Differential pre-mRNA splicing regulates Nnat isoforms in the hypothalamus after gastric bypass surgery in mice.

BACKGROUND: Neuronatin (NNAT) is an endoplasmic reticulum proteolipid implicated in intracellular signalling. Nnat is highly-expressed in the hypothalamus, where it is acutely regulated by nutrients and leptin. Nnat pre-mRNA is differentially spliced to create Nnat-α and -ß isoforms. Genetic variation of NNAT is associated with severe obesity. Currently, little is known about the long-term regulation of Nnat. METHODS: Expression of Nnat isoforms were examined in the hypothalamus of mice in response to acute fast/feed, chronic caloric restriction, diet-induced obesity and modified gastric bypass surgery. Nnat expression was assessed in the central nervous system and gastrointestinal tissues. RTqPCR was used to determine isoform-specific expression of Nnat mRNA. RESULTS: Hypothalamic expression of both Nnat isoforms was comparably decreased by overnight and 24-h fasting. Nnat expression was unaltered in diet-induced obesity, or subsequent switch to a calorie restricted diet. Nnat isoforms showed differential expression in the hypothalamus but not brainstem after bypass surgery. Hypothalamic Nnat-ß expression was significantly reduced after bypass compared with sham surgery (P = 0.003), and was positively correlated with post-operative weight-loss (R(2) = 0.38, P = 0.01). In contrast, Nnat-α expression was not suppressed after bypass surgery (P = 0.19), and expression did not correlate with reduction in weight after surgery (R(2) = 0.06, P = 0.34). Hypothalamic expression of Nnat-ß correlated weakly with circulating leptin, but neither isoform correlated with fasting gut hormone levels post- surgery. Nnat expression was detected in brainstem, brown-adipose tissue, stomach and small intestine. CONCLUSIONS: Nnat expression in hypothalamus is regulated by short-term nutrient availability, but unaltered by diet-induced obesity or calorie restriction. While Nnat isoforms in the hypothalamus are co-ordinately regulated by acute nutrient supply, after modified gastric bypass surgery Nnat isoforms show differential expression. These results raise the possibility that in the radically altered nutrient and hormonal milieu created by bypass surgery, resultant differential splicing of Nnat pre-mRNA may contribute to weight-loss.

Dominant role of the p110beta isoform of PI3K over p110alpha in energy homeostasis regulation by POMC and AgRP neurons.

PI3K signaling is thought to mediate leptin and insulin action in hypothalamic pro-opiomelanocortin (POMC) and agouti-related protein (AgRP) neurons, key regulators of energy homeostasis, through largely unknown mechanisms. We inactivated either p110alpha or p110beta PI3K catalytic subunits in these neurons and demonstrate a dominant role for the latter in energy homeostasis regulation. In POMC neurons, p110beta inactivation prevented insulin- and leptin-stimulated electrophysiological responses. POMCp110beta null mice exhibited central leptin resistance, increased adiposity, and diet-induced obesity. In contrast, the response to leptin was not blocked in p110alpha-deficient POMC neurons. Accordingly, POMCp110alpha null mice displayed minimal energy homeostasis abnormalities. Similarly, in AgRP neurons, p110beta had a more important role than p110alpha. AgRPp110alpha null mice displayed normal energy homeostasis regulation, whereas AgRPp110beta null mice were lean, with increased leptin sensitivity and resistance to diet-induced obesity. These results demonstrate distinct metabolic roles for the p110alpha and p110beta isoforms of PI3K in hypothalamic energy regulation.