Abstract
Mutations in the pancreatic duodenal homeobox (PDX1) gene are associated with diabetes in humans. Pdx1-haploinsufficient mice also develop diabetes, but the molecular mechanism is unknown. To this end, we knocked down Pdx1 gene expression in mouse MIN6 insulinoma cells. Pdx1 suppression not only increased apoptotic cell death but also decreased cell proliferation, which was associated with a decrease in activity of mechanistic target of rapamycin complex 1 (mTORC1). We found that in Pdx1-deficient mice, tuberous sclerosis 1 (Tsc1) ablation in pancreatic β-cells restores β-cell mass, increases β-cell proliferation and size, decreases the number of TUNEL-positive cells and restores glucose tolerance after glucose challenge. In addition, Tsc1 ablation in pancreatic β-cells increases phosphorylation of initiation factor 4E-binding protein 1 (4E-BP1) phosphorylation and 40S ribosomal protein S6, two downstream targets of mTORC1 indicating that Tsc1 mediates mTORC1 downregulation induced by Pdx1 suppression. These results suggest that the Tsc1-mTORC1 pathway plays an important role in mediating the decrease in β-cell proliferation and growth and the reduction in β-cell mass that occurs in Pdx1-deficient diabetes. Thus, mTORC1 may be target for therapeutic interventions in diabetes associated with reductions in β-cell mass.
Introduction
A decrease in pancreatic β-cell mass is associated with diabetes mellitus. Patients with type 1 diabetes have a dramatic reduction in β-cell mass, leading to insulin insufficiency and hyperglycemia. In type 2 diabetes, insulin resistance causes a compensatory expansion of β-cells and increased plasma insulin levels (Uchizono et al. 2009, Feng et al. 2016). However, diabetes eventually develops as β-cell mass decreases. Therefore, approaches to increase functional pancreatic β-cell mass may lead to improved therapeutic options for diabetes treatment. β-cell death, β-cell size and β-cell proliferation are the three major mechanisms to regulate pancreatic β-cell mass. β-cell replication maintains functional β-cell mass in adult mice and humans (Dor et al. 2004, Meier et al. 2008). Although several studies have shown primary effects on β-cell proliferation and growth following a variety of genetic or pharmacologic interventions (Bonner-Weir et al. 2010, Fiaschi-Taesch et al. 2010, Fiaschi-Taesch et al. 2013), the molecular mechanism underlying β-cell proliferation and growth need further clarification.
The mammalian target of rapamycin (mTOR), a serine/threonine kinase, is a master regulator of cellular metabolism and promotes cell size in response to environmental stimuli. mTOR forms two distinct signaling complexes, mTOR complex 1 (mTORC1, rapamycin sensitive) and mTOR complex 2 (mTORC2, rapamycin insensitive), by binding with multiple companion proteins such as mLST8, Tti1/Tel2, DEPTOR, RAPTOR and PRAS40 (Lee et al. 2007). mTORC1 integrates various stimuli and signaling networks to stimulate synthesis of protein, lipid and nucleotides and block catabolic processes such as autophagy at the post-translational and transcriptional levels (Jewell & Guan 2013, Shimobayashi & Hall 2014). The growth factor/PI3K/AKT signaling pathway is involved in regulating both mTORC1 and mTORC2. The tuberous sclerosis (TSC) tumor suppressor complex (TSC1/TSC2) is the most important upstream negative regulator of mTORC1. Activated AKT directly phosphorylates and inhibits TSC1/2 (Huang & Manning 2008). AKT-dependent phosphorylation results in dissociation of TSC1/2 from the lysosome, where the Ras homolog enriched in brain (Rheb) is localized, promoting Rheb activation. Since GTP-bound Rheb is a potent mTORC1 activator, inhibition of TSC1/2 by AKT-dependent phosphorylation results in mTORC1 activation (Kim & Guan 2015). Rapamycin not only inhibits β-cell proliferation in vitro but also blocks the effects of glucose and AKT activation on β-cell mass and proliferation by inhibition of mTOR in β-cells (Balcazar et al. 2009, Blandino-Rosano et al. 2012). Mice lacking Tsc1 in pancreatic β-cells (Rip-Tsc1 KO mice) develop obesity due to hypothalamic Tsc1 excision in older Rip-Tsc1cKO animals, but young animals displayed a prominent gain-of-function β-cell phenotype prior to the onset of obesity. The young Rip-Tsc1KO animals displayed improved glucose tolerance due to mTOR-mediated enhancement of β-cell size, mass and insulin production (Mori et al. 2009a , b ).
Pdx1 (pancreas and duodenal homeobox-1) plays an essential role in pancreas development, β-cell function and survival. Islet-specific disruption of Pdx1 causes impaired insulin release, glucose intolerance and diabetes (Ahlgren et al. 1998, Johnson et al. 2003). Humans with heterozygous missense and frame shift mutations of the Pdx1 gene develop reductions in insulin secretion resulting in one form of maturity-onset diabetes of the young (MODY 4). Previous studies from our laboratory and others have shown that islets from heterozygous Pdx1 +/ − mice are reduced in number, smaller in size and show increased susceptibility to cell death (Fujimoto et al. 2010a , b , Ren et al. 2014a ). The present study was undertaken to determine whether mTORC1 plays a role in mediating pancreatic β-cell proliferation and growth in Pdx1 +/ − mice.
