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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.

Carnosine, which is abundant in meat, is a dipeptide composed of β-alanine and histidine, known to afford various health benefits. It has been suggested that carnosine can elicit an anti-obesity effect via induction and activation of brown/beige adipocytes responsible for non-shivering thermogenesis. However, the relationship between carnosine and brown/beige adipocytes has not been comprehensively elucidated. We hypothesized that β-alanine directly modulates brown/beige adipogenesis and performed an in vitro assessment to test this hypothesis. HB2 brown preadipocytes were differentiated using insulin from day 0. Cells were treated with various concentrations of β-alanine (12.5-100 μM) during adipogenesis (days 0-8) and differentiation (days 8-10). Then, cells were further stimulated with or without forskolin, an activator of the cAMP-dependent protein kinase pathway, on day 8 or day 10 for 4 h before harvesting. We observed that HB2 cells expressed molecules related to the transport and signal transduction of β-alanine. Treatment with β-alanine during brown adipogenesis dose-dependently enhanced forskolin-induced Ucp1 expression; this was not observed in differentiated brown adipocytes. Consistent with these findings, treatment with β-alanine during days 0-8 increased phosphorylation levels of CREB in forskolin-treated HB2 cells. In addition, β-alanine treatment during brown adipogenesis increased the expression of Pparα, known to induce brown/beige adipogenesis, in a dose-dependent manner. These findings revealed that β-alanine could target HB2 adipogenic cells and enhance forskolin-induced Ucp1 expression during brown adipogenesis, possibly by accelerating phosphorylation and activation of CREB. Thus, β-alanine, a carnosine-constituting amino acid, might directly act on brown adipogenic cells to stimulate energy expenditure.

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.

Activin B, a homodimer of the inhibin βB subunit, acts as a regulator of gonadal function and as an adipokine. To clarify the role of activin B in dogs, we characterized the canine inhibin βB gene and signalling pathways regulated by the canine inhibin βB. Using 5'- and 3'-rapid amplification of cDNA end (RACE) and RT-PCR on RNA isolated from the ovary of dogs, we identified short and long forms of the inhibin βB gene. Immunoreactive inhibin βB molecules were detected at ~25 and ~14 kDa under nonreducing and reducing conditions, respectively, in culture supernatants from HEK293 cells transfected with a plasmid containing the long form of the inhibin βB gene, indicating activin B production and secretion. Similar to human and murine activin B, the canine activin B-stimulated transcriptions of reporter genes, CAGA-luc and Hepcidin-luc, regulated by the canonical activin/transforming growth factor-β (TGF-β) and bone morphogenetic protein (BMP) pathway, respectively. Activin B-induced CAGA-luc transcription was not detected in ALK7-deficient MDCK canine-derived cells; however, the forced expression of ALK7 resulted in the activin B-dependent expression in MDCK cells. Unexpectedly, the activin B-induced activation of the BMP pathway was partially blocked by the inhibition of endogenous activin/TGF-β receptor activity. The present study identified an experimentally isolated long form of the canine inhibin βB gene producing activin B that transactivates BMP- and activin/TGF-β-regulated gene expression.

Hepcidin negatively regulates the circulating iron levels by inhibiting the intestinal absorption of iron as well as iron release from macrophages. Hepcidin activity is largely determined by its expression, which is regulated at the transcriptional level. Hepcidin transcription is induced not only by the iron status-related bone morphogenetic protein (BMP)-2/6, but also by inflammatory cytokines, such as interleukin (IL)-1β and IL-6. The present study reveals that the microphthalmia (MiT)/transcription factor E (TFE) family members are novel regulators of hepcidin transcription. Melanocyte-inducing transcription factor (MITF)-A, a member of the MiT/TFE family, was identified as a positive regulator of hepcidin transcription via stimulus screening for transcription regulators. An E-box (5'-CATGTG-3') spanning nt-645 to nt-640 of the murine hepcidin promoter was identified as an MITF-A-responsive element. Responsiveness to MITF-A on hepcidin transcription decreased when the cells were stimulated with BMP2 or IL-1β. These results suggest a functional interaction between the MITF pathway and BMP- or IL-1β-mediated signaling. TFEB and TFE3, members of the MiT/TFE family, also stimulated hepcidin transcription, but the main region responsible for hepcidin transcription was distinct from that induced by MITF-A. The region spanning nt-581 to nt-526 was involved in TFEB/TFE3-mediated hepcidin transcription. Considering that members of the MiT/TFE family act as regulators of starvation-induced lysosomal biogenesis, hepcidin expression may be controlled by additional pathways apart from those identified so far.

