An improved mouse model that rapidly develops fibrosis in non‐alcoholic steatohepatitis

Non‐alcoholic steatohepatitis (NASH) is a progressive fibrotic disease, the pathogenesis of which has not been fully elucidated. One of the most common models used in NASH research is a nutritional model where NASH is induced by feeding a diet deficient in both methionine and choline. However, the dietary methionine‐/choline‐deficient model in mice can cause severe weight loss and liver atrophy, which are not characteristics of NASH seen in human patients. Exclusive, long‐term feeding with a high‐fat diet (HFD) produced fatty liver and obesity in mice, but the HFD for several months did not affect fibrosis. We aimed to establish a mouse model of NASH with fibrosis by optimizing the methionine content in the HFD. Male mice were fed a choline‐deficient, L‐amino acid‐defined, high‐fat diet (CDAHFD) consisting of 60 kcal% fat and 0.1% methionine by weight. After 1–14 weeks of being fed CDAHFD, the mice were killed. C57BL/6J mice maintained or gained weight when fed CDAHFD, while A/J mice showed a steady decline in body weight (of up to 20% of initial weight). In both strains of mice, plasma levels of alanine aminotransferase increased from week 1, when hepatic steatosis was also observed. By week 6, C57BL/6J mice had developed enlarged fatty liver with fibrosis as assessed by Masson's trichrome staining and by hydroxyproline assay. Therefore, this improved CDAHFD model may be a mouse model of rapidly progressive liver fibrosis and be potentially useful for better understanding human NASH disease and in the development of efficient therapies for this condition.     

Animal models of non‐alcoholic fatty liver disease: current perspectives and recent advances

Non‐alcoholic fatty liver disease (NAFLD) is a continuous spectrum of diseases characterized by excessive lipid accumulation in hepatocytes. NAFLD progresses from simple liver steatosis to non‐alcoholic steatohepatitis and, in more severe cases, to liver fibrosis, cirrhosis, and hepatocellular carcinoma (HCC). Because of its growing worldwide prevalence, various animal models that mirror both the histopathology and the pathophysiology of each stage of human NAFLD have been developed. The selection of appropriate animal models continues to be one of the key questions faced in this field. This review presents a critical analysis of the histopathology and pathogenesis of NAFLD, the most frequently used and recently developed animal models for each stage of NAFLD and NAFLD‐induced HCC, the main mechanisms involved in the experimental pathogenesis of NAFLD in different animal models, and a brief summary of recent therapeutic targets found by the use of animal models. Integrating the data from human disease with those from animal studies indicates that, although current animal models provide critical guidance in understanding specific stages of NAFLD pathogenesis and progression, further research is necessary to develop more accurate models that better mimic the disease spectrum, in order to provide both increased mechanistic understanding and identification/testing of novel therapeutic approaches. © 2016 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland.     

Clinicopathological correlations in a series of adult patients with non‐alcoholic fatty liver disease

Possible correlations among clinical data, serum aminotransferase levels and histological features were assessed in a series of 37 adult patients with non‐alcoholic fatty liver disease (NAFLD), consisting of nine patients with fatty liver (FL) and 28 with non‐alcoholic steatohepatitis (NASH). In each liver biopsy, the NAFLD activity score (NAS) and the stage of fibrosis were determined. Additionally, the number of Kupffer cell aggregates (microgranulomas) per centimeter of biopsy length (MG/cm ratio) was assessed on immunohistochemical stains for CD68 antigen. Definite NASH (NAS ≥ 5) was strongly correlated with serum aspartate aminotransferase (AST) level (P= 0.003), stage of fibrosis (P= 0.003) and age (P= 0.014). On multivariate analysis, age >46 years and AST level above normal values were found to be independent clinical predictors of established NASH. The MG/cm ratio increased from control liver to FL to NASH (P < 0.001), and was correlated with the NAS (P= 0.003) and with the stage of fibrosis (P= 0.004), but not with the serum aminotransferase levels. In conclusion, persistent AST elevation in patients with suspected NAFLD should be an indication for liver biopsy, in order to determine the severity of necroinflammatory activity and the stage of fibrosis. Microgranuloma counting may represent a useful complementary marker of necroinflammatory activity in patients with NAFLD.     

