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

ZnT8 is a zinc transporter that is closely associated with both type 1 and type 2 diabetes mellitus (T1DM and T2DM). It is an important autoantigen in patients with T1DM (1). Meanwhile its gene polymorphism has also been identified as a risk factor for T2DM (2), suggesting an important physiological function of ZnT8 in metabolic disease progression. ZnT8 is highly abundant in pancreatic β-cells (3). Several colonies of global and β- or α-cell–specific ZnT8 knockout mice have been generated to investigate its effects on insulin granule morphology, insulin secretion, and systemic glucose metabolism (4–6). Global ZnT8 knockout mice exhibit an exacerbation of diet-induced obesity and glucose intolerance compared with wild-type mice (4,6). Unexpectedly, this phenotype was not observed in mice that lack ZnT8, specifically in β- or α-cells (6). This discrepancy strongly implies that the presence of ZnT8 in non–β/α-cell or extrapancreatic tissues plays a critical role in organism energy homeostasis. Since ZnT8 is negligible in hypothalamus, fat, and skeletal muscle (6), we speculated that ZnT8 might be expressed in other endocrine tissues or cells.

The gastrointestinal (GI) tract contains the largest number of endocrine cells. Many GI hormones play critical roles in glucose homeostasis (7). 5-hydroxytryptamine (5-HT [serotonin]) is the most prevalent GI hormone 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, adipose tissues, and pancreas, only produce a small amount of 5-HT (8). The initial and rate-limiting step of 5-HT synthesis is catalyzed by tryptophan hydroxylase (TPH). There are two isoforms of TPHs: TPH1 in the peripheral tissues and TPH2 in the central nervous system (CNS) and enteric neurons (9). The 5-HT system possesses complex bioactivities mediated by various types of 5-HT receptors expressed in different tissues. In the CNS, 5-HT acts as a neurotransmitter to regulate appetite, emotions, sleep, and systemic metabolism through the sympathetic nervous system (10). The function of peripheral 5-HT is relatively less clear compared with its central role. The classical action of peripheral 5-HT includes regulation of GI functions, such as motility, secretion, sensation, modulation of platelet coagulation, and bone density (11). Interestingly, obesity increases peripheral 5-HT levels (12), and the genetic polymorphism of TPH1 is associated with obesity (13). Recent studies have found that peripheral 5-HT promotes white adipose tissue (WAT) lipogenesis and inhibits brown adipose tissue (BAT) adaptive thermogenesis (14,15). Genetic deficiency or pharmacological inhibition of 5-HT synthesis enzyme TPH1 in mice leads to a resistance to diet-induced obesity and glucose intolerance (14,15), suggesting that peripheral 5-HT is an important regulator of lipid metabolism and systemic energy homeostasis.

In this study, we generated a new strain of ZnT8 knockout mice using transcription activator-like effector nuclease (TALEN) technology. We examined the presence of ZnT8 in enteroendocrine cells (EECs) and its role in 5-HT biogenesis and lipid metabolism using cell biological and transgenic techniques. We also observed an unexpected change in colon morphology, function, and microbiota in ZnT8-deficient mice, which may contribute to the increased sensitivity of diet-induced obesity and T2DM.

ZnT8 knockout (Slc30a8^−/−) mice were generated by Cyagen Biosciences Inc. (Guangzhou, China). Exon 3 of Slc30a8 gene was selected as the target site. TALEN mRNAs generated by in vitro transcription were then injected into fertilized eggs from the C57BL/6N mouse strain for knockout mouse production. The founders were genotyped by PCR followed by DNA sequencing analysis. The positive founders were bred to the next generation, which was genotyped by PCR and DNA sequencing analysis.

For the high-fat diet (HFD) treatment experiment, male mice (aged 6–8 weeks) were fed either a normal chow diet (ND) or an HFD (45% fat calories, D12451, or 60% fat calories, D12492; Research Diets). For the TPH inhibitor injection experiment, PBS or 4-chloro-dl-phenylalanine methyl ester hydrochloride (PCPA) (300 mg/kg body weight) (C3635; Sigma-Aldrich) was administered as a daily intraperitoneal injection. All animal experiments were undertaken with the approval of the Scientific Investigation Board of the Health Science Center of Shenzhen University.

