Page 1 of 57 Diabetes 1 Deficiency of ZnT8 promotes adiposity and metabolic dysfunction by 2 increasing peripheral serotonin production 3 Running title: ZnT8 regulates peripheral serotonin production 4 5 Zhuo Mao1, Hui Lin1, Wen Su1, Jinghui Li1, Minsi Zhou1, Zhuoran Li1, Beibei 6 Zhou2, Qing Yang1, Mingyan Zhou1, Ke Pan2, Jinhan He3, * and Weizhen Zhang 1,4, * 7 8 1. Center for Diabetes, Obesity and Metabolism, Department of Physiology, 9 Shenzhen University Health Science Center, Shenzhen, Guangdong province, China. 10 2. Institute for Advanced Study, Shenzhen University, Shenzhen, Guangdong 11 province, China. 12 3. Department of Pharmacy, State Key Laboratory of Biotherapy and Cancer 13 Center, West China Hospital of Sichuan University, Chengdu, Sichuan, China. 14 4. Department of Physiology and Pathophysiology, School of Basic Science, 15 Peking University Health Science Center, Beijing, China. 16 17 Correspondence to 18 Dr. Weizhen Zhang 19 Center for Diabetes, Obesity and Metabolism, Department of Physiology, 20 Shenzhen University Health Science Center, Shenzhen, Guangdong province, China. 21 Department of Physiology and Pathophysiology, School of Basic Science, Peking 22 University Health Science Center, Beijing, China. Diabetes Publish Ahead of Print, published online April 1, 2019 Diabetes Page 2 of 57 23 Email: [email protected] 24 Tel: +86-15010909001 25 26 Dr. Jinhan He 27 Department of Pharmacy, State Key Laboratory of Biotherapy and Cancer Center, 28 West China Hospital of Sichuan University, Chengdu, Sichuan, China. 29 E-mail: [email protected] 30 Tel.: +86-28-85426416 31 32 Word count: 7071 33 Figures: 8 (color) 34 Supplemental Figures: 11 35 Supplemental Tables: 1 Page 3 of 57 Diabetes 37 Abstract 38 ZnT8 is a zinc transporter enriched in the pancreatic beta cells and its 39 polymorphism is associated with increased susceptibility to type 2 diabetes. However, 40 the exact role of ZnT8 in systemic energy metabolism remains elusive. In this study, 41 we found that ZnT8 knockout mice displayed increased adiposity without obvious 42 weight gain. We also observed the intestinal tract morphology, motility and gut 43 microbiota were changed in ZnT8 knockout mice. Further study demonstrated that 44 ZnT8 was expressed in enteroendocrine cells, especially in 5-HT positive 45 enterochromffin cells. Lack of ZnT8 resulted in an elevated circulating 5-HT level 46 owing to enhanced expression of tryptophan hydroxylase 1. Blocking 5-HT synthesis 47 in ZnT8 deficient mice restored adiposity, high-fat diet-induced obesity and glucose 48 intolerance. Moreover, overexpression of human ZnT8 diabetes high risk allele 49 R325W increased 5-HT levels relative to the low risk allele in RIN14B cells. Our 50 study revealed an unexpected role of ZnT8 in regulating peripheral 5-HT biogenesis 51 and intestinal microenvironment, which might contribute to the increased risk of 52 obesity and type 2 diabetes. 53 54 Key words: ZnT8; 5-HT; obesity; adiposity; type 2 diabetes, intestinal hormones; 55 energy metabolism Diabetes Page 4 of 57 57 ZnT8 is a zinc transporter which is closely associated with both type 1 and type 2 58 diabetes mellitus (DM). It is an important autoantigen in T1DM patients [1]. 59 Meanwhile its gene polymorphism has also been identified as a risk factor for T2DM 60 [2], suggesting an important physiological function of ZnT8 in metabolic disease 61 progression. ZnT8 is highly abundant in pancreatic beta cells [3]. Several colonies of 62 global and beta cell or alpha cell specific ZnT8 knockout mice have been generated to 63 investigate its effects on insulin granule morphology, insulin secretion and systemic 64 glucose metabolism [4-6]. Global ZnT8 knockout mice exhibit an exacerbation of 65 diet-induced obesity and glucose intolerance compared to wild-type mice [4, 6]. 66 Unexpectedly, this phenotype was not observed in mice that lack ZnT8 specifically in 67 beta cells or alpha cells [6]. This discrepancy strongly implies the presence of ZnT8 in 68 non-beta/alpha cell or extrapancreatic tissues plays a critical role in organism energy 69 homeostasis. Since ZnT8 is negligible in hypothalamus, fat and skeletal muscle [6], 70 we therefore speculate that ZnT8 may be expressed in other endocrine tissues or cells. 71 Gastrointestinal (GI) tract contains the largest number of various endocrine cells. 72 Many GI hormones play critical roles in glucose homeostasis [7]. 73 5-hydroxytryptamine (5-HT, serotonin) is the most prevalent GI hormone which 74 exhibits both central and peripheral functions. More than 90% of 5-HT is synthesized 75 in and released from the enterochromaffin cells (ECCs). Other tissues, such as 76 neurons, adipose tissues and pancreas, only produces a small amount of 5-HT [8]. The 77 initial and rate-limiting step of 5-HT synthesis is catalyzed by the tryptophan 78 hydroxylase (TPH). There are two isoforms of TPHs, TPH1 in the peripheral tissues Page 5 of 57 Diabetes 79 and TPH2 in the central nervous system (CNS) and enteric neurons [9]. 