Neuronal control of intra-abdominal and subcutaneous white adipose tissue and the role of estrogens in the brain

Fat is not just fat. Obesity is a heterogeneous condition in that fat distribution differs between individuals as do the pathological sequelae of obesity, collectively termed metabolic syndrome. Metabolic syndrome is more significantly related to the amount of intra-abdominal white adipose tissue (WAT), whereas subcutaneous WAT is relatively benign. Males and postmenopausal females have greater amounts of intra-abdominal WAT and a higher risk of developing metabolic syndrome compared to premenopausal females. However, the mechanisms controlling fat distribution are poorly understood. Retroperitoneal white adipose tissue (rWAT) and subcutaneous (inguinal) white adipose tissue (iWAT) are both innervated and regulated by sympathetic efferents, but the distribution and identity of the cells in the brain that regulate sympathetic outflow are poorly characterised. The first aim of this thesis was to use two isogenic strains of a neurotropic virus (pseudorabies-Bartha) tagged with either green or red fluorescent reporters to identify cells in the brain that project to rWAT and/or iWAT. These viruses were injected into separate WAT depots in male and female Sprague-Dawley rats. All virally-labelled brain regions contained neurons projecting to either and both WAT depots. Neurons polysynaptically projecting to rWAT were the most abundant in males whereas females contained a greater proportion of neurons polysynaptically projecting to iWAT via private lines and collateral branches. The second aim was to characterise the neurons that polysynaptically project to rWAT and/or iWAT in order to determine the expression patterns of neurochemicals and receptors. Retrogradely labelled neurons in the central nervous system were characterised by immunohistochemistry on brain sections and polymerase chain reaction on individual neurons isolated using laser capture microscopy. Retrogradely labelled neurons directed to WAT expressed estrogen receptor-α (ER) and fewer neurons to subcutaneous WAT expressed ERα in males. Regardless of sex, projections from the arcuate nucleus were predominantly from pro-opiomelanocortin (POMC) cells, with a notable lack of projections from agouti-related protein-expressing neurons. Within the lateral hypothalamus, neurons directed to rWAT and iWAT expressed orexin and melanin-concentrating hormone (MCH), but male rats had a predominance of MCH directed to iWAT. In conclusion, the neurochemical substrates that project through polysynaptic pathways to iWAT and rWAT are different in male and female rats suggesting that metabolic regulation of rWAT and iWAT is sexually dimorphic. A sexual dimorphism exists with respect to WAT deposition, whereby males have more intra-abdominal WAT and females have greater amounts of subcutaneous WAT. Estrogens and the estrogen receptor alpha subtype (ERα) are known to regulate body weight, glucose regulation and energy expenditure independent of food intake. However, the extent to which this control is mediated by central ERα is poorly understood. Therefore, the final aim of the work was to examine the role of central ERα on fat pad mass, fat distribution and glucose regulation using mice with a specific knockout of ERα in post mitotic neurons in the brain. Specifically, CamKIIα-iCre BAC and ERα floxed transgenic mice were used to generate homozygous knockout (KO; completely lacking central ERα), heterozygous KO (partly lacking ERα) and control (ERα completely intact) mice. Both male and female mice lacking ERα were mildly glucose intolerant despite no differences in body weight, food intake or insulin tolerance. There was a trend toward increases in both intra-abdominal and subcutaneous fat pad mass in central neuronal ERα KO male mice. However, gonad-intact homozygous KO mice had increased levels of sex hormones potentially confounding and/or masking the results. Therefore, mice were gonadectomised and a subgroup of gonadectomised mice of each genotype was treated with β-estradiol (E2). Ovariectomised homozygous KO mice treated with E2 showed elevated levels of fat, an attenuated effect of E2 on fat distribution and alterations in genes involved in energy balance regulation. Conversely, castrated male homozygous KO mice showed mild glucose intolerance and lower levels of POMC gene expression, but no alterations in body weight, food intake or fat were observed compared to castrated male control mice. E2 treatment completely abolished these differences in castrated male mice illustrating a strong dependence on the signaling of estrogens in the periphery. In conclusion, central ERα is important for mediating effects of estrogens on fat pad mass and distribution in females, but central ERα only plays a minor role in regulating glucose homeostasis in males consistent with the idea that metabolic regulation is sexually dimorphic. Collectively this data add significantly to our understanding of the neurochemical blueprint of pathways directed to different fat compartments and specifically, the importance of estrogens acting on central ERα in the determination of the size and distribution of fat. Differences in the latter, in addition to the control of glucose regulation, appear to be essentially sexually dimorphic.