The rabbit anti-ZnT8 polyclonal antibody was generated against the synthetic mouse ZnT8 peptide (KPVNKDQCPGDRPEHPEAGGIYH, 29–51 amino acids). Antibodies against insulin, chromogranin A (CgA), uncoupling protein 1 (UCP1), β3-adrenergic receptor (AR), gastric inhibitory polypeptide, and glucagon-like peptide 1 were from Abcam. Anti–tyrosine hydroxylase (TH) antibody was from Millipore. The tubulin, actin, and GAPDH antibodies were from Proteintech. Alexa Fluor 488 and 594 dyes were obtained from Molecular Probes. The horseradish peroxidase–labeled secondary antibody was purchased from Amersham BioSciences (GE Healthcare).

The dissected tissues were fixed with 4% paraformaldehyde in PBS for 16 h at 4°C. The samples were sequentially dehydrated and embedded in paraffin. Tissue samples then were sectioned at a 6-μm thickness and used for standard hematoxylin-eosin staining and quantification. The quantification was determined by Image J software. Immunohistochemistry and immunofluorescent staining were performed following general protocols. Images were obtained by Nikon Eclipse Ti microscope.

For Oil Red O staining, frozen liver sections were washed in PBS once and fixed with 4% paraformaldehyde in PBS for 15 min at room temperature and then washed three times with PBS. The sections were incubated in 60% isopropyl alcohol and then stained with filtered Oil Red O solution (3 mg/mL) for 30 min and rinsed twice with distilled water. Periodic acid Schiff (PAS) staining for glycogen in the liver and goblet cells in the colon was performed using a commercial kit (Solarbio, Beijing, China) according to the manufacturer’s instructions.

ZnT8 group mice were starved for 16 h, followed by an oral glucose infusion or intraperitoneal glucose injection (1.5 g/kg body weight). The blood glucose levels were measured from the tail vein before and at 15, 30, 60, 90, and 120 min after injection using a glucometer (Accu-Chek; Roche).

ZnT8 group mice were fasted for 4 h before insulin tolerance testing and then received an injection of human regular insulin (0.5 units/kg body weight). Blood glucose levels were recorded before and at 15, 30, 60, 90, and 120 min after injection using a glucometer (Accu-Chek).

Serum total triglycerides, total cholesterol, and nonesterified fatty acid were measured using commercial kits (Biosino Biotechnology and Wako Chemicals, respectively). Serum 5-HT levels were measured using the mouse ELISA kit (Enzo Life Sciences). Insulin levels were analyzed using the mouse ultrasensitive insulin kit (Alpco), and catecholamine levels were measured using ELISA kits (BioVision) according to the manufacturer’s instructions.

The RIN14B cells (purchased from Zeye Bio-Tech, Shanghai, China) were cultured in high-glucose DMEM (Gibco) containing 10% FBS (Gibco) in a humidified incubator with 5% CO₂ at 37°C. Cells were plated at optimal densities and grown for 24 h and then transfected with plasmids using Lipofectamine 3000 (Thermo Fisher Scientific) according to the manufacturer’s instructions.

A solution of 6% carmine red (300 μL) (Sigma-Aldrich) suspended in 0.5% methylcellulose (Sigma-Aldrich) was administered by gavage through a 21-gauge round-tip feeding needle. The time at which gavage took place was recorded as T0. After gavage, fecal pellets were monitored at 10-min intervals for the presence of carmine red. Total GI transit time was considered as the interval between T0 and the time of first observance of carmine red in stool.

Stool samples freshly collected from each mouse were immediately frozen at −20°C and transported to the laboratory with ice pack. Metagenomic sequencing and analysis were performed by Novogene Bioinformatics Technology Co., Ltd. (Beijing, China).

Measurements of energy expenditure, respiratory exchange ratio, indirect calorimetry, and physical activity using metabolic cages (Columbus Instruments) were performed by the Biomedical Research Institute of Nanjing University (Nanjing, China). Whole-body composition of ZnT8 group mice was analyzed by EchoMRI.

The isolated islets were incubated in the 0.1 mg/mL dithizone (Sigma) solution at 37°C for 15 min. After washing with Hanks’ balanced salt solution, the islets were examined with a stereomicroscope.

