Objective: Arcuate proopiomelanocortin (POMC) neurons are critical nodes in the control of body weight. Often characterized simply as direct targets for leptin, recent data suggest a more complex architecture.

Methods: Using single cell RNA sequencing, we have generated an atlas of gene expression in murine POMC neurons.

Results: Of 163 neurons, 118 expressed high levels of Pomc with little/no Agrp expression and were considered “canonical” POMC neurons (P⁺). The other 45/163 expressed low levels of Pomc and high levels of Agrp (A⁺P₊). Unbiased clustering analysis of P⁺ neurons revealed four different classes, each with distinct cell surface receptor gene expression profiles. Further, only 12% (14/118) of P⁺ neurons expressed the leptin receptor (Lepr) compared with 58% (26/45) of A⁺P₊ neurons. In contrast, the insulin receptor (Insr) was expressed at similar frequency on P⁺ and A⁺P₊ neurons (64% and 55%, respectively).

Conclusions: These data reveal arcuate POMC neurons to be a highly heterogeneous population.

Objective: The GH/IGF-1 axis has important roles in growth and metabolism. GH and GH receptor (GHR) are active in the central nervous system (CNS) and are crucial in regulating several aspects of metabolism. In the hypothalamus, there is a high abundance of GH-responsive cells, but the role of GH signaling in hypothalamic neurons is unknown. Previous work has demonstrated that the Ghr gene is highly expressed in LepRb neurons. Given that leptin is a key regulator of energy balance by acting on leptin receptor (LepRb)-expressing neurons, we tested the hypothesis that LepRb neurons represent an important site for GHR signaling to control body homeostasis.

Methods: To determine the importance of GHR signaling in LepRb neurons, we utilized Cre/loxP technology to ablate GHR expression in LepRb neurons (Lepr^EYFPΔGHR). The mice were generated by crossing the Lepr^cre on the cre-inducible ROSA26-EYFP mice to GHR^L/L mice. Parameters of body composition and glucose homeostasis were evaluated.

Results: Our results demonstrate that the sites with GHR and LepRb co-expression include ARH, DMH, and LHA neurons. Leptin action was not altered in Lepr^EYFPΔGHR mice; however, GH-induced pStat5-IR in LepRb neurons was significantly reduced in these mice. Serum IGF-1 and GH levels were unaltered, and we found no evidence that GHR signaling regulates food intake and body weight in LepRb neurons. In contrast, diminished GHR signaling in LepRb neurons impaired hepatic insulin sensitivity and peripheral lipid metabolism. This was paralleled with a failure to suppress expression of the gluconeogenic genes and impaired hepatic insulin signaling in Lepr^EYFPΔGHR mice.

Conclusions: These findings suggest the existence of GHR-leptin neurocircuitry that plays an important role in the GHR-mediated regulation of glucose metabolism irrespective of feeding.

Objective: The endocannabinoid system hypertonicity features obesity. Excess circulating 2-arachidonoylglycerol was variously associated with obesity-related metabolic impairment; however, unstandardized experimental and analytical settings have clouded its usefulness as a dysmetabolism biomarker. We aimed at assessing the influence of body mass index (BMI), menopause in women, and aging in men on 2-arachidonoylglycerol relationship with metabolic parameters.

Methods: Adult, unmedicated women (premenopausal (preMW): n = 103; menopausal (MW): n = 81) and men (n = 144) were stratified in normal weight (NW; BMI: 18.5–24.9 kg/m²), overweight (OW; BMI: 25.0–29.9 kg/m²), and obese (OB; BMI ≥ 30.0 kg/m²) classes. Anthropometric and metabolic parameters were determined. Plasma 2-arachidonoylglycerol was measured by a validated liquid chromatography-mass spectrometry assay.

