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京都大学 / 新制・課程博士 / 博士(医学) / 甲第24091号 / 医博第4867号 / 新制||医||1059(附属図書館) / 京都大学大学院医学研究科医学専攻 / (主査)教授 林 康紀, 教授 髙橋 良輔, 教授 渡邉 大 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

The objectives of this research were to characterize insulin-like growth factor-I (IGF-I) gene expression in cattle, to determine how IGF-I gene expression is affected by nutritional intake and growth hormone (GH) in cattle, and to identify the regulatory DNA region that mediates GH stimulation of IGF-I gene expression. It was found that transcription of the IGF-I gene in cattle was initiated from both exon 1 and exon 2, generating class 1 and class 2 IGF-I mRNA, respectively. Both classes of IGF-I mRNA appeared to be ubiquitously expressed, with the highest level in liver and with class 1 being more abundant than class 2 in all tissues examined. Class 1 IGF-I mRNA may be also translated more efficiently than class 2 IGF-I mRNA. Liver expression of IGF-I mRNA was decreased (P < 0.01) by food deprivation in cattle, and this decrease was due to an equivalent decrease in both classes of IGF-I mRNA. Liver expression of IGF-I mRNA was increased (P < 0.01) by GH, and this increase resulted mainly from increased expression of class 2 IGF-I mRNA. Using cotransfection analyses, a ~700 bp chromosomal region ~75 kb 5' from the first exon of the human IGF-I gene was found to enhance reporter gene expression in the presence of constitutively active signal transducer and activator of transcription 5 (STAT5) proteins, transcription factors that are known to be essential for GH-increased IGF-I gene expression. This 700 bp DNA region contains two STAT5-binding sites that appear to be conserved in mammals including cattle. Electrophoretic mobility shift assays and cotransfection analyses confirmed their ability to bind to STAT5 proteins and to mediate STAT5 activation of gene expression, respectively. Chromatin immunoprecipitation assays indicated that overexpressed constitutively active STAT5b protein bound to the chromosomal region containing these two STAT5-binding sites in Hep G2 cells, and this binding was associated with increased expression of IGF-I mRNA. These two STAT5-binding sites were also able to mediate GH-induced STAT5 activation of gene expression in reconstituted GH-responsive cells. These results together suggest that the distal DNA region that contains two STAT5-binding sites may mediate GH-induced STAT5 activation of IGF-I gene transcription in vivo. / Ph. D.

The overall objective of this dissertation research was to understand the mechanisms by which serum insulin-like growth factor-I (IGF-I) is regulated by nutritional intake in cattle. Two studies were conducted to achieve this objective. In the first study, effects of feeding levels on basal and growth hormone (GH)-stimulated serum concentrations of IGF-I, IGF binding protein-3 (IGFBP-3) and acid-labile subunit (ALS), and their mRNA expression in the liver were determined in beef cows. It was found that increased nutritional intake did not alter basal concentrations of serum IGF-I, IGFBP-3 or ALS, or their mRNA expression in the liver. However, under increased nutritional intake, GH administration stimulated a greater increase in serum IGF-I concentration, and this greater increase was not due to reduced degradation of IGF-I in serum. Increased nutritional intake did not enhance GH-stimulated IGF-I mRNA expression in the liver, but it increased the amount of IGF-I mRNA associated with polysomes, suggesting that liver translation of IGF-I mRNA is enhanced under increased nutritional intake. Under increased nutritional intake, GH also stimulated greater increases in serum IGFBP-3 and ALS concentrations, but these greater increases were not due to greater expression or translation of their mRNAs in the liver. Taken together, these results suggest that translation of GH-stimulated IGF-I mRNA in the liver is enhanced under increased nutritional intake and this enhancement may be partially responsible for the greater GH-stimulated increase in serum IGF-I concentration. These results also suggest that the greater GH-stimulated increases in serum IGFBP-3 and ALS may be secondary to the greater increase in serum IGF-I because increased IGF-I may increase the formation of IGF-I/IGFBP-3/ALS complexes, thereby increasing the retention of IGFBP-3 and ALS in the blood. In the second study, the effects of food deprivation on serum IGF-I concentration in steers and the underlying mechanism were determined. It was found that food deprivation decreased serum IGF-I concentration and that this decrease was not due to increased IGF-I degradation in serum. Food deprivation decreased liver IGF-I mRNA expression, and this decrease was associated with decreased expression of GH receptor (GHR) mRNA and protein in the liver. Food deprivation was also associated with increased mRNA expression of two inhibitors of the GHR signaling pathway, suppressor of cytokine signaling-2 (SOCS2) and cytokine-inducible SH2 protein (CIS). These results suggest that decreased IGF-I gene expression in the liver may be at least partially responsible for the decrease in circulating IGF-I concentration during food deprivation, and that the former decrease may be due to increased expression of SOCS2 and CIS, and decreased expression of GHR in the liver. Overall, this dissertation research indicates that multiple mechanisms are involved in nutritional regulation of circulating IGF-I concentration in cattle. / Ph. D.