Materials and methods
MIN6 cell culture, quantification of mRNA levels and lentivirus-mediated shRNA expression
MIN6 cell culture, RNA isolation and first-strand cDNA synthesis and preparation of pLKO.1-Pdx1 shRNA lentivirus were performed as previously described (Ren et al. 2014a ). TaqMan assay numbers were Hmbs, Mm00660262; Pdx1, Mm00435565; Tsc1, Mm00452208_m1 and Tsc2, Mm00442004_m1. Lentivirus was added to the medium on day 1. The blots were probed with antibodies against Pdx1 (07-696; Millipore), α-tubulin (T6199, Sigma), Actin (A-3853; Sigma), Tsc1 (6935; Cell Signaling), Tsc2 (4308, Cell Signaling), Phospho-S6 ribosomal protein (Ser240/244) (5364, Cell Signaling), S6 ribosomal protein (2217, Cell Signaling), 4E-BP1 (9644, Cell Signaling) and Phospho-4E-BP1 (Ser65) (9451, Cell Signaling), Phospho-AKT(Thr308) and Phospho-AKT (Ser473) (9916, Cell Signaling Sampler Kit), Phospho-GSK3β (Ser9) (9322, Cell Signaling) and GSK3β (9315, Cell Signaling). Phospho-S6 ribosomal protein blocking peptide is from Cell Signaling Technology (1220). Antibody detection was accomplished using enhanced chemiluminescence (PerkinElmer) and LAS-3000 Imaging system (FUJIFILM). The integrated density of each band was measured using NIH ImageJ software (Bethesda, MD, USA).
Immunofluorescence (IFC) and immunohistochemistry (IHC)
Pancreas tissue was harvested following transcardiac perfusion with 4% paraformaldehyde and fixed in 4% paraformaldehyde. Pancreatic sections were stained with antibodies against insulin and glucagon, phosphorylated S6 (p-S6; Ser235/236 for immunohistochemistry (IHC) and immunofluorescence (IFC), Cell Signaling). Nascent protein synthesis was visualized using the Click-iT protein synthesis assay kit (C10428, Life Technologies).
Isolation primary mouse pancreatic islets
Mouse islets were isolated by using collagenase and filtration as previously described (Johnson et al. 2003).
Tamoxifen administration
In this study, over a 5-day period, 4-week-old male mice were injected intraperitoneally with three doses of 2.5 mg tamoxifen (Sigma, T5648) freshly dissolved in corn oil at 10 mg/mL (Wicksteed et al. 2010).
Autocrine effect of released insulin
To determine the influence of an autocrine effect of released insulin on cell death, we incubated MIN6 cells with 2.5 mM and 25 mM glucose serum-free DMEM. One day after Pdx1 knockdown (KD) in MIN6 cells, MIN6 cells were serum and glucose deprived for 24 h (DMEM, 2.5 mM glucose, no serum), and then maintained in culture in DMEM (no serum) supplemented with 2.5 or 25 mM glucose for 48 h. Then, the cell death was determined by propidium iodide (PI) staining.
In vivo characterization of mice
The Pdx1 +/− mice have been previously described (Ren et al. 2014a ). Tsc1 flox/flox mice were purchased from the Jackson Laboratory and MIP-Cre/ERT (here referred to Cre) mice were provided by Louis Philipson (University of Chicago) (Wicksteed et al. 2010). Mice were maintained on the mixed genetic background (C57Bl/6 × 129Sv). Male mice were fed a high-fat diet (HFD) containing 42% fat (Harlan Laboratories Inc., Indianapolis, IN, USA) from 5 weeks of age (1 week after tamoxifen injection) and provided with water ad libitum as previously described (Ren et al. 2014a ). Intraperitoneal glucose tolerance tests (GTTs) were performed on mice after a 5-h fast (2 g/kg dextrose) at age of 17 weeks (12 weeks for HFD). Insulin levels were measured after 5-h fasting and 10 min after glucose challenge. Insulin tolerance tests were performed after a 5-h fast by administering human recombinant insulin (0.75 U/kg). We quantified β-cell area from anti-insulin-stained pancreas sections counterstained with hematoxylin using the intensity thresholding function of the integrated morphometry package in ImageJ. BrdU (Sigma-Aldrich) was injected intraperitoneally (100 mg/kg) every 24 h, starting 3 days before killing, three injections in total (Stolovich-Rain et al. 2012). TUNEL labeling, Ki-67 staining, BrdU labeling and β-cell size measurement were performed as previously described (Chintinne et al. 2012, Ren et al. 2014a ). For Ki67 staining and BrdU, at least 20,000 β-cells or 100 islets were counted. For TUNEL staining, more than 10,000 β-cells were counted. Only Ki67 or BrdU and insulin double-positive cells were counted as Ki67+ or BrdU+ β-cell in islets. All animal experiments in this study were performed under protocols approved by the University of Chicago Animal Studies Committee and were conducted in accordance with National Institutes of Health guidelines for the care and use of animals in research.
Rapamycin treatment studies
Rapamycin (Sigma) was initially dissolved in 100% ethanol, stored at −20°C and further diluted in an aqueous solution of 5.2% Tween-80 and 5.2% PEG 400 (final ethanol concentration 2%) immediately before use (Mori et al. 2009b ). Four-week-old mice were injected with tamoxifen. After being on HFD for 4 weeks, these mice were injected with rapamycin intraperitoneally (1 mg/kg body weight every other day) for 4 weeks with HFD. The mice were fed HFD for 12 weeks.
Imaging studies of pancreatic islets
Formalin-fixed pancreas sections underwent antigen retrieval in boiling citrate buffer (pH 6.0) for 10 min before labeling with antibodies against insulin (A0564; Dako), glucagon (G2654; Sigma-Aldrich) and DAPI (P-36931; Invitrogen).
Statistical analysis
ANOVA was used to assess the statistical significance of differences. Differences were considered significant when P < 0.05. In all experiments, the number of asterisks is used to designate the following levels of statistical significance: ***P < 0.001, **P < 0.01, *P < 0.05 compared with control group or WT group. #### P < 0.001, ## P < 0.01, # P < 0.05 compared with Pdx1 +/ − Tsc1 F/F Cre − group. Results are presented as mean ± s.e.m. Each experiment was repeats at least three times.
