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Published in final edited form as: Metabolism. 2018 Jan 7;82:100–110. doi: 10.1016/j.metabol.2017.12.017

Distinct roles of systemic and local actions of insulin on pancreatic β-cells

Takumi Kitamoto a,b,c, Kenichi Sakurai d, Eun Young Lee a, Koutaro Yokote b, Domenico Accili c, Takashi Miki a,*
PMCID: PMC7391221  NIHMSID: NIHMS1602713  PMID: 29320716

Abstract

Objective

Pancreatic β-cell mass and function are critical in glucose homeostasis. Their regulatory mechanisms have been studied principally under experimental conditions of reduced β-cell numbers, such as β-cell ablation and partial pancreatectomy. In the present study, we generated an opposite mouse model with an excessive amount of ectopic β-cells, and analyzed its consequence on β-cell mass and survival.

Methods

Mice underwent sub-renal transplantation (SRT) of pseudo-islets generated from a pancreatic β-cell line MIN6 or intra-pancreatic transplantation (IPT) of MIN6 cells, and morphological and functional changes of their endocrine pancreata were analyzed. Cellular fate of pancreatic β-cells after transplantation was traced using RipCre:Rosa26-tdTomato mice. By using MIN6 cells, we evaluated the roles of extracellular glucose, membrane potential, and insulin signaling on β-cell survival.

Results

SRT mice developed severe, progressive hypoglycemia associated with marked reduction in insulin-positive (Ins+) cell mass and apparent increase in apoptotic Ins+ cells. In in vitro experiments of MIN6 cells, insulin signaling blockade potently induced cell death, suggesting that local insulin action is required for β-cell survival.In fact, IPT(i.e. transplantation closeto endogenous β-cells)resulted in fewer apoptotic Ins+ cells compared with those induced by SRT. On the other hand, β-cell mass was decreased in proportion to the decrease in blood glucose levels in both SRT and IPT mice, suggesting a contribution of hypoglycemia induced by systemic hyperinsulinemia.

Conclusion

Insulin plays distinct roles in β-cell survival and β-cell mass regulation through its local and systemic actions on β-cells, respectively.

Keywords: β-Cell survival, β-cell mass regulation, Hypoglycemia, Insulin signaling

1. Introduction

Type 2 diabetes mellitus (T2DM) is a global public health problem that brings on morbidity, mortality, and a major economic burden. Although life-style modification [1,2] and various drugs [3] have been proposed as treatment options, more effective prevention and therapeutic strategy have been still necessary. T2DM is a metabolic disorder characterized by chronic hyperglycemia, caused by defective insulin secretion from pancreatic β-cells and/or defective insulin action [4–6]. However, impairment of insulin secretion and/or action arises prior to the onset of hyperglycemia, and the decline in β-cell function has been shown to be progressive in a clinical study of a Pima Indian population in whom the transition from impaired glucose tolerance to T2DM was monitored in a cohort study [7]. In 2003, Butler et al. found that pancreatic β-cell mass significantly decreased in patients with impaired fasting glucose as well as T2DM [8], suggesting that the importance of maintaining appropriate β-cell mass. Accordingly, we hypothesized that β-cell mass and function might be regulated dynamically in response to both its surplus andshortage through anintricate mechanism. The regulatory mechanism against β-cell mass reduction has been examined in several animal models, in which β-cell mass is acutely decreased by experimental procedures, such as partial pancreatectomy [9,10]and β-cell ablation by toxins [11–13]. After these procedures, β-cells have been shown to proliferate and recover their mass. On the contrary, the effect of an excess amount of β-cell on its mass and function remains unknown. To clarify this, pseudo-islets generated from the β-cell line MIN6 were transplanted into the sub-renal capsule of wild-type mice (SRT mice). Approximately 2 weeks after transplantation, SRT mice developed severe, progressive, and eventually lethal hypoglycemia following growth of the transplants. Histological analysis of these severely hypoglycemic mice revealedthat endogenous pancreatic β-cell mass was markedly reduced and that the number of apoptotic β-cells was increased. Using this mouse model, we examined the regulatory mechanism of β-cell mass and its function in detail.

