Sanja Sädevirta

Regulation of Angiopoietin-Like Proteins (ANGPTLs) 3 and 8 by Insulin.

OBJECTIVE Circulating ANGPTL8 has recently been used as a marker of insulin action. We studied expression and insulin regulation of ANGPTL8 and ANGPTL3 in vivo and in vitro. DESIGN AND METHODS Expression of ANGPTL8 and ANGPTL3 was studied in 34 paired samples of human liver and adipose tissue. Effects of insulin on 1) plasma concentrations and adipose tissue expression of ANGPTL8 and ANGPTL3 (in vivo 6-h euglycemic hyperinsulinemia; n = 18), and 2) ANGPTL8 and ANGPTL3 gene and protein expression in immortalized human hepatocytes (IHH) and adipocytes were measured. Effect of ANGPTL3 on secretion of ANGPTL8 in cells stably overexpressing ANGPTL3, -8, or both was determined. RESULTS ANGPTL3 was only expressed in the liver, whereas ANGPTL8 was expressed in both tissues. In vivo hyperinsulinemia significantly decreased both plasma ANGPTL8 and ANGPTL3 at 3 and 6 hours. Insulin increased ANGPTL8 expression in human adipose tissue 14- and 18-fold at 3 and 6 hours and ANGPTL8 was the most insulin-responsive transcript on microarray. Insulin also increased ANPGTL8 in cultured adipocytes and IHH but the protein mainly remained intracellular. In vitro in IHH, insulin decreased ANGPTL3 gene expression and secretion of ANGPTL3 into growth medium. Overexpression of ANGPTL8 in CHO cells did not result in its release into culture medium while abundant secretion occurred in cells co-expressing ANGPTL3 and -8. CONCLUSIONS Insulin decreases plasma ANGPTL3 by decreasing ANGPTL3 expression in the liver. Insulin markedly increases ANGPTL8 in adipose tissue and the liver but not in plasma. These data show that measurement of plasma ANGPTL3 but not -8 reflects insulin action in target tissues.

Liver Fat Content and Hepatic Insulin Sensitivity in Overweight Patients With Type 1 Diabetes

OBJECTIVES Patients with type 1 diabetes mellitus (T1DM) lack the portal/peripheral insulin gradient, which might diminish insulin stimulation of hepatic lipogenesis and protect against development of nonalcoholic fatty liver disease (NAFLD). We compared liver fat content and insulin sensitivity of hepatic glucose production and lipolysis between overweight T1DM patients and nondiabetic subjects. MATERIALS AND METHODS We compared 32 overweight adult T1DM patients and 32 nondiabetic subjects matched for age, body mass index (BMI), and gender. Liver fat content was measured using proton magnetic resonance spectroscopy ((1)H-MRS), body composition by magnetic resonance imaging, and insulin sensitivity using the euglycemic-hyperinsulinemic clamp technique (insulin 0.4 mU/kg · min combined with infusion of D-[3-(3)H]glucose). We also hypothesized that low liver fat might protect from obesity-associated increases in insulin requirements and, therefore, determined insulin requirements across BMI categories in 3164 T1DM patients. RESULTS Liver fat content was significantly lower in T1DM patients than in nondiabetic subjects (0.6% [25th-75th quartiles, 0.3%-1.1%] vs 9.0% [3.0%-18.0%]; P < .001). The endogenous rate of glucose production (R(a)) during euglycemic hyperinsulinemia was significantly lower (0.4 [-0.7 to 0.8] mg/kg fat-free mass · min vs 0.9 [0.2-1.6] fat-free mass · min; P = .012) and the percent suppression of endogenous Ra by insulin was significantly greater (89% [78%-112%] vs 77% [50%-94%]; p = .009) in T1DM patients than in nondiabetic subjects. Serum nonesterified fatty acid concentrations during euglycemic hyperinsulinemia were significantly lower (78.5 [33.0-155.0] vs 306 [200.0-438.0] μmol/L; P < .001) and the percent suppression of nonesterified fatty acids significantly higher (89.1% [78.6%-93.3%] vs 51.4% [36.5%-71.1%]; P < .001) in T1DM patients than in nondiabetic subjects. Insulin doses were similar across BMI categories. CONCLUSIONS T1DM patients might be protected from steatosis and hepatic insulin resistance. Obesity may not increase insulin requirements in T1DM.

