Trimethylamine N-Oxide Response to a Mixed Macronutrient Tolerance Test in a Cohort of Healthy United States Adults.

Plasma trimethylamine n-oxide (TMAO) concentration increases in responses to feeding TMAO, choline, phosphatidylcholine, L-carnitine, and betaine but it is unknown whether concentrations change following a mixed macronutrient tolerance test (MMTT) with limited amounts of TMAO precursors. In this proof-of-concept study, we provided healthy female and male adults (n = 97) ranging in age (18-65 years) and BMI (18-44 kg/m2) a MMTT (60% fat, 25% sucrose; 42% of a standard 2000 kilo calorie diet) and recorded their metabolic response at fasting and at 30 min, 3 h, and 6 h postprandially. We quantified total exposure to TMAO (AUC-TMAO) and classified individuals by the blood draw at which they experienced their maximal TMAO concentration (TMAO-response groups). We related AUC-TMAO to the 16S rRNA microbiome, to two SNPs in the exons of the FMO3 gene (rs2266782, G>A, p.Glu158Lys; and rs2266780, A>G, p.Glu308Gly), and to a priori plasma metabolites. We observed varying TMAO responses (timing and magnitude) and identified a sex by age interaction such that AUC-TMAO increased with age in females but not in males (p-value = 0.0112). Few relationships between AUC-TMAO and the fecal microbiome and FMO3 genotype were identified. We observed a strong correlation between AUC-TMAO and TNF-α that depended on TMAO-response group. These findings promote precision nutrition and have important ramifications for the eating behavior of adults who could benefit from reducing TMAO exposure, and for understanding factors that generate plasma TMAO.

Identification of novel compound heterozygous variants in the SLC30A7 (ZNT7) gene in two French brothers with stunted growth, testicular hypoplasia and bone marrow failure.

Zinc is an essential trace mineral. Dietary zinc deficiency results in stunted growth, skin lesions, hypogonadism and frequent infections in humans. Mice genetically lacking Slc30a7 suffer from mild zinc deficiency and are prone to development of prostate cancer and insulin resistance. Disease-causing variants or mutations in the human SLC30A7 (ZNT7) gene have not been previously reported. Here, we describe two-boy siblings from a French family with stunted growth, testicular hypoplasia and bone marrow failure. Exome sequencing revealed compound heterozygous variants in ZNT7 consisting of NM_133496.5:c.21dup; p.Asp8ArgfsTer3 and c.842 + 15 T > C inherited from their unaffected mother and father, respectively. The c.21dup variant led to a premature stop codon generated in exon 1 of the ZNT7 coding sequence. RNA-seq analysis demonstrated that the c.842 + 15 T > C variant resulted in a leaky mRNA splicing event generating a premature stop codon right after the splicing donor site of exon 8. Moreover, the expression of ZNT7 protein was remarkably reduced by 80-96% in the affected brothers compared to the control cells. These findings strongly suggest that biallelic variants in SLC30A7 should be considered as a cause of growth retardation, testicular hypoplasia and syndromic bone marrow failure.

Effects of a genetic variant rs13266634 in the zinc transporter 8 gene (SLC30A8) on insulin and lipid levels before and after a high-fat mixed macronutrient tolerance test in U.S. adults.

