Diabetes Volume 68, June 2019 1197

Deficiency of ZnT8 Promotes Adiposity and Metabolic Dysfunction by Increasing Peripheral Serotonin Production

Zhuo Mao,1 Hui Lin,1 Wen Su,1 Jinghui Li,1 Minsi Zhou,1 Zhuoran Li,1 Beibei Zhou,2 Qing Yang,1 Mingyan Zhou,1 Ke Pan,2 Jinhan He,3 and Weizhen Zhang1,4

Diabetes 2019;68:1197–1209 | https://doi.org/10.2337/db18-1321

ZnT8 is a zinc transporter enriched in pancreatic b-cells, important physiological function of ZnT8 in metabolic and its polymorphism is associated with increased sus- disease progression. ZnT8 is highly abundant in pancreatic ceptibility to . However, the exact role of b-cells (3). Several colonies of global and b-ora-cell– ZnT8 in systemic energy metabolism remains elusive. In specific ZnT8 knockout mice have been generated to in- this study, we found that ZnT8 knockout mice displayed vestigate its effects on granule morphology, insulin increased adiposity without obvious weight gain. We – secretion, and systemic glucose metabolism (4 6). STUDIES OBESITY also observed that the intestinal tract morphology, Global ZnT8 knockout mice exhibit an exacerbation of motility, and gut microbiota were changed in ZnT8 diet-induced obesity and glucose intolerance compared knockout mice. Further study demonstrated that ZnT8 with wild-type mice (4,6). Unexpectedly, this phenotype was expressed in enteroendocrine cells, especially in was not observed in mice that lack ZnT8, specifically in b- 5-hydroxytryptamine (5-HT)–positive enterochromaffin or a-cells (6). This discrepancy strongly implies that the cells. Lack of ZnT8 resulted in an elevated circulating presence of ZnT8 in non–b/a-cell or extrapancreatic tis- 5-HT level owing to enhanced expression of tryptophan hydroxylase 1. Blocking 5-HT synthesis in ZnT8-deficient sues plays a critical role in organism energy homeostasis. mice restored adiposity, high-fat diet–induced obesity, Since ZnT8 is negligible in hypothalamus, fat, and skeletal and glucose intolerance. Moreover, overexpression of muscle (6), we speculated that ZnT8 might be expressed in human ZnT8 diabetes high-risk R325W increased other endocrine tissues or cells. 5-HT levels relative to the low-risk allele in RIN14B cells. The gastrointestinal (GI) tract contains the largest Our study revealed an unexpected role of ZnT8 in reg- number of endocrine cells. Many GI hormones play critical ulating peripheral 5-HT biogenesis and intestinal micro- roles in glucose homeostasis (7). 5-hydroxytryptamine environment, which might contribute to the increased (5-HT [serotonin]) is the most prevalent GI hormone risk of obesity and type 2 diabetes. that exerts both central and peripheral functions. More than 90% of 5-HT is synthesized in and released from the enterochromaffin cells. Other tissues, such as neurons, ZnT8 is a zinc transporter that is closely associated with adipose tissues, and pancreas, only produce a small both type 1 and type 2 diabetes mellitus (T1DM and amount of 5-HT (8). The initial and rate-limiting step of T2DM). It is an important autoantigen in patients with 5-HT synthesis is catalyzed by tryptophan hydroxylase T1DM (1). Meanwhile its polymorphism has also (TPH). There are two isoforms of TPHs: TPH1 in the been identified as a risk factor for T2DM (2), suggesting an peripheral tissues and TPH2 in the central nervous system

1Center for Diabetes, Obesity and Metabolism, Department of Physiology, Shenz- Received 14 December 2018 and accepted 4 March 2019 hen University Health Science Center, Shenzhen, Guangdong Province, China This article contains Supplementary Data online at http://diabetes 2 Institute for Advanced Study, Shenzhen University, Shenzhen, Guangdong .diabetesjournals.org/lookup/suppl/doi:10.2337/db18-1321/-/DC1. Province, China © 2019 by the American Diabetes Association. Readers may use this article as 3Department of Pharmacy, State Key Laboratory of Biotherapy and Cancer Center, long as the work is properly cited, the use is educational and not for profit, and the West China Hospital of Sichuan University, Chengdu, Sichuan, China work is not altered. More information is available at http://www.diabetesjournals 4Department of Physiology and Pathophysiology, School of Basic Science, Peking .org/content/license. University Health Science Center, Beijing, China Corresponding author: Weizhen Zhang, [email protected], or Jinhan He, [email protected] 1198 ZnT8 Regulates Peripheral Serotonin Production Diabetes Volume 68, June 2019