The tissues or RIN14B cells were quickly harvested, rinsed with cold PBS, and homogenized in cold radioimmunoprecipitation assay buffer (150 mmol/L NaCl, 1% Triton X-100, 1% sodium deoxycholate, 0.1% SDS, 50 mmol/L Tris-HCl, and 2 mmol/L EDTA, pH 7.4) supplemented with protease inhibitor cocktail (Roche). A total of 40 μg of protein was loaded onto SDS-PAGE gels and electrophoretically transferred to polyvinylidene fluoride membranes (Bio-Rad). Transferred membranes were blocked with 5% nonfat milk in Tris-buffered saline with 0.1% Tween 20 and then incubated with primary antibodies at 4°C overnight. After washing with Tris-buffered saline with 0.1% Tween 20, membranes were incubated with secondary antibodies and developed with SuperSignal West Pico Chemiluminescent Substrate (Thermo Fisher Scientific). Signals were detected by the Amersham Imager 600 (GE Healthcare).

Total RNA was isolated from mouse tissues or RIN14B cells using Direct-zol RNA MiniPrep Kit (R2052; Zymo Research). One microgram of total RNA was used for reverse transcription using PrimeScript RT Master Mix (Takara Bio). SYBR Green–based real-time PCR was performed using LightCycler 96 (Roche) with SYBR Premix Ex Taq II (Takara Bio). The quantity of mRNA was calculated using the ΔΔCt method. All reactions were performed in duplicate. The primers used for quantitative PCR are listed in Supplementary Table 1.

All results are presented as mean ± SEM. Data were analyzed with Student t test or one-way ANOVA followed by Bonferroni multiple comparison test. P < 0.05 was considered statistically significant.

To investigate the mechanism by which ZnT8 increases T2DM sensitivity, we generated ZnT8 knockout mice (Slc30a8^−/− mice) on the C57BL/6N genetic background using TALEN technology (Supplementary Fig. 1A) by deleting 2 base pairs in exon 3. We verified the newly generated ZnT8 knockout line by analyzing the pancreatic expression and function of ZnT8. Loss of ZnT8 mRNA and protein expression were first confirmed in ZnT8 knockout pancreas (Supplementary Fig. 1B–D). Then dithizone staining of isolated islets from Slc30a8^−/− mice demonstrated a marked reduction of intensity representing zinc depletion, indicating the functional inactivity of ZnT8 protein in pancreatic islets (Supplementary Fig. 1E).

The overall morphology and growth curve were similar between wild-type and Slc30a8^−/− mice (Fig. 1A). However, we observed a significant increase in fat mass in both male and female Slc30a8^−/− mice, whereas lean mass showed a trend of reduction (Fig. 1B). The WAT mass, including epididymal WAT (eWAT) and subcutaneous WAT (scWAT), was significantly increased in Slc30a8^−/− mice (Fig. 1C and D). This alteration resulted from increased adipocyte size (Fig. 1E and F). Furthermore, the expression levels of genes related to lipid synthesis, fatty acid uptake, and lipolysis were significantly increased, whereas lipogenesis and fatty acid oxidation–relevant genes remained unchanged (Fig. 1G–K).

In addition to WAT expansion, there was an increase of lipid accumulation in BAT as evidenced by increased mass and unilocular fat droplets in ZnT8 knockout mice (Fig. 2A and B). Most genes related to thermogenesis, including Ucp1 and Pgc1α, remained unchanged (Fig. 2G and H). The lipogenesis and lipid synthesis genes were increased, whereas the fatty acid oxidation–related genes were decreased (Fig. 2C–F). Unexpectedly, we found a significant reduction of β3-AR protein level in BAT (Fig. 2H). The neuronal marker TH remained unchanged, suggesting an intact neuron innervation to the BAT (Fig. 2H). Total sympathetic tone was normal as measured by serum catecholamine (including dopamine, norepinephrine, and epinephrine) (Supplementary Fig. 2).

Liver is another important organ for lipid metabolism. Although a previous study did not detect excessive lipid or glycogen deposition in Slc30a8^−/− liver (16), we found that the lipid and glycogen contents were significantly increased in our ZnT8 knockout liver (Fig. 3A–C). The hepatic triglyceride level but not total cholesterol level was elevated (Fig. 3D and E). All these data suggest that ZnT8 deficiency increases fat accumulation in adipose tissues and liver, which may contribute for an increased propensity for metabolic derangement under stress conditions.