2. Methods

2.1. Reagents

Hydroxyl-2-naphthalenylmethylphosphonic acid triscetoxymethyl ester (HNMPA) was purchased from Millipore (Billerica, MA, USA). Insulin and diazoxide were purchased from Sigma Aldrich (St. Louis, MO, USA). Corning® Matrigel® Growth Factor Reduced Basement Membrane Matrix was purchased from Corning Inc. (Corning, NY, USA).

2.2. Animals

We generated Rip-Cre;Rosa26tdTomato/+ (hereinafter Rip-Cre:Rosa26-tdTomato) mice with a genetic backgroundof C57BL/6Jbycrossbreeding promoter-driven Cre transgenic mice (Rip-Cre)[14] with fluorescent protein (Tomato)-reporter mice (Rosa26tdTomato/+) for a cell lineage tracing experiment. Eight-week-old C57BL/6J mice and Rip-Cre: Rosa26-tdTomato mice were subjected to transplantation experiments. The mice were housed in a climate controlled room with a temperature of 23 ± 3 °C, humidity of 55 ± 15%, and a 12 h light/12 h dark cycle, and were fed standard laboratory chow (CE-2) (Clea Japan Inc., Tokyo, Japan) ad libitum. Blood glucose was measured at indicated time points (16:00–18:00) using Glutestmint (Sanwa Kagaku Kenkyusho Co. Ltd., Nagoya, Japan). Serum insulin levels were measured using an ultrasensitive mouse insulin ELISA kit (Morinaga Institute of Biological Science, Yokohama, Japan).

All animal experiments were approved by the Animal Care Committee of Chiba University.

2.3. Cell Culture

MIN6 (K20) cells, a pancreatic β-cell line with a genetic background of C57BL/6J [15], were cultured in 25 mM glucose (HG)-Dulbecco's modified eagle medium (DMEM) supplemented with 10% fetal bovine serum (FBS) as previously described [16]. INS-1 cells [17] and βTC3 cells [18], other insulin-secreting cell lines, were cultured in RPMI-1640 and HG-DMEM supplemented with 10% FBS, respectively. Mouse aortic endothelial(MAE) cells [19]were cultured in Ham's F-12 medium supplemented with 10% FBS, and simian kidney fibroblast-like (COS-1) cells [20] were cultured in HG-DMEM containing 10% FBS.

2.4. Generating Pseudo-islets

To generate pseudo-islets, 20 μl MIN6 suspension (1.5 × 104 cells/ ml) in HG-DMEM was cultured in a hanging drop for 3 days, and the cell spheres were then transferred to a gelatin (1%)-coated dish and cultured for another 3 days before being used for transplantation.

2.5. Pseudo-islets Transplantation

During surgery, mice were anesthetized by inhalation of 1.7–1.9% isoflurane (DS Pharma Animal Health Co. Ltd., Osaka, Japan) under sterile conditions. For transplantation into the sub-renal capsule, the capsules of the kidneys were incised, and 150 pseudo-islets were implanted around the upper pole of the left kidney in eight-week-old female mice. For transplantation into the pancreas, MIN6 cells (1 × 105) were mixed with growth factor reduced matrigel and injected directly into the pancreas.

2.6. Histological Analysis

Excised pancreata were fixed and embedded in paraffin, and 4 μm sections were cut. For quantitative analysis of β-cell mass, we examined all regions of the whole pancreas in sections spaced 160 μm apart from each other. Pancreatic sections were immunostained using antibodies listed in Supplementary Table 1. The immunoreactivity was visualized either by the immunoperoxidase method with 3,3-diaminobenzidine (DAB) or by the immunofluorescence method. For calculating the area of a specific cell type(i.e. insulin-positive or Tomato-positive cell), the area positive for its immunoreactivity was detected and measured using a BA-8100 microscope and BZ-II Analyzer software (Keyence, Osaka, Japan). Total mass of a specific cell type was determined by multiplying the ratio of the cell area (the sum of all sections)-to-pancreas area (the sum of all sections) by total weight of the pancreas [16]. The average cellular size of a specific cell type was calculated from the ratios of cellular area-to-the number of the cell nuclei in each islet. The average cell size was measured using at least 30 islet sections per each mouse. Histological images of the islets were obtained by FV10i confocal microscope (Olympus, Tokyo, Japan).

