Diabetes Volume 68, January 2019 81

SREBP1c-PAX4 Axis Mediates Pancreatic b-Cell Compensatory Responses Upon Metabolic Stress

Gung Lee,1 Hagoon Jang,1 Ye Young Kim,1 Sung Sik Choe,1 Jinuk Kong,1 Injae Hwang,1 Jeu Park,1 Seung-Soon Im,2 and Jae Bum Kim1

Diabetes 2019;68:81–94 | https://doi.org/10.2337/db18-0556

SREBP1c is a key for de novo lipo- or hyperglycemia in obesity, pancreatic islets adaptively genesis. Although SREBP1c is expressed in pancreatic elevate the level of serum insulin through a b-cell com- islets, its physiological roles in pancreatic b-cells are pensatory mechanism. This process includes the expansion largely unknown. In this study, we demonstrate that of b-cell mass and the augmentation of insulin production b SREBP1c regulates -cell compensation under meta- (3,4). Although b-cell neogenesis and protection from bolic stress. SREBP1c expression level was augmented apoptosis can contribute to b-cell mass expansion, accu- in pancreatic islets from obese and diabetic animals. In mulating evidence suggests that an increase in b-cell pro- pancreatic b-cells, SREBP1c activation promoted the

b liferation would be the principal mechanism of postnatal STUDIES ISLET expression of cell cycle and stimulated -cell b b proliferation through its novel target , PAX4. Com- -cell growth (5,6). Thus, the potentiation of -cell pro- pared with SREBP1c+/+ mice, SREBP1c2/2 mice showed liferation is important to maintain glucose homeostasis glucose intolerance with low insulin levels. Moreover, and to protect against the onset of diabetes. b-cells from SREBP1c2/2 mice exhibited reduced SREBP1c is a basic helix-loop-helix tran- capacity to proliferate and secrete insulin. Conversely, scription factor that governs de novo lipogenesis (7–10). transplantation of SREBP1c-overexpressing islets re- During the postprandial state, insulin activates SREBP1c stored insulin levels and relieved hyperglycemia in strep- via AKT ( kinase B) and mTORC1 (mammalian tozotocin-induced diabetic animals. Collectively, these target of rapamycin C1) (11,12). SREBP1c stimulates de data suggest that pancreatic SREBP1c is a key player novo lipogenesis by elevating the expression of lipogenic in mediating b-cell compensatory responses in obesity. target genes including fatty acid synthase, stearoyl-CoA desaturase 1, and acetyl-CoA carboxylase (12–14). More- over, SREBP1c has been implicated in lipid metabolisms of Pancreatic islets regulate glucose homeostasis through the pancreatic islets (15,16). For instance, it has been reported secretion of several hormones, particularly glucagon and that the activation of SREBP1c by liver X agonist insulin. Under hypoglycemic conditions, glucagon is re- increases intracellular lipid accumulation in pancreatic leased from pancreatic a-cells to promote hepatic glyco- islets (16). SREBP1 is associated with cell growth through genolysis and gluconeogenesis to increase blood glucose lipid metabolism and/or cell cycle progression in cer- levels (1). In contrast, insulin secretion from pancreatic tain cell types (17,18). However, it remains unclear b-cells is stimulated by hyperglycemia. Insulin effectively whether SREBP1c might be associated with pancreatic b-cell lowers the level of blood glucose via glucose uptake into proliferation. peripheral tissues and hinders hepatic glucose production. Paired box 4 (PAX4) belongs to the PAX gene family, On the other hand, metabolic stresses such as obesity and a group of transcription factors that carry out essential insulin resistance increase insulin demand and disrupt roles in embryogenesis as well as in cellular plasticity glucose homeostasis (2). In response to chronic fuel surfeit in adults (19). PAX4 is mainly expressed in endocrine

