Weak response of bovine hepcidin induction to iron through decreased expression of Smad4.

Hepcidin negatively regulates systemic iron levels by inhibiting iron entry into the circulation. Hepcidin production is increased in response to an increase in systemic iron via the activation of the bone morphogenetic protein (BMP) pathway. Regulation of hepcidin expression by iron status has been proposed on the basis of evidence mainly from rodents and humans. We evaluated the effect of iron administration on plasma hepcidin concentrations in calves and the expression of bovine hepcidin by the BMP pathway in a cell culture study. Hematocrit as well as levels of blood hemoglobin and plasma iron were lower than the reference level in calves aged 1-4 weeks. Although intramuscular administration of iron increased iron-related parameters, plasma hepcidin concentrations were unaffected. Treatment with BMP6 increased hepcidin expression in human liver-derived cells but not in bovine liver-derived cells. A luciferase-based reporter assay revealed that Smad4 was required for hepcidin reporter transcription induced by Smad1. The reporter activity of hepcidin was lower in the cells transfected with bovine Smad4 than in those transfected with murine Smad4. The lower expression levels of bovine Smad4 were responsible for the lower activity of the hepcidin reporter, which might be due to the instability of bovine Smad4 mRNA. In fact, the endogenous Smad4 protein levels were lower in bovine cells than in human and murine cells. Smad4 also confers TGF-ß/activin-mediated signaling. Induction of TGF-ß-responsive genes was also lower after treatment with TGF-ß1 in bovine hepatocytes than in human hepatoma cells. We revealed the unique regulation of bovine hepcidin expression and the characteristic TGF-ß family signaling mediated by bovine Smad4. The present study suggests that knowledge of the regulatory expression of hepcidin as well as TGF-ß family signaling obtained in murine and human cells is not always applicable to bovine cells.

Regulatory mechanisms underlying interleukin-6 expression in murine brown adipocytes.

Three types of adipocytes, white, brown, and beige, regulate the systemic energy balance through the storage and expenditure of chemical energy. In addition, adipocytes produce various bioactive molecules known as adipokines. In contrast to white adipocyte-derived molecules, less information is available on the adipokines produced by brown adipocytes (batokine). This study explored the regulatory expression of interleukin (IL)-6 in cell culture studies. Norepinephrine or a nonselective ß-adrenergic receptor agonist increased the expression of IL-6 in primary brown adipocytes and HB2 brown adipocytes. Treatment with forskolin (Fsk), an activator of the cAMP-dependent protein kinase (PKA) pathway (downstream signaling of the ß-adrenergic receptor), efficiently stimulated IL-6 expression in brown adipocytes and myotubes. Phosphorylated CREB and phosphorylated p38 MAP kinase levels were increased in Fsk-treated brown adipocytes within 5 min. In contrast, a long-term (∼60 min and ∼4 h) treatment with Fsk was required for increase in STAT3 phosphorylation and C/EBPß expression, respectively. The PKA, p38 MAP kinase, STAT3, and C/EBPß pathways are required for the maximal IL-6 expression induced by Fsk, which were verified by use of various inhibitors of these signal pathways. Vitamin C enhanced Fsk-induced IL-6 expression through the extracellular signal-regulated kinase activity. The present study provides basic information on the regulatory expression of IL-6 in activated brown adipocytes.

Enhancement of vitamin C-induced myogenesis by inhibition of extracellular signal-regulated kinase (ERK) 1/2 pathway.

Myogenesis is a complex process that is regulated by a variety of factors. We have previously shown that vitamin C and mild endoplasmic reticulum stress synergistically enhance myogenesis. The present study evaluated the effects of vitamin C (ascorbic acid (AsA) and AsA 2-phosphate (AsAp)) and extracellular signal-regulated kinase (ERK) 1/2 pathway on myogenesis. Treatment with U0126, an inhibitor of MEK1/2 that phosphorylates and activates ERK1/2, during the differentiation, increased the mRNA levels of Myod and Myog with an increase in the protein level of myosin heavy chain (MYH)1/2. Treatment with AsA or AsAp alone had minimal effects on myogenesis in C2C12 cells. However, combination treatment with vitamin C and U0126 greatly enhanced myogenesis; the number of thick and long myotubes was increased, and the expression of MYH1/2 was also increased. PD98059, another MEK1/2 inhibitor, also enhanced myogenesis in combination with vitamin C. These results indicate that relief of endogenous ERK1/2 activity enhances vitamin C-mediated myogenesis, suggesting a functional interaction between endogenous ERK1/2 activity and vitamin C. In addition, inhibition of p38 mitogen-activated protein kinase repressed myogenesis in the presence of vitamin C. Thus, vitamin C is a conditional factor that modulates myogenesis.

