The Effect of Testosterone Upon the Urate Reabsorptive Transport System in Mouse Kidney

It is hypothesized that hyperuricemia in males is caused by androgen-induced urate reabsorptive transport system in the kidney. The expression of urate transporter 1 (Urat1), sodium-coupled monocarboxylate transporter 1 (Smct1) and glucose transporter 9 (Glut9) were investigated in orchiectomized mice with or without testosterone replacement. Testosterone enhanced mRNA and protein levels of Smct1 while those of Glut9 were attenuated. Although the mRNA level of Urat1 was enhanced by testosterone, the corresponding levels of Urat1 protein remained unaffected. Thus, the induction of Smct1 by testosterone is a candidate mechanism underlying hyperuricemia in males.     

The effect of female hormones upon urate transport systems in the mouse kidney

Increased levels of serum urate in postmenopausal women are thought to be caused by a change in renal urate elimination associated with the loss of female hormones. In this study, we investigated the regulation of renal urate transporter expression by female hormones using ovariectomized mice with or without hormone replacement. Estradiol suppressed the protein levels of urate reabsorptive transporters urate transporter 1 and glucose transporter 9 (Urat1 and Glut9), and that of urate efflux transporter ATP-binding cassette sub-family G member 2 (Abcg2). Progesterone suppressed protein levels of sodium-coupled monocarboxylate transporter 1 (Smct1). However, neither estradiol nor progesterone influenced the respective levels of mRNA.     

ABCG2 Dysfunction Increases Serum Uric Acid by Decreased Intestinal Urate Excretion

ATP-binding cassette transporter G2 (ABCG2), also known as breast cancer resistance protein (BCRP), is identified as a high-capacity urate exporter and its dysfunction has an association with serum uric acid (SUA) levels and gout/hyperuricemia risk. However, pathophysiologically important pathway(s) responsible for the ABCG2-mediated urate excretion were unknown. In this study, we investigated how ABCG2 dysfunction affected the urate excretion pathways. First, we revealed that mouse Abcg2 mediates urate transport using the membrane vesicle system. The export process by mouse Abcg2 was ATP-dependent and not saturable under the physiological concentration of urate. Then, we characterized the excretion of urate into urine, bile, and intestinal lumen using in vivo mouse model. SUA of Abcg2-knockout mice was significantly higher than that of control mice. Under this condition, the renal urate excretion was increased in Abcg2-knockout mice, whereas the urate excretion from the intestine was decreased to less than a half. Biliary urate excretion showed no significant difference regardless of Abcg2 genotype. From these results, we estimated the relative contribution of each pathway to total urate excretion; in wild-type mice, the renal excretion pathway contributes approximately two-thirds, the intestinal excretion pathway contributes one-third of the total urate excretion, and the urate excretion into bile is minor. Decreased intestinal excretion could account for the increased SUA of Abcg2-knockout mice. Thus, ABCG2 is suggested to have an important role in extra-renal urate excretion, especially in intestinal excretion. Accordingly, increased SUA in patients with ABCG2 dysfunction could be explained by the decreased excretion of urate from the intestine.     

Inhibition of intestinal bile acid transporter Slc10a2 improves triglyceride metabolism and normalizes elevated plasma glucose levels in mice

Interruption of the enterohepatic circulation of bile acids increases cholesterol catabolism, thereby stimulating hepatic cholesterol synthesis from acetate. We hypothesized that such treatment should lower the hepatic acetate pool which may alter triglyceride and glucose metabolism. We explored this using mice deficient of the ileal sodium-dependent BA transporter (Slc10a2) and ob/ob mice treated with a specific inhibitor of Slc10a2. Plasma TG levels were reduced in Slc10a2-deficient mice, and when challenged with a sucrose-rich diet, they displayed a reduced response in hepatic TG production as observed from the mRNA levels of several key enzymes in fatty acid synthesis. This effect was paralleled by a diminished induction of mature sterol regulatory element-binding protein 1c (Srebp1c). Unexpectedly, the SR-diet induced intestinal fibroblast growth factor (FGF) 15 mRNA and normalized bile acid synthesis in Slc10a2-/- mice. Pharmacologic inhibition of Slc10a2 in diabetic ob/ob mice reduced serum glucose, insulin and TGs, as well as hepatic mRNA levels of Srebp1c and its target genes. These responses are contrary to those reported following treatment of mice with a bile acid binding resin. Moreover, when key metabolic signal transduction pathways in the liver were investigated, those of Mek1/2-Erk1/2 and Akt were blunted after treatment of ob/ob mice with the Slc10a2 inhibitor. It is concluded that abrogation of Slc10a2 reduces hepatic Srebp1c activity and serum TGs, and in the diabetic ob/ob model it also reduces glucose and insulin levels. Hence, targeting of Slc10a2 may be a promising strategy to treat hypertriglyceridemia and diabetes.     