Results
The expression levels of Tsc1 are increased in Pdx1-deficient β-cells
Our previous studies showed Pdx1 +/ − mice develop diabetes due to decreasing β-cell mass, which is related to β-cell death (Ren et al. 2014b ). To investigate whether Pdx1 also affect β-cell proliferation, β-cell proliferation in islets from WT and Pdx1 +/ − mice was determined by Ki67 staining and BrdU labeling. Ki67+ and BrdU+ β-cells were significantly decreased in Pdx1 +/ − mice compared to WT mice (P < 0.001 and P < 0.05 respectively, Fig. 1A). These results indicate that Pdx1 deficiency in mice significantly decreases β-cell proliferation. The effects of Pdx1 on TSC-mTORC1 pathway in MIN6 cells were next determined since this pathway is known to play a major role in hormonal and nutritional signals that regulate cell size and proliferation. Pdx1 was knocked down in MIN 6 cells using shRNA lentivirus. Indeed, Pdx1 suppression inhibited mTORC1 activity as shown by decreasing phosphorylation of 4E-BP and S6 (Fig. 1B), both molecules are downstream effectors of mTORC1. Phosphorylation of 4E-BP and S6 were decreased by 70% and 50%, respectively (Fig. 1B). Since a reduction in phosphorylation of S6 may suggest a reduced translational activity, we determined the effect of Pdx1 KD on insulin transcription and translation. Pdx1 KD in MIN6 cells induced a significant reduction of mRNA levels of insulin by 44.5% (P < 0.05, Fig. 1C). The protein levels of proinsulin were decreased by 52.9% in Pdx1 KD MIN6 cells. These results indicate that Pdx1 deficiency also induces a reduction of insulin translation by 8%. In consistent with these results, Pdx1 KD in MIN6 cells significantly decreased nascent protein synthesis by 14% (Fig. 1C). As the TSC tumor suppressor complex (TSC1/TSC2) is a critical negative regulator of mTORC1, we studied the effect of Pdx1 suppression on expression levels of Tsc1 and Tsc2. The results showed that Pdx1 suppression induced an increase in Tsc1 mRNA levels by 49% (P < 0.05, Fig. 1D). However, Pdx1 KD had no effect on Tsc2 mRNA levels (Fig. 1D). Interestingly, both Tsc1 and Tsc2 protein levels were increased in Pdx1 KD cells (Fig. 1E). Pdx1 suppression induced an increase in Tsc1 protein levels by 140% (P < 0.05) and Tsc2 protein levels by 50% (P < 0.05), respectively (Fig. 1E). IFC staining also clearly showed elevated TSC1 expression in Pdx1 +/ − islets (Fig. 1F). These results indicate that Pdx1 suppression elevates Tsc1 and Tsc2 expression.
The expression levels of Tsc1 are increased in Pdx1 +/ − β-cells. (A) Ki67 staining and BrdU labeling of Pdx1 +/ − β-cells demonstrating a significant reduction in 15-week HFD-fed Pdx1 +/ − mice. ***P < 0.001 compared to WT. (B) Pdx1 suppression inhibits mTORC1 activity. (C) Pdx1 suppression leads to an inhibition of insulin transcription and translation. (D) Pdx1 suppression induces an increase in mRNA (D) and protein levels of Tsc1 (E) in MIN6 cells. The mRNA levels of Tsc2 are not changed. Three days after Pdx1 shRNA lentivirus infection in MIN6 cells, mRNA and proteins levels were analyzed by QRT-PCR (C and D) and Western blot (B, C and E), respectively. *P < 0.05 compared to control group. Nascent protein synthesis was detected by Click-iT protein synthesis assay kit according to the instruction. (F) Elevated Tsc1 expression in 20-week old Pdx1 +/ − islets. Immunofluorescence staining of the pancreatic sections from mice with 15-week HFD was performed with Tsc1 antibody. The scale bar represents 20 µm.
Citation: Journal of Endocrinology 238, 2; 10.1530/JOE-18-0015
Effect of Tsc1 ablation in adult Pdx1+/− mice
To investigate the function of the TSC-mTOR pathway in pancreatic β-cells, we established a mouse model with pancreatic β-cell-specific deletion of Tsc1. Mice containing homozygous floxed Tsc1 alleles (Tsc1 F/F ) were mated with the Cre transgenic line driven by the mouse Ins1 promoter (MIP-Cre/ERT, here refers to Cre). Pancreatic islets from Pdx1 +/ − Tsc1 F/F Cre − mice were small and contained reduced numbers of insulin-containing β-cells (Fig. 2A). Pancreatic islets from Tsc1 F/F Cre + mice appeared bigger than those from Tsc1 F/F Cre − mice (Fig. 2A). β-cells in Pdx1 +/ − Tsc1 F/F Cre − islets also exhibited increased TUNEL labeling (P < 0.05) (Fig. 2B). However, this parameter was not significantly decreased in Tsc1 F/F Cre + and Pdx1+/− Tsc1 F/F Cre + mice compared to Tsc1 F/F Cre − or Pdx1 +/ − Tsc1 F/F Cre − , respectively (Fig. 2B). To determine whether Tsc1 ablation affects β-cell proliferation, islets were stained for the proliferative marker Ki-67. Compared to Tsc1 F/F Cre − , β-cell proliferation was decreased in Pdx1 +/ − Tsc1 F/F Cre − islets (P < 0.001) and was significantly improved by Tsc1 ablation in Pdx1 +/ − Tsc1 F/F Cre + islets (P < 0.05, Fig. 2C). The Tsc1 F/F Cre + mice showed an increase in β-cell mass by 138% compared to that in Tsc1 F/F Cre − mice (P < 0.001, Fig. 2D). β-cell mass in Pdx1 +/ − Tsc1 F/F Cre + mice was restored to the value in Tsc1 F/F Cre − mice and β-cell mass approximately doubled compared to Pdx1 +/ − Tsc1 F/F Cre − mice (P < 0.05) (Fig. 2D). Given enlargement of islets observed in the p-S6 staining and to determine whether the increase in islet mass reflected increased cell size in addition to cell proliferation, we also measured the size of individual β-cells. We found that β-cells in Tsc1 F/F Cre + islets were significantly larger than those in Tsc1 F/F Cre − islets. β-cell size increased by 87% (P < 0.05). β-cell size increased by 73% in Pdx1 +/ − Tsc1 F/F Cre + islets compared to Pdx1 +/ − Tsc1 F/F Cre − islets (P < 0.05, Fig. 2E). In contrast, although β-cell size was smaller in Pdx1 +/ − Tsc1 F/F Cre − islets, we did not observe significant differences in β-cell size between Tsc1 F/F Cre − and Pdx1 +/ − Tsc1 F/F Cre − islets. These results indicate that Tsc1 ablation in β-cells decreases β-cell apoptosis, increases β-cell proliferation, β-cell size and β-cell mass.