Increased adiposity results from increased energy intake or decreased energy expenditure. Interestingly, metabolic profiling of ZnT8 littermate mice showed that food consumption and energy expenditure characterized by oxygen consumption, total activity, and heat generation remained unchanged (Supplementary Fig. 3). Moreover, the serum inflammation markers (Supplementary Fig. 4) and most obesity/diabetes-related hormones (Supplementary Fig. 5) remained unaltered in ZnT8 knockout mice.

We also examined glucose homeostasis. Fasting blood glucose and insulin tolerance were comparable between these two genotypes of mice (Fig. 4A and B). Unexpectedly, oral glucose tolerance was significantly impaired in Slc30a8^−/− mice, whereas intraperitoneal glucose tolerance remained unaltered (Fig. 4C and D). This result suggests a possible involvement of the GI tract in lipid and glucose derangement in Slc30a8^−/− mice.

When we carefully examined the GI tract, we found that the intestinal tract from the ZnT8 knockout mouse was significantly thickened, especially the proximal colon (Fig. 5A and B), whereas no difference of the whole GI tract length was observed (Supplementary Fig. 6A and B). Proliferation analysis of PCNA staining showed a remarkable increased number and intensity of positively stained cells (Fig. 5C). In addition, TUNEL assay for apoptosis identified a smaller number of apoptotic cells in the mucosal layer of ZnT8 knockout colon (Fig. 5D). These results suggest that ZnT8 deficiency promoted proliferation and inhibited apoptosis in ZnT8 knockout colon. Interestingly, we also observed an increased volume of mucosal goblet cells and observed a significant increase of the enzyme carbonic anhydrase 1 (CA1) in ZnT8 knockout colon (Fig. 5E and F). CA1 is a zinc metalloenzyme that catalyzes the reversible hydration of CO₂. Increased CA1 may disturb the colonic acid-base balance in ZnT8 knockout mice. In addition to the morphology and biochemical changes, ZnT8 knockout mice also had a slower intestinal motility under physiological conditions (Fig. 5G).

Gut microbiota is an important factor regulating the intestinal tract microenvironment and energy homeostasis. Therefore, we assessed the effects of ZnT8 deficiency on gut microbiota composition by sequencing the fecal bacterial 16S rRNA V3 and V4 region. ZnT8 deficiency significantly decreased richness and diversity of gut microbiota as indicated by decreased operational taxonomic unit number and the observed species (Supplementary Fig. 7A–D). However, the microbiota change pattern was a protective pattern for host metabolism, with increased relative abundance of Bacteroidetes and Verrucomicrobia together with reduced ratios of Proteobacteria and Deferribacteres. The Firmicutes abundance was not significantly changed (Supplementary Fig. 7E and F). Taken together, these results suggest that deficiency of ZnT8 resulted in a significant change in colon morphology, biochemical environment, motility, and microbiota.

Next, we sought to examine whether ZnT8 was present in the intestinal tract by immunofluorescent staining. ZnT8 immunoreactivity was observed in the epithelial layer but not in the smooth muscle layer or lamina propria in the intestine. The positively stained cells were triangular and scattered in the enterocytes, mainly in the proximal colon (Fig. 6A). We costained EEC marker CgA and other hormone markers with ZnT8 antibody. ZnT8 signal was observed mainly in cells positive for CgA and 5-HT (Fig. 6B and Supplementary Fig. 8A), whereas a small proportion of ZnT8 colocalized with gastric inhibitory polypeptide (Supplementary Fig. 8B). We did not observe a colocalization between ZnT8 and glucagon-like peptide 1 or polypeptide YY in the intestines of wild-type mice, and no ZnT8 signal was observed in the intestinal tract of ZnT8 knockout mice (Supplementary Fig. 8A).

5-HT is the most prevalent GI hormone, and the GI tract is the major source for peripheral 5-HT. More than 90% of circulating 5-HT is synthesized in and secreted from the GI tract (17). Notably, the staining intensity and number of 5-HT cells in the colon were remarkably increased in ZnT8 knockout mice (Fig. 6C and D). The serum 5-HT level in ZnT8 knockout mice was also significantly higher than in wild-type mice (Fig. 6E). TPH1 is responsible for synthesizing 5-HT in the peripheral tissue. Both mRNA and protein levels of TPH1 in the proximal colon were significantly increased (Fig. 6F and G). These results suggest that ZnT8 was expressed in the EECs and that deficiency of ZnT8 increased peripheral 5-HT by elevating the TPH1 protein level.

