The gut signals to AGRP-expressing cells of the pituitary to control glucose homeostasis.

Glucose homeostasis can be improved after bariatric surgery, which alters bile flow and stimulates gut hormone secretion, particularly FGF15/19. FGFR1 expression in AGRP-expressing cells is required for bile acids' ability to improve glucose control. We show that the mouse Agrp gene has 3 promoter/enhancer regions that direct transcription of each of their own AGRP transcripts. One of these Agrp promoters/enhancers, Agrp-B, is regulated by bile acids. We generated an Agrp-B knockin FLP/knockout allele. AGRP-B-expressing cells are found in endocrine cells of the pars tuberalis and coexpress diacylglycerol lipase B - an endocannabinoid biosynthetic enzyme - distinct from pars tuberalis thyrotropes. AGRP-B expression is also found in the folliculostellate cells of the pituitary's anterior lobe. Mice without AGRP-B were protected from glucose intolerance induced by high-fat feeding but not from excess weight gain. Chemogenetic inhibition of AGRP-B cells improved glucose tolerance by enhancing glucose-stimulated insulin secretion. Inhibition of the AGRP-B cells also caused weight loss. The improved glucose tolerance and reduced body weight persisted up to 6 weeks after cessation of the DREADD-mediated inhibition, suggesting the presence of a biological switch for glucose homeostasis that is regulated by long-term stability of food availability.

Cyclin-dependent kinase 4 is a preclinical target for diet-induced obesity.

When obesity is caused by consumption of a high-fat diet, the tumor suppressor pRb is phosphoinactivated in the neurons of the mediobasal hypothalamus, a brain area critical for energy-balance regulation. However, the functional relevance of pRb phosphoinactivation in the mediobasal hypothalamus to diet-induced obesity remains unknown. Here, we show that inhibiting pRb phosphorylation in the mediobasal hypothalamus can prevent and treat diet-induced obesity in mice. Expressing an unphosphorylable pRb nonselectively in the mediobasal hypothalamus or conditionally in anorexigenic POMC neurons inhibits diet-induced obesity. Intracerebroventricular delivery of US Food and Drug Administration-approved (FDA-approved) cyclin-dependent kinase 4 (CDK4) inhibitor abemaciclib inhibits pRb phosphorylation in the mediobasal hypothalamus and prevents diet-induced obesity. Oral administration of abemaciclib at doses approved for human use reduces fat mass in diet-induced obese mice by increasing lipid oxidation without significantly reducing lean mass. With analysis of recent literature identifying CDK4 as the most abundantly expressed neuronal CDK in the mediobasal hypothalamus, our work uncovers CDK4 as the major kinase for hypothalamic pRb phosphoinactivation and a highly effective central antiobesity target. As three CDK4/6 inhibitors have recently received FDA approval for life-long breast cancer therapy, our study provides a preclinical basis for their expedient repurposing for obesity management.

TXNIP in Agrp neurons regulates adiposity, energy expenditure, and central leptin sensitivity.

Thioredoxin interacting protein (TXNIP) has recently been described as a key regulator of energy metabolism through pleiotropic actions that include nutrient sensing in the mediobasal hypothalamus (MBH). However, the role of TXNIP in neurochemically specific hypothalamic subpopulations and the circuits downstream from MBH TXNIP engaged to regulate energy homeostasis remain unexplored. To evaluate the metabolic role of TXNIP activity specifically within arcuate Agrp neurons, we generated Agrp-specific TXNIP gain-of-function and loss-of-function mouse models using Agrp-Ires-cre mice, TXNIP (flox/flox) mice, and a lentivector expressing the human TXNIP isoform conditionally in the presence of Cre recombinase. Overexpression of TXNIP in Agrp neurons predisposed to diet-induced obesity and adipose tissue storage by decreasing energy expenditure and spontaneous locomotion, without affecting food intake. Conversely, Agrp neuronal TXNIP deletion protected against diet-induced obesity and adipose tissue storage by increasing energy expenditure and spontaneous locomotion, also without affecting food intake. TXNIP overexpression in Agrp neurons did not primarily affect glycemic control, whereas deletion of TXNIP in Agrp neurons improved fasting glucose levels and glucose tolerance independently of its effects on body weight and adiposity. Bidirectional manipulation of TXNIP expression induced reciprocal changes in central leptin sensitivity and the neural regulation of lipolysis. Together, these results identify a critical role for TXNIP in Agrp neurons in mediating diet-induced obesity through the regulation of energy expenditure and adipose tissue metabolism, independently of food intake. They also reveal a previously unidentified role for Agrp neurons in the brain-adipose axis.