The matricellular protein SPARC/osteonectin as a newly identified factor up-regulated in obesity

Alterations in the expression level of genes may contribute to the development and pathophysiology of obesity. To find genes differentially expressed in adipose tissue during obesity, we performed suppression subtractive hybridization on epididymal fat mRNA from goldthioglucose (GTG) obese mice and from their lean littermates. We identified the secreted protein acidic and rich in cysteine (SPARC), a protein that mediates cell-matrix interactions and plays a role in modulation of cell adhesion, differentiation, and angiogenesis. SPARC mRNA expression in adipose tissue was markedly increased (between 3- and 6-fold) in three different models of obesity, i.e. GTG mice, ob/ob mice, and AKR mice, after 6 weeks of a high fat diet. Immunoblotting of adipocyte extracts revealed a similar increase in protein level. Using a SPARC-specific ELISA, we demonstrated that SPARC is secreted by isolated adipocytes. We found that insulin administration to mice increased SPARC mRNA in the adipose tissue. Food deprivation had no effect on SPARC expression, but after high fat refeeding SPARC mRNA levels were significantly increased. Our results reveal both hormonal and nutritional regulation of SPARC expression in the adipocyte, and importantly, its alteration in obesity. Finally, we show that purified SPARC increased mRNA levels of plasminogen activator inhibitor 1 (PAI-1) in cultured rat adipose tissue suggesting that elevated adipocyte expression of SPARC might contribute to the abnormal expression of PAI-1 observed in obesity. We propose that SPARC is a newly identified autocrine/paracrine factor that could affect key functions in adipose tissue physiology and pathology.     

Selective up-regulation of fatty acid uptake by adipocytes characterizes both genetic and diet-induced obesity in rodents.

Long chain fatty acid transport is selectively up-regulated in adipocytes of Zucker fatty rats, diverting fatty acids from sites of oxidation toward storage in adipose tissue. To determine whether this is a general feature of obesity, we studied [(3)H]oleate uptake by adipocytes and hepatocytes from 1) homozygous male obese (ob), diabetic (db), fat (fat), and tubby (tub) mice and from 2) male Harlan Sprague-Dawley rats fed for 7 weeks a diet containing 55% of calories from fat. V(max) and K(m) were compared with controls of the appropriate background strain (C57BL/6J or C57BLKS) or diet (13% of calories from fat). V(max) for adipocyte fatty acid uptake was increased 5-6-fold in ob, db, fat, and tub mice versus controls (p < 0.001), whereas no differences were seen in the corresponding hepatocytes. Similar changes occurred in fat-fed rats. Of three membrane fatty acid transporters expressed in adipocytes, plasma membrane fatty acid-binding protein mRNA was increased 9-11-fold in ob and db, which lack a competent leptin/leptin receptor system, but was not increased in fat and tub, i.e. in strains with normal leptin signaling capability; fatty acid translocase mRNA was increased 2.2-6.5-fold in tub, ob, and fat adipocytes, but not in db adipocytes; and only marginal changes in fatty acid transport protein 1 mRNA were found in any of the mutant strains. Adipocyte fatty acid uptake is generally increased in murine obesity models, but up-regulation of individual transporters depends on the specific pathophysiology. Leptin may normally down-regulate expression of plasma membrane fatty acid binding protein.     