To determine whether peripheral 5-HT contributes to the metabolic effects of ZnT8 deficiency, we treated ZnT8 group mice with the specific TPH inhibitor, PCPA. Daily injection of PCPA for 4 weeks attenuated the increase in body weight and oral glucose intolerance, which was markedly elevated in Slc30a8^−/− mice fed an ND (Fig. 7A and B). ZnT8 knockout mice fed an HFD showed a significant increase in body weight, fat mass, and hepatic steatosis compared with the wild-type mice (Supplementary Fig. 9A and B). Hyperglycemia, hyperinsulinemia, and hyperlipidemia were also observed (Supplementary Fig. 9C–F). PCPA effectively rescued the diet-induced obese and glucose intolerance phenotypes of Slc30a8^−/− mice. It prevented the increase in body weight, fat mass, and glucose intolerance (Fig. 7C–F and Supplementary Fig. 10A–G). PCPA demonstrated negligible effects on food intake in wild-type and Slc30a8^−/− mice whether they were fed an ND or HFD (Fig. 7G). These results suggest that reducing peripheral 5-HT level was able to, at least partially, rescue the increase in adiposity and related glucose intolerance in ZnT8-deficient mice fed either an ND or an HFD.

To investigate how ZnT8 regulates 5-HT level, we used RIN14B cells as the in vitro cellular model. RIN14B cell is a rat δ-cell line and has been used for studying 5-HT secretion as a proper cellular model (18). δ-Cells are known to produce somatostatin and are expressed in the pancreatic islets as well as in the intestines. We detected the presence of ZnT8 mRNA by quantitative PCR analysis in RIN14B cells. When endogenous ZnT8 was knocked down by small interfering RNA transfection (Fig. 8A), the TPH1 protein level was increased almost threefold (Fig. 8B). ELISA analysis showed that silencing ZnT8 significantly increased 5-HT level in the cell lysate as well as in the medium (Fig. 8C and D). These results suggest that ZnT8 deficiency increased the expression of TPH1 and, hence, the synthesis of 5-HT.

ZnT8 mediates the zinc transport across cell membranes (19). We therefore analyzed whether an altered zinc concentration affects levels of TPH1 and 5-HT. Addition of ZnSO₄ in the medium significantly increased the expression level of TPH1 and 5-HT in the medium. Depletion of zinc ions by zinc chelator N,N,N′,N′-tetrakis (2-pyridinylmethyl)-1,2-ethanediamine (TPEN) reduced the TPH1 mRNA and protein levels, although 5-HT secretion in the medium was not significantly changed (Fig. 8E–G). These results suggest that ZnT8 deficiency increased the TPH1 level probably by changing the intracellular level of zinc ion.

Genomic studies have identified that the risk C allele (rs13266634) of ZnT8 gene, which encodes an arginine (R) in place of a tryptophan (W), is associated with T2DM and BMI (20,21). Recent studies found that the T2DM risk R325 ZnT8 variant had a higher zinc transport activity (22) and that human islets with the R325 ZnT8 variant had a higher zinc content (23). Therefore, we tested whether the human T2DM risk allele altered 5-HT biosynthesis. We generated human ZnT8 wild-type and mutant (R325W) plasmids and transfected them into RIN14B cells. Interestingly, ZnT8 R325W mutant overexpression significantly increased the 5-HT level in the medium (Fig. 8I), but no change of 5-HT or TPH1 level in the cell lysate was detected (Fig. 8H and Supplementary Fig. 11). These results suggest that the diabetes risk ZnT8 allele could affect 5-HT levels but with a distinctive mechanism.

The present study demonstrates that ZnT8 regulates 5-HT biogenesis, which is critical for organism lipid and energy metabolism (Fig. 8J). This conclusion is supported by the following observations: 1) Deficiency of ZnT8 increased lipid accumulation in adipose tissues and liver without obvious body weight gain, 2) ZnT8 was detected in the EEC of the GI tract, 3) deficiency of ZnT8 increased peripheral 5-HT levels through elevating TPH1 protein levels, 4) pharmacological inhibition of 5-HT synthesis effectively reversed diet-induced body weight increase and glucose intolerance in ZnT8 knockout mice, and 5) in RIN14B cells, downregulation of ZnT8 promoted 5-HT synthesis and secretion. Interestingly, overexpression of diabetes high-risk allele of human SLC30A8 increased 5-HT secretion in the medium. These effects could be mimicked by administration of zinc ion.

