Knockdown of Acid-sensing Ion Channel 1a in the PVN Promotes Metabolic Disturbances in Male Mice

Endocrinology

2022 Oct 01

Wang, W;Xu, M;Yue, J;Zhang, Q;Nie, X;Jin, Y;Zhang, Z;
PMID: 35894166 | DOI: 10.1210/endocr/bqac115

Increasing incidence of metabolic disturbances has become a severe public healthcare problem. Ion channels and receptors in the paraventricular nucleus (PVN) of the hypothalamus serve vital roles in modulating neuronal activities and endocrine functions, which are linked to the regulation of energy balance and glucose metabolism. In this study, we found that acid-sensing ion channel 1a (ASIC1a), a Ca2+-permeable cationic ion channel was localized in the PVN. Knockdown of ASIC1a in this region led to significant body weight gain, glucose intolerance, and insulin resistance. Pharmacological inhibition of ASIC1a resulted in an increase in food intake and a decrease in energy expenditure. Our findings suggest ASIC1a in the PVN as a potential new target for the therapeutic intervention of metabolic disorders.

Cell selectivity in succinate receptor SUCNR1/GPR91 signaling in skeletal muscle

American journal of physiology. Endocrinology and metabolism

2023 Feb 22

Abdelmoez, AM;Dmytriyeva, O;Zurke, YX;Trauelsen, M;Marica, AA;Savikj, M;Smith, JAB;Monaco, C;Schwartz, TW;Krook, A;Pillon, NJ;
PMID: 36812387 | DOI: 10.1152/ajpendo.00009.2023

Succinate is released by skeletal muscle during exercise and activates SUCNR1/GPR91. Signaling of SUCNR1 is involved in metabolite-sensing paracrine communication in skeletal muscle during exercise. However, the specific cell types responding to succinate and the directionality of communication are unclear. We aim to characterize the expression of SUCNR1 in human skeletal muscle. De novo analysis of transcriptomic datasets demonstrated that SUCNR1 mRNA is expressed in immune, adipose, and liver tissues, but scarce in skeletal muscle. In human tissues, SUCNR1 mRNA was associated with macrophage markers. Single-cell RNA sequencing and fluorescent RNAscope demonstrated that in human skeletal muscle, SUCNR1 mRNA is not expressed in muscle fibers but coincided with macrophage populations. Human M2-polarized macrophages exhibit high levels of SUCNR1 mRNA and stimulation with selective agonists of SUCNR1 triggered Gq- and Gi-coupled signaling. Primary human skeletal muscle cells were unresponsive to SUCNR1 agonists. In conclusion, SUCNR1 is not expressed in muscle cells and its role in the adaptive response of skeletal muscle to exercise is most likely mediated via paracrine mechanisms involving M2-like macrophages within the muscle.

Paracrine relationship between incretin hormones and endogenous 5-hydroxytryptamine in the small and large intestine

Neurogastroenterology and motility : the official journal of the European Gastrointestinal Motility Society

2023 Apr 03

Tough, IR;Lund, ML;Patel, BA;Schwartz, TW;Cox, HM;
PMID: 37010838 | DOI: 10.1111/nmo.14589

Enterochromaffin (EC) cell-derived 5-hydroxytryptamine (5-HT) is a mediator of toxin-induced reflexes, initiating emesis via vagal and central 5-HT3 receptors. The amine is also involved in gastrointestinal (GI) reflexes that are prosecretory and promotile, and recently 5-HT's roles in chemosensation in the distal bowel have been described. We set out to establish the efficacy of 5-HT signaling, local 5-HT levels and pharmacology in discrete regions of the mouse small and large intestine. We also investigated the inter-relationships between incretin hormones, glucagon-like peptide-1 (GLP-1) and gastric inhibitory polypeptide (GIP) and endogenous 5-HT in mucosal and motility assays.Adult mouse GI mucosae were mounted in Ussing chambers and area-specific studies were performed to establish the 5-HT3 and 5-HT4 pharmacology, the sidedness of responses, and the inter-relationships between incretins and endogenous 5-HT. Natural fecal pellet transit in vitro and full-length GI transit in vivo were also measured.We observed the greatest level of tonic and exogenous 5-HT-induced ion transport and highest levels of 5-HT in ascending colon mucosa. Here both 5-HT3 and 5-HT4 receptors were involved but elsewhere in the GI tract epithelial basolateral 5-HT4 receptors mediate 5-HT's prosecretory effect. Exendin-4 and GIP induced 5-HT release in the ascending colon, while L cell-derived PYY also contributed to GIP mucosal effects in the descending colon. Both peptides slowed colonic transit.We provide functional evidence for paracrine interplay between 5-HT, GLP-1 and GIP, particularly in the colonic mucosal region. Basolateral epithelial 5-HT4 receptors mediated both 5-HT and incretin mucosal responses in healthy colon.

