Perilla extract improves frequent urination in spontaneously hypertensive rats with enhancement of the urothelial presence and anti-inflammatory effects.

OBJECTIVE: To investigate the effects of perilla extract on urinary symptoms in spontaneously hypertensive rats as a model of spontaneous overactive bladder. METHODS: Spontaneously hypertensive rats were randomly divided into two groups and fed either a control diet or a perilla extract-containing diet. Cystometry, gene expression and histological analyses were carried out to evaluate the effects of perilla extract after 2-week feeding of either the control or the perilla extract diet. The expression of inflammation-related genes in the human urothelial cell line HT-1376 and the normal human bladder epithelial cell was measured after the treatment with perillaldehyde, the main component of perilla extract, or perillic acid, the final metabolite of perillaldehyde. RESULTS: A significant 27% increase in the micturition interval and decreased expression of nerve growth factor, tumor necrosis factor-α, interleukin-1ß and transient receptor potential V1 were observed in the perilla group compared with the control group. The level of uroplakin 3A was 40% higher in the perilla group than in the control group. The urothelium in the control group was thin or defective, but it was almost completely intact in the perilla group. Perillaldehyde and perillic acid suppressed the induction of nerve growth factor and tumor necrosis factor-α by interleukin-1ß in HT-1376 and normal human bladder epithelial cells. CONCLUSIONS: The present findings suggest that perilla extract improves frequent urination, and this improvement seems to be mediated, at least in part, by enhancement of the urothelial presence and by the anti-inflammatory effects of perilla.

Alteration of amiloride-sensitive salt taste nerve responses in aldosterone/NaCl-induced hypertensive rats.

Salt taste sensitivity is related to physiological condition, and declined in hypertensive patients. However, little is known about the mechanism underlying changes in salt taste sensitivity during the development of hypertension. This is largely due to lack of an appropriate animal model which shows the decline of salt taste sensitivity caused by hypertension. Previous studies have suggested that one of main causes of salt-sensitive hypertension is dysfunction of the renin-angiotensin-aldosterone system (RAAS). To examine the involvement of RAAS in modulation of salt taste sensitivity, we utilized aldosterone/NaCl-treated rats as a well-established model of salt-sensitive hypertension caused by RAAS dysfunction. Amount of sodium intake in aldosterone/NaCl-treated rats was higher than that in control rats. In addition to behavioral changes, the amiloride-sensitive salt taste nerve responses in aldosterone/NaCl-treated rats were remarkably lower by approximately 90% than those in the other groups. Moreover, αENaC mRNA expression in the epithelium of circumvallate papillae was significantly low in aldosterone/NaCl-treated rats. Thus, RAAS modulates salt taste system as is case in hypertensive patients. This report is to our knowledge the first to describe an animal model with decline of amiloride-sensitive salt taste nerve responses by RAAS dysfunction-mediated salt-sensitive hypertension.

Dietary phospholipids ameliorate fructose-induced hepatic lipid and metabolic abnormalities in rats.

Overconsumption of fructose results in hepatic dyslipidemia, which has a documented correlation with metabolic syndrome. We examined whether the ingestion of phospholipids (PL) from soybeans prevents fructose-induced metabolic abnormalities. Rats were fed either a fructose-free diet (C), a 60% fructose diet (F), or a 60% fructose plus 3% PL diet (F-PL) for 10 wk. At wk 8, plasma glucose concentrations after glucose loading were significantly higher in rats fed the F diet than in rats fed the C and F-PL diets, which did not differ from one another. The concentrations of hepatic TG, diglycerides, ceramides, and oleates in rats fed the F diet for 10 wk was significantly higher than those in rats fed the C diet. The increases were prevented by concurrent PL ingestion; concentrations did not differ between the F-PL and C groups. Dietary fructose increased the mRNA expression of SREBP1, ChREBP, and genes related to lipogenesis. PL completely inhibited these increases. Furthermore, reflecting the difference at the mRNA level, lipogenic enzyme activities were greater in rats fed the F diet than in rats fed the C diet, and PL ingestion suppressed the increased activities by fructose feeding. Treatment of cultured Hep-G2 cells with fructose for 24 h increased the levels of SREBP1 and ChREBP nuclear proteins, which were suppressed by culture with purified PL components, especially phosphatidylethanolamine and phosphatidylinositol. These findings indicate that PL prevents fructose-induced metabolic abnormalities in association with alterations of the hepatic lipid profile by inhibiting de novo lipogenesis.