Tsc1 ablation preserves β-cells mass in adult Pdx1 +/ − mice. (A) Islet morphology in adult mice on a HFD for 12 weeks; anti-insulin and anti-glucagon antibodies were used to stain β-cells (red) and α cells (green), respectively. The scale bar represents 20 µm. (B and C) TUNEL, cleaved caspase3 staining and Ki67+ labeling of pancreatic β-cells in 12-week HFD-fed mice. Quantitative TUNEL, cleaved caspase3 (B) and Ki67+ β-cells (C) data are shown. *P < 0.05 and ***P < 0.001 compared to the Tsc1 F/F Cre − mice (n = 3-5 per group). # P < 0.05 compared to Pdx1+/− Tsc1F/FCre− mice. (D) Histological analysis of pancreatic islets in 12-week HFD-fed mice and quantitation of group data for β-cells mass are shown (n = 3-5 per group). *P < 0.05, **P < 0.01 compared to the Tsc1 F/F Cre − mice. # P < 0.05 compared to Pdx1 +/ − Tsc1 F/F Cre − mice. (E) β-cell size increases in 12-week HFD-fed mice Pdx1 +/ − Tsc1 F/F Cre + mice. Pancreatic sections were stained with anti-insulin antibody to determine the size of individual β-cells. The data showed an average size of ≥2000 β-cells from each of three mice of each genotype. *P < 0.05 compared to Tsc1 F/F cre − mice and # P < 0.05 compared to Pdx1 +/ − Tsc1 F/F Cre − mice. Values are mean ± s.e.m.
Citation: Journal of Endocrinology 238, 2; 10.1530/JOE-18-0015
Tsc1 gene ablation prevents diabetes in Pdx1+/− mice
To rule out the effect of Cre itself on mice, we first determined the effect of Cre on the Pdx1 +/ − mice. We performed GTTs on these mice with HFD. The results showed that both Pdx1 +/ − Cre − and Pdx1 +/ − Cre + mice showed glucose intolerance and had a similar response to glucose (Supplementary Fig. 1, see section on supplementary data given at the end of this article). In addition, compared to Cre + mice, Pdx1 +/ − Cre + mice significantly increased blood glucose levels (P < 0.01 and P < 0.001) (Supplementary Fig. 1). Thus, MIP-Cre/ER transgene itself is not affecting β-cell function in response to glucose. To determine whether enhanced β-cell size and proliferation due to Tsc1 ablation prevents the diabetic phenotype in Pdx1 +/ − mice, we performed GTT and insulin tolerance tests (ITTs) on Pdx1 +/ − Tsc1 F/F Cre − , Pdx1 +/ − Tsc1 F/F Cre + mice. Male Pdx1 +/ − Tsc1 F/F Cre − mice on HFD develop increased fasting blood glucose and impaired glucose clearance (Fig. 3A and B). Fasting blood glucose levels were increased in Pdx1+/− Tsc1 F/F Cre − mice compared to Tsc1 F/F Cre − mice on HFD for 11 weeks (P < 0.001) (Fig. 3A). However, Pdx1 +/ − Tsc1 F/F Cre + mice exhibited significantly lower fasting blood glucose (Fig. 3A and B). The blood glucose in Pdx1 +/ − Tsc1 F/F Cre + mice was decreased by 34% compared with that in Pdx1 +/ − Tsc1 F/F Cre − mice (Fig. 3A). The Pdx1 +/ − Tsc1 F/F Cre + mice demonstrated improved glucose tolerance and had normal glucose tolerance compared to Tsc1 F/F Cre − mice, indicating that Tsc1 ablation prevents diabetes in Pdx1 +/ − Tsc1 F/F Cre − mice (Fig. 3B). The area under the blood glucose curve (AUC) decreased by 51% in Pdx1 +/ − Tsc1 F/F Cre + mice compared to Pdx1 +/ − Tsc1 F/F Cre − mice (P < 0.001) (Fig. 3C). The response to exogenous insulin was significantly increased in Pdx1 +/ − Tsc1 F/F Cre + and Tsc1 F/F Cre + mice compared to Pdx1 +/ − Tsc1 F/F Cre − and Tsc1 F/F Cre − (both P < 0.05), respectively. The reduction in blood glucose in Pdx1 +/ − Tsc1 F/F Cre + mice after insulin administration was similar to Tsc1 F/F Cre + mice (Fig. 3D). At 14 weeks on HFD, fasting insulin levels in Pdx1 +/ − Tsc1 F/F Cre − mice were 45% of the values in Tsc1 F/F Cre − animals (Fig. 3E), and these levels were increased in Pdx1 +/ − Tsc1 F/F Cre + mice both under basal conditions (P < 0.001) and following glucose challenge (P < 0.001) (Fig. 3E). There were no significant differences in body weight in the four groups of mice (Supplementary Fig. 2). Together with the above results, we conclude that Tsc1 ablation in β-cells prevents the diabetic phenotype in the Pdx1 +/ − Tsc1 F/F Cre − mouse by reducing apoptotic β-cell death, increasing β-cell proliferation, preserving glucose tolerance and increasing the response to exogenous insulin.