Leptin induces the hepatic high density lipoprotein receptor scavenger receptor B type I (SR-BI) but not cholesterol 7alpha-hydroxylase (Cyp7a1) in leptin-deficient (ob/ob) mice

Cholesterol elimination from the body involves reverse cholesterol transport from peripheral tissues in which the elimination of high density lipoprotein (HDL) and low density lipoprotein (LDL) cholesterol by the liver and subsequent biliary excretion as free cholesterol and bile acids are important. In situations of peripheral fat and cholesterol accumulation, such as obesity, these pathways may be overloaded, contributing to increased cholesterol deposition. Leptin has an important role in obesity, suppressing food intake and increasing energy expenditure. This hormone, which is absent in genetically obese ob/ob mice, is also thought to be involved in the coordination of lipid excretion pathways, although available data are somewhat inconsistent. We therefore studied the expression of the hepatic HDL receptor, scavenger receptor class B type I (SR-BI), and the LDL receptor as well as the rate-limiting enzyme in bile acid synthesis, cholesterol 7alpha-hydroxylase (Cyp7a1), in leptin-deficient ob/ob mice and their wild-type controls. In ob/ob mice, protein levels of both LDL receptor and SR-BI were reduced, whereas LDL receptor mRNA levels were increased and those of SR-BI were reduced, regardless of challenge with a 2% cholesterol diet. In ob/ob mice, the enzymatic activity and mRNA for Cyp7a1 were reduced, and the increase in response to dietary cholesterol was blunted. Upon short-term (2 days) treatment with leptin, a dose-dependent increase was seen in the SR-BI protein and mRNA, whereas the Cyp7a1 protein and mRNA were reduced. Our findings indicate that leptin is an important regulator of hepatic SR-BI expression and, thus, HDL cholesterol levels, whereas it does not stimulate Cyp7a1 and bile acid synthesis.     

Urat1-Uox double knockout mice are experimental animal models of renal hypouricemia and exercise-induced acute kidney injury

ABSTRACT

Renal hypouricemia (RHUC) is a hereditary disease characterized by a low level of plasma urate but with normal urinary urate excretion. RHUC type 1 is caused by mutations of the urate transporter URAT1 gene (SLC22A12). However, the plasma urate levels of URAT1 knockout mice are no different from those of wild-type mice. In the present study, a double knockout mouse, in which the URAT1 and uricase (Uox) genes were deleted (Urat1-Uox-DKO), were used as an experimental animal model of RHUC type 1 to investigate RHUC and excise-induced acute kidney injury (EIAKI). Mice were given a variable content of allopurinol for one week followed by HPLC measurement of urate and creatinine concentrations in spot urine and blood from the tail. The urinary excretion of urate in Urat1-Uox-DKO mice was approximately 25 times higher than those of humans. With allopurinol, the plasma urate levels of Urat1-Uox-DKO mice were lower than those of Uox-KO mice. There were no differences in the urinary urate excretions between Urat1-Uox-DKO and Uox-KO mice administered with 9 mg allopurinol /100 g feed. In the absence of allopurinol, plasma creatinine levels of some Urat1-Uox-DKO mice were higher than those of Uox-KO mice. Consequently, hypouricemia and normouricosuria may indicate that the Urat1-Uox-DKO mouse administered with allopurinol may represent a suitable animal model of RHUC type 1. Urat1-Uox-DKO mice without allopurinol exhibited acute kidney injury, thus providing additional benefit as a potential animal model for EIAKI. Finally, our data indicate that allopurinol appears to provide prophylactic effects for EIAKI.     

Glucose catabolic gene mRNA levels in skeletal muscle exhibit non-coordinate expression in hyperglycemic mice.

To assess the correlation between hyperglycemia and glucose catabolic gene levels in diabetic and healthy mice, we determined mRNA levels of pivotal proteins such as glucose transporters, hexokinase II, glycogen synthase, glutamine:fructose-6-phosphate amidotransferase and uncoupling proteins. Both KK and KKAy mice showed marked decreases of Glut1 and Glut4 mRNA levels in soleus compared to C57BL; db/db and ob/ob mice exhibited significantly decreased Glut4 mRNA levels, but not Glut1, in soleus. KK and KKAy mice showed a decrease of soleus HKII gene level, which may indicate decreased intracellular catabolism of glucose. Likewise, GS mRNA level was decreased in soleus muscle tissue in KK and KKAy mice. GFAT mRNA levels was no different between hyperglycemic and normoglycemic mice. In contrast, UCP2 and UCP3 mRNA levels were higher in KK and KKAy mice. Conversely, db/db and ob/ob mice showed a significant decrease in UCP3 mRNA. Individual correlation analysis indicated that the decrease in Glut4 gene levels was only observed in hyperglycemic mice. The more important observation is that the glucose catabolic genes do not exhibit any clear coordinate expression. Abnormal expression of glucose catabolic genes may contribute to hyperglycemia and muscle insulin resistance in these four strains.     

