Reduction in the activity of the a-melanocyte-stimulating hormone (a-MSH) system causes obesity, and infusions of a-MSH can produce satiety, raising the possibility that a-MSH may mediate physiological satiety signals. Since a-MSH is coded for by the pro-opiomelanocortin (POMC) gene, we examined if POMC gene expression would be inhibited by fasting in normal mice or in models of obesity characterized by leptin insufficiency (ob/ob} or leptin insensitivity (dfe/db). In wild-type mice, hypothalamic POMC mRNA was decreased >60% after a 2-day fast and was positively correlated with leptin mRNA. Similarly, compared with controls, POMC mRNA was decreased by at least 60% in both db/db and ob/ob mice. POMC mRNA was negatively correlated with both neuropeptide Y (NPY) and melanin-concentrating hormone (MCH) mRNA. Finally, treatment of both male and female ob/ob mice with leptin stimulated hypothalamic POMC mRNA by about threefold. These results suggest that impairment in production, processing, or responsiveness to a-MSH may be a common feature of obesity and that hypothalamic POMC neurons, stimulated by leptin, may constitute a link between leptin and the melanocortin system. Diabetes 47:294-297, 1997

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Mutations in the obese (ob)

Exogenous administration of the neuropeptide oxytocin reliably facilitates sexual behavior in the female rat and exposure to estrogen increases oxytocin receptor (OTR) binding in the ventromedial nucleus (VMN) of the hypothalamus. We have used a novel approach to investigate the role of hypothalamic OTR in controlling behavior by infusing antisense oligodeoxy-nucleotides (oligo) to the 5’-region of the human OTR mRNA into the VMN of hormonally primed rats. Control infusions consisted of a scrambled-sequence oligo that had little or no homology to known mRNAs. OTR antisense oligo infusion significantly reduced lordosis frequency and intensity in females primed with estrogen. There was also a significantly greater number of rejection behaviors exhibited by antisense-oligo-infused estrogen-treated females versus controls and no evidence of decreased locomotion by either treatment. In contrast to the effects in estrogen-primed-females, when females were primed to be sexually receptive with estrogen plus progesterone, OTR antisense-oligo infusion had no effect on sexual behavior. The lack of effectiveness of OTR antisense oligo in females primed with progesterone may be the result of the action of this steroid on other neurotransmitter systems that also facilitate lordosis and thereby override a deficit in oxytocin binding. Alternatively, via previously described mechanisms, progesterone may enhance the effectiveness of oxytocin binding at its receptor. In vitro receptor autoradiography in estrogen-primed females indicated a 31% reduction in VMN OTR binding in the vicinity of the cannula tip in antisense-oligo-infused females compared to controls. There was no significant difference in the level of OTR binding in the central nucleus of the amygdala. In addition, OTR antisense-oligo-infused females exhibited a significant reduction in food intake as demonstrated by weight loss and a reduced appetite for sweetened milk compared to scrambled-oligo-infused controls. Since VMN damage results in increased food intake, these results indicate a lack of damage to the VMN as a result of the OTR antisense infusions.

We have previously used a defective herpes simplex virus vector to express a foreign gene in the adult rat brain. One application of this technology would be the in vivo analysis of promoter function in brain after de novo transfer, which would allow the rapid generation of vectors with localized application in a broad range of mammalian species while avoiding influences of other nearby promoters. A 2.7-kb fragment of the rat preproenkephalin promoter was placed upstream of the bacterial lacZ gene in our herpes simplex virus amplicon. A restricted pattern of lacZ expression was observed in vivo, which follows previously observed patterns of endogenous preproenkephalin expression. These results, from the direct gene transfer into an adult animal brain for in vivo promoter analysis, demonstrate that sequence information that influences restricted expression of preproenkephalin is located within 2.7 kb upstream of transcriptional initiation. lacZ expression was also observed in rat brain for 2 months after direct transfer, and PCR analysis confirmed the continued presence of amplicon DNA in lacZ-positive sections. Restricted and long-term expression observed with an endogenous promoter has important implications for gene therapy using viral vectors.The herpes simplex virus (HSV) amplicon was developed as a eukaryotic cloning-expression vector (1-4). The amplicon is a plasmid that contains an HSV origin of DNA replication and cleavage/packaging signal, but by itself expresses no

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The factors regulating leptin mRNA under physiological conditions have not been fully elucidated, although both insulin and glucose have been implicated. Since, in male mice, plasma glucose decreases with age without a change in body weight or insulin, aging mice constitute a model to examine effects of glucose independent of effects of insulin or body weight. Therefore, we measured leptin mRNA in adipose tissue of 6-, 15- and 24-month-old C57BL/6J male mice, sacrificed either after a 48 h fast (nutritional deprivation) or 15 min after an intraperitoneal injection of glucose (2 mg/g body weight) (nutritional stimulation). There was a significant effect of both age and nutritional status on leptin mRNA, correlated with effects of age and nutritional status on plasma glucose. Leptin mRNA correlated with body weight, plasma glucose and plasma insulin. After statistically removing effects of plasma glucose, the remaining effects of age, nutritional status, and plasma insulin on leptin mRNA were no longer significant. However, after statistically removing effects of plasma insulin, the remaining effects of age, nutritional status, and plasma glucose continued to be significant. When nutrition-deprived and nutrition-stimulated mice were analyzed separately, plasma glucose significantly correlated with leptin mRNA in both groups, but body weight and plasma insulin correlated with leptin mRNA only in nutrition-deprived mice. When mice at each age were analyzed separately, glucose correlated with leptin mRNA at every age, and after statistical removal of the effects of glucose, the remaining effects of insulin on leptin mRNA were no longer significant at any age. These results support the hypothesis that plasma glucose is important in the regulation of leptin gene expression.