The recently developed histological scoring system for non-alcoholic fatty liver disease (NAFLD) by the NASH Clinical Research Network (NASH-CRN) has been widely used in clinical settings, but is increasingly employed in preclinical research as well. However, it has not been systematically analyzed whether the human scoring system can directly be converted to preclinical rodent models. To analyze this, we systematically compared human NAFLD liver pathology, using human liver biopsies, with liver pathology of several NAFLD mouse models. Based upon the features pertaining to mouse NAFLD, we aimed at establishing a modified generic scoring system that is applicable to broad spectrum of rodent models. The histopathology of NAFLD was analyzed in several different mouse models of NAFLD to define generic criteria for histological assessment (preclinical scoring system). For validation of this scoring system, 36 slides of mouse livers, covering the whole spectrum of NAFLD, were blindly analyzed by ten observers. Additionally, the livers were blindly scored by one observer during two separate assessments longer than 3 months apart. The criteria macrovesicular steatosis, microvesicular steatosis, hepatocellular hypertrophy, inflammation and fibrosis were generally applicable to rodent NAFLD. The inter-observer reproducibility (evaluated using the Intraclass Correlation Coefficient) between the ten observers was high for the analysis of macrovesicular steatosis and microvesicular steatosis (ICC = 0.784 and 0.776, all p<0.001, respectively) and moderate for the analysis of hypertrophy and inflammation (ICC = 0.685 and 0.650, all p<0.001, respectively). The intra-observer reproducibility between the different observations of one observer was high for the analysis of macrovesicular steatosis, microvesicular steatosis and hypertrophy (ICC = 0.871, 0.871 and 0.896, all p<0.001, respectively) and very high for the analysis of inflammation (ICC = 0.931, p<0.001). We established a simple NAFLD scoring system with high reproducibility that is applicable for different rodent models and for all stages of NAFLD etiology.

OBJECTIVE—We recently showed that intracerebroventricular infusion of neuropeptide Y (NPY) hampers inhibition of endogenous glucose production (EGP) by insulin in mice. The downstream mechanisms responsible for these effects of NPY remain to be elucidated. Therefore, the aim of this study was to establish whether intracerebroventricular NPY administration modulates the suppressive action of insulin on EGP via hepatic sympathetic or parasympathetic innervation. RESEARCH DESIGN AND METHODS—The effects of a continuous intracerebroventricular infusion of NPY on glucose turnover were determined in rats during a hyperinsulinemic-euglycemic clamp. Either rats were sham operated, or the liver was sympathetically (hepatic sympathectomy) or parasympathetically (hepatic parasympathectomy) denervated. RESULTS—Sympathectomy or parasympathectomy did not affect the capacity of insulin to suppress EGP in intracerebroventricular vehicle–infused animals (50 ± 8 vs. 49 ± 6 vs. 55 ± 6%, in hepatic sympathectomy vs. hepatic parasympathectomy vs. sham, respectively). Intracerebroventricular infusion of NPY significantly hampered the suppression of EGP by insulin in sham-denervated animals (29 ± 9 vs. 55 ± 6% for NPY/sham vs. vehicle/sham, respectively, P = 0.038). Selective sympathetic denervation of the liver completely blocked the effect of intracerebroventricular NPY administration on insulin action to suppress EGP (NPY/hepatic sympathectomy, 57 ± 7%), whereas selective parasympathetic denervation had no effect (NPY/hepatic parasympathectomy, 29 ± 7%). CONCLUSIONS—Intracerebroventricular administration of NPY acutely induces insulin resistance of EGP via activation of sympathetic output to the liver.

Non-alcoholic steatohepatitis (NASH) has become one of the most common liver diseases and is still without approved pharmacotherapy. Lifestyle interventions using exercise and diet change remain the current treatment of choice and even a small weight loss (5-7%) can already have a beneficial effect on NASH. However, the underlying molecular mechanisms of exercise and diet interventions remain largely elusive, and it is unclear whether they exert their health effects via similar or different pathways. Ldlr-/-.Leiden mice received a high fat diet (HFD) for 30 weeks to establish a severe state of NASH/fibrosis with simultaneous atherosclerosis development. Groups of mice were then either left untreated (control group) or were treated for 20 weeks with exercise (running wheel), diet change (switch to a low fat chow diet) or the combination thereof. The liver and distant organs including heart, white adipose tissue (WAT) and muscle were histologically examined. Comprehensive transcriptome analysis of liver, WAT and muscle revealed the organ-specific effects of exercise and diet and defined the underlying pathways. Exercise and dietary change significantly reduced body weight, fat mass, adipocyte size and improved myosteatosis and muscle function with additive effects of combination treatment. WAT inflammation was significantly improved by diet change, tended to be reduced with exercise, and combination therapy had no additive effect. Hepatic steatosis and inflammation were almost fully reversed by exercise and diet change, while hepatic fibrosis tended to be improved with exercise and was significantly improved with diet change. Additive effects for the combination therapy were shown for liver steatosis and associated liver lipids, and atherosclerosis, but not for hepatic inflammation and fibrosis. Pathway analysis revealed complementary effects on metabolic pathways and lipid handling processes, thereby substantiating the added value of combined lifestyle treatment. Exercise, diet change and the combination thereof can reverse established NASH/fibrosis in obese Ldlr-/-.Leiden mice. In addition, the lifestyle interventions had beneficial effects on atherosclerosis, WAT inflammation and muscle function. For steatosis and other parameters related to adiposity or lipid metabolism, exercise and dietary change affected more distinct pathways that acted complementary when the interventions were combined resulting in an additive effect for the combination therapy on important endpoints including NASH and atherosclerosis. For inflammation, exercise and diet change shared several underlying pathways resulting in a net similar effect when the interventions were combined.

Non-alcoholic steatohepatitis (NASH) is a fast-growing liver disorder that is associated with an increased incidence of cardiovascular disease and type 2 diabetes. Animal models adequately mimicking this condition are scarce. We herein investigate whether Ldlr−/−. Leiden mice on different high-fat diets represent a suitable NASH model. Ldlr−/−. Leiden mice were fed a healthy chow diet or fed a high-fat diet (HFD) containing lard or a fast food diet (FFD) containing milk fat. Additionally, the response to treatment with obeticholic acid (OCA) was evaluated. Both high-fat diets induced obesity, hyperlipidemia, hyperinsulinemia, and increased alanine aminotransferase (ALT) and aspartate aminotransferase (AST) levels. Mice on both diets developed progressive macro- and microvesicular steatosis, hepatic inflammation, and fibrosis, along with atherosclerosis. HFD induced more severe hyperinsulinemia, while FFD induced more severe hepatic inflammation with advanced (F3) bridging fibrosis, as well as more severe atherosclerosis. OCA treatment significantly reduced hepatic inflammation and fibrosis, and it did not affect atherosclerosis. Hepatic transcriptome analysis was compared with human NASH and illustrated similarity. The present study defines a translational model of NASH with progressive liver fibrosis and simultaneous atherosclerosis development. By adaptation of the fat content of the diet, either insulin resistance (HFD) or hepatic inflammation and fibrosis (FFD) can be aggravated.