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

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.
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fig6: 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.

Mentions: In order to confirm firstly the deletion of LepRb in target cells, genomic DNA was harvested from several different tissues and brain areas and PCR was performed using primers flanking the LoxP sites (Methods; Figure 6A). The presence of the recombined allele was clearly detected in islets, and to a lesser extent in the olfactory bulb, the nucleus tractus solitarius (NTS) and intestine. Correspondingly, in mice carrying the Rosa26.tdRFP reporter, red fluorescence was clearly apparent in the majority of glucagon-positive α cells (Figure 6B), but not in Rosa26.tdRFP mice lacking iGluCre (Figure 6C). In the presence of the Cre transgene, recombination occurred in the vast majority (∼80%) of glucagon-positive islet cells, but also in a minority (∼20%) of insulin-positive β cells (Figure 6B,D).


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)

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

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

fig6: 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.
Mentions: In order to confirm firstly the deletion of LepRb in target cells, genomic DNA was harvested from several different tissues and brain areas and PCR was performed using primers flanking the LoxP sites (Methods; Figure 6A). The presence of the recombined allele was clearly detected in islets, and to a lesser extent in the olfactory bulb, the nucleus tractus solitarius (NTS) and intestine. Correspondingly, in mice carrying the Rosa26.tdRFP reporter, red fluorescence was clearly apparent in the majority of glucagon-positive α cells (Figure 6B), but not in Rosa26.tdRFP mice lacking iGluCre (Figure 6C). In the presence of the Cre transgene, recombination occurred in the vast majority (∼80%) of glucagon-positive islet cells, but also in a minority (∼20%) of insulin-positive β cells (Figure 6B,D).

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