Vitronectin deficiency attenuates hepatic fibrosis in a non‐alcoholic steatohepatitis‐induced mouse model

Vitronectin (VN), an extracellular matrix protein, is a promising immune biomarker of non‐alcoholic steatohepatitis (NASH); however, its precise function remains unclear. This study investigated how VN deficiency contributes to the development of NASH. Towards this aim, wild‐type (WT) and VN?/? mice were fed with a choline‐deficient, L‐amino acid‐defined, high‐fat diet (CDAHFD) for 6 and 10 weeks to induce NASH, and the livers were isolated. In WT mice fed with CDAHFD for 6 and 10 weeks, the expression of Vn mRNA and protein was up‐regulated compared with that in mice fed with the MF control diet, indicating that VN is regulated in NASH condition. VN?/? mice showed decreased picrosirius red staining in the liver area and Col1a2 mRNA expression levels, compared with WT mice, indicating that the severity of hepatic fibrosis is attenuated in the CDAHFD‐fed VN?/? mice. In addition, VN deficiency did not affect the area of lipid droplets in haematoxylin‐eosin staining and the mRNA expression levels of fatty acid synthases, Srebp, Acc and Fas in the CDAHFD‐fed mice. Moreover, VN deficiency decreased the inflammation score and the mRNA expression levels of Cd11b and F4/80, macrophage markers, as well as Tnf‐α and Il‐1β, inflammatory cytokines in the CDAHFD‐fed mice. Furthermore, VN deficiency decreased the protein and mRNA expression levels of α‐smooth muscle actin in the CDAHFD‐fed mice, suggesting that VN deficiency inhibits the activation of hepatic stellate cells (HSCs). Our findings indicate that VN contributes to the development of fibrosis in the NASH model mice via modulation of the inflammatory reaction and activation of HSCs.     

Epoxyeicosatrienoic acids alleviate methionine‐choline‐deficient diet–induced non‐alcoholic steatohepatitis in mice

The epoxyeicosatrienoic acids (EETs) are products of cytochrome P450 epoxygenases and have recently been found to have an anti‐inflammatory activity. However, the role of EETs in non‐alcoholic steatohepatitis has not been fully understood. In this study, we investigated the protective role of EETs in methionine‐choline‐deficient (MCD) diet–induced non‐alcoholic steatohepatitis (NASH) in mice and the potential mechanisms. We used 1‐trifluoromethoxyphenyl‐3‐(1‐propionylpiperidin‐4‐yl)urea(TPPU), a soluble epoxide hydrolase inhibitor, to increase the endogenous EET level in mice. Upon TPPU treatment, the liver steatosis and inflammatory damage were significantly ameliorated in mice with steatohepatitis, paralleled by the downregulation of pro‐inflammatory cytokines (TNF‐α, IL‐1β, IL‐6) as well as chemokines (CXCL1, MCP‐1). Compared with untreated NASH mice, mRNA levels of sterol regulatory element binding protein 1c (SREBP1c) and inflammation relevant adhesion molecules (ICAM‐1, VCAM‐1) were downregulated, whereas mRNA level of peroxisome proliferator–activated receptor α(PPAR‐α) was elevated in TPPU‐treated mice. In vitro, 11,12‐EET treatment remarkably attenuated free fatty acid (FFA)–induced inflammation in HepG2 and THP‐1 cells. Further, 11,12‐EET inhibited the activation of NF‐κB signalling pathway in macrophages from mice with steatohepatitis. Collectively, these results suggest that EETs play a protective role and alleviate the MCD diet–induced steatohepatitis in mice mainly by downregulating activation of NF‐κB pathway in macrophages.     