2.7. Cell Viability Assay

Cells were seeded in 96 well plates (1.5 × 104 cells/well) 2 days before the experiment. For cell viability assay, MIN6 cells were cultured for 12 h in DMEM containing 1 mM glucose, sodium pyruvate (1 mM), and L-glutamine (4 mM) or in HG-DMEM supplemented with diazoxide (200 μM; Sigma) or insulin (1 μM; Sigma). HNMPA (100 μM) was added 2 h before sampling. HNMPA treatment was also performed with other cell types. Cell viability was determined using Cell Counting Kit-8 (Dojindo Laboratories, Kumamoto, Japan), and the viability was expressed as arbitrary units [A.U.(%)]. Experiments using the same protocol were repeated three times to ascertain reproducibility.

2.8. Quantitative Real-time PCR

Quantitative real-time PCR was performed under standardized protocol as previously described [21]. The primers used were Ddit3:Fw5'-GAGCTGGAAGCCTGGTATGA-3': Rev 5'-ACGCAGGGTCAAGAGTAGTG-3' and Hprt:Fw5'-GCGTCGTGATTAGCGATGA-3': Rev 5'-ATGGCCTCCCA TCTCCTT-3'.

2.9. Western Blot Analyses

Western blot analysis was carried out with MIN6 cells after treatment with low glucose (1 mM for 12 h) or HNMPA (10 μM for 12 h). The cells were lysed in sonication buffer [20 mM HEPES pH 7.5, 150 mM NaCl, 25 mM EDTA, 1% NP-40, 10% glycerol, 1 mM sodium vanadate, 1 mM phenylmethylsulfonyl fluoride (PMSF) and protease and phosphatase inhibitors]. Thirty microgram protein was subjected to SDS-PAGE. Antibodies against the following proteins were used (all from Cell Signaling): cleaved caspase-3 (1:1000) and β-actin (1:1000).

2.10. Statistical Analysis

Values are represented as means ± SEM, and tests were performed using SAS version 9.3 (SAS Institute). Comparisons between two groups were assessed using unpaired Studenťs t-test for normally distributed variables. Analysis of multiple comparisons was made using one-way ANOVA followed by Bonferroni post-hoc test. To investigate the relationship between two variables, Pearson's correlation coefficient was used. P values were considered significant at P < 0.05.

3. Results

3.1. Transplantation of Pseudo-islets Into Sub-renal Capsule Evokes Marked Reduction in Pancreatic β-cell Mass

To induce a state of excessive β-cell mass, we transplanted 150 pseudo-islets into the sub-renal capsule of a wild-type mouse. About 2–4 weeks after transplantation, the mice with sub-renal transplantation (SRT mice) developed severe hypoglycemia (Fig. 1A). To evaluate the ability of the transplants on glucose stimulated insulin secretion (GSIS) in SRT mice, we performed oral glucose tolerance test in 2 h fasted SRT mice on day 14 after transplantation and found that the mice exhibited much lower glycemic levels during oral glucose tolerance along with an excessive GSIS (Suppl. Fig. 1).

Fig. 1.

Fig. 1.

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Transplantation of pseudo-islets into sub-renal capsule influences pancreatic β-cell mass and survival. (A) Representative transition of blood glucose levels in a SRT mouse. (B) Comparison of representative immunofluorescence images between control mouse and SRT mouse having <300 AUCGlc. (C,D) Relationship between Ins+ cell mass or size and AUCGlc [control mice; white circles (n = 5) and SRT mice; black circles (n = 11)]. Correlation coefficient is calculated only with SRT mice. (E) Relationship between the frequency of TUNEL+ cells in Ins+ cells and AUCGlc. (F–H) Quantification of (C–E) (control mice vs. SRT mice with <300 AUCGlc). Data are mean ± SEM. r = Pearson's correlation coefficient. **P< 0.01 vs. control mice, Scale bars = 20 μm.