1National Creative Research Initiatives Center for Adipose Tissue Remodeling, This article contains Supplementary Data online at http://diabetes Institute of Molecular Biology and Genetics, Department of Biological Sciences, .diabetesjournals.org/lookup/suppl/doi:10.2337/db18-0556/-/DC1. Seoul National University, Seoul, South Korea © 2018 by the American Diabetes Association. Readers may use this article as 2 Department of Physiology and Medical Research Center, Keimyung University long as the work is properly cited, the use is educational and not for profit, and the School of Medicine, Daegu, Republic of Korea work is not altered. More information is available at http://www.diabetesjournals Corresponding author: Jae Bum Kim, [email protected] .org/content/license. Received 18 May 2018 and accepted 3 October 2018 82 SREBP1c-PAX4 Axis in b-Cell Compensation Diabetes Volume 68, January 2019 pancreas where it plays an essential role to induce differ- using the sensitive colorimetric assay for viable cells entiation toward b-cells (20). Also, PAX4 has been impli- according to the manufacturer protocol (#CK04-11; cated in b-cell plasticity of adult pancreatic islets. For Dojindo Molecular Technologies). example, it has been revealed that ectopic expression of PAX4 in human or murine islets enhances b-cell prolifer- Quantitative Real-time PCR ation by stimulating the transcription of cell cycle genes Total RNAs were isolated from MIN6 and aTC1–6 cells (21–23). Moreover, the overexpression of PAX4 protects using TRIzol reagent (Invitrogen). Islet RNAs were pre- b-cells from apoptosis induced by streptozotocin (STZ) pared using the RNeasy Mini Kit (Qiagen). Subsequently, (24). Recently, it has been reported that mutations of the equal amounts of RNA were synthesized to cDNA using PAX4 gene are associated with type 1 and 2 diabetes as well RevertAid reverse transcriptase (#EP0441; Thermo Fisher as ketosis-prone diabetes in various ethnic groups (25). Scientific). Relative amounts of mRNA were calculated by Although the correlation between PAX4 dysfunction and using a CFX Real-Time Quantitative PCR Detection System diabetes has been demonstrated, the regulatory mecha- (Bio-Rad) after normalization to cyclophilin mRNA. The nism of PAX4 expression remains elusive. primer sequences used are listed in Supplementary Table 1. +/+ In this study, we investigated SREBP1c and 2/2 SREBP1c mice under normal chow diet (NCD) or Preparation of Recombinant Adenovirus a high-fat diet (HFD) to understand the roles of SREBP1c Adenoviral plasmid were constructed as previously de- in pancreatic islets. We demonstrate that pancreatic scribed (26). cDNAs for SREBP1c and PAX4 were incor- SREBP1c plays an important role in b-cell compensatory porated into an AdTrack-CMV shuttle vector and an responses upon metabolic stress, accompanied with regu- Ad-Easy vector. Adenoviruses were amplified in human lating b-cell proliferation and survival. In pancreatic islets, embryonic kidney 293A (HEK293A) cells and isolated by we identified that PAX4 could act as a downstream medi- CsCl density centrifugation. Green fluorescent protein ator of SREBP1c to control b-cell growth. In addition, was coexpressed from an independent promoter with transplantation of SREBP1c-overexpressing primary islets inserted cDNA. Empty virus expressing only the gene ameliorated glucose intolerance in diabetic animals, im- for green fluorescent protein served as control (MOCK). plying that elevated expression of SREBP1c would poten- b tiate -cell function. Collectively, our data suggest that the Cell Culture and siRNA SREBP1c-PAX4 axis would play a pivotal role in compen- MIN6 cells were cultured in DMEM medium containing satory responses of pancreatic islets under metabolic 15% FBS (HyClone). HEK293T, aTC1–6 cells were cultured stress. in DMEM containing 10% FBS. Harvested primary islets were cultured in RPMI 1640 medium (HyClone) with 10% RESEARCH DESIGN AND METHODS FBS. Two kinds of siRNAs were designed and produced Animals and Treatment by Bioneer (Daejeon, South Korea). The antisense siPAX4 2/2 SREBP1c mice were provided by Dr. Jay D. Horton at sequences were as follows: antisense, UGGUACUCCUCA- the University of Texas Southwestern Medical Center CAGAAGG and ACUGUCAAAUAGAGGCCUC. siRNA was (Dallas, TX). db/+ and db/db male mice were obtained transfected using lipofectamine-iMAX (Thermo Fisher 2/2 from Daehan Bio (Seoul, Korea). SREBP1c and Scientific) into cell lines. +/+ SREBP1 littermates were maintained on a NCD (Zeigler) for 8 weeks. They were fed with NCD or 60% HFD (Re- Immunohistochemistry search Diets Inc.) for 12 weeks. For fasted/refed experi- The pancreata were isolated from mice, fixed in 4% para- ments, blood samples were collected after fasting for 16 h formaldehyde, and embedded in paraffin block. Paraffin and refeeding for 2 h. Mouse tissue specimens were blocks were cut into 5-mm-thick sections with 30-mm immediately stored at 280°C. For the glucose tolerance intervals. Pancreas sections from NCD- or HFD-fed mice test (GTT), mice were fasted for 16 h and intraperitoneally and db/db mice were stained with anti-SREBP1 (SC-367; 2 injected with glucose (1 g $ kg 1 body weight for mice). Santa Cruz Biotechnology), anti-insulin (ab7842; Abcam), Blood glucose levels were measured in tail vein blood anti-pax4 (ab42450; Abcam), anti-glucagon (ab10983; samples by using a Contour TS Blood Glucose Meter Abcam), and anti-Ki67-FITC (11-5698-80; eBioscience) anti- (Bayer). For the insulin tolerance test (ITT), ad libitum bodies. Species-specific secondary antibodies staining or mice were intraperitoneally injected with insulin (1 unit/kg diaminobenzidine staining was followed. For the islet mor- body weight for mice). All experiments with mice were phology analysis, specimens were viewed on a confocal LSM approved by the Seoul National University Institutional 700 System (Carl Zeiss). The areas of the images were Animal Care and Use Committee. processed and measured using ImageJ software.