Tsc1 gene ablation prevents diabetes in Pdx1 +/ − mice. (A) Overnight fasting glucose levels were measured in male mice on HFD for 13 weeks (n = 8–12). ***P < 0.001 compared to the Tsc1 F/F Cre − mice. ### P < 0.01 compared to Pdx1 +/ − Tsc1 F/F Cre − mice. (B) Blood glucose levels after intraperitoneal injection of dextrose (1 g/kg) in the male mice at 13 weeks with HFD. *P < 0.05, **P < 0.01, ***P < 0.001 compared to the Tsc1 F/F Cre − mice. ## P < 0.01, ### P < 0.001 compared to Pdx1 +/ − Tsc1 F/F Cre − mice (n = 8–22). (C) Area under the blood glucose curves (AUC) using the data from B in the four mouse groups designated. ***P < 0.001 compared to the Tsc1 F/F Cre − mice. ### P < 0.001 compared to Pdx1 +/ − Tsc1 F/F Cre − mice. (D) Glucose levels in response to 0.75 U/kg body weight insulin in the four male mouse groups designated at 14 weeks with HFD (n = 12-22). *P < 0.05, Tsc1 F/F Cre + mice compared to Tsc1 F/F Cre − mice; # P < 0.05, Pdx1 +/ − Tsc1 F/F Cre + compared to Pdx1 +/ − Tsc1 F/F Cre − mice. (E) Insulin levels measured fasting and 10 min after intraperitoneal dextrose in mice at 14 weeks with HFD. *P < 0.05, **P < 0.01, ***P < 0.001 compared to the Tsc1 F/F Cre − mice. ### P < 0.001 compared to Pdx1 +/ − Tsc1 F/F Cre − mice. Values are mean ± s.e.m.
Citation: Journal of Endocrinology 238, 2; 10.1530/JOE-18-0015
β-cell-specific Tsc1 deletion activates mTORC1 in islets
Tsc1 mRNA levels were assayed in islets by QRT-PCR after tamoxifen treatment. Tamoxifen treatment significantly reduced Tsc1 mRNA levels by 68% and 65% in islets from Tsc1 F/F Cre + mice on normal chow (P < 0.001, Supplementary Fig. 3A) and HFD (P < 0.001, Fig. 4A), respectively. To determine the molecular mechanism by which Tsc1 deletion in β-cells prevents diabetes induced by Pdx1 deficiency, activation of mTORC1 was measured. We determined the activation status of mTORC1 in islets by immunoblotting for S6 and 4E-BP phosphorylation (p-S6 and p-4E-BP), which serve as two convenient downstream readouts for mTORC1. Loss of Tsc1 was associated with increased p-S6 and p-4E-BP in Tsc1 F/F Cre + islets (Supplementary Fig. 3B), indicating that mTORC1 was activated in the mutant islets. p-4E-BP and p-S6 were increased by 31% and 23-fold, respectively, in islets from Tsc1 F/F Cre + mice compared to those in islets from Tsc1 F/F Cre − mice (P < 0.001) (Supplementary Fig. 3C). The data indicate that deletion of TSC1 from mice on a normal chow induce activation of mTORC1. Next, we determined whether loss of Tsc1 in Pdx1-deficient mice on HFD can prevent diabetes due to activation of mTORC1 in β-cells. Due to increasing β-cell death and decreasing β-cell size and proliferation, we could not isolate sufficient numbers of islets from Pdx1 +/ − Tsc1 F/F Cre − mice on HFD for immunoblotting. However, both p-4E-BP and p-S6 were significantly increased in islets from Pdx1 +/ − Tsc1 F/F Cre + mice (Fig. 4B). Interestingly, p-S6 from Pdx1 +/ − Tsc1 F/F Cre + islets was increased by more than 40-fold compared to that from Tsc1 F/F Cre − islets, which was an even greater elevation than observed in Tsc1 F/F Cre + islets (Fig. 4C).
β-cell-specific Tsc1 deletion activates mammalian target of rapamycin (mTOR) complex 1 in islets. (A) Tsc1 mRNA levels were measured by QRT-PCR in islets from mice on HFD for 13 weeks. ***P < 0.001 compared to control group. (B) Elevated 4E-BP and S6 phosphorylation in Pdx1 +/ − Tsc1 F/F cre + islets. Immunoblot analysis of phospho-4E-BP (p-4E-BP), 4E-BP, phospho-S6 (Ser240/244, p-S6), S6, Tsc1 and Tubulin are shown. Islets were isolated from independent male mice on HFD for 15 weeks. (C) Bar graphs represent quantification using densitometry of the relative amounts of the indicated proteins determined by Western blots in D. ***P < 0.001 compared to the Tsc1 F/F Cre − mice.
Citation: Journal of Endocrinology 238, 2; 10.1530/JOE-18-0015
To further determine whether Tsc1 ablation in β-cells induces elevated S6 phosphorylation, S6 phosphorylation was determined in β-cells from Tsc1 F/F Cre + and Pdx1 +/ − Tsc1 F/F Cre + mice by IFC and IHC methods. First, we determine the specificity of p-S6 antibody. The results showed that p-S6 was increased in islets from Tsc1 F/F Cre + mice. However, p-S6 antibody-specific blocking peptide prevented pS6 antibody from recognizing the p-S6 in Tsc1 F/F Cre + islets (Fig. 5A). These results indicate that P-S6 antibody is specific to detect phosphorylation of S6 in islets. Immunostaining for p-S6 within the pancreas showed that mTORC1 was activated only in the β-cells from Tsc1 F/F Cre + and Pdx1 +/ − Tsc1 F/F Cre + mice not in the β-cells from Tsc1 F/F Cre − and Pdx1 +/ − Tsc1 F/F Cre − mice and not the neighboring cells (Fig. 5B and C), which is consistent with the cell-autonomous effect of Tsc1 on mTORC1. These data also revealed large islets, suggesting the expansion of islet mass in the Tsc1 F/F Cre + and Pdx1 +/ − Tsc1 F/F Cre + mice.