Identification of diacylglycerol acyltransferase inhibitors from Rosa centifolia petals.

Diacylglycerol acyltransferase (DGAT) catalyzes the final step of triacylglycerol (TAG) synthesis, and is considered as a potential target to control hypertriglyceridemia or other metabolic disorders. In this study, we found that the extract of rose petals suppressed TAG synthesis in cultured cells, and that the extract showed DGAT inhibitory action in a dose-dependent manner. Fractionation of the rose extract revealed that the DGAT inhibitory substances in the extract were ellagitannins; among them rugosin B, and D, and eusupinin A inhibited DGAT activity by 96, 82, and 84% respectively, at 10 µM. These substances did not inhibit the activities of other hepatic microsomal enzymes, glucose-6-phosphatase and HMG-CoA reductase, or pancreatic lipase, suggesting that ellagitannins inhibit DGAT preferentially. In an oral fat load test using mice, postprandial plasma TAG increase was suppressed by rose extract; TAG levels 2 h after the fat load were significantly lower in mice administered a fat emulsion containing rose extract than in control mice (446.3 ± 33.1 vs 345.3 ± 25.0 mg/dL, control vs rose extract group; P < 0.05). These results suggest that rose ellagitannins or rose extract could be beneficial in controlling lipid metabolism and used to improve metabolic disorders.

Responses of muscle mass, strength and gene transcripts to long-term heat stress in healthy human subjects.

The present study was performed to investigate the effects of long-term heat stress on mass, strength and gene expression profile of human skeletal muscles without exercise training. Eight healthy men were subjected to 10-week application of heat stress, which was performed for the quadriceps muscles for 8 h/day and 4 days/week by using a heat- and steam-generating sheet. Maximum isometric force during knee extension of the heated leg significantly increased after heat stress (~5.8%, P < 0.05). Mean cross-sectional areas (CSAs) of vastus lateralis (VL, ~2.7%) and rectus femoris (~6.1%) muscles, as well as fiber CSA (8.3%) in VL, in the heated leg were also significantly increased (P < 0.05). Statistical analysis of microarrays (SAM) revealed that 10 weeks of heat stress increased the transcript level of 925 genes and decreased that of 1,300 genes, and gene function clustering analysis (Database for Annotation, Visualization and Integrated Discovery: DAVID) showed that these regulated transcripts stemmed from diverse functional categories. Transcript level of ubiquinol-cytochrome c reductase binding protein (UQCRB) was significantly increased by 10 weeks of heat stress (~3.0 folds). UQCRB is classified as one of the oxidative phosphorylation-associated genes, suggesting that heat stress can stimulate ATP synthesis. These results suggested that long-term application of heat stress could be effective in increasing the muscle strength associated with hypertrophy without exercise training.

Effects of coumestrol on lipid and glucose metabolism as a farnesoid X receptor ligand.

In the course of an effort to identify novel agonists of the farnesoid X receptor (FXR), coumestrol was determined to be one such ligand. Reporter and in vitro coactivator interaction assays revealed that coumestrol bound and activated FXR. Treatment of Hep G2 cells with coumestrol stimulated the expression of FXR target genes, thereby regulating the expression of target genes of the liver X receptor and hepatocyte nuclear factor-4alpha. Through these actions, coumestrol is expected to exert beneficial effects on lipid and glucose metabolism.