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-β (TGF-β)/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-β/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-β 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.

Previous studies suggest a negative relationship between hepatic oxidative stress and productivity in beef cattle. Uncoupling protein 2 (UCP2) is involved in the disappearance of reactive oxygen species, suggesting the defensive role of UCP2 against oxidative stress. The present study examined the relationship between oxidative stress and expression levels of UCP2/Ucp2 in cultured human and mouse liver-derived cells. We also explored factors regulating bovine Ucp2 transcription. As oxidative stress inducers, hydrogen peroxide, ethanol, and cumene hydroperoxide (CmHP) were used. Expression levels of hemoxygenase 1 (HMOX1), a representative gene induced by oxidative stress, were not affected by any oxidative stress inducers in HepG2 human liver-derived cells. The levels of UCP2 mRNA were also unaffected by the oxidative stress inducers. Treatment with CmHP increased expression of Hmox1 in Hepa1-6 mouse liver-derived cells, but Ucp2 expression was not changed. Stimulus screening for regulator of transcription (SSRT) revealed that expression of p50 or p65, transcription factors conferring response to oxidative stress, did not stimulate bovine Ucp2 transcrition in HepG2 cells. SSRT also showed 11 molecules that induced Ucp2 transcription more than 4-fold; among them, endoplasmic reticulum (ER) stress-related transcription factors such as XBP1, c-JUN, JUNB, and C/EBPβ were identified. However, treatment with ER stress inducers did not increase Ucp2 expression in HepG2 and Hepa1-6 cells. The present results suggest that 1) neither oxidative stress nor ER stress induces Ucp2 expression in liver-derived cells, and 2) Ucp2 transcription is stimulated by several transcription factors.

DNA methylation of enhancers and promoters generally inhibits gene transcription. DNA methylation occurs predominantly at the dinucleotide CpG, a methyl group that is covalently bonded to cytosine. We have previously demonstrated tissue-restricted expression of the uncoupling protein 1 ( Ucp1) in common carp. Here, we characterized the methylation status of the upstream region of the transcriptional initiation site of the carp Ucp1 gene in the liver, brain, kidney, skeletal muscle, and scales. In addition, we explored the direct role of methylation on Ucp1 transcription. Ucp1 expression was higher in the liver than that in other tissues including the kidney, skeletal muscle, and scales. The extent of methylation at nt −2178 and nt −2103 was lower in the liver and kidney than that in the brain, skeletal muscle, and scales. In addition, methylation at the upstream proximal-region of the Ucp1 gene was generally less frequent in the liver compared with that in the other organs. The transcriptional activation assay using the CpG-free luciferase-based reporter suggested that the methylation of the distal and proximal regions of the carp Ucp1 gene did not affect Ucp1 transcription. Unexpectedly, mutation of cytidylic acid to guanylic acid at nt −108 decreased Ucp1 promoter activity. The present results reveal that the status of DNA methylation of the upstream region of the carp Ucp1 gene is different among different tissues, but suggest that the DNA methylation do not directly repress the transcription of Ucp1.

Beige adipocytes and brown adipocytes can generate heat by using mitochondrial uncoupling protein 1 (Ucp1), a thermogenic protein. Browning/beiging is the emergence of beige adipocytes in white adipose tissues (WAT) for cold acclimatization. Here we show the existence of brown/beige adipocytes in retro-orbital WAT in mice. Histologically, Ucp1-positive cells with multilocular lipid droplets were abundant in retro-orbital WAT of immature mice; those cells decreased in number with age. However, Ucp1-positive adipocytes with multilocular lipid droplets emerged in retro-orbital WAT in adult mice, due to cold exposure as short as 3 h. Consistent with this observation, the expression level of Ucp1 mRNA was enhanced in tissues upon cold exposure. Furthermore, eye surface temperature remained within a physiological range during cold challenge. RT-qPCR suggested a mixed phenotype of brown and beige adipocytes in retro-orbital WAT. Transmission electron microscopic observation showed multiple lipid droplets and numerous mitochondria with high cristae density in retro-orbital WAT cells from both control and cold-exposed mice. Our results suggest that warming of the orbital cavity by browning/beiging in retro-orbital WAT is a protective mechanism against cold cataract caused by lowered lens temperature.

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.