Factors affecting the induction of uncoupling protein 1 in C2C12 myogenic cells.

Brown/beige adipocytes, which are derived from skeletal muscle/smooth muscle-lineage cells, consume excess energy as heat through the expression of mitochondrial uncoupling protein 1 (UCP1). Previous studies have shown that forced expression of PR/SET domain (PRDM)-16 or early B-cell factor (EBF)-2 induced UCP1-positive adipocytes in C2C12 myogenic cells. Here, we explored the culture conditions to induce Ucp1 expression in C2C12 cells without introducing exogenous genes. Treatment with rosiglitazone (a peroxisome proliferator-activated receptor (PPAR)-γ agonist), GW501516 (a PPARδ agonist), and bone morphogenetic protein (BMP)-7 for 8 days efficiently increased Ucp1 expression in response to treatment with forskolin, an activator of the protein kinase A pathway. BMP7 dose-dependently increased forskolin-induced Ucp1 expression in the presence of rosiglitazone and GW501516; however, GW501516 was not required for Ucp1 induction. Additionally, the structurally related proteins, BMP6 and BMP9, efficiently increased forskolin-induced Ucp1 expression in rosiglitazone-treated cells. UCP1 protein was localized in cells with lipid droplets, but adipocytes were not always positive for UCP1. Continuous treatment with BMP7 was needed for the efficient induction of Ucp1 by forskolin treatment. Significant expression of Prdm16 was not detected, irrespective of the treatment, and treatment with rosiglitazone, GW501516, and BMP7 did not affect the expression levels of Ebf2. Fibroblast growth factor receptor (Fgfr)-3 expression levels were increased by BMP9 in rosiglitazone-treated cells, and molecules that upregulate Fgfr3 transcription partly overlapped with those that stimulate Ucp1 transcription. The present results provide basic information on the practical differentiation of myogenic cells to brown adipocytes.

Stimulation of myogenesis by ascorbic acid and capsaicin.

Myogenesis is a complex process regulated by several factors. This study evaluated the functional interaction between vitamin C and a high dose of capsaicin (a potential endoplasmic reticulum (ER) stress inducer) on myogenesis. After the induction of differentiation, treatment with ascorbic acid or ascorbic acid phosphate (AsAp) alone had minimal effects on myogenesis in C2C12 cells. However, treatment with capsaicin (300 µM) in undifferentiated C2C12 cells increased the expression levels of genes related to ER stress as well as oxidative stress. Myogenesis was effectively enhanced in C2C12 cells treated with a combination of capsaicin (300 µM) for one day before differentiation stimulation and AsAp for four days post-differentiation; subsequently, thick and long myotubes formed, and the expression levels of myosin heavy chain (MYH) 1/2 and Myh1, Myh4, and Myh7 increased. Considering that mild ER stress stimulates myogenesis, AsAp may elicit myogenesis through the alleviation of oxidative stress-induced negative effects in capsaicin-pretreated cells. The enhanced expression of Myh1 and Myh4 coincided with the expression of Col1a1, a type I collagen, suggesting that the fine-tuning of the myogenic cell microenvironment is responsible for efficient myogenesis. Our results indicate that vitamin C is a potential stimulator of myogenesis in cells, depending on the cell context.

Regulatory expression of uncoupling protein 1 and its related genes by endogenous activity of the transforming growth factor-ß family in bovine myogenic cells.