(CNS) and enteric neurons (9). The 5-HT system possesses insulin, chromogranin A (CgA), uncoupling 1 (UCP1), complex bioactivities mediated by various types of 5-HT b3-adrenergic receptor (AR), gastric inhibitory polypeptide, receptors expressed in different tissues. In the CNS, 5-HT and glucagon-like peptide 1 were from Abcam. Anti– acts as a neurotransmitter to regulate appetite, emotions, tyrosine hydroxylase (TH) antibody was from Millipore. sleep, and systemic metabolism through the sympathetic The tubulin, actin, and GAPDH antibodies were from nervous system (10). The function of peripheral 5-HT is Proteintech. Alexa Fluor 488 and 594 dyes were obtained relatively less clear compared with its central role. The from Molecular Probes. The horseradish peroxidase– classical action of peripheral 5-HT includes regulation of GI labeled secondary antibody was purchased from Amer- functions, such as motility, secretion, sensation, modula- sham BioSciences (GE Healthcare). tion of platelet coagulation, and bone density (11). In- terestingly, obesity increases peripheral 5-HT levels (12), Histology and Immunohistochemistry/ and the genetic polymorphism of TPH1 is associated with Immunofluorescent Staining fi obesity (13). Recent studies have found that peripheral The dissected tissues were xed with 4% paraformalde- 5-HT promotes white adipose tissue (WAT) lipogenesis hyde in PBS for 16 h at 4°C. The samples were sequentially fi and inhibits brown adipose tissue (BAT) adaptive thermo- dehydrated and embedded in paraf n. Tissue samples then m genesis (14,15). Genetic deficiency or pharmacological in- were sectioned at a 6- m thickness and used for standard fi hibition of 5-HT synthesis enzyme TPH1 in mice leads to hematoxylin-eosin staining and quanti cation. The quan- fi a resistance to diet-induced obesity and glucose intoler- ti cation was determined by Image J software. Immuno- fl ance (14,15), suggesting that peripheral 5-HT is an im- histochemistry and immuno uorescent staining were portant regulator of lipid metabolism and systemic energy performed following general protocols. Images were homeostasis. obtained by Nikon Eclipse Ti microscope. In this study, we generated a new strain of ZnT8 Oil Red O and PAS Staining knockout mice using transcription activator-like effector For Oil Red O staining, frozen liver sections were washed nuclease (TALEN) technology. We examined the presence in PBS once and fixed with 4% paraformaldehyde in PBS of ZnT8 in enteroendocrine cells (EECs) and its role in 5-HT biogenesis and lipid metabolism using cell biological for 15 min at room temperature and then washed three times with PBS. The sections were incubated in 60% and transgenic techniques. We also observed an unex- isopropyl alcohol and then stained with filtered Oil Red O pected change in colon morphology, function, and micro- solution (3 mg/mL) for 30 min and rinsed twice with biota in ZnT8-deficient mice, which may contribute to the distilled water. Periodic acid Schiff (PAS) staining for increased sensitivity of diet-induced obesity and T2DM. glycogen in the liver and goblet cells in the colon was RESEARCH DESIGN AND METHODS performed using a commercial kit (Solarbio, Beijing, China) according to the manufacturer’s instructions. Animals 2/2 ZnT8 knockout (Slc30a8 ) mice were generated by Cya- Oral and Intraperitoneal Glucose Tolerance Tests gen Biosciences Inc. (Guangzhou, China). Exon 3 of Slc30a8 ZnT8groupmicewerestarvedfor16h,followedby gene was selected as the target site. TALEN mRNAs gen- an oral glucose infusion or intraperitoneal glucose in- erated by in vitro transcription were then injected into jection (1.5 g/kg body weight). The blood glucose levels fertilized eggs from the C57BL/6N mouse strain for knock- were measured from the tail vein before and at 15, 30, out mouse production. The founders were genotyped by 60, 90, and 120 min after injection using a glucometer PCR followed by DNA sequencing analysis. The positive (Accu-Chek; Roche). founders were bred to the next generation, which was genotyped by PCR and DNA sequencing analysis. Insulin Tolerance Test For the high-fat diet (HFD) treatment experiment, male ZnT8 group mice were fasted for 4 h before insulin mice (aged 6–8 weeks) were fed either a normal chow diet tolerance testing and then received an injection of human (ND) or an HFD (45% fat calories, D12451, or 60% fat regular insulin (0.5 units/kg body weight). Blood glucose calories, D12492; Research Diets). For the TPH inhibitor levels were recorded before and at 15, 30, 60, 90, and injection experiment, PBS or 4-chloro-DL-phenylalanine 120 min after injection using a glucometer (Accu-Chek). methyl ester hydrochloride (PCPA) (300 mg/kg body weight) (C3635; Sigma-Aldrich) was administered as a daily intra- Blood Biochemistry and ELISA peritoneal injection. All animal experiments were under- Serum total triglycerides, total cholesterol, and nonester- taken with the approval of the Scientific Investigation Board ified fatty acid were measured using commercial kits of the Health Science Center of Shenzhen University. (Biosino Biotechnology and Wako Chemicals, respectively). Serum 5-HT levels were measured using the mouse ELISA Antibody kit (Enzo Life Sciences). Insulin levels were analyzed using The rabbit anti-ZnT8 polyclonal antibody was generated the mouse ultrasensitive insulin kit (Alpco), and catechol- against the synthetic mouse ZnT8 peptide (KPVNKDQCPG- amine levels were measured using ELISA kits (BioVision) DRPEHPEAGGIYH, 29–51 amino acids). Antibodies against according to the manufacturer’s instructions. diabetes.diabetesjournals.org Mao and Associates 1199

