little is known about the downstream targets responsible for these effects. To elucidate transcriptional targets of CB1 signaling, we performed microarrays on hepatic RNA isolated from DMSO (control), IDFP and AM251/IDFP-treated mice. The gene for the secreted glycoprotein lipocalin 2 (lcn2), which has been implicated in obesity and insulin resistance, was among those most responsive to alterations in CB1 signaling. The expression pattern of IDFP mice segregated from DMSO mice in hierarchal cluster analysis and AM251 pre-administration reduced (>50%) the majority (303 of 533) of the IDFP induced alterations. Pathway analysis revealed that IDFP altered expression of genes involved in lipid, fatty acid and steroid metabolism, the acute phase response, and amino acid metabolism in a CB1-dependent manner. PCR confirmed array results of key target genes in multiple independent experiments. Overall, we show that acute IDFP treatment induces hepatic TG accumulation and insulin resistance, at least in part through the CB1 receptor, and identify novel cannabinoid responsive genes.

VLDL hydrolysis by LPL activates PPAR-alpha through generation of unbound fatty acids.

Recent evidence suggests that lipoproteins serve as circulating reservoirs of peroxisomal proliferator activated receptor (PPAR) ligands that are accessible through lipolysis. The present study was conducted to determine the biochemical basis of PPAR-alpha activation by lipolysis products and their contribution to PPAR-alpha function in vivo. PPAR-alpha activation was measured in bovine aortic endothelial cells following treatment with human plasma, VLDL lipolysis products, or oleic acid. While plasma failed to activate PPAR-alpha, oleic acid performed similarly to VLDL lipolysis products. Therefore, fatty acids are likely to be the PPAR-alpha ligands generated by VLDL lipolysis. Indeed, unbound fatty acid concentration determined PPAR-alpha activation regardless of fatty acid source, with PPAR-alpha activation occurring only at unbound fatty acid concentrations that are unachievable under physiological conditions without lipase action. In mice, a synthetic lipase inhibitor (poloxamer-407) attenuated fasting-induced changes in expression of PPAR-alpha target genes. Apolipoprotein CIII (apoCIII), an endogenous inhibitor of lipoprotein and hepatic lipase, regulated access to the lipoprotein pool of PPAR-alpha ligands, because addition of exogenous apoCIII inhibited, and removal of endogenous apoCIII potentiated, lipolytic PPAR-alpha activation. These data suggest that the PPAR-alpha response is generated by unbound fatty acids released locally by lipase activity and not by circulating plasma fatty acids.

Calorie restriction increases fatty acid synthesis and whole body fat oxidation rates.

Calorie restriction (CR) increases longevity and retards the development of many chronic diseases, but the underlying metabolic signals are poorly understood. Increased fatty acid (FA) oxidation and reduced FA synthesis have been hypothesized to be important metabolic adaptations to CR. However, at metabolic steady state, FA oxidation must match FA intake plus synthesis; moreover, FA intake is low, not high, during CR. Therefore, it is not clear how FA dynamics are altered during CR. Accordingly, we measured food intake patterns, whole body fuel selection, endogenous FA synthesis, and gene expression in mice on CR. Within 2 days of CR being started, a shift to a cyclic, diurnal pattern of whole body FA metabolism occurred, with an initial phase of elevated endogenous FA synthesis [respiratory exchange ratio (RER) >1.10, lasting 4-6 h after food provision], followed by a prolonged phase of FA oxidation (RER = 0.70, lasting 18-20 h). CR mice oxidized four times as much fat per day as ad libitum (AL)-fed controls (367 +/- 19 vs. 97 +/- 14 mg/day, P < 0.001) despite reduced energy intake from fat. This increase in FA oxidation was balanced by a threefold increase in adipose tissue FA synthesis compared with AL. Expression of FA synthase and acetyl-CoA carboxylase mRNA were increased in adipose and liver in a time-dependent manner. We conclude that CR induces a surprising metabolic pattern characterized by periods of elevated FA synthesis alternating with periods of FA oxidation disproportionate to dietary FA intake. This pattern may have implications for oxidative damage and disease risk.

Overactive endocannabinoid signaling impairs apolipoprotein E-mediated clearance of triglyceride-rich lipoproteins.

The endocannabinoid (EC) system regulates food intake and energy metabolism. Cannabinoid receptor type 1 (CB1) antagonists show promise in the treatment of obesity and its metabolic consequences. Although the reduction in adiposity resulting from therapy with CB1 antagonists may not account fully for the concomitant improvements in dyslipidemia, direct effects of overactive EC signaling on plasma lipoprotein metabolism have not been documented. The present study used a chemical approach to evaluate the direct effects of increased EC signaling in mice by inducing acute elevations of endogenously produced cannabinoids through pharmacological inhibition of their enzymatic hydrolysis by isopropyl dodecylfluorophosphonate (IDFP). Acute IDFP treatment increased plasma levels of triglyceride (TG) (2.0- to 3.1-fold) and cholesterol (1.3- to 1.4-fold) in conjunction with an accumulation in plasma of apolipoprotein (apo)E-depleted TG-rich lipoproteins. These changes did not occur in either CB1-null or apoE-null mice, were prevented by pretreatment with CB1 antagonists, and were not associated with reduced hepatic apoE gene expression. Although IDFP treatment increased hepatic mRNA levels of lipogenic genes (Srebp1 and Fas), there was no effect on TG secretion into plasma. Instead, IDFP treatment impaired clearance of an intravenously administered TG emulsion, despite increased postheparin lipoprotein lipase activity. Therefore, overactive EC signaling elicits an increase in plasma triglyceride levels associated with reduced plasma TG clearance and an accumulation in plasma of apoE-depleted TG-rich lipoproteins. These findings suggest a role of CB1 activation in the pathogenesis of obesity-related hypertriglyceridemia and underscore the potential efficacy of CB1 antagonists in treating metabolic disease.