Non‐alcoholic fatty liver disease – histological scoring systems: a large cohort single‐center,evaluation study

Non‐alcoholic fatty liver disease (NAFLD) is an increasingly common cause of chronic liver disease. Till date, liver biopsy remains the gold standard for identification and quantification of the wide histological spectra of NAFLD. Histological scorings are very useful and widely applied for the diagnosis and management in clinical trials and follow‐up studies of non‐alcoholic steatohepatitis (NASH). However, in view of scarce published literature, there is a need to evaluate them in large cohort of NAFLD. This study was aimed to evaluate the two histological scoring systems (NAS‐CRN, SAF) in the diagnosis of NAFLD and to assess the role of histological characteristics as injury markers in NAFLD. Retrospective histological study of liver biopsies of 1000 patients diagnosed as NAFLD, between 2010 and 2016, was conducted. Histopathologic evaluation and semiquantiative scoring based on NAS‐CRN and SAF algorithm and their correlation with serum aminotransferase and fibrosis were performed. Liver biopsies were classified according to the NAS‐CRN scoring, as NAS <3 (not NASH) in 72 (7.2%), NAS 3–4 (borderline NASH) in 310 (31%), and NAS ≥5 (definite NASH) in 618 (61.8%), and SAF classified 117 (11.7%) not NASH and 883 (88.3%) definite NASH. There was excellent concordance for definite NASH and not NASH; however, 88.06% of borderline NASH was classified as NASH by SAF. 76.39% by NAS and 78.63% by SAF algorithm who were diagnosed as not NASH showed the presence of fibrosis; however, higher stages of fibrosis were significantly more prevalent in definite NASH, excluding burnt‐out cirrhosis. Serum ALT was significantly associated with increasing stages of fibrosis (p < 0.001) and the three categories (not NASH, borderline NASH, and definite NASH) when classified as with/without fibrosis (p < 0.001). Steatosis of higher grades, more ballooned cells, and more foci of Lobular Inflammation were found in significantly higher proportion of patients with NASH (p < 0.001), with higher fibrosis stages (p < 0.001) and higher serum ALT levels (p < 0.001). NAFLD classifications based on histological scoring NAS‐CRN and SAF algorithm are concordant for the category of definite NASH and not NASH, while borderline NASH shows discrepant interpretation. There was highly significant correlation between the NAS and SAF categories with high grades of histological characteristics, with serum ALT and with higher stages of fibrosis. Exclusion of fibrosis is a limitation with both scores.     

Therapeutic reversal of chronic alcohol‐related steatohepatitis with the ceramide inhibitor myriocin

Alcohol‐related liver disease (ALD) is associated with steatohepatitis and insulin resistance. Insulin resistance impairs growth and disrupts lipid metabolism in hepatocytes. Dysregulated lipid metabolism promotes ceramide accumulation and oxidative stress, leading to lipotoxic states that activate endoplasmic reticulum (ER) stress pathways and worsen inflammation and insulin resistance. In a rat model of chronic alcohol feeding, we characterized the effects of a ceramide inhibitor, myriocin, on the histopathological and ultrastructural features of steatohepatitis, and the biochemical and molecular indices of hepatic steatosis, insulin resistance and ER stress. Myriocin reduced the severity of alcohol‐related steatohepatitis including the abundance and sizes of lipid droplets and mitochondria, inflammation and architectural disruption of the ER. In addition, myriocin‐mediated reductions in hepatic lipid and ceramide levels were associated with constitutive enhancement of insulin signalling through the insulin receptor and IRS‐2, reduced hepatic oxidative stress and modulation of ER stress signalling mechanisms. In conclusion, ceramide accumulation in liver mediates tissue injury, insulin resistance and lipotoxicity in ALD. Reducing hepatic ceramide levels can help restore the structural and functional integrity of the liver in chronic ALD due to amelioration of insulin resistance and ER stress. However, additional measures are needed to protect the liver from alcohol‐induced necroinflammatory responses vis‐à‐vis continued alcohol abuse.     

Hepatocellular carcinoma in a mouse model fed a choline‐deficient,L‐amino acid‐defined,high‐fat diet