Cell Culture and Transfection RNA Extraction and Quantitative RT-PCR The RIN14B cells (purchased from Zeye Bio-Tech, Shanghai, Total RNA was isolated from mouse tissues or RIN14B cells China) were cultured in high-glucose DMEM (Gibco) containing using Direct-zol RNA MiniPrep Kit (R2052; Zymo Research). 10% FBS (Gibco) in a humidified incubator with 5% CO2 at One microgram of total RNA was used for reverse tran- 37°C. Cells were plated at optimal densities and grown for scription using PrimeScript RT Master Mix (Takara Bio). 24 h and then transfected with plasmids using Lipofect- SYBR Green–based real-time PCR was performed using amine 3000 (Thermo Fisher Scientific) according to the LightCycler 96 (Roche) with SYBR Premix Ex Taq II (Takara manufacturer’s instructions. Bio). The quantity of mRNA was calculated using the DDCt method. All reactions were performed in duplicate. The Total GI Transit Time primers used for quantitative PCR are listed in Supple- A solution of 6% carmine red (300 mL) (Sigma-Aldrich) mentary Table 1. suspended in 0.5% methylcellulose (Sigma-Aldrich) was administered by gavage through a 21-gauge round-tip Statistical Analysis feeding needle. The time at which gavage took place was All results are presented as mean 6 SEM. Data were recorded as T0. After gavage, fecal pellets were monitored analyzed with Student t test or one-way ANOVA followed at 10-min intervals for the presence of carmine red. Total by Bonferroni multiple comparison test. P , 0.05 was GI transit time was considered as the interval between T0 considered statistically significant. and the time of first observance of carmine red in stool. RESULTS Fecal Microbiota Sequencing 2/2 Slc30a8 Mice Display Increased Adiposity Stool samples freshly collected from each mouse were 2 To investigate the mechanism by which ZnT8 increases immediately frozen at 20°C and transported to the labo- T2DM sensitivity, we generated ZnT8 knockout mice 2 2 ratory with ice pack. Metagenomic sequencing and analysis (Slc30a8 / mice) on the C57BL/6N genetic background were performed by Novogene Bioinformatics Technology using TALEN technology (Supplementary Fig. 1A) by de- Co., Ltd. (Beijing, China). leting 2 base pairs in exon 3. We verified the newly generated ZnT8 knockout line by analyzing the pancreatic Metabolic Cage Studies and Body Composition expression and function of ZnT8. Loss of ZnT8 mRNA and Measurements of energy expenditure, respiratory ex- protein expression were first confirmed in ZnT8 knockout change ratio, indirect calorimetry, and physical activity pancreas (Supplementary Fig. 1B–D). Then dithizone using metabolic cages (Columbus Instruments) were per- 2 2 staining of isolated islets from Slc30a8 / mice demon- formed by the Biomedical Research Institute of Nanjing strated a marked reduction of intensity representing zinc University (Nanjing, China). Whole-body composition of depletion, indicating the functional inactivity of ZnT8 ZnT8 group mice was analyzed by EchoMRI. protein in pancreatic islets (Supplementary Fig. 1E). Dithizone Staining The overall morphology and growth curve were similar 2/2 The isolated islets were incubated in the 0.1 mg/mL between wild-type and Slc30a8 mice (Fig. 1A). How- fi dithizone (Sigma) solution at 37°C for 15 min. After ever, we observed a signi cant increase in fat mass in both 2/2 washing with Hanks’ balanced salt solution, the islets male and female Slc30a8 mice, whereas lean mass were examined with a stereomicroscope. showed a trend of reduction (Fig. 1B). The WAT mass, including epididymal WAT (eWAT) and subcutaneous WAT 2 2 Western Blotting (scWAT), was significantly increased in Slc30a8 / mice The tissues or RIN14B cells were quickly harvested, rinsed (Fig. 1C and D). This alteration resulted from increased with cold PBS, and homogenized in cold radioimmuno- adipocyte size (Fig. 1E and F). Furthermore, the expression precipitation assay buffer (150 mmol/L NaCl, 1% Triton levels of related to lipid synthesis, fatty acid uptake, X-100, 1% sodium deoxycholate, 0.1% SDS, 50 mmol/L and lipolysis were significantly increased, whereas lipogen- Tris-HCl, and 2 mmol/L EDTA, pH 7.4) supplemented with esis and fatty acid oxidation–relevant genes remained protease inhibitor cocktail (Roche). A total of 40 mgof unchanged (Fig. 1G–K). protein was loaded onto SDS-PAGE gels and electropho- In addition to WAT expansion, there was an increase of retically transferred to polyvinylidene fluoride membranes lipid accumulation in BAT as evidenced by increased mass (Bio-Rad). Transferred membranes were blocked with 5% and unilocular fat droplets in ZnT8 knockout mice (Fig. 2A nonfat milk in Tris-buffered saline with 0.1% Tween and B). Most genes related to thermogenesis, including 20 and then incubated with primary antibodies at 4°C Ucp1 and Pgc1a, remained unchanged (Fig. 2G and H). The overnight. After washing with Tris-buffered saline with lipogenesis and lipid synthesis genes were increased, 0.1% Tween 20, membranes were incubated with second- whereas the fatty acid oxidation–related genes were de- ary antibodies and developed with SuperSignal West Pico creased (Fig. 2C–F). Unexpectedly, we found a significant Chemiluminescent Substrate (Thermo Fisher Scientific). reduction of b3-AR protein level in BAT (Fig. 2H). The Signals were detected by the Amersham Imager 600 (GE neuronal marker TH remained unchanged, suggesting an Healthcare). intact neuron innervation to the BAT (Fig. 2H). Total 1200 ZnT8 Regulates Peripheral Serotonin Production Diabetes Volume 68, June 2019

