The hypothalamus plays a central role in monitoring and regulating systemic glucose metabolism. The brain is enriched with phospholipids containing poly-unsaturated fatty acids, which are biologically active in physiological regulation. Here, we show that intraperitoneal glucose injection induces changes in hypothalamic distribution and amounts of phospholipids, especially arachidonic-acid-containing phospholipids, that are then metabolized to produce prostaglandins. Knockdown of cytosolic phospholipase A2 (cPLA2), a key enzyme for generating arachidonic acid from phospholipids, in the hypothalamic ventromedial nucleus (VMH), lowers insulin sensitivity in muscles during regular chow diet (RCD) feeding. Conversely, the down-regulation of glucose metabolism by high fat diet (HFD) feeding is improved by knockdown of cPLA2 in the VMH through changing hepatic insulin sensitivity and hypothalamic inflammation. Our data suggest that cPLA2-mediated hypothalamic phospholipid metabolism is critical for controlling systemic glucose metabolism during RCD, while continuous activation of the same pathway to produce prostaglandins during HFD deteriorates glucose metabolism.

The regulation of food intake is a major research area in the study of obesity, which plays a key role in the development of metabolic syndrome. Gene targeting studies have clarified the roles of hypothalamic neurons in feeding behavior, but the deletion of a gene has a long-term effect on neurophysiology. Our understanding of short-term changes such as appetite under physiological conditions is therefore still limited. Targeted recombination in active populations (TRAP) is a newly developed method for labeling active neurons by using tamoxifen-inducible Cre recombination controlled by the promoter of activity-regulated cytoskeleton-associated protein (Arc/Arg3.1), a member of immediate early genes. Transgenic mice for TRAP were fasted overnight, re-fed with normal diet, and injected with 4-hydroxytamoxifen 1 h after the refeeding to label the active neurons. The role of labeled neurons was examined by expressing excitatory or inhibitory designer receptors exclusively activated by designer drugs (DREADDs). The labeled neurons were extracted and RNA sequencing was performed to identify genes that are specifically expressed in these neurons. Fasting-refeeding activated and labeled neurons in the compact part of the dorsomedial hypothalamus (DMH) that project to the paraventricular hypothalamic nucleus. Chemogenetic activation of the labeled DMH neurons decreased food intake and developed place preference, an indicator of positive valence. Chemogenetic activation or inhibition of these neurons had no influence on the whole-body glucose metabolism. The labeled DMH neurons expressed prodynorphin (pdyn), gastrin-releasing peptide (GRP), cholecystokinin (CCK), and thyrotropin-releasing hormone receptor (Trhr) genes. We identified a novel cell type of DMH neurons that can inhibit food intake and promote feeding-induced positive valence. Our study provides insight into the role of DMH and its molecular mechanism in the regulation of appetite and emotion.

Glucose is a significant energy resource for maintaining physiological activities, including body temperature homeostasis, and glucose homeostasis is tightly regulated in mammals. Although ambient temperature tunes glucose metabolism to maintain euthermia, the significance of body temperature in metabolic regulation remains unclear owing to strict thermoregulation. Activation of Qrfp neurons in the preoptic area induced a harmless hypothermic state known as Q-neuron–induced hypothermia and hypometabolism (QIH), which is suitable for studying glucose metabolism under hypothermia. In this study, we observed that QIH mice had hyperinsulinemia and insulin resistance. This glucose hypometabolic state was abolished by increasing the body temperature to euthermia. Moreover, QIH-mediated inappetence and locomotor inactivity were recovered in euthermia QIH mice. These results indicate that body temperature is considerably more powerful than ambient temperature in regulating glucose metabolism and behavior, and the glucose hypometabolism in QIH is secondary to hypothermia rather than modulated by Qrfp neurons.

