Urocortin3 mediates somatostatin-dependent negative feedback control of insulin secretion

Journal name:
Nature Medicine
Volume:
21,
Pages:
769–776
Year published:
DOI:
doi:10.1038/nm.3872
Received
Accepted
Published online

Abstract

The peptide hormone urocortin3 (Ucn3) is abundantly expressed by mature beta cells, yet its physiological role is unknown. Here we demonstrate that Ucn3 is stored and co-released with insulin and potentiates glucose-stimulated somatostatin secretion via cognate receptors on delta cells. Further, we found that islets lacking endogenous Ucn3 have fewer delta cells, reduced somatostatin content, impaired somatostatin secretion, and exaggerated insulin release, and that these defects are rectified by treatment with synthetic Ucn3 in vitro. Our observations indicate that the paracrine actions of Ucn3 activate a negative feedback loop that promotes somatostatin release to ensure the timely reduction of insulin secretion upon normalization of plasma glucose. Moreover, Ucn3 is markedly depleted from beta cells in mouse and macaque models of diabetes and in human diabetic islets. This suggests that Ucn3 is a key contributor to stable glycemic control, whose reduction during diabetes aggravates glycemic volatility and contributes to the pathophysiology of this disease.

At a glance

Figures

  1. Ucn3 is a paracrine factor expressed by mouse beta cells.
    Figure 1: Ucn3 is a paracrine factor expressed by mouse beta cells.

    (a) Quantification of Ucn3 expression relative to that of all genes that encode secreted factors in wild-type mouse islets. (b) Comparison of islet Ucn3 peptide content to that of other islet hormones. (c) Comparison of Ucn3 and insulin secretion from mouse islets in response to glucose (1,000 islets per well; Ucn3 molecular weight, 4,174 Da; insulin molecular weight, 5,785 Da). (d) Ucn3 and insulin colocalize in secretory granules of mouse beta cells by super-resolution structured illumination microscopy (SR-SIM) with thresholded Manders' coefficients for insulin and Ucn3 of 62.7% and 59.6%, respectively. (e,f) Images of an Ins1-H2b-mCherry reporter line crossed to either an Sst-Cre (e) or a Gcg-Cre (f) allele and a floxed YFP reporter. (g,h) FACS purification of mCherry+ beta cells and delta (g) or alpha (h) cells. (i) Gene expression by qPCR of Crhr2 and a panel of established alpha, beta and delta cell markers. AU, arbitrary units. Scale bars, 10 μm (d) and 50 μm (e,f). Error bars show mean ± s.e.m.

  2. Ucn3-null islets are deficient in delta cell number and somatostatin secretion.
    Figure 2: Ucn3-null islets are deficient in delta cell number and somatostatin secretion.

    (ad) Transcriptome analysis of Ucn3-null islets compared to wild-type littermates for Ucn3 (a) Sst (b), Hhex (c) and Rbp4 (d) (n = 3); data normalized to 1 × 107 reads. (e) Somatostatin, insulin and glucagon content of Ucn3-null and control islets (n = 4). (f) Relative delta, beta and alpha cell numbers of Ucn3-null and control islets (n = 3 for control, 4 for Ucn3-null, 5−7 islets per animal). (g) Somatostatin secretion from Ucn3-null islets compared to control islets (n = 4, 95 islets per well). (h,i) Insulin secretion from Ucn3-null islets compared to control islets in static incubation (h) (n = 4, 7 islets per well) and in islet perfusion (i) (n = 2, 150 islets per chamber, representative of two experiments). Significance determined by Student's t-test (e,f), two-way analysis of variance (ANOVA) for treatment and genotype followed by Holm–Sidak's multiple comparison test (g,h), or two-way ANOVA for genotype and its interaction with time for each block (i). Error bars show mean ± s.e.m.; *P < 0.05, **P < 0.01, ***P < 0.001.

  3. Endogenous Ucn3 promotes somatostatin-mediated negative feedback.
    Figure 3: Endogenous Ucn3 promotes somatostatin-mediated negative feedback.

