George B. Karkanias

Hypothalamic insulin signaling is required for inhibition of glucose production.

Circulating insulin inhibits endogenous glucose production. Here we report that bidirectional changes in hypothalamic insulin signaling affect glucose production. The infusion of either insulin or a small-molecule insulin mimetic in the third cerebral ventricle suppressed glucose production independent of circulating levels of insulin and of other glucoregulatory hormones. Conversely, central antagonism of insulin signaling impaired the ability of circulating insulin to inhibit glucose production. Finally, third-cerebral-ventricle administration of inhibitors of ATP-sensitive potassium channels, but not of antagonists of the central melanocortin receptors, also blunted the effect of hyperinsulinemia on glucose production. These results reveal a new site of action of insulin on glucose production and suggest that hypothalamic insulin resistance can contribute to hyperglycemia in type 2 diabetes mellitus.

Decreasing hypothalamic insulin receptors causes hyperphagia and insulin resistance in rats

We investigated the role of hypothalamic insulin signaling in the regulation of energy balance and insulin action in rats through selective decreases in insulin receptor expression in discrete hypothalamic nuclei. We generated an antisense oligodeoxynucleotide directed against the insulin receptor precursor protein and administered this directly into the third cerebral ventricle. Immunostaining of rat brains after 7-day administration of the oligodeoxynucleotide showed a selective decrease of insulin receptor protein within cells in the medial portion of the arcuate nucleus (decreased by ∼80% as compared to rats treated with a control oligodeoxynucleotide). Insulin receptors in other hypothalamic and extra-hypothalamic areas were not affected. This selective decrease in hypothalamic insulin receptor protein was accompanied by rapid onset of hyperphagia and increased fat mass. During insulin-clamp studies, physiological hyperinsulinemia decreased glucose production by 55% in rats treated with control oligodeoxynucleotides but by only 25% in rats treated with insulin receptor antisense oligodeoxynucleotides. Thus, insulin receptors in discrete areas of the hypothalamus have a physiological role in the control of food intake, fat mass and hepatic action of insulin.

Central melanocortin receptors regulate insulin action

Energy balance and insulin action are tightly coregulated. Leptin regulates energy intake and expenditure partly by modulation of the melanocortin pathway in the hypothalamus. Here we demonstrate potent effects of the melanocortin pathway on insulin action and body distribution of adiposity. Conscious rats received week-long infusions of either a melanocortin receptor agonist, alpha-melanocyte-stimulating hormone (alpha-MSH), or antagonist, SHU9119, in the third cerebral ventricle while food intake was maintained constant in each group. alpha-MSH decreased intra-abdominal fat and markedly enhanced the actions of insulin on both glucose uptake and production, while SHU9119 exerted opposite effects. Our findings elucidate a neuroendocrine network that is likely to play a central role in the coupling of energy intake and insulin action.

Intracerebroventricular Leptin Regulates Hepatic but Not Peripheral Glucose Fluxes

Acute intravenous infusions of leptin markedly alter hepatic glucose fluxes (Rossetti, L., Massillon, D., Barzilai, N., Vuguin, P., Chen, W., Hawkins, M., Wu, J., and Wang, J. (1997)J. Biol. Chem. 272, 27758–22763). Here we examine whether intracerebroventricular (ICV) leptin administration regulates peripheral and hepatic insulin action. Recombinant mouse leptin (n = 14; 0.02 or 1 μg/kg·h) or vehicle (n = 9) were administered ICV for 6 h to conscious rats, and insulin action was determined by insulin (3 milliunits/kg·min) clamp and tracer dilution techniques. During physiologic hyperinsulinemia (∼65 microunits/ml), the rates of glucose uptake (R d , 20.1 ± 0.6 and 23.1 ± 0.7 versus 21.7 ± 0.6 mg/kg·min;p = NS), glycolysis and glycogen synthesis were similar in rats receiving low- and high-dose leptin versusvehicle. ICV leptin resulted in a 2–3-fold increase in hepatic phosphoenolpyruvate carboxykinase mRNA levels. Glycogenolysis and PEP-gluconeogenesis (2.1 ± 0.3 mg/kg·min) contributed similarly to endogenous glucose production (GP) in the vehicle-infused group. However, gluconeogenesis accounted for ∼80% of GP in both groups receiving ICV leptin, while hepatic glycogenolysis was markedly suppressed (0.7 ± 0.3 and 1.2 ± 0.3 versus2.2 ± 0.4 mg/kg·min, in rats receiving low- and high-dose leptin versus vehicle, respectively; p < 0.01). In summary, short-term ICV leptin administration: 1) failed to affect peripheral insulin action, but 2) induced a striking re-distribution of intrahepatic glucose fluxes. The latter effect largely reproduced that of leptin given systemically at much higher doses. Thus, the regulation of hepatic glucose fluxes by leptin is largely mediated via its central receptors.