Results: 2-arachidonoylglycerol level was raised by menopause (P < 0.001) and by obesity in preMW (P < 0.001) and in men (P = 0.019). In the overall cohorts, 2-arachidonoylglycerol displayed BMI-independent relationships with dyslipidemia (preMW, MW and men), insulin resistance (MW and men), and hypertension (men), but not with waist circumference. Within preMW BMI classes, 2-arachidonoylglycerol correlations were found with triglycerides (P = 0.020) and total cholesterol (TC; P = 0.040) in OB women. In MW, 2-arachidonoylglycerol correlation with triglycerides was found in NW (P = 0.001) and OW (P = 0.034), but not in OB class. Moreover, we found 2-arachidonoylglycerol correlations with TC (P = 0.003), glucose (P < 0.001), and HOMA-IR (P = 0.035) specific for NW MW class. In men, 2-arachidonoylglycerol correlated with triglycerides in NW, OW (both P < 0.001), and OB (P = 0.029), with SBP (P = 0.023) and diastolic BP (DBP; P = 0.048) in OB, and with TC (P < 0.001) in OW class. In NW class 2-arachidonoylglycerol correlations were found with insulin (P = 0.003) and HOMA-IR (P = 0.001), both enhanced by aging (both P = 0.004), and with glucose (P = 0.015) and HDL (P = 0.004).

Conclusions: Plasma 2AG is a biomarker of clustering metabolic dysfunctions, especially in lean men and menopausal women, and could be of help in identifying subjects with elevated cardiometabolic risk despite a healthy anthropometric appearance.

Objective: Alterations in sphingolipid and ceramide metabolism have been associated with various diseases, including nonalcoholic fatty liver disease (NAFLD). Acid sphingomyelinase (ASM) converts the membrane lipid sphingomyelin to ceramide, thereby affecting membrane composition and domain formation. We investigated the ways in which the Asm knockout (Smpd1^−/−) genotype affects diet-induced NAFLD.

Methods: Smpd1^−/− mice and wild type controls were fed either a standard or Western diet (WD) for 6 weeks. Liver and adipose tissue morphology and mRNA expression were assessed. Quantitative proteome analysis of liver tissue was performed. Expression of selected genes was quantified in adipose and liver tissue of obese NAFLD patients.

Results: Although Smpd1^−/− mice exhibited basal steatosis with normal chow, no aggravation of NAFLD-type injury was observed with a Western diet. This protective effect was associated with the absence of adipocyte hypertrophy and the increased expression of genes associated with brown adipocyte differentiation. In white adipose tissue from obese patients with NAFLD, no expression of these genes was detectable. To further elucidate which pathways in liver tissue may be affected by Smpd1^−/−, we performed an unbiased proteome analysis. Protein expression in WD-fed Smpd1^−/− mice indicated a reduction in Rictor (mTORC2) activity; this reduction was confirmed by diminished Akt phosphorylation and altered mRNA expression of Rictor target genes.

Conclusions: These findings indicate that the protective effect of Asm deficiency on diet-induced steatosis is conferred by alterations in adipocyte morphology and lipid metabolism and by reductions in Rictor activation.

Objective: In the hypothalamic arcuate nucleus (ARC), orexigenic agouti-related peptide (AgRP) neurons regulate feeding behavior and energy homeostasis, functions connected to bone metabolism. The 3-phosphoinositide-dependent protein kinase-1 (PDK1) serves as a major signaling molecule particularly for leptin and insulin in AgRP neurons. We asked whether PDK1 in AGRP neurons also contributes to bone metabolism.

Methods: We generated AgRP neuron-specific PDK1 knockout (Agrp Pdk1^−/−) mice and those with additional AgRP neuron-specific expression of transactivation-defective FoxO1 (Agrp Pdk1^−/− Δ256Foxo1). Bone metabolism in KO and WT mice was analyzed by quantitative computed tomography (QCT), bone histomorphometry, measurement of plasma biomarkers, and qPCR analysis of peptides.

