Validation of computed tomography as a diagnostic tool in guinea pigs with non-alcoholic fatty liver disease.

Non-alcoholic fatty liver disease (NAFLD) and subsequent steatohepatitis (NASH) is the most common cause of liver disease and liver transplantation in humans. Affecting millions of patients worldwide, diagnosis relies on a biopsy, not without risk to the patient, and emphasises the need for improved diagnostic measures to determine and monitor disease progression. Despite intensive research, approved pharmacological treatment modalities are few, underlining that animal models with increased translational validity are important to advance preclinical drug development. This study validates the applicability of computed tomography (CT) as a non-invasive diagnostic tool for the assessment of liver steatosis in a guinea pig model of NAFLD/NASH. Guinea pigs with induced NAFLD or NASH were compared to healthy controls at two separate time points: week 16, serving as baseline measure, and week 25 to monitor disease progression over time. The animals were subsequently euthanised, and samples were collected to confirm disease stage. The data showed a strong negative correlation between liver triglycerides and Hounsfield unit (HU) values (R2 = 0.8157; p < 0.0001). A significant difference in histopathological scoring and HU values between grade 0 and more advanced stages of steatosis was recorded (p < 0.001), although the degree of liver fibrosis could not be accurately evaluated by differences in HU. In conclusion, the present study validates CT scanning for the determination of hepatic steatosis in guinea pigs, and it strongly supports the technique as a relevant non-invasive diagnostic tool in this species.

Dietary Long-Chain Fatty Acids Accelerate Metabolic Dysfunction in Guinea Pigs with Non-Alcoholic Steatohepatitis.

The composition of dietary fatty acids may be important for the development and progression of metabolic syndrome and non-alcoholic steatohepatitis (NASH). This study investigated the effect of two high-fat diets based on coconut oil, containing predominantly medium-chain fatty acids (MCFA), or cocoa butter, containing mainly long-chain fatty acids (LCFA), on glucose homeostasis and NASH in guinea pigs following 16 and 32 weeks of diet. At week 16, glucose intolerance was increased in the LCFA animals compared to the MCFA animals (p < 0.001), with both groups differing from the controls by week 32 (p < 0.0001), supported by increased hemoglobin A1c (p < 0.05). NASH was present in both high-fat groups from week 16, with advancing fibrosis appearing more progressive in the LCFA animals at week 16. In agreement, gene expression showed overall increased expression of NASH target genes in the LCFA animals compared to the MCFA animals at weeks 16 and 32 (p < 0.05 and p < 0.0001, respectively). The LCFA animals also displayed increased plasma uric acid at both time points (p < 0.05), a phenomenon linked to NASH in humans. In conclusion, this study reports that a diet high in LCFA promotes metabolic imbalance and may accelerate NASH-associated hepatic fibrosis. This highlights the importance of a critical evaluation of fatty acid composition when investigating NASH-associated endpoints.

Differential Effects of Dietary Components on Glucose Intolerance and Non-Alcoholic Steatohepatitis.

Pharmacological treatment modalities for non-alcoholic fatty liver disease (NAFLD) and steatohepatitis (NASH) are scarce, and discoveries are challenged by lack of predictive animal models adequately reflecting severe human disease stages and co-morbidities such as obesity and type 2 diabetes. To mimic human NAFLD/NASH etiology, many preclinical models rely on specific dietary components, though metabolism may differ considerably between species, potentially affecting outcomes and limiting comparability between studies. Consequently, understanding the physiological effects of dietary components is critical for high translational validity. This study investigated the effects of high fat, cholesterol, and carbohydrate sources on NASH development and metabolic outcomes in guinea pigs. Diet groups (n = 8/group) included: low-fat low-starch (LF-LSt), low-fat high-starch (LF-HSt), high-fat (HF) or HF with 4.2%, or 8.4% sugar water supplementation. The results showed that caloric compensation in HF animals supplied with sugar water led to reduced feed intake and a milder NASH phenotype compared to HF. The HF group displayed advanced NASH, weight gain and glucose intolerance compared to LF-LSt animals, but not LF-HSt, indicating an undesirable effect of starch in the control diet. Our findings support the HF guinea pig as a model of advanced NASH and highlights the importance in considering carbohydrate sources in preclinical studies of NAFLD.

Extracellular Vesicles as Drivers of Non-Alcoholic Fatty Liver Disease: Small Particles with Big Impact.