Alpha cell TXNIP deletion improves diabetes-associated hyperglycemia and hyperglucagonemia

Endocrinology

2022 Aug 12

Lu, B;Chen, J;Xu, G;Grayson, TB;Jing, G;Jo, S;Shalev, A;
PMID: 35957590 | DOI: 10.1210/endocr/bqac133

Thioredoxin-interacting protein (Txnip) has emerged as a key factor in pancreatic beta cell biology and its upregulation by glucose and diabetes contributes to the impairment in functional beta cell mass and glucose homeostasis. In addition, beta cell deletion of Txnip protects against diabetes in different mouse models. However, while Txnip is ubiquitously expressed, its role in pancreatic alpha cells has remained elusive. We therefore now generated an alpha cell Txnip knockout (aTKO) mouse and assessed the effects on glucose homeostasis. While no significant changes were observed on regular chow, after a 30-week high-fat diet, aTKO animals showed improvement in glucose tolerance and lower blood glucose levels compared to their control littermates. Moreover, in the context of streptozotocin (STZ)-induced diabetes, aTKO mice showed significantly lower blood glucose levels compared to controls. While serum insulin levels were reduced in both control and aTKO mice, STZ-diabetes significantly increased glucagon levels in control mice, but this effect was blunted in aTKO mice. Moreover, glucagon secretion from aTKO islets was >2-fold lower than from control islets, while insulin secretion was unchanged in aTKO islets. At the same time, no change in alpha cell or beta cell numbers or mass was observed and glucagon and insulin expression and content were comparable in isolated islets from aTKO and control mice. Thus, together the current studies suggest that downregulation of alpha cell Txnip is associated with reduced glucagon secretion and that this may contribute to the glucose-lowering effects observed in diabetic aTKO mice.

Steroidogenic factor 1 regulates transcription of the inhibin B co-receptor in pituitary gonadotrope cells

Endocrinology

2022 Aug 12

Lin, YF;Schang, G;Buddle, ERS;Schultz, H;Willis, TL;Ruf-Zamojski, F;Zamojski, M;Mendelev, N;Boehm, U;Sealfon, SC;Andoniadou, CL;Bernard, DJ;
PMID: 35957608 | DOI: 10.1210/endocr/bqac131

The inhibins control reproduction by suppressing follicle-stimulating hormone synthesis in pituitary gonadotrope cells. The newly discovered inhibin B co-receptor, TGFBR3L, is selectively and highly expressed in gonadotropes in both mice and humans. Here, we describe our initial characterization of mechanisms controlling cell-specific Tgfbr3l/TGFBR3L transcription. We identified two steroidogenic factor 1 (SF-1 or NR5A1) cis-elements in the proximal Tgfbr3l promoter in mice. SF-1 induction of murine Tgfbr3l promoter-reporter activity was inhibited by mutations in one or both sites in heterologous cells. In homologous cells, mutation of these cis-elements or depletion of endogenous SF-1 similarly decreased reporter activity. We observed nearly identical results when using a human TGFBR3L promoter-reporter. The Tgfbr3l gene was tightly compacted and Tgfbr3l mRNA expression was essentially absent in gonadotropes of SF-1 (Nr5a1) conditional knockout mice. During murine embryonic development, Tgfbr3l precedes Nr5a1 expression, though the two transcripts are fully co-localized by embryonic day 18.5 and thereafter. Collectively, these data indicate that SF-1 directly regulates Tgfbr3l/TGFBR3L transcription and is required for post-natal expression of the gene in gonadotropes.