Pancreata of SRT mice were sampled at differential time points with various degrees of hypoglycemia; the endogenous β-cell mass, as assessed by insulin immunoreactivity, was found to be decreased in the mice exposed to sustained, severe hypoglycemia. The pancreatic islets of SRT mice with severe hypoglycemia were small in size and irregular in shape and contained fewer insulin-positive (Ins+) cells along with reduced immunoreactivity (Fig. 1B). The β-cell mass of SRT mice was found to be proportional to the sum (area under the curve) of glycemia for 7 days before sampling (AUCGlc) (Fig. 1C). To consider the cause of the reduction of β-cell mass, we quantified cellular size and apoptosis of Ins+ cells (Fig. 1D, E) in SRT mice. Histological analysis revealed that Ins+ cell size, as well as its mass, was decreased proportionally to the decrease in AUCGlc (Fig. 1D). By contrast, the frequency of apoptotic Ins+ cells, as assessed by TUNEL staining, was much greater only under severely hypoglycemic (<<300 AUGlc ) conditions (Fig. 1E).

Quantitative analyses of SRT mice having 300 AUCGlcG revealed that c the Ins+ cell mass was decreased 18.3% (Fig. 1F), Ins+ cell size was d-dcreased 54.8% (Fig. 1G), and the frequency of TUNEL-positive (TUNEL+) cells was increased 15 fold (Fig. 1H) compared with those of the controls.

3.2. Glucose and Insulin Signals are Essential for β-cell Survival; Hyperpolarization Does not Trigger Cell Death

We then examined the mechanism of β-cell apoptosis in SRT mice with <300 AUCGlc. The pancreatic β-cell is electrically excitable, and is reported to require extracellular glucose [22,23], β-cell firing [24], and insulin signaling [25,26] for its survival and/or its proliferation. To evaluate the effects of these factors on β-cell survival, we treated MIN6 cells with low glucose, the ATP-sensitive K+ channel opener diazoxide, high dose of exogenous insulin, or the insulin receptor specific tyrosine kinase inhibitor HNMPA for 12 h, 12 h, 12 h, and 2 h, respectively. Morphological observation by detecting dead cells with trypan blue suggested that cell death was induced mildly by low glucose and fiercely by HNMPA (data not shown). Cell death was then assessed by quantifying the surviving cells after exposure to each stimulus (Fig. 2A). The surviving cell number was mildly but significantly decreased by low glucose treatment. By contrast, treatment with diazoxide or a high concentration of insulin failed to reduce the cell number. Importantly, cell number was markedly reduced by HNMPA treatment.

Fig. 2.

Fig. 2.

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Insulin signaling is essential for cell survival specifically in pancreatic β-cells. (A) Viability of control [MIN6 cells cultured in HG-DMEM (HG)] and MIN6 cells with low (1 mM) glucose (LG), diazoxide (DZ), 1 μM insulin (INS), and HNMPA treatment. (B) mRNA expression levels of Ddit3 in MIN6 cells with each treatment. (C-D) Cell viability assay and mRNA expression levels of Ddit3 for MIN6 cells cultured in HG or LG with/without glutamine. (E) TUNEL staining of MIN6 cells treated by low glucose or HNMPA. (F) Western blot analysis of cleaved caspase-3 (C-Cas-3) levels in the same condition as (E). (G) Cell viability assay for MIN6, INS-1, βTC3, COS-1, and MAE cells with HNMPA treatment. All values represent means ± SEM. *P<0.05, **P<0.01, ***P<0.001.