Nonalcoholic fatty liver disease (NAFLD) is becoming the leading chronic liver disease, negatively affecting the lives of millions of patients worldwide. The complex pathogenesis involves crosstalk between multiple cellular networks, but how the intricate communication between these cells drives disease progression remains to be further elucidated. Furthermore, the disease is not limited to the liver and includes the reprogramming of distant cell populations in different organs. Extracellular vesicles (EVs) have gained increased attention as mediators of cellular communication. EVs carry specific cargos that can act as disease-specific signals both locally and systemically. Focusing on NAFLD advancing to steatohepatitis (NASH), this review provides an update on current experimental and clinical findings of the potential role of EVs in hepatic inflammation and fibrosis, the main contributors to progressive NASH. Particular attention is placed on the characteristics of EV cargos and potential specificity to disease stages, with putative value as disease markers and treatment targets for future investigations.

The effect of acetylsalicylic acid and pentoxifylline in guinea pigs with non-alcoholic steatohepatitis.

Therapeutic options are urgently needed for non-alcoholic fatty liver disease (NAFLD), but development is time-consuming and costly. In contrast, drug repurposing offers the advantages of re-applying compounds that are already approved, thereby reducing cost. Acetylsalicylic acid (ASA) and pentoxifylline (PTX) have shown promise for treatment of NAFLD, but have not yet been tested in combination. Guinea pigs were fed a high-fat diet for 16 weeks and then continued on the diet while being treated with ASA, PTX or ASA+PTX for 8 weeks. Chow-fed animals served as healthy controls. Guinea pigs were CT scanned before intervention start and at intervention end. Animals without steatosis (ie NAFLD) at week 16 were excluded from the data analysis. ASA and PTX alone or in combination did not improve hepatic steatosis, ballooning, inflammation or fibrosis nor did the treatments affect liver enzymes (aminotransferases and alkaline phosphatase) or circulating lipids. Liver triglyceride levels, relative liver weight and hepatic mRNA expression of monocyte chemoattractant protein 1, interleukin 8 and platelet-derived growth factor b were nominally decreased. Thus, in the current study, treatment with ASA and PTX alone or in combination for 8 weeks did not ameliorate NASH or hepatic fibrosis in guinea pigs.

Molecular drivers of non-alcoholic steatohepatitis are sustained in mild-to-late fibrosis progression in a guinea pig model.

Hepatic fibrosis increases mortality in humans with non-alcoholic steatohepatitis (NASH), but it remains unclear how fibrosis stage and progression affect the pathogenic mechanisms of NASH. This study investigates the transcriptional regulation and the impact of fibrosis stage, of pathways relating to hepatic lipid and cholesterol homeostasis, inflammation and fibrosis using RT-qPCR in the guinea pig NASH model. Animals were fed a chow (4% fat), a high-fat (20% fat, 0.35% cholesterol) or high-fat/high-sucrose (20% fat, 15% sucrose, 0.35% cholesterol) diet for 16 or 25 weeks (n = 7/group/time point). High-fat diets induced NASH. In NASH, markers of hepatic de novo lipogenesis were enhanced (e.g. FASN, > twofold, p < 0.05) while markers of mitochondrial, peroxisomal and cytochrome fatty acid oxidation were reduced (e.g. CPT1A > twofold, p < 0.05). Markers of fatty acid uptake were unaltered or decreased. Likewise, expression of cholesterol uptake and synthesis markers were decreased, whereas genes relating to lipid and cholesterol export were unaltered. Inflammatory and chemotactic cytokines were enhanced alongside fibrogenic pathways including increased hepatic stellate cell activation and migration, matrix deposition (e.g. MCP1, TNFα, ß-PDGF and Col1a1, > threefold, p < 0.05) and decreased matrix degradation. Fibrosis stage (mild vs. severe) and progression did generally not affect the expression of the investigated pathways. This suggests that liver dysfunction at the transcriptional level is induced early and maintained throughout fibrosis progression, allowing potential treatments to target dysregulated pathways already at early disease stages. As the guinea pig NASH model mimics several aspects of human molecular pathophysiology, these results may be used to increase the current understanding of NASH pathology and explore future treatment targets.

Molecular mechanisms of hepatic lipid accumulation in non-alcoholic fatty liver disease.

Non-alcoholic fatty liver disease (NAFLD) is currently the world's most common liver disease, estimated to affect up to one-fourth of the population. Hallmarked by hepatic steatosis, NAFLD is associated with a multitude of detrimental effects and increased mortality. This narrative review investigates the molecular mechanisms of hepatic steatosis in NAFLD, focusing on the four major pathways contributing to lipid homeostasis in the liver. Hepatic steatosis is a consequence of lipid acquisition exceeding lipid disposal, i.e., the uptake of fatty acids and de novo lipogenesis surpassing fatty acid oxidation and export. In NAFLD, hepatic uptake and de novo lipogenesis are increased, while a compensatory enhancement of fatty acid oxidation is insufficient in normalizing lipid levels and may even promote cellular damage and disease progression by inducing oxidative stress, especially with compromised mitochondrial function and increased oxidation in peroxisomes and cytochromes. While lipid export initially increases, it plateaus and may even decrease with disease progression, sustaining the accumulation of lipids. Fueled by lipo-apoptosis, hepatic steatosis leads to systemic metabolic disarray that adversely affects multiple organs, placing abnormal lipid metabolism associated with NAFLD in close relation to many of the current life-style-related diseases.

