The adipocyte-derived hormone adiponectin regulates glucose and lipid metabolism and influences the risk for developing obesity, type 2 diabetes, and cardiovascular disease. Adiponectin binds to two different seven-transmembrane domain receptors termed AdipoR1 and AdipoR2. To study the physiological importance of these receptors, AdipoR1 gene knockout mice (AdipoR1 Ϫ/Ϫ ) and AdipoR2 gene knockout mice (AdipoR2 Ϫ/Ϫ ) were generated. AdipoR1 Ϫ/Ϫ mice showed increased adiposity associated with decreased glucose tolerance, spontaneous locomotor activity, and energy expenditure. However, AdipoR2Ϫ/Ϫ mice were lean and resistant to high-fat diet-induced obesity associated with improved glucose tolerance and higher spontaneous locomotor activity and energy expenditure and reduced plasma cholesterol levels. Thus, AdipoR1 and AdipoR2 are clearly involved in energy metabolism but have opposing effects. Diabetes 56:583-593, 2007

Objective-We investigated the role of adipocyte differentiation-related protein (ADRP) in triglyceride turnover and in the secretion of very low-density lipoprotein (VLDL) from McA-RH7777 cells and primary rat hepatocytes. Methods and Results-An increase in the expression of ADRP increased triglyceride accumulation in cytosolic lipid droplets and prevented the incorporation of fatty acids into secretable triglycerides, thereby reducing the secretion of triglycerides as well as of apolipoprotein B-100 (apoB-100) and apoB-48 VLDL. The ability of ADRP to block the secretion of apoB-100 VLDL1 decreased with increasing quantities of fatty acids in the medium, indicating a saturable process and emphasizing the importance of sequestering of fatty acids for the effect of ADRP on VLDL secretion. Knockdown (small interfering RNA) of ADRP decreased the pool of cytosolic lipid droplets but increased only the secretion of apoB-48 VLDL1. Additionally, there was an increased flow of fatty acids into ␤-oxidation. Conclusions-ADRP is essential for the accumulation of triglycerides in cytosolic lipid droplets. An increase in ADRP prevents the formation of VLDL by diverting fatty acids from the VLDL assembly pathway into cytosolic triglycerides, whereas a decrease of the protein increases the sorting of fatty acids to ␤-oxidation and promotes the secretion of apoB-48 VLDL1. Key Words: adipose differentiation-related protein Ⅲ cytosolic lipid droplets Ⅲ apolipoproteins B Ⅲ ␤-oxidation Ⅲ small interfering RNA C ytosolic lipid droplets are ubiquitous organelles involved in the storage and turnover of neutral lipids such as triglycerides. Several proteins have been identified on these droplets, the most well known being the PAT domain proteins, 1-3 including the perilipins, adipocyte differentiationrelated protein (ADRP or adipophilin) and Tip 47. ADRP, which is ubiquitously expressed, 4 has a central role in the formation of lipid droplets. 5 These droplets are assembled at the microsomal membrane by an insulin-dependent process 6 that requires phospholipase D1, extracellular signal regulated kinase 2, and the motor protein dynein. 6,7 The assembly process involves the formation of small primordial droplets, 7 which grow in size by a fusion process that is dependent on intact microtubules 8 and dynein. 6 The assembly of very-low density lipoproteins (VLDLs) 9 -12 starts with the cotranslational lipidation of apolipoprotein B-100 (apoB-100), forming a pre-VLDL particle. VLDL2 (Svedberg flotation [sf] units 20 to 60) is formed from pre-VLDL by additional lipidation, 13 whereas VLDL1 (sf 60 to 80) is formed from VLDL2 by a mechanism that is dependent on an ADP ribosylation factor 1-controlled sorting/transport process 14 and involves the addition of a bulk load of lipids to the particle. 12,13 The triglycerides used in this assembly process are largely derived from triglycerides in cytosolic lipid droplets. 15,16 In this article, we demonstrate that an increase in ADRP promotes the storage of triglycerides in cytosolic lipid dropl...

Summary Insulin resistance is a hallmark of diabetes and an unmet clinical need. Insulin inhibits hepatic glucose production and promotes lipogenesis by suppressing FOXO1-dependent activation of G6pase and inhibition of Glucokinase, respectively. The tight coupling of these events poses a dual conundrum: mechanistically, as the FOXO1 corepressor of Glucokinase is unknown; and clinically, as inhibition of glucose production is predicted to increase lipogenesis. Here we report that SIN3A is the insulin-sensitive FOXO1 corepressor of Glucokinase. Genetic ablation of SIN3A abolishes nutrient regulation of Glucokinase, without affecting other FOXO1 target genes, and lowers glycemia without concurrent steatosis. To extend this work, we executed a small molecule screen and discovered selective inhibitors of FOXO-dependent glucose production devoid of lipogenic activity in hepatocytes. In addition to identifying a novel mode of insulin action, these data raise the possibility of developing selective modulators of unliganded transcription factors to dial out adverse effects of insulin sensitizers.