The overall objective of this research was to further understand the mechanism by which growth hormone (GH) stimulates milk production in cattle. Three studies were conducted toward this objective. In the first study, the effects of GH and insulin-like growth factor-I (IGF-I), a major mediator of GH action in vivo, on cell proliferation, nutrient transport, and milk protein gene expression in bovine mammary epithelial cell line MAC-T cells were determined. GH increased (P < 0.01) expression of four major milk protein genes in MAC-T cells transfected with GHR expression plasmid. Cotransfection analyses indicated that GH also stimulated (P < 0.01) luciferase reporter gene expression from the promoters of the four milk protein genes in MAC-T cells. These findings together with the fact that GHR mRNA and protein are expressed in the epithelial cells of the bovine mammary gland suggest that GH may directly stimulate milk protein gene expression in the mammary gland. This study also showed that IGF-I increased the proliferation (P < 0.01) and amino acid transport (P < 0.05) in MAC-T cells. Because GH is known to stimulate IGF-I production in animals, IGF-I-mediated mammary epithelial cell proliferation and amino acid uptake may be additional mechanisms by which GH increases milk production in cattle. In the second study, the role of connective tissue growth factor (CTGF) on IGF-I-stimulated proliferation of MAC-T cells was investigated. A microarray analysis revealed that IGF-I decreased CTGF mRNA expression in MAC-T cells (P < 0.01). This effect of IGF-I was further found to be mediated through the PI-3 kinase/Akt signaling pathway from the IGF-I receptor (IGF-IR). CTGF alone stimulated MAC-T cell proliferation (P < 0.01). However, together with IGF-I, CTGF attenuated the proliferating effect of IGF-I on MAC-T cells, and this attenuation was reversed by additional IGF-I. Therefore, IGF-I inhibition of CTGF expression may benefit IGF-I stimulation of MAC-T cell proliferation. CTGF had no effect on IGF-I-induced phosphorylation of IGF-IR or total IGF-IR expression in MAC-T cells, suggesting that CTGF may attenuate IGF-I stimulation of MAC-T cell proliferation through a postreceptor inhibition of the IGF-IR signaling pathway. In the third study, whether a milk yield-associated T/A polymorphism in exon 8 of the bovine GHR gene affected GHR signaling was determined. It was found that the two corresponding GHR variants did not differ in mediating GH induction of gene expression, suggesting that the two GHR variants are not functionally different and hence are unlikely to mediate different effects of GH on milk production. In summary, the results of this dissertation research suggest that GH may directly stimulate milk protein gene expression and indirectly stimulate mammary epithelial cell proliferation and amino acid uptake through IGF-I, thereby stimulating milk production in cattle. The results also suggest that IGF-I stimulation of mammary epithelia cell proliferation may involve an inhibition of CTGF expression in the cells. / Ph. D.

Diets rich in hydrolyzable carbohydrates induce a hyperglycemic/insulinemic response and may increase the incidence of metabolic disorders associated with some types of laminitis, exertional rhabdomyolysis and osteochondrosis in horses. This study applied the minimal model of glucose and insulin dynamics to determine the effect of diet on metabolites and hormones that regulate glucose metabolism in young horses. Twelve Thoroughbred foals were raised on pasture and supplemented twice daily with a feed high in either sugar and starch (SS) or fat and fiber (FF). As weanlings (age 199 ± 19 d, weight 274 ± 18 kg), the subjects underwent a modified frequent sampling intravenous glucose tolerance test during which they remained in stalls and had access to grass hay and water ad libitum. Samples were colleted at -60, -45, 1, 2, 3, 4, 5, 6, 7, 8, 10, 12, 14, 16, 19, 22, 23, 24, 25, 27, 30, 35, 40, 50, 60 , 70 , 80, 90, 100, 120, 150, 180, 210, 240, 270, 300, 330 and 360 min with a glucose bolus of 300 mg/kg BW at 0 min and an insulin bolus of 1.5 mU/kg BW at 20 min. Plasma was analyzed for glucose, insulin, growth hormone (GH) and insulin-like growth factor-I (IGF-I) concentrations. Insulin sensitivity, glucose effectiveness, acute insulin response to glucose and disposition index were derived using Minmod Millennium and WinSAAM software. Diet groups were compared using the non-parametric Kruskal-Wallis test or the sign test. Time interactions were compared using a mixed model with repeated effects. Rank-ordered linear regression was used for correlations. Basal glucose did not differ between groups (P = 0.75). There was nearly a trend towards higher basal (P = 0.11), and median insulin was higher in the sugar and starch foals at all 36 sample points (P = 0.030). The basal glucose:basal insulin ratio for the sugar and starch supplemented foals was lower than for fat and fiber foals (P = 0.025). Insulin sensitivity (SI) was lower in foals fed sugar and starch than foals fed fat and fiber (P = 0.007). Acute insulin response to glucose was directly correlated to weight (r = 0.78; P = 0.003) and inversely correlated with SI (r = -0.55; P = 0.067). The glucose:insulin ratio was directly correlated to SI (r = 0.92; P < 0.001). Growth hormone concentrations were increased from basal from 19 to 180 min after the glucose dose (P < 0.05). Basal IGF-I was higher (P = 0.006) in the SS group compared to the FF group. Concentrations of total IGF-I increased with time (P = 0.002) in the SS group. The change in IGF-I concentration from baseline to the end of the study was positively correlated (r = 0.72; P = 0.008) to the area under the insulin curve from 0 to 80 min. Basal IGF-I was inversely correlated to SI (r = 0.71; P = 0.015). These results show that the metabolic response to a diet high in hydrolyzable carbohydrates differs from the response to a fat and fiber meal resembling forage. Weanlings adapted to meals high in glucose equivalents have higher insulin and IGF-I secretion as compared to foals adapted to a fat and fiber feed, possibly contributing to lower insulin sensitivity observed in these foals. Such deviations may contribute to metabolic dysfunction and osteochondrosis in horses fed grain diets. / Master of Science