Figure 1—Slc30a82/2 mice display increased adiposity. A: Body weight of male and female mice (n = 6 per genotype) over the course of the study. B: Body composition of 12-week-old male mice (n = 7 per genotype) and 12-week-old female mice (n = 6 per genotype). C: The weight- to-body weight ratio of eWAT and subcutaneous (sc)WAT in wild-type (WT) and Slc30a82/2 mice (n = 5 per genotype). D: Representative hematoxylin-eosin images of eWAT from WT and Slc30a82/2 mice. E and F: Average and distribution of eWAT fat cell size from 8-week-old male wild-type and Slc30a82/2 mice (n = 5 per genotype). G–K: The mRNA expression level of lipogenesis, lipid synthesis, fatty acid oxidation, lipolysis, and fatty acid uptake in eWAT from WT or Slc30a82/2 mice. Data are mean 6 SEM. *P , 0.05, **P , 0.01, ***P , 0.001 by unpaired Student t test.

sympathetic tone was normal as measured by serum was elevated (Fig. 3D and E). All these data suggest catecholamine (including dopamine, norepinephrine, and that ZnT8 deficiency increases fat accumulation in adi- epinephrine) (Supplementary Fig. 2). pose tissues and liver, which may contribute for an Liver is another important organ for lipid metabolism. increased propensity for metabolic derangement under Although a previous study did not detect excessive lipid stress conditions. 2 2 or glycogen deposition in Slc30a8 / liver (16), we found Increased adiposity results from increased energy that the lipid and glycogen contents were significantly intake or decreased energy expenditure. Interestingly, increased in our ZnT8 knockout liver (Fig. 3A–C). The metabolic profiling of ZnT8 littermate mice showed that hepatic triglyceride level but not total cholesterol level food consumption and energy expenditure characterized diabetes.diabetesjournals.org Mao and Associates 1201

Figure 2—Slc30a82/2 mice have increased lipid deposition in BAT. A: The weight-to-body weight ratio of BAT in wild-type and Slc30a82/2 mice (n = 5 per genotype). B: Representative hematoxylin-eosin images of BAT from wild-type (WT) and Slc30a82/2 mice. C–G:ThemRNAexpression level of lipogenesis, lipid synthesis, lipolysis, fatty acid oxidation, and thermogenesis in BAT from WT and Slc30a82/2 mice (n =5pergenotype).H: Western blot and quantification of UCP1, b3-AR, and TH in WT and Slc30a82/2 mice. Data are mean 6 SEM. *P , 0.05 by unpaired Student t test.

by oxygen consumption, total activity, and heat generation and most obesity/diabetes-related hormones (Supple- remained unchanged (Supplementary Fig. 3). Moreover, mentary Fig. 5) remained unaltered in ZnT8 knock- the serum inflammation markers (Supplementary Fig. 4) out mice. 1202 ZnT8 Regulates Peripheral Serotonin Production Diabetes Volume 68, June 2019

Figure 3—Slc30a82/2 mice have increased lipid and glycogen deposition in the liver. A: Gross morphology (top) and representative hematoxylin-eosin images (bottom) of liver from wild-type (WT) and Slc30a82/2 mice. B: Representative images and quantitative analysis of Oil red O staining. C: Representative images and quantitative analysis of PAS staining. D and E: Hepatic total triglycerides (TG) and total cholesterol (TC) levels in WT and Slc30a82/2 mice. Data are mean 6 SEM. *P , 0.05 by unpaired Student t test.