Long term Westernized diet leads to region-specific changes in brain signaling mechanisms.

Western diets, high in fat and energy, are associated with cognitive deficits in humans and animal models, but the underlying mechanisms are not fully elucidated. This includes whether diet-induced dyslipidemia per se negatively impacts brain signaling. Here we investigate the effects of dyslipidemia induced by two high fat diets with or without high sucrose on hippocampal and frontal cortical oxidative stress, brain-derived neurotrophic factor (BDNF) and down-stream markers of synaptic plasticity, as well as alterations in monoaminergic neurotransmitter levels. A high fat diet was associated with decreased antioxidant status (vitamin C), increased serotonin in the frontal cortex, and increased ratio of phosphorylated Ca2+/calmodulin-dependent protein kinase II in the hippocampus, while a high fat and sucrose diet decreased levels of vitamin C in the frontal cortex and BDNF in the hippocampus. Markers of dyslipidemia correlated significantly with cerebral vitamin C levels, monoaminergic neurotransmitters and metabolites in the frontal cortex, but not in the hippocampus. Thus, a high fat diet caused regional alterations in antioxidant levels, neurochemistry and molecular markers in the non-obese dyslipidemic guinea pig.

High-fat but not sucrose intake is essential for induction of dyslipidemia and non-alcoholic steatohepatitis in guinea pigs.

BACKGROUND: Non-alcoholic fatty liver disease (NAFLD) and dyslipidemia are closely related. Diet plays an important role in the progression of these diseases, but the role of specific dietary components is not completely understood. Therefore, we investigated the role of dietary sucrose and fat/cholesterol on the development of dyslipidemia and NAFLD. METHODS: Seventy female guinea pigs were block-randomized (based on weight) into five groups and fed a normal chow diet (control: 4 % fat), a very high-sucrose diet (vHS: 4 % fat, 25 % sucrose), a high-fat diet (HF: 20 % fat, 0.35 % cholesterol), a high-fat/high-sucrose diet (HFHS: 20 % fat, 15 % sucrose, 0.35 % cholesterol) or a high-fat/very high-sucrose diet (HFvHS: 20 % fat, 25 % sucrose, 0.35 % cholesterol) for 16 and 25 weeks. RESULTS: All three high-fat diets induced dyslipidemia with increased concentrations of plasma cholesterol (p < 0.0001), LDL-C (p < 0.0001) and VLDL-C (p < 0.05) compared to control and vHS. Contrary to this, plasma triglycerides were increased in control and vHS compared to high-fat fed animals (p < 0.01), while circulating levels of free fatty acids were even between groups. Histological evaluation of liver sections revealed non-alcoholic steatohepatitis (NASH) with progressive inflammation and bridging fibrosis in high-fat fed animals. Accordingly, hepatic triglycerides (p < 0.05) and cholesterol (p < 0.0001) was increased alongside elevated levels of alanine and aspartate aminotransferase (p < 0.01) compared to control and vHS. CONCLUSION: Collectively, our results suggest that intake of fat and cholesterol, but not sucrose, are the main factors driving the development and progression of dyslipidemia and NAFLD/NASH.

Does vitamin C deficiency promote fatty liver disease development?

Obesity and the subsequent reprogramming of the white adipose tissue are linked to human disease-complexes including metabolic syndrome and concurrent non-alcoholic fatty liver disease (NAFLD) and non-alcoholic steatohepatitis (NASH). The dietary imposed dyslipidemia promotes redox imbalance by the generation of excess levels of reactive oxygen species and induces adipocyte dysfunction and reprogramming, leading to a low grade systemic inflammation and ectopic lipid deposition, e.g., in the liver, hereby promoting a vicious circle in which dietary factors initiate a metabolic change that further exacerbates the negative consequences of an adverse life-style. Large epidemiological studies and findings from controlled in vivo animal studies have provided evidence supporting an association between poor vitamin C (VitC) status and propagation of life-style associated diseases. In addition, overweight per se has been shown to result in reduced plasma VitC, and the distribution of body fat in obesity has been shown to have an inverse relationship with VitC plasma levels. Recently, a number of epidemiological studies have indicated a VitC intake below the recommended daily allowance (RDA) in NAFLD-patients, suggesting an association between dietary habits, disease and VitC deficiency. In the general population, VitC deficiency (defined as a plasma concentration below 23 µM) affects around 10% of adults, however, this prevalence is increased by an adverse life-style, deficiency potentially playing a broader role in disease progression in specific subgroups. This review discusses the currently available data from human surveys and experimental models in search of a putative role of VitC deficiency in the development of NAFLD and NASH.