BACKGROUND: The common C-allele of rs13266634 (c.973C>T or p.Arg325Trp) in SLC30A8 (ZNT8) is associated with increased risk of type 2 diabetes. While previous studies have examined the correlation of the variant with insulin and glucose metabolism, the effects of this variant on insulin and lipid responses after a lipid challenge in humans remain elusive. The goal of this study was to determine whether the C-allele had an impact on an individual's risk to metabolic syndromes in U.S. adults. METHOD: We studied the genotypes of rs13266634 in 349 individuals aged between 18 and 65 y with BMI ranging from 18.5 to 45 kg/m2. The subjects were evaluated for insulin, glucose, HbA1c, ghrelin, and lipid profiles before and after a high-fat mixed macronutrient tolerance test (MMTT). RESULTS: We found that the effects of variants rs13266634 on glucose and lipid metabolism were sex-dimorphic, greater impact on males than on females. Insulin incremental area under the curve (AUC) after MMTT was significantly decreased in men with the CC genotype (p < 0.05). Men with the CC genotype also had the lowest fasting non-esterified fatty acid (NEFA) concentrations. On the other hand, the TT genotype was associated with a slower triglyceride removal from the circulation in men after MMTT. The reduced triglyceride removal was also observed in subjects with BMI ≥ 30 carrying either the heterozygous or homozygous T-allele. Nevertheless, the SNP had little effect on fasting or postprandial blood glucose and cholesterol concentrations. CONCLUSION: We conclude that the CC genotype negatively affects insulin response after MMTT while the T-allele may negatively influence lipolysis during fasting and postprandial blood triglyceride removal in men and obese subjects, a novel finding in this study.

SNPs in apolipoproteins contribute to sex-dependent differences in blood lipids before and after a high-fat dietary challenge in healthy U.S. adults.

BACKGROUND: The effect of genetic polymorphisms on fasting blood lipid levels have been widely studied but the effects of these within the context of a high-fat meal challenge remain less characterized. The current study aimed to investigate the association of SNPs in lipoprotein-related genes with blood lipid profiles in healthy adults in the U.S. METHODS: Subjects (n = 393) between 18-66 years of age with BMIs ranging from 18.5-45 kg/m2 were enrolled the cross-sectional Nutritional Phenotyping Study. Among them, 349 subjects (men: 48%; women: 52%) gave consent for genotyping. SNPs in APOA5, APOB, APOC3, APOE, and LDLR were assessed. The association between lipid markers and genotypes was tested separately for each SNP with analysis of variance (ANOVA), adjusted for sex, age, and BMI. We also examined two-factor interactions between SNPs and sex, age, or BMI. RESULTS: Women carrying the C allele of rs3135506 in APOA5 or men carrying the C allele of rs429358 in APOE had reduced HDL-cholesterol levels during fasting and postprandially. The C allele in APOE was also correlated to increased LDL-C levels. The TT genotype of rs2854116 in APOC3 was associated with elevated total cholesterol. Additive effect of the risk alleles of APOA5 and APOE or APOC3 and APOE was detected. Nevertheless, the tested SNPs had little impact on the postprandial triglyceride responses to the high-fat challenge meal. We found no significant effects of SNPs in APOB (rs1042034) or LDLR (rs2228671) on triglycerides, cholesterol, or free fatty acid levels. CONCLUSIONS: In healthy adults, fasting and postprandial cholesterol levels are strongly correlated with the tested APOA5, APOE, and APOC3 genotypes. Sex contributes to the genetic impact of the tested SNPs on lipid profiles. TRIAL REGISTRATION: ClinicalTrials.gov, NCT02367287. Registered February 20, 2015, https://clinicaltrials.gov/ct2/show/NCT02367287 .

The Znt7-null mutation has sex dependent effects on the gut microbiota and goblet cell population in the mouse colon.

Cellular homeostasis of zinc, an essential element for living organisms, is tightly regulated by a family of zinc transporters. The zinc transporter 7, ZnT7, is highly expressed on the membrane of the Golgi complex of intestinal epithelial cells and goblet cells. It has previously been shown that Znt7 knockout leads to zinc deficiency and decreased weight gain in C57BL/6 mice on a defined diet. However, effects within the colon are unknown. Given the expression profile of Znt7, we set out to analyze the changes in mucin density and gut microbial composition in the mouse large intestine induced by Znt7 knockout. We fed a semi-purified diet containing 30 mg Zn/kg to Znt7-/- mice with their heterozygous and wild type littermates and found a sex specific effect on colonic mucin density, goblet cell number, and microbiome composition. In male mice Znt7 knockout led to increased goblet cell number and mucin density but had little effect on gut microbiome composition. However, in female mice Znt7 knockout was associated with decreased goblet cell number and mucin density, with increased proportions of the microbial taxa, Allobaculum, relative to wild type. The gut microbial composition was correlated with mucin density in both sexes. These findings suggest that a sex-specific relationship exists between zinc homeostasis, mucin production and the microbial community composition within the colon.

