Reducing neuronal nitric oxide synthase (nNOS) expression in the ventromedial hypothalamus (VMH) increases body weight and blood glucose levels while decreasing physical activity in female mice.

Glucose-inhibited (GI) neurons of the ventromedial hypothalamus (VMH) depend on neuronal nitric oxide synthase (nNOS) and AMP-activated protein kinase (AMPK) for activation in low glucose. The Lopez laboratory has shown that the effects of estrogen on brown fat thermogenesis and white fat browning require inhibition of VMH AMPK. This effect of estrogen was mediated by downstream lateral hypothalamus (LH) orexin neurons 1,2 . We previously showed that estrogen inhibits activation of GI neurons in low glucose by inhibiting AMPK 3 . Thus, we hypothesized that VMH AMPK- and nNOS-dependent GI neurons project to and inhibit orexin neurons. Estrogen inhibition of AMPK in GI neurons would then disinhibit orexin neurons and stimulate brown fat thermogenesis and white fat browning, leading to decreased body weight. To test this hypothesis, we reduced VMH nNOS expression using nNOS shRNA in female mice and measured body weight, adiposity, body temperature, white and brown fat uncoupling protein (UCP1; an index of thermogenesis and browning), locomotor activity, and blood glucose levels. Surprisingly, we saw no effect of reduced VMH nNOS expression on body temperature or UCP1. Instead, body weight and adiposity increased by 30% over 2 weeks post injection of nNOS shRNA. This was associated with increased blood glucose levels and decreased locomotor activity. We also found that VMH nNOS-GI neurons project to the LH. However, stimulation of VMH-LH projections increased excitatory glutamate input onto orexin neurons. Thus, our data do not support our original hypothesis. Excitation of orexin neurons has previously been shown to increase physical activity, leading to decreased blood glucose and body weight 4 . We now hypothesize that VMH nNOS-GI neurons play a role in this latter function of orexin neurons.

Lateral hypothalamus hypocretin/orexin glucose-inhibited neurons promote food seeking after calorie restriction.

OBJECTIVE: The present study tests the hypothesis that changes in the glucose sensitivity of lateral hypothalamus (LH) hypocretin/orexin glucose-inhibited (GI) neurons following weight loss leads to glutamate plasticity on ventral tegmental area (VTA) dopamine neurons and drives food seeking behavior. METHODS: C57BL/6J mice were calorie restricted to a 15% body weight loss and maintained at that body weight for 1 week. The glucose sensitivity of LH hypocretin/orexin GI and VTA dopamine neurons was measured using whole cell patch clamp recordings in brain slices. Food seeking behavior was assessed using conditioned place preference (CPP). RESULTS: 1-week maintenance of calorie restricted 15% body weight loss reduced glucose inhibition of hypocretin/orexin GI neurons resulting in increased neuronal activation with reduced glycemia. The effect of decreased glucose on hypocretin/orexin GI neuronal activation was blocked by pertussis toxin (inhibitor of G-protein coupled receptor subunit Gαi/o) and Rp-cAMP (inhibitor of protein kinase A, PKA). This suggests that glucose sensitivity is mediated by the Gαi/o-adenylyl cyclase-cAMP-PKA signaling pathway. The excitatory effect of the hunger hormone, ghrelin, on hcrt/ox neurons was also blocked by Rp-cAMP suggesting that hormonal signals of metabolic status may converge on the glucose sensing pathway. Food restriction and weight loss increased glutamate synaptic strength (indexed by increased AMPA/NMDA receptor current ratio) on VTA dopamine neurons and the motivation to seek food (indexed by CPP). Chemogenetic inhibition of hypocretin/orexin neurons during caloric restriction and weight loss prevented these changes in glutamate plasticity and food seeking behavior. CONCLUSIONS: We hypothesize that this change in the glucose sensitivity of hypocretin/orexin GI neurons may drive, in part, food seeking behavior following caloric restriction.

The Antinarcolepsy Drug Modafinil Reverses Hypoglycemia Unawareness and Normalizes Glucose Sensing of Orexin Neurons in Male Mice.

Perifornical hypothalamus (PFH) orexin glucose-inhibited (GI) neurons that facilitate arousal have been implicated in hypoglycemia awareness. Mice lacking orexin exhibit narcolepsy, and orexin mediates the effect of the antinarcolepsy drug modafinil. Thus, hypoglycemia awareness may require a certain level of arousal for awareness of the sympathetic symptoms of hypoglycemia (e.g., tremors, anxiety). Recurrent hypoglycemia (RH) causes hypoglycemia unawareness. We hypothesize that RH impairs the glucose sensitivity of PFH orexin GI neurons and that modafinil normalizes glucose sensitivity of these neurons and restores hypoglycemia awareness after RH. Using patch-clamp recording, we found that RH enhanced glucose inhibition of PFH orexin GI neurons in male mice, thereby blunting activation of these neurons in low-glucose conditions. We then used a modified conditioned place preference behavioral test to demonstrate that modafinil reversed hypoglycemia unawareness in male mice after RH. Similarly, modafinil restored normal glucose sensitivity to PFH orexin GI neurons. We conclude that impaired glucose sensitivity of PFH orexin GI neurons plays a role in hypoglycemia unawareness and that normalizing their glucose sensitivity after RH is associated with restoration of hypoglycemia awareness. This suggests that the glucose sensitivity of PFH orexin GI neurons is a therapeutic target for preventing hypoglycemia unawareness.

Insulin actions on hypothalamic glucose-sensing neurones.

Subsequent to the discovery of insulin 100 years ago, great strides have been made in understanding its function, especially in the brain. It is now clear that insulin is a critical regulator of the neuronal circuitry controlling energy balance and glucose homeostasis. This review focuses on the effects of insulin and diabetes on the activity and glucose sensitivity of hypothalamic glucose-sensing neurones. We highlight the role of electrophysiological data in understanding how insulin regulates glucose-sensing neurones. A brief introduction describing the benefits and limitations of the major electrophysiological techniques used to investigate glucose-sensing neurones is provided. The mechanisms by which hypothalamic neurones sense glucose are discussed with an emphasis on those glucose-sensing neurones already shown to be modulated by insulin. Next, the literature pertaining to how insulin alters the activity and glucose sensitivity of these hypothalamic glucose-sensing neurones is described. In addition, the effects of impaired insulin signalling during diabetes and the ramifications of insulin-induced hypoglycaemia on hypothalamic glucose-sensing neurones are covered. To the extent that it is known, we present hypotheses concerning the mechanisms underlying the effects of these insulin-related pathologies. To conclude, electrophysiological data from the hippocampus are evaluated aiming to provide clues regarding how insulin might influence neuronal plasticity in glucose-sensing neurones. Although much has been accomplished subsequent to the discovery of insulin, the work described in our review suggests that the regulation of central glucose sensing by this hormone is both important and understudied.