Uncoupling protein 1 (UCP1) is responsible for non-shivering thermogenesis, with restricted expression in brown/beige adipocytes in humans and rodents. We have previously shown an unexpected expression of UCP1 in bovine skeletal muscles. This study evaluated factors affecting Ucp1 gene expression in cultured bovine myogenic cells. Myosatellite cells, which were isolated from the bovine musculus longissimus cervicis, were induced to differentiate into myotubes in the presence of 2% horse serum. Previous studies using murine brown/beige adipocytes revealed that Ucp1 expression levels are directly increased by forskolin and all-trans retinoic acid (RA). The transforming growth factor-ß (TGF-ß)/activin pathway negatively regulated Ucp1 expression, whereas activation of the bone morphogenetic protein (BMP) pathway indirectly increases Ucp1 expression through the stimulation of brown/beige adipogenesis. Neither forskolin nor RA significantly affected Ucp1 mRNA levels in bovine myogenic cells. A-83-01, an inhibitor of the TGF-ß/activin pathway, stimulated myogenesis in these cells. A-83-01 significantly increased the expression of some brown fat signature genes such as Pgc-1α, Cox7a1, and Dio2, with a quantitative but not significant increase in the expression of Ucp1. Treatment with LDN-193189, an inhibitor of the BMP pathway, did not affect the differentiation of bovine myosatellite cells. Rather, LDN-193189 increased Ucp1 mRNA levels without modulating the levels of other brown/beige adipocyte-related genes. The current results indicate that the regulation of Ucp1 expression in bovine myogenic cells is distinct from that in murine brown/beige adipocytes, which has been more intensely characterized. SIGNIFICANCE OF THE STUDY: We previously reported unexpected expression of Ucp1 in bovine muscle tissues; Ucp1 expression has been known to be detected predominantly in brown/beige adipocytes. This study examined regulatory expression of bovine Ucp1 in myogenic cells. Consistent with the changes in expression levels of brown/beige adipocyte-selective genes, Ucp1 expression tended to be increased by inhibition of endogenous TGF-ß activity. In contrast, inhibition of endogenous BMP significantly increased Ucp1 expression without affecting brown/beige adipocyte-selective gene expression. The current results indicate that regulatory expression of Ucp1 in bovine myogenic cells is distinct from that in murine brown/beige adipocytes that is more intensely characterized.

Magnesium bioavailability of dried and thinly shaved kombu in rats.

BACKGROUND: Magnesium (Mg) is highly bioavailable in kombu compared with other edible seaweeds. However, a considerable amount of Mg is lost during industrial processing and cooking of kombu. We hypothesized that thinly shaved kombu (TSK), a traditional Japanese kombu product, is a suitable Mg source for daily diets because TSK hardly loses Mg during processing. Rats were fed diets containing TSK or magnesium oxide (MgO) to satisfy 25%, 50%, 75%, or 100% of their Mg requirements. We determined the relative Mg bioavailability of TSK compared to MgO and examined factors affecting Mg bioavailability in TSK. RESULTS: The relative bioavailability of Mg in TSK compared with MgO was calculated as 92.3%, 111.4%, and 87.2% from apparent absorption, urinary excretion, and femoral concentration of Mg, respectively. The ultrafiltrable Mg concentration was lower in the cecal content of rats given TSK than those given MgO. However, the mRNA expression of TRPM6, an Mg channel responsible for Mg absorption, was higher in the cecum of rats given TSK than those given MgO. CONCLUSION: Enhancement of TRPM6 expression in the large intestine negates the low bioaccessibility of Mg in TSK, and thus TSK shows Mg bioavailability comparable with MgO. © 2020 Society of Chemical Industry.

Metabolic changes in adipose tissues in response to ß3 -adrenergic receptor activation in mice.

