Proc Natl Acad Sci U S A.
Okamoto H, Cavino K, Na E, Krumm E, Kim SY, Cheng X, Murphy AJ, Yancopoulos GD, Gromada J.
PMID: 28115707 | DOI: 10.1073/pnas.1621069114
Inactivating mutations in the insulin receptor results in extreme insulin resistance. The resulting hyperglycemia is very difficult to treat, and patients are at risk for early morbidity and mortality from complications of diabetes. We used the insulin receptor antagonist S961 to induce severe insulin resistance, hyperglycemia, and ketonemia in mice. Using this model, we show that glucagon receptor (GCGR) inhibition with a monoclonal antibody normalized blood glucose and β-hydroxybutyrate levels. Insulin receptor antagonism increased pancreatic β-cell mass threefold. Normalization of blood glucose levels with GCGR-blocking antibody unexpectedly doubled β-cell mass relative to that observed with S961 alone and 5.8-fold over control. GCGR antibody blockage expanded α-cell mass 5.7-fold, and S961 had no additional effects. Collectively, these data show that GCGR antibody inhibition represents a potential therapeutic option for treatment of patients with extreme insulin-resistance syndromes.
Proc Natl Acad Sci U S A.
Kleiner S, Gomez D, Megra B, Na E, Bhavsar R, Cavino K, Xin Y, Rojas J, Dominguez-Gutierrez G, Zambrowicz B, Carrat G, Chabosseau P, Hu M, Murphy AJ, Yancopoulos GD, Rutter GA, Gromada J.
PMID: 30038024 | DOI: 10.1073/pnas.1721418115
SLC30A8 encodes a zinc transporter that is primarily expressed in the pancreatic islets of Langerhans. In β-cells it transports zinc into insulin-containing secretory granules. Loss-of-function (LOF) mutations in SLC30A8 protect against type 2 diabetes in humans. In this study, we generated a knockin mouse model carrying one of the most common human LOF mutations for SLC30A8, R138X. The R138X mice had normal body weight, glucose tolerance, and pancreatic β-cell mass. Interestingly, in hyperglycemic conditions induced by the insulin receptor antagonist S961, the R138X mice showed a 50% increase in insulin secretion. This effect was not associated with enhanced β-cell proliferation or mass. Our data suggest that the SLC30A8 R138X LOF mutation may exert beneficial effects on glucose metabolism by increasing the capacity of β-cells to secrete insulin under hyperglycemic conditions.
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.
Proceedings of the National Academy of Sciences of the United States of America
Beumer, J;Bauzá-Martinez, J;Veth, TS;Geurts, V;Boot, C;Gilliam-Vigh, H;Poulsen, SS;Knop, FK;Wu, W;Clevers, H;
PMID: 36343264 | DOI: 10.1073/pnas.2212057119
Enteroendocrine cells (EECs) secrete hormones in response to ingested nutrients to control physiological processes such as appetite and insulin release. EEC hormones are synthesized as large proproteins that undergo proteolytic processing to generate bioactive peptides. Mutations in EEC-enriched proteases are associated with endocrinopathies. Due to the relative rarity of EECs and a paucity of in vitro models, intestinal prohormone processing remains challenging to assess. Here, human gut organoids in which EECs can efficiently be induced are subjected to CRISPR-Cas9-mediated modification of EEC-expressed endopeptidase and exopeptidase genes. We employ mass spectrometry-based analyses to monitor peptide processing and identify glucagon production in intestinal EECs, stimulated upon bone morphogenic protein (BMP) signaling. We map the substrates and products of major EECs endo- and exopeptidases. Our studies provide a comprehensive description of peptide hormones produced by human EECs and define the roles of specific proteases in their generation.