In Vitro Cell Proliferation Assay Chromatin Immunoprecipitation Assay Cell proliferation rates were assayed using a Cell Counting Chromatin immunoprecipitation (ChIP) assays were con- Kit-8 reagent. Briefly, cell growth curves were generated ducted as previously described (26). Briefly, extracted diabetes.diabetesjournals.org Lee and Associates 83 from total cell lysates were immunoprecipitated insulin level was analyzed (fasting [16 h]/refeeding [2 h]), with anti-SREBP1 antibody (557036; BD Biosciences) or and a GTT was performed (fasting 16 h). IgG (sc-2025; Santa Cruz Biotechnology) for 2 h. Pre- cipitated DNA fragments were analyzed by quantitative Apoptosis Assay real-time PCR (qRT-PCR) using primer sets that encom- Apoptosis rates were determined using an in situ cell death passed the sterol regulatory element–containing region detection kit (12156792910; Roche) according to the of the mouse PAX4 gene promoter and non–sterol manufacturer protocol. Fluorescein-labeling images were fl regulatory element negative control region. The primer taken by a uorescence microscope (Olympus) using an excitation wavelength of 450–500 nm and a detection sequences for the ChIP assay were as follows: sense, 59- wavelength of 515–565 nm. Peroxidase-labeling images GTATAATTGTGAGCAGATGGCG-39 and antisense, 59- were monitored under a microscope, and statistical anal- GGCCTAGCAAGCCCAAA-39; negative control primer: 9 9 yses of the obtained images were performed using sense, 5 -TTCCAGGCAAGAACTCACCT-3 and antisense, LSM510 software (Carl Zeiss). 59-TATCGTTTCCCAGCCATCA-39. Cell Cycle Analysis Mouse Islet Studies Trypsinized pancreatic islets were washed with PBS and The pancreatic islets were isolated from male mice using fixed and permeabilized using Fixation/Permeabilization the intraductal collagenase technique. The pancreas was Concentrate (eBioscience) for 30 min. Fixed cells were dissected from the surrounding tissues and digested in incubated with propidium iodide solution containing a shaking bath for 12 min at 37°C with collagenase P 0.1% Nonidet P-40, 100 mg/mL RNase, and 2.5 mg/mL fi (Roche). The islets were puri ed using Ficoll gradient propidium iodide for 30 min. Stained cells were analyzed ’ solutions (29%, 24%, and 15% [w/v] in Hanks balanced and quantified for each stage by flow cytometry using salt solution). After centrifugation for 20 min, the islets a FACSCanto II System (BD Biosciences). were collected and washed with cold Hanks’ balanced salt solution. The isolated islets were cultured in RPMI Statistical Analysis 1460 medium supplemented with 10% FBS (Gibco). For Sample sizes were chosen based on pilot experiments that ex vivo experiments or transplantation, primary islets ensured adequate statistical power with similar variances. were incubated with adenovirus (40 multiplicity of in- Multiple comparisons were performed by one-way ANOVA, fection) for 12 h in RPMI 1640 serum-free medium and or by two-way ANOVA when two conditions were in- replaced to RPMI 1640 medium with 10% FBS media for volved. Statistical significance was assessed by the 2 days. For static glucose-stimulated insulin secretion Student t test and are presented as the mean 6 SD assays with mouse islets, ;20 islets were hand picked, determined from at least three independent experiments. incubated for 2 h in Krebs-Ringer bicarbonate buffer at Values of P , 0.05 were considered to be statistically 37°C, 5% CO , and then incubated for 60 min in 2.8 2 significant differences. All n values defined in the legends mmol/L glucose in the presence or absence of 30 mmol/L refer to biological replicates, unless otherwise indicated. If KCl or 22.2 mmol/L glucose. Secreted insulin was mea- sured using an insulin ELISA kit (Morinaga Ultra Sensitive technical failures such as the failure of intraperitoneal injection occurred, these samples were excluded from Mouse Insulin ELISA kit; Morinaga Institute of Biolog- fi ical Science), and normalized to the total amounts of the nal analysis. protein. Total insulin contents of the pancreas were extracted with acidic ethanol (0.2 mol/L HCl in 75% ethanol) RESULTS andmeasuredwithaninsulinELISAkit. SREBP1c Expression Is Upregulated in Pancreatic Islets of Obese and Diabetic Animals Primary Islet Transplantation It has been well established that SREBP1c is closely asso- Transplantation of primary islets was performed as pre- ciated with metabolic disorders, including obesity and viously described (27). Mice were intraperitoneally injec- insulin resistance (28,29). However, it is not thoroughly ted 150 mg/kg STZ. After 3 days of injections, primary understood whether SREBP1c might be involved in insulin islets were transplanted into recipient mice showing metabolism in obese pancreatic islets. To address this, we hyperglycemia .350 mg/dL. For islet transplantation, investigated the levels of SREBP1c protein and mRNA in ;200 freshly isolated islets were aspirated into a 200-mL pancreatic islets. In lean mice, SREBP1c protein was abun- pipette tip, and the pipette tip was connected to a sil- dantly expressed in the nuclei of insulin-positive b-cells icon tube (internal diameter 3 outer diameter = 0.58 3 (Fig. 1A). In pancreatic islets, the levels of SREBP1c protein 0.965 mm; Becton Dickinson). Under anesthesia, the and mRNA were higher in HFD-fed obese mice than in right kidney of the recipient mouse was exposed through NCD-fed lean mice (Fig. 1A–C). Similarly, pancreatic ex- an incision and primary islets were injected under the pression of SREBP1c was upregulated in db/db mice com- outersurfaceofthekidney.Thetubewasremoved,and pared with nondiabetic db/+ mice (Fig. 1D–F). In contrast, the capsulotomy was cauterized. After transplantation, the SREBP1a, another SREBP1 isoform, was not significantly 84 SREBP1c-PAX4 Axis in b-Cell Compensation Diabetes Volume 68, January 2019

Figure 1—SREBP1c is upregulated in the pancreatic islets of obese and diabetic animals. A: Immunofluorescence staining of pancreatic sections of C57BL/6J mice on NCD (n = 5) or HFD (n = 5) using anti-SREBP1 (red) and anti-insulin (green) antibodies. Scale bar, 20 mm. B: Relative level of SREBP1 in pancreata of NCD and HFD. In the insulin-positive area, the size of the SREBP1-positive area was measured using ImageJ. C: qRT-PCR analysis of SREBP1c mRNA expression in pancreatic islets of NCD- and HFD-fed mice. D: Immunofluorescence staining of pancreatic sections of lean (db/+)(n = 4) and db/db (n = 5) mice using anti-SREBP1 (red) and anti-insulin (green) antibodies. Scale bar, 20 mm. E: Relative levels of SREBP1 in pancreases of db/+ mice and db/db mice. F: qRT-PCR analysis of SREBP1c mRNA expression in pancreatic islets of db/+ and db/db mice. The data represent the mean 6 SD. *P , 0.05; **P , 0.01 vs. lean (db/+) mice.