We also examined glucose homeostasis. Fasting blood a slower intestinal motility under physiological condi- glucose and insulin tolerance were comparable between tions (Fig. 5G). these two genotypes of mice (Fig. 4A and B). Unexpectedly, Gut microbiota is an important factor regulating the oral glucose tolerance was significantly impaired in intestinal tract microenvironment and energy homeostasis. 2 2 Slc30a8 / mice, whereas intraperitoneal glucose toler- Therefore, we assessed the effects of ZnT8 deficiency on ance remained unaltered (Fig. 4C and D). This result gut microbiota composition by sequencing the fecal bac- suggests a possible involvement of the GI tract in lipid terial 16S rRNA V3 and V4 region. ZnT8 deficiency 2 2 and glucose derangement in Slc30a8 / mice. significantly decreased richness and diversity of gut microbiota as indicated by decreased operational taxo- ZnT8 Knockout Mice Have Altered Intestinal nomic unit number and the observed species (Supplemen- Morphology and Motility tary Fig. 7A–D). However, the microbiota change pattern When we carefully examined the GI tract, we found that was a protective pattern for host metabolism, with in- the intestinal tract from the ZnT8 knockout mouse was creased relative abundance of Bacteroidetes and fi signi cantly thickened, especially the proximal colon (Fig. Verrucomicrobia together with reduced ratios of Proteobac- 5A and B), whereas no difference of the whole GI tract teria and Deferribacteres. The Firmicutes abundance was length was observed (Supplementary Fig. 6A and B). Pro- not significantly changed (Supplementary Fig. 7E and liferation analysis of PCNA staining showed a remarkable F). Taken together, these results suggest that deficiency of increased number and intensity of positively stained ZnT8 resulted in a significant change in colon morphology, cells (Fig. 5C). In addition, TUNEL assay for apoptosis biochemical environment, motility, and microbiota. identified a smaller number of apoptotic cells in the mucosal layer of ZnT8 knockout colon (Fig. 5D). These ZnT8 Is Expressed in EECs and Regulates Peripheral results suggest that ZnT8 deficiency promoted prolifer- 5-HT Levels ation and inhibited apoptosis in ZnT8 knockout colon. Next, we sought to examine whether ZnT8 was present in Interestingly, we also observed an increased volume of the intestinal tract by immunofluorescent staining. ZnT8 mucosal goblet cells and observed a significant increase immunoreactivity was observed in the epithelial layer but of the enzyme carbonic anhydrase 1 (CA1) in ZnT8 not in the smooth muscle layer or lamina propria in the knockout colon (Fig. 5E and F). CA1 is a zinc metal- intestine. The positively stained cells were triangular and loenzyme that catalyzes the reversible hydration of CO2. scattered in the enterocytes, mainly in the proximal colon Increased CA1 may disturb the colonic acid-base balance (Fig. 6A). We costained EEC marker CgA and other hor- in ZnT8 knockout mice. In addition to the morphology mone markers with ZnT8 antibody. ZnT8 signal was and biochemical changes, ZnT8 knockout mice also had observed mainly in cells positive for CgA and 5-HT (Fig. diabetes.diabetesjournals.org Mao and Associates 1203

Figure 4—Slc30a82/2 mice have a normal intraperitoneal glucose tolerance test (IPGTT) but abnormal oral glucose tolerance test (OGTT). A: Fasting blood glucose level (n = 8 per genotype). B: Insulin tolerance test (ITT) and area under the curve analysis (n = 6 per genotype). C: IPGTT and area under the curve analysis (n = 6 per genotype). D: OGTT and area under the curve analysis (n = 6 per genotype). Data are mean 6 SEM. *P , 0.05 by unpaired Student t test.

6B and Supplementary Fig. 8A), whereas a small propor- significant increase in body weight, fat mass, and hepatic tion of ZnT8 colocalized with gastric inhibitory poly- steatosis compared with the wild-type mice (Supplemen- peptide (Supplementary Fig. 8B). We did not observe tary Fig. 9A and B). Hyperglycemia, hyperinsulinemia, and a colocalization between ZnT8 and glucagon-like peptide hyperlipidemia were also observed (Supplementary Fig. 1 or polypeptide YY in the intestines of wild-type mice, and 9C–F). PCPA effectively rescued the diet-induced obese 2 2 no ZnT8 signal was observed in the intestinal tract of ZnT8 and glucose intolerance phenotypes of Slc30a8 / mice. It knockout mice (Supplementary Fig. 8A). prevented the increase in body weight, fat mass, and 5-HT is the most prevalent GI hormone, and the GI glucose intolerance (Fig. 7C–F and Supplementary Fig. tract is the major source for peripheral 5-HT. More than 10A–G). PCPA demonstrated negligible effects on food 2 2 90% of circulating 5-HT is synthesized in and secreted intake in wild-type and Slc30a8 / mice whether they from the GI tract (17). Notably, the staining intensity and were fed an ND or HFD (Fig. 7G). These results suggest number of 5-HT cells in the colon were remarkably in- that reducing peripheral 5-HT level was able to, at least creased in ZnT8 knockout mice (Fig. 6C and D). The serum partially, rescue the increase in adiposity and related 5-HT level in ZnT8 knockout mice was also significantly glucose intolerance in ZnT8-deficient mice fed either an higher than in wild-type mice (Fig. 6E). TPH1 is respon- ND or an HFD. sible for synthesizing 5-HT in the peripheral tissue. Both mRNA and protein levels of TPH1 in the proximal colon ZnT8 Deficiency Increases 5-HT Biosynthesis Through were significantly increased (Fig. 6F and G). These results Elevating TPH1 Level in RIN14B Cells suggest that ZnT8 was expressed in the EECs and that To investigate how ZnT8 regulates 5-HT level, we used deficiency of ZnT8 increased peripheral 5-HT by elevating RIN14B cells as the in vitro cellular model. RIN14B cell is d the TPH1 protein level. a rat -cell line and has been used for studying 5-HT secretion as a proper cellular model (18). d-Cells are known Reversal of Metabolic Dysfunction by Inhibition of 5-HT to produce somatostatin and are expressed in the pancre- Synthesis in ZnT8-Deficient Mice atic islets as well as in the intestines. We detected the To determine whether peripheral 5-HT contributes to the presence of ZnT8 mRNA by quantitative PCR analysis in metabolic effects of ZnT8 deficiency, we treated ZnT8 RIN14B cells. When endogenous ZnT8 was knocked down group mice with the specific TPH inhibitor, PCPA. Daily by small interfering RNA transfection (Fig. 8A), the TPH1 injection of PCPA for 4 weeks attenuated the increase protein level was increased almost threefold (Fig. 8B). in body weight and oral glucose intolerance, which was ELISA analysis showed that silencing ZnT8 significantly 2 2 markedly elevated in Slc30a8 / mice fed an ND (Fig. 7A increased 5-HT level in the cell lysate as well as in the and B). ZnT8 knockout mice fed an HFD showed a medium (Fig. 8C and D). These results suggest that ZnT8 1204 ZnT8 Regulates Peripheral Serotonin Production Diabetes Volume 68, June 2019