5-HT system 80 possesses complex bioactivities mediated by different types of 5-HT receptor (5HTR) 81 expressed in various tissues. In the CNS, 5-HT acts as a neurotransmitter to regulate 82 appetite, emotions, sleep, and systemic metabolism through sympathetic nervous 83 system (SNS) [10]. The peripheral 5-HT function is relatively less clear compared to 84 its central role. The classical action of peripheral 5-HT includes regulation of GI 85 functions such as motility, secretion, sensation, modulation of platelet coagulation and 86 bone density [11]. Interestingly, obesity increases peripheral 5-HT level [12]. And 87 genetic polymorphism of TPH1 is associated with obesity [13]. Recent studies have 88 found that peripheral 5-HT promotes white adipose tissue (WAT) lipogenesis and 89 inhibits brown adipose tissues (BAT) adaptive thermogenesis [14, 15]. Genetic 90 deficiency or pharmacological inhibition of 5-HT synthesis enzyme TPH1 in mice 91 leads to a resistance to diet-induced obesity and glucose intolerance [14, 15], 92 suggesting that peripheral 5-HT is an important regulator of lipid metabolism and 93 systemic energy homeostasis. 94 In this study, we generated a new strain of ZnT8 knockout mice using TALEN 95 technology. We examined the presence of ZnT8 in enteroendocrine cells (EECs) and 96 its role in 5-HT biogenesis and lipid metabolism using cell biological and transgenic 97 techniques. We also observed an unexpected change in colon morphology, function 98 and microbiota in ZnT8 deficient mice which may contribute to the increased 99 sensitivity of diet-induced obesity and T2DM. Diabetes Page 6 of 57 100 Research Design and Methods 101 Animals 102 ZnT8 knockout mice (Slc30a8-/- mice) were generated by Cyagen Biosciences 103 Inc. (Guangzhou, China). Exon 3 of Slc30a8 gene was selected as the target site. 104 TALEN mRNAs generated by in vitro transcription were then injected into fertilized 105 eggs from C57BL/6N strain for knockout mouse production. The founders were 106 genotyped by PCR followed by DNA sequencing analysis. The positive founders 107 were breeding to the next generation which was genotyped by PCR and DNA 108 sequencing analysis. 109 For high-fat diet (HFD) treatment experiment, male mice (aged 6-8 weeks old) 110 were fed either a normal chow diet (ND) or an HFD (45 % fat calories, Research 111 Diets D12451 or 60 % fat calories, Research Diets D12492). For TPH inhibitor 112 injection experiment, PBS or 4-Chloro-DL-phenylalanine methyl ester hydrochloride 113 (PCPA, C3635, Sigma-Aldrich) (300 mg/kg BW) was administered as a daily 114 intraperitoneal injection. All animal experiments were undertaken with the approval 115 of the Scientific Investigation Board of Health Science Center of Shenzhen University 116 (Shenzhen, Guangdong Province, China). 117 Antibody 118 The rabbit anti-ZnT8 polyclonal antibody was generated against the synthetic 119 mouse ZnT8 peptide (KPVNKDQCPGDRPEHPEAGGIYH, 29-51 aa). The 120 antibodies against insulin, chromogranin A, UCP1, Beta3 AR, GIP and GLP-1 were 121 from Abcam. Anti TH antibody was from Millipore. The tubulin, actin and GAPDH Page 7 of 57 Diabetes 122 antibodies were from Proteintech. The Alexa Fluor 488 and Alexa Fluor 594 dye were 123 obtained from Molecular Probes. The horseradish peroxidase (HRP)-labeled 124 secondary antibody was purchased from Amersham BioSciences (GE Healthcare). 125 Histology and Immunofluorescent staining 126 The dissected tissues were fixed with 4% paraformaldehyde (PFA) in PBS for 16 127 hrs at 4°C. The samples were sequentially dehydrated and embedded in paraffin. Then 128 the tissue samples were sectioned at a 6-μm thickness and were used for standard 129 H&E staining and quantification. The quantification was determined by the Image J 130 software. Immunochemistry/immunofluorescent staining were performed following 131 general protocols. Images were obtained by Nikon Eclipse Ti microscope. 132 Oil Red O staining and PAS staining 133 The frozen liver sections were washed in PBS once and were fixed with 4% PFA 134 in PBS for 15 min at room temperature and then washed three times with PBS. The 135 sections were incubated in the 60% isopropyl alcohol and then stained with filtered 136 Oil Red O solution (1.5 mg/mL) for 30 min and rinsed twice with distilled water. 137 Periodic Acid Schiff （PAS）staining for glycogen in the liver and goblet cells in 138 the colon were performed using the commercial kit (Solarbio, Beijing) following the 139 manual instruction. 140 Oral/intraperitoneal glucose tolerance test (OGTT/IPGTT) 141 ZnT8 group mice were first starved for 16 hrs, followed by an oral glucose 142 infusion or intraperitoneal glucose injection (1.5 g/kg body weight). The blood 143 glucose levels were measured from the tail vein before and at 15, 30, 60, 90 and 120 Diabetes Page 8 of 57 144 min after injection using a glucometer (Accu-check, Roche).