Brown and beige adipocytes dissipate energy as heat. Thus, the activation of brown adipocytes and the emergence of beige adipocytes in white adipose tissue (WAT) are suggested to be useful for preventing and treating obesity. Although ß3 -adrenergic receptor activation is known to stimulate lipolysis and activation of brown and beige adipocytes, fat depot-dependent changes in metabolite concentrations are not fully elucidated. The current study examined the effect of treatment with CL-316,243, a ß3 -adrenergic receptor agonist, on the relative abundance of metabolites in interscapular brown adipose tissue (iBAT), inguinal WAT (ingWAT), and epididymal WAT (epiWAT). Intraperitoneal injection of CL-316,243 (1 mg/kg) for 3 consecutive days increased the relative abundance of several glycolysis-related metabolites in all examined fat depots. The cellular concentrations of metabolites involved in the citric acid cycle and of free amino acids were also increased in epiWAT by CL-316,243. CL-316,243 increased the expression levels of several enzymes and transporters related to glucose metabolism and amino acid catabolism in ingWAT and iBAT but not in epiWAT. CL-316,243 also induced the emergence of more beige adipocytes in ingWAT than in epiWAT. Furthermore, adipocytes surrounded by macrophages were detected in the epiWAT of mice given CL-316,243. The current study reveals the fat depot-dependent modulation of cellular metabolites in CL-316,243-treated mice, presumably resulting from differential regulation of cell metabolism in different cell populations.

Relationships between expression levels of genes related to adipogenesis and adipocyte function in dogs.

There are three kinds of adipocytes; white adipocytes accumulate excess energy as fat, whereas brown/beige adipocytes dissipate energy through expression of uncoupling protein 1 (UCP1). Obesity, a feature of excess accumulation of white adipocytes in a body, is one of the risk factors for onset of various diseases in dogs. As the first step to explore adipose genes related to dog obesity, we examined relationships among mRNA levels of putative molecules related to adipogenesis and function of adipocytes in fat of hospitalized dogs. Gonadal adipose tissues were collected from a total of 29 dogs, and the gene expression levels were examined by quantitative RT-PCR analysis. The multicollinearity analysis revealed that body condition score (BCS), which reflects adiposity, did not correlate with expression levels of any genes but correlated with age of dog. Bone morphogenetic protein (BMP) pathway stimulates not only commitment of mesenchymal stem cells to white adipocyte-lineage cells but also brown/beige adipogenesis. Some relationships between expression levels of BMP receptors were significant; especially, expression levels of activin receptor-like kinase (Alk) 3 (a BMP type I receptor) positively related to those of Alk2 (another BMP type I receptor), activin receptor type II (ActRII) A (a type II receptor to transmit BMP signal), ActRIIB (another type II receptor to transmit BMP signal) and BMP receptor type 2 (Bmpr2). PR domain containing 16 (Prdm16) expression levels strongly correlated with expression levels of ActRIIB. Although PRDM16 is known to stimulate brown/beige adipogenesis, expression levels of Ucp1 did not correlate with those of Prdm16. On the other hand, expression levels of Ucp1 correlated with those of Alk6. The present study suggests close relationships among adipose expressions of BMP signal components, and the relationships of expression levels of BMP receptor and those of Prdm16 or Ucp1 in dogs. Further studies using more dogs with various BCS potentially lead to identification of adipose factors to relate with adiposity in dogs.

Chronic retinoic acid treatment induces differentiation and changes in the metabolite levels of brown (pre)adipocytes.

Dietary vitamin A status affects energy metabolism. The present study explored the effect of all-trans retinoic acid (ATRA) on the expression levels of molecules and metabolites of brown adipocytes. Chronic ATRA treatment was initiated during the early stage (days 0-8) or late stage (days 8-12) of adipogenesis. Treatment with ATRA during the early and late stage of adipogenesis resulted in an increase in the expression level of Ucp1 and Cidea, genes highly expressed in brown adipocytes, on day 8 and day 12, respectively, whereas expression of Pgc-1α, another gene expressed during brown adipogenesis, was unaffected by ATRA. Non-targeted metabolomic analyses indicated that the pathways related to the glucose metabolism were affected by ATRA, irrespective of the differentiation stage. Cellular levels of glucose 6-phosphate, fructose 6-phosphate, citric acid, and succinic acid decreased after ATRA treatment on days 8 and 12. In contrast, glucose level was higher in ATRA-treated cells on day 8, but it was lower on day 12. ATRA decreased the cellular level of aconitic acid, fumaric acid, and malic acid on day 12 but not on day 8. Furthermore, ATRA increased the expression level of Hxk2 and downregulated the expressions of G6pdh and Pfkl/Pfkp on day 8 but not on day 12. Together, the results indicate that the chronic treatment with ATRA stimulated the formation of activated brown adipocytes, eventually leading to alterations in the levels of cellular metabolites related to glucose metabolism. SIGNIFICANCE OF THE STUDY: Significance of the study treatment with all-trans retinoic acid (ATRA) during the early and late stage of adipogenesis increased the expression of Ucp1 and Cidea, genes highly expressed in brown adipocytes, on day 8 and day 12. Cellular levels of glucose 6-phosphate, fructose 6-phosphate, citric acid, and succinic acid decreased after ATRA treatment on days 8 and 12. In contrast, glucose level was higher in ATRA-treated cells on day 8, but it was lower on day 12. The present results indicate that ATRA stimulated the formation of activated brown adipocytes, eventually leading to alterations in the levels of cellular metabolites related to glucose metabolism.

