Recently, persistent organic pollutants such as polychlorinated biphenyls (PCBs) were classified as “metabolic disruptors” for their suspected roles is altering metabolic and energy homeostasis through bioaccumulation in liver and adipose tissues. Among PCBs, a specific congener, 3,3',4,4',5-pentachlorobiphenyl (PCB126), is a potent arylhydrocarbon receptor (AhR) agonist and elicits toxicity similar to the classic dioxin, 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). PCB126 levels found in human blood are particularly associated with diabetes and nonalcoholic fatty liver disease (NAFLD) in humans, however the mechanisms are unclear. We hypothesized that the accumulation of PCB126 disrupts carbohydrate and lipid metabolism by altering the functions of liver and adipose tissues. Hence, our objective was to characterize PCB126 induced-metabolic disruption and the underlying molecular mechanisms that cause toxicity. Separate animal studies were performed using a rat model to understand the time- and dose-dependent effects after PCB126 administration. The chronology of PCB126 toxicity showed early decreases in serum glucose level at 9 h, worsened in a time-dependent way until the end of the study at 12 d. Lipid accumulation and the liver pathology also worsened over time between 3 d and 12 d post administration. These observed effects in the liver were also found to be dose-dependent. The decrease in serum glucose was a result of a decrease in the transcript levels of gluconeogenic and glycogenolytic enzymes, necessary for hepatic glucose production and hence the maintenance of steady glucose levels in the blood. Phosphoenolpyruvate carboxykinase (PEPCK-C), the rate limiting enzyme of gluconeogenesis, was found to be significantly decreased upon exposure to PCB126. The expression levels of peroxisome proliferator-activated receptor alpha (Pparα) and some of its targets involved in fatty acid oxidation were also found to be time and dose-dependently decreased upon exposure to PCB126. In an attempt to understand the molecular targets that may cause these dual effects on both gluconeogenic and fatty acid oxidation, we found that PCB126 significantly decreases phosphorylation of the cAMP response element-binding protein (CREB). CREB is a nuclear transcription factor that is activated in the liver through phosphorylation; to switch-on the transcription of enzymes that catalyze gluconeogenesis and fatty acid oxidation, in order to meet energy demands, especially during fasting. Further, to understand the toxicity of PCB126 on adipose tissue, a human pre-adipocyte model that can be differentiated into mature adipocytes was used. In these studies, we found that exposure of preadipocytes to PCB126 resulted in a significant reduction in their ability to differentiate into adipocytes. This results in decreased lipid accumulation in the adipocyte. Reduction in the differentiation by PCB126 was associated with down regulation in transcript levels of a key adipocyte transcription factor, PPARγ and its transcriptional targets necessary for adipogenesis and adipocyte function. These inhibitory effects of PCB126 on the regulation of PPARγ and the initiation of adipogenesis were mediated through activation of AhR. Overall, this work shows that PCB126 disrupts nutrient homeostasis through its effects on the function of target tissues; liver and adipose. PCB126 significantly alters the nutrient homeostasis through its effects on gluconeogenesis and fatty-acid oxidation necessary for glucose and energy regulation during fasting. In addition, PCB126 interrupts the storage functions of adipose tissue by inhibiting adipogenesis and thus disrupts lipid storage and distribution

