Sang-gun Roh

Diverse intracellular signalling systems used by growth hormone-releasing hormone in regulating voltage-gated Ca2+ or K channels in pituitary somatotropes.

Influx of Ca2+ via Ca2+ channels is the major step triggering exocytosis of pituitary somatotropes to release growth hormone (GH). Voltage‐gated Ca2+ and K+ channels, the primary determinants of the influx of Ca2+, are regulated by GH‐releasing hormone (GHRH) through G‐protein‐coupled intracellular signalling systems. Using whole‐cell patch‐clamp techniques, the changes of the Ca2+ and K+ currents in primary cultured ovine and human somatotropes were recorded. Growth hormone‐releasing hormone (10 nmol/L) increased both L‐ and T‐type voltage‐gated Ca2+ currents. Inhibition of the cAMP/protein kinase A (PKA) pathway by either Rp‐cAMP or H89 blocked this increase in both L‐ and T‐type Ca2+ currents. Growth hormone‐releasing hormone also decreased voltage‐gated transient (IA) and delayed rectified (IK) K+ currents. Protein kinase C (PKC) inhibitors, such as calphostin C, chelerythrine or downregulation of PKC, blocked the effect of GHRH on K+ currents, whereas an acute activation of PKC by phorbol 12,13‐dibutyrate (1 μmol/L) mimicked the effect of GHRH. Intracellular dialysis of a specific PKC inhibitor (PKC19–36) also prevented the reduction in K+ currents by GHRH. It is therefore concluded that GHRH increases voltage‐gated Ca2+ currents via cAMP/PKA, but decreases voltage‐gated K+ currents via the PKC signalling system. The GHRH‐induced alteration of Ca2+ and K+ currents augments the influx of Ca2+, leading to an increase in [Ca2+]i and the GH secretion.

Cloning, expression analysis, and regulatory mechanisms of bovine chemerin and chemerin receptor.

Recently, we reported that chemerin, a new adipokine, is highly expressed in the adipose tissue, up-regulated during adipocyte differentiation, and regulates adipogenesis via its own receptor in mice. The objectives of this study were to clone chemerin and its receptor from the adipose tissues of Japanese Black cattle and to investigate the expression of these genes in 16 different tissues. We compared the gene expression of chemerin and its receptor between adipocytes and stromal-vascular (S-V) cells (non-adipocytes) prepared from subcutaneous adipose tissue. In addition, we investigated the mRNA expression levels of chemerin and its receptor in bovine differentiated adipocytes. The DNA sequences of bovine chemerin and its receptor were determined, and they were found to be highly homologous to those of humans, mice, and pigs. The amino acid sequences predicted for the full-length cDNA of bovine chemerin and its receptor were also similar to those of humans, mice, and pigs, suggesting that these genes have similar functions. Bovine chemerin mRNA was highly expressed in the adipose and liver tissues, and the transcripts of chemerin receptor were widely expressed in several tissues including adipose, muscle, liver, and brain tissues. The expression of bovine chemerin mRNA was higher in adipocytes than in S-V cells prepared from adipose tissue. The transcripts of chemerin and its receptor were up-regulated during adipocyte differentiation. Treatment with tumor necrosis factor (TNF)-alpha (10 ng/mL) in bovine differentiated adipocytes increased the mRNA expression of chemerin and its receptor. These results indicate that chemerin, a new adipokine highly expressed in the adipocytes of bovine adipose tissue, is the TNF-alpha-up-regulated gene with a role in adipogenesis.

Oxytocin Receptor in the Hypothalamus Is Sufficient to Rescue Normal Thermoregulatory Function in Male Oxytocin Receptor Knockout Mice

Oxytocin (OXT) and OXT receptor (OXTR) have been implicated in the regulation of energy homeostasis, but the detailed mechanism is still unclear. We recently showed late-onset obesity and impaired cold-induced thermogenesis in male OXTR knockout (Oxtr(-/-)) mice. Here we demonstrate that the OXTR in the hypothalamus has important functions in thermoregulation. Male Oxtr(-/-) mice failed to maintain their body temperatures during exposure to a cold environment. Oxtr(-/-) mice also showed decreased neuronal activation in the thermoregulatory hypothalamic region during cold exposure. Normal cold-induced thermogenesis was recovered in Oxtr(-/-) mice by restoring OXTR to the hypothalamus with an adeno-associated virus-Oxtr vector. In addition, brown adipose tissue (BAT) in Oxtr(-/-) mice contained larger lipid droplets in both 10- and 20-week-old compared with BAT from age-matched Oxtr(+/+) control mice. In BAT, the expression level of β3-adrenergic receptor at normal temperature was lower in Oxtr(-/-) mice than that in control mice. In contrast, α2A-adrenergic receptor expression level was higher in BAT from Oxtr(-/-) mice in both normal and cold temperatures. Because β3- and α2A-adrenergic receptors are known to have opposite effects on the thermoregulation, the imbalance of adrenergic receptors is suspected to affect this dysfunction in the thermoregulation. Our study is the first to demonstrate that the central OXT/OXTR system plays important roles in the regulation of body temperature homeostasis.