Dietary fish oil upregulates intestinal lipid metabolism and reduces body weight gain in C57BL/6J mice.

Fish oils (FO) rich in (n-3) PUFA exert hypolipidemic and antiobesity effects in association with modulated hepatic lipid metabolism. We recently demonstrated the possible involvement of intestinal lipid metabolism in the development of obesity. In this study, we examined the effect of FO ingestion on intestinal lipid metabolism in relation to obesity. When diet-induced obesity-prone C57BL/6J mice were fed an 8% FO, high-fat (30%) diet for 5 mo, body weight gain was significantly reduced compared with mice fed a 30% triacylglycerol (TG) diet without FO. In addition to modulating messenger RNA (mRNA) levels in the liver, FO ingestion for 2 wk affected the intestinal mRNA levels of lipid metabolism-related genes; those of carnitine palmitoyltransferase 1a, cytochrome P450 4A10, and malic enzyme were significantly higher in mice fed the 8% FO diet compared with mice fed the 30% TG diet. Northern blot analysis revealed that the expression levels of most lipid metabolism-related genes in the small intestine of mice fed the 8% FO diet were comparable to those in the liver. Furthermore, reflecting the difference at the mRNA level, FO ingestion affected lipid metabolism-related enzyme activity; fatty acid beta-oxidation, omega-oxidation, and malic enzyme activities in the small intestine of mice fed the 8% FO diet were 1.2-, 1.6-, and 1.7-fold those in mice fed the 30% TG diet, respectively. These findings suggest that an upregulation of intestinal lipid metabolism is associated with the antiobesity effect of FO.

Differential regulation of intestinal lipid metabolism-related genes in obesity-resistant A/J vs. obesity-prone C57BL/6J mice.

The effects of high-fat (HF) feeding on gene expression in the small intestine were examined using obesity-resistant A/J mice and obesity-prone C57BL/6J (B6) mice. Both strains of mice were maintained on low-fat (LF; 5% fat) or HF (30% fat) diets for 2 wk. Quantitative reverse transcription-PCR analysis revealed that lipid metabolism-related genes, including carnitine palmitoyltransferase (CPT) I, liver fatty acid binding protein, pyruvate dehydrogenase kinase-4, and NADP(+)-dependent cytosolic malic enzyme, were upregulated by HF feeding in both strains of mice. The upregulated gene expression levels were higher in A/J mice than in B6 mice, suggesting more active lipid metabolism in the small intestine of A/J mice. The prominent upregulation of the lipid metabolism-related genes were specific to the small intestine; the expression levels were little or unchanged in the liver, muscle, and white adipose tissue. The increase by HF feeding and predominant expression of the intestinal lipid metabolism-related genes in A/J mice were reflected in the enzyme activities; malic enzyme, CPT, and beta-oxidation activities were increased by HF feeding, and the upregulated malic enzyme and CPT activities were significantly higher in obesity-resistant A/J mice compared with those in obesity-prone B6 mice. These findings suggest that intestinal lipid metabolism is associated with susceptibility to obesity.

Dietary diacylglycerol reduces postprandial hyperlipidemia and ameliorates glucose intolerance in Otsuka Long-Evans Tokushima Fatty (OLETF) rats.