Figure 5—Slc30a82/2 mice have altered intestinal tract morphology and motility. A: Representative hematoxylin-eosin (H&E) images of proximal colon from wild-type (WT) and Slc30a82/2 mice. B: Quantitative analysis of proximal colon diameter (n = 5 per genotype). C: Representative immunostaining and quantitative analysis of PCNA in WT and Slc30a82/2 proximal colon. D: Immunohistochemical staining and quantification of TUNEL assay in WT and Slc30a82/2 proximal colon (n = 3 per genotype). Arrowheads indicate positive staining signals. E: Representative H&E and PAS staining of goblet cells in mucosal layer from WT and Slc30a82/2 proximal colon. F: Representative immunostaining and quantitative analysis of CA1 in WT and Slc30a82/2 proximal colon. G: Total intestinal transit time of wild-type and Slc30a82/2 mice (n = 5 per genotype). Data are mean 6 SEM. *P , 0.05, **P , 0.01, ***P , 0.001 by unpaired Student t test.

deficiency increased the expression of TPH1 and, hence, (22) and that human islets with the R325 ZnT8 variant had the synthesis of 5-HT. a higher zinc content (23). Therefore, we tested whether ZnT8 mediates the zinc transport across cell mem- the human T2DM risk allele altered 5-HT biosynthesis. We branes (19). We therefore analyzed whether an altered generated human ZnT8 wild-type and mutant (R325W) zinc concentration affects levels of TPH1 and 5-HT. Ad- plasmids and transfected them into RIN14B cells. Inter- dition of ZnSO4 in the medium significantly increased the estingly, ZnT8 R325W mutant overexpression signifi- expression level of TPH1 and 5-HT in the medium. De- cantly increased the 5-HT level in the medium (Fig. 8I), pletion of zinc ions by zinc chelator N,N,N9,N9-tetrakis (2- but no change of 5-HT or TPH1 level in the cell lysate was pyridinylmethyl)-1,2-ethanediamine (TPEN) reduced the detected (Fig. 8H and Supplementary Fig. 11). These TPH1 mRNA and protein levels, although 5-HT secretion results suggest that the diabetes risk ZnT8 allele could in the medium was not significantly changed (Fig. 8E–G). affect 5-HT levels but with a distinctive mechanism. These results suggest that ZnT8 deficiency increased the TPH1 level probably by changing the intracellular level of DISCUSSION zinc ion. The present study demonstrates that ZnT8 regulates 5-HT Genomic studies have identified that the risk C allele biogenesis, which is critical for organism lipid and energy (rs13266634) of ZnT8 gene, which encodes an arginine (R) metabolism (Fig. 8J). This conclusion is supported by the in place of a tryptophan (W), is associated with T2DM and following observations: 1)Deficiency of ZnT8 increased BMI (20,21). Recent studies found that the T2DM risk lipid accumulation in adipose tissues and liver without R325 ZnT8 variant had a higher zinc transport activity obvious body weight gain, 2) ZnT8 was detected in the EEC diabetes.diabetesjournals.org Mao and Associates 1205

Figure 6—ZnT8 is expressed in EECs and regulates peripheral 5-HT level. A: Representative immunofluorescent staining of ZnT8 in duodenum and proximal colon. B: Representative immunofluorescent staining and quantification of overlap between 5-HT–expressing cells and ZnT8-expressing cells in proximal colon. C: Representative immunohistochemical staining and quantitative analysis of 5-HT intensity in proximal colon. D: Representative immunofluorescent staining and quantitative analysis of 5-HT–positive cell number in proximal colon. E: Serum 5-HT level (n = 5 per genotype). F: mRNA analysis of 5-HT metabolism-related genes. 36B4 was used as the reference gene. G: Western blot analysis and quantification of TPH1 and CgA in wild-type (WT) and Slc30a82/2 proximal colon. Tubulin was used as the loading control. Data are mean 6 SEM. *P , 0.05, **P , 0.01, ***P , 0.001 by unpaired Student t test.

