Glucagon regulates hepatic kisspeptin to impair insulin secretion - PubMed (original) (raw)

. 2014 Apr 1;19(4):667-81.

doi: 10.1016/j.cmet.2014.03.005.

Prosenjit Mondal 1, Andrew Wolfe 2, Laura C Alonso 3, Rachel Stamateris 3, Benny W T Ong 1, Owen C Lim 1, Kil S Yang 1, Sally Radovick 4, Horacio J Novaira 4, Emily A Farber 5, Charles R Farber 5, Stephen D Turner 6, Mehboob A Hussain 7

Affiliations

Glucagon regulates hepatic kisspeptin to impair insulin secretion

Woo-Jin Song et al. Cell Metab. 2014.

Abstract

Early in the pathogenesis of type 2 diabetes mellitus (T2DM), dysregulated glucagon secretion from pancreatic α cells occurs prior to impaired glucose-stimulated insulin secretion (GSIS) from β cells. However, whether hyperglucagonemia is causally linked to β cell dysfunction remains unclear. Here we show that glucagon stimulates via cAMP-PKA-CREB signaling hepatic production of the neuropeptide kisspeptin1, which acts on β cells to suppress GSIS. Synthetic kisspeptin suppresses GSIS in vivo in mice and from isolated islets in a kisspeptin1 receptor-dependent manner. Kisspeptin1 is increased in livers and in serum from humans with T2DM and from mouse models of diabetes mellitus. Importantly, liver Kiss1 knockdown in hyperglucagonemic, glucose-intolerant, high-fat-diet fed, and Lepr(db/db) mice augments GSIS and improves glucose tolerance. These observations indicate a hormonal circuit between the liver and the endocrine pancreas in glycemia regulation and suggest in T2DM a sequential link between hyperglucagonemia via hepatic kisspeptin1 to impaired insulin secretion.

Copyright © 2014 Elsevier Inc. All rights reserved.

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Figures

Figure 1

Figure 1. Comparison between L-Δprkar1a and D-glucose mice identifies Kiss1

A (left) Representative liver IB of prkar1afl/fl and L-Δprkar1a 4 days after adenovirus treatment. L-Δprkar1a mice show Prkar1a ablation and increased pCREB (right) Liver IB from Sal- and D-glucose mice shows unaltered Prkar subtypes, Pkac, pCREB. B Fasting glucose levels in prkar1afl/fl, L-Δprkar1a, Sal- and D-glucose mice. Prkar1afl/fl and Sal- mice have similar fasting glucose; D-glucose infusion achieves fasting glucose similar to L-Δprkar1a mice (mean±SEM, * P<0.05). C (top) plasma glucose and (bottom) serum insulin during ipGTT in prkar1afl/fl, L-Δprkar1a, Sal- and D-glucose mice. L-Δprkar1a mice exhibit impaired GT (top) and GSIS (bottom). D-glucose mice have mildly impaired GT and robust GSIS. Prkar1a fl/fl and Sal-mice have similar GT and GSIS (mean±SEM, * P<0.05). D qRT-PCR of indicated genes of gluconeogenic program in prkar1afl/fl, L-Δprkar1a, Sal- and D-glucose-mouse livers. (top) gluconeogenic program is upregulated in L-Δprkar1a as compared to prkar1afl/fl mice; (bottom) gluconeogenic program is downregulated in D-glucose as compared to saline-mice (mean±SEM, * P<0.05).. E GSIS of WT mouse islets cultured in serum free media conditioned with plasma of prkar1afl/fl or L-Δprkar1a mice. Prkar1afl/fl plasma does not affect GSIS. L-Δprkar1a plasma at 1:10 but not at 1:100 dilution suppresses GSIS (mean±SEM, * P<0.05). F Volcano plot of gene expression analysis of liver from prkar1afl/fl and L-Δprkar1a mice. Significant upregulation of Kiss1 transcript is detected in L-Δprkar1a mice. G (top) qRT-PCR of Kiss1 transcript and (bottom) IB in liver tissue from mice with indicated liver genetic complement or intravenous infusion. L-Δprkar1a liver shows increased Kiss1 transcript and kisspeptin protein. D-glucose mice show Kiss1 downregulation as compared to controls (mean±SEM, * P<0.05).

