Despite the high prevalence of nonalcoholic fatty liver disease (NAFLD), little is known of its pathogenesis based on study of human liver samples. By the use of Affymetrix GeneChips (17,601 genes), we investigated gene expression in the human liver of subjects with extreme steatosis due to NAFLD without histological signs of inflammation (liver fat 66.0 +/- 6.8%) and in subjects with low liver fat content (6.4 +/- 2.7%). The data were analyzed by using sequence-based reannotation of Affymetrix probes and a robust model-based normalization method. We identified genes involved in hepatic glucose and lipid metabolism, insulin signaling, inflammation, coagulation, and cell adhesion to be significantly associated with liver fat content. In addition, genes involved in ceramide signaling (MAP2K4) and metabolism (UGCG) were found to be positively associated with liver fat content. Genes involved in lipid metabolism (PLIN, ACADM), fatty acid transport (FABP4, CD36), amino acid catabolism (BCAT1), and inflammation (CCL2) were validated by real-time PCR and were found to be upregulated in subjects with high liver fat content. The data show that multiple changes in gene expression characterize simple steatosis.

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Human genomic DNA fragments containing catechol 0-methyltransferase (COMT) sequences were isolated and the exon-intron structure analysed by sequencing, PCR and comparing to the human COMT cDNA sequences. The gene contains six exons, of which exons 1 and 2 are noncoding. MB-ATG and S-ATG codons, responsible for the initiation of translation of the membranebound (MB) and soluble (S) forms of the enzyme, are located in exon 3. Two distinct COMTspecific transcripts, 1.3 kb and 1.5 kb, were detected in various human tissues and cell lines. Different quantities of the shorter COMT-specific mRNA in the tissues studied suggest a tissue-specific regulation of the COMT gene at transcriptional level. Mapping of the 5' ends of the COMT mRNAs showed that transcription initiates at multiple sites in two separate DNA regions, which are preceded by functional promoter sequences. The proximal promoter (Pl), located between the two translation initiation codons and extending approximately 200 bp upstream of the MB-ATG initiation codon, apparently gives rise to the 1.3-kb S-COMT mRNA (S-mRNA). The distal promoter (P2) is located in a DNA fragment in front of and partly overlapping the transcription-start region of the 1.5-kb transcript, suggesting that it controls the expression of this MB-mRNA. Similarities between the rat and human COMT gene promoters are analyzed.

OBJECTIVE-The objective of this study is to quantitate expression of genes possibly contributing to insulin resistance and fat deposition in the human liver.RESEARCH DESIGN AND METHODS-A total of 24 subjects who had varying amounts of histologically determined fat in the liver ranging from normal (n ϭ 8) to steatosis due to a nonalcoholic fatty liver (NAFL) (n ϭ 16) were studied. The mRNA concentrations of 21 candidate genes associated with fatty acid metabolism, inflammation, and insulin sensitivity were quantitated in liver biopsies using real-time PCR. In addition, the subjects were characterized with respect to body composition and circulating markers of insulin sensitivity. . PPAR␥ coactivator 1 (PGC1) was significantly lower in subjects with NAFL than in those without. Genes significantly associated with obesity included nine genes: plasminogen activator inhibitor 1, PPAR␥, PPAR␦, MCP-1, CCL3 (macrophage inflammatory protein [MIP]-1␣), PPAR␥2, carnitine palmitoyltransferase (CPT1A), FABP4, and FABP5. The following parameters were associated with liver fat independent of obesity: serum adiponectin, insulin, C-peptide, and HDL cholesterol concentrations and the mRNA concentrations of MCP-1, MIP-1␣, ACSL4, FABP4, FABP5, and LPL. RESULTS-TheCONCLUSIONS-Genes involved in fatty acid partitioning and binding, lipolysis, and monocyte/macrophage recruitment and inflammation are overexpressed in the human fatty liver.

Aims/hypothesis It has recently been suggested that the rs738409 G allele in PNPLA3, which encodes adiponutrin, is strongly associated with increased liver fat content in three different ethnic groups. The aims of the present study were as follows: (1) to try to replicate these findings in European individuals with quantitative measures of hepatic fat content; (2) to study whether the polymorphism influences hepatic and adipose tissue insulin sensitivity; and (3) to investigate whether PNPLA3 expression is altered in the human fatty liver. Methods We genotyped 291 Finnish individuals in whom liver fat had been measured using proton magnetic resonance spectroscopy. Hepatic PNPLA3 expression was measured in 32 participants. Hepatic and adipose tissue insulin sensitivities were measured using a euglycaemic-hyperinsulinaemic (insulin infusion 0.3 mU kg −1 min Results The rs738409 G allele in PNPLA3 was associated with increased quantitative measures of liver fat content (p=0.011) and serum aspartate aminotransferase concentrations (p=0.002) independently of age, sex and BMI. Fasting serum insulin and hepatic and adipose tissue insulin sensitivity were related to liver fat content independently of genotype status. PNPLA3 mRNA expression in the liver was positively related to obesity (r=0.62, p<0.0001) and to liver fat content (r=0.58, p=0.025) in participants who were not morbidly obese (BMI <40 kg/m 2 ). Conclusions/interpretation A common variant in PNPLA3 increases the risk of hepatic steatosis in humans.