La sleeve gastrectomie (SG) est une opération qui consiste à réduire le volume de l’estomac. L’hypothèse que d’autres mécanismes indépendants de la perte de poids sont impliqués dans l’amélioration des complications métaboliques de l’obésité après SG a été émise. L’effet de la SG chez des souris soumis à un régime High Fat Diet a été étudié chez trois groupes d’animaux : SG, sham pair fed (SPF) et sham. Le test de tolérance au glucose montrait une amélioration de l’insulinorésistance des animaux SG à J23. Au niveau hépatique les animaux SG montraient une diminution significative de la stéatose. Il existe donc des mécanismes améliorant les complications hépatiques et métaboliques de l’obésité qui sont en partie indépendants de la réduction de l’apport calorique. Dans le second volet nous avons étudié l’évolution à long terme des lésions hépatiques liées à la NASH chez des patients obèses morbides avec une NASH lors de la chirurgie bariatrique. Dix patients d’une cohorte prospective ont été inclus. La deuxième biopsie a été réalisée à une médiane de 57 mois après le RYGB. La perte de poids moyenne était de –13,3 points de l’IMC lors du suivi. La rémission du syndrome métabolique et du diabète a été observée chez 71,6 % et 100 % des patients respectivement. Le NAS score a été amélioré chez tous les patients. Le taux sérique moyen du fragment clivé de la cytokératine 18 (M30), marqueur de l’apoptose hépatocytaire, était significativement abaissé. Le RYGB a permis une amélioration à long terme des lésions hépatocytaires liées à la NASH chez les patients obèses morbides. L’amélioration post-opératoire de la souffrance hépatocytaire corrèle avec la baisse du taux sérique du M30. / The mechanisms responsible for weight loss and improvement of metabolic disturbances have not been completely elucidated. We investigated the effect of sleeve gastrectomy (SG) on body weight, adipose tissue depots, glucose tolerance, and liver steatosis independent of reduced caloric intake in high-fat-diet-induced obese mice. Mice fed a high fat diet were divided into 3 groups: SG, sham-operated ad libitum fed and sham-operated pair fed. SG mice showed improved glucose tolerance and lower levels of liver steatosis. This was associated with a decrease in the ratios of the weight of pancreas, epididymal and inguinal adipose tissues to body weight. Reduced white adipose tissue inflammation, modification of adipose tissue development, and ectopic fat are potential mechanisms that may account for the reduced caloric intake independent effects of SG. We also investigated long-term impact of RYGB surgery on liver complications in morbidly obese patients with NASH. Ten morbidly obese patients with biopsy-proven NASH were followed after RYGB and underwent a second liver biopsy. The median interval between the RYGB and second liver biopsy was 57 months. Clinical and biological data were obtained at baseline and ≥40 months after RYGB. RYGB was associated with significant weight loss, improved hepatic steatosis, resolution of hepatic inflammation and hepatocyte ballooning. Hepatocyte apoptosis, as evaluated by serum K18 fragment improved within the first year and at 57 months. Hepatic fibrosis resolved in 90% of cases. RYGB in morbidly obese patients with NASH is associated with a long-term beneficial impact on hepatic steatosis, inflammation, injury and, possibly, fibrosis.

Hepatitis C virus (HCV) infects millions of people worldwide and is one of the leading causes of liver damage. Infection with HCV is strongly correlated with an increased risk of steatosis, or fatty liver disease, which is caused by a build-up of fat deposits in hepatocytes. All genotypes of HCV appear to cause some degree of steatosis in approximately 50% of infected individuals, especially in the presence of contributing host factors such as diabetes, obesity and alcoholism. However, approximately 70% of genotype 3a infections exhibit steatosis. Furthermore, successful clearance of the genotype 3a virus results in eradication of the steatosis, suggesting the genotype 3a virus may be able to directly cause steatosis.<p> Research suggests a role for the core protein of HCV, which forms the capsid of the virus, in the alteration of lipid metabolism pathways during infection. As such, I hypothesized that: 1) HCV alters lipid metabolism pathways and causes the build up of lipid in hepatocytes and the development of steatosis; 2) HCV-3a core protein has a differential or increased effect on these pathways in comparison to 1b core protein; and 3) other HCV proteins could also play a role in the altering of lipid metabolism. My research characterized the subcellular localization on lipid droplets of the HCV-3a core protein in comparison to HCV-1b core protein. It was found that HCV-3a core causes increased transcriptional activity from the Fatty Acid Synthase (FAS) promoter, an important enzyme involved in the synthesis of triglycerides in hepatocytes. In addition, one specific amino acid of HCV-3a core was determined to be partially responsible for this effect. Further research determined that the effect of HCV-3a core on FAS was dependent on the transcription factor Sterol Response Element Binding Protein-1 (SREBP-1) and the presence of HCV-3a core increased the processing and activity of SREBP-1. HCV core was also able to increase activity of Akt 1 and Akt2; inhibition of Akt activity resulted in decreased SREBP-1 activity thereby indicating that HCV core partially mediates SREBP-1 via Akt. Further experiments examined the role of another HCV protein, NS2, in these same lipid metabolism pathways. NS2 was also able to increase transcription from the FAS promoter via SREBP-1, suggesting that this HCV protein may also be important in the development of steatosis during HCV infection.<p> The evidence provided in these studies shows a very important role for HCV in altering lipid metabolism during infection that may lead to the development of steatosis. Current research suggests that the SREBP-1 pathway may be critical in the life cycle of the virus and these studies have provided important information on how lipid metabolism pathways are being changed by the virus. Hopefully this work can help identify potential treatment options for HCV that can slow down disease progression by preventing the development of steatosis or by decreasing viral replication.