Activin E, a secreted peptide encoded by the inhibin/activin βE subunit gene, is a member of the transforming growth factor-β superfamily, which is predominantly expressed in the liver. Recent reports have suggested that activin E plays a role in energy homeostasis as a hepatokine. Here, using transgenic mice overexpressing activin E under the control of the β-actin promoter, we demonstrate that activin E controls energy metabolism through brown/beige adipocytes. The glucose tolerance test and insulin tolerance test showed that the insulin sensitivity was improved in the transgenic mice. Furthermore, the mice had a high body temperature compared with wild-type mice. The transgenic brown adipose tissue and mesenteric white adipose tissue showed upregulation of uncoupling protein 1, which enables energy dissipation as heat by uncoupling oxidative phosphorylation from ATP production. Present results indicate that activin E activates energy expenditure through brown/beige adipocyte activation, suggesting that activin E has high potential for obesity therapy.

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.

This was originally identified as an antimicrobial peptide, and later characterized as an iron-regulatory hormone. Hepcidin negatively regulates serum iron through the inhibition of intestinal iron absorption and iron recycling by macrophages.

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.

Iron is essential for a variety of physiological processes. Hepatic iron overload acts as a trigger for the progression of hepatic steatosis to nonalcoholic steatohepatitis and hepatocellular carcinoma. In the present study, we aimed to study the effects of iron overload on cellular responses in hepatocytes. Rat primary hepatocytes (RPH), mouse primary hepatocytes (MPH), HepG2 human hepatoma cells and Hepa1-6 mouse hepatoma cells were treated with FeCl3. Treatment with FeCl3 effectively increased iron accumulation in primary hepatocytes. Expression levels of molecules involved in cellular signaling such as AMPK pathway, TGF-β family pathway, and MAP kinase pathway were decreased by FeCl3 treatment in RPH. Cell viability in response to FeCl3 treatment was decreased in RPH but not in HepG2 and Hepa1-6 cells. Treatment with FeCl3 also decreased expression level of LC-3B, a marker of autophagy in RPH but not in liver-derived cell lines. Ultrastructural observations revealed that cell death resembling ferroptosis and necrosis was induced upon FeCl3 treatment in RPH. The expression level of genes involved in iron transport varied among different liver-derived cells- iron is thought to be efficiently incorporated as free Fe2+ in primary hepatocytes, whereas transferrin-iron is the main route for iron uptake in HepG2 cells. The present study reveals specific cellular responses in different liver-derived cells as a consequence of iron overload.

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 β-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 17β-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 17β-estradiol during the early stage of brown adipogenesis enhanced the responsiveness to isoproterenol (Iso), an agonist for the β-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.

Brown adipocyte activation or beige adipocyte emergence in white adipose tissue (WAT) increases energy expenditure, leading to a reduction in body fat mass and improved glucose metabolism. We found that activin E functions as a hepatokine that enhances thermogenesis in response to cold exposure through beige adipocyte emergence in inguinal WAT (ingWAT). Hepatic activin E overexpression activated thermogenesis through Ucp1 upregulation in ingWAT and other adipose tissues including interscapular brown adipose tissue and mesenteric WAT. Hepatic activin E-transgenic mice exhibited improved insulin sensitivity. Inhibin βE gene silencing inhibited cold-induced Ucp1 induction in ingWAT. Furthermore, in vitro experiments suggested that activin E directly stimulated expression of Ucp1 and Fgf21, which was mediated by transforming growth factor-β or activin type I receptors. We uncovered a function of activin E to stimulate energy expenditure through brown and beige adipocyte activation, suggesting a possible preventive or therapeutic target for obesity., アクチビンEが脂肪燃焼細胞の増加を促進することを解明 --肥満解消の新たなプレイヤーを発見--. 京都大学プレスリリース. 2018-11-02.

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 β3 -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 β3 -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.