Figure 2

Figure 2. Glucagon and insulin counter-regulate liver Kiss1 expression

A qRT-PCR of Kiss1 and Pck1 in isolated mouse hepatocytes exposed to indicated treatment. Glucagon stimulates, insulin suppresses both genes (mean±SEM, *P<0.05). B qRT-PCR of Kiss1 in isolated mouse hepatocytes exposed to vehicle (DMSO) or fsk/IBMX. cAMP stimulation stimulates Kiss1 expression (mean±SEM, *P<0.05).. C Representative IB of cultured mouse hepatocytes after treatment with PBS, glucagon, insulin or INS plus GCGN. Glucagon stimulates kisspeptin, insulin treatment has little effect on already low kisspeptin. Insulin counterregulates glucagon stimulation of kisspeptin. D qRT-PCR of Kiss1 in liver tissue of WT mice after in vivo ip treatment with vehicle (PBS) or glucagon. Glucagon treatment stimulates Kiss1 in liver (mean±SEM, *P<0.05). E Representative liver IB in WT mice after in vivo ip treatment with PBS or glucagon. Glucagon increases liver kisspeptin1 (mean±SEM, *P<0.05). F qRT-PCR of Kiss1 in liver of ad lib fed, O/N fasted, and refed WT mice. Fasting stimulates liver Kiss1 expression, refeeding supresses elevated Kiss1 (mean±SEM, *P<0.05). G Liver IB from ad lib fed, O/N fasted, and refed WT mice. Fasting stimulates liver kisspeptin1. H qRT-PCR of Kiss1 in liver of L-ΔGcgr mice after ip PBS or glucagon. Glucagon does to stimulate Kiss1 in L-ΔGcgr mice (mean±SEM, * P<0.05). I (top) Representative liver IB from Gcgrfl/fl and L-ΔGcgr mice. L-ΔGcgr lack GCGR. (bottom) Liver IB from Gcgrfl/fl and L-ΔGcgr mice after ip treatment with PBS or glucagon. Baseline kisspeptin1 is similar in Gcgrfl/fl and L-ΔGcgr mouse liver. Glucagon treatment stimulates kisspeptin1 in Gcgrfl/fl but not in L-ΔGcgr mice. J qRT-PCR of Kiss1 in liver of ad lib fed and O/N fasted L-ΔGcgr mice. O/N fast does not stimulate Kiss1 in L-ΔGcgr mouse liver (mean±SEM, *P<0.05). K Representative liver IB from ad lib fed and O/N fasted Gcgrfl/fl and L-ΔGcgr mice. Baseline kisspeptin1 is similar in Gcgrfl/fl and L-ΔGcgr livers. Fasting stimulates kisspeptin1 in Gcgr fl/fl but not in L-ΔGcgr liver. L qRT-PCR of Kiss1 in liver of Insrfl/fl and L-ΔInsr mice. Liver Insr ablation does not affect Kiss1 expression (mean±SEM, *P<0.05). M Representative liver IB of Insrfl/fl and L-ΔInsr mice. L-ΔInsr liver lacks insulin receptor immunoreactivity. N qRT-PCR of Kiss1 in liver of Insrfl/fl and L-ΔInsr mice after ip treatment with vehicle PBS or glucagon. Glucagon stimulates Kiss1 in Insrfl/fl mice and more so in L-ΔInsr liver (mean±SEM, *P<0.05). O Representative liver IB of Insrfl/fl and L-ΔInsr mice after ip PBS or glucagon. Glucagon stimulates stronger liver kisspeptin1 production in L-ΔInsr than in Insrfl/fl liver.