ZnT8, a transmembrane protein mediating the transport of zinc ion, is mainly expressed in pancreatic islets. Its physiological function has been related to the secretion of islet hormones and glucose homeostasis (24). Genomic analyses have shown that the risk C allele (rs13266634) of ZnT8 gene is associated with BMI and T2DM (2,20,21,25). The metabolic function of ZnT8 has long been proposed to occur through its regulation of the secretion of islet hormones, specifically insulin. This concept has been recently challenged by studies using transgenic techniques. Although global deletion of ZnT8 leads to significant diet-induced obesity and impaired glucose tolerance, the exacerbation was not observed in β-cell–specific ZnT8 knockout mice (6). These results indicate an alternative mechanism underlying the regulation of glucose metabolism by ZnT8. Our study revealed that ZnT8 contributed to the modulation of 5-HT biosynthesis in intestinal EEC cells, which subsequently altered systemic glucose and lipid metabolism. Deficiency of ZnT8 led to a significant increase in colonic TPH1 and circulating 5-HT level. Since >90% of circulating 5-HT is derived from the GI tract, we assume that the elevation of circulating 5-HT primarily results from a deficiency of ZnT8 in the gut rather than in other tissues such as adipose tissue and enteric neurons.

The influence of ZnT8 on lipid metabolism remains largely unexplored. Deficiency of ZnT8 in mice leads to an increase of adiposity under ND conditions, which is profoundly aggravated under HFD conditions. Our observations suggest that elevated circulating 5-HT contributed to the obesity and glucose dysfunction. Consistently, recent studies have demonstrated that gut-derived 5-HT inhibits lipolysis and thermogenesis in eWAT and BAT and promotes hepatic glucose production, leading to obesity and dysglycemia (14,15). Our study further confirmed that blockade of TPH reversed the increased adiposity and glucose intolerance in ZnT8 knockout mice. In our ZnT8-deficient mice, we also observed an increase in lipid and glycogen deposition in the liver, although Tamaki et al. (16) did not show significant changes in liver. Previously, several groups have reported that ZnT8-deficient mice have substantial hypersecretion of insulin from pancreatic β-cells (5,16). It is reasonable to speculate that the insulin directly flows into the liver through the portal vein, causing lipid and glycogen deposition in the liver. The discrepancy may be due to the different mouse model with different genetic background or other environmental factors, causing distinct liver sensitivity to the elevated insulin.

Up to now, there have been six ZnT8 knockout mouse studies (4–6,26–28). There was a discrepancy of metabolic phenotypes, including glucose tolerance, insulin sensitivity, and insulin secretion capability, under either ND or HFD conditions. There must be other factors influencing the effects of Slc30a8 deletion. Environmental conditions, such as diets and intestinal microbial composition, might be important. In our ZnT8 knockout mouse model, we observed a significant change in intestinal tract morphology and motility. The altered gut microenvironment could lead to increased harvest from the diet, changes in fatty acid metabolism, gut 5-HT secretion, and the intestinal barrier. Intestinal 5-HT production is also affected by gut microbes (29,30). As of now, we still do not know whether it is a direct effect of intestinal ZnT8 on 5-HT biosynthesis or an indirect effect caused by the disturbed gut microenvironment or microbiota. Moreover, we cannot exclude the existence and effects of ZnT8 in other tissues, such as immune cells, the CNS, or the enteric nervous system, which may contribute to the disturbed 5-HT biogenesis and systemic metabolic dysregulation. To exclude these confounding factors, the intestinal-specific ZnT8 deletion mouse could be a better model for investigating the role of ZnT8 in the intestinal tract.