Association of Lactase Persistence Genotypes (rs4988235) and Ethnicity with Dairy Intake in a Healthy U.S. Population.

Lactase persistence (LP) is a trait in which lactose can be digested throughout adulthood, while lactase non-persistence (LNP) can cause lactose intolerance and influence dairy consumption. One single nucleotide polymorphism (SNP ID: rs4988235) is often used as a predictor for dairy intake, since it is responsible for LP in people in European descent, and can occur in other ethnic groups. The objective of this study was to determine whether rs4988235 genotypes and ethnicity influence reported dairy consumption in the United States (U.S.). A food frequency questionnaire (FFQ) and multiple Automated Self-Administered 24-h recalls (ASA24®) were used to measure habitual and recent intake, respectively, of total dairy, cheese, cow's milk, plant-based alternative milk, and yogurt in a multi-ethnic U.S. cohort genotyped for rs4988235. Within Caucasian subjects, LP individuals reported consuming more recent total dairy and habitual total cow's milk intake. For subjects of all ethnicities, LP individuals consumed more cheese (FFQ p = 0.043, ASA24 p = 0.012) and recent total dairy (ASA24 p = 0.005). For both dietary assessments, Caucasians consumed more cheese than all non-Caucasians (FFQ p = 0.036, ASA24 p = 0.002) independent of genotype, as well as more recent intake of yogurt (ASA24 p = 0.042). LP subjects consumed more total cow's milk than LNP, but only when accounting for whether subjects were Caucasian or not (FFQ p = 0.015). Fluid milk and alternative plant-based milk consumption were not associated with genotypes or ethnicity. Our results show that both LP genotype and ethnicity influence the intake of some dairy products in a multi-ethnic U.S. cohort, but the ability of rs4988235 genotypes to predict intake may depend on ethnic background, the specific dairy product, and whether intake is reported on a habitual or recent basis. Therefore, ethnicity and the dietary assessment method should also be considered when determining the suitability of rs4988235 as a proxy for dairy intake.

Effects of Bacillus aryabhattai TBRC8450 on vibriosis resistance and immune enhancement in Pacific white shrimp, Litopenaeus vannamei.

The use of probiotics in aquaculture is a practical alternative to promote animal health and disease prevention. Meanwhile, this practice can also reduce the use of prophylactic antibiotics. The purpose of this study was to identify candidate probiotics that could control pathogen populations in host's gastrointestinal (GI) tract and stimulate host immunity in shrimp aquaculture. Bacillus aryabhattai TBRC8450, a bacterial strain isolated from the environment in a shrimp farm, has an antimicrobial activity against many pathogenic strains of Vibrio harveyi and V. parahaemolyticus. Supplementation of B. aryabhattai to Pacific white shrimp (Litopenaeus vannamei) not only decreased the abundance of Vibrio populations, but also shifted the bacterial community in the shrimp GI tract. We found that supplementation of B. aryabhattai triggered shrimp innate immunity and antioxidant activities. mRNA expression of genes encoding microbial peptides and antioxidant enzymes, including C-type lectin, penaeidin-3, heat shock protein 60, thioredoxin, and ferritin, was significantly upregulated in the hepatopancreas of shrimp fed B. aryabhattai. Furthermore, phenoloxidase activity in the hemocytes and the total antioxidant activity in the plasma were increased, indicating enhanced immune and antioxidant responses at the systemic level. In contrast, supplementation of B. aryabhattai had no effect on the total hemocyte count and superoxide dismutase activity in the plasma and hepatopancreas. Importantly, a pathogen challenge test using V. harveyi 1562 showed a significant increase in survival rates of shrimp fed B. aryabhattai compared to the control group. Our findings suggest that B. aryabhattai TBRC8450 can likely be used as a probiotic to reduce the population of V. harveyi in the shrimp GI tract and to enhance shrimp innate immunity and antioxidant capacity for vibriosis resistance in shrimp aquaculture.