elevated in pancreatic islets of HFD-fed obese mice and increased blood glucose under the refed condition (Fig. 2B 2/2 db/db mice (Supplementary Fig. 1A and B). These results and C). Because refed SREBP1c mice exhibited reduced suggest that SREBP1c expression would be upregulated levels of serum insulin, we hypothesized that SREBP1c in obese and diabetic pancreatic islets. deficiency might affect systemic glucose homeostasis. To +/+ 2/2 test this, SREBP1c and SREBP1c mice were sub- SREBP1c-Deficient Mice Exhibit Glucose Intolerance jectedtoaGTTandanITT.UponNCDorHFDfeeding, 2/2 Given that pancreatic b-cell dysregulation leads to im- SREBP1c mice were shown to be glucose intolerant +/+ paired glucose homeostasis, we raised the question of compared with SREBP1c mice (Fig. 2D and E). How- 2/2 whether SREBP1c deficiency may alter glucose and/or ever, NCD-fed SREBP1c mice did not exhibit signif- insulin metabolism. To answer this, we investigated met- icant differences in ITT (Fig. 2F), implying that decreased abolic phenotypes of SREBP1c-deficient mice upon being levels of postprandial insulin in SREBP1c-deficient mice +/+ 2/2 fed a NCD or HFD. Both SREBP1c and SREBP1c mice might be uncoupled to insulin sensitivity in lean animals. showed similar body weight gains with NCD or HFD Together, these findingssuggestthatSREBP1cdeficiency feeding (Fig. 2A). Nonetheless, NCD- or HFD-fed might alter the function of pancreatic islets, leading to 2/2 SREBP1c mice showed decreased serum insulin and glucose intolerance. diabetes.diabetesjournals.org Lee and Associates 85

Figure 2—SREBP1c-deficient mice exhibit glucose intolerance. A: SREBP1c+/+ mice and SREBP1c2/2 mice (n = 5/group) were fed either NCD or HFD. Body weight was measured during the experimental period. *P , 0.05 vs. HFD-fed SREBP1c+/+ mice. B: Levels of serum insulin (left panel) and blood glucose (right panel) in fasted (16 h)/refed (2 h) NCD-fed mice. *P , 0.05 vs. NCD-fed SREBP1c+/+ mice. C: Levels of serum insulin (left panel) and blood glucose (right panel) of fasted (16 h)/refed (2 h) HFD-fed mice. **P , 0.01 vs. HFD-fed SREBP1c+/+ mice. D: Intraperitoneal GTT and area under the curve (AUC) analysis of NCD-fed SREBP1c+/+ and SREBP1c2/2 mice. *P , 0.05 vs. SREBP1c+/+ mice. E: Intraperitoneal GTT in HFD-fed mice. The data represent mean 6 SD. *P , 0.05, **P , 0.01 vs. HFD-fed SREBP1c+/+ mice. F: ITT and AUC analysis of SREBP1c+/+ and SREBP1c2/2 mice.

2/2 SREBP1c Deficiency Leads to a Decrease in Pancreatic insulincontentsinthepancreataofSREBP1c mice +/+ b-Cell Mass were lower than those in the pancreata of SREBP1c Reduced b-cell mass has been considered to be one of the mice (Fig. 3C). In addition, the numbers of pancreatic +/+ major characteristics of diabetic pancreatic islets (30). To islet clusters were not different between SREBP1c and 2/2 determine whether impaired glucose tolerance in SREBP1c mice (Fig. 3D). These data imply that a de- 2/2 SREBP1c mice might result from decreased b-cell crease in b-cell numbers in islet clusters might confer 2/2 mass, we histologically analyzed pancreatic islets. In low insulin contents in the pancreata of SREBP1c the embryo stage, there was no significant difference mice. in the development or distribution of insulin-positive Because b-cell proliferation and apoptosis are crucial +/+ 2/2 cells from SREBP1c and SREBP1c mice (Supple- factors to decide b-cell mass (6,31), we next investigated mentary Fig. 2A and B), implying that SREBP1c deficiency what factors may be involved in the reduction of b-cell 2/2 might have little, if any, effect on b-cell differentiation mass in SREBP1c mice. As shown in Fig. 3E and F, the during embryonic development. In adult stage, the in- number of Ki67-positive b-cells, which represent prolifer- 2/2 sulin-positive b-cell area in SREBP1c mice was smaller ating b-cells, was lower in pancreatic islets from NCD- and +/+ 2/2 +/+ than that in SREBP1c mice (Fig. 3A and B). Although HFD-fed SREBP1c mice compared with SREBP1c the insulin-positive b-cell area was expanded by HFD mice. Moreover, mRNA levels of cell cycle–related genes 2/2 feeding, the extent of increase was much less in HFD-fed were declined in pancreatic islets from SREBP1c mice 2/2 SREBP1c mice.Inaccordancewiththesedata,total (Supplementary Fig. 3), indicating that SREBP1c deficiency 86 SREBP1c-PAX4 Axis in b-Cell Compensation Diabetes Volume 68, January 2019

