Endoplasmic reticulum stress does not contribute to steatohepatitis in obese and insulin resistant high-fat diet fed foz/foz mice

Non-alcoholic fatty liver (steatosis) and steatohepatitis [non-alcoholic steatohepatitis (NASH)] are hepatic complications of the metabolic syndrome. Endoplasmic reticulum (ER) stress is proposed as a crucial disease mechanism in obese and insulin-resistant animals (such as ob/ob mice) with simple steatosis, but its role in NASH remains controversial. We therefore evaluated the role of ER stress as a disease mechanism in foz/foz mice, which develop both the metabolic and histological features that mimic human NASH. We explored ER stress markers in the liver of foz/foz mice in response to a high-fat diet (HFD) at several time points. We then evaluated the effect of treatment with an ER stress inducer tunicamycin, or conversely with the ER protectant tauroursodeoxycholic acid (TUDCA), on the metabolic and hepatic features. foz/foz mice are obese, glucose intolerant and develop NASH characterized by steatosis, inflammation, ballooned hepatocytes and apoptosis from 6 weeks of HFD feeding. This was not associated with activation of the upstream unfolded protein response [phospho-eukaryotic initiation factor 2α (eIF2α), inositol-requiring enzyme 1α (IRE1α) activity and spliced X-box-binding protein 1 (Xbp1)]. Activation of c-Jun N-terminal kinase (JNK) and up-regulation of activating transcription factor-4 (Atf4) and CCAAT/enhancer-binding protein-homologous protein (Chop) transcripts were however compatible with a ‘pathological’ response to ER stress. We tested this by using intervention experiments. Induction of chronic ER stress failed to worsen obesity, glucose intolerance and NASH pathology in HFD-fed foz/foz mice. In addition, the ER protectant TUDCA, although reducing steatosis, failed to improve glucose intolerance, hepatic inflammation and apoptosis in HFD-fed foz/foz mice. These results show that signals driving hepatic inflammation, apoptosis and insulin resistance are independent of ER stress in obese diabetic mice with steatohepatitis

Defective adaptive thermogenesis contributes to metabolic syndrome and liver steatosis in obese mice

Abstract Fatty liver diseases are complications of the metabolic syndrome associated with obesity, insulin resistance and low grade inflammation. Our aim was to uncover mechanisms contributing to hepatic complications in this setting. We used foz/foz mice prone to obesity, insulin resistance and progressive fibrosing non-alcoholic steatohepatitis (NASH). Foz/foz mice are hyperphagic but wild-type (WT)-matched calorie intake failed to protect against obesity, adipose inflammation and glucose intolerance. Obese foz/foz mice had similar physical activity level but reduced energy expenditure. Thermogenic adaptation to high-fat diet (HFD) or to cold exposure was severely impaired in foz/foz mice compared with HFD-fed WT littermates due to lower sympathetic tone in their brown adipose tissue (BAT). Intermittent cold exposure (ICE) restored BAT function and thereby improved glucose tolerance, decreased fat mass and liver steatosis. We conclude that failure of BAT adaptation drives the metabolic complications of obesity in foz/foz mice, including development of liver steatosis. Induction of endogenous BAT function had a significant therapeutic impact on obesity, glucose tolerance and liver complications and is a potential new avenue for therapy of non-alcoholic fatty liver disease (NAFLD)