Obesity-induced upregulation of myocardial endothelin-1 expression is mediated by leptin

Several studies have shown that leptin, the product of the obese gene, may link obesity with cardiovascular diseases, and in particular with cardiac hypertrophy. In vitro studies suggest that the mechanism by which leptin causes cardiac hypertrophy involves the upregulation of endogenous endothelin-1 (ET-1), a potent vasoconstrictor and mitogen. Whether obesity-associated hyperleptinemia causes an increase in myocardial ET-1 expression in vivo remains unclear. To address this issue, we fed mice with a high-fat diet and analyzed serum levels of ET-1 and ET-1 mRNA in the heart. We found that in mice fed a high-fat diet, serum ET-1, myocardial ET-1, leptin and leptin receptor mRNA were all elevated. In contrast, in leptin-deficient obese (ob/ob) mice, both serum and myocardial ET-1 levels were not higher than in wild type mice. These findings suggest that upregulation of myocardial ET-1 by obesity is mediated by leptin.     

PPARα信号通路激活抵抗高脂和Leptin缺失诱导的肥胖和脂肪肝

脂肪酰基辅酶A氧化酶1(acyl-coenzyme A oxidase 1,Acox1)缺失可通过内源性配体激活过氧化物酶体增殖物激活受体α(peroxisome proliferator-activated receptor-α,PPARα)及其调控的信号通路,从而减轻肥胖基因leptin突变型(ob/ob)小鼠的肥胖和脂肪肝症状,但提高了其肝癌发生率.为进一步研究PPARα信号通路在高脂日粮和leptin缺失诱导的脂肪肝形成过程中的作用,本研究以野生型、Acox1-/-、ob/ob和Acox1Δob/ob小鼠为模型,用正常日粮或60%高脂日粮饲喂10个月.结果显示,正常日粮或高脂日粮饲喂情况下,Acox1-/-和Acox1Δob/ob小鼠的体重、白色脂肪细胞体积、棕色脂肪组织含量及肝脏脂肪含量均分别显著低于WT和ob/ob小鼠.溴化脱氧尿嘧啶核苷(Brdurd)及烯酰辅酶A水合酶(L-PBE)免疫组化染色结果显示Acox1-/-和Acox1Δob/ob小鼠肝脏内肝细胞增殖及L-PBE活性、肝脏重量及其占体重的百分比均显著高于WT和ob/ob小鼠.正常日粮饲喂的WT、Acox1-/-、ob/ob和Acox1Δob/ob小鼠肝癌发生率分别为0%、100%、0%和4%,高脂日粮饲喂后,其肝癌发生率分别为0%、100%、2.9%和100%.Q-PCR结果显示Acox1-/-和Acox1Δob/ob小鼠肝脏内L-PBE、Cyp4a3、Akr1b10、ap2等基因的表达水平显著高于WT和ob/ob小鼠.综上所述,PPARα信号通路激活可以抵抗高脂日粮和leptin缺失诱导的肥胖和脂肪肝,但脂质过氧化反应可能通过Nrf2-Akr1b10信号通路促进了肝癌发生.     