Background Expression of hepcidin, a hormone produced by hepatocytes which negatively regulates the circulating iron levels, is known to be positively regulated by BMP6, a member of transforming growth factor (TGF)-β family. Previous studies have shown that iron status is sensed by sinusoidal endothelial cells of hepatic lamina, leading to the modulation of BMP6 expression. Methods ISOS-1, HUVEC, F-2, and SK-HEP1 endothelial cells were treated with either iron or 2,2′-dipyridyl (2DP), a cell-permeable iron-chelator, and expression level of Bmp6 was examined. To identify factors affecting Bmp6 transcription, stimulus screening for regulator of transcription (SSRT) was developed. Results Treatment with iron slightly increased the expression levels of Bmp6, while 2DP unexpectedly increased Bmp6 expression in a dose-dependent manner. 2DP-induced Bmp6 expression was resistant to co-treatment with iron. 2DP-induced Bmp6 expression was also detected in HUVEC, F-2 cells, and SK-HEP1 cells. Luciferase-based reporter assays indicated that forced expression of JunB increased the transcription of Bmp6. 2DP induced phosphorylation of JunB; co-treatment with SP600125 blocked the 2DP-induced Bmp6 expression partially. JunB-induced Bmp6 transcription was not affected by mutations of putative JunB-responsive elements. Some endoplasmic reticulum stress inducers increased the expression of Bmp6. SSRT revealed pathways regulating Bmp6 transcription positively and negatively. Hepa1–6 liver cells and C2C12 myogenic cells were prone to 2DP induced Bmp6 expression. Conclusions The present study reveals non‑iron-regulated Bmp6 expression in endothelial cells. General significance Regulatory expression of Bmp6 may be important as a key step for fine tuning of BMP activity.

The data presented here are related to the research article entitled “Inducible brown/beige adipocytes in retro-orbital adipose tissues” [1]. Brown and beige adipocytes dissipate stored energy through the generation of heat by using mitochondrial uncoupling protein 1 (Ucp1), which is a mammalian brown/beige adipocyte-specific protein that promotes proton leakage across the inner mitochondrial membrane. Both cells up-regulate Ucp1 expression in response to β-adrenergic receptor activation such as cold exposure. Here, we provide data on induction of Ucp1 positive cells in retro-orbital white adipose tissues (WAT) from cold exposed both male and female mice. The fluctuation of eye surface temperature was monitored during the cold exposure. In addition, distribution of tyrosine hydroxylase positive bundles was observed in the retro-orbital WAT from the mice. Keywords: Eye, Metabolism, Ocular disorder, Retrobulbar, Thermogenesis

Long-distance transportation has negative impacts on production and health in cattle. Feed and water are routinely deprived from cattle during transportation. We investigated whether niacin supplementation could improve niacin nutrition and mitigate the adverse effect of transportation with feed and water deprivation in steer calves. We also studied the adverse effect of feed and water deprivation in nontransported steer calves. Twelve calves were assigned to feed and water deprivation for 2 days, or full access to feed and water in experiment 1. Ten calves were assigned to 2-day transportation with feed and water deprivation, or the transportation with feed and water deprivation, but with supplementation of rumen-protected niacin at 100 g/day per head in experiment 2. Bodyweight was measured and blood was collected for 32 days in each experiment. Feed and water deprivation temporarily decreased serum glucose concentrations and bodyweight gain. Transportation with deprivation of feed and water caused a temporal decrease in bodyweight gain and serum albumin concentration, and a continuous decrease in serum glucose and total cholesterol concentrations, which was suppressed by niacin supplementation. Niacin supplementation increased blood niacin concentration. These results suggest that niacin supplementation mitigates adverse effects of transportation with feed and water deprivation in steer calves.

With longevity, the prevalence of osteoporosis, which occurs when the activity of osteoclast surpasses that of osteoblasts, has increased in dogs. However, limited information is available on canine osteoclastogenesis. We herein described culture conditions to induce osteoclasts from canine bone marrow cells, and identified factors affecting canine osteoclastogenesis. Tartrate-resistant acid phosphatase-positive multinucleated cells were efficiently formed in a culture of bone marrow mononuclear cells with macrophage colony-stimulating factor (M-CSF 25 ng/mL) for 3 days and a subsequent culture in the presence of M-CSF (25 ng/mL) and soluble receptor activator of NF-κB ligand (RANKL 50 ng/mL) for 4 days. We previously reported in a murine cell system that gene induction of the E isoform of microphthalmia-associated transcription factor (Mitf-E) was required and sufficient for osteoclastogenesis, while transforming growth factor-β (TGF-β) enhanced RANKL-induced Mitf-E expression and osteoclastogenesis. Mitf-E expression also increased during RANKL-induced osteoclastogenesis in canine cells; however, TGF-β down-regulated Mitf-E expression and osteoclastogenesis, indicating a species-dependent response. The results of the present study show that, consistent with murine cells, M-CSF and soluble RANKL enable canine bone marrow cells to differentiate into osteoclasts, and Mitf-E expression is induced during osteoclastogenesis. However, the role of TGF-β in osteoclast formation is distinct between murine and canine cells, suggesting the necessity of analyses using canine cells to examine the factors affecting canine osteoclastogenesis.