Monitoring of white striping and wooden breast cases and impacts on quality of breast meat collected from commercial broilers (Gallus gallus).

OBJECTIVE: This study aimed at investigating white striping (WS) and wooden breast (WB) cases in breast meat collected from commercial broilers. METHODS: A total of 183 breast samples were collected from male Ross 308 broilers slaughtered at the age of 6 weeks (n = 100) and 7 weeks (n = 83). The breasts were subjected to meat defect inspection, meat quality determination and histology evaluation. RESULTS: Of 183, 4 breasts from 6-week-old broilers were classified as non-defective while the others exhibited the WS lesion. Among the 6-week-old birds, the defective samples from the medium size birds (carcass weight ≤2.5 kg) showed mild to moderate WS degree with no altered meat quality. Some of the breasts from the 6-week-old birds with carcass weight above 2.5 kg exhibited WB in accompanied with the WS condition. Besides of a reduction of protein content, increases in collagen matter and pH values in the defective samples (p<0.05), no other impaired quality indices were detected within this group. All 7-week-old broilers yielded carcasses weighing above 2.5 kg and showed abnormal characteristics with progressive severity. The breasts affected with severe WS and WB showed the greatest cook loss, hardness, springiness and chewiness (p<0.05). Development of WB induced significantly increased drip loss in the samples (p<0.05). Histology indicated necrotic events in the defective myofibers. Based on logistic regression, increasing percent breast weight by one unit enhanced the chance of WS and WB development with advanced severity by 50.9% and 61.0%, respectively. Delayed slaughter age from 6 to 7 weeks increased the likelihood of obtaining increased WS severity by 56.3%. CONCLUSION: Cases of WS and WB defects in Southeast Asia have been revealed. Despite few cases of the severe WS and WB, such abnormal conditions significantly impaired technological properties and nutritional quality of broiler breasts.

Aberrant fatty acid metabolism in skeletal muscle contributes to insulin resistance in zinc transporter 7 (znt7)-knockout mice.

ZnT7 (Slc30a7) is a widely expressed zinc transporter involved in sequestration of zinc into the Golgi apparatus and vesicular compartments. znt7-knockout (KO) mice are mildly zinc-deficient and lean. Despite their lean phenotype, adult male znt7-KO mice are prone to insulin resistance. We hypothesized that fat partitioning from adipose to nonadipose tissues causes insulin resistance in znt7-KO mice. Here, we used biological and biochemical methods, including fatty acid and oxylipin profiling, EM, immunohistochemistry, quantitative RT-PCR, and Western blot analysis, to identify the underlying mechanism of insulin resistance in znt7-KO mice. We found that insulin resistance in this model was primarily associated with increased intracellular fatty acid levels in the skeletal muscle, which promoted intracellular lipid accumulation and production of bioactive lipid mediators, such as 12,13-dihydroxyoctadecanoic acid (12,13-DiHOME) and 12-hydroxyeicosatetraenoic acid (12-HETE). The expression of fatty acid-binding protein 3 (Fabp3) was dramatically up-regulated in the znt7-KO muscle cells accompanied by increased expression of Cd36, Slc27a1, and Slc27a4, the three major fatty acid transporters in the skeletal muscle. We also demonstrated that znt7-KO muscle cells had increased fatty acid oxidative capacity, indicated by enlarged mitochondria and increased mRNA or protein expression of key enzymes involved in the fatty acid mitochondrial shuttle and ß-oxidation. We conclude that increased fatty acid uptake in the znt7-KO skeletal muscle is a key factor that contributes to the excessive intracellular lipid deposit and elevated production of bioactive lipid mediators. These mediators may play pivotal roles in oxidative stress and inflammation, leading to insulin resistance.

SLC30A family expression in the pancreatic islets of humans and mice: cellular localization in the ß-cells.