Post translational modifications in histone proteins are transmissible changes that are not coded for in the DNA sequence itself but have a significant affect in the control of gene expression. Eukaryotic transcription is a regulated process, and acetylation plays a major role in this regulation. Deranged equilibrium of histone acetylation can lead to alteration in chromatin structure and transcriptional dysregulation of genes that are involved in the control of proliferation, cell-cycle progression, differentiation and or apoptosis. Evidence shows that high glucose conditions mimicking diabetes can increase histone acetylation and augment the inflammatory gene expression. Recent advances also highlight the involvement of altered histone acetylation in gastrointestinal carcinogenesis or hyperacetylation in amelioration of experimental colitis. However, the role of histone acetylation under obesity conditions is not yet known. Therefore in the present study, western blot analysis in the liver of Zucker obese versus lean rats was performed to determine the pattern and level of H3 and H4 acetylation (both in nuclear and homogenate fractions) at specific lysine (K) in pathological state of hepatic steatosis The same technique was also applied in the liver of obese rats fed higher amounts of vitamin B6 (OH) versus those fed normal amounts of vitamin B6 (ON) to assess if hyper-acetylation can be a protective response to hepatic steatosis. In both experimental models, it was also of interest to elucidate the expression of anti- and pro- apoptotic factor Bcl-2 and Bax in respect to histone acetylation. It was observed that, in liver homogenate fractions in control animals (LC/OC), there was a higher level of histone H3 acetylation at (K9, K14) and H4 acetylation at K5 in the obese animals. In contrast, the nuclear level of H3 and H4 acetylation at the same lysine residues was considerably higher in the lean and lower in the obese animals. Obese animals contained lower liver preneoplastic lesions as well as liver weight as a result of higher amounts of vitamin B6, had significantly higher H3 acetylation at K9 and K14 and H4 acetylation at K5, in both homogenate and nuclear fractions. However, histone acetylation was not detected for histone H4 at lysine 12 (K12) in either control group (LC/OC) or obese with different B6 diet group (OH/ON). Nevertheless, global histone H3 and H4 acetylation in both homogenate and nuclear fractions, was slightly higher in the lean rats and obese rats fed higher amounts of B6. By using the western blot technique, the level of anti- and pro- apoptotic Bcl-2 and Bax were also evaluated. The moderately higher level expression of anti-apoptotic Bcl2 protein was found in lean animals, whereas the expression of pro-apoptotic Bax was significantly higher in obese animals. Furthermore, anti-apoptotic Bcl2 protein expression was slightly higher in the obese rats fed normal amounts of B6 diet; but, pro-apoptotic Bax was higher in the obese rats fed higher amounts of vitamin B6. This is the first study which shows that hyperacetylation of histones in liver nuclei can be correlated with amelioration of hepatic steatotis. Histone acetylation and B6 rich diet might be involved in the regulation of biological availability of key apoptotic proteins, which, in turn, can possibly modify the severity of the disease.