Effect of molting on the concentration of plasma 25-hydroxyvitamin D in captive African penguins (Spheniscus demersus).

Feed intake and body weight are drastically altered in penguins during peri-molting period, and molting is known to affect the nutritional status of vitamin A and E. Although vitamin D status is not known in penguins during peri-molting period, vitamin D intake is supposed to be remarkably altered. The objective of the present study was to clarify the alterations in plasma 25-hydroxyvitamin D (25(OH)D) concentration, the most reliable biomarker for assessing vitamin D status, and vitamin D intake during peri-molting period. Blood samples were collected from seven adult male African penguins (Spheniscus demersus) in the control period, pre-molting period, early-molting period, and late-molting period. The dietary content of vitamin D and calcium (Ca) were higher than that of the estimated requirements. Feed intake increased in the pre-molting period and drastically decreased during the molting periods. Body weight increased in the pre-molting period, followed by the loss of weight towards the end of the experiment. Although vitamin D and Ca intakes decreased during the molting periods, plasma 25(OH)D concentration increased during the molting periods and the increase in plasma Ca concentration was also observed in the late-molting period. These results suggest that the reduction in body fat induced by reducing feed intake stimulated the release of vitamin D from body fat, which increased plasma 25(OH)D and Ca concentrations in molting penguins. Penguins are unlikely to suffer from typical hypervitaminosis D even during molting and vitamin D toxicity is not a realistic problem in penguins because of the short duration of molting.

Interleukin-1ß (IL-1ß) transcriptionally activates hepcidin by inducing CCAAT enhancer-binding protein δ (C/EBPδ) expression in hepatocytes.

Hepcidin is a liver-derived hormone that negatively regulates serum iron levels and is mainly regulated at the transcriptional level. Previous studies have clarified that in addition to hepatic iron levels, inflammation also efficiently increases hepatic hepcidin expression. The principle regions responsible for efficient hepcidin transcription are bone morphogenetic protein-responsive elements (BMP-REs) 1 and 2 as well as the signal transducer and activator of transcription 3-binding site (STAT-BS). Here, we show that the proinflammatory cytokine interleukin-1ß (IL-1ß) efficiently increases hepcidin expression in human HepG2 liver-derived cells and primary mouse hepatocytes. The primary region responsible for IL-1ß-mediated hepcidin transcription was the putative CCAAT enhancer-binding protein (C/EBP)-binding site (C/EBP-BS) at the hepcidin promoter spanning nucleotides -329 to -320. IL-1ß induces the expression of C/EBPδ but neither C/EBPα nor C/EBPß in hepatocytes, and C/EBPδ bound to the C/EBP-BS in an IL-1ß-dependent manner. Lipopolysaccharide (LPS) induced the expression of IL-1ß in Kupffer cells and hepatocytes in the mouse liver; furthermore, the culture supernatants from the macrophage-like cell line RAW264.7 treated with LPS potentiated the stimulation of hepcidin expression in hepatocytes. The present study reveals that: 1) inflammation induces IL-1ß production in Kupffer cells and hepatocytes; 2) IL-1ß increases C/EBPδ expression in hepatocytes; and 3) induction of C/EBPδ activates hepcidin transcription via the C/EBP-BS that has been uncharacterized yet. In cooperation with the other pathways activated by inflammation, IL-1ß pathway stimulation leads to excess production of hepcidin, which could be causative to anemia of inflammation.