Diabetes mellitus (DM) is caused by insufficient insulin release from the pancreatic β-cells (Type1 DM) and insulin sensitivity in muscles, liver, and adipose tissues (Type2 DM). Insulin injection treats DM patients but leads to hypoglycemia as a side effect. Cortisol and catecholamines are released to activate glucose production from the liver to recover hypoglycemia, called counter-regulatory responses (CRR). In DM research using rodent models, glucose tolerance tests and 2-deoxy-glucose injection are used to measure insulin release and CRR, respectively. However, blood glucose concentrations change persistently during experiments, causing difficulties in assessing net insulin release and CRR. This article describes a method in which blood glucose is kept at 250 mg/dL or 50 mg/dL in conscious mice to compare the release of insulin and CRR hormones, respectively. Polyethylene tubing is implanted in the mice's carotid artery and jugular vein, and the mice are allowed to recover from the surgery. The jugular vein tubing is connected to a Hamilton syringe with a syringe pump to enable insulin or glucose infusion at a constant and variable rate. The carotid artery tubing is for blood collection. For the hyperglycemic clamp, 30% glucose is infused into the vein, and blood glucose levels are measured from the arterial blood every 5 min or 10 min. The infusion rate of 30% glucose is increased until the blood glucose level becomes 250 mg/dL. Blood is collected to measure insulin concentrations. For hypoglycemic clamp, 10 mU/kg/min insulin is infused together with 30% glucose, whose infusion rate is variable to maintain 50 mg/dL of blood glucose level. Blood is collected to measure counter-regulatory hormones when both glucose infusion and blood glucose reach a steady state. Both hyperglycemic and hypoglycemic clamps have the same surgical procedure and experimental setups. Thus, this method is useful for researchers of systemic glucose metabolism.

The hypothalamus monitors blood glucose levels and regulates glucose production in the liver. In response to hypoglycemia, glucose-inhibited (GI) neurons trigger counterregulatory responses (CRRs), which stimulate the release of glucagon, epinephrine, and cortisol to elevate blood glucose. Recurrent hypoglycemia (RH), however, reduces the effectiveness of these CRRs. This study examined the role of hypothalamic prostaglandins in glucose recovery during acute hypoglycemia and RH. Imaging mass spectrometry and liquid chromatography/mass spectrometry showed phospholipid and prostaglandin levels in the hypothalamus of C57BL mice were changed after insulin or 2-deoxy-glucose administration. Ibuprofen, a nonsteroidal anti-inflammatory drug, was infused into the ventromedial hypothalamus (VMH) to analyze its effect on glucose production during hypoglycemia, revealing that prostaglandin inhibition decreased glucagon secretion. Additionally, RH-treated mice decreased glucagon release and glucose production during hypoglycemia. Inhibiting prostaglandin production via shRNA against cytosolic phospholipase A2 (cPLA2) in the hypothalamus restored CRRs diminished by RH via increasing glucagon sensitivity. These findings suggest that hypothalamic prostaglandins play a critical role in glucose recovery from acute hypoglycemia by activating VMH neurons and are also crucial for the attenuation of CRRs during RH. Article Highlights:

The hypothalamus monitors blood glucose levels and regulates glucose production in the liver. In response to hypoglycemia, glucose-inhibited (GI) neurons trigger counter-regulatory responses (CRRs), which stimulate the release of glucagon, epinephrine, and cortisol to elevate blood glucose. Recurrent hypoglycemia (RH), however, reduces the effectiveness of these CRRs. This study examined the role of hypothalamic prostaglandins in glucose recovery during acute hypoglycemia and RH. Using imaging mass spectrometry and liquid chromatography/mass spectrometry, phospholipid and prostaglandin levels in the hypothalamus of C57BL mice were increased following insulin or 2-deoxy-glucose administration. Ibuprofen, a nonsteroidal anti-inflammatory drug (NSAID), was infused into the ventromedial hypothalamus (VMH) to analyze its effect on glucose production during hypoglycemia, revealing that prostaglandin inhibition decreased glucagon secretion. Additionally, RH-treated mice decreased glucagon release and glucose production during hypoglycemia. Inhibiting prostaglandin production via short-hairpin RNA against cytosolic phospholipase A2 (cPLA2) in the hypothalamus restored CRRs diminished by RH via increasing glucagon sensitivity. These findings suggest that hypothalamic prostaglandins play a critical role in glucose recovery from acute hypoglycemia by activating VMH neurons and are also crucial for the attenuation of CRRs during RH. Graphical Article Highlights: Insulin decreases arachidonic acid-containing phospholipids and increases prostaglandin production in the hypothalamus. Prostaglandin in the hypothalamus increases glucagon secretion and glucose production during hypoglycemia. Recurrent hypoglycemia decreases glucagon secretion and glucose production during hypoglycemia. Prostaglandin in the hypothalamus during recurrent hypoglycemia decreases glucagon sensitivity and glucose production.