There are several plausible mechanisms through which intestinal ZnT8 regulates circulating levels of 5-HT. First, ZnT8 regulates the zinc level within EEC cells. Zinc is a critical cofactor for >300 proteins and enzymes. It plays essential roles in various biochemical processes, including tryptophan metabolism (31). ZnT8 is a zinc transporter that mediates zinc export from the intracellular to the extracellular or cellular organelles (19). Previous studies reported that ZnT8 is expressed on the plasma membrane and may mediate bidirectional transport of zinc ions (32). Ablation of ZnT8 alters intracellular or vesicular zinc levels in β-cells (5). We speculate that a similar increase of cytosolic zinc level occurs in EEC cells after ZnT8 deletion, hence stimulating 5-HT biosynthesis. In support of this concept, TPH1 is significantly increased in the intestinal mucosa of ZnT8 knockout mice, and silence of ZnT8 gene increases 5-HT levels in RIN14B cells. Another potential mechanism relates to the secretion of 5-HT granules. Lack of zinc ion in the granule may lead to a looser crystalline structure, which probably affects the storage and exocytosis process of 5-HT.

Although a number of ZnT8 knockout rodent models consistently displayed increased sensitivity of T2DM and impaired glucose-induced insulin secretion in vivo (33), several loss-of-function mutations of SLC30A8 in humans confer protection from human T2DM (34). Later transgenic mouse models that harbor the human ZnT8 truncation mutant have increased insulin secretion (35). Increased 5-HT level may account for this discrepancy. 5-HT increases insulin secretion through modulating insulin granule exocytosis (36,37). During pregnancy, the expression of islet 5-HT is profoundly increased and regulates pancreatic β-cell mass and increased glucose-induced insulin secretion (38,39). Upon a metabolic stress condition, such as HFD feeding, 5-HT is also significantly increased (12) and enhances glucose-induced insulin secretion (40). Consequently, an elevated insulin level leads to lipid deposition in the adipose tissue and lipid/glycogen production in liver.

Interestingly, overexpression of diabetes high-risk allele ZnT8 R325 variant increased 5-HT secretion in RIN14B cells. However, TPH1 protein level and 5-HT in the cell lysate were not significantly changed. This suggests that different from total loss of function in ZnT8 deficiency, the mutant form of human ZnT8 may interfere with the exocytosis rather than the biosynthesis of 5-HT. The exact mechanism by which diabetes risk ZnT8 mutant alters the exocytosis process still needs further investigation.

Normal weight obesity means higher fat mass but normal BMI. These groups of patients have received more and more attention since they have similar risks for serious metabolic disorders as obese people, but they are easily ignored by clinicians and themselves. Interestingly, two genome-wide association studies have shown that the human SLC30A8 risk allele confers higher risk of developing T2DM in a lower BMI population or nonobese subjects (21,41). These findings suggest that ZnT8 and 5-HT levels are important factors that determine the percentage of body fat regardless of body weight. Our findings further support this concept. ZnT8 knockout mice demonstrated a significant increase of fat mass without body weight gain, which was reminiscent of normal weight obesity in humans. Since the lean mass remained unchanged, one possibility of increased fat mass without body weight gain is due to loss of bone density because 5-HT elevation has been known to cause osteoporosis (42).

In summary, our study identified the presence of ZnT8 in 5-HT–positive EEC cells. Deficiency of ZnT8 enhanced 5-HT biosynthesis through modulating TPH1 level. GI-derived 5-HT acted on the peripheral organs to promote lipid deposition and obesity. Targeting intestinal ZnT8 may provide an alternative strategy for the intervention of obesity and its associated metabolic dysfunction, such as hepatic steatosis and T2DM.

Acknowledgments. The authors acknowledge Chaowei Zhu, Weiqi Wu, and Ruolu Bao (Center for Diabetes, Obesity and Metabolism, Shenzhen University Health Science Center) for technical support.

Funding. This work was funded by the National Key R&D Program of China (2017YFC0908900), the National Natural Science Foundation of China (81500619, 81730020, 81870405), the Natural Science Foundation of Guangdong Province (2016A030310040), and the Shenzhen Science and Technology Project (JCYJ20160422091658982, JCYJ20160422153856130).

Duality of Interest. No potential conflicts of interest relevant to this article were reported.

Author Contributions. Z.M. designed and performed most of the experiments. 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 K.P. performed the experiments and assisted with data analysis. W.Z. is the guarantor of this work and, as such, had full access to all the data and takes responsibility of the integrity of the data and the accuracy of the data analysis.