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.

Vitamin C Deficiency Exacerbates Dysfunction of Atherosclerotic Coronary Arteries in Guinea Pigs Fed a High-Fat Diet.

Vitamin C (vitC) deficiency has been associated with an increased risk of cardiovascular disease; while several putative mechanistic links have been proposed, functional evidence supporting a causal relationship is scarce. In this study, we investigated how vitC deficiency affects coronary artery vasomotor function and the development of coronary atherosclerotic plaques in guinea pigs subjected to chronic dyslipidemia by a high-fat diet regime. Female Hartley guinea pigs were fed either a control (low-fat diet and sufficient vitC) (N = 8) or a high-fat diet with either sufficient (N = 8) or deficient (N = 10) vitC for 32 weeks. Guinea pigs subjected to the high-fat diet developed significant atherosclerotic plaques in their coronary arteries, with no quantitative effect of vitC deficiency. In isolated coronary arteries, vasomotor responses to potassium, carbachol, nitric oxide, or bradykinin were studied in a wire myograph. Carbachol, bradykinin, and nitric oxide mediated relaxation in the coronary arteries of the control group. While vasorelaxation to carbachol and nitric oxide was preserved in the two high-fat diet groups, bradykinin-induced vasorelaxation was abolished. Interestingly, bradykinin induced a significant contraction in coronary arteries from vitC-deficient guinea pigs (p < 0.05). The bradykinin-induced contraction was unaffected by L-NAME but significantly inhibited by both indomethacin and vitC, suggesting that, during vitC deficiency, increased release of arachidonic acid metabolites and vascular oxidative stress are involved in the constrictor effects mediated by bradykinin. In conclusion, the present study shows supporting evidence that poor vitC status negatively affects coronary artery function.

Modelling Nonalcoholic Steatohepatitis In Vivo-A Close Transcriptomic Similarity Supports the Guinea Pig Disease Model.

The successful development of effective treatments against nonalcoholic steatohepatitis (NASH) is significantly set back by the limited availability of predictive preclinical models, thereby delaying and reducing patient recovery. Uniquely, the guinea pig NASH model develops hepatic histopathology and fibrosis resembling that of human patients, supported by similarities in selected cellular pathways. The high-throughput sequencing of guinea pig livers with fibrotic NASH (n = 6) and matched controls (n = 6) showed a clear separation of the transcriptomic profile between NASH and control animals. A comparison to NASH patients with mild disease (GSE126848) revealed a 45.2% overlap in differentially expressed genes, while pathway analysis showed a 34% match between the top 50 enriched pathways in patients with advanced NASH (GSE49541) and guinea pigs. Gene set enrichment analysis highlighted the similarity to human patients (GSE49541), also when compared to three murine models (GSE52748, GSE38141, GSE67680), and leading edge genes THRSP, CCL20 and CD44 were highly expressed in both guinea pigs and NASH patients. Nine candidate genes were identified as highly correlated with hepatic fibrosis (correlation coefficient > 0.8), and showed a similar expression pattern in NASH patients. Of these, two candidate genes (VWF and SERPINB9) encode secreted factors, warranting further investigations as potential biomarkers of human NASH progression. This study demonstrates key similarities in guinea pig and human NASH, supporting increased predictability when translating research findings to human patients.

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.

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.

Vitamin C Deficiency May Delay Diet-Induced NASH Regression in the Guinea Pig.

Oxidative stress is directly linked to non-alcoholic fatty liver disease (NAFLD) and the progression to steaotohepatitis (NASH). Thus, a beneficial role of antioxidants in delaying disease progression and/or accelerating recovery may be expected, as corroborated by recommendations of, e.g., vitamin E supplementation to patients. This study investigated the effect of vitamin C deficiency-often resulting from poor diets low in fruits and vegetables and high in fat-combined with/without a change to a low fat diet on NAFLD/NASH phenotype and hepatic transcriptome in the guinea pig NASH model. Vitamin C deficiency per se did not accelerate disease induction. However, the results showed an effect of the diet change on the resolution of hepatic histopathological hallmarks (steatosis, inflammation, and ballooning) (p < 0.05 or less) and indicated a positive effect of a high vitamin C intake when combined with a low fat diet. Our data show that a diet change is important in NASH regression and suggest that a poor vitamin C status delays the reversion towards a healthy hepatic transcriptome and phenotype. In conclusion, the findings support a beneficial role of adequate vitamin C intake in the regression of NASH and may indicate that vitamin C supplementation in addition to lifestyle modifications could accelerate recovery in NASH patients with poor vitamin C status.

Atorvastatin and Vitamin E Accelerates NASH Resolution by Dietary Intervention in a Preclinical Guinea Pig Model.

