Asako Takenaka

Paraquat-induced Oxidative Stress Represses Phosphatidylinositol 3-Kinase Activities Leading to Impaired Glucose Uptake in 3T3-L1 Adipocytes

Accumulated evidence indicates that oxidative stress causes and/or promotes insulin resistance; however, the mechanism by which this occurs is not fully understood. This study was undertaken to elucidate the molecular mechanism by which oxidative stress induced by paraquat impairs insulin-dependent glucose uptake in 3T3-L1 adipocytes. We confirmed that paraquat-induced oxidative stress decreased glucose transporter 4 (GLUT4) translocation to the cell surface, resulting in repression of insulin-dependent 2-deoxyglucose uptake. Under these conditions, oxidative stress did not affect insulin-dependent tyrosine phosphorylation of insulin receptor, insulin receptor substrate (IRS)-1 and -2, or binding of the phosphatidylinositol 3′-OH kinase (PI 3-kinase) p85 regulatory subunit or p110α catalytic subunit to each IRS. In contrast, we found that oxidative stress induced by paraquat inhibited activities of PI 3-kinase bound to IRSs and also inhibited phosphorylation of Akt, the downstream serine/threonine kinase that has been shown to play an essential role in insulin-dependent translocation of GLUT4 to the plasma membrane. Overexpression of active form Akt (myr-Akt) restored inhibition of insulin-dependent glucose uptake by paraquat, indicating that paraquat-induced oxidative stress inhibits insulin signals upstream of Akt. Paraquat treatment with and without insulin treatment decreased the activity of class Ia PI 3-kinases p110α and p110β that are mainly expressed in 3T3-L1 adipocytes. However, paraquat treatment did not repress the activity of the PI 3-kinase p110α mutated at Cys90 in the p85 binding region. These results indicate that the PI 3-kinase p110 is a possible primary target of paraquat-induced oxidative stress to reduce the PI 3-kinase activity and impaired glucose uptake in 3T3-L1 adipocytes.

Effect of protein nutrition on the mRNA content of insulin-like growth factor-binding protein-1 in liver and kidney of rats

Effect of quantity and nutritional quality of dietary proteins on the content of mRNA of insulin-like growth factor-binding protein-1 (IGFBP-1) was studied in rat liver and kidney. IGFBP-1 mRNA content per unit RNA increased in liver and kidney of rats fed on a protein-free diet and in those of fasted rats compared with that in the rats fed on a casein diet. When rats were given a gluten diet for 7 d, IGFBP-1 mRNA content in liver did not change significantly but that in kidney increased considerably compared with that in those organs of the rats fed on the casein diet. Because IGFBP-1 mRNA has been demonstrated both in liver parenchymal and non-parenchymal cells (Takenaka et al. 1991), the effect of the protein-free diet on these two types of cells has been studied. An increase in IGFBP-1 mRNA content under protein deprivation was observed in both liver parenchymal and non-parenchymal cells, suggesting that these two types of cells are regulated in a similar mode as far as IGFBP-1 mRNA content is concerned. The physiological and nutritional significance of the previously stated results on protein anabolism are discussed when considered together with our previous observations on the plasma concentrations of IGF-1 (Takahashi et al. 1990) and IGFBP (Umezawa et al. 1991) and insulin-like growth factor-1 mRNA content in liver (Miura et al. 1991).

Effects of Growth Hormone and Insulin-Like Growth Factor 1 (IGF-1) Treatments on the Nitrogen Metabolism and Hepatic IGF-1-Messenger RNA Expression in Postoperative Parenterally Fed Rats

BACKGROUND Few studies have made direct comparisons of the metabolic effects of growth hormone (GH) and insulin-like growth factor 1 (IGF-1). We have assessed the dose-dependent effects of GH and IGF-1 treatments on nitrogen metabolism, intestinal structure, and hepatic IGF-1-messenger RNA (mRNA) expression in postoperative parenterally fed rats. METHODS Rats were maintained on total parenteral nutrition (TPN) for 3 days after gastrectomy. GH (0.4 or 0.8 IU/kg/d) or IGF-1 (1,2, or 4 mg/kg/d) was infused throughout the experimental period. Anabolic effects of GH and IGF-1 were assessed by body weight change, nitrogen excretion, and whole-body protein turnover. Organ weights, intestinal structure, plasma IGF-1 levels and hepatic IGF-1-mRNA contents were also determined. RESULTS Both GH and IGF-1 attenuated body weight loss and nitrogen excretion and increased whole-body protein synthesis and spleen weight. These observations suggest that the anabolic effects of 1 mg/kg/d of IGF-1 were equivalent to those of 0.66 IU/kg/d of GH. IGF-1, but not GH, reduced atrophy of the intestinal mucosa. GH treatment increased hepatic IGF-1-mRNA and the plasma IGF-1 level, whereas IGF-1 treatment increased the plasma IGF-1 level with no change in the hepatic IGF-1-mRNA content. CONCLUSIONS Administration of GH or IGF-1 attenuates catabolism after surgery. The anabolic effects of 1 mg/kg/d of IGF-1 are equivalent to those of 0.66 IU/kg/d of GH. IGF-1 reduces intestinal mucosal atrophy. GH increases hepatic IGF-1-mRNA and the plasma IGF-1 level.