Figure 3

Figure 3. In L-Δprkar1a mice liver Kiss1 knockdown derepresses GSIS and ameliorates glucose tolerance despite continued upregulated gluconeogenesis

A (top) Liver kisspeptin1 IB, (bottom left) liver qRT-PCR of Kiss1 mRNA, and (bottom right) plasma kisspeptin1 in L-Δprkar1a mice 3 days after Adv-scr or -Kiss1 shRNA treatment. Liver kisspeptin1 protein, Kiss1 mRNA and plasma kisspeptin1 are reduced after Kiss1 Kiss1 L-Δprkar1a mice (mean±SEM, *P<0.05). B (top) pCREB and total CREB IB, (bottom) qRT-PCR of Ppargc1a and Pck1 in liver of L-Δprkar1a mice 3 days after Adv-scr shRNA or Adv-Kiss1 shRNA treatment. CREB phosphorylation and total CREB protein are unaffected by Kiss1 knockdown in L-Δprkar1a liver. Ppargc1a and Pck1 mRNA levels are upregulated in L-Δprkar1a as compared to prkar1afl/fl livers and are unaffected by liver Kiss1 knockdown in L-Δprkar1a mice (mean±SEM, *P<0.05). C In vivo ChIP of CREB occupancy on CRE1 and CRE2 within the Kiss1 promoter in liver samples. CREB occupancy on Kiss1 CRE1 and 2 in L-Δprkar1a liver is increased as compared to prkar1afl/fl liver and unaffected by Kiss1 knockdown in L-Δprkar1a mice (mean±SEM, *P<0.05). D ipPCT in fed prkar1afl/fl and L-Δprkar1a mice 3 days after Adv-scr or -Kiss1 shRNA treatment. Gluconeogensis activity is increased in L-Δprkar1a as compared to prkar1afl/fl mice. Gluconeogenesis activity in L-Δprkar1a mice is unaffected by Kiss1 knockdown (mean±SEM, *P<0.05). E ipITT in L-Δprkar1a mice 3 days after treatment with Adv-scr or -Kiss1 shRNA. Peripheral insulin sensitivity in L-Δprkar1a mice is unaffected by Kiss1 knockdown (mean±SEM, *P<0.05). F ipGTT in L-Δprkar1a mice 3 days after treatment with Adv-scr or -Kiss1 shRNA. L-Δprkar1a mice with Kiss1 knockdown show improved GT as compared to controls (mean±SEM, *P<0.05). G Serum insulin during ipGTT in L-Δprkar1a mice 3 days after treatment with Adv-scr or -Kiss1 shRNA. GSIS is augmented in L-Δprkar1a mice after liver Kiss1 knockdown as compared to controls (mean±SEM, *P<0.05). H Body weight in L-Δprkar1a mice 3 days after treatment with Adv-scr or -Kiss1 shRNA. Body weight in L-Δprkar1a mice is unaffected by Kiss1 knockdown. I Caloric intake in L-Δprkar1a mice during 3 days after treatment with Adv-scr or -Kiss1 shRNA. Caloric intake in L-Δprkar1a mice is unaffected by Kiss1 knockdown.

Figure 4

Figure 4. Kisspeptin1 at nanomolar concentrations inhibits GSIS in a Kiss1R-dependent manner. Pancreas Kiss1R is located in β-cells