Post translational modifications in histone proteins are transmissible changes that are not coded for in the DNA sequence itself but have a significant affect in the control of gene expression. Eukaryotic transcription is a regulated process, and acetylation plays a major role in this regulation. Deranged equilibrium of histone acetylation can lead to alteration in chromatin structure and transcriptional dysregulation of genes that are involved in the control of proliferation, cell-cycle progression, differentiation and or apoptosis. Evidence shows that high glucose conditions mimicking diabetes can increase histone acetylation and augment the inflammatory gene expression. Recent advances also highlight the involvement of altered histone acetylation in gastrointestinal carcinogenesis or hyperacetylation in amelioration of experimental colitis. However, the role of histone acetylation under obesity conditions is not yet known. Therefore in the present study, western blot analysis in the liver of Zucker obese versus lean rats was performed to determine the pattern and level of H3 and H4 acetylation (both in nuclear and homogenate fractions) at specific lysine (K) in pathological state of hepatic steatosis The same technique was also applied in the liver of obese rats fed higher amounts of vitamin B6 (OH) versus those fed normal amounts of vitamin B6 (ON) to assess if hyper-acetylation can be a protective response to hepatic steatosis. In both experimental models, it was also of interest to elucidate the expression of anti- and pro- apoptotic factor Bcl-2 and Bax in respect to histone acetylation. It was observed that, in liver homogenate fractions in control animals (LC/OC), there was a higher level of histone H3 acetylation at (K9, K14) and H4 acetylation at K5 in the obese animals. In contrast, the nuclear level of H3 and H4 acetylation at the same lysine residues was considerably higher in the lean and lower in the obese animals. Obese animals contained lower liver preneoplastic lesions as well as liver weight as a result of higher amounts of vitamin B6, had significantly higher H3 acetylation at K9 and K14 and H4 acetylation at K5, in both homogenate and nuclear fractions. However, histone acetylation was not detected for histone H4 at lysine 12 (K12) in either control group (LC/OC) or obese with different B6 diet group (OH/ON). Nevertheless, global histone H3 and H4 acetylation in both homogenate and nuclear fractions, was slightly higher in the lean rats and obese rats fed higher amounts of B6. By using the western blot technique, the level of anti- and pro- apoptotic Bcl-2 and Bax were also evaluated. The moderately higher level expression of anti-apoptotic Bcl2 protein was found in lean animals, whereas the expression of pro-apoptotic Bax was significantly higher in obese animals. Furthermore, anti-apoptotic Bcl2 protein expression was slightly higher in the obese rats fed normal amounts of B6 diet; but, pro-apoptotic Bax was higher in the obese rats fed higher amounts of vitamin B6. This is the first study which shows that hyperacetylation of histones in liver nuclei can be correlated with amelioration of hepatic steatotis. Histone acetylation and B6 rich diet might be involved in the regulation of biological availability of key apoptotic proteins, which, in turn, can possibly modify the severity of the disease.

Hepatitis C virus (HCV) infects millions of people worldwide and is one of the leading causes of liver damage. Infection with HCV is strongly correlated with an increased risk of steatosis, or fatty liver disease, which is caused by a build-up of fat deposits in hepatocytes. All genotypes of HCV appear to cause some degree of steatosis in approximately 50% of infected individuals, especially in the presence of contributing host factors such as diabetes, obesity and alcoholism. However, approximately 70% of genotype 3a infections exhibit steatosis. Furthermore, successful clearance of the genotype 3a virus results in eradication of the steatosis, suggesting the genotype 3a virus may be able to directly cause steatosis.<p> Research suggests a role for the core protein of HCV, which forms the capsid of the virus, in the alteration of lipid metabolism pathways during infection. As such, I hypothesized that: 1) HCV alters lipid metabolism pathways and causes the build up of lipid in hepatocytes and the development of steatosis; 2) HCV-3a core protein has a differential or increased effect on these pathways in comparison to 1b core protein; and 3) other HCV proteins could also play a role in the altering of lipid metabolism. My research characterized the subcellular localization on lipid droplets of the HCV-3a core protein in comparison to HCV-1b core protein. It was found that HCV-3a core causes increased transcriptional activity from the Fatty Acid Synthase (FAS) promoter, an important enzyme involved in the synthesis of triglycerides in hepatocytes. In addition, one specific amino acid of HCV-3a core was determined to be partially responsible for this effect. Further research determined that the effect of HCV-3a core on FAS was dependent on the transcription factor Sterol Response Element Binding Protein-1 (SREBP-1) and the presence of HCV-3a core increased the processing and activity of SREBP-1. HCV core was also able to increase activity of Akt 1 and Akt2; inhibition of Akt activity resulted in decreased SREBP-1 activity thereby indicating that HCV core partially mediates SREBP-1 via Akt. Further experiments examined the role of another HCV protein, NS2, in these same lipid metabolism pathways. NS2 was also able to increase transcription from the FAS promoter via SREBP-1, suggesting that this HCV protein may also be important in the development of steatosis during HCV infection.<p> The evidence provided in these studies shows a very important role for HCV in altering lipid metabolism during infection that may lead to the development of steatosis. Current research suggests that the SREBP-1 pathway may be critical in the life cycle of the virus and these studies have provided important information on how lipid metabolism pathways are being changed by the virus. Hopefully this work can help identify potential treatment options for HCV that can slow down disease progression by preventing the development of steatosis or by decreasing viral replication.