Figure 3—SREBP1c-deficient mice exhibited reduced pancreatic b-cell area. A: Immunohistochemistry analysis of pancreatic sections of SREBP1c+/+ mice and SREBP1c2/2 mice using anti-insulin (dark brown) antibody and diaminobenzidine staining. Magnification 340. B: Relative islet size of SREBP1c+/+ mice and SREBP1c2/2 mice. Relative islet size was calculated by dividing the insulin-positive area by the pancreatic area. C: Pancreatic insulin contents. Data represented the mean 6 SD; *P , 0.05, **P , 0.01 vs. NCD-fed SREBP1c+/+ mice; #P , 0.05, ##P , 0.01 vs. HFD-fed SREBP1c+/+ mice. D: Insulin-positive islets were counted in pancreatic sections of SREBP1c+/+ and SREBP1c2/2 mice using anti-insulin (green) antibodies (n =5pergroup).E:Immunofluorescence staining of pancreatic sections of SREBP1c+/+ mice and SREBP1c2/2 mice using anti-Ki67 (red) and anti-insulin (green) antibodies. The white arrows represent Ki67-positive cells. n = 5 per group. Scale bars = 20 mm. F: Ratio of Ki67-positive cells to total cells as identified by DAPI staining. *P , 0.05, **P , 0.01 vs. NCD-fed SREBP1c+/+ mice; ##P , 0.01 vs. HFD-fed SREBP1c+/+ mice. G: Immunofluorescence staining of pancreatic sections of SREBP1c+/+ mice and SREBP1c2/2 mice using TUNEL assay kit. The white arrows represent TUNEL-positive cells. n = 5/group. Scale bars = 20 mm. H: Ratio of TUNEL-positive cells to total cells as identified by DAPI staining. Scale bar, 20 mm. *P , 0.05 vs. NCD-fed SREBP1c+/+ mice; #P , 0.05 vs. HFD-fed SREBP1c+/+ mice. diabetes.diabetesjournals.org Lee and Associates 87 might lead to deterioration of b-cell proliferation. On the overexpressing MIN6 b-cells. Although ectopic expres- other hand, the number of TUNEL-positive apoptotic sion of SREBP1c in pancreatic b-cells upregulated ex- 2/2 b-cells in SREBP1c mice was higher than that of pression of cell cycle genes and secretion-related genes, +/+ SREBP1c mice (Fig. 3G and H). Together, these results PAX4 suppression attenuated the effects of SREBP1c on propose that SREBP1c deficiency would decrease b-cell (Fig. 5A). Consistently, PAX4 knock- mass, accompanied with reduced b-cell proliferation and down downregulated the rate of SREBP1c-induced b-cell increased b-cell apoptosis. proliferation (Fig. 5B). Moreover, SREBP1c overexpres- sion elevated insulin secretion upon high glucose stim- SREBP1c Regulates PAX4 Expression in Pancreatic uli, whereas PAX4 suppression diminished this increase Islets (Fig. 5C), indicating that PAX4 would be a downstream Since SREBP1c is a transcriptional activator, we hypoth- player of SREBP1c-mediated b-cell proliferation and esized that SREBP1c could modulate b-cell proliferation secretory function. In accordance with these data, by regulating b-cell–specifictargetgenes.Toidentify PAX4 overexpression rescued mRNA expression of cell SREBP1c target genes in b-cells, we scrutinized gene cycle genes and secretion-related genes whose expres- 2/2 expression profiles in SREBP1c-overexpressing b-cell sion was downregulated in pancreatic islets of SREBP1c lines and primary islets (Supplementary Fig. 4A). Among mice (Fig. 5D). Taken together, these data evidently sug- several candidates, we found that PAX4 might be a po- gest that the SREBP1c-PAX4 axis could promote b-cell tential target gene of SREBP1c. Previously, it has been proliferation and insulin secretion by regulating gene reported that PAX4 is selectively expressed in pancreatic expression. endocrine cells and plays critical roles in the proliferation and survival of b-cells (21). As shown in Fig. 4A and B,the SREBP1c in Transplanted Islets Helps to Ameliorate level of PAX4 protein was reduced in pancreatic islets of Glucose Intolerance in Diabetic Animals 2/2 NCD- or HFD-fed SREBP1c mice. Consistently, the level To test the idea that SREBP1c might modulate the phys- of PAX4 mRNA was decreased in pancreatic islets from iological functions of pancreatic islets, we infected primary 2/2 +/+ SREBP1c mice compared with SREBP1c mice (Fig. 4C). islets with SREBP1c adenovirus (Ad-SREBP1c). In primary As the proximal promoter regions of the PAX4 gene contain islets, SREBP1c overexpression stimulated mRNA levels of putative binding sites for SREBP1c (Supplementary Fig. cell cycle genes and secretion-related genes (Fig. 6A). 4B), we decided to test the idea whether SREBP1c might Consistently, ectopic expression of SREBP1c in pancreatic upregulate the promoter activity of the PAX4 gene. As islets led to an increased S and G2/M phase population showninFig.4D, ectopic expression of SREBP1c trans- (Supplementary Fig. 6). Moreover, SREBP1c-overexpressing activated mouse PAX4 promoter activity in a luciferase islets exhibited augmented insulin secretion upon reporter assay. Accordingly, ChIP assays with SREBP1 glucose stimulation (Fig. 6B). To further investigate antibody revealed physical bindings of SREBP1 to the whether SREBP1c would mediate functional compensation promoter of mouse PAX4 gene (Fig. 4E). Moreover, the of pancreatic islets and restore systemic glucose metabolism levelofPAX4mRNAwasincreasedbySREBP1cover- in diabetic animals, we transplanted SREBP1c-overexpressing expression in b-cells (Fig. 4F), indicating that PAX4 primary islets into the STZ-induced diabetic mice and would be a novel target gene of SREBP1c in pancreatic analyzed glucose metabolism. As expected, STZ potently islets. destroyed b-cells and led to hyperglycemia, whereas transplantation of wild-type primary islets infected SREBP1c Stimulates b-Cell Proliferation and Insulin with MOCK adenovirus relieved hyperglycemia in di- Secretion via PAX4 abetic mice (Fig. 6C). More importantly, there was It has been reported that pancreatic PAX4 plays profound a stronger lowering effect on blood glucose in the roles in b-cell proliferation and insulin secretion SREBP1c-overexpressing islet-transplanted group (Fig. (21,22). To examine whether PAX4 could modulate 6C). In addition, islet transplantation restored the se- b-cell proliferation, we analyzed gene expression with rum insulin level, which was further potentiated by PAX4 modulation using siRNA or adenovirus. Consis- SREBP1c-overexpressing islet transplantation (Fig. tent with previous reports (21,22), suppression of PAX4 6D). Next, we investigated the effects of islet trans- +/+ 2/2 via siRNA decreased the growth rate of b-cells and plantation from SREBP1c and SREBP1c mice reduced the expression of cell cycle genes (Supplemen- on glucose metabolism. In GTT analysis, the SREBP1c tary Fig. 5A–C). In contrast, PAX4 overexpression in- knockout islet-transplanted group exhibited glucose in- creased b-cell proliferation and elevated the expression tolerance compared with the SREBP1c wild-type islet- 2/2 of cell cycle genes (Supplementary Fig. 5D and E), in- transplanted group (Fig. 6E). Moreover, the SREBP1c dicating that PAX4 expression has a positive correlation islet transplantation group showed decreased serum with b-cell proliferation. Next, to investigate whether insulin levels after 30 min of glucose challenge (Fig. PAX4 would indeed act as a downstream mediator of 6F). Collectively, these data suggest that SREBP1c would SREBP1c in b-cell proliferation and insulin secretion, we be crucial for the quantity and quality of pancreatic examined the effects of PAX4 suppression in SREBP1c- islets. 88 SREBP1c-PAX4 Axis in b-Cell Compensation Diabetes Volume 68, January 2019