Changes in circulating adiponectin and metabolic hormone concentrations during periparturient and lactation periods in Holstein dairy cows.

Although our previous report demonstrated that adiponectin and AdipoR1 gene expressions changed among different lactation stages in the bovine mammary gland, its in vivo kinetics remain unclear in ruminant animals. In this study, we investigated the changes in circulating concentrations of adiponectin, as well as other metabolic hormones and metabolites, (i) during the periparturient period and (ii) among different lactation stages, in Holstein dairy cows. In experiment 1, serum adiponectin concentrations increased after parturition. Serum insulin concentrations were lower in the postpartum than prepartum period, whereas serum growth hormone (GH) concentrations increased in the postpartum period. Serum nonesterified fatty acids (NEFA) levels were increased during the postpartum period and were dependent on the parity. In experiment 2, there was no significant difference in plasma adiponectin concentrations among lactational stages. Plasma insulin concentrations tended to be lower in early lactation while plasma GH levels tended to be higher. Plasma NEFA concentrations were significantly lower in mid- and late-lactation stages than non-lactation stages. These findings indicate that elevation of serum adiponectin might be involved in energy metabolism just around parturition, and might exert its action through regulation of receptor expression levels in target tissues in each lactational stage in Holstein dairy cows.

Trigonelline attenuates the adipocyte differentiation and lipid accumulation in 3T3-L1 cells

Trigonelline is a natural alkaloid mainly found in Trigonella Foenum Graecum (fenugreek) Fabaceae and other edible plants with a variety of medicinal applications. Therefore, we investigated the molecular mechanism of trigonelline (TG) on the inhibition of adipocyte differentiation and lipid accumulation in 3T3-L1 cells. Trigonelline suppressed lipid droplet accumulation in a concentration (75 and 100 μM) dependent manner. Treatment of adipocyte with of TG down regulates the peroxisome proliferator-activated receptor (PPARγ) and CCAAT element binding protein (C/EBP-α) mRNA expression, which leads to further down regulation of other gene such as adiponectin, adipogenin, leptin, resistin and adipocyte fatty acid binding protein (aP2) as compared with respective control cells on 5th and 10th day of differentiation. Further, addition of triognelline along with troglitazone to the adipocyte attenuated the troglitazone effects on PPARγ mediated differentiation and lipid accumulation in 3T3-L1 cells. Trigonelline might compete against troglitazone for its binding to the PPARγ. In addition, adipocyte treated with trigonelline and isoproterenol separately. Isoproterenol, a lipolytic agent which inhibits the fatty acid synthase and GLUT-4 transporter expression via cAMP mediated pathway, we found that similar magnitude response of fatty acid synthase and GLUT-4 transporter expression in trigonelline treated adipocyte. These results suggest that the trigonelline inhibits the adipogenesis by its influences on the expression PPARγ, which leads to subsequent down regulation of PPAR-γ mediated pathway during adipogenesis. Our findings provide key approach to the mechanism underlying the anti-adipogenic activity of trigonelline.

Chemerin analog regulates energy metabolism in sheep

Accumulating data suggest a relationship between chemerin and energy metabolism. Our group previously described gene cloning, expression analysis and the regulatory mechanism of chemerin and its own receptor in mice and cattle. The objective of the present study was to investigate the physiological effect of chemerin on endocrine changes and energy metabolism in sheep using a biologically stable chemerin analog. The chemerin analog was intravenously administrated (100 or 500 µg/head) to sheep, and plasma insulin and metabolites (glucose, nonesterified fatty acids (NEFA), triglyceride, total cholesterol and high-density lipoprotein (HDL) cholesterol) were analyzed. The chemerin analog dramatically increased the insulin levels, and glucose levels were decreased. NEFA levels were slightly decreased at 20 min but then increased gradually from 60 to 180 min after analog administration. In addition, injection of the chemerin analog immediately increased triglyceride and total cholesterol but not HDL levels. These results suggested that chemerin analog regulated insulin secretion related to glucose metabolism and the release of triglycerides in sheep in vivo. This study provides new information about endocrine and metabolic changes in response to chemerin in sheep.