Imbalance between demand and supply of ATP, including that in hypoxia, has been reported to induce ER stress and oxidative stress, resulting in cellular apoptosis [27,28]. Considering that intracellular ATP concentrations in pancreatic β-cells should be decreased in SRT mice with severe hypoglycemia, we examined whether low glucose treatment of MIN6 cells induces the expression of CHOP (Ddit3), the central player of ER stress-induced apoptotic cell death [29]. As expected, low glucose significantly increased gene expression of CHOP (Fig. 2B). In addition, removal of glutamine, another important ATP-sustaining substrate, from the medium significantly potentiated low glucose-induced cell death (Fig. 2C) and CHOP expression (Fig. 2D). By contrast, for diazoxide suppressed CHOP expression, a high concentration of insulin did not affect CHOP expression. HNMPA mildly induced CHOP expression regardless of its lethal effect (Fig. 2B). Treatment of the cells with low glucose and HNMPA increased TUNEL-positive cells (Fig. 2E), suggesting that shortage in glucose supply and/or defective insulin signaling in β-cells could induce cell apoptosis.

However, cleaved caspase-3, one of the central players in apoptosis [30], was detected only under low glucose treatment (Fig. 2F). We also examined whether HNMPA-induced cell death is an event that occurs specifically in insulin-secreting β-cells as a consequence of a blockade of autocrine insulin action. For this purpose, we repeated the same experiment in other cell lines: INS-1 cells, βTC cells, COS-1 cells and MAE cells. Interestingly, HNMPA treatment did not induce cell death in either COS-1 cells or MAE cells, while the cell death was induced in two other insulin secreting cell lines, INS-1 and βTC cells (Fig. 2G), suggesting that insulin signaling has a role in cell survival specifically in pancreatic β-cells.

3.3. Auto/Paracrine Insulin Signal Input to a β-cell From Neighboring β-cells is Required for its Survival

Our results in MIN6 cells reveal that attenuation of insulin signaling in pancreatic β-cells triggers their cellular death. In SRT mice with <300 AUCGlc, the extracellular insulin concentrations in the vicinity of endogenous β-cells may be diminished due to cessation of insulin secretion. Accordingly, apoptotic cell death in β-cells of SRT mice could be attributable to attenuated insulin signaling. To test this, we transplanted the MIN6 cells directly into the pancreas (rather thanthesub-renal capsule) to elevate the extracellular insulin concentrations around the β-cells and evaluated the frequency of β-cell apoptosis.

By using MIN6 cells stably expressing green fluorescent protein (GFP), we confirmed successful intra-pancreatic transplantation (IPT) of MIN6 cells, as demonstrated by formation of GFP-positive MIN6 clusters (Fig. 3A). Due to the very thin serous membrane of the pancreas, we could not secure sufficient subserosal space to place the pseudo-islets. We therefore transplanted the comparable cell number of dispersed MIN6 cells directly to the pancreas (Suppl. Fig. 2). After IPT, we confirmed successful in vivo formation of pseudo-islets with a structure resembling those generated in culture dishes in vitro. We found that the morphology of pseudo-islets in IPT resembles that in SRT and that IPT mice exhibited progressive hypoglycemia with a time-course similar to that of SRT mice (Suppl. Fig. 3). In order to confirm that similar degrees of hypoglycemia and hyperinsulinemia were induced in SRT and IPT mice, we measured the blood glucose and serum insulin levels at several AUCGlc levels (Supple Fig. 4). As expected, we found that marked hyperinsulinemia was induced under severe hypoglycemia (<300 AUCGlc) similarly in SRT and IPT mice (Fig. 3B). Next, IPT mice were subjected to the same analyses as SRT mice. Similar to SRT mice, IPT mice showed a decrease in cell mass (Fig. 3C) and cellular size (Fig. 3D) of Ins+ cells as well as an increase in apoptotic β-cells (TUNEL+/Ins+ cells) (Fig. 3E) under hypoglycemia. As expected, when compared with those in SRT mice, TUNEL+ cells were detected less frequently in hypoglycemic (<<300 AUGlc ) IPT mice, the relative c abundance of TUNEL+ cells (in Ins+ cells) being significantly (P<b 0.001) lower in IPT mice (1.22±± 0.16%) than in SRT mice (2.83±± 0.29%) (Fig. 3F).

Fig. 3.

Fig. 3.