Yoon, JS;Sasaki, S;Velghe, J;Lee, MYY;Winata, H;Nian, C;Lynn, FC;
PMID: 35616696 | DOI: 10.1007/s00125-022-05718-1
Pancreatic islets depend on cytosolic calcium (Ca2+) to trigger the secretion of glucoregulatory hormones and trigger transcriptional regulation of genes important for islet response to stimuli. To date, there has not been an attempt to profile Ca2+-regulated gene expression in all islet cell types. Our aim was to construct a large single-cell transcriptomic dataset from human islets exposed to conditions that would acutely induce or inhibit intracellular Ca2+ signalling, while preserving biological heterogeneity.We exposed intact human islets from three donors to the following conditions: (1) 2.8 mmol/l glucose; (2) 16 mmol/l glucose and 40 mmol/l KCl to maximally stimulate Ca2+ signalling; and (3) 16 mmol/l glucose, 40 mmol/l KCl and 5 mmol/l EGTA (Ca2+ chelator) to inhibit Ca2+ signalling, for 1 h. We sequenced 68,650 cells from all islet cell types, and further subsetted the cells to form an endocrine cell-specific dataset of 59,373 cells expressing INS, GCG, SST or PPY. We compared transcriptomes across conditions to determine the differentially expressed Ca2+-regulated genes in each endocrine cell type, and in each endocrine cell subcluster of alpha and beta cells.Based on the number of Ca2+-regulated genes, we found that each alpha and beta cell cluster had a different magnitude of Ca2+ response. We also showed that polyhormonal clusters expressing both INS and GCG, or both INS and SST, are defined by Ca2+-regulated genes specific to each cluster. Finally, we identified the gene PCDH7 from the beta cell clusters that had the highest number of Ca2+-regulated genes, and showed that cells expressing cell surface PCDH7 protein have enhanced glucose-stimulated insulin secretory function.Here we use our large-scale, multi-condition, single-cell dataset to show that human islets have cell-type-specific Ca2+-regulated gene expression profiles, some of them specific to subpopulations. In our dataset, we identify PCDH7 as a novel marker of beta cells having an increased number of Ca2+-regulated genes and enhanced insulin secretory function.A searchable and user-friendly format of the data in this study, specifically designed for rapid mining of single-cell RNA sequencing data, is available at https://lynnlab.shinyapps.io/Human_Islet_Atlas/ . The raw data files are available at NCBI Gene Expression Omnibus (GSE196715).
Wollam J, Riopel M, Xu YJ, Johnson AMF, Ofrecio JM, Ying W, El Ouarrat D, Chan LS, Han AW, Mahmood NA, Ryan CN, Lee YS, Watrous JD, Chordia MD, Pan D, Jain M, Olefsky JM.
PMID: 31010956 | DOI: 10.2337/db18-1307
The composition of the gastrointestinal (GI) microbiota and associated metabolites changes dramatically with diet and the development of obesity. Although many correlations have been described, specific mechanistic links between these changes and glucose homeostasis remain to be defined. Here we show that blood and intestinal levels of the microbiota-produced N-formyl peptide, formyl-methionyl-leucyl-phenylalanine (fMLF), are elevated in high fat diet (HFD)-induced obese mice. Genetic or pharmacological inhibition of the N-formyl peptide receptor Fpr1 leads to increased insulin levels and improved glucose tolerance, dependent upon glucagon-like peptide-1 (GLP-1). Obese Fpr1-knockout (Fpr1-KO) mice also display an altered microbiome, exemplifying the dynamic relationship between host metabolism and microbiota. Overall, we describe a new mechanism by which the gut microbiota can modulate glucose metabolism, providing a potential approach for treatment of metabolic disease.
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.