Figure 4—SREBP1c regulates PAX4 expression as a pancreatic target gene. A: Immunofluorescence staining of pancreatic sections of SREBP1c+/+ mice and SREBP1c2/2 mice using anti-PAX4 (red) and anti-insulin (green) antibodies (n = 5/group). Scale bar, 20 mm. B: Relative abundance of PAX4-positive signals in the insulin-positive area. ImageJ was used for analysis. C: qRT-PCR analysis of PAX4 mRNA expression in pancreatic islets of SREBP1c+/+ mice and SREBP1c2/2 mice. *P , 0.05, **P , 0.01 vs. NCD-fed SREBP1c+/+ mice; #P , 0.05, ##P , 0.01 vs. HFD-fed SREBP1c+/+ mice. D: Luciferase activity of the PAX4 promoter was measured after cotransfection with expression plasmids encoding either SREBP1c or mock in HEK293T cells. Total cell lysates were subjected to luciferase and b-galactosidase assays. **P , 0.01 vs. mock. E: ChIP assay, showing PAX4 promoter occupancy by SREBP1 in MIN6 cells. F: Relative mRNA level of PAX4 gene in MIN6 cell lines with adenovirus infection. **P , 0.01 vs. Ad-MOCK.

SREBP1c-PAX4 Axis Is Involved in Glucagon reported that PAX4 is involved in the suppression of Metabolism glucagon expression and the transdifferentiation of Dysregulated interaction between pancreatic endocrine a-cells into b-cells (34). Thus, we decided to test whether cells and hyperglucagonemia has been recognized as an- the SREBP1c-PAX4 axis might also affect glucagon metab- +/+ other contributor to imbalanced glucose homeostasis in olism in pancreatic islets. Relative to SREBP1c mice, 2/2 patients with diabetes (32,33). In addition, it has been SREBP1c mice exhibited higher levels of serum diabetes.diabetesjournals.org Lee and Associates 89

Figure 5—SREBP1c regulates b-cell proliferation and function via PAX4. A: Relative mRNA levels of cell cycle genes were analyzed in MIN6 cells after cotransfection with adenovirus and siRNA. *P , 0.05; **P , 0.01 vs. siNC + Ad-MOCK; #P , 0.05 vs. siNC + Ad-SREBP1c. B: Growth curves of MIN6 cells. Cells were counted at 24-h intervals after cotransfection with adenovirus and siRNA. **P , 0.01. C: Relative level of secreted insulin (insulin in conditioned media/total insulin contents [%]) in MIN6 cells after cotransfection with adenovirus and siRNA. MIN6 cells were incubated in 2.8 or 22.2 mmol/L glucose in Krebs-Ringer bicarbonate buffer. *P , 0.05. D: Relative mRNA levels of cell cycle genes in pancreatic islets of SREBP1c2/2 mice with adenovirus infection. *P , 0.05; **P , 0.01 vs. Ad-MOCK. Pcna, proliferating cell nuclear antigen.

glucagon (Fig. 7A) and glucagon mRNA (Fig. 7B). Accord- DISCUSSION ingly, the degree of glucagon-positive a-cells was higher in 2/2 +/+ To resolve hyperglycemia upon metabolic stress, pancre- SREBP1c mice than in SREBP1c mice (Fig. 7C and D). atic islets adaptively increase the level of serum insulin Furthermore, the overexpression of either SREBP1c or through b-cell compensation. In this regard, the capacity PAX4 downregulated the level of glucagon mRNA in of b-cell compensation is crucial for glucose homeostasis a-cell lines (Fig. 7E), implying that the SREBP1c-PAX4 and protection against type 2 diabetes (35). Nonetheless, axis might be a negative regulator of glucagon expression the mechanisms underlying b-cell compensation are in pancreatic a-cells. not thoroughly understood. Our data provide compelling 90 SREBP1c-PAX4 Axis in b-Cell Compensation Diabetes Volume 68, January 2019

Figure 6—SREBP1c in transplanted islets helps to ameliorate glucose intolerance in diabetic animals. A: Relative mRNA levels of cell cycle genes and soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE)–related genes in primary islets with adenovirus infection. *P , 0.05; **P , 0.01 vs. Ad-MOCK. B: Glucose-stimulated insulin secretion studies using primary islets after transfection with adenovirus in response to low glucose (2.8 mmol/L) and high glucose (22.2 mmol/L). *P , 0.05 vs. Ad-MOCK. C and D: Primary islets were transplanted into STZ-induced diabetic mice. C: Blood glucose levels of from STZ-control, Ad-MOCK islet–transplanted group, Ad-SREBP1c islet–transplanted group after transplantation. D: Serum insulin levels from fasted (16 h)/refed (2 h) mice of STZ-control, Ad-MOCK islet– transplanted group, and Ad-SREBP1c islet–transplanted group. n = 3/group. *P , 0.05 vs. Ad-MOCK islet transplantation. E: Intraperitoneal GTT of islet transplantation groups. Primary islets from SREBP1c+/+ and SREBP1c2/2 mice. n = 3 per group. *P , 0.05; **P , 0.01 vs. SREBP1c+/+ islet–transplanted group. F: Serum insulin levels after glucose challenge in mice of Sham-control, SREBP1c+/+ islet– transplanted group, SREBP1c2/2 islet–transplanted group. n = 3/group. *P , 0.05 vs. SREBP1c+/+ islet–transplanted group.