    (a) Somatostatin secretion from wild-type mouse islets in response to Ucn3 or its antagonist Ast2B (n values in each bar, 100 islets per well). (b) Interactions between Ucn3 and diazoxide or isradipine on somatostatin (top, n values in each bar, 50 or 100 islets per well) or insulin (bottom, n = 7, 12 islets per well) secretion. (c) Ucn3 amplifies somatostatin secretion induced by tolbutamide (n values in each bar, 80 or 50 islets per well). (d,e) Inhibition of endogenous Ucn3 by Ast2B acutely de-represses insulin secretion (d) via reduced somatostatin release (e) (n = 3, 150 (d) or 270 (e) islets per chamber). (f) Ast2B enhances exendin-4 (Ex4)–induced insulin secretion (n = 6, 12 islets per well). (g,h) Ucn3 impairs glucose tolerance (g) and suppresses glucose-stimulated plasma insulin (h) in vivo, whereas Ast2B has no effect (n = 7 for saline and Ucn3 groups, 6 for Ast2B). (i) Ucn3-mediated glucose intolerance is prevented by somatostatin antagonists (n = 5). (j) Effects of UCN3 and Ast2B on insulin secretion from human islets (n values in each bar, 50 islets/well; normalized secretion across two (left) or three (right) individual donors. Significance determined by one-way ANOVA followed by Student's t-test with Welch's correction for unequal variance as necessary (ac,f,h,j), two-way ANOVA for the interaction of treatment and time for each block followed by the comparison of individual time points by Student's t-test (d,e) or two-way ANOVA for treatment and its interaction with time followed by Holm–Sidak's multiple comparison test (g,i). NS, not significant; *P < 0.05, **P < 0.01, ***P < 0.001. Error bars show mean ± s.e.m.

  4. Ucn3 marks mature beta cells and aggravates hyperglycemia.
    Figure 4: Ucn3 marks mature beta cells and aggravates hyperglycemia.

    (ac) Ucn3 immunoreactivity (a) and gene expression (b) (n = 9 controls, 8 ob/ob) in islets from ob/ob and db/db mice (c). (d) Somatostatin release from ob/ob islets compared to lean controls in response to Ast2B. (e) Loss of Ucn3 correlates with increased glycemic volatility and extended period length in ob/ob animals compared to in lean controls. n values reflect the number of data points, P values reflect differences in variance between animals, see Supplementary Table 2. (f) Doxycycline-inducible Ucn3-overexpressing mice (Supplementary Fig. 5) crossed on the ob/ob background facilitate the restoration of Ucn3 expression by beta cells. (g) Effect of Ucn3 induction on plasma glucose and body weight in doxycycline-inducible Ucn3-overexpressing mice. (h) Doxycycline administration to pregnant dams from E10.5 onward induces Ucn3 prematurely in bitransgenic but not control offspring at P2. (i,j) Effect of premature Ucn3 induction on plasma glucose (i) before and after endogenous Ucn3 is expressed in all beta cells at 3 weeks of age (j). Scale bars, 50 μm in all panels except the detail in f, lower right (10 μm). Significance determined by Student's t-test (b), one-way ANOVA followed by Student's t-test with Welch's correction as necessary (d), and linear regression between groups before and after induction with Student's t-test to compare the glucose values immediately before and after induction (g). Significance of Ucn3 induction in i determined by Student's t-test for P2 and P10 groups and by two-way ANOVA for treatment and time for older ages. Error bars show mean ± s.d. in e and mean ± s.e.m. in all other panels; *P < 0.05, **P < 0.01, ***P < 0.001.

  5. UCN3 is lost from the beta cells of individuals with type 2 diabetes and pre-diabetic macaques.
    Figure 5: UCN3 is lost from the beta cells of individuals with type 2 diabetes and pre-diabetic macaques.

    (a,b) UCN3 in the islets of a 20-year-old nondiabetic donor with a healthy BMI (a), and in a 37-year-old morbidly obese type 2 diabetic donor (b). We applied masks based on insulin (red) and glucagon (white) to isolate UCN3 staining in beta and alpha cells. Data for 16 additional human donors with and without diagnosed type 2 diabetes are presented in Supplementary Figure 7 and support these observations. (ce) UCN3 staining in macaques on a control diet (c) and on a high-fat diet and classified as either 'diet-resistant' (d) or 'diet-sensitive' (e). Similar data for 14 additional macaques across these cohorts are presented in Supplementary Figure 8 and support these observations. Scale bars, 50 μm.