Effects of Na-butyrate supplementation in milk formula on plasma concentrations of GH and insulin, and on rumen papilla development in calves

Although the growth-promoting action of sodium-butyrate (Na-butyrate) used as a feed additive has been observed in calves and pigs, the precise mechanisms involved remain to be clarified. In this study, pre-weaning calves were given milk formula (MF) supplemented with butyrate for 6 weeks to investigate its effects on postprandial changes in the plasma concentrations of metabolic hormones, and, simultaneously, on growth performance, the weight of the digestive organs and rumen papilla development. Ingestion of MF increased (P<0.05) the plasma concentrations of GH and insulin as well as the glucose level, but decreased the non-esterified fatty acid concentration. Na-butyrate supplementation in MF or in lactose solution (with the same quantity of lactose contained in the MF, 5%) suppressed the increase in plasma insulin and GH concentrations, and the plasma IGF1 level was not changed. The length of the rumen papilla and the weight of the perirenal fat tended to increase in the calves fed with Na-butyrate-supplemented MF, but the weight of the liver, spleen, and stomach were not changed. In addition, there was no difference in the expression of mRNA for sodium-dependent glucose transporter-1 in the small intestinal epithelial tissues. We conclude that the accelerated growth performance related to the intake of Na-butyrate used as a feed additive reported previously in several species is partly due to improved insulin sensitivity and a better digestive functional development. These data could be applicable to animal and human nutrition.

Gene expression and hormonal regulation of adiponectin and its receptors in bovine mammary gland and mammary epithelial cells

Although the functions of adiponectin, a differentiated adipocyte-derived hormone, in regulating glucose and fatty acid metabolism are regulated by two subtypes of adiponectin receptors (AdipoRs; AdipoR1 and AdipoR2), those in ruminants remain unclear. Therefore we examined the messenger RNA (mRNA) expression levels of adiponectin and its receptors in various bovine tissues and mammary glands among different lactation stages, and the effects of lactogenic hormones (insulin, dexamethasone and prolactin) and growth hormone (GH) on mRNA expression of the AdipoRs in cultured bovine mammary epithelial cells (BMEC). AdipoRs mRNAs were widely expressed in various bovine tissues, but adiponectin mRNA expression was significantly higher in adipose tissue than in other tissues. In the mammary gland, although adiponectin mRNA expression was significantly decreased at lactation, AdipoR1 mRNA expression was significantly higher at peak lactation than at the dry-off stage. In BMEC, lactogenic hormones and GH upregulated AdipoR2 mRNA expression but did not change that of AdipoR1. In conclusion, adiponectin and its receptor mRNA were expressed in various bovine tissues and the adiponectin mRNA level was decreased during lactation. These results suggest that adiponectin and its receptors ware changed in mammary glands by lactation and that AdipoRs mRNA expression was regulated by different pathways in BMEC.

The Regulation of Chemerin and CMKLR1 Genes Expression by TNF-α, Adiponectin, and Chemerin Analog in Bovine Differentiated Adipocytes

Adipokines, adipocyte-derived protein, have important roles in various kinds of physiology including energy homeostasis. Chemerin, one of adipocyte-derived adipokines, is highly expressed in differentiated adipocytes and is known to induce macrophage chemotaxis and glucose intolerance. The objective of the present study was to investigate the changes of chemerin and the chemokine-like-receptor 1 (CMKLR1) gene expression levels during differentiation of the bovine adipocyte and in differentiated adipocytes treated with tumor necrosis factor-α (TNF-α), adiponectin, leptin, and chemerin (peptide analog). The expression levels of the chemerin gene increased at d 6 and 12 of the differentiation period accompanied by increased cytoplasm lipid droplets. From d 6 onward, peroxisome proliferator-activated receptor-γ2 (PPAR-γ2) gene expression levels were significantly higher than that of d 0 and 3. In contrast, CMKLR1 expression levels decreased at the end of the differentiation period. In fully differentiated adipocytes (i.e. at d 12), the treatment of TNF-α and adiponectin upregulated both chemerin and CMKLR1 gene expression levels, although leptin did not show such effects. Moreover, chemerin analog treatment was shown to upregulate chemerin gene expression levels regardless of doses. These results suggest that the expression of chemerin in bovine adipocyte might be regulated by chemerin itself and other adipokines, which indicates its possible role in modulating the adipokine secretions in adipose tissues.

The Regulation of Oxytocin Receptor Gene Expression during Adipogenesis

Although it has been reported that oxytocin stimulates lipolysis in adipocytes, changes in the expression of oxytocin receptor (OTR) mRNA in adipogenesis are still unknown. The present study aimed to investigate the expression of OTR mRNA during adipocyte differentiation and fat accumulation in adipocytes. OTR mRNA was highly expressed in adipocytes prepared from mouse adipose tissues compared to stromal‐vascular cells. OTR mRNA expression was increased during the adipocyte differentiation of 3T3‐L1 cells. OTR expression levels were higher in subcutaneous and epididymal adipose tissues of 14‐week‐old male mice compared to 7‐week‐old male mice. Levels of OTR mRNA expression were higher in adipose tissues at four different sites of mice fed a high‐fat diet than in those of mice fed a normal diet. The OTR expression level was also increased by refeeding for 4 h after fasting for 16 h. Oxytocin significantly induced lipolysis in 3T3‐L1 adipocytes. In conclusion, a new regulatory mechanism is demonstrated for oxytocin to control the differentiation and fat accumulation in adipocytes via activation of OTR as a part of the hypothalamic‐pituitary‐adipose axis.