Jukka Westerbacka

Overproduction of large VLDL particles is driven by increased liver fat content in man

Aims/hypothesisWe determined whether hepatic fat content and plasma adiponectin concentration regulate VLDL1 production.MethodsA multicompartment model was used to simultaneously determine the kinetic parameters of triglycerides (TGs) and apolipoprotein B (ApoB) in VLDL1 and VLDL2 after a bolus of [2H3]leucine and [2H5]glycerol in ten men with type 2 diabetes and in 18 non-diabetic men. Liver fat content was determined by proton spectroscopy and intra-abdominal fat content by MRI.ResultsUnivariate regression analysis showed that liver fat content, intra-abdominal fat volume, plasma glucose, insulin and HOMA-IR (homeostasis model assessment of insulin resistance) correlated with VLDL1 TG and ApoB production. However, only liver fat and plasma glucose were significant in multiple regression models, emphasising the critical role of substrate fluxes and lipid availability in the liver as the driving force for overproduction of VLDL1 in subjects with type 2 diabetes. Despite negative correlations with fasting TG levels, liver fat content, and VLDL1 TG and ApoB pool sizes, adiponectin was not linked to VLDL1 TG or ApoB production and thus was not a predictor of VLDL1 production. However, adiponectin correlated negatively with the removal rates of VLDL1 TG and ApoB.Conclusions/interpretationWe propose that the metabolic effect of insulin resistance, partly mediated by depressed plasma adiponectin levels, increases fatty acid flux from adipose tissue to the liver and induces the accumulation of fat in the liver. Elevated plasma glucose can further increase hepatic fat content through multiple pathways, resulting in overproduction of VLDL1 particles and leading to the characteristic dyslipidaemia associated with type 2 diabetes.

Gene expression in human NAFLD

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.

Genes Involved in Fatty Acid Partitioning and Binding, Lipolysis, Monocyte/Macrophage Recruitment, and Inflammation Are Overexpressed in the Human Fatty Liver of Insulin-Resistant Subjects

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 = 8) to steatosis due to a nonalcoholic fatty liver (NAFL) (n = 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. RESULTS—The following genes were significantly upregulated in NAFL: peroxisome proliferator–activated receptor (PPAR)γ2 (2.8-fold), the monocyte-attracting chemokine CCL2 (monocyte chemoattractant protein [MCP]-1, 1.8-fold), and four genes associated with fatty acid metabolism (acyl-CoA synthetase long-chain family member 4 [ACSL4] [2.8-fold], fatty acid binding protein [FABP]4 [3.9-fold], FABP5 [2.5-fold], and lipoprotein lipase [LPL] [3.6-fold]). PPARγ 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, PPARγ, PPARδ, MCP-1, CCL3 (macrophage inflammatory protein [MIP]-1α), PPARγ2, 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-1α, ACSL4, FABP4, FABP5, and LPL. CONCLUSIONS—Genes involved in fatty acid partitioning and binding, lipolysis, and monocyte/macrophage recruitment and inflammation are overexpressed in the human fatty liver.

Adipose tissue inflammation and increased ceramide content characterize subjects with high liver fat content independent of obesity.

OBJECTIVE— We sought to determine whether adipose tissue is inflamed in individuals with increased liver fat (LFAT) independently of obesity. RESEARCH DESIGN AND METHODS— A total of 20 nondiabetic, healthy, obese women were divided into normal and high LFAT groups based on their median LFAT level (2.3 ± 0.3 vs. 14.4 ± 2.9%). Surgical subcutaneous adipose tissue biopsies were studied using quantitative PCR, immunohistochemistry, and a lipidomics approach to search for putative mediators of insulin resistance and inflammation. The groups were matched for age and BMI. The high LFAT group had increased insulin (P = 0.0025) and lower HDL cholesterol (P = 0.02) concentrations. RESULTS— Expression levels of the macrophage marker CD68, the chemokines monocyte chemoattractant protein-1 and macrophage inflammatory protein-1α, and plasminogen activator inhibitor-1 were significantly increased, and those of peroxisome proliferator–activated receptor-γ and adiponectin decreased in the high LFAT group. CD68 expression correlated with the number of macrophages and crown-like structures (multiple macrophages fused around dead adipocytes). Concentrations of 154 lipid species in adipose tissue revealed several differences between the groups, with the most striking being increased concentrations of triacylglycerols, particularly long chain, and ceramides, specifically Cer(d18:1/24:1) (P = 0.01), in the high LFAT group. Expression of sphingomyelinases SMPD1 and SMPD3 were also significantly increased in the high compared with normal LFAT group. CONCLUSIONS— Adipose tissue is infiltrated with macrophages, and its content of long-chain triacylglycerols and ceramides is increased in subjects with increased LFAT compared with equally obese subjects with normal LFAT content. Ceramides or their metabolites could contribute to adverse effects of long-chain fatty acids on insulin resistance and inflammation.