JNK facilitates IL-1ß-induced hepcidin transcription via JunB activation.

Hepcidin, a liver-derived hormone, negatively regulates circulating iron levels through an increase in its expression in response to iron overload. Inflammation also increases production of hepcidin, potentially leading to inflammatory anemia. We previously revealed that proinflammatory cytokine interleukin (IL)-1ß increased hepcidin expression through its transcriptional stimulation in hepatocytes. Induction of CCAAT-enhancer-binding protein (C/EBP) δ and IL-6 in response to IL-1ß treatment stimulated hepcidin transcription via the C/EBP-binding site (C/EBP-BS) and signal transducer and activator of transcription (STAT)-BS on the hepcidin promoter, respectively. Here, we show an additional pathway responsible for IL-1ß-induced hepcidin transcription. IL-1ß stimulated phosphorylation of c-Jun N-terminal kinase (JNK) and its substrates c-Jun and JunB. SP600125, a JNK inhibitor, blocked IL-1ß-induced phosphorylation of c-Jun and JunB as well as IL-1ß-induced expression and transcription of hepcidin. Reporter assays for hepcidin transcription revealed that reporters with mutations of cAMP response element (CRE) site B, a putative Jun binding element, decreased responsiveness to IL-1ß, and that activated JunB, but not c-Jun, conferred IL-1ß-induced hepcidin transcription. Furthermore, binding of JunB to hepcidin promoter was increased by IL-1ß. The present study indicated that IL-1ß activates JNK and subsequently stimulates JunB activation, leading to hepcidin transcription via CRE site B on the hepcidin promoter. The present experiment provides novel insights into the molecular mechanisms underlying induction of hepcidin by inflammation and alteration of iron homeostasis.

Role of estradiol and testosterone in Ucp1 expression in brown/beige adipocytes.

Activity of brown/beige adipocytes is higher in women than in men. The expression level of uncoupling protein 1 (UCP1) is largely consistent with the thermogenic activity in brown/beige adipocytes. The present study examined the direct effects of sex hormones on Ucp1 expression in brown adipocytes and beige adipocytes, which were differentiated from HB2 brown preadipocytes and 3T3-L1 white preadipocytes, respectively; treatment with estradiol or testosterone was used during the early (days 0-8) or late stage (days 8-12) of brown adipogenesis and beige adipogenesis. On day 8 or day 12, cells were treated with or without isoproterenol (Iso), an agonist for the ß-adrenergic receptor, for 4 hours. Furthermore, the sex of cells was examined; the sex-determining region y gene, which is located on the y chromosome, was present in HB2 cells, but not in 3T3-L1 cells, suggesting that HB2 cells and 3T3-L1 cells are male and female cells, respectively. Treatment with 17ß-estradiol during the early stage of brown adipogenesis enhanced the responsiveness to Iso on Ucp1 induction, whereas treatment during the late stage of brown adipogenesis decreased Ucp1 expression in unstimulated brown adipocytes. Estradiol decreased Iso-induced Ucp1 expression during the early stage of beige adipogenesis. Treatment with testosterone during the early stage of brown adipogenesis did not affect Ucp1 expression but increased the responsiveness to Iso on Ucp1 induction by the treatment during the late stage of brown adipogenesis. The present results suggest that sex hormones modulate the expression level of Ucp1 in brown/beige adipocytes in a stage-dependent manner. Direct effects of sex hormones in brown/beige adipogenesis were evaluated. Treatment with 17ß-estradiol during the early stage of brown adipogenesis enhanced the responsiveness to isoproterenol (Iso), an agonist for the ß-adrenergic receptor, on Ucp1 induction, whereas treatment during the late stage of brown adipogenesis decreased Ucp1 expression in unstimulated brown adipocytes. Estradiol decreased Iso-induced Ucp1 expression during the early stage of beige adipogenesis. Testosterone during the late stage of brown adipogenesis increased the responsiveness to Iso on Ucp1 induction. Sex hormones modulate the expression level of Ucp1 in brown/beige adipocytes in a stage-dependent manner.

WITHDRAWN: Modulation of adipocyte function by the TGF-ß family.

