Diet induced obesity and Type 2 diabetes affect an alarming number of people in the world. Patients exhibit a number of associated co-morbidities that require treatment and reduce life expectancy. The development of Type 2 diabetes results from a chronic disruption of glucose homeostasis as a consequence of environmental and genetic factors. The regulation of hypothalamic metabolic signalling is understood to be critical to the dynamic and long-term maintenance of glucose homeostasis by virtue of its influence on peripheral effectors (pancreas, liver, muscle and fat). Prolonged periods of inflammation observed in the development of diet-induced obesity and diabetes and accumulating evidence suggests this may negatively affect the maintenance of these neuroendocrine networks.
Interleukin 6 (IL-6) is a pleiotropic cytokine involved in the acute, as well as the
resolving and adaptive phase of inflammatory responses. The IL-6 receptor has
recently been shown to exhibit extensive localization in key regions of the human and rodent hypothalamus involved in glucose and energy homeostasis regulation. Recent work at Prof. Rory McCrimmon's lab investigating the role of IL-6 in the development of hypoglycaemia unawareness in Type 1 Diabetes identified the development of a glucose-sensing defect in hypothalamic glucose-sensing mouse cultures (GT1-7) in response to antecedent IL-6. These studies provided preliminary evidence to suggest that inflammatory cytokines such as IL-6 could directly modulate hypothalamic glucose sensing neurons and therefore might influence whole body glucose and energy homeostasis.
The body of work contained in this thesis sets out to examine the hypothesis that down-regulation of central IL-6 signaling would disturb the ability of mammals to regulate energy homeostasis, and would exacerbate defects in glucose clearance, insulin sensitivity and body composition typically observed in during the development of Type 2 Diabetes through chronic energy excess. To test this hypothesis cre-lox technology was used to generate a CRE-mediated nervous-system specific NesCreIL-6R knock-down (KD) mouse. Chronic administration of high-fat diet (>40% fat) in mice is commonly used to model the effects of diet-induced obesity and the progression to type 2 diabetes in humans. To examine the effects of this intervention in the development of diet induced obesity and diabetes, age (8-10 weeks old) and sex-matched KD and control animals on standard chow (SC -15% fat) and high fat diet (HFD - 60% fat) were characterized using an array of in vivo metabolic phenotyping tests over a period of 20 weeks.
To generate a brain-specific IL-6R KD, heterozygous B6.Cg-Tg (Nes-cre) 1Kln/J
mice were crossed with B6 (SJL)-Il6ratm.1.1Drew/J animals (IL-6Rflox/flox). The Nestin promoter exhibits brain-specific expression, while the IL-6R gene is flanked by 2 loxP sites in IL-6Rflox/flox mice that would theoretically allow for brain-specific disruption of the receptor gene in offspring of this cross. Incorporation of the CRE gene in NesCre+ mice, and the presence of loxP sites in the genome of IL-6Rflox/flox colony were confirmed by standard PCR (Figures 1.1, 1.2). Offspring of the cross bearing the CRE gene were classified as KD, while the rest were considered wild-type littermates. Disruption of the IL-6R gene was confirmed at the mRNA level using real-time PCR (Figure 1.3.2), with KD mice exhibiting down-regulation of the gene at the hypothalamus (~50%) and hippocampus (~35%). The effects of the NesCremediated down-regulation of the IL-6R were also examined using in vivo and ex vivo approaches, in relation to the induction of the STAT3 phosphorylation typically observed in response to IL-6. Administration of IL-6 in the periphery failed to induce STAT3 phosphorylation in the hypothalamus and hippocampus of KD mice, even though similar responses to control animals were observed in peripheral organs (Figures 1.4, 1.5). Furthermore, examination of IL-6R protein expression by immunohistochemistry, confirmed decreases in the number of IL-6R-positive neuronal cells in NesCreIL-6R KDs compared to control mice (Figures 1.7.1-4).
Brain-specific IL-6R down-regulation in NesCreIL-6R KDs was associated with a
dramatic suppression of in vivo GSIS (Figure 2.5.4), but was of little consequence to the blood glucose response to insulin (Figures 2.4.1-3). Examination of insulin and glucagon immunohistochemistry and ex vivo hormone release, KD and control islets were found to produce and release similar levels of the hormones in isolation, implicating a central component in the effect observed in vivo (Figures 2.8.1-3). Body weight was closely matched between KD and control animals on SC diet, while dramatic increases observed in control animals on HFD were mirrored in HF-fed KDs (Figure 2.1). Paradoxically, KD SC mice were significantly leaner than control mice, despite being hyperphagic (Figures 2.2.1-5, 2.3, 2.9.1), hypoactive and hypothermogenic (Figure 2.6.2-4). Hyperphagia and suppression of energy expenditure was also observed in the HF-fed KDs, but in this case the leaner phenotype was progressively reversed through the course of 20 weeks of diet (Figures 2.2.1-5, 2.6.2-4). Examination of a separate cohort of SC mice indicated that increases in food intake and body fat in response to chronic diet, were not a consequence of differences in leptin sensitivity between KD and control animals (Figures 2.9.1-2).
Collectively, data presented in this thesis suggest central IL-6 to play a fundamental role in the ability of the body to release insulin and regulate glycaemia in response to a glucose challenge. KD mice exhibited increased food intake and decreased energy expenditure and activity, that were only associated with increased body fat following chronic HF-feeding. This would suggest that increases in peripheral IL-6 observed in obese patients are part of a protective response to chronic energy excess, acting centrally to promote energy expenditure at least in part through increases in circulating insulin and thermogenic fat oxidation.