The relationship between mineral concentrations, and the relationship of mineral concentrations with physicochemical characteristics in muscles were investigated using the Longissimus thoracis (LT) muscle of 44 Japanese Black steers. We determined moisture content, fat content, meat color, fatty acid composition and mineral concentrations in the LT muscle. Magnesium (Mg), potassium (K) and zinc (Zn) concentrations had negative correlations with fat content, but sodium (Na), manganese (Mn), copper (Cu) and molybdenum (Mo) concentrations were not correlated with fat content. The concentrations of Mg, Mn, Fe, Cu and Zn largely and positively contributed to the first principal component of mineral concentrations. Because the red muscle was rich in these minerals compared to the white muscle, the variation of these minerals probably results from the abundance of red fibers in the LT muscle. The concentration of K was positively correlated with moisture content but Na concentration was not related to moisture content, suggesting that the intracellular fluid volume can largely affect moisture content. The results of the present experiment suggest that mineral concentrations reflect some traits such as not only fat content but also the composition of myofiber type and the intracellular fluid volume in the LT muscle of Japanese Black cattle.

Long-distance transportation is sometimes inevitable in the beef industry because of the geographic separation of major breeding and fattening areas. Long-distance transportation negatively impacts production and health of cattle, which may, at least partly, result from the disturbance of metabolism during and after transportation. However, alteration of metabolism remains elusive in transported cattle. We investigated the effects of transportation on the metabolomic profiles of Holstein steer calves. Non-targeted analysis of serum concentrations of low molecular weight metabolites was performed by gas chromatography mass spectrometry. Transportation affected 38 metabolites in the serum. A pathway analysis suggested that 26, 10, and 10 pathways were affected immediately after transportation, and 3 and 7 days after transportation, respectively. Some pathways were disturbed only immediately after transportation, likely because of feed and water withdrawal during transit. Nicotinate and nicotinamide metabolism, and citric acid cycle were affected for 3 days after transportation, whereas propionate metabolism, phenylalanine and tyrosine metabolism were affected throughout the experiment. Four pathways were not affected immediately after transportation, but were altered thereafter. These results suggested that many metabolic pathways had marked perturbations during transportation. Metabolites such as citric acid, propionate, tyrosine and niacin can be candidate supplements for mitigating transportation-induced adverse effects.

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-1β (IL-1β) efficiently increases hepcidin expression in human HepG2 liver-derived cells and primary mouse hepatocytes. The primary region responsible for IL-1β-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-1β induces the expression of C/EBPδ but neither C/EBPα nor C/EBPβ in hepatocytes, and C/EBPδ bound to the C/EBP-BS in an IL-1β-dependent manner. Lipopolysaccharide (LPS) induced the expression of IL-1β 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-1β production in Kupffer cells and hepatocytes; 2) IL-1β 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-1β pathway stimulation leads to excess production of hepcidin, which could be causative to anemia of inflammation.

Expression of uncoupling protein (Ucp) 2 but not Ucp3 in subcutaneous fat was significantly higher in cattle fed the concentrate diet than in those fed the roughage diet. Ucp2 expression in mesenteric fat was higher in cattle fed the vitamin A-deficient diet than in those fed the control diet.

Uncoupling protein 1 (Ucp1) is predominantly expressed in brown/beige adipocytes in mammals. Although myogenic cells have been suggested to commit to a brown adipocyte lineage through the induction of Prdm16 expression, Prdm16 is also expressed in skeletal muscle. Thus, we examined expression of Ucp1 in bovine myogenic cells. Considering that Ucp1 is a principle molecule that induces energy expenditure in brown/beige adipocytes, expression of Ucp1 is not preferable in beef cattle because of potential decrease in energy (fattening) efficiency. The RT-PCR analyses revealed the expression of Ucp1 in the skeletal muscle of cattle; expression levels were markedly lower than those in the brown fat of calves. Immunohistochemical analyses showed that Ucp1 surrounded muscle fibers, but not adipocytes residing in skeletal muscle. Myosatellite cells cultured in myogenic medium showed an increase in the expression levels of myogenic regulatory factors ( < 0.05), while those in cells cultured in adipogenic medium were decreased ( < 0.05). The Ucp1 expression was also detected in myosatellite cells; expression levels were greater in cells after myogenic culture for 12 d than in those after myogenic culture for 6 d ( < 0.05) and were decreased when cells were cultured in adipogenic medium ( < 0.05). The Prdm16 expression was not affected by culture conditions, suggesting that the expression of Ucp1 is not regulated by that of Prdm16. The results of the present study provide an insight into the unexpected expression of Ucp1 in bovine skeletal muscle, which suggests the necessity for further studies on Ucp1-mediated energy expenditure in bovine skeletal muscle.

