Panu K. Luukkonen

The MBOAT7 variant rs641738 alters hepatic phosphatidylinositols and increases severity of non-alcoholic fatty liver disease in humans

The MBOAT7 variant rs641738 alters hepatic phosphatidylinositols and increases severity of non-alcoholic fatty liver disease in humans

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.

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.

Heterogeneity of non‐alcoholic fatty liver disease

It has only recently dawned on us that obesity is even a bigger threat to our liver health than alcohol although those enjoying culinary delights often both overeat and drink. We have learnt that the metabolic syndrome (MetS) develops particularly in those obese individuals, who deposit excess fat in the liver. In this type of NAFLD (‘Obese/Metabolic NAFLD’), insulin does not normally inhibit the production of glucose or VLDL from the liver. This leads to hyperglycaemia and hyperinsulinaemia, and an increase in serum triglycerides, which in turn lowers HDL cholesterol (1). The stimulatory effect of insulin on de novo lipogenesis (DNL) remains insulin sensitive, leading to increased DNL in hyperinsulinaemic subjects (2). Although ‘Metabolic NAFLD’ can occasionally be found in non-obese subjects without predisposing NAFLD genes, overweight and inactivity remain the most important risk factors for this type of NAFLD (Fig. 1). The discovery of the common (30–50% of all subjects) I148M gene variant in PNPLA3 in 2008 in the Dallas Heart Study was groundbreaking. The variant increases liver fat content without having adverse effects on body weight or features of IR or the MetS (3). This finding has been confirmed in at least nine genome-wide association studies and over 100 other studies (1, 4). Impressive progress in genetics of a common disorder. The gene variant has also been shown, even in meta-analyses, to increase the risk of NASH, fibrosis, cirrhosis and hepatocellular carcinoma (4, 5). Its impact on liver disease is not restricted to NAFLD as it also increases steatosis and fibrosis in alcoholic liver disease, hepatitis B and C, Wilson disease and in liver transplant recipients (6–11). In a recent article published in this journal, Park et al. (12) analysed risk factors for NAFLD in a crosssectional study, which included 1363 apparently healthy 30–60-year-old Korean men. Compared to the participants of the population-based Dallas Heart Study (mean BMI 30 kg/m), the Korean men were lean (mean BMI 25 kg/m), in keeping with a greater risk of NAFLD in Asians compared to Europid Americans at any given BMI (1). The I148M gene variant increased the risk of NAFLD by 1.4-fold. The prevalence of NAFLD diagnosed by ultrasound was 47% in those with and 39% in those without the gene variant. Subjects with different genotypes had similar BMI, subcutaneous and visceral fat and IR as measured by HOMA-IR (12), in keeping with the many previous studies showing that the gene variant increases liver fat, but has no effect on body weight or features of IR. Thus, liver fat differs in the face of similar BMI. When Park et al. adjusted the BMIs in the groups differing with respect to the PNPLA3 genotype with liver fat (NAFLD status as measured by ultrasound), the subjects with the gene variant were leaner than those without. Thus, BMI differed when liver fat was forced to be similar. Carriers of the gene variant with NAFLD (‘PNPLA3 NAFLD’) were leaner than those with NAFLD attributable to obesity (‘Metabolic/Obese NAFLD’). The study of Park et al. is important as it emphasizes that non-obese subjects may have NAFLD and that these patients often carry the I148M gene variant. The study does not, however, show that the I148M variant regulates body weight as the indices of adiposity did not differ between the PNPLA3 genotypes in this or previous studies (3, 4). In mice, overexpression of the human I148M gene variant in the liver increases liver triglyceride content, whereas overexpression in adipose tissue has no effect on liver fat content, body weight, fat distribution or adipose tissue triglyceride content, morphology or gene expression (13). The rate of DNL, which produces saturated, IR-inducing fatty acids (14), is lower in homozygous PNPLA3 I148M variant allele compared to non-carriers (15). The circulating profile of triglycerides is markedly different between PNPLA3 I148M carriers with NAFLD compared to subjects that do not carry this gene variant (16). Overexpression of the I148M gene variant in the mouse liver is not accompanied by changes in circulating glucose or insulin or lipid concentrations. In vitro in hepatocytes, Li et al. found the I148M gene variant to decrease the rate of lipolysis of intra-hepatocellular triglycerides., whereas Kumari et al. (17) found the mutated human I148M protein to increase triglyceride synthesis. It is currently unclear whether the I148M variant inhibits triglyceride breakdown or stimulates triglyceride synthesis in vivo in humans or both. The I148M gene variant is truly international. It is associated with NAFLD in Europids, Mexican Americans,

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.