Hepatocellular carcinoma (HCC) is a common cancer worldwide and represents the outcome of the natural history of chronic liver disease. The growing rates of HCC may be partially attributable to increased numbers of people with non‐alcoholic fatty liver disease (NAFLD) and non‐alcoholic steatohepatitis (NASH). However, details of the liver‐specific molecular mechanisms responsible for the NAFLD–NASH–HCC progression remain unclear, and mouse models that can be used to explore the exact factors that influence the progression of NAFLD/NASH to the more chronic stages of liver disease and subsequent HCC are not yet fully established. We have previously reported a choline‐deficient, L‐amino acid‐defined, high‐fat diet (CDAHFD) as a dietary NASH model with rapidly progressive liver fibrosis in mice. The current study in C57BL/6J mice fed CDAHFD provided evidence for the chronic persistence of advanced hepatic fibrosis in NASH and disease progression towards HCC in a period of 36 weeks. When mice fed CDAHFD were switched back to a standard diet, hepatic steatosis was normalized and NAFLD activity score improved, but HCC incidence increased and the phenotype of fibrosis‐associated HCC development was observed. Moreover, when mice continued to be fed CDAHFD for 60 weeks, HCC further developed without severe body weight loss or carcinogenesis in other organs. The autochthonous tumours showed a variety of histological features and architectural patterns including trabecular, pseudoglandular and solid growth. The CDAHFD mouse model might be a useful tool for studying the development of HCC from NAFLD/NASH, and potentially useful for better understanding pathological changes during hepatocarcinogenesis.     

Nonalcoholic steatohepatitis: histologic features and clinical correlations with 30 blinded biopsy specimens

Thirty overweight patients with clinically characterized and biopsy proven nonalcoholic steatohepatitis (NASH) were enrolled in a 48-week treatment trial with rosiglitazone, a peroxisome proliferator-activator receptor (PPAR)-gamma agonist that enhances insulin sensitivity. Improvement in laboratory liver tests, insulin resistance and liver fat content were documented; blinded biopsy review demonstrated decreases in necroinflammatory activity or grade and in individual components of grade, and changes in the relationship of lobular and portal inflammation as well as in the nature of perisinusoidal fibrosis. The current study identified correlations of histological features of the protocol entry biopsy specimens with contemporaneous laboratory and imaging tests. Significant correlations with histologically assessed steatosis were liver fat, evaluated by computed tomography (P = 0.001); mean HbA1C, a measure of glycemic control (P = 0.004); and QUICKI, a measure of insulin sensitivity (P = 0.05). Histologically determined grades of steatohepatitis (SH) correlated with HbA1C (P = 0.01), and a trend toward elevated fasting glucose levels was seen. No subject in the study was cirrhotic at entry; fibrosis scores of the 30 subjects did not significantly correlate with age, gender, body mass index, or clinical tests. All subjects underwent 3 biopsies (prior, entry, and posttreatment), and all had undergone a prior biopsy with diagnostic SH. By blinded analysis, 7 study entry biopsy specimens did not fulfill published strict criteria for SH. Laboratory results from these subjects included normal fasting glucose level and, compared with the 23 subjects with criteria for SH, lower mean alanine aminotransferase and aspartate aminotransferase levels (P = 0.02 for both), less insulin resistance (P = 0.03), and lower mean HbA1C (P = 0.001). We conclude that biopsy findings determined by blinded analysis correlated with image-detected steatosis, laboratory markers of hepatic inflammation, insulin resistance, and long-term glycemia; the findings confirm the usefulness of strict histological criteria in the evaluation of NASH.     

The insulin‐like growth factor system and sarcomas

Sarcomas are a diverse group of malignant mesenchymal tumours arising from bone and soft tissues. The identification of critical cellular signalling pathways in sarcomas is an important issue for the development of new targeted therapies. This review highlights the experimental and clinical evidence supporting the role of the insulin‐like growth factor (IGF) signalling system in the cellular transformation and progression of several types of sarcoma, including rhabdomyosarcoma, synovial sarcoma, leiomyosarcoma, Ewing's sarcoma and osteosarcoma. Preclinical data suggest that the IGF system could be a promising target for therapy in these sarcomas. Currently, therapies interrupting IGF signalling have been or are being developed. In recent phase 1 clinical studies with humanized monoclonal antibodies directed against IGF receptor type 1 (IGF‐1R), objective tumour responses were observed in several patients with Ewing's sarcoma, encouraging further clinical testing in Ewing's sarcoma and other sarcoma (sub)types. Moreover, the occasional occurrence of paraneoplastic hypoglycaemia as a result of the secretion of incompletely processed forms of pro‐IGF‐II by sarcomas is discussed. Copyright © 2008 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.     