This article has been withdrawn at the request of the author(s) and/or editor. The Publisher apologizes for any inconvenience this may cause. The full Elsevier Policy on Article Withdrawal can be found at https://www.elsevier.com/about/our-business/policies/article-withdrawal.

Fluctuations in metabolite content in the liver of magnesium-deficient rats.

Mg deficiency induces various metabolic disturbances including glucose metabolism in the liver. However, no comprehensive information is currently available on the metabolic pathways affected by Mg deficiency. The present study examined metabolite content in the liver of Mg-deficient rats using a metabolomic analysis. In this study, 4-week-old, male Sprague-Dawley rats were fed a control diet or a Mg-deficient diet for 8 weeks. The metabolomic analysis identified 105 metabolites in the liver, and significant differences were observed in the hepatic contents for thirty-three metabolites between the two groups. An analysis by MetaboAnalyst, a web-based metabolome data analysis tool, indicated that the Mg deficiency affected taurine/hypotaurine metabolism, methionine metabolism and glycine/serine/threonine metabolism; taurine, hypotaurine, glycine, serine and threonine contents were increased by Mg deficiency, whereas the amounts of 2-ketobutyric acid (a metabolite produced by the catabolism of cystathionine or threonine) and 5'-methylthioadenosine (a metabolite involved in spermidine synthesis) were decreased. The amount of glucose 6-phosphate, a hub metabolite of glycolysis/gluconeogenesis and the pentose phosphate pathway, was significantly decreased in Mg-deficient rats. Mg deficiency also decreased metabolite contents from the citric acid cycle, including citric acid, fumaric acid and malic acid. Aberrant metabolism may be related to the allosteric regulation of enzymes; the mRNA levels of enzymes were generally similar between the two groups. The present study suggests that the Mg deficiency-mediated modulation of hepatic metabolism is as yet uncharacterised.

Direct action of capsaicin in brown adipogenesis and activation of brown adipocytes.

The ingestion of capsaicin, the principle pungent component of red and chili peppers, induces thermogenesis, in part, through the activation of brown adipocytes expressing genes related to mitochondrial biogenesis and uncoupling such as peroxisome proliferator-activated receptor (Ppar) γ coactivator-1α (Pgc-1α) and uncoupling protein 1 (Ucp1). Capsaicin has been suggested to induce the activation of brown adipocytes, which is mediated by the stimulation of sympathetic nerves. However, capsaicin may directly affect the differentiation of brown preadipocytes, brown adipocyte function, or both, through its significant absorption. We herein demonstrated that Trpv1, a capsaicin receptor, is expressed in brown adipose tissue, and that its expression level is increased during the differentiation of HB2 brown preadipocytes. Furthermore, capsaicin induced calcium influx in brown preadipocytes. A treatment with capsaicin in the early stage of brown adipogenesis did not affect lipid accumulation or the expression levels of Fabp4 (a gene expressed in mature adipocytes), Pparγ2 (a master regulator of adipogenesis) or brown adipocyte-selective genes. In contrast, a treatment with capsaicin in the late stage of brown adipogenesis slightly increased the expression levels of Fabp4, Pparγ2 and Pgc-1α. Although capsaicin did not affect the basal expression level of Ucp1, Ucp1 induction by forskolin was partially inhibited by capsaicin, irrespective of the dose of capsaicin. The results of the present study suggest the direct effects of capsaicin on brown adipocytes or in the late stage of brown adipogenesis.

Modulation of brown adipocyte activity by milk by-products: Stimulation of brown adipogenesis by buttermilk.

Brown adipocytes dissipate chemical energy in the form of heat through the expression of mitochondrial uncoupling protein 1 (Ucp1); Ucp1 expression is further upregulated by the stimulation of ß-adrenergic receptors in brown adipocytes. An increase in energy expenditure by activated brown adipocytes potentially contributes to the prevention of or therapeutics for obesity. The present study examined the effects of milk by-products, buttermilk and butter oil, on brown adipogenesis and the function of brown adipocytes. The treatment with buttermilk modulated brown adipogenesis, depending on the product tested; during brown adipogenesis, buttermilk 1 inhibited the differentiation of HB2 brown preadipocytes. In contrast, buttermilk 3 and 5 increased the expression of Ucp1 in the absence of isoproterenol (Iso), a ß-adrenergic receptor agonist, suggesting the stimulation of brown adipogenesis. In addition, the Iso-induced expression of Ucp1 was enhanced by buttermilk 2 and 3. The treatment with buttermilk did not affect the basal or induced expression of Ucp1 by Iso in HB2 brown adipocytes, except for buttermilk 5, which increased the basal expression of Ucp1. Conversely, butter oil did not significantly affect the expression of Ucp1, irrespective of the cell phase of HB2 cells, ie, treatment during brown adipogenesis or of brown adipocytes. The results of the present study indicate that buttermilk is a regulator of brown adipogenesis and suggest its usefulness as a potential food material for antiobesity.