Zinc is a vital co-factor for insulin metabolism in the pancreatic ß-cell, involved in synthesis, maturation, and crystallization. Two families of zinc transporters, namely SLC30A (ZNT) and SLC39A (ZIP) are involved in maintaining cellular zinc homeostasis in mammalian cells. Single nuclear polymorphisms or mutations in zinc transporters have been associated with insulin resistance and risk of type 2 diabetes (T2D) in both humans and mice. Thus, mice can be useful for studying the underlying mechanisms of zinc-associated risk of T2D development. To determine potential differences in zinc transporter expression and cellular localization in the pancreatic ß-cells between humans and mice, we examined all members (ZNT1-10) of the ZNT family in pancreatic islets and in ß-cell lines derived from both species using immunohistochemistry and immunofluorescence microscopic analysis. We found that there were no substantial differences in the expression of nine ZNT proteins in the human and mouse islets and ß-cells with exception of ZNT3, which was only detected in human ß-cells, but not in mouse ß-cells. Moreover, we found that ZNT2 was localized on the cell surface of both human and mouse ß-cells, suggesting a role of ZNT2 in direct export of zinc out of the ß-cell. Together, our study suggests functional conservations of the ZNT proteins between humans and mice. We believe that our results are of interest for future studies in the association of zinc metabolism with risk of T2D in humans using mouse models.

ZNT7 binds to CD40 and influences CD154-triggered p38 MAPK activity in B lymphocytes-a possible regulatory mechanism for zinc in immune function.

Zinc deficiency impairs the immune system leading to frequent infections. Although zinc is known to play critical roles in maintaining healthy immune function, the underlying molecular targets are largely unknown. In this study, we demonstrate that zinc is important for the CD154-CD40-mediated activation of downstream signaling pathways in human B lymphocytes. CD40 is a receptor localized on the cell surface of many immune cells, including B lymphocytes. It binds to CD154, a membrane protein expressed on antigen-activated T helper (Th) lymphocytes. This CD154-CD40 interaction leads to B-cell activation. We showed that cellular zinc deficiency impaired the CD154-CD40-mediated p38 mitogen-activated protein kinase (p38 MAPK) phosphorylation. We also showed that zinc supplemental treatment of B lymphocytes had limited effect on this CD40-mediated p38 MAPK signaling. Most importantly, we demonstrated that the zinc transporter protein zinc transporter 7 (ZNT7) interacted with CD40 using immunoprecipitation analyses. ZNT7 knockdown in B lymphocytes had a negative effect on the cell surface expression of CD40. Consequently, the CD40-mediated p38 MAPK signaling transduction was down-regulated in ZNT7 KD B lymphocytes. Conversely, this p38 MAPK signaling activity was up-regulated by overexpression (OE) of ZNT7 in B lymphocytes. Moreover, we found that ZNT7 knockdown in B lymphocytes constitutively up- and down-regulated the inhibitor of i kappa B kinase and AKT serine/threonine kinase phosphorylation, respectively, which implies the activation of survival signaling in ZNT7 KD B cells. We conclude that CD40 is the target molecule for ZNT7 in regulation of immune function of B lymphocytes.

Zinc transporter 7 deficiency affects lipid synthesis in adipocytes by inhibiting insulin-dependent Akt activation and glucose uptake.

Mice deficient for zinc transporter 7 protein (ZnT7) are mildly zinc deficient with low body weight gain and body fat accumulation. To investigate the underlying mechanism of ZnT7 deficiency in body adiposity, we examined fatty acid composition and insulin sensitivity in visceral (epididymal) and subcutaneous fat pads from Znt7 knockout and control mice. We showed that ZnT7 deficiency had adverse effects on fatty acid metabolism and insulin action in subcutaneous fat but not in epididymal fat in mice, consistent with the ZnT7 protein expression pattern in adipose tissues. Importantly, we found that the expression of ZnT7 protein was induced by lipogenic differentiation and reached a peak when the adipocyte was fully differentiated in mouse 3T3-L1 adipocytes. We demonstrated, using Znt7 knockdown (Znt7KD) 3T3-L1 adipocytes, that reduction in Znt7 expression blunted activations of the signal transduction pathways that regulated both basal and insulin-stimulated glucose uptake in adipocytes, resulting in low glucose uptake and lipid accumulation. The expression of the signaling mediators critical for the initiation of pre-adipocyte differentiation, including Pparγ and C/Ebpα, appeared not to be affected by Znt7KD in 3T3-L1 adipocytes. These findings strongly suggest a role for ZnT7 in adipocyte lipogenesis.

