The leptin receptor has no role in delta-cell control of beta-cell function in the mouse

Abstract

Introduction: Leptin inhibits insulin secretion from isolated islets from multiple species, but the cell type that mediates this process remains elusive. Several mouse models have been used to explore this question. Ablation of the leptin receptor (Lepr) throughout the pancreatic epithelium results in altered glucose homeostasis and ex vivo insulin secretion and Ca2+ dynamics. However, Lepr removal from neither alpha nor beta cells mimics this result. Moreover, scRNAseq data has revealed an enrichment of LEPR in human islet delta cells.

Methods: We confirmed LEPR upregulation in human delta cells by performing RNAseq on fixed, sorted beta and delta cells. We then used a mouse model to test whether delta cells mediate the diminished glucose-stimulated insulin secretion in response to leptin.

Results: Ablation of Lepr within mouse delta cells did not change glucose homeostasis or insulin secretion, whether mice were fed a chow or high-fat diet. We further show, using a publicly available scRNAseq dataset, that islet cells expressing Lepr lie within endothelial cell clusters.

Conclusions: In mice, leptin does not influence beta-cell function through delta cells.

Keywords: beta cells; beta-cell activity; delta cells; differential expression (DE); leptin receptor (LEPR); mouse models.

Figure 1

Testing whether leptin mediates inhibition of insulin secretion through the delta cell. (A) RNAseq of sorted mouse alpha, beta, and delta cells has revealed the expression of all the components involved in canonical and amplifying pathways of insulin secretion, except insulin itself, in the delta cell. These components include a glucose transporter, voltage-gated potassium channels, glucokinase, and adenylate cyclase. Delta cells are also regulated by paracrine interactions by other endocrine cells, including by Urocortin 3 and by GLP1 (–34). Although cytosolic Ca²⁺ may be secondary to cAMP in regulating delta-cell function, Ca²⁺ concentration correlates with somatostatin secretion. In human islets, the leptin receptor is enriched within the delta cell population. We, therefore, hypothesized that leptin receptor signaling enhances SST release by activating a PKC isoform that enhances Ca_Δv activity and increases intracellular Ca²⁺ (pathway denoted by black arrows). Proposed leptin receptor signaling is shown with black arrows while established signaling pathways in the delta cell are depicted with grey arrows. (B) Mouse model: Sst^rtTA ;Tet-O-Cre;Lepr^fl/fl (Lepr^Δδ ) and littermate control mice were placed on doxycycline from eight weeks to ten weeks of age. Mice were assigned to a high-fat or normal chow diet at ten weeks of age. GTTs were performed at eight, 14, and 36 weeks of age, corresponding to before dox treatment, and four and 26 weeks after doxycycline cessation.

Figure 2

Lepr recombination and expression analysis. (A) Schematic of Lepr^fl allele and primers used to assess DNA recombination of the loxP elements. (B, C) PCR was performed on DNA collected from fixed pancreatic sections. F1/R1 primers amplify unrecombined DNA (B), while F1/R2 primers result in a ~400bp product after Lepr^fl recombination (C). All samples tested had one allele of Sst^rtTA. Samples 1 and 7 were heterozygous for Lepr^fl , and Samples 2,3,4,5,6, and 8 were homozygous for Lepr^fl . Samples 1-4 were Tet-O-Cre-negative, while Samples 5-8 were Tet-O-Cre-positive. D) qPCR analysis of Lepr expression in whole islets from Lepr^Δδ or control mice.

Figure 3

Lepr^Δδ mice display unaltered weight, beta-cell mass, and islet morphology. (A, B) Body weight for (A) males and (B) females on chow diet. (C, D) Body weight for (C) males and (D) females on high-fat diet. E-F) Beta-cell mass at 36 weeks of age for males (E) and females (F) on HFD. (G, H) Representative image of islets from control (G) and Lepr^Δδ (H) mice taken at 400X.

Figure 4

Lepr^Δδ male mice display normal glucose tolerance on chow and high-fat diet. (A-J) GTTs (A, C, E, G, I) and corresponding area-under-the-curve analysis (B, D, F, H, J) for male Lepr^Δδ mice prior to doxycycline administration (A, B), at 14 weeks of age and four weeks after doxycycline removal and placement on a chow diet (C, D), at 36 weeks and six months after doxycycline removal and placement on a chow diet (E, F), at 14 weeks of age and four weeks after doxycycline removal and placement on a high-fat diet (G, H), and at 36 weeks of age and 26 weeks (6 months) after doxycycline removal and placement on a high-fat diet.

Figure 5

Lepr^Δδ female mice display normal glucose tolerance on chow and high-fat diet. (A-J) GTTs (A, C, E, G, I) and corresponding area-under-the-curve analysis (B, D, F, H, J) for female Lepr^Δδ mice prior to doxycycline administration (A, B), at 14 weeks of age and four weeks after doxycycline removal and placement on a chow diet (C, D), at 36 weeks and six months after doxycycline removal and placement on a chow diet (E, F), at 14 weeks of age and four weeks after doxycycline removal and placement on a high-fat diet (G, H), and at 36 weeks of age and 26 weeks (6 months) after doxycycline removal and placement on a high-fat diet.

Figure 6

Male and female Lepr^Δδ mice on a high-fat diet display normal glucose tolerance in response to a reduced glucose load. Glucose tolerance tests using 1g/kg glucose were performed before doxycycline treatment (A, B, E, F) and at 14 weeks of age in male (C, D) and female (E-H) Lepr^Δδ and control mice on a HFD diet starting at 10 weeks of age. Glucose curves (A, C, E, G) and area-under-the-curve (B, D, F, H) are shown.

Figure 7

Islets from Lepr^Δ mice display normal insulin secretion in response to high glucose. Isolated islets from 36-week old male (A, B) and female (C, D) Lepr^Δδ and control mice were subjected to perifusions (A, C) at 0 mM glucose (0 G), 16 mM glucose (16 G), and with KCl. Both males (A, B) and females (C, D) were examined. Area-under-the-curve for 16 mM glucose is shown (B, D).

Figure 8

Lepr is not expressed in mouse delta cells. (A, B) RNAseq reads for Lepr (A) and Pdx1 (B) in sorted alpha, beta, and delta cells were obtained from publicly available data. An inset for exon 1 Pdx1 expression in alpha cells at a zoomed-in scale is included to demonstrate the appearance of lower-expressed genes. (C) scRNAseq expression in mouse islets cells for Lepr, Sst, Pdx1, Ins, and two endothelial markers, Tie1 and Pecam, was obtained from the public database PanglaoDB (https://panglaodb.se SRA745567). (D) scRNAseq expression in human islet cells for LEPR, SSST, PDX1, INS, TIE1, and PECAM was obtained from PanglaoDB (SRA701877).