Brown adipose tissue in metabolic syndrome, NAFLD progression and management

Non-alcoholic fatty liver disease (NAFLD) is a progressive disease associated with insulin resistance and obesity. The spectrum of NAFLD ranges from simple steatosis to non-alcoholic steatohepatitis (NASH) characterized by steatosis, inflammation, hepatocyte injuries and progressive fibrosis. No treatment has been proven efficacious except for lifestyle modifications coupling physical exercise with weight reduction. Better understanding NASH pathogenesis should light on new mechanisms involved in NAFLD progression and thereby on new targets to counteract NAFLD development. High-fat diet (HFD)-fed foz/foz mice, a strain of mice carrying a mutation in a ciliary protein, represent a unique mouse model of NASH as they develop both the metabolic context and the histological features of human NASH. Their unique metabolic phenotype has been linked to hyperphagia resulting from abnormal ciliary function in the central nervous system but the links between obesity, metabolic syndrome and fibrosing NASH are not elucidated. My aim was thus to unveil mechanisms contributing to the phenotype in this model. In this doctoral thesis, we performed a pair-feeding experiment to determine whether the metabolic and liver alterations in foz/foz mice were only related to an increase in food intake. Caloric restriction failed to protect foz/foz mice against obesity, adipose inflammation and glucose intolerance. Obese foz/foz mice had similar physical activity level but reduced energy expenditure compared to their wild-type (WT) littermates. We identified an altered thermogenic adaptation to HFD or a cold exposure in foz/foz mice compared to HFD-fed WT littermates hence decreasing energy substrate utilization. Defective adaptive thermogenesis was due to a lower sympathetic tone in brown adipose tissue (BAT). Sympathetic stimulation by intermittent cold exposure restored BAT function in foz/foz mice and thereby improved glucose tolerance, adiposity and hepatic steatosis. Next, in interventional experiments, we evaluated whether increasing non-shivering thermogenesis could prevent and/or improve pre-existing NASH. In foz/foz mice with metabolic syndrome and liver steatosis, β3 adrenergic receptor (β3AR) agonist improved BAT function and induced browning of white adipose tissue. Increased thermogenic capacity was associated with a better glucose tolerance, a decreased NAFLD activity score and decreased transaminases levels with no change in body weight. When initiated after the onset of NASH in foz/foz mice, β3AR agonist treatment restored BAT function and increased glucose tolerance but had no impact on liver pathology compared to untreated mice. Similarly, β3AR agonist had no therapeutic effect when administrated for 4 weeks on methione and choline deficient diet-induced NASH neither in C57Bl6 nor in obese and diabetic db/db mice. As boosting BAT activity had no impact of body weight loss, we reasoned that increased energy substrate mobilization through weight loss therapy could divert lipids towards burning in the BAT and improve NASH. When coupled with caloric restriction, β3AR agonist enhanced weight loss and glucose tolerance compared to mice with caloric restriction only. In addition, the combined therapy, but not food restriction alone, decreased hepatic fat content, hepatocyte ballooning and NAFLD activity score, thus improving pre-established NASH. In conclusion, our findings indicate that failure of BAT adaptation drives the metabolic complications of obesity in foz/foz mice, including development of NAFLD. Increasing thermogenic capacities prevents NAFLD progression but is not effective to cure pre-existing NASH. Nonetheless, when coupled with weight loss therapy, BAT stimulation provides additional therapeutic advantage in NASH management and opens new possibilities to improve liver histology in NAFLD patients.(BIFA - Sciences biomédicales et pharmaceutiques) -- UCL, 201

Brown adipose tissue: a potential target in the fight against obesity and the metabolic syndrome.

BAT (brown adipose tissue) is the main site of thermogenesis in mammals. It is essential to ensure thermoregulation in newborns. It is also found in (some) adult humans. Its capacity to oxidize fatty acids and glucose without ATP production contributes to energy expenditure and glucose homoeostasis. Brown fat activation has thus emerged as an attractive therapeutic target for the treatment of obesity and the metabolic syndrome. In the present review, we integrate the recent advances on the metabolic role of BAT and its relation with other tissues as well as its potential contribution to fighting obesity and the metabolic syndrome

Increased non-shivering thermogenesis had preventive but no therapeutic effects on non-alcoholic steatohepatitis

Background and Aims: Non-alcoholic steatohepatitis (NASH) is the progressive form of non-alcoholic fatty liver disease spectrum. No treatment has been proven efficacious except for lifestyle modifications coupling physical exercise with weight reduction. We recently identified defective adaptive thermogenesis as a contributing factor to obesity and metabolic syndrome in foz/foz mice. We now aim to test whether increased non-shivering thermogenesis prevent and/or improve pre-existing NASH in mice. Methods: A HFD for 4 or 8weeks induced a metabolic syndromewith fatty liver or NASH, respectively in male foz/foz mice. Mice were randomized and treated with a beta 3-adrenergic receptor (B3AR) POSTER PRESENTATIONS Journal of Hepatology 2017 vol. 66 | S333–S542 S433 agonist (CL-316,243–1mg/kg/day) to enhance thermogenic capacities or with vehicle (untreated) together with HFD for 2 or 4 weeks, respectively. C57Bl6 and db/db mice were fed a methionine and choline deficient (MCD) diet to induce NASH and treated with B3AR agonist for 4 additional weeks (n = 6–8/group). Results: In foz/foz mice with metabolic syndrome and liver steatosis, B3AR agonist improved brown adipose tissue (BAT) function assessed by increased cAMP and UCP1 BAT contents and upregulation of thermogenic genes (UCP1, DIO2). It also caused browning of white adipose tissue. All this resulted in increased tolerance to cold exposure and was associated with a better glucose tolerance (p < 0.05), a decreased NAS score (2 ± 1.3 vs 3.7 ± 1.6; p < 0.05) and decreased transaminases levels (p < 0.05) with no change in body weight. When treatment was initiated after the onset of NASH (NAS score = 5 ± 1.15) in foz/foz mice, B3AR agonist treatment restored BAT function, induced a slight 2%weight loss (p < 0.05), increased glucose tolerance (p < 0.001) but had no on impact liver pathology (NAS score 5.6 ± 2.1 vs 6.7 ± 1.3; ALT 286 ± 117 vs 396 ± 190 U/L) compared to untreated mice. Similarly, B3AR agonist has no therapeutic effect when administrated for 4 weeks on MCD-induced NASH whether in C57Bl6 or in obese and diabetics db/db mice. Conclusions: B3AR agonist treatment improved BAT function and glucose tolerance, prevented the progression of a simple steatosis to NASH but was not sufficient to cure a pre-established NASH, supporting previous observation that control over metabolic syndrome is insufficient to treat NASH. In our study, B3AR agonist caused no major weight loss and therefore, it will be of interest to evaluate whether BAT stimulation offers an additional advantage over weight loss therapy in NASH management