Journal of Neuroendocrinology

2022 Aug 19

Decourt, C;Connolly, G;Ancel, C;Inglis, M;Anderson, G;
| DOI: 10.1111/jne.13190

Agouti-related peptide (AgRP) neurons are thought to indirectly regulate the activity of hypothalamic gonadotrophin-releasing hormone neurons which control fertility. AgRP neurons also drive caloric intake and are modulated by metabolically-relevant hormones, providing a link to the hypothalamic-pituitary-gonadal axis. In mice expressing Cre-dependant designer receptors (DREADDs) in AgRP neurons, we activated or silenced these neurons in vivo using the synthetic ligand clozapine-N-oxide (CNO) to observe the effect of AgRP neuron activity on timing of puberty. To validate these animals, we chronically treated both stimulatory (hM3Dq) and inhibitory (hM4Di) DREADD × AgRP-Cre mice with CNO, observing a pronounced increase and decrease of food intake, respectively, consistent with the known orexigenic effects of these neurons. RNAscope was performed to visually confirm the activation of AgRP neurons. Puberty onset was assessed in males and females. There was no effect on preputial separation in males or vaginal opening and first oestrus in females after CNO treatment from day 26 to 30 to chronically modulate AgRP neurons. Next, to determine whether the delay in puberty onset occurring in response to neonatal underfeeding could be overcome by inhibiting AgRP neuronal activity, mice were raised in large (neonatally underfed) or normal litter sizes. The delay in puberty from underfeeding was completely reversed in CNO-treated AgRP-hM4Di male mice. These data highlight the inhibitory role of AgRP neurons to delay puberty onset when undernutrition occurs during the neonatal period, at least in male mice.

Adulthood bisphenol A exposure induces anxiety in male mice via downregulation of alpha-1D adrenergic receptor in paraventricular thalamus

Ecotoxicology and environmental safety

2023 Jun 29

Ji, R;Cui, M;Zhou, D;Pan, X;Xie, Y;Wu, X;Liang, X;Zhang, H;Song, W;
PMID: 37392660 | DOI: 10.1016/j.ecoenv.2023.115205

Bisphenol A (BPA), a ubiquitous endocrine disrupting chemical, is widely used in household plastic products. Large amounts of evidence indicate prenatal and postnatal BPA exposure causes neurodevelopmental disorders such as anxiety and autism. However, the neuronal mechanisms underlying the neurotoxic effects of adulthood BPA exposure remain poorly understood. Here, we provided evidences that adult mice treated with BPA (0.45 mg/kg/day) during 3 weeks exhibited sex-specific anxiety like behaviors. We demonstrated that the BPA-induced anxiety in male mice, but not in female mice, was closely associated with hyperactivity of glutamatergic neurons in the paraventricular thalamus (PVT). Acute chemogenetic activation of PVT glutamatergic neurons caused similar effects on anxiety as observed in male mice exposed to BPA. In contrast, acute chemogenetic inhibition of PVT glutamatergic neurons reduced BPA-induced anxiety in male mice. Concomitantly, the BPA-induced anxiety was related with a down-regulation of alpha-1D adrenergic receptor in the PVT. Taken together, the present study indicated a previously unknown target region in the brain for neurotoxic effects of BPA on anxiety and implicated a possible molecular mechanism of action.

Deletion of androgen receptors from kisspeptin neurons prevents PCOS features in a letrozole mouse model

Endocrinology

2023 May 16

Decourt, C;Watanabe, Y;Evans, MC;Inglis, MA;Fisher, LC;Jasoni, CL;Campbell, RE;Anderson, GM;
PMID: 37191144 | DOI: 10.1210/endocr/bqad077

Polycystic ovarian syndrome (PCOS) is the leading cause of anovulatory infertility and is a heterogenous condition associated with a range of reproductive and metabolic impairments. While its etiology remains unclear, hyperandrogenism and impaired steroid negative feedback have been identified as key factors underpinning the development of PCOS-like features both clinically and in animal models. We tested the hypothesis that androgen signaling in kisspeptin-expressing neurons, which are key drivers of the neuroendocrine reproductive axis, is critically involved in PCOS pathogenesis. To this end, we used a previously validated letrozole (LET)-induced hyperandrogenic mouse model of PCOS in conjunction with Cre-lox technology to generate female mice exhibiting kisspeptin-specific deletion of androgen receptor (KARKO mice) to test whether LET-treated KARKO females are protected from the development of reproductive and metabolic PCOS-like features. LET-treated mice exhibited hyperandrogenism, and KARKO mice exhibited a significant reduction in the coexpression of kisspeptin and androgen receptor mRNA compared to controls. In support of our hypothesis, LET-treated KARKO mice exhibited improved estrous cyclicity, ovarian morphology, and insulin sensitivity in comparison to LET-treated control females. However, KARKO mice were not fully protected from the effects of LET-induced hyperandrogenism and still exhibited reduced corpora lutea numbers and increased body weight gain. These data indicate that increased androgen signaling in kisspeptin-expressing neurons plays a critical role in PCOS pathogenesis, but highlight that other mechanisms are also involved.