of the GI tract, 3)deficiency of ZnT8 increased peripheral mechanism underlying the regulation of glucose metabo- 5-HT levels through elevating TPH1 protein levels, 4) lism by ZnT8. Our study revealed that ZnT8 contributed to pharmacological inhibition of 5-HT synthesis effectively the modulation of 5-HT biosynthesis in intestinal EEC reversed diet-induced body weight increase and glucose cells, which subsequently altered systemic glucose and lipid intolerance in ZnT8 knockout mice, and 5) in RIN14B cells, metabolism. Deficiency of ZnT8 led to a significant in- downregulation of ZnT8 promoted 5-HT synthesis and crease in colonic TPH1 and circulating 5-HT level. Since secretion. Interestingly, overexpression of diabetes high- .90% of circulating 5-HT is derived from the GI tract, we risk allele of human SLC30A8 increased 5-HT secretion in assume that the elevation of circulating 5-HT primarily the medium. These effects could be mimicked by admin- results from a deficiency of ZnT8 in the gut rather than in istration of zinc ion. other tissues such as adipose tissue and enteric neurons. ZnT8, a transmembrane protein mediating the trans- The influence of ZnT8 on lipid metabolism remains port of zinc ion, is mainly expressed in pancreatic islets. Its largely unexplored. Deficiency of ZnT8 in mice leads to an physiological function has been related to the secretion of increase of adiposity under ND conditions, which is pro- islet hormones and glucose homeostasis (24). Genomic foundly aggravated under HFD conditions. Our observa- analyses have shown that the risk C allele (rs13266634) of tions suggest that elevated circulating 5-HT contributed to ZnT8 gene is associated with BMI and T2DM (2,20,21,25). the obesity and glucose dysfunction. Consistently, recent The metabolic function of ZnT8 has long been proposed studies have demonstrated that gut-derived 5-HT inhibits to occur through its regulation of the secretion of islet lipolysis and thermogenesis in eWAT and BAT and pro- hormones, specifically insulin. This concept has been re- motes hepatic glucose production, leading to obesity and cently challenged by studies using transgenic techniques. dysglycemia (14,15). Our study further confirmed that Although global deletion of ZnT8 leads to significant blockade of TPH reversed the increased adiposity and diet-induced obesity and impaired glucose tolerance, the glucose intolerance in ZnT8 knockout mice. In our exacerbation was not observed in b-cell–specific ZnT8 ZnT8-deficient mice, we also observed an increase in lipid knockout mice (6). These results indicate an alternative and glycogen deposition in the liver, although Tamaki et al. 1206 ZnT8 Regulates Peripheral Serotonin Production Diabetes Volume 68, June 2019

Figure 7—Inhibition of 5-HT synthesis in ZnT8-deficient mice reverses metabolic dysfunction. A and B: Wild-type (WT) and Slc30a82/2 mice fed an ND received daily intraperitoneal PCPA injection (300 mg/kg body weight [BW]) for 4 weeks, starting at the age of 7 weeks. A: BW over the course of the study. B: Oral glucose tolerance test (OGTT) and area under the curve analysis after PCPA injection for 4 weeks (n =5–7 per genotype). C–F: WT and Slc30a82/2 mice fed an HFD (60 % of kcal fat) starting at the age of 6 weeks received daily intraperitoneal PCPA injections (300 mg/kg BW) for 4 weeks starting at the age of 12 weeks (n =5–7 per genotype). C: BW over the course of the study. D:BW change after PCPA injection from the age of 12 weeks to the age of 16 weeks. E: Serum 5-HT level. F: Intraperitoneal glucose tolerance test (IPGTT) and area under the curve analysis after PCPA injection for 3 weeks. G: Average food intake of WT and Slc30a82/2 mice as indicated. Data are mean 6 SEM. *P , 0.05, **P , 0.01, ***P , 0.001 by unpaired Student t test. d, day; KO, knockout.

(16) did not show significant changes in liver. Previously, phenotypes, including glucose tolerance, insulin sensitiv- several groups have reported that ZnT8-deficient mice ity, and insulin secretion capability, under either ND or have substantial hypersecretion of insulin from pancreatic HFD conditions. There must be other factors influencing b-cells (5,16). It is reasonable to speculate that the insulin the effects of Slc30a8 deletion. Environmental conditions, directly flows into the liver through the portal vein, such as diets and intestinal microbial composition, might causing lipid and glycogen deposition in the liver. The be important. In our ZnT8 knockout mouse model, we discrepancy may be due to the different mouse model with observed a significant change in intestinal tract morphol- different genetic background or other environmental ogy and motility. The altered gut microenvironment could factors, causing distinct liver sensitivity to the elevated lead to increased harvest from the diet, changes in fatty insulin. acid metabolism, gut 5-HT secretion, and the intestinal Up to now, there have been six ZnT8 knockout mouse barrier. Intestinal 5-HT production is also affected by gut studies (4–6,26–28). There was a discrepancy of metabolic microbes (29,30). As of now, we still do not know whether diabetes.diabetesjournals.org Mao and Associates 1207

