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. 2015 Jun 25;4(9):619-30.
doi: 10.1016/j.molmet.2015.06.007. eCollection 2015 Sep.

Limited impact on glucose homeostasis of leptin receptor deletion from insulin- or proglucagon-expressing cells

Helen Soedling  1 , David J Hodson  1 , Alice E Adrianssens  2 , Fiona M Gribble  2 , Frank Reimann  2 , Stefan Trapp  3 , Guy A Rutter  1
Affiliations

Limited impact on glucose homeostasis of leptin receptor deletion from insulin- or proglucagon-expressing cells

Helen Soedling et al. Mol Metab. .

Abstract

Aims/hypothesis: The adipose tissue-derived hormone leptin plays an important role in the maintenance of body weight and glucose homeostasis. Leptin mediates its effects by interaction with leptin receptors (LepRb), which are highly expressed in the hypothalamus and other brain centres, and at lower levels in the periphery. Previous studies have used relatively promiscuous or inefficient Cre deleter strains, respectively, to explore the roles of LepR in pancreatic β and α cells. Here, we use two newly-developed Cre lines to explore the role of leptin signalling in insulin and proglucagon-expressing cells.

Methods: Leptin receptor expression was measured in isolated mouse islets and highly-purified islet cells by RNASeq and quantitative RT-PCR. Mice lacking leptin signalling in pancreatic β, or in α and other proglucagon-expressing cells, were generated using Ins1Cre- or iGluCre-mediated recombination respectively of flox'd leptin receptor alleles. In vivo glucose homeostasis, changes in body weight, pancreatic histology and hormone secretion from isolated islets were assessed using standard techniques.

Results: Leptin receptor mRNA levels were at or below the level of detection in wild-type adult mouse isolated islets and purified cells, and leptin signalling to Stat3 phosphorylation was undetectable. Whereas male mice further deleted for leptin receptors in β cells exhibited no abnormalities in glucose tolerance up to 16 weeks of age, females transiently displayed improved glucose tolerance at 8 weeks (11.2 ± 3.2% decrease in area under curve; p < 0.05), and improved (39.0 ± 13.0%, P < 0.05) glucose-stimulated insulin secretion in vitro. No differences were seen between genotypes in body weight, fasting glucose or β/α cell ratio. Deletion of LepR from α-cells, a minority of β cells, and a subset of proglucagon-expressing cells in the brain, exerted no effects on body weight, glucose or insulin tolerance, nor on pancreatic hormone secretion assessed in vivo and in vitro.

Conclusions/interpretation: The use here of a highly selective Cre recombinase indicates that leptin signalling plays a relatively minor, age- and sex-dependent role in the control of β cell function in the mouse. No in vivo role for leptin receptors on α cells, nor in other proglucagon-expressing cells, was detected in this study.

Keywords: AUC, area under the curve; Diabetes; GLP-1; GTT, ITT, glucose and insulin tolerance test, respectively; Glucagon; IP, intraperitoneal; Insulin; KATP, ATP-sensitive K+ channel; L-cell; Leptin; NTS, nucleus tractus solitarius; [Ca2+]i, intracellular free Ca2+ concentration; α cell; β cell.