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 β-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 β-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.

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.

Hepcidin expression is determined through transcriptional regulation by systemic iron status. However, acute or chronic inflammation also increases the expression of hepcidin, which is associated with the dysregulation of iron metabolism in pathological conditions. Interleukin (IL)-6 has been suggested to be a principal molecule to confer inflammation-related hepcidin transcription, which is mediated via signal transducer and activator of transcription (STAT)-binding site on the hepcidin promoter. Recently, it has been uncovered that another pro-inflammatory cytokine IL-1β stimulates hepcidin expression through the distinct mechanism underlying IL-6-mediated hepcidin transcription. In addition to IL-6 induction, IL-1β stimulates expression of CCAAT-enhancer-binding protein (C/EBP)δ, a transcription factor, leading to transcriptional activation of hepcidin via C/EBP-binding site on the hepcidin promoter. Thus, hepcidin transcription is stimulated through multiple elements in response to proinflammatory cytokines. Relationships between increased production of IL-1β and dysregulated iron metabolism have been suggested in various diseases, which may be linked to overproduction of hepcidin.

Hepcidin expression is determined through transcriptional regulation by systemic iron status. However, acute or chronic inflammation also increases the expression of hepcidin, which is associated with the dysregulation of iron metabolism in pathological conditions. Interleukin (IL)-6 has been suggested to be a principal molecule to confer inflammation-related hepcidin transcription, which is mediated via signal transducer and activator of transcription (STAT)-binding site on the hepcidin promoter. Recently, it has been uncovered that another pro-inflammatory cytokine IL-1β stimulates hepcidin expression through the distinct mechanism underlying IL-6-mediated hepcidin transcription. In addition to IL-6 induction, IL-1β stimulates expression of CCAAT-enhancer-binding protein (C/EBP)δ, a transcription factor, leading to transcriptional activation of hepcidin via C/EBP-binding site on the hepcidin promoter. Thus, hepcidin transcription is stimulated through multiple elements in response to proinflammatory cytokines. Relationships between increased production of IL-1β and dysregulated iron metabolism have been suggested in various diseases, which may be linked to overproduction of hepcidin.

Four Japanese Black steers (16 months of age) were assigned to a 4 × 4 Latin square design to investigate the effect of graded levels of sweet-potato condensed distillers solubles (SCDS) in their diets on intake and urinary excretion of minerals. The four diets consisted of 0%, 10%, 20% and 30% (dry matter (DM) basis) SCDS, with SCDS replacing commercial concentrate (CC). Intake of K, Cl, S, P and Mg increased linearly with increasing SCDS content. Urinary pH increased linearly with increasing dietary SCDS content. SCDS feeding increased urinary K concentrations (linear and quadratic effects). Urinary concentrations of Cl increased linearly with increasing SCDS content. In contrast, urinary concentrations of Mg decreased with increasing SCDS content. Feeding of SCDS did not apparently affect urinary NH3 ,P, Na or Ca concentrations. These results suggest that high SCDS feeding is not a risk for crystallization of minerals leading to the formation of magnesium-phosphate type calculi: although SCDS contains large amounts of P and Mg, high SCDS feeding decreased the Mg concentration and did not affect the P concentration in urine. Additionally, high SCDS feeding had no apparent effects on plasma concentrations of Na, K, Cl, Ca or inorganic P.

Although the insulin-mediated cell signaling pathway has been extensively examined, changes in the cellular content of metabolites currently remain unclear. We herein examined metabolite contents in 3T3-L1 adipocytes treated with insulin using a metabolomic analysis. Fifty-four compounds were detected, and the contents of metabolites from the citric acid cycle increased in response to the insulin treatment for 4 h, which was sensitive to U0126 and LY294002, inhibitors for mitogen-activated protein kinase kinase-1 and phosphoinositide 3-kinase, respectively. The cellular contents of fumaric acid and malic acid were increased more by insulin than those of citric acid and succinic acid. Time-course changes in metabolites from the citric acid cycle exhibited oscillations with a 2-h cycle. A metabolic pathway analysis also indicated that insulin affected the metabolism of alanine, aspartate and glutamate, as well as that of arginine and proline. The contents of free amino acids were slightly decreased by the insulin treatment, while the co-treatment with U0126 and LY294002 abrogated these insulin-mediated decreases. The present study revealed the unexpected accumulation of citric acid cycle metabolites in adipocytes by insulin. Our results indicate the usefulness of metabolomic analyses for obtaining a more comprehensive understanding of the regulation of metabolic pathways in cell-culture systems.