A Representative IB for Kiss1R mouse brain, HEK 293T cells, mouse islets, INS1 rat insulinoma cells, Min6 mouse insulinoma cells and human islets. Mouse brain, mouse islets, insulinoma cells and human islets express Kiss1R. HEK 293T cells do not expess Kiss1R. B (left) Immunohistochemistry for insulin, glucagon and Kiss1R in pancreas from Kiss1Rfl/fl mice. Kiss1R immunoreactivity colocalizes with insulin-positive β-cells but not with glucagon-positive α-cells. 20× magnification. Pseudocoloring: red: glucagon, green: insulin, yellow: Kiss1R, blue: nucleus counter-stain with DAPI (left bottom) inset of previous image at 40× magnification. (right top) Immunohistochemistry for insulin, glucagon and Kiss1R in pancreas from Panc-ΔKiss1R mice. Kiss1R immunoreactivity is lacking in Panc-ΔKiss1R islet. (right bottom) Representative islet IB from Kiss1Rfl/fl and Panc-ΔKiss1R mice. Kiss1R is absent in Panc-ΔKiss1R islets. C ipGTT in Kiss1Rfl/fl and Panc-ΔKiss1R mice during ip co-injection of PBS and glucose. (top) GT is similar in Kiss1Rfl/fl and Panc-ΔKiss1R mice. (bottom) baseline fasting glucose is slightly elevated in Panc-ΔKiss1R mice as compared to Kiss1Rfl/fl littermates. In vivo GSIS is similar in Kiss1Rfl/fl and Panc-ΔKiss1R mice (mean±SEM, *P<0.05). D ipGTT in Kiss1Rfl/fl and Panc-ΔKiss1R mice during ip co-injection of 10 nM K54 and glucose. K54 impairs GT (top) and GSIS (bottom) in Kiss1Rfl/fl but not in Panc-ΔKiss1R mice (mean±SEM, *P<0.05). E (top) Dose response curve of K54 and K10 inhibition of GSIS from WT mouse islets during static at 10 or 20 mM glucose. Both K54 and K10 inhibit GSIS in a dose-dependent manner from 0 to 100 nM at both 10 and 20 mM glucose; (bottom) GSIS from Kiss1R fl/fl and Panc-ΔKiss1R islets treated with PBS K10 or K54. K10 or K54 (both 10 nM) inhibit GSIS from Kiss1R fl/fl but not from Panc-ΔKiss1R islets. F cAMP synthesis and GSIS in response to K54 and to incretin analogue exendin-4 (E4) in Kiss1R fl/fl and Panc-ΔKiss1R islets. (top) K54 impairs cAMP synthesis in Kiss1Rfl/fl but not in Panc-ΔKiss1R islets. E4 stimulates cAMP synthesis similarly in both Kiss1Rfl/fl and in Panc-ΔKiss1R islets. K10 reduces E4-stimulated cAMP levels in Kiss1Rfl/fl but not in Panc-ΔKiss1R islets. (bottom) During static incubation of mouse islets, K54 impairs GSIS and also E4 potentiation of GSIS from islets in a dose dependent manner (mean±SEM, *P<0.05). G Islet perifusion assay in WT islets in response to K54 and to E4. (top) K54 impairs both first and second phasea of GSIS and (bottom) E4-potentiated first and second phase GSIS; End of perifusion shows similar insulin release upon KCL induced depolarization (mean±SEM, *P<0.05). H Area under the curve (AUC) of (top) first and (bottom) second phase GSIS from WT mouse islets treated with PBS, K54, E4 or K54+E4. K54 inhibits both first and second phases of GSIS and E4 potentiated GSIS (mean±SEM, *P<0.05).

Figure 5

Figure 5. Liver Kisspeptin1 expression and plasma kisspeptin levels are elevated in mouse models of DM and in humans with T2DM

A (top) qRT-PCR for Kiss1 in liver tissue; and (bottom) liver IB for kisspeptin1 in SD, HFD fed and Leprdb/db mice. Both Kiss1 mRNA and kisspepetin1 protein are increased in HFD fed mice and found at higher levels in db/db mouse livers (mean±SEM, *P<0.05). B Plasma kisspeptin1 in SD, HFD fed and Leprdb/db mice. Plasma kisspeptin1 is increased in (left) HFD fed and (right) in Leprdb/db mice as compared to SD fed littermates (mean±SEM, *P<0.05). C GSIS from cultured (left) Kiss1Rfl/fl and (right) Panc-ΔKiss1R mouse islets in media conditioned with plasma from HFD fed and Leprdb/db mice. GSIS from Kiss1Rfl/fl is suppressed during culture in media conditioned with HFD fed or db/db plasma at 1:10 dilution but not at 1:100 dilution. GSIS from Panc-ΔKiss1R islets is unaffected by media conditioned with plasma of HFD fed or Leprdb/db mice (mean±SEM, *P<0.05). D Representative liver IB for kisppeptin1 in humans without DM and humans with T2DM. Humans with T2DM exhibit varying degrees of kisspeptin immunoreactivity in liver tissue. E Plasma kisspeptin1 in humans without DM and with T2DM. Plasma kisspeptin1 levels are elevated in humans with T2DM as compared to humans without diabetes (mean±SEM, *P<0.05). F GSIS from cultured (left) Kiss1Rfl/fl and (right) Panc-ΔKiss1R mouse islets in media conditioned with plasma from humans with T2DM and without DM. GSIS from Kiss1Rfl/fl but not from Panc-ΔKiss1R islets is suppressed during culture in media conditioned with T2DM plasma (mean±SEM, *P<0.05).