Elevated S6 phosphorylation in Tsc1 F/F Cre + and Pdx1 +/ − Tsc1 F/F Cre + islets. (A) Specificity of p-S6 (Ser235/236) antibody. For immunohistochemistry, 2 µL peptide was added to 1 µL antibody used in 100 µL total volume. The mixture was incubated for 30 min prior to adding the entire volume to the slide. Double labeling of the pancreatic sections from mice on HFD for 15 weeks was performed with p-S6 (Ser235/236) antibody and insulin antibody. The scale bar represents 100 µm. (B) S6 phosphorylation was quantified in β-cells from Tsc1 F/F Cre + and Pdx1 +/ − Tsc1 F/F Cre + mice. Double labeling of the pancreatic sections from mice on HFD for 15 weeks was performed with p-S6 (Ser235/236) antibody and insulin antibody. The scale bar represents 100 µm. (C) Elevated S6 phosphorylation in Tsc1 F/F Cre + and Pdx1 +/ − Tsc1 F/F Cre + islets. Immunostaining of the pancreatic sections from mice on HFD for 15 weeks was performed with p-S6 (Ser235/236) antibody. The scale bar represents 200 µm (low mag., low magnification) and 100 µm (high mag., high magnification).
Citation: Journal of Endocrinology 238, 2; 10.1530/JOE-18-0015
Rapamycin decreased mTORC1 activity in β-cells from Tsc1F/FCre+ and Pdx1+/−Tsc1F/FCre+ mice
To further test the effects of mTORC1 activation on pancreatic β-cells, we treated 8-week-old mutant and control mice with vehicle and rapamycin, a specific mTORC1 inhibitor, at 1 mg/kg every other day for 4 weeks. The vehicle itself has no effect on the mice (Supplementary Fig. 4). The response of the mice after vehicle treatment to glucose and insulin is similar to Fig. 3B. However, after rapamycin treatment, fasting and feeding glucose levels were not significantly different between Tsc1 F/F Cre − and Tsc1 F/F Cre + mice (Fig. 6A and B). Rapamycin treatment significantly increased glucose levels in Pdx1 +/ − Tsc1 F/F Cre + mice compared to vehicle treatment (Supplementary Fig. 4). After rapamycin treatment, Pdx1 +/ − Tsc1 F/F Cre + mice showed similar feeding and fasting glucose levels compared to Pdx1 +/ − Tsc1 F/F Cre − (Fig. 6A and B). However, rapamycin-treated Pdx1 +/ − Tsc1 F/F Cre − mice showed impaired glucose tolerance compared to Pdx1 +/ − Tsc1 F/F Cre + mice (Fig. 6C). Indeed, after rapamycin treatment, the AUC was significantly higher in Pdx1 +/ − Tsc1 F/F Cre + mice than in Pdx1 +/ − Tsc1 F/F Cre − mice (P < 0.05) (Fig. 6D), while the Tsc1 F/F Cre − and Tsc1 F/F Cre + mice showed similar AUC (P > 0.05) (Fig. 6D). Indeed, rapamycin treatment inhibited the increase in phosphorylation of S6 in Tsc1 F/F Cre + and Pdx1 +/− Tsc1 F/F Cre + islets (Fig. 6E). Thus, rapamycin treatment abrogated the increased mTORC1 activity in the mutant pancreatic β-cells. The effects of treating mutant animals with rapamycin were attributable to the inhibition of the increase of mTORC1 activity in β-cells.
Rapamycin decreased mTORC1 activity in β-cells from Tsc1 F/F Cre + and Pdx1 +/ − Tsc1 F/F Cre + mice. (A) Glucose levels after feeding. (B) Glucose levels after fasting. The glucose levels were measured in male mice with rapamycin treatment on HFD for 11 weeks (A) and 12 weeks (B) (n = 8–10). ns, not significant. (C) Blood glucose levels after intraperitoneal injection of dextrose (1 g/kg) in the male mice with rapamycin treatment on HFD for 12 weeks. *P < 0.05 compared to the Tsc1 F/F Cre − mice (n = 8-9). (D) Area under the blood glucose curves (AUC) using the data from C in the four mouse groups designated. ns, not significant. (E) Rapamycin treatment inhibits S6 phosphorylation. Double labeling of the pancreatic sections from mice on HFD for 15 weeks was performed with p-S6 (Ser235/236) antibody and insulin antibody. The scale bar represents 100 µm. Values are mean ± s.e.m.
Citation: Journal of Endocrinology 238, 2; 10.1530/JOE-18-0015
Pdx1 suppression decreases AKT activity
To determine the mechanism by which Pdx1 suppression decreases mTORC1, we knocked down Pdx1 in MIN6 cells. Since both AKT and GSK3β are involved in regulating mTORC1 activity, the phosphorylation of AKT and GSK3β were determined. The results showed that IGF-1 rapidly stimulated Akt phosphorylation in control cells by 4.7-fold, but Pdx1 KD significantly decreased IGF-1-induced Akt phosphorylation by 68% (Fig. 7A). To determine the possibility of an autocrine effect of released insulin, the cell death was determined in Pdx1 KD MIN6 cells. The results showed that Pdx1 KD induced a significant increase in cell death in MIN6 cells with 2.5 mM glucose (Fig. 7B). The increasing glucose concentrations resulted in inhibition of cell death around by 10% (Fig. 7B). The results indicate that the autocrine released insulin due to higher glucose increases cell survival. Pdx1 KD also significantly decreased insulin release and content (Fig. 7C). In addition, Pdx1 KD induced a decrease in GSK3β protein levels, whereas phosphorylation of GSK3β at Ser9 was not significantly different from the control cells (Fig. 7D). The model of Pdx1 in regulating Tsc-mTORC1 pathway is shown in Fig. 7E. Pdx1 suppression decreases AKT activity, and increases Tsc1 and Tsc2, leading to the decrease in mTORC1-mediated β-cell size and proliferation.