ESTRADIOL REGULATION OF ALPHA 1B-ADRENOCEPTOR MRNA IN FEMALE RAT HYPOTHALAMUS-PREOPTIC AREA

Estradiol treatment for 48 h increases the density of α1B‐adrenoceptors in the hypothalamus‐preoptic area of ovariectomized female rats by five‐ to six‐fold. Present studies tested the hypothesis that estradiol elevation of hypothalamus‐preoptic area α1B‐adrenoceptor density is correlated with increased levels of mRNA for this receptor. We developed a semiquantitative reverse transcriptase‐polymerase chain reaction (RT‐PCR) protocol for measuring brain α1b‐adrenoceptor mRNA. The primers chosen yielded the predicted 409 base pair PCR product when used to amplify authentic α1b‐adrenoceptor cDNA. The identity of the RT‐PCR products from rat brain was confirmed by restriction digest analysis and sequencing. Moreover, there was a good correlation between the levels of α1b‐adrenoceptor mRNA measured by RT‐PCR in liver, whole brain and cerebellum with previous measurements using Northern blots and RNAse protection assays. We then performed RT‐PCR on total RNA from hypothalamic‐preoptic area tissue taken from ovariectomized control rats and from ovariectomized rats injected once or twice with 2 μg of estradiol benzoate at 24 or 24 and 48 h before sacrifice. Exposure to estradiol for either 24 or 48 h significantly increased levels of α1b‐adrenoceptor mRNA by 86–110% in the hypothalamus‐preoptic area of ovariectomized female rats when compared to oil‐treated controls. We also examined whether estradiol regulates α1b‐adrenoceptor mRNA in the cortex. Cortical α1b‐adrenoceptor mRNA levels were reduced to approximately 20% of control levels when measured 24 h after hormone injection. A similar decrease in cortical α1b‐adrenoceptor mRNA was observed 48 h after estrogen administration. In summary, estradiol treatment significantly increases the level of α1b‐adrenoceptor mRNA in the hypothalamus‐preoptic area, a brain region involved in the control of reproductive function. In the cortex, a brain region with relatively few estrogen receptors, the same estrogen treatment reducesα1b‐adrenoceptor mRNA levels.

Estrogen regulation of noradrenergic signaling in the hypothalamus

Hypothalamic circuits utilizing the monoamine neurotransmitter norepinephrine (NE) may be key elements upon which the ovarian steroids estradiol (E2) and progesterone (P) act to regulate female reproductive behavior. Recent studies have focused on the modulation of hypothalamic NE release by E2 and P treatments that facilitate sexual behavior. Brain microdialysis studies suggest that oxytocin, a neuropeptide known to enhance lordosis when infused into the ventromedial hypothalamus (VMH) of E2 + P-primed females, modulates NE release in the VMH. Systemic administration of oxytocin reliably enhances extracellular NE levels in the VMH of animals primed with moderate doses of both E2 and P. Thus, ovarian steroids may facilitate female sexual behavior in part by promoting oxytocin-induced NE release in the VMH. Studies examining the release of 3H-NE from superfused hypothalamic slices indicate that estrogen treatment also facilitates NE neurotransmission by attenuating alpha 2-adrenergic receptor-mediated inhibition of NE release. Hypothalamic alpha 2-adrenergic receptors are not downregulated by estrogen, suggesting that brain adrenoceptor function can be modulated by E2 independent of changes in receptor density. A model is proposed wherein E2 and P enhance hypothalamic NE release, leading to increased excitability of VMH neuronal activity and the expression of lordosis behavior.