Role of a TPA-responsive element in hepcidin transcription induced by the bone morphogenetic protein pathway.

Systemic iron balance is governed by the liver-derived peptide hormone hepcidin. The transcription of hepcidin is primarily regulated by the bone morphogenetic protein (BMP) and inflammatory cytokine pathways through the BMP-response element (BMP-RE) and STAT-binding site, respectively. In addition to these elements, we previously identified a TPA-responsive element (TRE) in the hepcidin promoter and showed that it mediated the transcriptional activation of hepcidin through activator protein (AP)-1 induced by serum. In the present study, we examined the role of TRE in the BMP-induced transcription of hepcidin in HepG2 liver cells. The serum treatment increased the basal transcription of hepcidin; however, responsiveness to the expression of ALK3(QD), a constitutively active BMP type I receptor, was unaffected. Consistent with these results, mutations in TRE in the hepcidin promoter decreased basal transcription, whereas responsiveness to the expression of ALK3(QD) remained unchanged. HepG2 cells significantly expressed AP-1 components in the basal state, whereas BMP did not up-regulate the expression of these components. The expression of c-fos enhanced the basal transcription of hepcidin as well as ALK3(QD)-mediated hepcidin transcription, whereas that of dominant-negative c-fos decreased hepcidin transcription. The results of the present study suggested that the cis-elements of the hepcidin promoter, BMP-RE and TRE, individually transmitted BMP-mediated and AP-1-mediated signals, respectively, whereas transcription was synergistically increased by the stimulation of BMP-RE and TRE.

Regulation of brown adipogenesis by the Tgf-ß family: involvement of Srebp1c in Tgf-ß- and Activin-induced inhibition of adipogenesis.

BACKGROUND: Brown adipocytes generate heat through the expression of mitochondrial Ucp1. Compared with the information on the regulatory differentiation of white preadipocytes, the factors affecting brown adipogenesis are not as well understood. The present study examined the roles of the Tgf-ß family members Bmp, Tgf-ß and Activin during differentiation of HB2 brown preadipocytes. METHODS: Endogenous Bmp activity and effects of exogenous Tgf-ß family members were examined. Role of Srebp1c in brown adipogenesis was further explored. RESULTS: Although Bmp7 has been suggested to be a potent stimulator of brown adipogenesis, it affected neither the expression of brown adipocyte-selective genes nor Ucp1 induction in response to a ß adrenergic receptor agonist. Unlike in 3T3-L1 white preadipocytes, endogenous Bmp activity was not required for brown adipogenesis; treatment with inhibitors of the Bmp pathway did not affect differentiation of preadipocytes. Administration of Tgf-ß1 or Activin A efficiently decreased the insulin-induced expression of brown adipocyte-selective genes. Tgf-ß1 and Activin A decreased the expression of Pparγ2 and C/ebpα, suggesting the inhibition of adipogenesis. The Tgf-ß- and Activin-induced inhibition of brown adipogenesis was mediated by the repression of Srebp1c expression; Tgf-ß1 and Activin A blocked Srebp1c gene induction in response to the differentiation induction, and knock-down of Srebp1 expression inhibited brown adipogenesis. CONCLUSION: Endogenous Bmp is dispensable for brown adipogenesis, and Srebp1c is indispensable, which is negatively regulated by Tgf-ß and Activin. GENERAL SIGNIFICANCE: Control of activity of the Tgf-ß family is potentially useful for maintenance of energy homeostasis through manipulation of brown adipogenesis.