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β-cell death is suppressed by MIN6 cells transplantation to pancreas.(A) Discrimination of MIN6 cells from endogenous β-cells by double immunofluorescence staining of GFP (green) and insulin (red). The pancreas and the transplant are circled by solid and dotted-lines, respectively. (B) The blood glucose and insulin levels of SRT and IPT mice having <300 AUCGlc [Control mice; white circle (n = 5), SRT mice; black circle (n = 5), and IPT mice; black triangle (n = 5)]. (C, D) Relationship between Ins+ cell mass or size and AUCGlc [control mice; white circle (n = 5) and IPT mice; black triangle (n = 12)]. Correlation coefficient is calculated with only IPT mice. (E) Relationship between the frequency of TUNEL+ cells in Ins+ cells and AUCGlc.(F–H) Quantification of (C–E) (control mice, SRT mice having <300 AUCGlc, and IPT mice having <300 AUCGlc). Data are mean ± SEM. r = Pearson's correlation coefficient. *P<0.05, **P<0.01.

3.4. MIN6 Cell Transplantation to Pancreas (Rather Than to Kidney) Protects Endogenous β-cells From Cell Death, but Deprives Them of Insulin Immunoreactivity

Surprisingly, in spite of the lesser apoptosis in Ins+ cells and similar Ins+ cell size of hypoglycemic (<<300 AUGlc )IP Tmice(Fig. 3F, H), their c Ins+ cell mass was rather decreased when compared with that of SRT mice (Fig. 3G). This apparent contradiction might be explained by insulin depletion in β-cells of IPT mice but not by increase in β-cell death. To assess this possibility, we employed a lineage tracing technique to specifically mark β-cells using Rip-Cre:Rosa26-tdTomato, a mouse permanently expressing the red fluorescent protein Tomato in most (~80%) β-cells (Fig. 4A). As the possible expression of hGH encoded in the transgene cassette of Rip-Cre mice [31] might influence their phenotype or islet morphology, Rip-Cre genotype (either positive or negative) was matched among control, SRT, and IPT mice in each experiment.

Fig. 4.

Fig. 4.

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β-cells in IPT mice are protected from cell death in comparison with those in SRT mice; insulin immunoreactivity is suppressed. (A) Scheme of Rip-Cre:Rosa26-tdTomato mice. (B) Quantitative analysis of Tomato+ cell mass of control, SRT, and IPT mice (n =4–5 each group).(C) Representative images showing co-staining for Tomato (red) and insulin (green) in Rip-Cre:Rosa26-tdTomato mice with SRT or IPT. Data are mean ± SEM. Scale bars = 20 μm *P< 0.05,**?< 0.01.

Quantitative analyses in Rip-Cre:Rosa26-tdTomato mice (Fig. 4A) reveled that the decrease in Tomato+ cell mass was significantly milder after IPT than after SRT (Fig. 4B). We then examined insulin immunoreactivity in Tomato+ cells (i.e., cells that once had expressed the insulin gene) of IPT-treated Rip-Cre:Rosa26-tdTomato mice. Interestingly, insulin-negative but Tomato-positive (Ins−/Tomato+) cells were frequently found after IPT, but only occasionally after SRT (Fig. 4C).

3.5. Expression of MafA and Subcellular-localization of Pdx1 was Altered Similarly in SRT and IPT Mice With Severe Hypoglycemia

The appearance of Ins−/Tomato+ cells in IPT-treated Rip-Cre: Rosa26-tdTomato mice (Fig. 4C) prompted us to evaluate protein expression of the critical genes in β-cells: MafA and Pdx1. Immunostating of pancreata of IPT-treated Rip-Cre:Rosa26-tdTomato mice revealed a marked decrease in MafA expression and cytoplasmic distribution of Pdx1 in Tomato+ cells (Fig. 5A, B). Similar change was also observed in SRT mice (Fig. 5A, B).

Fig. 5.

Fig. 5.

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Both SRT and IPT show decreased expression level of MafA and altered cytoplasmic distribution of Pdx1. (A and B) Representative images showing co-staining for Tomato (red) and MafA (green, A) or Pdx1 (green, B) in each mouse.Scale bars = 20 μm.