Effects of leptin and melanocortin signaling interactions on pubertal development and reproduction.

Leptin and melanocortin signaling control ingestive behavior, energy balance, and substrate utilization, but only leptin signaling defects cause hypothalamic hypogonadism and infertility. Although GnRH neurons do not express leptin receptors, leptin influences GnRH neuron activity via regulation of immediate downstream mediators including the neuropeptides neuropeptide Y and the melanocortin agonist and antagonist, α-MSH, agouti-related peptide, respectively. Here we show that modulation of melanocortin signaling in female db/db mice through ablation of agouti-related peptide, or heterozygosity of melanocortin 4 receptor, restores the timing of pubertal onset, fertility, and lactation. Additionally, melanocortin 4 receptor activation increases action potential firing and induces c-Fos expression in GnRH neurons, providing further evidence that melanocortin signaling influences GnRH neuron activity. These studies thus establish melanocortin signaling as an important component in the leptin-mediated regulation of GnRH neuron activity, initiation of puberty and fertility.

Complete rescue of obesity, diabetes, and infertility in db/db mice by neuron-specific LEPR-B transgenes.

We have generated mice that carry a neuron-specific leptin receptor (LEPR) transgene whose expression is driven by the rat synapsin I promoter synapsin-LEPR B (SYN-LEPR-B). We have also generated mice that are compound hemizygotes for the transgenes SYN-LEPR-B and neuron-specific enolase-LEPR B (NSE-LEPR-B). We observed a degree of correction in db/db mice that are hemizygous (Syn db/db) and homozygous (Syn/Syn db/db) for the SYN-LEPR-B transgene similar to that previously reported for the NSE-LEPR-B transgene. We also show complete correction of the obesity and related phenotypes of db/db mice that are hemizygous for both NSE-LEPR-B and SYN-LEPR-B transgenes (Nse+Syn db/db). Body composition, insulin sensitivity, and cold tolerance were completely normalized in Nse+Syn db/db mice at 12 weeks of age compared with lean controls. In situ hybridization for LEPR B isoform expression in Nse+Syn db/db mice showed robust expression in the energy homeostasis-relevant regions of the hypothalamus. Expression of 3 neuropeptide genes, agouti-related peptide (Agrp), neuropeptide Y (Npy), and proopiomelanocortin (Pomc), was fully normalized in dual transgenic db/db mice. The 2 transgenes in concert conferred normal fertility to male and female db/db mice. Male mice with partial peripheral deletion of Lepr, induced in the periweaning phase, did not show alterations in body composition or mass. In summary, we show that brain-specific leptin signaling is sufficient to reverse the obesity, diabetes, and infertility of db/db mice.

Neuronal deletion of Lepr elicits diabesity in mice without affecting cold tolerance or fertility.

Leptin signaling in the brain regulates energy intake and expenditure. To test the degree of functional neuronal leptin signaling required for the maintenance of body composition, fertility, and cold tolerance, transgenic mice expressing Cre in neurons (CaMKIIalpha-Cre) were crossed to mice carrying a floxed leptin receptor (Lepr) allele to generate mice with neuron-specific deletion of Lepr in approximately 50% (C F/F mice) and approximately 75% (C Delta17/F mice) of hypothalamic neurons. Leptin receptor (LEPR)-deficient mice (Delta17/Delta17) with heat-shock-Cre-mediated global Lepr deletion served as obese controls. At 16 wk, male C F/F, C Delta17/F, and Delta17/Delta17 mice were 13.2 (P < 0.05), 45.0, and 55.9% (P < 0.001) heavier, respectively, than lean controls, whereas females showed 31.6, 68.8, and 160.7% increases in body mass (P < 0.001). Significant increases in total fat mass (C F/F: P < 0.01; C Delta17/F and Delta17/Delta17:P < 0.001 vs. sex-matched, lean controls), and serum leptin concentrations (P < 0.001 vs. controls) were present in proportion to Lepr deletion. Male C Delta17/F mice had significant elevations in basal serum insulin concentrations (P < 0.001 vs. controls) and were glucose intolerant, as measured by glucose tolerance test (AUC P < 0.01 vs. controls). In contrast with previous observations in mice null for LEPR signaling, C F/F and C Delta17/F mice were fertile and cold tolerant. These findings support the hypothesis that body weight, adiposity, serum leptin concentrations, and glucose intolerance are proportional to hypothalamic LEPR deficiency. However, fertility and cold tolerance remain intact unless hypothalamic LEPR deficiency is complete.