Biancolin, AD;Jeong, H;Mak, KWY;Yuan, Z;Brubaker, PL;
PMID: 35876276 | DOI: 10.1210/endocr/bqac118
Metabolism and circadian rhythms are intimately linked, with circadian glucagon-like peptide-1 (GLP-1) secretion by the intestinal L-cell entraining rhythmic insulin release. GLP-1 secretion has been explored in the context of obesogenic diets, but never in a rodent model of type 2 diabetes (T2D). There is also considerable disagreement regarding GLP-1 levels in human T2D. Furthermore, recent evidence has demonstrated decreased expression of the β-cell exocytotic protein, secretagogin in T2D. To extend these findings to the L-cell, we administered OGTTs at 6 time points in 4 hour intervals to the high-fat diet/streptozotocin (HFD-STZ) mouse model of T2D. This revealed a 10-fold increase in peak GLP-1 secretion with a phase shift of the peak from the normal feeding period into the fasting-phase. This was accompanied by impairments in the rhythms of glucose, glucagon, mucosal clock genes (Arntl and Cry2) and Scgn. Immunostaining revealed that L-cell GLP-1 intensity was increased in the HFD-STZ model, as was the proportion of L-cells that expressed SCGN; however, this was not found in L-cells from humans with T2D, which exhibited decreased GLP-1 staining but maintained their SCGN expression. Gcg expression in isolated L-cells was increased along with pathways relating to GLP-1 secretion and electron transport chain activity in the HFD-STZ condition. Further investigation into the mechanisms responsible for this increase in GLP-1 secretion may give insights into therapies directed towards upregulating endogenous GLP-1 secretion.
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.
Petersen, JE;Pedersen, MH;Dmytriyeva, O;Nellemose, E;Arora, T;Engelstoft, MS;Asher, WB;Javitch, JA;Schwartz, TW;Trauelsen, M;
PMID: 37348738 | DOI: 10.1016/j.molmet.2023.101757
Free fatty acid receptor 1 (FFAR1) is highly expressed in enteroendocrine cells of the small intestine and pancreatic beta cells, where FFAR1 agonists function as GLP-1 and insulin secretagogues, respectively. Most efficacious are so-called second-generation synthetic agonists such as AM5262, which, in contrast to endogenous long-chain fatty acids are able to signal through both IP3/Ca2+ and cAMP pathways. Whereas IP3 signaling is to be expected for the mainly Gq-coupled FFAR1, the mechanism behind FFAR1-induced cAMP accumulation remains unclear, although originally proposed to be Gs mediated.When stimulated with AM5262, we observe that FFAR1 can activate the majority of the Gα proteins, except - surprisingly - members of the Gs family. AM5262-induced FFAR1-mediated transcriptional activation through cAMP response element (CREB) was blocked by the specific Gq inhibitor, YM253890. Furthermore, in Gq-deficient cells no CREB signal was observed unless Gq or G11 was reintroduced by transfection. By qPCR we determined that adenylate cyclase 2 (Adcy2) was highly expressed and enriched relative to the nine other Adcys in pro-glucagon expressing enteroendocrine cells. Co-transfection with ADCY2 increased the FFAR1-induced cAMP response 4-5-fold in WT HEK293 cells, an effect fully inhibited by YM253890. Moreover, co-transfection with ADCY2 had no effect in Gq-deficient cells without reintroduction of either Gq or G11. Importantly, although both AM5262/FFAR1 and isoproterenol/β2 adrenergic receptor (β2AR) induced cAMP production was lost in Gs-deficient cells, only the β2AR response was rescued by Gs transfection, whereas co-transfection with ADCY2 was required to rescue the FFAR1 cAMP response. In situ hybridization demonstrated a high degree of co-expression of ADCY2 and FFAR1 in enteroendocrine cells throughout the intestine. Finally, in the enteroendocrine STC-1 and GLUTag cell lines AM5262-induced cAMP accumulation and GLP-1 secretion were both blocked by YM253890.Our results show that Gq signaling is responsible not only for the IP3/Ca2+ but also the cAMP response, which together are required for the highly efficacious hormone secretion induced by second-generation FFAR1 agonists - and that ADCY2 presumably mediates the Gq-driven cAMP response.