3.6. Transplant Removal Induced Functional and Morphological Recovery of the Residual β-cells in SRT Mice

We next examined whether the deterioration of β-cells in hypoglycemic SRT mice is reversible. Hypoglycemic (<300 AUCGlc)SRT mice were subjected to transplant removal by nephrectomy, which induced a rapid and drastic shift in glycemic levels from hypoglycemia to a transient (<4 days) hyperglycemia (peaking at 431.4 ± 75.0 mg/dl, n = 5), followed by sustained normoglycemia (Fig. 6A). Since the time-course of glycemia strongly suggests functional recovery of endogenous β-cells, we next examined morphological changes of islets in nephrectomized-SRT mice on day 4. We found that the attenuated immunoreactivity of insulin and MafA and cytoplasmic expression of Pdx1 in SRT mice was normalized 4 days after nephrectomy (Fig. 6B, C). Morphological analyses of the islets revealed a significant (2.2 fold) increase in Ins+ cell mass (Fig. 6D) and enlargement of Ins+ cell size (Fig. 6E), which accords with normalization of glycemic control.

Fig. 6.

Fig. 6.

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β-cell function and mass are recovered after transplant removal. (A)Blood glucose levels before and after transplant removal. An arrow indicates the time point of nephrectomy and sacrificing of each mouse. (B and C) Representative images showing staining for insulin (red) and MafA (green, B) or Pdx1 (green, C) in control and SRT mice 4 days after transplant removal. (D and E) Comparison of β-cell mass and size among control (white circle), SRT mice having <300 AUCGlc (black circle), and SRT mice after transplantation removal. Data are mean ± SEM. Scale bars = 20 μm. *P<0.05, **P<0.01.

4. Discussion

In the present study, using mice transplanted with pseudo-islets of MIN6 cells, we found that the cell mass and function of endogenous β-cells is substantially jeopardized by the existence of excessive β-cells. Interestingly, although SRT mice exhibited both increased β-cell apoptosis and decreased β-cell mass, the two phenotypes may well be induced by distinct mechanisms, as the former occurs in proportion to the decrease of AUCGlc while the latter occurs only under severely hypoglycemic conditions.

In accord with the previous finding [22], our data showed that apoptosis of MIN6 cell was induced by low glucose in in vitro experiments and that β-cell death was induced in vivo both in SRT and IPT mice in response to severe hypoglycemia. Nevertheless,the attenuated apoptotic β-cell death in IPT mice (compared with SRT mice having a similar level of hypoglycemia) strongly suggests that this difference can be attributed to other humoral factors, most likely those released from the transplanted β-cells, such as insulin, Zn2+ [32], islet amyloid polypeptide (IAPP) [33], and GABA [34]. Among them, insulin signaling in β-cells might play a critical role, as blockade of insulin signaling by the insulin receptor antagonist HNMPA markedly induced apoptotic cell death in insulin producing cells (Fig. 2G).

The importance of insulin signaling on β-cell function and mass has been demonstrated in many studies. In particular, analyses of genetically engineered mice deficient in any of the insulin signaling molecules, such as the insulin receptor [35], IRS-2 [26,36–38], Akt [39], PDK1 [40], or mTOR [41], revealed that the signaling is essential for both β-cell survival and its function [26,37].

On the other hand, the physiological relevance of insulin signaling in β-cells has long been questioned by many researchers. Rhodes et al. question the concept of a physiological role of autocrineaction of insulin on β-cells [42]. However, our present findings of attenuated β-cell apoptosis in IPT mice and induction of MIN6 cell death by the insulin receptor antagonist strongly suggest that insulin secreted from neighboring β-cells may play an important role in β-cell survival.

To our surprise, Ins−/Tomato+ cells (β-cells without insulin immunoreactivity) were frequently detected in IPT mice, but only sparsely in SRT mice (Fig. 4C). Interestingly, Goginashvili et al. reported that pancreatic β-cells evoke insulin granule destruction under metabolic deprivation through a p38δ/PKD1 signalingpathway [43].PKD1 is inactivated by nutrient deprivation via p38δ and inactivated PKD1 suppresses insulin secretion. Endogenous β-cells in our SRT and IPT mice may elicit granule destruction through a mechanism similar to that reported by Goginashvili et al., while a larger number of the β-cells in IPT mice were considered to survive by virtue of insulin secreted from transplanted pseudo-islets.

