OBJECTIVE Nonalcoholic fatty liver disease (i.e., increased intrahepatic triglyceride [IHTG] content), predisposes to type 2 diabetes and cardiovascular disease. Adipose tissue lipolysis and hepatic de novo lipogenesis (DNL) are the main pathways contributing to IHTG. We hypothesized that dietary macronutrient composition influences the pathways, mediators, and magnitude of weight gain-induced changes in IHTG. RESEARCH DESIGN AND METHODS We overfed 38 overweight subjects (age 48 ± 2 years, BMI 31 ± 1 kg/m2, liver fat 4.7 ± 0.9%) 1,000 extra kcal/day of saturated (SAT) or unsaturated (UNSAT) fat or simple sugars (CARB) for 3 weeks. We measured IHTG (1H-MRS), pathways contributing to IHTG (lipolysis ([2H5]glycerol) and DNL (2H2O) basally and during euglycemic hyperinsulinemia), insulin resistance, endotoxemia, plasma ceramides, and adipose tissue gene expression at 0 and 3 weeks. RESULTS Overfeeding SAT increased IHTG more (+55%) than UNSAT (+15%, P < 0.05). CARB increased IHTG (+33%) by stimulating DNL (+98%). SAT significantly increased while UNSAT decreased lipolysis. SAT induced insulin resistance and endotoxemia and significantly increased multiple plasma ceramides. The diets had distinct effects on adipose tissue gene expression. CONCLUSIONS Macronutrient composition of excess energy influences pathways of IHTG: CARB increases DNL, while SAT increases and UNSAT decreases lipolysis. SAT induced the greatest increase in IHTG, insulin resistance, and harmful ceramides. Decreased intakes of SAT could be beneficial in reducing IHTG and the associated risk of diabetes.

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Non-alcoholic fatty liver disease (NAFLD) covers a spectrum of liver disease from simple steatosis to non-alcoholic steatohepatitis (NASH) and cirrhosis. NAFLD is commonly associated with features of the metabolic/insulin resistance syndrome ('Metabolic/Obese NAFLD') and may therefore predict type 2 diabetes (T2DM). For this review, we searched for prospective studies examining whether NAFLD predicts T2DM, and if so, whether this occurs independently of factors such as age and obesity. These studies included NAFLD diagnosed by ultrasonography (n = 6) or liver enzymes (n = 14). All ultrasonography studies found NAFLD to predict the risk of T2DM independently of age, and in 4 out of 6 studies NAFLD was also a predictor independently of BMI. NAFLD was a predictor of T2DM in all 14 studies where NAFLD was diagnosed by liver enzymes. In 12 of these studies, ALT or AST or GGT were significant predictors of T2DM risk, independently of age and BMI. NAFLD, however, is heterogeneous and may also be caused by common genetic variants. The I148M variant in PNPLA3 and the E167K variant in TM6SF2 are both associated with increased liver fat content, but not features of the metabolic/insulin resistance syndrome. These genetic forms of NAFLD predict NASH and cirrhosis but not T2DM. Taken together these data imply that 'Metabolic/Obese NAFLD' predicts T2DM independently of age and obesity and support the role of hepatic insulin resistance in the pathogenesis of this disease.