Linking cellular zinc status to body weight and fat mass: mapping quantitative trait loci in Znt7 knockout mice.

Zinc transporter 7 (Znt7, Slc30a7) knockout (KO) mice display abnormalities in body weight gain and body adiposity. Regulation of body weight and body fat accumulation is complex, involving multiple genetic and environmental factors. To understand how zinc homeostasis influences body weight and fat deposit and to identify quantitative trait loci (QTLs) that link zinc metabolism to growth and adiposity, we conducted a genome-wide mapping study using male F2 Znt7 KO mice and wild-type (WT) littermates with a mixed 129P1/ReJ and C57BL/6J genetic background. The mice were fed a semi-purified diet containing 30-mg Zn/kg diet at weaning. Body weights and fat pad weights including epididymal, retroperitoneal, and femoral subcutaneous fat pads were measured at 16 weeks of age. We detected two significant QTLs (p < 0.05) for body weight and fat deposit. One was in the F2 Znt7 KO population and the other in the F2 WT population. In Znt7 KO mice, the body weight and fat deposit was significantly linked to a locus on chromosome 7 ranging from 64.3 to 78.3 Mb. In WT mice, a significant linkage of retroperitoneal fat mass was found on chromosome 8 between 14.5 and 63.5 Mb. In addition, several other suggestive QTLs (p < 0.63) for body weight and fat accumulation were detected in Znt7 KO and WT mice. In conclusion, the QTLs identified in this study may provide new hints to uncover the genes linking cellular zinc status to growth and body fat accumulation.

Znt7-null mice are more susceptible to diet-induced glucose intolerance and insulin resistance.

The Znt7 gene encodes a ubiquitously expressed zinc transporter that is involved in transporting cytoplasmic zinc into the Golgi apparatus and a ZnT7-containing vesicular compartment. Overexpression of ZnT7 in the pancreatic ß-cell stimulates insulin synthesis and secretion through regulation of insulin gene transcription. In this study, we demonstrate that ZnT7 is expressed in the mouse skeletal muscle. The activity of the insulin signaling pathway was down-regulated in myocytes isolated from the femoral muscle of Znt7 knock-out (KO) mice. High fat diet consumption (45% kcal) induced weight gain in male Znt7 KO mice but not female Znt7 KO mice. Male Znt7 KO mice fed the high fat diet at 5 weeks of age for 10 weeks exhibited hyperglycemia in the non-fasting state. Oral glucose tolerance tests revealed that male Znt7 KO mice fed the high fat diet had severe glucose intolerance. Insulin tolerance tests showed that male Znt7 KO mice were insulin-resistant. Diet-induced insulin resistance in male Znt7 KO mice was paralleled by a reduction in mRNA expression of Insr, Irs2, and Akt1 in the primary skeletal myotubes isolated from the KO mice. Overexpression of ZnT7 in a rat skeletal muscle cell line (L6) increased Irs2 mRNA expression, Irs2 and Akt phosphorylation, and glucose uptake. We conclude that a combination of decreased insulin secretion and increased insulin resistance accounts for the glucose intolerance observed in Znt7 KO mice.

Dietary strawberry powder reduces blood glucose concentrations in obese and lean C57BL/6 mice, and selectively lowers plasma C-reactive protein in lean mice.