Results: In Agrp Pdk1^−/− female mice aged 6 weeks, compared with Agrp Cre mice, both stature and femur length were shorter while body weight was unchanged. Cortical bone mineral density (BMD) and cancellous BMD in the femur decreased, and bone formation was delayed. Furthermore, plasma GH and IGF-1 levels were reduced in parallel with decreased mRNA expressions for GH in pituitary and GHRH in ARC. Osteoblast activity was suppressed and osteoclast activity was enhanced. These changes in stature, BMD and GH level were rescued in Agrp Pdk1^−/− Δ256Foxo1 mice, suggesting that the bone abnormalities and impaired GH release were mediated by enhanced Foxo1 due to deletion of PDK1.

Conclusions: This study reveals a novel role of PDK1-Foxo1 pathway of AgRP neurons in controlling bone metabolism primarily via GHRH-GH-IGF-1 axis.

Objective: Obesity is a major health threat that affects men and women equally. Despite this fact, weight-loss potential of pharmacotherapies is typically first evaluated in male mouse models of diet-induced obesity (DIO). To address this disparity we herein determined whether a monomeric peptide with agonism at the receptors for glucagon-like peptide 1 (GLP-1), glucose-dependent insulinotropic polypeptide (GIP), and glucagon is equally efficient in correcting DIO, dyslipidemia, and glucose metabolism in DIO female mice as it has been previously established for DIO male mice.

Methods: Female C57BL/6J mice and a cohort of fatmass-matched C57BL/6J male mice were treated for 27 days via subcutaneous injections with either the GLP-1/GIP/glucagon triagonist or PBS. A second cohort of C57BL/6J male mice was included to match the females in the duration of the high-fat, high-sugar diet (HFD) exposure.

Results: Our results show that GLP-1/GIP/glucagon triple agonism inhibits food intake and decreases body weight and body fat mass with comparable potency in male and female mice that have been matched for body fat mass. Treatment improved dyslipidemia in both sexes and reversed diet-induced steatohepatitis to a larger extent in female mice compared to male mice.

Conclusions: We herein show that a recently developed unimolecular peptide triagonist is equally efficient in both sexes, suggesting that this polypharmaceutical strategy might be a relevant alternative to bariatric surgery for the treatment of obesity and related metabolic disorders.

Objective: The mechanistic target of rapamycin (mTOR) is a serine/threonine kinase that functions into distinct protein complexes (mTORC1 and mTORC2) that regulates growth and metabolism. DEP-domain containing mTOR-interacting protein (DEPTOR) is part of these complexes and is known to reduce their activity. Whether DEPTOR loss affects metabolism and organismal growth in vivo has never been tested.

Methods: We have generated a conditional transgenic mouse allowing the tissue-specific deletion of DEPTOR. This model was crossed with CMV-cre mice or Albumin-cre mice to generate either whole-body or liver-specific DEPTOR knockout (KO) mice.

Results: Whole-body DEPTOR KO mice are viable, fertile, normal in size, and do not display any gross physical and metabolic abnormalities. To circumvent possible compensatory mechanisms linked to the early and systemic loss of DEPTOR, we have deleted DEPTOR specifically in the liver, a tissue in which DEPTOR protein is expressed and affected in response to mTOR activation. Liver-specific DEPTOR null mice showed a reduction in circulating glucose upon fasting versus control mice. This effect was not associated with change in hepatic gluconeogenesis potential but was linked to a sustained reduction in circulating glucose during insulin tolerance tests. In addition to the reduction in glycemia, liver-specific DEPTOR KO mice had reduced hepatic glycogen content when fasted. We showed that loss of DEPTOR cell-autonomously increased oxidative metabolism in hepatocytes, an effect associated with increased cytochrome c expression but independent of changes in mitochondrial content or in the expression of genes controlling oxidative metabolism. We found that liver-specific DEPTOR KO mice showed sustained mTORC1 activation upon fasting, and that acute treatment with rapamycin was sufficient to normalize glycemia in these mice.

Conclusions: We propose a model in which hepatic DEPTOR accelerates the inhibition of mTORC1 during the transition to fasting to adjust metabolism to the nutritional status.