Figure 6

Figure 6. Hyperlucagonemia is linked to liver kisspeptin1 production in HFD fed and Leprdb/db mice

HFD fed mice: Panels A–F Leprdb/db mice: Panels G–M A, G Plasma glucagon levels in the fed state 60 min after treatment with GAI or GAC. Plasma glucagon levels remain unchanged after GAI or GAC treament. B, H Representative liver IB for pCREB, total CREB and kisspeptin1 in GAI and GAC treated mice. Phospho-CREB is reduced in mice treated with GAI but not GAC. C, I qRT-PCR of indicated genes of the gluconeogenic program in livers of GAI or GAC treated mice. GAI but not GAC treatment downregulates Pparg1a, Src1, G6P and Pck1 mRNA (mean±SEM, *P<0.05). D, J qRT-PCR of Kiss1 in livers of GAI and GAC treated mice. GAI but not GAC treatment downregulates liver Kiss1 mRNA (mean±SEM, *P<0.05). E, K In vivo ChIP of CREB occupancy on (left) CRE1 and (right) CRE2 half-sites of the Kiss1 promoter in livers of GAI or GAC treated SD mice, HFD and Leprdb/db mice. GAI reduces CREB occupancy on Kiss1 CRE 1 & 2 to levels similar to those in control mice (mean±SEM, *P<0.05). F, L ipGTT in GAI or GAC treated mice. GAI treatment improves GT as compared to GAC treatment (mean±SEM, *P<0.05).

Figure 7

Figure 7. Kiss1 shRNA knockdown in vivo in livers of in HFD and Leprdb/db mice de-represses GSIS and glucose tolerance

HFD mice: Panels A–H Leprdb/db mice: Panels I–P A, I Representative liver IB of pCREB, total CREB and kisspeptin1 3 days after treatment with Adv-scr or -Kiss1 shRNA. Liver pCREB is not affected and liver kisspeptin1 protein is reduced by Kiss1 knockdown. B, J qRT-PCR of Kiss1 in livers 3 days after treatment with Adv-scr or -Kiss1 shRNA. Adv-Kiss1 shRNA downregulates liver Kiss1 mRNA levels as compared to Adv-scr shRNA treatment (mean±SEM, *P<0.05). C, K Plasma kisspeptin1 levels 3 days after treatment with Adv-scr or -Kiss1 shRNA. Liver Kiss1 knockdown reduces plasma kisspeptin1 (mean±SEM, *P<0.05). D, L Plasma glucagon levels 3 days after treatment with Adv-scr or -Kiss1 shRNA. Liver Kiss1 knockdown does not change plasma glucagon levels (mean±SEM0. E, M Caloric intake in during 3 days after treatment with Adv-scr or -Kiss1 shRNA. Caloric intake is unaffected by Kiss1 knockdown (mean±SEM). F, N ipGTT 3 days after treatment with Adv-scr or -Kiss1 shRNA. GT is improved after Kiss1 knockdown (mean±SEM, *P<0.05). G, O GSIS during ipGTT 3 days after treatment with Adv-scr or -Kiss1 shRNA. GSIS is improved after Kiss1 knockdown (mean±SEM, *P<0.05). H, P ipITT 3 days after treatment with Adv-scr or -Kiss1 shRNA. Insulin tolerance is not different after Kiss1 knockdown.

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