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

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.
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fig1: 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.

Mentions: We first assessed expression of leptin receptors in isolated islets and highly purified preparations of islet cells from wild-type mice (Materials and Methods) using RNASeq and quantitative RT-PCR. LepR mRNA was detected at 0.2 RPKM in whole islets [27], at the lower 39th centile of all messages, and 0.23, 0.036 and 0 RPKM in purified β, δ and α and cells, respectively (n = 3, 2 and 4 separate preparations, respectively). Correspondingly, whereas qRT-PCR amplified LepR mRNA from whole islets (not shown), this approach failed to amplify a product from any of the three isolated cell preparations (Figure 1A–C). By contrast, the primers used efficiently amplified LepR mRNA from hypothalamus-derived polyA + mRNA (Figure 1D). Consistent with low levels of LepR protein in islets, staining of islets with fluorescently-labelled Cy3-leptin [12] failed to reveal consistent signals above background (results not shown). To further test the hypothesis that LepR signalling is low or undetectable in pancreatic islet cells, we exposed isolated islets from wild type C57BL6 mice to either leptin, or to a cytokine mixture (IL1-β and TNF-α) as a positive control, and monitored Stat3 phosphorylation on Tyr 705 using immunocytochemistry and laser-scanning confocal imaging. Whereas the cytokine mixture caused a robust increase in Stat3 phosphorylation as anticipated, no effects were observed upon addition of leptin, even at the highest dose (10 nM) tested (Figure 1E).


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)

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

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

fig1: 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.
Mentions: We first assessed expression of leptin receptors in isolated islets and highly purified preparations of islet cells from wild-type mice (Materials and Methods) using RNASeq and quantitative RT-PCR. LepR mRNA was detected at 0.2 RPKM in whole islets [27], at the lower 39th centile of all messages, and 0.23, 0.036 and 0 RPKM in purified β, δ and α and cells, respectively (n = 3, 2 and 4 separate preparations, respectively). Correspondingly, whereas qRT-PCR amplified LepR mRNA from whole islets (not shown), this approach failed to amplify a product from any of the three isolated cell preparations (Figure 1A–C). By contrast, the primers used efficiently amplified LepR mRNA from hypothalamus-derived polyA + mRNA (Figure 1D). Consistent with low levels of LepR protein in islets, staining of islets with fluorescently-labelled Cy3-leptin [12] failed to reveal consistent signals above background (results not shown). To further test the hypothesis that LepR signalling is low or undetectable in pancreatic islet cells, we exposed isolated islets from wild type C57BL6 mice to either leptin, or to a cytokine mixture (IL1-β and TNF-α) as a positive control, and monitored Stat3 phosphorylation on Tyr 705 using immunocytochemistry and laser-scanning confocal imaging. Whereas the cytokine mixture caused a robust increase in Stat3 phosphorylation as anticipated, no effects were observed upon addition of leptin, even at the highest dose (10 nM) tested (Figure 1E).

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