Aims/hypothesis The rs738409 C>G single-nucleotide polymorphism in PNPLA3 leads to a missense mutation (I148M) which increases liver fat but does not cause insulin resistance. We hypothesised that patients with non-alcoholic fatty liver disease (NAFLD) due to the PNPLA3 variant ('PNPLA3 NAFLD'=PNPLA3-148MM) do not have adipose tissue (AT) inflammation in contrast with those with NAFLD due to obesity ('obese NAFLD'). Methods Biopsy specimens of AT were taken, and PNPLA3 genotype and liver fat ( 1 H-magnetic resonance spectroscopy) were determined in 82 volunteers, who were divided into groups based on either median BMI (obese 36.2± 0.7 kg/m 2 ; non-obese 26.0±0.4 kg/m 2 ) or PNPLA3 genotype. All groups were similar with respect to age and sex.The PNPLA3 subgroups were equally obese (PNPLA3-148MM, 31.1 ± 1.3 kg/m 2 ; PNPLA3-148II, 31.2 ± 0.8 kg/m 2 ), while the obese and non-obese subgroups had similar PNPLA3 genotype distribution. Gene expression of proinflammatory (MCP-1, CD68) and anti-inflammatory (Twist1, ADIPOQ) markers was measured using quantitative real-time RT-PCR. Results Liver fat was similarly increased in obese NAFLD (9.5±1.3% vs 5.1±0.9%, obese vs non-obese, p=0.007) and PNPLA3 NAFLD (11.4± 1.7% vs 5.3± 0.8%, PNPLA3-148MM vs PNPLA3-148II, p<0.001). Fasting serum insulin was higher in the obese than the non-obese group (76±6 vs 47±6 pmol/l, p<0.001), but similar in PNPLA3-148MM and PNPLA3-148II (60±8 vs 62±5 pmol/l, NS). In obese vs non-obese, MCP-1 and CD68 mRNAs were upregulated, whereas those of Twist1 and ADIPOQ were significantly downregulated. AT gene expression of MCP-1, CD68, Twist1 and ADIPOQ was similar in PNPLA3-148MM and PNPLA3-148II groups. Conclusions/interpretation PNPLA3 NAFLD is characterised by an increase in liver fat but no insulin resistance or AT inflammation, while obese NAFLD has all three of these features. Keywords

Liver fat can be non-invasively measured by proton magnetic resonance spectroscopy (1H-MRS) and fibrosis estimated as stiffness using transient elastography (FibroScan). There are no longitudinal data on changes in liver fat in Europids or on predictors of liver stiffness using these methods. We determined liver fat (1H-MRS) and clinical characteristics including features of insulin resistance at baseline and after a median follow-up period of 11.3 (range 7.3–13.4) years in 97 Finnish subjects. Liver stiffness was measured at 11.3 years. Liver fat content decreased by 5% (p < 0.05) over time. Values at baseline and 11.3 years were closely interrelated (r = 0.81, p < 0.001). Baseline liver fat (OR 1.32; 95%CI: 1.15–1.50) and change in BMI (OR 1.67; 95%CI: 1.24–2.25) were independent predictors of liver fat at 11.3 years (AUROC 0.90; 95%CI: 0.83–0.96). Baseline liver fat (AUROC 0.84; 95%CI: 0.76–0.92) predicted liver fat at 11.3 years more accurately than routinely available parameters (AUROC 0.76; 95%CI: 0.65–0.86, p = 0.02). At 11.3 years, 29% of the subjects had increased liver stiffness. Baseline liver fat (OR 2.17; 95%CI: 1.05–4.46) was an independent predictor of increased liver stiffness. These data show that liver fat is more important than the associated metabolic abnormalities as the predictor of future liver fat and fibrosis.

Increased liver fat may be caused by insulin resistance and adipose tissue inflammation or by the common I148M variant in PNPLA3 at rs738409, which lacks both of these features. We hypothesised that obesity/insulin resistance rather than liver fat increases circulating coagulation factor activities. We measured plasma prothrombin time (PT, Owren method), activated partial thromboplastin time (APTT), activities of several coagulation factors, VWF:RCo and fibrinogen, and D-dimer concentration in 92 subjects divided into groups based on insulin sensitivity [insulin-resistant ('IR') versus insulin-sensitive ('IS')] and PNPLA3 genotype (PNPLA3 vs PNPLA3). Liver fat content (H-MRS) was similarly increased in 'IR' (13 ± 1 %) and PNPLA3 (12 ± 2 %) as compared to 'IS' (6 ± 1 %, p<0.05) and PNPLA3 (8 ± 1 %, p<0.05), respectively. FVIII, FIX, FXIII, fibrinogen and VWF:RCo activities were increased, and PT and APTT shortened in 'IR' versus 'IS', in contrast to these factors being similar between PNPLA3 and PNPLA3 groups. In subjects undergoing a liver biopsy and entirely lacking the I148M variant, insulin-resistant subjects had higher hepatic expression of F8, F9 and FGG than equally obese insulin-sensitive subjects. Expression of pro-inflammatory genes in adipose tissue correlated positively with PT (% of normal), circulating FVIII, FIX, FXI, VWR:RCo and fibrinogen, and expression of anti-inflammatory genes negatively with PT (%), FIX and fibrinogen. We conclude that obesity/insulin resistance rather than an increase in liver fat is associated with a procoagulant plasma profile. This reflects adipose tissue inflammation and increased hepatic production of coagulation factors and their susceptibility for activation.

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