OBJECTIVE: The aim of the present study was to determine the effects of dietary diacylglycerol (DG) on the metabolism of lipids and glucose in type II diabetic Otsuka Long-Evans Tokushima Fatty (OLETF) rats. METHODS: In experiment 1, the rats were orally administered 10 mL/kg of a triacylglycerol (TG) or DG emulsion (15% [w/v] oil), and the subsequent changes in the serum lipid levels were compared. In experiment 2, the rats were fed diets containing 15% DG or TG oil. After 22 weeks, the serum levels of lipids, glucose, and cytokines were determined. In addition, an oral glucose tolerance test (OGTT) was performed on the rats. RESULTS: Administration of an oral fat load caused marked hypertriglyceridemia with a peak at 2 h. Oral DG loading reduced the serum TG increase; the difference between the groups was significant at 4 and 6 h (P < 0.05). Diacylglycerol also markedly reduced the serum free fatty acid concentration increase due to the fat load. After 22 weeks of feeding, dietary DG reduced serum TG levels in the non-fasting state. Moreover, an OGTT revealed enhanced glucose disposal in the DG-fed rats compared with the TG-fed rats. Serum levels of adiponectin, an important insulin-sensitizing adipocytokine, were higher in the DG-fed rats than in the TG-fed rats (P < 0.05). In addition, DG-feeding reduced serum levels of C-reactive protein, a cardiovascular risk factor (P < 0.05). CONCLUSION: These results suggested that dietary DG improves lipid metabolism and glucose tolerance, and retards the progress of diabetes mellitus in OLETF rats.

Decreased levels of covalently bound ceramide are associated with ultraviolet B-induced perturbation of the skin barrier.

Although ultraviolet B (UVB) irradiation perturbs the skin barrier, little is known about the mechanism(s) with respect to the metabolism of ceramide (Cer). We examined changes in intercellular lipids in murine stratum corneum following UVB irradiation. A single UVB (75 mJ per cm(2)) irradiation caused a significant increase in transepidermal water loss, which plateaued at day 4. In parallel, covalently bound Cer was significantly decreased with the greatest decrease at days 3-4. In contrast, the levels of other free, non-bound lipids (including Cer or acylceramides) were significantly increased for Cer, or remained unchanged at day 4 compared with non-irradiated controls. RT-PCR analysis demonstrated a significant decrease in mRNA encoding transglutaminase-1 (TGase1). The peak occurred 2-4 d after a single UVB irradiation, a time when covalently bound Cer was significantly downregulated in concert with the disruption of the skin barrier. Furthermore, UVB-induced epidermal hyperplasia occurred to the greatest extent between 2 and 4 d following UVB irradiation. These results suggest that decreases in covalently bound Cer in the stratum corneum are mediated via the downregulation of TGase-1 as well as by the rapid induction of epidermal hyperplasia, which is attributable to the perturbation of the skin barrier induced by UVB irradiation.

Disturbed secretion of mutant adiponectin associated with the metabolic syndrome.

Adiponectin, an adipocyte-derived protein, consists of collagen-like fibrous and complement C1q-like globular domains, and circulates in human plasma in a multimeric form. The protein exhibits anti-diabetic and anti-atherogenic activities. However, adiponectin plasma concentrations are low in obese subjects, and hypoadiponectinemia is associated with the metabolic syndrome, which is a cluster of insulin resistance, type 2 diabetes mellitus, hypertension, and dyslipidemia. We have recently reported a missense mutation in the adiponectin gene, in which isoleucine at position 164 in the globular domain is substituted with threonine (I164T). Subjects with this mutation showed markedly low level of plasma adiponectin and clinical features of the metabolic syndrome. Here, we examined the molecular characteristics of the mutant protein associated with a genetic cause of hypoadiponectinemia. The current study revealed (1) the mutant protein showed an oligomerization state similar to the wild-type as determined by gel filtration chromatography and, (2) the mutant protein exhibited normal insulin-sensitizing activity, but (3) pulse-chase study showed abnormal secretion of the mutant protein from adipose tissues. Our results suggest that I164T mutation is associated with hypoadiponectinemia through disturbed secretion into plasma, which may contribute to the development of the metabolic syndrome.

Digestion and assimilation features of dietary DAG in the rat small intestine.

