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

Soedling H, Hodson DJ, Adrianssens AE, Gribble FM, Reimann F, Trapp S, Rutter GA - Mol Metab (2015)

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Section of Cell Biology and Functional Genomics, Division of Diabetes, Endocrinology and Metabolism, Department of Medicine, Imperial College London, du Cane Road, London W12 0NN, UK.

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.

No MeSH data available.


Related in: MedlinePlus

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 [44] 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.
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fig5: 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 [44] 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.

Mentions: Deletion of LepRb from β-cells using a RIP2Cre has been reported to impair glucose-dependent Ca2+ signals [47]. However, this observation was ascribed partly to the effects of the RIP2Cre transgene itself [31], as well as to the loss of LepRb. Contrary to the latter findings, deletion of LepRb with Pdx1.Cre potentiated glucose-induced Ca2+ oscillations. To examine the effect of LepRb deletion more selectively in the β cell we therefore monitored changes in intracellular free Ca2+ ([Ca2+]i) in response to high glucose, or depolarisation with KCl, in islets from 8 weeks old female Ins1CreLepRKO or control mouse islets. Islets lacking LepRb in β cells displayed a tendency towards lower [Ca2+]i increases in response to high glucose (Figure 5A), though this difference was not significant when hundreds of single cells were individually assessed within each micro-organ to exclude outliers which confound signal analysis (i.e. glucose non-responsive cells, and those cells displaying large and sustained increases indicative of apoptosis). Likewise, no changes in response to stimulation with KCl (not shown), or in β cell-β cell connectivity (Figure 5B,C), [44], were observed in Ins1CreLepRKO mice versus control littermates suggesting that β cell population responses to glucose remain coordinated even in the absence of LepRb signalling.


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

Soedling H, Hodson DJ, Adrianssens AE, Gribble FM, Reimann F, Trapp S, Rutter GA - Mol Metab (2015)

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 [44] 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.
© Copyright Policy - CC BY
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4563029&req=5

fig5: 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 [44] 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.
Mentions: Deletion of LepRb from β-cells using a RIP2Cre has been reported to impair glucose-dependent Ca2+ signals [47]. However, this observation was ascribed partly to the effects of the RIP2Cre transgene itself [31], as well as to the loss of LepRb. Contrary to the latter findings, deletion of LepRb with Pdx1.Cre potentiated glucose-induced Ca2+ oscillations. To examine the effect of LepRb deletion more selectively in the β cell we therefore monitored changes in intracellular free Ca2+ ([Ca2+]i) in response to high glucose, or depolarisation with KCl, in islets from 8 weeks old female Ins1CreLepRKO or control mouse islets. Islets lacking LepRb in β cells displayed a tendency towards lower [Ca2+]i increases in response to high glucose (Figure 5A), though this difference was not significant when hundreds of single cells were individually assessed within each micro-organ to exclude outliers which confound signal analysis (i.e. glucose non-responsive cells, and those cells displaying large and sustained increases indicative of apoptosis). Likewise, no changes in response to stimulation with KCl (not shown), or in β cell-β cell connectivity (Figure 5B,C), [44], were observed in Ins1CreLepRKO mice versus control littermates suggesting that β cell population responses to glucose remain coordinated even in the absence of LepRb signalling.

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Section of Cell Biology and Functional Genomics, Division of Diabetes, Endocrinology and Metabolism, Department of Medicine, Imperial College London, du Cane Road, London W12 0NN, UK.

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.

No MeSH data available.


Related in: MedlinePlus