The purpose of the present study was to test the anti-inflammatory and blood glucose (BG)-regulating capacity of strawberries in a mouse model of diet-induced obesity. A total of thirty-six male C57BL/6J mice were randomly divided into four groups (nine mice per group). Mice were fed a low-fat diet (LF, 13 % fat), the LF supplemented with 2·6 % freeze-dried strawberry powder (LFSB), a high-fat diet (HF, 44 % fat) or the HF supplemented with 2·6 % strawberry powder (HFSB). Blood samples were collected to measure BG, inflammation and systemic markers for endocrine function of pancreas and adipose tissue. Splenocytes were harvested at the end of the study and activated with either anti-cluster of differentiation (CD) 3/anti-CD28 antibodies or lipopolysaccharide to test immune responsiveness. The HF increased non-fasted BG, insulin, soluble intracellular adhesion molecule-1, E-selectin, leptin, resistin and plasminogen activator protein-1 (P < 0·05). High dietary fat decreased IL-4 production from activated splenocytes (P < 0·05). BG concentrations were lower in the mice supplemented with SB (10·64 mmol/l) compared to the non-supplemented mice (11·37 mmol/l; P = 0·0022). BG values were approximately 6·5 % lower in the supplemented mice. Additionally, SB lowered plasma C-reactive protein in the LFSB group compared to the other three groups (P < 0·05). The dietary intake of SB approximated one human serving of strawberries. These results, although modest, support a promising role for dietary strawberries in reducing the risks associated with obesity and diabetes, and regulating the levels of inflammatory markers in non-obese individuals.

A null-mutation in the Znt7 gene accelerates prostate tumor formation in a transgenic adenocarcinoma mouse prostate model.

Decrease of cellular zinc in the epithelium of the prostate has been implicated in the development of prostate cancer. To investigate whether ZnT7, a zinc transporter involved in intracellular zinc accumulation, played a role in prostate cancer development, we generated and characterized a transgenic adenocarcinoma mouse prostate (TRAMP) model with a Znt7-null genetic background. TRAMP mice (TRAMP/Znt7(-/-) and TRAMP/Znt7(+/+)) were euthanized at 6, 8, 16, and 28 weeks of age for histopathological analysis of the prostates and for the presence of prostate tumors and metastasis. At 6 and 8 weeks of age, TRAMP/Znt7(-/-) mice displayed higher frequencies of low grade prostatic intraepithelial neoplasia (PIN) and high grade PIN, respectively, in the prostates than the age-matched TRAMP/Znt7(+/+) mice. At 16 weeks of age, 33% TRAMP/Znt7(-/-) mice had prostate tumors and one half of the mice with prostate tumors had tumor metastasized to the draining lymph nodes while no prostate tumor was detected in the control TRAMP mice. By 28 weeks, 67% TRAMP/Znt7(-/-) mice developed prostate tumors while only 22% control TRAMP mice had prostate tumors. Furthermore, apoptosis was reduced in the prostates of TRAMP/Znt7(-/-) mice. In conclusion, a null-mutation of the Znt7 gene accelerates prostate tumor formation in TRAMP mice.

Over-expression of ZnT7 increases insulin synthesis and secretion in pancreatic beta-cells by promoting insulin gene transcription.

The mechanism by which zinc regulates insulin synthesis and secretion in pancreatic beta-cells is still unclear. Cellular zinc homeostasis is largely maintained by zinc transporters and intracellular zinc binding proteins. In this study, we demonstrated that zinc transporter 7 (ZnT7, Slc30a7) was co-expressed with insulin in the islet of Langerhans in the mouse pancreas. In RIN5mF cells (rat insulinoma cells), ZnT7 was found mainly residing in the perinuclear region of the cell, which is consistent with its Golgi apparatus localization. Over-expression of ZnT7 in RIN5mF cells increased the total cellular insulin content leading to a high basal insulin secretion. Furthermore, glucose-induced insulin secretion was not altered in RIN5mF cells over-expressing ZnT7. Quantitative RT-PCR and (35)S metabolic labeling analysis demonstrated that over-expression of ZnT7 in RIN5mF cells led to an increase of insulin mRNA expression and subsequent insulin protein synthesis in the cell. Metal-responsive elements (MREs) were identified in the promoter regions of the Ins1 and Ins2 genes. Mtf1, a metal-responsive transcription factor, was shown to specifically bind to the MRE in the Ins genes and activated the insulin gene transcription. Together, the data strongly suggest that ZnT7 plays an important role in regulating insulin expression by modulating Mtf1 transcriptional activity.