Despite affecting millions of patients worldwide, no pharmacological treatment has yet proved effective against non-alcoholic steatohepatitis (NASH) induced liver fibrosis. Current guidelines recommend lifestyle modifications including reductions in dietary energy intake. Recently, therapy with atorvastatin and vitamin E (vitE) has been recommended, although clinical studies on the resolution of hepatic fibrosis are inconclusive. Targeting NASH-induced hepatic end-points, this study evaluated the effects of atorvastatin and vitE alone or in combination with a dietary intervention in the guinea pig NASH model. Guinea pigs (n = 72) received 20 weeks of high fat feeding before allocating to four groups: continued HF feeding (HF), HF diet with atorvastatin and vitE (HF+), low-fat diet (LF) and low-fat with atorvastatin and vitE (LF+), for four or eight weeks of intervention. Both LF and LF+ decreased liver weight, cholesterol and plasma dyslipidemia. LF+ further improved hepatic histopathological hallmarks (p < 0.05), liver injury markers aspartate aminotransferase (AST) and alanine aminotransferase (ALT) (p < 0.05) and reduced the expression of target genes of hepatic inflammation and fibrosis (p < 0.05), underlining an increased effect on NASH resolution in this group. Collectively, the data support an overall beneficial effect of diet change, and indicate that atorvastatin and vitE therapy combined with a diet change act synergistically in improving NASH-induced endpoints.

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.

A role of peroxisome proliferator-activated receptor γ in non-alcoholic fatty liver disease.

Non-alcoholic fatty liver disease is becoming a major health burden, as prevalence increases and there are no approved treatment options. Thiazolidinediones target the nuclear receptor peroxisome proliferator-activated receptor γ (PPARγ) and have been investigated in several clinical trials for their potential in treating non-alcoholic fatty liver disease (NAFLD) and non-alcoholic steatohepatitis (NASH). PPARγ has specialized roles in distinct tissues and cell types, and although the primary function of PPARγ is in adipose tissue, where the highest expression levels are observed, hepatic expression levels of PPARγ are significantly increased in patients with NAFLD. Thus, NAFLD patients receiving treatment with PPARγ agonists might have a liver response apart from the one in adipose tissue. Owing to the different roles of PPARγ, new treatment strategies include development of compounds harnessing the beneficial effects of PPARγ while restricting PPARγ unwanted effects such as adipogenesis resulting in weight gain. Furthermore, dual or pan agonists targeting two or more of the PPARs have shown promising results in pre-clinical research and some are currently proceeding to clinical trials. This MiniReview explores adipose- and liver-specific actions of PPARγ, and how this knowledge may contribute in the search for new treatment modalities in NAFLD/NASH.

Liraglutide Decreases Hepatic Inflammation and Injury in Advanced Lean Non-Alcoholic Steatohepatitis.

Although commonly associated with obesity, non-alcoholic fatty liver disease (NAFLD) is also present in the lean population representing a unique disease phenotype. Affecting 25% of the world's population, NAFLD is associated with increased mortality especially when progressed to non-alcoholic steatohepatitis (NASH). However, no approved pharmacological treatments exist. Current research focuses mainly on NASH associated with obesity, leaving the effectiveness of promising treatments in lean NASH virtually unknown. This study therefore aimed to evaluate the effect of liraglutide (glucagon-like peptide 1 analogue) and dietary intervention, alone and in combination, in guinea pigs with non-obese NASH. After 20 weeks of high-fat feeding (20% fat, 15% sucrose, 0.35% cholesterol), 40 female guinea pigs were block-randomized based on weight into four groups receiving one of four treatments for 4 weeks: continued high-fat diet (HF, control), high-fat diet and liraglutide treatment (HFL), chow diet (4% fat, 0% sucrose, 0% cholesterol; HFC) or chow diet and liraglutide treatment (HFCL). High-fat feeding induced NASH with severe fibrosis. Liraglutide decreased inflammation (p < 0.05) and hepatocyte ballooning (p < 0.05), while increasing hepatic α-tocopherol (p = 0.0154). Dietary intervention did not improve liver histopathology significantly, but decreased liver weight (p = 0.004), plasma total cholesterol (p = 0.0175), LDL-cholesterol (p = 0.0063), VLDL-cholesterol (p = 0.0034), hepatic cholesterol (p < 0.0001) and increased hepatic vitamin C (p = 0.0099). Combined liraglutide and dietary intervention induced a rapid weight loss, necessitating periodical liraglutide dose adjustment/discontinuation, limiting the strength of the findings from this group. Collectively, this pre-clinical study supports the beneficial effect of liraglutide on NASH and extends this notion to lean NASH.