Expression of aggrecan(ases) during murine preadipocyte differentiation and adipose tissue development

The expression and potential functional role of aggrecan in adipogenesis and adipose tissue development was investigated in murine models of obesity. Aggrecan, as well as the two aggrecanases ADAMTS-4 and ADAMTS-5 (A Disintegrin And Metalloproteinase with Thrombospondin motif) mRNAs, are expressed in subcutaneous (SC) and gonadal (GON) adipose tissues of mice. Their presence was confirmed by western blotting using adipose tissue extracts. In mice with nutritionally induced obesity (high fat diet) as well as in lean controls, aggrecan mRNA expression was downregulated whereas ADAMTS-4 and ADAMTS-5 were upregulated with time. In mice with genetically determined obesity (ob/ob), ADAMTS-5 mRNA was upregulated in both SC and GON adipose tissues, as compared to wild-type (WT) mice (p<0.001). Enhanced aggrecanase expression levels in these tissues were associated with significantly elevated levels of G1-NITEGE, a degradation product of aggrecan. Thus, aggrecan levels were high at the early stages of adipose tissue development in mice, whereas its production decreased and its degradation increased during development of obesity. A functional role of aggrecan in promoting early stages of adipogenesis is supported by the findings that it stimulated the in vitro differentiation of 3T3-F442A preadipocytes and the de novo in vivo accumulation of fat in Matrigel plaques injected into WT mice. Proteoglycans in the extracellular matrix of adipose tissue, such as aggrecan, may contribute to the regulation of lipid uptake and obesity in mice.     

Genomic organization and regulation by dietary fat of the uncoupling protein 3 and 2 genes

Uncoupling protein-1 (UCP1) dissipates the transmitochondrial proton gradient as heat. UCP2 and UCP3 are two recently discovered homologues that also have uncoupling activity and thus presumably have a role in energy homeostasis. We now report the genomic structure of murine UCP3 (7 exons) and UCP2 (8 exons). UCP3 is approximately 8 kilobases upstream of UCP2. An UCP3 variant mRNA, UCP3S, was also found and characterized. The effect of a high fat diet (45% versus 10%) on UCP3 and UCP2 mRNA levels was measured. Eating the 45% fat diet for eight weeks caused greater weight gain in AKR and C57BL/6J mice than in the obesity-resistant A/J mice. The high fat diet increased muscle UCP3 expression twofold in C57BL/6J animals. UCP2 expression increased slightly on the 45% fat diet in white adipose of AKR mice, but not in A/J or C57BL/6J mice. In skeletal muscle, UCP2 expression showed little variation with diet. Thus, UCP2 and UCP3 expression levels change in response to diet-induced obesity, but the changes are modest and depend on the tissue and genotype. The data suggest that it is not a reduction in UCP2 or UCP3 expression that causes obesity in the susceptible mice.     

Comprehensive analysis of mechanism underlying hypouricemic effect of glucosyl hesperidin

Hyperuricemia is caused by hepatic overproduction of uric acid and/or underexcretion of urate from the kidneys and small intestine. Although increased intake of citrus fruits, a fructose-rich food, is associated with increased risk of gout in humans, hesperidin, a flavonoid naturally present in citrus fruits, reportedly reduces serum uric acid (SUA) levels by inhibiting xanthine oxidase (XOD) activity in rats. However, the effects of hesperidin on renal and intestinal urate excretion were previously unknown. In this study, we used glucosyl hesperidin (GH), which has greater bioavailability than hesperidin, to clarify comprehensive mechanisms underlying the hypouricemic effects of hesperidin in vivo. GH dose-dependently decreased SUA levels in mice with hyperuricemia induced by potassium oxonate and a fructose-rich diet, and inhibited XOD activity in the liver. GH decreased renal urate excretion without changes in kidney URAT1, ABCG2 or GLUT9 expressions, suggesting that reducing uric acid pool size by inhibiting XOD decreased renal urate excretion. We also found that GH had no effect on intestinal urate excretion or protein expression of ABCG2. Therefore, we concluded that GH exhibits a hypouricemic effect by inhibiting XOD activity in the liver without increasing renal or intestinal urate excretion. Of note, this is the first study to elucidate the effect of a flavonoid on intestinal urate excretion using a mice model, whose findings should prove useful in future food science research in the area of urate metabolism. Taking these findings together, GH may be useful for preventing hyperuricemia, especially in people with the overproduction type.     

Dietary modulation of circulating leptin levels: site-specific changes in fat deposition and ob mRNA expression.