Expression of the ZNT (SLC30) family members in the epithelium of the mouse prostate during sexual maturation.

A prostate contains approximately 10-fold higher zinc than other soft organs. The function of the prostate is to produce a zinc-enriched seminal fluid. To establish a protein expression profile for zinc transporters involved in zinc efflux and intracellular sequestration/storage in the mouse prostate during sexual maturation, ZNT expression were investigated by immunohistochemistry. Our study demonstrated that ZNT proteins were differentially expressed in the prostate during sexual maturation. ZNT1 was mainly detected on the lateral membrane of the epithelium. Other ZNTs examined resided intracellularly. Among differences were a staining of ZNT2/ZNT5 in the ER-rich area of the epithelium in the anterior lobe, a staining of ZNT2 along the lateral and apical membrane, a luminal border staining of ZNT4, a staining of ZNT5 in the Golgi area of the epithelium in the ventral lobe, a uniform expression of ZNT6 across the lobes and ages, and a staining of ZNT7 in all lobes across ages.

Znt7 (Slc30a7)-deficient mice display reduced body zinc status and body fat accumulation.

In vitro studies have demonstrated that ZNT7 is involved in transporting the cytoplasmic zinc into the Golgi apparatus of the cell for zinc storage or to be incorporated into newly synthesized zinc-requiring enzymes/proteins. To evaluate the physiological role of ZNT7, we created a mouse model of Znt7 deficiency by a gene-trap approach. Znt7-deficient mice were zinc-deficient based on their low zinc content in serum, liver, bone, kidney, and small intestine. In embryonic fibroblasts isolated from Znt7-deficient mice, cellular zinc was approximately 50% that of wild-type controls. Znt7-deficient mice also displayed some classic manifestations of dietary zinc deficiency, such as reduced food intake and poor body weight gain. However, the mutant mice did not show any sign of hair abnormality and dermatitis that are commonly associated with dietary zinc deficiency. A radioactive feeding study suggested that Znt7-deficient mice had reduced zinc absorption in the gut resulting in decreased zinc accumulations in other organs in the body. The poor growth found in Znt7-deficient mice could not be corrected by feeding the mutant mice with a diet containing 6-fold higher zinc (180 mg/kg) than the suggested adequate intake amount (30 mg/kg). Furthermore, the reduced body weight gain of the mutant mice was largely due to the decrease in body fat accumulation. We conclude that ZNT7 has essential functions in dietary zinc absorption and in regulation of body adiposity.

A di-leucine sorting signal in ZIP1 (SLC39A1) mediates endocytosis of the protein.

It has been demonstrated that the plasma membrane expression of ZIP1 is regulated by endocytic mechanisms. In the zinc-replete condition, the level of surface expressed ZIP1 is low due to the rapid internalization of ZIP1. The present study aimed to identify a sorting signal(s) in ZIP1 that mediated endocytosis of ZIP1. Four potential sorting signals (three di-leucine-and one tyrosine-based) were found by searching the eukaryotic linear motif resource for functional sites in proteins (http://elm.eu.org). Site-directed mutagenesis and immunofluorescence microscopic analyses demonstrated that the di-leucine sorting signal, ETRALL144-149, located in the variable loop region of ZIP1, was required for the ZIP1 internalization and lysosomal degradation. Substitutions of alanines for the di-leucine residues (LL148,149/AA) severely impaired the internalization of ZIP1 and subsequent protein degradation, leading to an accumulation of the mutant ZIP1 on the cell surface, as well as inside the cell. Using chimeric proteins composed of an alpha-chain of interleukin-2 receptor fused to the peptides derived from the variable loop region of ZIP1, we found that the di-leucine sorting signal of ZIP1 was required and sufficient for endocytosis of the chimeric proteins.