Mitochondrial genome architecture in non‐alcoholic fatty liver disease

Non‐alcoholic fatty liver disease (NAFLD) is associated with mitochondrial dysfunction, a decreased liver mitochondrial DNA (mtDNA) content, and impaired energy metabolism. To understand the clinical implications of mtDNA diversity in the biology of NAFLD, we applied deep‐coverage whole sequencing of the liver mitochondrial genomes. We used a multistage study design, including a discovery phase, a phenotype‐oriented study to assess the mutational burden in patients with steatohepatitis at different stages of liver fibrosis, and a replication study to validate findings in loci of interest. We also assessed the potential protein‐level impact of the observed mutations. To determine whether the observed changes are tissue‐specific, we compared the liver and the corresponding peripheral blood entire mitochondrial genomes. The nuclear genes POLG and POLG2 (mitochondrial DNA polymerase‐γ) were also sequenced. We observed that the liver mtDNA of patients with NAFLD harbours complex genomes with a significantly higher mutational (1.28‐fold) rate and degree of heteroplasmy than in controls. The analysis of liver mitochondrial genomes of patients with different degrees of fibrosis revealed that the disease severity is associated with an overall 1.4‐fold increase in mutation rate, including mutations in genes of the oxidative phosphorylation (OXPHOS) chain. Significant differences in gene and protein expression patterns were observed in association with the cumulative number of OXPHOS polymorphic sites. We observed a high degree of homology (～98%) between the blood and liver mitochondrial genomes. A missense POLG p.Gln1236His variant was associated with liver mtDNA copy number. In conclusion, we have demonstrated that OXPHOS genes contain the highest number of hotspot positions associated with a more severe phenotype. The variability of the mitochondrial genomes probably originates from a common germline source; hence, it may explain a fraction of the ‘missing heritability’ of NAFLD. Copyright © 2016 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.     

Variation in characteristics of islets of Langerhans in insulin‐resistant,diabetic and non‐diabetic‐rat strains

Assessment of the histopathological and plasma biochemical characteristics of diabetic and non‐diabetic rat strains [Han and AP Wistar, lean and obese Zucker Fatty (ZF), and lean and obese Zucker Diabetic Fatty (ZDF) rats] was performed at 6 or 14 weeks of age. Wistar and lean ZF and ZDF rats showed no or minimal islet pathology or plasma biochemical alterations at both timepoints. Obese ZFs were euglycaemic at both timepoints and mildly and severely hyperinsulinaemic at 6 and 14 weeks respectively. Islet morphology was normal at 6 weeks but at 14 weeks, islet hyperplasia was present with a minority showing degenerative changes namely, β‐cell vacuolation, vascular congestion and haemorrhage with minimal mononuclear cell and T lymphocytic infiltration. Obese ZDFs were euglycaemic and moderately hyperinsulinaemic at 6 weeks and severely hyperglycaemic with minor hypoinsulinaemia at 14 weeks. Obese ZDFs at 6 weeks showed mainly normal islets with some displaying degeneration (ranging from β‐cell vacuolation alone to the features described above). At 14 weeks, islet degeneration was more severe and widespread: β‐cell death was present in numerous islets at low level. Islet β‐cell numbers were reduced or absent (with associated reduction in insulin immunostaining) within the islets that now consisted predominantly of fibroblasts, collagen and mononuclear cells. Fibroproliferation consisting of smooth muscle actin‐α‐positive tissue was associated with mononuclear cell infiltration. Some fibrous scars were visible indicative of lost islets. Islet degeneration in obese ZF and ZDF rats was not accompanied by a reduction in β‐cell proliferation or in compensatory proliferation of β‐cell neogenic clusters. In the light of recent reports of adaptive and inflammation‐mediated degenerative changes in human non‐insulin dependent diabetes mellitus (NIDDM) islets, the hypertrophy/hyperplasia of β‐cells and islet degeneration involving infiltration by monocyte/macrophages in obese ZF and obese ZDF rats respectively offers substantial potential for elucidation of the processes involved.     