Continuous Grading of Early Fibrosis in NAFLD Using Label-Free Imaging: A Proof-of- Concept Study

Background and Aims Early detection of fibrosis is important in identifying individuals at risk for advanced liver disease in non-alcoholic fatty liver disease (NAFLD). We tested whether second-harmonic generation (SHG) and coherent anti-Stokes Raman scattering (CARS) microscopy, detecting fibrillar collagen and fat in a label-free manner, might allow automated and sensitive quantification of early fibrosis in NAFLD. Methods We analyzed 32 surgical biopsies from patients covering histological fibrosis stages 0–4, using multimodal label-free microscopy. Native samples were visualized by SHG and CARS imaging for detecting fibrillar collagen and fat. Furthermore, we developed a method for quantitative assessment of early fibrosis using automated analysis of SHG signals. Results We found that the SHG mean signal intensity correlated well with fibrosis stage and the mean CARS signal intensity with liver fat. Little overlap in SHG signal intensities between fibrosis stages 0 and 1 was observed. A specific fibrillar SHG signal was detected in the liver parenchyma outside portal areas in all samples histologically classified as having no fibrosis. This signal correlated with immunohistochemical location of fibrillar collagens I and III. Conclusions This study demonstrates that label-free SHG imaging detects fibrillar collagen deposition in NAFLD more sensitively than routine histological staging and enables observer-independent quantification of early fibrosis in NAFLD with continuous grading.

Novel hepatic microRNAs upregulated in human nonalcoholic fatty liver disease

MicroRNAs (miRNAs) control gene expression by reducing mRNA stability and translation. We aimed to identify alterations in human liver miRNA expression/function in nonalcoholic fatty liver disease (NAFLD). Subjects with the highest (median liver fat 30%, n = 15) and lowest (0%, n = 15) liver fat content were selected from >100 obese patients for miRNA profiling of liver biopsies on microarrays carrying probes for 1438 human miRNAs (a cross‐sectional study). Target mRNAs and pathways were predicted for the miRNAs most significantly upregulated in NAFLD, their cell‐type‐specific expression was investigated by quantitative PCR (qPCR), and the transcriptome of immortalized human hepatocytes (IHH) transfected with the miRNA with the highest number of predicted targets, miR‐576‐5p, was studied. The screen revealed 42 miRNAs up‐ and two downregulated in the NAFLD as compared to non‐NAFLD liver. The miRNAs differing most significantly between the groups, miR‐103a‐2*, miR‐106b, miR‐576‐5p, miRPlus‐I137*, miR‐892a, miR‐1282, miR‐3663‐5p, and miR‐3924, were all upregulated in NAFLD liver. Target pathways predicted for these miRNAs included ones involved in cancer, metabolic regulation, insulin signaling, and inflammation. Consistent transcriptome changes were observed in IHH transfected with miR‐576‐5p, and western analysis revealed a marked reduction of the RAC1 protein belonging to several miR‐576‐5p target pathways. To conclude, we identified 44 miRNAs differentially expressed in NAFLD versus non‐NAFLD liver, 42 of these being novel in the context of NAFLD. The study demonstrates that by applying a novel study set‐up and a broad‐coverage array platform one can reveal a wealth of previously undiscovered miRNA dysregulation in metabolic disease.

Saturated Fat Is More Metabolically Harmful for the Human Liver Than Unsaturated Fat or Simple Sugars

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.

Predictors of Liver Fat and Stiffness in Non-Alcoholic Fatty Liver Disease (NAFLD) – an 11-Year Prospective Study

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.