Objective Nonalcoholic fatty liver disease (NAFLD) is becoming a leading cause of advanced chronic liver disease. The progression of NAFLD, including nonalcoholic steatohepatitis (NASH), has a strong genetic component, and the most robust contributor is the patatin-like phospholipase domain-containing 3 ( PNPLA3 ) rs738409 encoding the 148M protein sequence variant. We hypothesized that suppressing the expression of the PNPLA3 148M mutant protein would exert a beneficial effect on the entire spectrum of NAFLD. Methods We examined the effects of liver-targeted GalNAc 3 -conjugated antisense oligonucleotide (ASO)-mediated silencing of Pnpla3 in a knock-in mouse model in which we introduced the human PNPLA3 I148M mutation. Results ASO-mediated silencing of Pnpla3 reduced liver steatosis ( p = 0.038) in homozygous Pnpla3 148M/M knock-in mutant mice but not in wild-type littermates fed a steatogenic high-sucrose diet. In mice fed a NASH-inducing diet, ASO-mediated silencing of Pnpla3 reduced liver steatosis score and NAFLD activity score independent of the Pnpla3 genotype, while reductions in liver inflammation score ( p = 0.018) and fibrosis stage ( p = 0.031) were observed only in the Pnpla3 knock-in 148M/M mutant mice. These responses were accompanied by reduced liver levels of Mcp1 ( p = 0.026) and Timp2 ( p = 0.007) specifically in the mutant knock-in mice. This may reduce levels of chemokine attracting inflammatory cells and increase the collagenolytic activity during tissue regeneration. Conclusion This study provides the first evidence that a Pnpla3 ASO therapy can improve all features of NAFLD, including liver fibrosis, and suppress the expression of a strong innate genetic risk factor, Pnpla3 148M, which may open up a precision medicine approach in NASH.

Background: Non-alcoholic steatohepatitis (NASH) is a severe form of non-alcoholic fatty liver disease (NAFLD) characterised by liver fat accumulation, inflammation and progressive fibrosis. Emerging data indicate that genetic susceptibility increases risks of NAFLD, NASH and NASH-related cirrhosis. Aims:To review NASH genetics and discuss the potential for precision medicine approaches to treatment. Method: PubMed search and inclusion of relevant literature. Results: Single-nucleotide polymorphisms in PNPLA3, TM6SF2, GCKR, MBOAT7 and HSD17B13 are clearly associated with NASH development or progression. These genetic variants are common and have moderate-to-large effect sizes for development of NAFLD, NASH and hepatocellular carcinoma (HCC). The genes play roles in lipid remodelling in lipid droplets, hepatic very low-density lipoprotein (VLDL) secretion and de novo lipogenesis. The PNPLA3 I148M variant (rs738409) has large effects, with approximately twofold increased odds of NAFLD and threefold increased odds of NASH and HCC per allele. Obesity interacts with PNPLA3 I148M to elevate liver fat content and increase rates of NASH. Although the isoleucine-to-methionine substitution at amino acid position 148 of the PNPLA3 enzyme inactivates its lipid remodelling activity, the effect of PNPLA3 I148M results from trans-repression of another lipase (ATGL/PNPLA2) by sequestration of a shared cofactor (CGI-58/ABHD5), leading to decreased hepatic lipolysis and VLDL secretion. In homozygous Pnpla3 I148M knock-in rodent models of NAFLD, targeted PNPLA3 mRNA knockdown reduces hepatic steatosis, inflammation and fibrosis. Conclusion:The emerging genetic and molecular understanding of NASH paves the way for novel interventions, including precision medicines that can modulate the activity of specific genes associated with NASH. and Sanofi. Rohit Loomba is co-founder of Liponexus, Inc and has served as a speaker, a consultant or an advisory board member for

