Student Number : 0101826W - M Med dissertation - School of Clinical Medicine - Faculty of Health Sciences / Non-alcoholic fatty liver disease (NAFLD) is a chronic liver disease with hepatic histology that resembles alcoholic liver disease. It is a frequent cause of chronic liver disease and is attracting increasing scientific attention worldwide. I explored the possibility that increased gastrointestinal alcohol production may have a role as a “second hit” in the pathogenesis of NAFLD in study subjects with the metabolic syndrome. In an attempt to investigate this hypothesis, this study looked at blood, urine and breath levels of alcohol in patients with the metabolic syndrome versus matched age and ethnic group healthy controls. Of the twenty study subjects, 80% had dyslipidaemia, 60% had hypertension and 70% had type 2 diabetes mellitus. Their mean BMI was 35.1±8.2 kg/m² (mean ± SD, P < 0.0001 versus controls). The serum aminotransferases were significantly elevated in the study subjects, their ALT levels being 57.4±44.79 U/L versus 17.4±4.60 U/L in the controls (95% CI 18.02 – 61.42, P < 0.001), and their AST levels 52.5±36.21 U/L versus 23.4±4.86 U/L in the controls (95% CI 11.99 – 46.20, P < 0.01). Seventy five percent of the study group had sonar features suggestive of fatty liver disease. Two adipocytokines, adiponectin and leptin, mediators of insulin resistance, an important factor in the development and progression of NAFLD, were also measured. Adiponectin levels were significantly lower (6875 ng/L versus 15475 ng/L; median value, P < 0.01), and leptin concentration levels significantly higher (13.56 ng/L versus 3.05 ng/L; median value, P < 0.05) in the study subjects than in the control group. Alcohol was detected in 60% of the study subjects, of which 35% tested positive for ethanol, 55% tested positive for methanol, and 30% tested positive for both ethanol and methanol. This was a statistically significant result, as none of the control group tested positive for any of the alcohols. The ethanol concentration in the study subjects’ blood was 7.14±3.28 mg% (mean ± SD), in their urine 3.71± 12.87 mg% (mean ± SD) whilst none was detected in their breath. The methanol concentration in the study subjects’ blood was 16.17±17.95 mg% (mean ± SD), in their urine 6.8± 13.58 mg% (mean ± SD) while their breath level was 2.05±3.19 mg (mean ± SD). This study therefore suggests that endogenous alcohol production may be indeed be involved in the pathogenesis of NAFLD in subjects with the metabolic syndrome. Not only ethanol but also methanol was detected in the subjects tested. Endogenous alcohol may therefore be responsible for the ‘second hit’ theory in the pathogenesis of NAFLD, and it is likely that formaldehyde, the metabolite of methanol may be a more potent toxin of hepatocyte injury as opposed to acetaldehyde, the metabolite of ethanol. The most likely source of the alcohol is from intestinal bacterial flora. These findings provide further insight into the pathogenesis of NALFD, suggesting other therapeutic alternatives such as the use of antibiotics and probiotics as a potential treatment strategy for NAFLD.

The objective of this study was to determine the effects of a structured weight loss program that included hypocaloric diet, exercise and dietary supplementation, on weight loss, metabolic syndrome risk factors and antioxidant levels in healthy overweight and obese females. Thirty-seven healthy overweight and obese women (BMI 29.5 ± 2.3 kg/m2, 41.1 ± 7.1 yrs) participated in this study. The subjects were randomized into one of two groups: an exercise, hypocaloric diet and antioxidant supplement (LifePak®; LSANT group, n=20) or an exercise, hypocaloric diet and appetite suppression supplement (HTP Complex® and TēGreen®; LSAS group, n=17). A significant weight loss occurred in both groups after 12 weeks (LSANT: -2.8 ± 2.8 kg and LSAS: -4.3 ± 2.7 kg, p < 0.001). Body fat mass, percent body fat, and waist circumference significantly improved in both groups (p < 0.05). No significant difference was found between the groups for weight loss (p > 0.05). However, a significant difference was found between the groups for body fat mass (LSANT: -1.8 ± 2.6 kg; LSAS: -3.4 ± 2.4 kg, p ≤ 0.05). Glucose, insulin and insulin resistance (HOMA-IR) were significantly decreased in the LSAS group (glucose: -5.0 ± 6.8 mg/dl, p=0.008; insulin: -2.6 ± 3.8 uIU/dl, p=0.013; and HOMA-IR: -0.7 ± 1.0, p=0.012) but not in the LSANT group (p > 0.05). There were no significant differences (p > 0.05) observed within or between the groups for cholesterol, triglycerides or LDL-c. HDL-c decreased significantly in the LSANT group (-2.9 ± 5.3 mg/dl, p=0.024) but not in the LSAS group (p > 0.05). Skin carotenoid scores (SCS) increased significantly within the LSANT group (LSANT: 10950 ± 8395 SCS, p < 0.001) but not the LSAS group (p > 0.05). Lifestyle intervention that involves a structured hypocaloric diet and increased physical activity results in weight loss and improvements in body composition. However, supplementation with an appetite suppressant (HTP Complex®) did not enhance weight loss beyond what was achieved with a structured lifestyle intervention. Antioxidant supplementation may be of benefit during a weight loss program that incorporates physical activity and a low energy diet.