Both MafA and Pdx1 participate synergistically in the transcriptional regulation of the insulin gene [44,45]. Transcription of MafA and nuclear localization of Pdx-1 protein are known to be regulated by extracellular glucose levels [46,47]. Accordingly, reduced MafA immunoreactivity in both SRT and IPT mice could be due to hypoglycemia. The mechanism of glucose-induced gene expression has been studied extensively in skeletal muscle, liver, and adipocytes, as well as β-cells [48]. However, we were unable to show the mechanism linking hypoglycemia with reduced immunoreactivity of MafA in β-cells of SRT and IPT mice in this study.

With regards to Pdx1, cytoplasmic distribution of Pdx1 in β-cells of both hypoglycemic SRT mice and IPT mice would also inhibit Pdx1 function, contributing to the suppression of insulin granule biogenesis. In addition to destruction of insulin granule, these mechanisms also might contribute to decreased insulin immunoreactivity in β-cells of SRT mice and IPT mice. Reduction in β-cell apoptosis by local insulin signaling in IPT mice but not in SRT mice may well explain the more frequent appearance of Ins−/Tomato+ cells in IPT mice than in SRT mice.

Normalization of hypoglycemia within 4 days after transplant removal suggested functional recovery of β-cells, although the recovery of Ins+ cell mass and Ins+ cell size remained marginal (~2 fold). Previously, Miyaura et al. conducted a similar experiment [49], and their findings were quite similar to ours except for the superior recovery of β-cell mass after removal of an insulinoma. Although the reason for this discrepancy is unknown at present, the difference in severity and/ or duration of the hypoglycemia might be involved.

5. Conclusion

In conclusion, our present study demonstrates that local insulin action on the β-cell is required for β-cell survival. Although the importance of insulin signaling in β-cells has been shown in various genetically engineered mouse models with defective insulin signaling, our study differs in showing that marked β-cell death can be induced in wild-type mice by severe, sustained hypoglycemia. Our study also shows that the normal blood glucose level is required for maintaining the normal β-cell mass through a mechanism independent from the β-cell loss due to hypoglycemia-induced cell apoptosis. Accordingly, insulin plays distinct roles in pancreatic β-cell survival and regulation of β-cell mass through direct and indirect action on β-cells.

Supplementary Material

Supplementary Table 1
Fig S1
Fig S2
Fig S3
Fig S4

Acknowledgments

We thank Drs. E. Mukai and M. Yoshida (Chiba University, Chiba, Japan) for their technical advice.

Funding

This study was supported by Grant-in-Aid from the Ministry of Education, Science, Sports, and Culture of Japan (16K08520 to T.M. and 16K00846 to EY.L.), JSPS Overseas Research Fellowships (685 to T.K.), and research support grants from Daiichi Sankyo Co. Ltd., Ono Pharmaceutical Co. Ltd., Mitsubishi Tanabe Pharma, and Takeda Pharmaceutical Co. Ltd.

Abbreviations

T2DM

type 2 diabetes mellitus

AUCGlc

area under the curve of glycemia

SRT

sub-renal transplantation

IPT

intra-pancreatic transplantation

GFP

green fluorescent protein

Footnotes

Declarations of Interest

None.

Supplementary data to this article can be found online at https://doi.org/10.1016/j.metabol.2017.12.017.

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Supplementary Table 1
NIHMS1602713-supplement-Supplementary_Table_1.docx (17.9KB, docx)
Fig S1
NIHMS1602713-supplement-Fig_S1.jpg (19.6KB, jpg)
Fig S2
NIHMS1602713-supplement-Fig_S2.jpg (25KB, jpg)
Fig S3
NIHMS1602713-supplement-Fig_S3.jpg (41.5KB, jpg)
Fig S4
NIHMS1602713-supplement-Fig_S4.jpg (63.9KB, jpg)

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