  6. Ucn3 promotes somatostatin secretion from delta cells in an incretin-like fashion.
    Figure 6: Ucn3 promotes somatostatin secretion from delta cells in an incretin-like fashion.

    (a) Insulin and other factors secreted by beta cells are generally considered inhibitory to glucagon secretion, whereas alpha cell hormones, paradoxically, stimulate insulin release. Ucn3 from human and mouse beta and human alpha cells is a paracrine signal that stimulates somatostatin secretion via Crhr2α receptors expressed by delta cells. This drives negative feedback and attenuates insulin and glucagon secretion once glucose homeostasis is restored. (b) Dependence of Ucn3-stimulated somatostatin secretion on KATP and L-type voltage-gated calcium channels suggests that delta cell–autonomous stimulus secretion coupling is required to trigger somatostatin release and is potentiated by Ucn3 acting through the Crhr2α expressed by delta cells. (c) While the term 'incretin' is sensu strictu reserved for hormones of gastrointestinal origins that potentiate glucose-stimulated insulin secretion, the actions of Ucn3 on the delta cell mechanistically resemble the actions of incretins on the beta cell as both cells respond to a class B GPCR peptide ligand to potentiate exocytosis under elevated ambient glucose conditions. ΔΨ↓, membrane depolarization. cAMP, cyclic adenosine monophosphate; Gip, glucose-dependent insulinotropic peptide; Gipr, Gip receptor; Glp1, glucagon-like peptide 1; Glp1r, Glp1 receptor; Prkac, protein kinase cAMP-dependent catalytic; Rapgef4, Rap guanine nucleotide exchange factor 4; Slc2a2, solute carrier family 2 member 2.

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Author information

Affiliations

  1. Department of Neurobiology, Physiology and Behavior, College of Biological Sciences, University of California, Davis, California, USA.

    • Talitha van der Meulen,
    • Anna E Hunter,
    • Christopher Cowing-Zitron &
    • Mark O Huising
  2. Clayton Foundation Laboratories for Peptide Biology, Salk Institute for Biological Studies, La Jolla, California, USA.

    • Talitha van der Meulen,
    • Cynthia J Donaldson,
    • Elena Cáceres,
    • Anna E Hunter &
    • Mark O Huising
  3. Division of Diabetes, Obesity and Metabolism, Oregon National Primate Research Center, Oregon Health & Science University, Beaverton, Oregon, USA.

    • Lynley D Pound &
    • Kevin L Grove
  4. Waitt Advanced Biophotonics Center, Salk Institute for Biological Studies, La Jolla, California, USA.

    • Michael W Adams
  5. Molecular Neurobiology Laboratory, Salk Institute for Biological Studies, La Jolla, California, USA.

    • Andreas Zembrzycki
  6. Department of Physiology and Membrane Biology, School of Medicine, University of California, Davis, Davis, California, USA.

    • Mark O Huising

Contributions

T.v.d.M. and M.O.H. validated the mouse models generated over the course of this study and designed all mouse experiments. T.v.d.M., E.C., A.E.H., A.Z. and M.O.H. performed mouse experiments. T.v.d.M., C.J.D., A.E.H. and M.O.H. performed many islet isolations. C.J.D. performed all insulin and somatostatin hormone measurements and coordinated the receipt of human islets. E.C. and M.O.H. designed, performed and analyzed the continuous glucose monitoring experiment. C.C.-Z. and M.O.H. performed bioinformatics analyses. T.v.d.M. and M.O.H. conducted immunolabeling and acquired all images. M.W.A. performed super-resolution structured illumination microscopy and supported image acquisition and analysis. L.D.P. and K.L.G. designed and conducted the nonhuman primate studies and provided the histological material used in this study. M.O.H. conceived the study, was responsible for its overall design and planning and wrote the article together with T.v.d.M.

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