Obese people with insulin resistance are at high risk of developing disease-related complications like heart attack and stroke. Recently, a significant amount of data has been published linking chronic inflammation with obesity and the etiology of the Metabolic syndrome (MetS). Scientists have found many of the same inflammatory pathways and pro-inflammatory molecules are involved in both conditions. In particular, recent studies have elucidated an important role for the inflammasome in the etiology of these diseases. Interfering with these chronic inflammatory processes may provide a new way to treat obesity. Pilot studies in animals and humans have shown positive outcomes using anti-inflammatory drugs for treatment of both obesity and MetS. One advantage to using anti-inflammatory drugs is that many are already clinically approved with known risk/benefit profiles. Trials to test their efficacy in MetS and obesity are thus feasible. If proven beneficial, these drugs could help treat a huge number of patients who do not currently have other safe options. In this thesis I propose that new drugs targeting the inflammasome components, such as caspase 1, may also show clinical benefit in the treatment of MetS and obesity. Also drugs that reduce activation of a subset of macrophages such as the M1 class may also prove useful in treatment of these conditions.

Patients suffering from hypertension often develop non-Insulin dependent diabetes mellitus (NIDDM), a condition caused by Insulin resistance. Though these patients have normal Insulin receptor (IR) and high levels of Insulin in blood, they fail to have IR-mediated signaling essential for glucose uptake and availability. NIDDM usually begins as insulin resistance, a condition in which Insulin Receptor (IR)-mediated signaling that leads to glucose uptake and glucose availability to cells is inhibited even in the presence of high levels of Insulin in blood. Mechanisms for the development of this Insulin resistance in patients suffering from hypertension are unclear. Angiotensin II (Ang II) hormone has been implicated in the pathogenesis of insulin resistance and inhibitors of Ang II receptor AT1 are shown to improve insulin sensitivity. Here we show that in the skeletal muscle tissue of SHR rats, Insulin Receptor (IR) beta- subunit forms a complex with the AT1 receptor and co-immunoprecipitates with IR-beta. Such strong AT1-IR association was not observed in normo-tensive rat tissue. To our knowledge this is the first report that shows AT1 can associate with IR-beta in mammalian tissue and that such association might play a role in the regulation of signaling by IR-beta. We further demonstrate that a 2-hour pre-incubation with Ang II (at concentrations 50pM to 1?ÝM) significantly inhibits 125I-insulin binding to IR in human cell line MCF-7. This effect was not seen when Ang II exposure was performed for shorter periods. The two-hour exposure to Ang II also led to the formation of a protein complex containing AT1 and IR-beta, similar to that seen in skeletal muscle tissue of SHR rats. Both AT1-IR association and differential tyrosine phosphorylation of IR-beta and associated proteins were inhibited when the cells were pre-treated with the AT1 antagonist losartan. These observations suggest that continuous presence of Ang II would result in sequestering IR in the AT1-IR complex and prevent IR from binding insulin. It also coincided with differential tyrosine-phosphorylation of IR beta-subunit and associated proteins, than that generated when IR was activated by insulin. Therefore, we infer that conformational alterations in IR caused by AT1-IR-beta association underlie the development of Ang II-induced insulin resistance. Based on these data we propose a model for AT1-mediated insulin resistance that involves receptor level interaction between the AT1 and the IR. Therefore, Insulin-independent, Ang II-induced tyrosine phosphorylation of IR prevents IR from binding Insulin and contributes to Insulin resistance. The observation that drugs that inhibit Angiotensin II converting enzyme (ACE), or activation of AT1 receptor, not only reduce hypertension, but also induce insulin sensitivity further supports the role for Ang II and AT1 in the development of NIDDM.

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An animal model to study complications resulting from childhood obesity is lacking. Our objective was to develop a porcine model for studying mechanisms underlying diet-induced childhood obesity. Pre-pubertal female pigs, age 35 d, were fed a high-energy diet (HED; n = 12), containing tallow and refined sugars, or a control corn-based diet (n = 11) for 16 wk. Initially, HED pigs self-regulated energy intake similar to controls, but, by wk 5, consumed more (P < 0.001) energy per kg body weight. At wk 15 and 22, pigs were subjected to an oral glucose tolerance test (OGTT); blood glucose increased (P < 0.05) in control pigs and returned to baseline levels within 60 min. HED pigs were hyperglycemic at time 0, and blood glucose did not return to baseline (P = 0.01), even 3 h post-challenge. During OGTT, glucose area under the curve was higher and insulin area under the curve was lower in HED pigs compared to controls (P = 0.001). Pigs given 6 wk of dietary intervention, consuming a control diet, marginally improved glucose area under the curve and LDL-cholesterol although insulin area under the curve was unaffected. Chronic HED intake increased (P < 0.05) subcutaneous, intramuscular, and perirenal fat deposition, and induced hyperglycemia, hypoinsulinemia, and low-density lipoprotein hypercholesterolemia; however, a 6 wk dietary intervention partially recovered a normal physiology. These data suggest pre-pubertal pigs fed HED are a viable animal model for studying childhood obesity. / Master of Science