Activation of brown adipose tissue enhances the efficacy of caloric restriction for treatment of nonalcoholic steatohepatitis.

Nonalcoholic steatohepatitis (NASH) is the form of nonalcoholic fatty liver disease that can evolve into cirrhosis. Lifestyle modifications achieving 10% weight loss reverse NASH, but there are no effective approved drug treatments. We previously identified defective adaptive thermogenesis as a factor contributing to metabolic syndrome and hepatic steatosis. We have now tested whether increasing nonshivering thermogenesis can improve preexisting NASH in mice. In high-fat diet-fed foz/foz mice with established NASH, treatment with β3AR agonist restored brown adipose tissue (BAT) function, decreased body weight, improved glucose tolerance, and reduced hepatic lipid content compared to untreated counterparts, but had no impact on liver inflammation or on nonalcoholic fatty liver disease activity score (NAS). Similarly, β3AR agonist did not alter liver pathology in other steatohepatitis models, including MCD diet-fed diabetic obese db/db mice. Caloric restriction alone alleviated the hepatic inflammatory signature in foz/foz mice. Addition of a β3AR agonist to mice subjected to caloric restriction enhanced weight loss and glucose tolerance, and improved liver steatosis, hepatocellular injury, and further reduced liver inflammation. These changes contributed to a significantly lower NAS score such as no (0/9) animals in this group fulfilled the criteria for NASH pathology compared to eight out of ten mice under caloric restriction alone. In conclusion, β3AR agonist counteracts features of the metabolic syndrome and alleviates steatosis, but does not reverse NASH. However, when coupled with weight loss therapy, BAT stimulation provides additional therapeutic advantages and reverses NASH

Defective adaptive thermogenesis contributes to metabolic syndrome and liver steatosis in obese mice.

Fatty liver diseases are complications of the metabolic syndrome associated with obesity, insulin resistance and low grade inflammation. Our aim was to uncover mechanisms contributing to hepatic complications in this setting. We used foz/foz mice prone to obesity, insulin resistance and progressive fibrosing NASH. Foz/foz mice are hyperphagic but WT-matched calorie intake failed to protect against obesity, adipose inflammation and glucose intolerance. Obese foz/foz mice had similar physical activity level but reduced energy expenditure. Thermogenic adaptation to high-fat diet or to a cold exposure was severely impaired in foz/foz mice compared to HFD-fed WT littermates due to lower sympathetic tone in their brown adipose tissue (BAT). Intermittent cold exposure restored BAT function and thereby improved glucose tolerance, decreased fat mass and liver steatosis. We conclude that failure of BAT adaptation drives the metabolic complications of obesity in foz/foz mice, including development of liver steatosis. Induction of endogenous BAT function had a significant therapeutic impact on obesity, glucose tolerance and liver complications and is a potential new avenue for therapy of non-alcoholic fatty liver disease

Lipid, fetuin-A and macrophage zonation in high fat diet foz-foz mice with non-alcoholic steatohepatitis