evidence that pancreatic SREBP1c could play a key role in and to secrete insulin upon increased insulin demands. regulating compensatory responses in b-cells. We found Particularly, SREBP1c modulated various gene expressions that SREBP1c expression upregulated pancreatic islets in b-cells through a novel target gene, PAX4. On the in obese and diabetic animals. In addition, pancreatic contrary, SREBP1c deficiency hindered this compensatory SREBP1c potentiated the capacity of b-cells to proliferate process, resulting in glucose intolerance (Fig. 8). Collectively, diabetes.diabetesjournals.org Lee and Associates 91

suggest that SREBP1c plays key roles in regulating b-cell +/+ mass and function. First, compared with SREBP1c mice, the b-cell area was smaller and total insulin contents was 2/2 lower in the pancreata of SREBP1c mice. Second, SREBP1c expression was upregulated in pancreatic islets of obese and diabetic animals. Third, SREBP1c overexpres- sion in b-cells stimulated mRNA levels of cell cycle genes and increased b-cell growth rate. Last, transplantation of SREBP1c-overexpressing islets ameliorated glucose intol- erance in diabetic mice. These findings unravel a novel mechanism in which pancreatic SREBP1c would play an important role in b-cell compensation. It has been reported (18) that SREBP1c is associated with lipid metabolism and cell cycle progression. For in- stance, SREBP1 influences cell growth through providing lipid metabolites as building blocks for cell division (17). Furthermore, SREBP1 is able to regulate expression of certain set of genes associated with cell cycle progression (18,36). To date, it remains unclear whether SREBP1c activation is involved in b-cell proliferation. Here, we demonstrate that SREBP1c augments b-cell proliferation through a novel target gene, PAX4. We observed that SREBP1c binds to the promoter of the PAX4 gene and transactivates its promoter in b-cells. Also, SREBP1c in- creased the expression of PAX4 mRNA and protein in pancreatic islets. Moreover, the expression levels of PAX4 and cell cycle genes were downregulated in pancreatic islets 2/2 of SREBP1c mice. Conversely, PAX4 upregulation res- cued the expression of cell cycle genes that were decreased in SREBP1c-deficient pancreatic islets. Given that PAX4 suppression significantly attenuated SREBP1c-induced b-cell growth, it is likely that SREBP1c could promote b-cell proliferation, at least in part, by upregulating PAX4. Figure 7—SREBP1c-PAX4 pathway is involved in glucagon metab- A SREBP1c+/+ SREBP1c2/2 Together, these data imply that the SREBP1c-PAX4 axis olism. : Serum glucagon levels in and b mice. n = 5/group. *P , 0.05 vs. NCD-fed SREBP1c+/+ mice. B: qRT- would play a key role in -cell proliferation. PCR analysis of glucagon mRNA expression in pancreatic islets of Dysregulation of two pancreatic hormones, character- 2/2 SREBP1c+/+ and SREBP1c mice. C and D: Relative sizes of the ized by insulin insufficiency and hyperglucagonemia, leads a SREBP1c+/+ 2/2 C -cell areas in and SREBP1c mice. : Immunohis- to hyperglycemia and contributes to increased diabetes tochemistry analysis of glucagon (dark brown) in pancreata sections of SREBP1c+/+ and SREBP1c2/2 mice. Magnification 340. D: Rel- incidence (37). Recently, it has been reported (25,38) that ative sizes of the a-cell area was calculated by dividing glucagon- single nucleotide polymorphisms and mutations of PAX4 positive area by total pancreatic area. *P , 0.05 vs. SREBP1c+/+ gene are closely associated with diabetes in numerous mice. E: qRT-PCR analysis of glucagon mRNA expression in aTC cell lines after adenovirus infection. *P , 0.05 vs. Ad-MOCK. ethnic groups. Since PAX4 has opposite roles in the de- velopment and function of pancreatic endocrine cells (39), we hypothesized that the dysregulation of the SREBP1c- PAX4 axis might be associated with an imbalance our findings propose that the SREBP1c-PAX4 axis would play of pancreatic endocrine cells. In pancreatic islets of 2/2 an essential role in b-cell compensation. SREBP1c mice, we observed that the degree of 2/2 SREBP1c mice exhibited normal growth and dis- TUNEL-positive apoptotic b-cells was elevated compared +/+ played body weight gain similar to that of age-matched with SREBP1c mice. Accordingly, the number of b-cells +/+ 2/2 2/2 SREBP1c mice. However, SREBP1c mice showed was decreased in SREBP1c mice. To our surprise, we postprandial hyperglycemia and glucose intolerance, could also detect increased numbers of pancreatic a-cells 2/2 whereas they were not defective to insulin sensitivity. and hyperglucagonemia in SREBP1c mice, implying 2/2 In pancreatic islets of SREBP1c mice, the levels of that SREBP1c deficiency may influence the ratio of two insulin secretion with glucose stimuli were decreased. primary pancreatic endocrine cells, such as a-cells and These data raised the possibility that SREBP1c deficiency b-cells. Intriguingly, PAX4 and SREBP1c overexpression might mediate pancreatic islet dysfunction, leading to suppressed glucagon expression in pancreatic a-cells. glucose intolerance. In this study, several lines of evidence Given that ectopic expression of PAX4 has dual roles 92 SREBP1c-PAX4 Axis in b-Cell Compensation Diabetes Volume 68, January 2019