Several recent studies have demonstrated that dietary DAG oil rich in 1,3-species suppresses the postprandial increase of serum TAG level and decreases body fat accumulation, compared with TAG oil. To clarify the mechanisms underlying the beneficial effects of DAG, we investigated the metabolic features of DAG in the small intestine with regard to the digestion pathway in the lumen and the TAG-synthesis pathway in the mucosa. When intraduodenally infused as an emulsion, TAG was digested to 1,2-DAG, 2-MAG, and FFA, whereas 1,3-DAG was digested to 1(3)-MAG and FFA. When assessed by the incorporation of [1-14C]linoleic acid in lipids, the mucosal TAG-synthesis was significantly reduced by DAG infusion compared with TAG infusion. However, the mucosal 1,3-DAG synthesis was remarkably increased in the DAG-infused rats. The total amount of mucosal 1,3-DAG was also increased (4.5-fold) after DAG infusion compared with that after TAG infusion. Next, we examined the synthesis pathway of 1,3-DAG. In cultures of the everted intestinal sacs, 1,3-DAG production required the presence of 1-MAG, suggesting that the 1,3-DAG synthesis was due to acylation of 1(3)-MAG in the DAG-infused rats. Furthermore, measurements of DAG acyltransferase activity indicated that 1,3-DAG was little utilized in TAG synthesis. These findings suggest that features of 1,3-DAG digestion and assimilation in the intestine may be responsible for the reduction of the postprandial serum TAG level by dietary DAG.

Coordinated regulation of fat-specific and liver-specific glycerol channels, aquaporin adipose and aquaporin 9.

Plasma glycerol is a major substrate for hepatic gluconeogenesis. Aquaporin adipose (AQPap/7), an adipose-specific glycerol channel, provides fat-derived glycerol into plasma. In the present study, we cloned the coding and promoter regions of mouse aquaporin 9 (AQP9), a liver-specific glycerol channel. Fasting and refeeding of mice increased and decreased hepatic AQP9 mRNA levels, respectively. Insulin deficiency induced by streptozotocin resulted in increased hepatic AQP9 mRNA. These changes in hepatic AQP9 mRNA were accompanied by those of hepatic gluconeogenic mRNAs and plasma glycerol levels. In cultured hepatocytes, insulin downregulated AQP9 mRNA. The AQP9 promoter contained the negative insulin response element TGTTTTC at -496/-502, similar to the promoter of the AQPap/7 gene. In contrast, in insulin-resistant db+/db+ mice, AQPap/7 mRNA in fat and AQP9 mRNA in liver were increased, despite hyperinsulinemia, with high plasma glycerol and glucose levels. Glycerol infusion in the db+/db+ mice augmented hepatic glucose output. Our results indicate that coordinated regulations of fat-specific AQPap/7 and liver-specific AQP9 should be crucial to determine glucose metabolism in physiology and insulin resistance.

Androgens decrease plasma adiponectin, an insulin-sensitizing adipocyte-derived protein.

Adiponectin, an adipose-specific secretory protein, exhibits antidiabetic and antiatherogenic properties. In the present study, we examined the effects of sex hormones on the regulation of adiponectin production. Plasma adiponectin concentrations were significantly lower in 442 men (age, 52.6 +/- 11.9 years [mean +/- SD]) than in 137 women (53.2 +/- 12.0 years) but not different between pre- and postmenopausal women. In mice, ovariectomy did not alter plasma adiponectin levels. In contrast, high levels of plasma adiponectin were found in castrated mice. Testosterone treatment reduced plasma adiponectin concentration in both sham-operated and castrated mice. In 3T3-L1 adipocytes, testosterone reduced adiponectin secretion into the culture media, using pulse-chase study. Castration-induced increase in plasma adiponectin was associated with a significant improvement of insulin sensitivity. Our results indicate that androgens decrease plasma adiponectin and that androgen-induced hypoadiponectinemia may be related to the high risks of insulin resistance and atherosclerosis in men.

Diet-induced insulin resistance in mice lacking adiponectin/ACRP30.