Introduction Non-alcoholic fatty liver disease (NAFLD) is characterized by steatosis (accumulation of triglycerides in the liver) and insulin resistance. A subgroup of patients can develop a more serious condition called non-alcoholic steatohepatitis (NASH) with increased risk of fibrosis development. Innate immunity, cell injury, lipid metabolism and severity of insulin resistance constitute potential mechanisms underlying disease progression. Fetuin-A , an emerging player in insulin resistance in type 2 diabetic patients, is described as a liver-derived protein increased in human NAFLD. Aim Here, we explore the effect of a high fat diet on the expression of fetuin-A and its relation with the development of steatosis, cell injury and liver macrophage (Kupffer cell) activation in a mouse model of obesity and NASH. Methods : Male foz/foz mice were fed a normal diet (ND) or a high fat diet (HFD) for 12 (long term HFD or LHFD) or 30 weeks (very long term HFD or VLHFD) (n=4/group) to induce early or definite fibrosing NASH, respectively. Liver tissue homogenates were prepared for Western blot protein studies and total RNA was extracted for gene expression analysis. Liver paraffin-embedded sections were used for hematoxylin and eosin staining, Sirius red staining and double immunofluorescence detection of F4/80 and fetuin-A. Results : Compared to foz/foz mice fed a ND, HFD-fed foz/foz mice developed obesity, insulin resistance and either steatosis (LHFD) or steatohepatitis with steatosis, hepatocyte ballooning, inflammation and fibrosis (VLHFD). In ND fed mice, fetuin-A staining was positive in the cytoplasm of zone 3 centrilobular hepatocytes while F4/80+ Kupffer cells were located in the sinusoids of the intermediate lobular zone 2. In LHFD fed mice, lipid deposition occurred in the hepatocytes of the zone 3 centrilobular areas. Fetuin-A protein was also located in the cytoplasm of these zone 3 centrilobular hepatocytes. F4/80+ macrophages distributed mainly in the sinusoids of the intermediate lobular zones 2, as seen in ND fed mice. However, liver m-RNA expression showed a 2-fold increased level of F4/80+ macrophage mRNA compared to ND (p<0.05), suggesting activation. In VLHD, we observed a loss of zonation of liver steatosis with the presence of fat loaded hepatocytes in all liver lobular zones. Fetuin-A was highest in periportal fat-ladden hepatocytes and next to inflammatory infiltrates. There was a 4-fold F4/80 mRNA increased level upon VLHFD compared to ND (p<0.05). Three types of F4/80+ cells were recognized on the morphology: elongated cells located in liver sinusoids compatible with liver resident Kupffer cells, cells forming lipogranuloma together with fat loaded hepatocytes and small inflammatory cells located in inflammatory foci compatible with recruited macrophages. Interestingly, F4/80+ cells from lipogranuloma were positive for fetuin-A protein staining. Liver fetuin-A mRNA levels remained unchanged either in LHFD or VLHFD compared to ND. Similarly, liver fetuin-A protein level was also stable under HFD. Conclusion : We demonstrate that VLHFD foz-foz mice develop NASH together with zonal changes of steatosis, liver macrophage activation and fetuin-A expression in fatty hepatocytes and macrophages. A shift of steatosis and fetuin-A from the centrilobular region in ND and LHFD to the periportal zone was observed in VLHFD, together with macrophage activation, recruitment and fetuin-A co-localization in macrophages forming the lipogranuloma. The stable liver fetuin-A protein level could be compatible with a redistribution of this protein and/or the profile of a secretory factor. Taken together, we could imagine that lipid deposition and macrophage infiltration may be important factors in the liver tissue remodeling observed during NASH development. Further work is planned to delineate whether fetuin-A presence in macrophages is linked with a production and/or a simple storage in those cells in this model as well as the role of this protein in NASH progression and insulin resistance pathogenesis

Defective adaptive thermogenesis contributes to metabolic syndrome and liver steatosis in obese mice

Fatty liver diseases are complications of the metabolic syndrome associated with obesity, insulin resistance and low grade inflammation. Our aim was to uncover mechanisms contributing to hepatic complications in this setting. We used foz/foz mice prone to obesity, insulin resistance and progressive fibrosing non-alcoholic steatohepatitis (NASH). Foz/foz mice are hyperphagic but wild-type (WT)-matched calorie intake failed to protect against obesity, adipose inflammation and glucose intolerance. Obese foz/foz mice had similar physical activity level but reduced energy expenditure. Thermogenic adaptation to high-fat diet (HFD) or to cold exposure was severely impaired in foz/foz mice compared with HFD-fed WT littermates due to lower sympathetic tone in their brown adipose tissue (BAT). Intermittent cold exposure (ICE) restored BAT function and thereby improved glucose tolerance, decreased fat mass and liver steatosis. We conclude that failure of BAT adaptation drives the metabolic complications of obesity in foz/foz mice, including development of liver steatosis. Induction of endogenous BAT function had a significant therapeutic impact on obesity, glucose tolerance and liver complications and is a potential new avenue for therapy of non-alcoholic fatty liver disease (NAFLD)This work was supported by the Communaute franc ´ ¸aise de Belgique – Actions de Recherche Concertees [grant number 12/17- ´ 047]; the Fund for Scientific Medical Research [grant number PDR T.1067.14] and unrestricted research grants from Gilead Belgium, Janssens Pharmaceutica Belgium, and Roche Belgium. I.A.L. is senior research associate with the Belgian National Fund for Scientific Research (FNRS) and fellowship to V.L. has been supported by the Institut de Recherche experimentale et Clinique (IREC) at the ´ Universite catholique de Louvain, Brussels, Belgium. The behavior ´ platform was supported by the Programme d’Excellence Marshall, Wallonia regio and Universite catholique de Louvain (Diane conven- ´ tion)