Members of the transforming growth factor-β (TGF-β) family function through Smad-dependent and Smad-independent pathways. The Smad-dependent pathway is stimulated through the phosphorylation of receptor-regulated Smad (R-Smad) and inhibited through the dephosphorylation of R-Smad or the gene induction of inhibitory Smad (I-Smad). Little information is available on the regulation of R-Smad gene expression. BMP4 potentiated the up-regulation of Smad8/9 expression in C2C12, H9c2, 3T3-L1, HepG2, B16, and primary fibroblasts. BMP4-induced Smad8/9 expression was cycloheximide-insensitive and LDN-193189-sensitive, suggesting a direct event mediated through BMP type I receptors. BMP4 transcriptionally stimulated the Smad8/9 gene, and BMP-responsive elements (BREs) spanning nt -121 to nt -44 are involved in the up-regulation of Smad8/9 expression in response to BMP4. Phosphorylated Smad1/5/8/9 specifically bound to the BREs of Smad8/9 gene. The present study reveals that Smad8/9 is a unique R-Smad regulated through the BMP pathway at the mRNA level. J. Cell. Biochem. 117: 1788-1796, 2016. © 2016 Wiley Periodicals, Inc.

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.

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 β-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 17β-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 17β-estradiol during the early stage of brown adipogenesis enhanced the responsiveness to isoproterenol (Iso), an agonist for the β-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.

Brown/beige adipocytes dissipate energy as heat. We previously showed that brown/beige adipocytes are present in white adipose tissue (WAT) of fattening cattle. The present study examined the effect of vitamin A restriction on mRNA expression of brown/beige adipocyte-related genes. In Japan, fattening cattle are conventionally fed a vitamin A-restricted diet to improve beef marbling. Twelve Japanese Black steers aged 10 mo were fed control feed (n = 6) or vitamin A-restricted feed (n = 6) for 20 mo. Subcutaneous WAT (scWAT) and mesenteric WAT (mesWAT) were collected, and mRNA expression levels of molecules related to the function of brown/beige adipocytes (Ucp1, Cidea, Dio2, Cox7a, and Cox8b) as well as transcriptional regulators related to brown/beige adipogenesis (Zfp516, Nfia, Prdm16, and Pgc-1α) were evaluated. The vitamin A restriction significantly increased or tended to increase expression levels of Cidea and Pgc-1α in scWAT, and Cidea, Dio2, and Nfia in mesWAT. Previous studies revealed that the bone morphogenetic protein (Bmp) pathway was responsible for commitment of mesenchymal stem cells to brown/beige adipocyte-lineage cells. The vitamin A restriction increased expression of Bmp7 and some Bmp receptors in WAT. The interrelationship between gene expression levels indicated that expression levels of Nfia, Prdm16, and Pgc-1α were closely related to those of genes related to the function of brown/beige adipocytes in scWAT. Also, expression levels of Nfia, Prdm16, and Pgc-1α were highly correlated with those of Alk3 in scWAT. In summary, the present results suggest that the vitamin A restriction increases the number or activity of brown/beige adipocytes through regulatory expression of transcriptional regulators to induce brown/beige adipogenesis, especially in scWAT of fattening cattle, which may be governed by the Bmp pathway.

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 β

We previously showed that brown (pre)adipocytes express Trpv1, a capsaicin receptor, and that capsaicin stimulates differentiation of brown preadipocytes in the late stages of brown adipogenesis. The present study revealed that treatment with 100 μM capsaicin stimulates brown adipogenesis by inducing endoplasmic reticulum (ER) stress. Treatment with capsaicin (100 μM) during brown adipogenesis enhanced lipid accumulation and the expression of Ucp1, a gene selectively expressed in brown adipocytes. Capsaicin treatment also caused an increase in the cytosolic calcium concentration even when extracellular calcium was removed. I-RTX, a Trpv1 inhibitor, did not modulate the increase in cytosolic calcium concentration, lipid accumulation or Ucp1 expression. Previous studies revealed that the release of calcium from the ER induces ER stress, leading to the conversion of X-box binding protein 1 (Xbp1) pre-mRNA to spliced Xbp1 (sXbp1) as well as the up-regulation of Chop expression. Capsaicin treatment increased the expression of sXbp1 and Chop in brown preadipocytes and did not enhance lipid accumulation or Ucp1 expression in Xbp1 knockdown cells. The present results describe a novel mechanism of brown adipogenesis regulation via ER stress that is induced by a supra-pharmacological concentration of capsaicin.