Figure 8—Depletion of ZnT8 in RIN14B cells increases TPH1 expression and 5-HT level. A–D: RIN14B cells were transfected with scramble or small interfering RNA against ZnT8 (siZnT8) for 48 h, and the cell lysates were collected for further analysis. A: mRNA expression analysis of Slc30a8 gene. B: Western blot analysis and quantification of TPH1. Tubulin was used as the loading control. C: 5-HT level in the cell lysates. D: 5-HT in the medium. E–G: RIN14B cells were treated with the zinc chelator TPEN or different concentration of ZnSO4 for 24 h and then collected for further analysis. E: Western blot analysis and quantification of TPH1. Actin was used as the loading control. F: mRNA expression analysis of Slc30a8 and Tph1 gene in RIN14B cells. G: 5-HT level in the medium. H and I: RIN14B cells were transfected with human ZnT8 (hZnT8) wild-type (WT), ZnT8 polymorphism variant R325 (mutant [Mut]), or pCDNA vector for 48 h. H: 5-HT level in the cell lysate. I: 5-HT in the medium. J: Proposed mechanisms by which ZnT8 deficiency regulates systemic energy balance through peripheral 5-HT production in EECs. Data are mean 6 SEM. *P , 0.05, **P , 0.01, ***P , 0.001 by unpaired Student t test or one-way ANOVA. Ctrl, control; OGTT, oral glucose tolerance test. 1208 ZnT8 Regulates Peripheral Serotonin Production Diabetes Volume 68, June 2019 it is a direct effect of intestinal ZnT8 on 5-HT biosynthesis ZnT8 mutant alters the exocytosis process still needs or an indirect effect caused by the disturbed gut micro- further investigation. environment or microbiota. Moreover, we cannot exclude Normal weight obesity means higher fat mass but normal the existence and effects of ZnT8 in other tissues, such as BMI. These groups of patients have received more and more immune cells, the CNS, or the enteric nervous system, attention since they have similar risks for serious meta- which may contribute to the disturbed 5-HT biogenesis bolic disorders as obese people, but they are easily ignored and systemic metabolic dysregulation. To exclude these by clinicians and themselves. Interestingly, two genome- confounding factors, the intestinal-specific ZnT8 deletion wide association studies have shown that the human mouse could be a better model for investigating the role of SLC30A8 risk allele confers higher risk of developing ZnT8 in the intestinal tract. T2DM in a lower BMI population or nonobese subjects There are several plausible mechanisms through which (21,41). These findings suggest that ZnT8 and 5-HT levels intestinal ZnT8 regulates circulating levels of 5-HT. First, are important factors that determine the percentage of ZnT8 regulates the zinc level within EEC cells. Zinc is body fat regardless of body weight. Our findings further a critical cofactor for .300 and enzymes. It plays support this concept. ZnT8 knockout mice demonstrated essential roles in various biochemical processes, including a significant increase of fat mass without body weight gain, tryptophan metabolism (31). ZnT8 is a zinc transporter which was reminiscent of normal weight obesity in that mediates zinc export from the intracellular to the humans. Since the lean mass remained unchanged, one extracellular or cellular organelles (19). Previous studies possibility of increased fat mass without body weight gain reported that ZnT8 is expressed on the plasma membrane is due to loss of bone density because 5-HT elevation has and may mediate bidirectional transport of zinc ions (32). been known to cause osteoporosis (42). Ablation of ZnT8 alters intracellular or vesicular zinc levels In summary, our study identifiedthepresenceof in b-cells (5). We speculate that a similar increase of ZnT8 in 5-HT–positive EEC cells. Deficiency of ZnT8 cytosolic zinc level occurs in EEC cells after ZnT8 deletion, enhanced 5-HT biosynthesis through modulating TPH1 hence stimulating 5-HT biosynthesis. In support of this level. GI-derived 5-HT acted on the peripheral organs concept, TPH1 is significantly increased in the intestinal to promote lipid deposition and obesity. Targeting mucosa of ZnT8 knockout mice, and silence of ZnT8 gene intestinal ZnT8 may provide an alternative strategy increases 5-HT levels in RIN14B cells. Another potential for the intervention of obesity and its associated mechanism relates to the secretion of 5-HT granules. Lack metabolic dysfunction, such as hepatic steatosis and of zinc ion in the granule may lead to a looser crystalline T2DM. structure, which probably affects the storage and exo- cytosis process of 5-HT. Acknowledgments. The authors acknowledge Chaowei Zhu, Weiqi Wu, Although a number of ZnT8 knockout rodent models and Ruolu Bao (Center for Diabetes, Obesity and Metabolism, Shenzhen University consistently displayed increased sensitivity of T2DM and Health Science Center) for technical support. impaired glucose-induced insulin secretion in vivo (33), Funding. This work was funded by the National Key R&D Program of China several loss-of-function mutations of SLC30A8 in humans (2017YFC0908900), the National Natural Science Foundation of China (81500619, confer protection from human T2DM (34). Later trans- 81730020, 81870405), the Natural Science Foundation of Guangdong Prov- genic mouse models that harbor the human ZnT8 trun- ince (2016A030310040), and the Shenzhen Science and Technology Project cation mutant have increased insulin secretion (35). (JCYJ20160422091658982, JCYJ20160422153856130). Increased 5-HT level may account for this discrepancy. Duality of Interest. No potential conflicts of interest relevant to this article 5-HT increases insulin secretion through modulating in- were reported. sulin granule exocytosis (36,37). During pregnancy, the Author Contributions. Z.M. designed and performed most of the experi- expression of islet 5-HT is profoundly increased and ments. Z.M., J.H., and W.Z. developed the study rationale, wrote the manuscript, and supervised the study. H.L., W.S., J.L., Mins.Z., Z.L., B.Z., Q.Y., Ming.Z., and regulates pancreatic b-cell mass and increased glucose- K.P. performed the experiments and assisted with data analysis. W.Z. is the induced insulin secretion (38,39). Upon a metabolic stress fi guarantor of this work and, as such, had full access to all the data and takes condition, such as HFD feeding, 5-HT is also signi cantly responsibility of the integrity of the data and the accuracy of the data analysis. increased (12) and enhances glucose-induced insulin se- cretion (40). 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