Figure 8—The SREBP1c-PAX4 axis is involved in b-cell compensation. Upon metabolic stress, SREBP1c expression is upregulated in pancreatic islets. SREBP1c upregulates PAX4 expression and b-cell proliferation, contributing to b-cell compensation. Conversely, SREBP1c-deficient mice exhibit insufficient b-cell compensation and fail to maintain glucose homeostasis under metabolic stress.

in enhancing b-cell survival and inhibiting glucagon ex- physiological range might have crucial roles in pancreatic pression (40), it is plausible to speculate that the SREBP1c- b-cells. Although we could not exclude the possibility that PAX4 axis could participate in the determination of ratio aberrant upregulation of SREBP1c might result in b-cell and functions of pancreatic endocrine cells. In future, it lipotoxicity under pathophysiological conditions, our needs to be elucidated whether developmental defects or in vivo data propose that an appropriate increase of indirect pathways might be attributable to SREBP1c-de- SREBP1c expression in obese pancreatic islets would be ficient pancreatic islets. Nonetheless, our data propose required for b-cell compensation. that dysregulated SREBP1c-PAX4 axis might be involved It has been previously proposed that deficiency of in pancreatic hormonal imbalance in obesity-induced SREBP1 does not cause significant effects on islet volume diabetes. or insulin contents in pancreas (44). It appears that a It has been reported that intracellular lipid metabolites previous report is somewhat contradictory to our finding 2/2 have various effects on b-cell functions and their pro- from SREBP1c mice. This discrepancy might result from liferation (41–43). Here, we found that SREBP1c expres- using different animal models. Here, we analyzed SREBP1c sion was enhanced in pancreatic islets of obese and isoform-specific knockout mice that have the SREBP1a diabetic mice. Accordingly, the level of triglycerides was isoform (46). Instead, Takahashi et al. (44) have examined 2/2 upregulated in obese pancreatic islets (Supplementary Fig. SREBP1 mice that do not have both isoforms such as 2/2 7). Previously, it has been suggested that an uncontrolled SREBP1a and SREBP1c. Unlike SREBP1c mice, it has 2/2 increase of SREBP1c expression could induce detrimental been reported that SREBP1 mice exhibited develop- effects on pancreatic b-cells, eventually leading to lipotox- mental defects and high lethality (50–85% died in utero at 2/2 icity and cell death (44,45). However, our in vivo data embryonic day 11) (47). Moreover, surviving SREBP1 showed that an increase of SREBP1c expression in pan- mice produced a truncated form of SREBP1 protein, which creatic islets of HFD-fed animals was not sufficient to might distort pancreatic phenotypes. provoke lipotoxicity. Instead, we could observe that b-cell In conclusion, we have newly identified the regulatory apoptosis was increased in SREBP1c-deficient islets, im- mechanism of b-cell compensation through the SREBP1c- plying that the moderate induction of SREBP1c within PAX4 axis. Our data suggest that pancreatic SREBP1c plays diabetes.diabetesjournals.org Lee and Associates 93 an important role in fine-tuning b-cell function upon 12. Kim JB, Sarraf P, Wright M, et al. Nutritional and insulin regulation of fatty insulin demands to confer systemic glucose homeostasis. acid synthetase and leptin gene expression through ADD1/SREBP1. J Clin Invest Given that the SREBP1c-PAX4 axis is crucial to maintain 1998;101:1–9 functional b-cell mass, it is likely that the regulation of 13. Shimano H, Yahagi N, Amemiya-Kudo M, et al. Sterol regulatory element- SREBP1c activity might be a potential target to treat binding protein-1 as a key transcription factor for nutritional induction of lipogenic – metabolic disease as well as pancreatic dysfunction. enzyme genes. J Biol Chem 1999;274:35832 35839 14. Latasa MJ, Moon YS, Kim KH, Sul HS. Nutritional regulation of the fatty acid synthase promoter in vivo: sterol regulatory element binding protein functions Acknowledgments. The authors thank the members of the laboratory of through an upstream region containing a sterol regulatory element. Proc Natl Acad adipocyte and metabolism research for helpful discussion. The authors also Sci U S A 2000;97:10619–10624 thank Dr. Jay Horton at the University of Texas Southwestern Medical Center for 15. Kakuma T, Lee Y, Higa M, et al. Leptin, troglitazone, and the expression of providing SREBP1c2/2 mice. sterol regulatory element binding proteins in liver and pancreatic islets. Proc Natl Funding. This work was supported by the National Creative Research Initiative Acad Sci U S A 2000;97:8536–8541 Program of the National Research Foundation funded by the Korea government 16. Choe SS, Choi AH, Lee JW, et al. Chronic activation of (Ministry of Science, ICT and Future Planning, 2011-0018312). G.L., Y.Y.K., and induces beta-cell apoptosis through hyperactivation of lipogenesis: liver X J.P. were supported by the BK21 program. receptor-mediated lipotoxicity in pancreatic beta-cells. Diabetes 2007;56: Duality of Interest. No potential conflicts of interest relevant to this article 1534–1543 were reported. 17. Bengoechea-Alonso MT, Ericsson J. Cdk1/cyclin B-mediated phosphoryla- Author Contributions. G.L. executed most experiments, designed the tion stabilizes SREBP1 during mitosis. Cell Cycle 2006;5:1708–1718 project, prepared the manuscript, contributed to performing animal experiments, 18. Lee JH, Jeon YG, Lee K-H, et al. 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