In order to study the effects of diet on fat distribution, circulating leptin levels and ob mRNA expression, diets of different macronutrient composition were fed to lean mice and gold thioglucose-obese mice. A high-fat diet and 2 high-carbohydrate diets, one containing mostly high-glycaemic-index starch and the other containing low-glycaemic-index starch were fed ad libitum for 10 weeks and were compared to standard laboratory chow. Weight gain was attenuated by feeding low-glycaemic-index starch in all mice and by feeding a high-fat diet in lean mice. Reduced adiposity was seen in lean mice fed low-glycaemic-index starch, whereas increased adiposity was seen in both lean and obese mice fed on the high-fat diet. Circulating leptin levels, when corrected for adiposity, were decreased in all mice fed either the high-fat diet or the low-GI diet. In epididymal fat pads, decreased ob mRNA expression was seen after both high-fat and high-glycaemic-index starch feeding. These results show that diet macronutrient composition contributes to the variability of circulating leptin levels by the combined effects of diet on fat distribution and on site-specific changes in ob mRNA expression.     

Inhibition of gastric inhibitory polypeptide signaling prevents obesity

Secretion of gastric inhibitory polypeptide (GIP), a duodenal hormone, is primarily induced by absorption of ingested fat. Here we describe a novel pathway of obesity promotion via GIP. Wild-type mice fed a high-fat diet exhibited both hypersecretion of GIP and extreme visceral and subcutaneous fat deposition with insulin resistance. In contrast, mice lacking the GIP receptor (Gipr(-/-)) fed a high-fat diet were clearly protected from both the obesity and the insulin resistance. Moreover, double-homozygous mice (Gipr(-/-), Lep(ob)/Lep(ob)) generated by crossbreeding Gipr(-/-) and obese ob/ob (Lep(ob)/Lep(ob)) mice gained less weight and had lower adiposity than Lep(ob)/Lep(ob) mice. The Gipr(-/-) mice had a lower respiratory quotient and used fat as the preferred energy substrate, and were thus resistant to obesity. Therefore, GIP directly links overnutrition to obesity and it is a potential target for anti-obesity drugs.     

Zinc supplementation aggravates body fat accumulation in genetically obese mice and dietary-obese mice

A perturbation of zinc metabolism has been noted in numerous laboratory animals with diabetes and obesity. The effects of zinc supplementation on body fat deposition in two types of experimental obese mice: genetically obese (ob/ob) mice and high-fat diet-induced ICR obese (HF) mice were investigated in this study. Their lean controls were +/? mice, and ICR on basal diet, respectively. The mice in the zinc-supplemented groups were administered 200 mg/kg zinc in their diets for 6 wk. Both the ob/ob mice and the HF mice, that were fed a diet containing a marginal zinc dosage (4–6 mg/kg), had lower zinc levels in their serum and carcass, and higher body fat content than their respective lean controls (p<0.01). After zinc supplementation, ob/ob mice and the HF mice significnatly (p<0.05) increased their body fat by 49.4% and 18.9%, respectively. This study revealed that body fat deposition can be aggravated by zinc supplementation in both types of obese mice. Zinc may be associated with the energy homeostasis of obesity, via its interaction with dietary fat consumption.     

Xanthine oxidoreductase is a regulator of adipogenesis and PPARgamma activity

In an effort to identify novel candidate regulators of adipogenesis, gene profiling of differentiating 3T3-L1 preadipocytes was analyzed using a novel algorithm. We report here the characterization of xanthine oxidoreductase (XOR) as a novel regulator of adipogenesis. XOR lies downstream of C/EBPbeta and upstream of PPARgamma, in the cascade of factors that control adipogenesis, and it regulates PPARgamma activity. In vitro, knockdown of XOR inhibits adipogenesis and PPARgamma activity while constitutive overexpression increases activity of the PPARgamma receptor in both adipocytes and preadipocytes. In vivo, XOR -/- mice demonstrate 50% reduction in adipose mass versus wild-type littermates while obese ob/ob mice exhibit increased concentrations of XOR mRNA and urate in the adipose tissue. We propose that XOR is a novel regulator of adipogenesis and of PPARgamma activity and essential for the regulation of fat accretion. Our results identify XOR as a potential therapeutic target for metabolic abnormalities beyond hyperuricemia.