OBJECTIVE-To determine whether 1) hepatic ceramide and diacylglycerol concentrations, 2) SCD1 activity, and 3) hepatic lipogenic index are increased in the human nonalcoholic fatty liver.RESEARCH DESIGN AND METHODS-We studied 16 subjects with (n ϭ 8) and without (n ϭ 8) histologically determined nonalcoholic fatty liver (NAFL ϩ and NAFL Ϫ ) matched for age, sex, and BMI. Hepatic concentrations of lipids and fatty acids were quantitated using ultra-performance liquid chromatography coupled to mass spectrometry and gas chromatography. RESULTS-The absolute (nmol/mg) hepatic concentrations of diacylglycerols but not ceramides were increased in the NAFL ϩ group compared with the NAFL Ϫ group. The livers of the NAFL ϩ group contained proportionally less long-chain polyunsaturated fatty acids as compared with the NAFL Ϫ group. Liver fat percent was positively related to hepatic stearoyl-CoA desaturase 1 (SCD1) activity index (r ϭ 0.70, P ϭ 0.003) and the hepatic lipogenic index (r ϭ 0.54, P ϭ 0.030). Hepatic SCD1 activity index was positively related to the concentrations of diacylglycerols (r ϭ 0.71, P ϭ 0.002) but not ceramides (r ϭ 0.07, NS).CONCLUSIONS-We conclude that diacylglycerols but not ceramides are increased in NAFL. The human fatty liver is also characterized by depletion of long polyunsaturated fatty acids in the liver and increases in hepatic SCD1 and lipogenic activities. Diabetes 58: [203][204][205][206][207][208] 2009 N onalcoholic fatty liver disease (NAFLD) is characterized by lipid accumulation in the liver (Ն10% of liver weight), which cannot be attributed to alcohol consumption or any other liver disease (1). NAFLD covers a range from simple nonalcoholic fatty liver (NAFL) to nonalcoholic steatohepatitis (NASH) and fibrosis (1). The fatty liver is resistant to the action of insulin to inhibit hepatic glucose (2,3) and VLDL (4) production, resulting in hyperglycemia and hypertriglyceridemia. The mechanisms underlying insulin resistance in human NAFLD are unclear. While triacylglycerols themselves are inert, lipid intermediates may act as important regulators of both oxidative stress (5) and insulin signaling (6). In vitro studies as well as studies in animals suggest that diacylglycerols, which are immediate precursors of triacylglycerols (7), can induce insulin resistance by activating specific isoforms of protein kinase C (PKC) (8,9). The concentrations of diacylglycerols have recently been shown to be increased in human NAFLD compared with subjects with normal liver histology (10). Ceramides are another class of reactive lipids that mediate saturated fat-induced insulin resistance (6). There are no data comparing ceramide and diacylglycerol concentrations in the human liver or relating them to hepatic fat content.Sources of hepatic lipids include dietary chylomicron remnants, free fatty acids released from either adipose tissue triacylglycerols or chylomicrons hydrolyzed at a rate in excess of what can be taken up by tissues (spillover), and de novo lipogenesis (11). Increased lipolysis is a...

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Ceramides may mediate saturated fat–induced insulin resistance, but there are no data comparing ceramide concentrations between human tissues. We therefore performed lipidomic analysis of human subcutaneous (SCfat) and intra‐abdominal (IAfat) adipose tissue, the liver, and serum in eight subjects. The liver contained (nmol/mg tissue) significantly more ceramides (1.5–3‐fold), sphingomyelins (7–8‐fold), phosphatidylethanolamines (10–11‐fold), lysophosphatidylcholines (7–12‐fold), less ether‐linked phosphatidylcholines (2–2.5‐fold) but similar amounts of diacylglycerols as compared to SCfat and IAfat. The amounts of ceramides and their synthetic precursors, such as palmitic (16:0) free fatty acids and sphingomyelins, differed considerably between the tissues. The liver contained proportionally more palmitic, stearic (18:0), and long polyunsaturated fatty acids than adipose tissues. Stearoyl‐CoA desaturase 1 (SCD1) activity reflected by serum, estimated from the 16:1/16:0‐ratio, was closely related to that in the liver (r = 0.86, P = 0.024) but not adipose tissues. This was also true for estimated elongase (18:1/16:1, r = 0.89, P = 0.01), and Δ5 (20:4/20:3, r = 0.89, P = 0.012) and Δ6 (18:3[n‐6]/18:2, r = 1.0, P < 0.001) desaturase activities. We conclude that the human liver contains higher concentrations of ceramides and saturated free fatty acids than either SCfat or IAfat.