Pdx1 suppression decrease AKT activity in MIN6 cells. (A) Pdx1 KD inhibits IGF-I-stimulated Akt activation in MIN6 cells. Three days after Pdx1 shRNA lentivirus infection in MIN6 cells, MIN6 cells were deprived of serum for 16 h. Cells were stimulated with IGF-1 (100 ng/mL) for 10 min prior to lysis. p-Akt and Akt were assessed by Western immunoblotting. One representative blot is shown. Graphs display densitometry measurements of relative levels and represent means ± s.e. of two independent experiments. *P < 0.05, ***P < 0.001, ### P < 0.001. (B) The effect of an autocrine effect of released insulin on cell death. One day after Pdx1 KD in MIN6 cells, MIN6 cells were serum and glucose deprived for 24 h (DMEM, 2.5 mM glucose, no serum) then maintained in culture in DMEM (no serum) supplemented with 2.5 or 25 mM glucose for 48 h. Then, the cell death was determined by PI staining. **P < 0.01, ***P < 0.001, ### P < 0.01. (C) Insulin secretion and content in Pdx1 KD MIN6 cells. Three days after culture with control or Pdx1 shRNA lentivirus, MIN6 cells were washed twice in DMEM without serum and glucose, and then were pre-incubated with 2.5 mM glucose for 1h prior to switching to 2.5 or 25 mM glucose for 1 h at 37°C. Insulin concentration in the medium and insulin content were measured by ELISA assay. (D) Three days after Pdx1 shRNA lentivirus infection in MIN6 cells, the proteins levels of Pdx1, p-GSK3β and GSK3β were determined by Western blot. Bar graphs represent quantification using densitometry of the relative amounts of the indicated proteins determined by Western Blots. (E) Model of Pdx1 in regulating Tsc1-mTORC1 pathway. Pdx1 suppression increases Tsc1 and Tsc2 and decreases AKT activity, leading to the decrease in mTORC1-mediated β-cell size and proliferation.
Citation: Journal of Endocrinology 238, 2; 10.1530/JOE-18-0015
Discussion
Our studies have demonstrated that Pdx1-deficient mice develop hyperglycemia associated with an increase in β-cell death and a decrease in β-cell mass (Ren et al. 2014a ). The present studies were undertaken to further define the molecular mechanisms responsible for the reduction in pancreatic β-cell mass induced by Pdx1 deficiency. The Pdx1 +/ − mice showed decreased β-cell mass associated with a failure of β-cell size and proliferation. To examine the role of mTOR in β-cell size and proliferation in Pdx1-deficient mice, we generated β-cell-specific Tsc1 KO mice, in which MIP-Cre/ER deletes the Tsc1 gene only in pancreatic β-cells and studied β-cell function in these animals. Although MIP-Cre/ER mice are known to possess abnormal β-cell size characteristics related to expression of the hGH minigene (Oropeza et al. 2015), the response of Pdx1 +/ − Cre + to glucose in this study was not different compared to Pdx1+/−Cre−, indicating that MIP-Cre/ER itself has minimal effect on mice. As expected, Tsc1 F/F Cre + mice manifested increased islet mass, β-cell size and insulin secretion. Our results are consistent with other studies (Rachdi et al. 2008, Shigeyama et al. 2008, Liu et al. 2009, Mori et al. 2009b , Wittenberg et al. 2016). Although β-cell proliferation was not significantly increased in Tsc1 F/F Cre + mice compared to Tsc1 F/F Cre − , Pdx1 +/ − Tsc1 F/F Cre + mice showed an increase in β-cell proliferation, which may be caused by inactivation of 4E-BP. Moreover, Pdx1 +/ − Tsc1 F/F Cre + mice showed an increase in β-cell size. Both increased β-cell size and proliferation may contribute to increased β-cell mass in Pdx1 +/ − Tsc1 F/F Cre + mice. Based on the data in Fig. 2D, β-cell mass is increased by 75% in Pdx1 +/ − Tsc1 F/F Cre + mice than that in Pdx1 +/ − Tsc1 F/F Cre − mice. β-cell size is increased by 70% in Pdx1 +/ − Tsc1 F/F Cre + mice compared to Pdx1 +/ − Tsc1 F/F Cre − mice. Therefore, the increase in β-cell size is the main cause of the almost complete recovery of β-cell mass by mTORC1 activation in Pdx1 deficiency in HFD feeding. In addition, the protein levels of p-S6 in Pdx1 +/ − Tsc1 F/F Cre + mouse islets are highly increased. It suggests that S6K activity is highly increased in these islets. Excessive and constant S6K activity leads to IRS2 downregulation that impairs survival and proliferation of β-cells (Shah et al. 2004). It is possible that remarkable increase in p-S6 in Pdx1 +/ − Tsc1 F/F Cre + mouse islets might impair the full recovery of β-cell proliferation and survival. Fasting hypoglycemia and deletion of Tsc1 in Pdx1-deficient mice increased plasma insulin to the levels of WT mice but not to the levels of Tsc1 F/F Cre + mice, which indicates that Tsc1 inhibition counteracted the Pdx1 deficiency to some extent, but there is still a remaining effect, which is not abrogated by Tsc1 deletion. The result also suggests that the regulation of insulin gene by Pdx1 may be Tsc1 independent. Moreover, rapamycin treatment has been shown to have mixed effects on β-cells, depending on the experimental conditions (28). Rapamycin treatment can cause reductions in β-cell size, mass, proliferation and insulin secretion alongside increases in apoptosis (28). In this study, rapamycin treatment reversed the improved glucose tolerance observed in Pdx1 +/ − Tsc1 F/F Cre + mice, while glucose tolerance was higher in all the Pdx1-deficient mice. Such results may be due to β-cell toxicity induced by rapamycin treatment and rapamycin may induce apoptosis in Pdx1-deficient β-cells. However, the mechanisms involved in inhibition of the TORC1 complex by rapamycin are complex. The individual contribution of 4EBP or S6K to these phenotypes could be further assessed by genetic experiments.