Here we investigated the biological functions of adiponectin/ACRP30, a fat-derived hormone, by disrupting the gene that encodes it in mice. Adiponectin/ACRP30-knockout (KO) mice showed delayed clearance of free fatty acid in plasma, low levels of fatty-acid transport protein 1 (FATP-1) mRNA in muscle, high levels of tumor necrosis factor-alpha (TNF-alpha) mRNA in adipose tissue and high plasma TNF-alpha concentrations. The KO mice exhibited severe diet-induced insulin resistance with reduced insulin-receptor substrate 1 (IRS-1)-associated phosphatidylinositol 3 kinase (PI3-kinase) activity in muscle. Viral mediated adiponectin/ACRP30 expression in KO mice reversed the reduction of FATP-1 mRNA, the increase of adipose TNF-alpha mRNA and the diet-induced insulin resistance. In cultured myocytes, TNF-alpha decreased FATP-1 mRNA, IRS-1-associated PI3-kinase activity and glucose uptake, whereas adiponectin increased these parameters. Our results indicate that adiponectin/ACRP30 deficiency and high TNF-alpha levels in KO mice reduced muscle FATP-1 mRNA and IRS-1-mediated insulin signaling, resulting in severe diet-induced insulin resistance.

Association of adiponectin mutation with type 2 diabetes: a candidate gene for the insulin resistance syndrome.

Adiponectin, also referred to as AdipoQ or ACRP30, is a plasma protein produced and secreted exclusively from adipose tissue. The protein contains a collagen-like domain and a C1q-like globular domain. A protease-generated globular segment enhances fatty acid oxidation in muscles, thereby modulating lipid and glucose metabolism. Plasma adiponectin levels are inversely correlated with the severity of insulin resistance. A recent genome-wide scan study mapped a susceptibility locus for type 2 diabetes and the metabolic syndrome to chromosome 3q27, where the adiponectin gene is located. Here, we screened Japanese patients with type 2 diabetes and age- and BMI-matched nondiabetic control subjects for mutations in adiponectin gene. We identified four missense mutations (R112C, I164T, R221S, and H241P) in the globular domain. Among these mutations, the frequency of I164T mutation was significantly higher in type 2 diabetic patients than in age- and BMI- matched control subjects (P < 0.01). Furthermore, plasma adiponectin concentrations of subjects carrying I164T mutation were lower than those of subjects without the mutation. All the subjects carrying I164T mutation showed some feature of metabolic syndrome, including hypertension, hyperlipidemia, diabetes, and atherosclerosis. Our findings suggest that I164T mutation is associated with low plasma adiponectin concentration and type 2 diabetes.

Human aquaporin adipose (AQPap) gene. Genomic structure, promoter analysis and functional mutation.

Aquaporin adipose (AQPap), which we identified from human adipose tissue, is a glycerol channel in adipocyte [Kishida et al. (2000) J. Biol. Chem. 275, 20896-20902]. In the current study, we determined the genomic structure of the human AQPap gene, and identified three AQPap-like genes that resembled (approximately 95%) AQPap, with little expression in human tissues. The AQPap promoter contained a putative peroxisome proliferator response element (PPRE) at -46 to -62, and a putative insulin response element (IRE) at -542/-536. Deletion of the PPRE abolished the pioglitazone-mediated induction of AQPap promoter activity in 3T3-L1 adipocytes. Deletion and single base pair substitution analysis of the IRE abolished the insulin-mediated suppression of the human AQPap gene. Analysis of AQPap sequence in human subjects revealed three missense mutations (R12C, V59L and G264V), and two silent mutations (A103A and G250G). The cRNA injection of the missense mutants into Xenopus oocytes revealed the absence of the activity to transport glycerol and water in the AQPap-G264V protein. In the subject homozygous for AQPap-G264V, exercise-induced increase in plasma glycerol was not observed in spite of the increased plasma noradrenaline. We suggest that AQPap is responsible for the increase of plasma glycerol during exercise in humans.