The Β-HYDROXYBUTYRATE-GPR109A Receptor Regulates Fasting-Induced Plasticity In The Mouse Adrenal Medulla

Endocrinology

2022 May 20

Gupta, R;Wang, M;Ma, Y;Offermanns, S;Whim, MD;
PMID: 35595517 | DOI: 10.1210/endocr/bqac077

During fasting, increased sympatho-adrenal activity leads to epinephrine release and multiple forms of plasticity within the adrenal medulla including an increase in the strength of the preganglionic → chromaffin cell synapse and elevated levels of AgRP, a peptidergic co-transmitter in chromaffin cells. Although these changes contribute to the sympathetic response, how fasting evokes this plasticity is not known. Here we report these effects involve activation of GPR109A (HCAR2). The endogenous agonist of this G protein-coupled receptor is β-hydroxybutyrate, a ketone body whose levels rise during fasting. In wild type animals, 24 hr fasting increased AgRP-ir in adrenal chromaffin cells but this effect was absent in GPR109A knockout mice. GPR109A agonists increased AgRP-ir in isolated chromaffin cells through a GPR109A- and pertussis toxin-sensitive pathway. Incubation of adrenal slices in nicotinic acid, a GPR109A agonist, mimicked the fasting-induced increase in the strength of the preganglionic → chromaffin cell synapse. Finally, RT-PCR experiments confirmed the mouse adrenal medulla contains GPR109A mRNA. These results are consistent with the activation of a GPR109A signaling pathway located within the adrenal gland. Because fasting evokes epinephrine release, which stimulates lipolysis and the production of β-hydroxybutyrate, our results indicate that chromaffin cells are components of an autonomic-adipose-hepatic feedback circuit. Coupling a change in adrenal physiology to a metabolite whose levels rise during fasting is presumably an efficient way to co-ordinate the homeostatic response to food deprivation.

Deletion of Androgen Receptor in LepRb Cells Improves Estrous Cycles in Prenatally Androgenized Mice

Endocrinology

2023 Jan 23

Cara, AL;Burger, LL;Beekly, BG;Allen, SJ;Henson, EL;Auchus, RJ;Myers, MG;Moenter, SM;Elias, CF;
PMID: 36683455 | DOI: 10.1210/endocr/bqad015

Androgens are steroid hormones crucial for sexual differentiation of the brain and reproductive function. In excess, however, androgens may decrease fertility as observed in polycystic ovary syndrome, a common endocrine disorder characterized by oligo/anovulation and/or polycystic ovaries. Hyperandrogenism may also disrupt energy homeostasis, inducing higher central adiposity, insulin resistance, and glucose intolerance, which may exacerbate reproductive dysfunction. Androgens bind to androgen receptors (AR), which are expressed in many reproductive and metabolic tissues, including brain sites that regulate the hypothalamo-pituitary-gonadal axis and energy homeostasis. The neuronal populations impacted by androgen excess, however, have not been defined. We and others have shown that, in mice, AR is highly expressed in leptin receptor (LepRb) neurons, particularly in the arcuate (ARH) and the ventral premammillary nuclei (PMv). Here, we assessed if LepRb neurons, which are critical in the central regulation of energy homeostasis and exert permissive actions on puberty and fertility, have a role in the pathogenesis of female hyperandrogenism. Prenatally androgenized (PNA) mice lacking AR in LepRb cells (LepRbΔAR) show no changes in body mass, body composition, glucose homeostasis, or sexual maturation. They show, however, a remarkable improvement of estrous cycles combined with normalization of ovary morphology compared to PNA controls. Our findings indicate that the prenatal androgenization effects on adult reproductive physiology (i.e., anestrus and anovulation) are mediated by a subpopulation of LepRb neurons directly sensitive to androgens. They also suggest that the effects of hyperandrogenism on sexual maturation and reproductive function in adult females are controlled by distinct neural circuits.