In our studies, we used MIP-Cre/ER to delete the Tsc1 gene in pancreatic β-cells. By contrast, others used Rip-Cre to generate conditional deletion of Tsc1 or Tsc2 in β-cells (Rachdi et al. 2008, Shigeyama et al. 2008, Mori et al. 2009b ). The Rip-Cre Tsc1 or Tsc2 mice showed an increase in islet mass, β-cell size and insulin content at a young age, but Rip-Cre Tsc1-knockout mice developed diabetes and β-cell failure after 40 weeks (Rachdi et al. 2008, Shigeyama et al. 2008, Mori et al. 2009b ). In addition, Rip-Cre Tsc1-knockout mice also developed obesity and insulin resistance. In contrast, our mice did not demonstrate effects on body weight, and the knockout mice maintained improved insulin sensitivity at least up to 5 months of age (not shown). These differences could be due at least in part to a functional effect of Tsc1 on the hypothalamus because Rip-Cre in contrast to MIP-Cre is also expressed in hypothalamus. The genetic background of the mice used may also affect the phenotypes since different genetic backgrounds affect insulin sensitivity and β-cell function (Kahn et al. 2012). Tsc1 F/F Cre + mice also showed an increase in β-cell proliferation besides cell size, which is consistent with the deletion of Tsc2 in β-cells (Rachdi et al. 2008). However, β-cell proliferation was not altered in the Rip-Cre Tsc1 KO mice. The results suggest that Tsc1 and Tsc2 may have different function in β-cells and the hypothalamus, although it is generally believed that Tsc1 and Tsc2 function together as a complex. Thus, additional studies are needed to determine precisely how mTORC1 acts upon 4E-BPs and S6K to modulate β-cell mass, proliferation and function.
Previous studies have established important roles of insulin signaling in maintaining β-cell size, proliferation and insulin secretion through regulating multiple pathways, including mTOR. mTOR is essential for cell size and proliferation and is part of two mTOR complexes (mTORC), mTORC1 and 2. mTORC1 activity is negatively regulated by TSC1, TSC2 and the small G protein Rheb (Bond 2016). This pathway integrates signals from growth factors and nutrients and controls growth, proliferation and metabolism by directly modulating 4E-BP and S6 kinases (S6K) and by indirectly attenuating Akt signaling via an mTORC1/S6K-mediated negative-feedback loop on IRS signaling. Tsc1 and Tsc2 mutations specifically activate mTORC1. In this study, we found that insulin expression levels and phosphorylation of AKT were significant decreased in Pdx1 KD MIN6 cells even under IGF-1 stimulation, which may be related to the decreased insulin secretion and insulin content shown in Fig. 7, which is consistent with other studies (Iype et al. 2005). Once activated by insulin, AKT can also directly activate mTORC1. In our previous study, Pdx1 deficiency induces an increase in Bim and Puma (Ren et al. 2014a ). Bim is a target of Akt/mTOR signaling. Direct phosphorylation at S87 attenuates Bim’s proapoptotic function and is mediated by PKB/Akt (Qi et al. 2006). Moreover, the transcription of Bim and Puma is under the negative control of the Akt/mTOR/FoxO axis (Gilley et al. 2003, You et al. 2006).
In addition, GSK3β is a negative factor for β-cell mass and function. GSk3β deficiency in Irs2 − / − mice preserved β-cell mass by reversing its negative effects on proliferation and apoptosis through increasing Pdx1 (Tanabe et al. 2008). Surprisingly, GSK3β was decreased in Pdx1 KD MIN6 cells. The decrease in GSK3β may be the feedback of Pdx1-deficient cells in order to maintain cell survival and proliferation. However, the mechanism for decreased GSK3β in Pdx1 KD cells needs to be further investigated. Thus, the effects of Pdx1 deficiency on mTORC1 activity increased Tsc1 and Tsc2 expressions and decreased AKT activity.
In conclusion, we have shown that Pdx1 deficiency induces a decrease in β-cell size and proliferation. mTORC1 plays an important role in regulating β-cell size and proliferation in Pdx1-deficient β-cells. Genetic ablation of Tsc1 in β-cells increases β-cell size, proliferation and insulin secretion and preserves β-cell mass in Pdx1 +/ − mice. These results suggest Tsc1 and mTORC1 may be targets for therapeutic interventions in diabetes associated with reductions in β-cell mass.
Supplementary data
This is linked to the online version of the paper at https://doi.org/10.1530/JOE-18-0015.
Declaration of interest
The authors declare that there is no conflict of interest that could be perceived as prejudicing the impartiality of the research reported.
Funding
This study was supported by P30-DK020595 (The University of Chicago and Diabetes Research and Training Center).
Author contribution statement
D R designed research; J S, L M, Y H and M L performed research; D R analyzed data; and D R wrote the paper.
Acknowledgements
The authors thank Dr Kenneth Polonsky for insightful suggestions, advice and discussions.
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