Overproduction of VLDL1 Driven by Hyperglycemia Is a Dominant Feature of Diabetic Dyslipidemia

Objective—We sought to compare the synthesis and metabolism of VLDL1 and VLDL2 in patients with type 2 diabetes mellitus (DM2) and nondiabetic subjects. Methods and Results—We used a novel multicompartmental model to simultaneously determine the kinetics of apolipoprotein (apo) B and triglyceride (TG) in VLDL1 and VLDL2 after a bolus injection of [2H3]leucine and [2H5]glycerol and to follow the catabolism and transfer of the lipoprotein particles. Our results show that the overproduction of VLDL particles in DM2 is explained by enhanced secretion of VLDL1 apoB and TG. Direct production of VLDL2 apoB and TG was not influenced by diabetes per se. The production rates of VLDL1 apoB and TG were closely related, as were the corresponding pool sizes. VLDL1 and VLDL2 compositions did not differ in subjects with DM2 and controls, and the TG to apoB ratio of newly synthesized particles was very similar in the 2 groups. Plasma glucose, insulin, and free fatty acids together explained 55% of the variation in VLDL1 TG production rate. Conclusion—Insulin resistance and DM2 are associated with excess hepatic production of VLDL1 particles similar in size and composition to those in nondiabetic subjects. We propose that hyperglycemia is the driving force that aggravates overproduction of VLDL1 in DM2.

Increased fat accumulation in the liver in HIV-infected patients with antiretroviral therapy-associated lipodystrophy

Objective: To determine liver fat content in patients with highly active antiretroviral therapy (HAART)-associated lipodystrophy. Background: Lipodystrophy in several animal models is associated with fat accumulation in insulin-sensitive tissues, such as the liver. This causes hyperinsulinaemia, dyslipidaemia and other features of insulin resistance. Design: A cross-sectional study. Subjects and methods: Three age- and weight-matched groups were compared: 25 HIV-positive men with HAART-associated lipodystrophy (HAART+LD+), nine HIV-positive men receiving HAART, but without lipodystrophy (HAART+LD−), and 35 HIV-negative healthy men (HIV−). Liver fat content was measured using proton spectroscopy. Intra-abdominal and subcutaneous fat were determined using magnetic resonance imaging. Results: Liver fat content was significantly higher in the HAART+LD+ (8 ± 10%) than the HIV− (5 ± 7%; P < 0.05) or the HAART+LD− (3 ± 5%; P < 0.01) group. Liver fat content correlated with serum fasting insulin in the HAART+LD+ (r = 0.47; P < 0.05) and HIV− groups (r = 0.65; P < 0.001), but not with the amount of intra-abdominal fat. Within the HAART+LD+ group, serum insulin did not correlate with the amount of intra-abdominal fat. The HAART+LD+ group had a lower serum leptin concentration when compared to the two other groups. Features of insulin resistance, including hepatic fat accumulation, were not found in HAART+LD− group. Conclusions: The severity of the insulin resistance syndrome in patients with HAART-associated lipodystrophy is related to the extent of fat accumulation in the liver rather than in the intra-abdominal region. Fat accumulation in the liver may therefore play a causative role in the development of insulin resistance in these patients.

Hepatic Stearoyl-CoA Desaturase (SCD)-1 Activity and Diacylglycerol but Not Ceramide Concentrations Are Increased in the Nonalcoholic Human Fatty Liver

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 = 8) and without (n = 8) histologically determined nonalcoholic fatty liver (NAFL+ and NAFL−) 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+ group compared with the NAFL− group. The livers of the NAFL+ group contained proportionally less long-chain polyunsaturated fatty acids as compared with the NAFL− group. Liver fat percent was positively related to hepatic stearoyl-CoA desaturase 1 (SCD1) activity index (r = 0.70, P = 0.003) and the hepatic lipogenic index (r = 0.54, P = 0.030). Hepatic SCD1 activity index was positively related to the concentrations of diacylglycerols (r = 0.71, P = 0.002) but not ceramides (r = 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.