Interleukin-17 exacerbates hepatic steatosis and inflammation in non-alcoholic fatty liver disease

Mechanisms associated with the progression of simple steatosis to non-alcoholic fatty liver disease (NAFLD) remain undefined. Regulatory T cells (T(regs)) play a critical role in regulating inflammatory processes in non-alcoholic steatohepatitis (NASH) and because T helper type 17 (Th17) functionally oppose T(reg)-mediated responses, this study focused on characterizing the role of Th17 cells using a NAFLD mouse model. C57BL/6 mice were fed either a normal diet (ND) or high fat (HF) diet for 8 weeks. Mice in the HF group had a significantly higher frequency of liver Th17 cells compared to ND-fed mice. Neutralization of interleukin (IL)-17 in HF mice ameliorated lipopolysaccharide (LPS)-induced liver injury reflected by decreased serum alanine aminotransferase (ALT) levels and reduced inflammatory cell infiltrates in the liver. In vitro, HepG2 cells cultured in the presence of free fatty acids (FFA; oleic acid and palmitic acid) for 24 h and IL-17 developed steatosis via insulin-signalling pathway interference. IL-17 and FFAs synergized to induce IL-6 production by HepG2 cells and murine primary hepatocytes which, in combination with transforming growth factor (TGF-β), expanded Th17 cells. It is likely that a similar process occurs in NASH patients, as there were significant levels of IL-17(+) cell infiltrates in NASH patient livers. The hepatic expression of Th17 cell-related genes [retinoid-related orphan receptor gamma (ROR)γt, IL-17, IL-21 and IL-23] was also increased significantly in NASH patients compared to healthy controls. Th17 cells and IL-17 were associated with hepatic steatosis and proinflammatory response in NAFLD and facilitated the transition from simple steatosis to steatohepatitis. Strategies designed to alter the balance between Th17 cells and T(regs) should be explored as a means of preventing progression to NASH and advanced liver diseases in NAFLD patients.     

Adoptive transfer of regulatory NKT lymphocytes ameliorates non-alcoholic steatohepatitis and glucose intolerance in ob/ob mice and is associated with intrahepatic CD8 trapping

The aim of this study was to determine the effect of adoptive transfer of regulatory natural killer T (NKT) lymphocytes on the metabolic disorder in leptin-deficient ob/ob mice, which feature depletion and defective function of NKT and CD4 lymphocytes. Leptin-deficient ob/ob mice were subjected to transplantation of 1 x 10(6) of either ob/ob or wild-type-derived NKT lymphocytes, or to transplantation of either ob/ob or wild-type-derived splenocytes. The effect on hepatic fat content was measured by magnetic resonance imaging (signal intensity index) and histology, using the steatohepatitis grading scale. The degree of glucose intolerance was measured by an oral glucose tolerance test (GTT). Adoptive transfer of wild-type or ob/ob-derived regulatory NKT cells led to a 12% decrease in hepatic fat content. A significant histological shift from macrosteatosis to microsteatosis was observed. Marked improvement in the GTT was noted in wild-type or ob/ob-derived NKT recipients. Metabolic effects were associated with a significant decrease in peripheral and intrahepatic CD4/CD8 lymphocyte ratios. Intrahepatic CD8 trapping was observed in all responders. Serum interleukin 10 levels decreased significantly. In conclusion, adoptive transfer of a relatively small number of regulatory NKT lymphocytes into ob/ob mice results in a significant reduction in hepatic fat content, a shift from macro to microsteatosis, and significant improvement in glucose intolerance. These effects were associated with decreased peripheral and intrahepatic CD4/CD8 ratios and decreased interleukin 10 levels. The results further support a role for regulatory NKT lymphocytes in the pathogenesis of non-alcoholic steatohepatitis in the leptin-deficient murine model.     

The epidemiology, pathogenesis and histopathology of fatty liver disease

Fatty liver disease includes non-alcoholic fatty liver disease (NAFLD) and alcoholic liver disease (ALD), each of which is increasing in prevalence. Each represents a histological spectrum that extends from isolated steatosis to steatohepatitis and cirrhosis. NAFLD is associated with obesity, diabetes, and insulin resistance, and is considered to be the liver manifestation of the metabolic syndrome. The pathogenesis of NAFLD and ALD involves cytokines, adipokines, oxidative stress, and apoptosis. Histopathology is the gold standard for assessing the severity of liver damage in NAFLD and ALD. We have reviewed the literature, and described and compared the epidemiology, natural disease history, pathogenesis and histopathology of NAFLD and ALD.     