Microsomal triglyceride transfer protein (MTP) is rate-limiting in the assembly and secretion of lipoproteins containing apolipoprotein (apo) B. Previously we demonstrated that Wy 14,643 (Wy), a peroxisome proliferator-activated receptor (PPAR) ␣ agonist, increases apoB-100 secretion despite decreased triglyceride synthesis. In this study, we sought to determine whether PPAR␣ activation increases MTP expression and activity. Treatment with Wy increased hepatic MTP expression and activity in rats and mice and increased MTP expression in primary cultures of rat and mouse hepatocytes. Addition of actinomycin D blocked this increase and the MTP promoter (؊136 to ؉67) containing a conserved DR1 element was activated by Wy, showing that PPAR␣ activates transcription of the gene. Wy did not affect MTP expression in the intestine or in cultured hepatocytes from PPAR␣-null mice. A retinoid X receptor agonist (9-cis-retinoic acid), but not a PPAR␥ agonist (rosiglitazone), increased MTP mRNA expression in cultured hepatocytes from both wild type and PPAR␣-null mice. In rat hepatocytes incubated with Wy, MTP mRNA levels increased between 6 and 24 h, and MTP protein expression and apoB-100 secretion increased between 24 and 72 h. In conclusion, PPAR␣ activation stimulates hepatic MTP expression via increased transcription of the Mtp gene. This effect is paralleled by a change in apoB-100 secretion, indicating that the effect of Wy on apoB-100 secretion is mediated by increased expression of MTP. The peroxisome proliferator-activated receptor (PPAR)1 ␣ is a nuclear receptor that controls the transcription of genes involved in several lipid metabolism pathways, such as ␤-oxidation and fatty acid uptake and transport, as well as lipoprotein production and clearance (1-3). PPAR␣ is expressed in tissues with a high degree of fatty acid catabolism, primarily liver, intestine, and skeletal muscle (1, 2, 4). PPAR␣ heterodimerizes with the retinoid X receptor ␣ (RXR␣), and this complex binds DR1 sequences that constitute PPAR response elements (for reviews, see Refs. 1, 2). The endogenous ligands for PPAR␣ are unsaturated fatty acids and eicosanoids, whereas hypolipidemic fibrates such as Wy 14,643 (Wy) are potent synthetic agonists (5). We recently found that PPAR␣ agonists increased apolipoprotein (apo) B-100 secretion 2-fold but did not change apoB-48 secretion (6). The increase occurred despite decreased triglyceride synthesis and unchanged apoB mRNA editing and could be explained by inhibition of the cotranslational degradation of apoB-100.Microsomal triglyceride transfer protein (MTP) catalyzes the transfer of neutral lipids to apoB and thus has a pivotal role in the assembly of apoB-containing lipoproteins (for reviews, see Refs. 7,8). The 97-kDa MTP protein that confers lipid transfer activity heterodimerizes with protein disulfide isomerase. Mutations in the Mtp gene cause abetalipoproteinemia, which is characterized by the inability to secrete apoB-containing lipoproteins (7,8). MTP influences apoB secretion through its eff...

Peroxisome proliferator-activated receptor (PPAR) alpha is a nuclear receptor that is mainly expressed in tissues with a high degree of fatty acid oxidation such as liver, heart, and skeletal muscle. Unsaturated fatty acids, their derivatives, and fibrates activate PPARalpha. Male rats are more responsive to fibrates than female rats. We therefore wanted to investigate if there is a sex difference in PPARalpha expression. Male rats had higher levels of hepatic PPARalpha mRNA and protein than female rats. Fasting increased hepatic PPARalpha mRNA levels to a similar degree in both sexes. Gonadectomy of male rats decreased PPARalpha mRNA expression to similar levels as in intact and gonadectomized female rats. Hypophysectomy increased hepatic PPARalpha mRNA and protein levels. The increase in PPARalpha mRNA after hypophysectomy was more pronounced in females than in males. GH treatment decreased PPARalpha mRNA and protein levels, but the sex-differentiated secretory pattern of GH does not determine the sex-differentiated expression of PPARalpha. The expression of PPARalpha mRNA in heart or soleus muscle was not influenced by gender, gonadectomy, hypophysectomy, or GH treatment. In summary, pituitary-dependent hormones specifically regulate hepatic PPARalpha expression. Sex hormones regulate the sex difference in hepatic PPARalpha levels, but not via the sexually dimorphic GH secretory pattern.

Glycerol-3-phosphate acyltransferase (GPAT) catalyses the first committed step in glycerolipid biosynthesis. The mitochondrial isoform (mtGPAT) is mainly expressed in liver, where it is highly regulated, indicating that mtGPAT may have a unique role in hepatic fatty acid metabolism. Because both mtGPAT and carnitine palmitoyl transferase-1 are located on the outer mitochondrial membrane, we hypothesized that mtGPAT directs fatty acyl-CoA away from ␤ -oxidation and toward glycerolipid synthesis. Adenoviralmediated overexpression of murine mtGPAT in primary cultures of rat hepatocytes increased mtGPAT activity 2.7-fold with no compensatory effect on microsomal GPAT activity. MtGPAT overexpression resulted in a dramatic 80% reduction in fatty acid oxidation and a significant increase in hepatic diacylglycerol and phospholipid biosynthesis. Following lipid loading of the cells, intracellular triacylglycerol biosynthesis was also induced by mtGPAT overexpression. Changing an invariant aspartic acid residue to a glycine [D235G] in mtGPAT resulted in an inactive enzyme, which helps define the active site required for mammalian mtGPAT function. To determine if obesity increases hepatic mtGPAT activity, two models of rodent obesity were examined and shown to have Ͼ 2-fold increased enzyme activity.Overall, these results support the concept that increased hepatic mtGPAT activity associated with obesity positively contributes to lipid disorders by reducing oxidative processes and promoting de novo glycerolipid synthesis. -Lindén, D., L. William-Olsson, M. Rhedin, A-K. Asztély, J. C. Clapham, and S. Schreyer. Overexpression of mitochondrial GPAT in rat hepatocytes leads to decreased fatty acid oxidation and increased glycerolipid biosynthesis.