Adrenal cortex size and homeostasis are regulated by gonadal hormones via androgen receptor/β-catenin signaling crosstalk

Disease models & mechanisms

2023 Apr 27

Lyraki, R;Grabek, A;Tison, A;Weerasinghe-Arachchige, LC;Peitzsch, M;Bechman, N;Youssef, SA;de Bruin, A;Bakker, ERM;Claessens, F;Chaboissier, MC;Schedl, A;
PMID: 37102205 | DOI: 10.1242/dmm.050053

Female bias is highly prevalent among adrenal cortex hyperplasia and neoplasia, but the reasons behind this phenomenon are poorly understood. In this article, we show that overexpression of the secreted WNT agonist R-spondin 1 leads to ectopic activation of WNT/β-catenin signaling and causes sex-specific adrenocortical hyperplasia in mice. While female adrenals show ectopic proliferation, male adrenals display excessive immune system activation and cortical thinning. Using a combination of genetic manipulations and hormonal treatment, we show that gonadal androgens suppress ectopic proliferation in the adrenal cortex and determine the selective regulation of WNT-related genes Axin2 and Wnt4. Notably, genetic removal of androgen receptor (AR) from adrenocortical cells restores the mitogenic effect of WNT/β-catenin signaling. This is the first demonstration that AR activity in the adrenal cortex determines susceptibility to canonical WNT signaling-induced hyperplasia.

Odor blocking of stress hormone responses

Scientific reports

2022 May 24

Lee, EJ;Saraiva, LR;Hanchate, NK;Ye, X;Asher, G;Ho, J;Buck, LB;
PMID: 35610316 | DOI: 10.1038/s41598-022-12663-x

Scents have been employed for millennia to allay stress, but whether or how they might do so is largely unknown. Fear and stress induce increases in blood stress hormones controlled by hypothalamic corticotropin releasing hormone neurons (CRHNs). Here, we report that two common odorants block mouse stress hormone responses to three potent stressors: physical restraint, predator odor, and male-male social confrontation. One odorant inhibits restraint and predator odor activation of excitatory neurons upstream of CRHNs in the bed nucleus of the stria terminalis (BNSTa). In addition, both activate inhibitory neurons upstream of CRHNs in the hypothalamic ventromedial nucleus (VMH) and silencing of VMH inhibitory neurons hinders odor blocking of stress. Together, these findings indicate that odor blocking can occur via two mechanisms: (1) Inhibition of excitatory neurons that transmit stress signals to CRHNs and (2) activation of inhibitory neurons that act directly or indirectly to inhibit stressor activation of CRHNs.

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	Description
sense Example: Hs-LAG3-sense	Standard probes for RNA detection are in antisense. Sense probe is reverse complent to the corresponding antisense probe.
Intron# Example: Mm-Htt-intron2	Probe targets the indicated intron in the target gene, commonly used for pre-mRNA detection
Pool/Pan Example: Hs-CD3-pool (Hs-CD3D, Hs-CD3E, Hs-CD3G)	A mixture of multiple probe sets targeting multiple genes or transcripts
No-XSp Example: Hs-PDGFB-No-XMm	Does not cross detect with the species (Sp)
XSp Example: Rn-Pde9a-XMm	designed to cross detect with the species (Sp)
O# Example: Mm-Islr-O1	Alternative design targeting different regions of the same transcript or isoforms
CDS Example: Hs-SLC31A-CDS	Probe targets the protein-coding sequence only
EnEm	Probe targets exons n and m
En-Em	Probe targets region from exon n to exon m
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tvn Example: Hs-LEPR-tv1	Designed to target transcript variant n
ORF Example: Hs-ACVRL1-ORF	Probe targets open reading frame
UTR Example: Hs-HTT-UTR-C3	Probe targets the untranslated region (non-protein-coding region) only
5UTR Example: Hs-GNRHR-5UTR	Probe targets the 5' untranslated region only
3UTR Example: Rn-Npy1r-3UTR	Probe targets the 3' untranslated region only
Pan Example: Pool	A mixture of multiple probe sets targeting multiple genes or transcripts

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