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Figures

Figure 1
Figure 1
Low levels of LepR mRNA and receptor signalling in purified islet cells and whole islets, respectively. Amplification plot of LepR (red) or Ppia (cyclophilin, green) using mRNA purified from mouse islet (A) β (B) α or (C) δ cells or (D) hypothalamus. (E) Immunofluorescence staining of pStat3 in whole islets after treatment with either leptin (10 nM), TNF-α and IL1-β, or control non-treated islets. Data are representative of two further experiments. Scale bar 52.5 μm. See the Materials and Methods section for further details.
Figure 2
Figure 2
Ins1Cre expression results in excision of the flox'd region of LepRb alleles selectively in islets. Genomic DNA was harvested from Ins1CreLepRKO and control LepRF/F animals and used as a template for PCR with the primers indicated (A). Predicted product sizes are 339 bp for the flox'd allele (primers b,c) and 207 bp for the excised allele (primers a,c) (B). Ins1CreLepRKO animals displayed a normal progression in bodyweight. Ins1CreLepRKO males (green line) and LepRF/F (black line) and below Ins1CreLepRKO females (red line) and LepRF/F (black line). (C). Blood glucose concentration after overnight fast in males (D) (Ins1CreLepRKO, n = 8, LepRF/F, n = 7) and females at 8 weeks (E) (Ins1CreLepRKO, n = 13, LepRF/F, n = 9). Plasma insulin levels in male mice (F) Ins1CreLepRKO, n = 10, LepRF/F n = 7, and in females (G) Ins1CreLepRKO, n = 9, LepRF/F, n = 7. β to α cell ratio in Ins1CreLepRKO and wild-type mice (H).
Figure 3
Figure 3
Ins1CreLepRKOfemales display improved glucose tolerance at the age of 8 weeks. (A) Blood glucose concentration following IPGTT in Ins1CreLepRKO (square symbol) and LepRF/F littermate controls (triangles) in 8-week old females: Ins1CreLepRKO, n = 13, LepRF/F, n = 9, (B) 16-week old females: Ins1CreLepRKO, n = 15, LepRF/F, n = 17, (C) 8-weeks old males: Ins1CreLepRKO, n = 8, LepRF/F, n = 7, (D) 16-week old males: Ins1CreLepRKO, n = 13, LepRF/F, n = 10. For all panels, the corresponding body weight and area under curve (AUC) are inset. Data are expressed as the average ± SEM. Statistical analysis was performed using two-way ANOVA *P < 0.05.
Figure 4
Figure 4
Enhanced glucose-stimulated insulin secretion in Ins1CreLepRKOfemales. (A) Blood glucose after IP injection of insulin in Ins1CreLepRKO (square symbol) and LepRF/F littermate controls (triangles) in males and females (n = 9–10 mice per genotype). (B) Plasma insulin levels after IP injection of glucose in males and females: Ins1CreLepRKO, n = 10, LepRF/F, n = 6. (C) Insulin secretion as assessed from isolated islet in response to glucose (LG = 3 mM, HG = 17 mM) and KCl (30 mM) in 30-week old female and male mice. n = 3 separate experiments involving 2–3 mice per genotype/experiment. Statistical analysis was performed using two-way ANOVA *P < 0.05.
Figure 5
Figure 5
Ins1CreLepRKOislets display impaired Ca2+responses to high glucose. Ca2+ recordings from control LepRF/F islets (black line) and Ins1CreLepRKO islets (red line) in response to either 11 mM glucose (A). Inset are area under the curve (AUC) and amplitudes (ΔF (Fmax-Fmin)) of the Ca2+ rises. Data are from 14 to 16 islets (3–4 mice) per genotype. (B,C) Connectivity analysis showing the proportion (%) of significantly correlated cell pairs during a step change from 3 to 11 mM glucose (i.e. the activity onsets), or at steady state in the continued presence of 11 mM glucose. Pseudocolor plots in (C) show the strength of connections, determined using by Pearson Correlation (Pearson R). Data are expressed as mean ± SEM. Statistical analysis was performed using two-way ANOVA and Student's t-test. *P < 0.05.
Figure 6
Figure 6
iGluCre expression results in recombination of LepRb flox'd alleles in the olfactory bulb, nucleus tractus solitarius, intestine and pancreatic islets. Genomic DNA was harvested from the tissues indicated and used as a template for PCR with primer pair a and c (as indicated in Figure 1A). Predicted product sizes are 625 bp for the flox'd allele and 207 bp for the excised allele. (A) PCR transcript of the excised alleles in iGluCreLepRKO and LepRF/F controls. Immunohistochemical analysis of pancreas from iGluCre:tdRFPStopFlox mice, bearing wild type LepR alleles (B) stained for RFP (red), glucagon (green) and DAPI (blue), and in control animals without Cre (tdRFPStopFlox) (C). Quantification of the percentage of RFP expressing α and β cells (D). n = 39 islets from three animals. Data are expressed as mean ± SEM.
Figure 7
Figure 7
iGluCreLepRKOmice display normal glucose tolerance and fasting blood glucose levels. (A) Body weight followed for 30 weeks in male (green) and female (red) iGluCreLepRKO and LepRF/F mice (n = 12–19 mice per genotype). (B) Fasting blood glucose levels in 8 week old males and females (n = 6–11 mice per genotype). (C,D) Blood glucose concentration after IPGTT in male and female iGluCreLepRKO (square symbol) and LepRF/F (green triangle) mice. Blood glucose concentration after ITT in males: (E) iGluCreLepRKO, n = 8, LepRF/F, n = 7; and females: (F) iGluCreLepRKO, n = 8, LepRF/F, n = 10. Plasma glucagon levels during ITT, iGluCreLepRKO, n = 6, LepRF/F, n = 4, (G), and glucagon release from isolated islets in the presence of the indicated glucose concentrations: LG = 0.5 mM, HG = 10 mM glucose; n = 6 animals per genotype (H). When present, leptin was added to 10 nM. Data are expressed as the mean ± SEM and statistical comparison was through two-way ANOVA, *p < 0.05. Other details are provided in the Materials and Methods section.
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