Calorie restriction increases insulin‐stimulated tyrosine phosphorylation of insulin receptor and insulin receptor substrate‐1 in rat skeletal muscle

A moderate reduction in calorie intake (calorie restriction, CR) improves insulin‐stimulated glucose transport in skeletal muscle. Therefore, we studied muscle insulin signalling in ad libitum (AL) and CR (~60% AL intake for 20 days) fed rats, which received a control injection (sterile water) or an insulin injection (30 U kg^–1 body weight). In control (not insulin‐treated) rats, there was no detectable tyrosine phosphorylation of insulin receptor (IR), regardless of diet; no diet effect on tyrosine phosphorylation of insulin receptor substrate‐1 (IRS1) or IRS1‐associated phosphatidylinositol 3‐kinase (PI3K) protein and 21% higher IRS1‐associated PI3K activity in AL vs. CR. In insulin‐treated rats, tyrosine‐phosphorylated IR was 79% higher for CR vs. AL; tyrosine‐phosphorylated IRS1 was 109% higher for CR vs. AL; IRS1‐associated PI3K protein and IRS1‐associated PI3K activity were unaffected by diet. Calorie restriction amplifies early insulin signalling steps without changing IRS1‐associated PI3K, suggesting enhanced glucose transport is mediated by altering: IRS1‐PI3K localization, PI3K associated with proteins other than IRS1 or post‐PI3K events.     

Role of non‐coding RNAs in pancreatic beta‐cell development and physiology

The progression of diabetes is accompanied by increasing demand to the beta‐cells to produce and secrete more insulin, requiring complex beta‐cell adaptations. Functionally active and ubiquitous non‐coding RNAs (ncRNAs) have the capacity to take part in such adaptations as they have been shown to be key regulatory molecules in various biological processes. In the pancreatic islets, the function of ncRNAs and their contribution to disease development is beginning to be understood. Here, we review the different classes of ncRNAs, such as long non‐coding RNAs (lncRNAs) and microRNAs (miRNAs), and their potential contribution to insulin secretion. A special focus will be on miRNAs and their regulatory function in beta‐cell physiology and insulin exocytosis. As important players in gene regulation, ncRNAs have huge potential in opening innovative therapeutic avenues against diabetes and associated complications.     

The induction of autoimmune hepatitis in the human leucocyte antigen‐DR4 non‐obese diabetic mice autoimmune hepatitis mouse model

Autoimmune hepatitis (AIH) is a chronic liver disease characterized by progressive inflammation, female preponderance and seropositivity for autoantibodies such as anti‐smooth muscle actin and/or anti‐nuclear, anti‐liver kidney microsomal type 1 (anti‐LKM1) and anti‐liver cytosol type 1 (anti‐LC1) in more than 80% of cases. AIH is linked strongly to several major histocompatibility complex (MHC) alleles, including human leucocyte antigen (HLA)‐DR3, ‐DR7 and ‐DR13. HLA‐DR4 has the second strongest association with adult AIH, after HLA‐DR3. We investigated the role of HLA‐DR4 in the development of AIH by immunization of HLA‐DR4 (DR4) transgenic non‐obese diabetic (NOD) mice with DNA coding for human CYP2D6/FTCD fusion autoantigen. Immunization of DR4 mice leads to sustained mild liver injury, as assessed biochemically by elevated alanine aminotransferase, histologically by interface hepatitis, plasma cell infiltration and mild fibrosis and immunologically by the development of anti‐LKM1/anti‐LC1 antibodies. In addition, livers from DR4 mice had fewer regulatory T cells (T_regs), which had decreased programmed death (PD)‐1 expression. Splenic T_regs from these mice also showed impaired inhibitory capacity. Furthermore, DR4 expression enhanced the activation status of CD8⁺ T cells, macrophages and dendritic cells in naive DR4 mice compared to naive wild‐type (WT) NOD mice. Our results demonstrate that HLA‐DR4 is a susceptibility factor for the development of AIH. Impaired suppressive function of T_regs and reduced PD‐1 expression may result in spontaneous activation of key immune cell subsets, such as antigen‐presenting cells and CD8⁺ T effectors, facilitating the induction of AIH and persistent liver damage.