Acute suppression of VLDL1 secretion rate by insulin is associated with hepatic fat content and insulin resistance

Aims/hypothesisOverproduction of VLDL1 seems to be the central pathophysiological feature of the dyslipidaemia associated with type 2 diabetes. We explored the relationship between liver fat and suppression of VLDL1 production by insulin in participants with a broad range of liver fat content.MethodsA multicompartmental model was used to determine the kinetic parameters of apolipoprotein B and TG in VLDL1 and VLDL2 after a bolus of [2H3]leucine and [2H5]glycerol during a hyperinsulinaemic–euglycaemic clamp in 20 male participants: eight with type 2 diabetes and 12 control volunteers. The participants were divided into two groups with low or high liver fat. All participants with diabetes were in the high liver-fat group.ResultsThe results showed a rapid drop in VLDL1-apolipoprotein B and -triacylglycerol secretion in participants with low liver fat during the insulin infusion. In contrast, participants with high liver fat showed no significant change in VLDL1 secretion. The VLDL1 suppression following insulin infusion correlated with the suppression of NEFA, and the ability of insulin to suppress the plasma NEFA was impaired in participants with high liver fat. A novel finding was an inverse response between VLDL1 and VLDL2 secretion in participants with low liver fat: VLDL1 secretion decreased acutely after insulin infusion whereas VLDL2 secretion increased.Conclusions/interpretationInsulin downregulates VLDL1 secretion and increases VLDL2 secretion in participants with low liver fat but fails to suppress VLDL1 secretion in participants with high liver fat, resulting in overproduction of VLDL1. Thus, liver fat is associated with lack of VLDL1 suppression in response to insulin.

Diminished Wave Reflection in the Aorta: A Novel Physiological Action of Insulin on Large Blood Vessels

Epidemiological data suggest that insulin may have direct effects on large-vessel function, but thus far insulin has only been shown, after prolonged infusions, to slowly decrease peripheral vascular resistance by increasing muscle blood flow. We determined whether physiological doses of insulin affect function of large arteries, before any changes in peripheral blood flow, in vivo using pulse wave analysis. Nine normal men were studied on 2 occasions: once during a 6-hour infusion of saline and once under normoglycemic hyperinsulinemic conditions (sequential 2-hour insulin infusions of 1, 2, and 5 mU/kg. min). Central aortic pressure waves were synthesized from those recorded in the periphery with the use of applanation tonometry and a validated reverse transfer function every 30 minutes. This allowed determination of central aortic augmentation (the pressure difference between early and late systolic pressure peaks) and augmentation index (augmentation expressed as a percentage of pulse pressure). Both augmentation and augmentation index decreased significantly within 1 hour after administration of insulin (P<0.001) but not saline. Systolic and diastolic blood pressure and heart rate remained unchanged for the first 2 hours. A significant increase in peripheral (forearm) blood flow was not observed until 2.5 hours after start of the insulin infusion. These data demonstrate that insulin, in normal subjects, rapidly decreases wave reflection in the aorta. This beneficial effect is consistent with increased distensibility or vasodilatation of large arteries. In contrast to the effect of insulin on peripheral blood flow, this action of insulin is observed under conditions in which both the insulin dose and duration of insulin exposure are physiological. Resistance to this action of insulin could provide a mechanism linking insulin resistance and conditions such as hypertension at the level of large arteries.

Intra-adipose sex steroid metabolism and body fat distribution in idiopathic human obesity

Objective  Causes of visceral fat accumulation include glucocorticoid excess or decreased oestrogen/androgen ratio either in plasma or within adipose tissue. In obese subjects, the intra‐adipose cortisol‐generating enzyme 11β‐hydroxysteroid dehydrogenase type 1 (11β‐HSD1) is increased, but information on sex steroid signalling is sparse. We aimed to test associations between body fat or fat distribution and mRNA transcript levels for androgen and oestrogen receptors and for enzymes metabolizing sex steroids in adipose tissue.