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.

Hepcidin plays a central role in systemic iron metabolism. The bone morphogenetic protein (BMP) pathway regulates expression of hepcidin through transcriptional activation via BMP-responsive elements (REs) 1 and 2 on the promoter. Previous studies also revealed that the BMP pathway stimulates transcription of its target genes via GC-rich sequences on the promoter. A search for GC-rich sequences on the hepcidin promoter indicated 13 regions across the distal (A to F), middle (G to I), and proximal (J to M) areas; among them, mutations of the GC-rich element found in regions B to D exhibited decreased responsiveness to ALK3(QD) expression in the presence of BMP-RE1 mutations, indicating necessity of the elements for full expression of hepcidin by the BMP pathway.

The liver is the organ that responds to nutritional disturbances including magnesium deficiency. The present study evaluated cellular responses to magnesium deficiency using model cells of the liver, namely, HepG2 cells as hepatocytes, RAW264.7 cells as Kupffer cells and human umbilical vein endothelial cells (HUVECs) as vascular endothelial cells; we examined effects of culture with magnesium deficient medium on cell responses in individual types of cells as well as interactive responses among cells. Metabolomic analyses indicated that magnesium deficiency differentially affected the cellular content of metabolites among HepG2 cells, RAW264.7 cells and HUVECs. The cellular content of the metabolites in HepG2 cells and HUVECs was also affected by the conditioned medium from RAW264.7 cells cultured with the magnesium-deficient media. The changes in HUVECs partly resembled those of the livers of magnesium-deficient rats previously described. RNA-seq analyses indicated that magnesium deficiency modulated the expression levels of molecules related to the ubiquitin-proteasome pathway and oxidative stress/antioxidant response in HepG2 cells and RAW264.7 cells, respectively. Furthermore, when HUVECs were co-cultured with RAW264.7 cells, lipopolysaccharide-induced expression of interleukin (IL)-1β and IL-6 was enhanced by magnesium deficiency, depending on the presence of RAW264.7 cells. The present study reveals that magnesium deficiency affects cellular metabolism in HepG2 liver cells, RAW264.7 macrophages and HUVECs, and that the modulation of cellular responses to extracellular magnesium deficiency in HUVECs depends on the presence of RAW264.7 cells. The complex responses in individual cells and through cell interactions partly explain the regulatory reaction to magnesium deficiency in the liver.

Expression of hepcidin, a central regulator of systemic iron metabolism, is transcriptionally regulated by the bone morphogenetic protein (BMP) pathway. However, the factors other than the BMP pathway also participate in the regulation of hepcidin expression. In the present study, we show that serum treatment increased hepcidin expression and transcription without inducing the phosphorylation of Smad1/5/8 in primary hepatocytes, HepG2 cells or Hepa1-6 cells. Co-treatment with LDN-193189, an inhibitor of the BMP type I receptor, abrogated this hepcidin induction. Reporter assays using mutated reporters revealed the involvement of the BMP response element-1 (BMP-RE1) and signal transducers and activator of transcription (STAT)- and activator protein (AP)-1-binding sites in serum-induced hepcidin transcription in HepG2 cells. Serum treatment induced the expression of the AP-1 components c-fos and junB in primary hepatocytes and HepG2 cells. Forced expression of c-fos or junB enhanced the response of hepcidin transcription to serum treatment. By contrast, the expression of dominant negative (dn)-c-fos and dn-junB decreased hepcidin transcription. The present study reveals that serum contains factors stimulating hepcidin transcription. Basal BMP activity is essential for the serum-induced hepcidin transcription, although serum treatment does not stimulate the BMP pathway. The induction of c-fos and junB by serum treatment stimulates hepcidin transcription, through possibly cooperation with BMP-mediated signaling. Considering that AP-1 is induced by various stimuli, the present results suggest that hepcidin expression is regulated by more diverse factors than had been previously considered.