Physiologic effect of leptin on insulin secretion is mediated mainly through central mechanisms

Leptin has been shown to decrease glucose‐stimulated insulin secretion in both in vivo and in vitro studies. As some of the effects of leptin have been elicited through both peripheral and central mechanisms, we assessed whether leptin modulates insulin secretion also through the central nervous system. We infused leptin or saline through implanted intracerebro‐ventricular (ICV) catheters to chronically catheterized, conscious rats (n=15), 2 h after initiation of hyperglycemic (∼11 mM) clamp. On ICV administration of leptin, there was a gradual and progressive decrease in plasma insulin levels by 52% with 30 ng (P<0.005) and by 28% with 20 ng (P<0.05) of leptin compared with ICV saline. The effect of 20 ng leptin ICV was replicated by intravenous (IV) leptin infusion that achieved physiological leptin levels of ∼17 ng/ml (n=5). When the melanocortin (MC) pathway was blocked with a nonselective MC‐3/4 antagonist SHU 9119 administered ICV, and either saline or leptin (n=12) was infused IV, intravenous leptin failed to produce a decrease in glucose‐stimulated insulin levels. We conclude that leptin decreases insulin levels by a predominantly central mechanism, probably via the melanocortin receptors; and peripheral leptin receptors on the β‐cells do not play a major role. The physiological features of this response suggest a possible role for leptin in the evolution of diabetes in overweight individuals.

Hormonal integration of neurochemical and sensory signals governing female reproductive behavior

This review focuses on findings from our laboratory regarding mechanisms by which the ovarian steroid hormones, estradiol (E2) and progesterone (P), act in the hypothalamus (HYP) to regulate the expression of lordosis, an important component of female reproductive behavior in rats and many other species. The first section summarizes recent work suggesting that cGMP, perhaps via P-receptor activation, may be an intracellular mediator of the facilitatory actions of a variety of hormones and neurotransmitters on lordosis behavior in E2-primed rats. In the second section, we focus on E2 and P regulation of norepinephrine (NE) neurotransmission in the HYP. We review evidence that ovarian hormones act both peripherally and centrally to determine whether NE is released in the HYP in response to copulatory stimuli. We also suggest that the steroid milieu determines the cellular responses of hypothalamic neurons to released NE, favoring the activation of pathways implicated in the facilitation of both lordosis behavior and the preovulatory gonadotropin surge. It is likely that E2 and P have similar actions on other neurotransmitter and neuromodulator systems, thereby maximizing the probability that females are sexually receptive during the periovulatory period.

Intracerebroventricular leptin regulates hepatic cholesterol metabolism.

To elucidate the control of hepatic cholesterol metabolism by leptin, rats were administered IV (intravenous) leptin, ICV (intracerebroventricular) leptin or saline. A single low dose of ICV leptin was as effective as a continuous IV infusion of high-dose leptin at decreasing the activities of 3-hydroxy-3-methylglutaryl-CoA reductase and cholesterol 7alpha-hydroxylase. These results indicate that the hepatic response to leptin is transduced via the central nervous system.

Effect of centrally administered insulin on gonadotropin-releasing hormone neuron activity and luteinizing hormone surge in the diabetic female rat.

Diabetic female rats have decreased ovulation, sexual behavior, and luteinizing hormone (LH) surges. Peripheral insulin treatment restores the phenotype to normal. We administered central insulin and analyzed serum LH during the time of the LH surge in diabetic and non-diabetic animals to determine if central insulin was sufficient to normalize the phenotype. We assessed the activity and number of hypothalamic gonadotropin-releasing hormone (GnRH) neurons by double label immunocytochemistry for c-Fos and GnRH to determine if decreased GnRH neuron activity or number could account for the diabetes-induced deficits in neuroendocrine function. All animals were ovariectomized and given estradiol and progesterone. Diabetic and control animals were given either intracerebroventricular (ICV) insulin or saline. In experiment I, serial blood collection was performed. In experiment II, animals were sacrificed and their brains were processed for immunocytochemistry during the presumed LH surge. Experiment I showed that diabetic, saline-treated animals were unable to trigger an LH surge. Central insulin restored LH production to control levels. Experiment II revealed similar numbers and activation of GnRH neurons in all four groups. Therefore, the diabetes-induced loss of the LH surge cannot be explained simply by a reduction of GnRH-expressing neurons or by a decrease in GnRH neuronal activity.