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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

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).
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fig2: 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).

Mentions: To ablate residual LepRb function selectively from pancreatic β cells, animals bearing alleles in which LoxP sites flank exon 17 of the leptin receptor gene [41] were crossed with animals harbouring the Ins1Cre transgene. Exon 17 includes the BOX1 domain required for leptin-induced JAK-STAT signalling [41]. Based on the use of reporter strains [27], this approach is expected to lead to recombination in the majority (>94%) of β cells, with <2% recombination in other islet cell types [27], in the resulting Ins1CreLepRKO mice. To confirm Cre-mediated excision of exon 17 (LepRbΔ17), genomic DNA was isolated from several tissues and PCR performed with two sets of primers as indicated in Figure 2A [41]. PCR amplification of DNA from islets derived from LepRbF/F or Ins1CreLepRKO animals generated a product of 339 bp and a band of 208 bp where Cre-mediated excision of exon 17 had occurred (Figure 2B). Whilst attempts were made to confirm deletion by measuring full-length and truncated LepRb mRNA, levels these were below the level of reliable quantitation both in LepRbF/F and Ins1CreLepRKO mice, as discussed above, and in line with the absence of a functional effect of leptin on islet pStat3 phosphorylation (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)

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).
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Related In: Results  -  Collection

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fig2: 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).
Mentions: To ablate residual LepRb function selectively from pancreatic β cells, animals bearing alleles in which LoxP sites flank exon 17 of the leptin receptor gene [41] were crossed with animals harbouring the Ins1Cre transgene. Exon 17 includes the BOX1 domain required for leptin-induced JAK-STAT signalling [41]. Based on the use of reporter strains [27], this approach is expected to lead to recombination in the majority (>94%) of β cells, with <2% recombination in other islet cell types [27], in the resulting Ins1CreLepRKO mice. To confirm Cre-mediated excision of exon 17 (LepRbΔ17), genomic DNA was isolated from several tissues and PCR performed with two sets of primers as indicated in Figure 2A [41]. PCR amplification of DNA from islets derived from LepRbF/F or Ins1CreLepRKO animals generated a product of 339 bp and a band of 208 bp where Cre-mediated excision of exon 17 had occurred (Figure 2B). Whilst attempts were made to confirm deletion by measuring full-length and truncated LepRb mRNA, levels these were below the level of reliable quantitation both in LepRbF/F and Ins1CreLepRKO mice, as discussed above, and in line with the absence of a functional effect of leptin on islet pStat3 phosphorylation (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