This study used the non-alcoholic steatohepatitis activity index (NAI), presence of fibrosis and Mallory-Denk bodies (MDBs), and quantification of glucose regulated protein 78 (GRP78) messenger ribonucleic acid (mRNA) as indicators of steatohepatitis development and recovery in the American mink (Neovison vison). Mink were fasted for 0, 1, 3, 5, or 7 days, and one group re-fed 28 days post 7-day fast. Liver NAI indicated that moderate fatty liver developed after 5 days of fasting. Liver recovery was achieved after the re-feeding period. There was no evidence of fibrosis or MDB formation. Upregulation of GRP78 was observed by day 7 of fasting indicating endoplasmic reticulum stress. This effect was greater in females. Results suggest that liver steatosis did not advance to steatohepatitis within a 7-day fast. However, should the length of fast be increased the mink may be at risk. Results also show that liver recovery from simple fatty liver is possible.

Objective: Glucagon-like peptide-1 receptor (GLP-1R) agonists have been shown to reduce atherosclerosis in non-diabetic mice. We hypothesized that treatment with GLP-1R agonists would reduce the development of atherosclerosis in diabetic Apoe-/- mice. Results: Exendin-4 treatment (10 nmol/kg/day) of high-fat diet-induced glucose-intolerant mice for 22 weeks did not significantly reduce oral glucose tolerance (P=0.62) or HbA1c (P=0.85), and did not reduce plaque size at the aortic sinus (P = 0.35). Taspoglutide treatment for 12 weeks (0.4-mg tablet/month) of diabetic mice reduced body weight (P<0.05), food intake (P<0.05), oral glucose tolerance (P<0.05), intrahepatic triglycerides (P<0.05) and cholesterol (P<0.001), and plasma IL-6 levels (P<0.01); increased insulin:glucose (P<0.05); and unaltered oral lipid tolerance (P=0.21), plasma triglycerides (P=0.45) or cholesterol (P=0.92). Nonetheless, taspoglutide unaltered aortic atherosclerosis (P=0.18, sinus; P=0.19, descending aorta) or macrophage infiltration (P=0.45, sinus; P=0.26, arch). Conclusions: GLP-1R activation in either glucose-intolerant or diabetic mice does not significantly modify the development of atherosclerosis.

Objective: Glucagon-like peptide-1 receptor (GLP-1R) agonists have been shown to reduce atherosclerosis in non-diabetic mice. We hypothesized that treatment with GLP-1R agonists would reduce the development of atherosclerosis in diabetic Apoe-/- mice. Results: Exendin-4 treatment (10 nmol/kg/day) of high-fat diet-induced glucose-intolerant mice for 22 weeks did not significantly reduce oral glucose tolerance (P=0.62) or HbA1c (P=0.85), and did not reduce plaque size at the aortic sinus (P = 0.35). Taspoglutide treatment for 12 weeks (0.4-mg tablet/month) of diabetic mice reduced body weight (P<0.05), food intake (P<0.05), oral glucose tolerance (P<0.05), intrahepatic triglycerides (P<0.05) and cholesterol (P<0.001), and plasma IL-6 levels (P<0.01); increased insulin:glucose (P<0.05); and unaltered oral lipid tolerance (P=0.21), plasma triglycerides (P=0.45) or cholesterol (P=0.92). Nonetheless, taspoglutide unaltered aortic atherosclerosis (P=0.18, sinus; P=0.19, descending aorta) or macrophage infiltration (P=0.45, sinus; P=0.26, arch). Conclusions: GLP-1R activation in either glucose-intolerant or diabetic mice does not significantly modify the development of atherosclerosis.

Despite inconsistent results in the literature, high protein diets are being promoted for the management of metabolic syndrome parameters primarily due to their proposed favorable effects on weight loss. Therefore, lean and fa/fa Zucker rats were given normal and high protein diets with varying protein sources for 12 weeks. A high protein diet with a mixture of animal and plant protein sources was the most effective for improving metabolic syndrome parameters, specifically insulin resistance and hepatic steatosis. A high protein soy diet was the second most effective diet, while a high protein casein diet demonstrated no benefits compared to the other two high protein diets and minimal benefits compared to a normal protein casein diet. Interestingly, high protein diets did not affect body weight regardless of protein source. These findings suggest that the source of protein within a high protein diet is critical for improving metabolic syndrome parameters and that improvements can be observed independent of weight loss.