Rapid increase in fibroblast growth factor 21 in protein malnutrition and its impact on growth and lipid metabolism

Protein malnutrition promotes hepatic steatosis, decreases insulin-like growth factor (IGF)-I production and retards growth. To identify new molecules involved in such changes, we conducted DNA microarray analysis on liver samples from rats fed an isoenergetic low-protein diet for 8 h. We identified the fibroblast growth factor 21 gene (Fgf21) as one of the most strongly up-regulated genes under conditions of acute protein malnutrition (P<0·05, false-discovery rate<0·001). In addition, amino acid deprivation increased Fgf21 mRNA levels in rat liver-derived RL-34 cells (P<0·01). These results suggested that amino acid limitation directly increases Fgf21 expression. FGF21 is a polypeptide hormone that regulates glucose and lipid metabolism. FGF21 also promotes a growth hormone-resistance state and suppresses IGF-I in transgenic mice. Therefore, to determine further whether Fgf21 up-regulation causes hepatic steatosis and growth retardation after IGF-I decrease in protein malnutrition, we fed an isoenergetic low-protein diet to Fgf21-knockout (KO) mice. Fgf21-KO did not rescue growth retardation and reduced plasma IGF-I concentration in these mice. Fgf21-KO mice showed greater epididymal white adipose tissue weight and increased hepatic TAG and cholesterol levels under protein malnutrition conditions (P<0·05). Overall, the results showed that protein deprivation directly increased Fgf21 expression. However, growth retardation and decreased IGF-I were not mediated by increased FGF21 expression in protein malnutrition. Furthermore, FGF21 up-regulation rather appears to have a protective effect against obesity and hepatic steatosis in protein-malnourished animals.

Insulin-Like Growth Factor 1 Has Beneficial Effects, Whereas Growth Hormone Has Limited Effects on Postoperative Protein Metabolism, Gut Integrity, and Splenic Weight in Rats With Chronic Mild Liver Injury

BACKGROUND Both growth hormone (GH) and insulin-like growth factor 1 (IGF-1) improve protein metabolism after surgical insult in subjects without liver disease. However, these effects in chronic liver injury, in which the GH-IGF-1 axis is impaired, have not been investigated. We examined the anabolic effects of GH and IGF-1 after gastrectomy in rats with chronic mild liver injury. METHODS Rats with chronic mild liver injury induced by thioacetamide were used. After gastrectomy, the rats were randomized into vehicle control, GH, and IGF-1 groups. In the latter two groups, 0.8 IU/kg/d of GH or 4 mg/kg/d of IGF-1 was infused for 72 hours. Anabolic effects were assessed by body weight change, 3-methylhistidine (3-MH) excretion, nitrogen excretion, and whole-body protein turnover. Organ weights, plasma levels of glucose, insulin, and IGF-1, tissue IGF-1 levels, hepatic messenger RNA (mRNA) content, and intestinal structure were also determined. RESULTS Both GH and IGF-1 decreased nitrogen excretion. IGF-1, but not GH, increased postoperative body weight, whole-body protein turnover, and splenic weight. IGF-1 reduced atrophy of the intestinal mucosa. GH treatment increased hepatic IGF-1-mRNA and the plasma IGF-1 level, whereas IGF-1 treatment increased the plasma IGF-1 level with no change in the hepatic IGF-1-mRNA content. There were no significant differences in plasma glucose or insulin levels among the three groups. Neither GH nor IGF-1 affected the gastrocnemius muscle IGF-1 level. CONCLUSIONS IGF-1 has beneficial effects, whereas GH has only limited effects on post-operative protein metabolism, gut integrity, and splenic weight in chronic mild liver injury.

Effect of protein restriction on the messenger RNA contents of bone-matrix proteins, insulin-like growth factors and insulin-like growth factor binding proteins in femur of ovariectomized rats

It has been reported that loss of ovarian oestrogen after menopause or by ovariectomy causes osteoporosis. In order to elucidate the effect of dietary protein restriction on bone metabolism after ovariectomy, we fed ovariectomized young female rats on a casein-based diet (50 g/kg diet (protein restriction) or 200 g/kg diet (control)) for 3 weeks and measured mRNA contents of bone-matrix proteins such as osteocalcin, osteopontin and alpha 1 type I collagen, insulin-like growth factors (IGF) and IGF-binding proteins (IGFBP) in femur. Ovariectomy decreased the weight of fat-free dry bone and increased urinary excretion of pyridinium cross-links significantly, although dietary protein restriction did not affect them. Neither ovariectomy nor protein restriction affected the content of mRNA of osteopontin and osteocalcin; however, ovariectomy increased and protein restriction extensively decreased the alpha 1 type I collagen mRNA content in bone tissues. Ovariectomy increased IGF-I mRNA only in the rats fed on the control diet. Conversely, protein restriction increased and ovariectomy decreased the IGF-II mRNA content in femur. Furthermore, the contents of IGFBP-2, IGFBP-4 and IGFBP-5 mRNA increased, but the content of IGFBP-3 mRNA decreased in femur of the rats fed on the protein-restricted diet. In particular, ovariectomy decreased the IGFBP-2 mRNA content in the protein-restricted rats and the IGFBP-6 mRNA content in the rats fed on the control diet. These results clearly show that the mRNA for some of the proteins which have been shown to be involved in bone formation are regulated by both quantity of dietary proteins and ovarian hormones.

Effect of Various Radical Generators on Insulin-Dependent Regulation of Hepatic Gene Expression

Oxidative stress is recognized to be associated with the development of insulin resistance. Although free radicals are generated in various ways in vivo, very few radical generators have been used to investigate the effect of oxidative stress on cellular insulin signaling. In order to compare the effect of radical generators with different sites and durations of radical formation on liver insulin action, primary cultured rat hepatocytes were incubated with radical generators and insulin-dependent regulation of gene expression was examined. The hydrophobic 2,2′-azobis(2,4-dimethylvaleronitrile) (AMVN) radical and H2O2 increased plasma membrane damage, and the hydrophilic 2-2′-azobis(2-amidinopropane) dihydrochloride (AAPH) radical and buthionine sulfoxyimine (BSO) increased oxidation of intracellular substances. Paraquat (PQ) and H2O2 inhibited insulin-dependent repression of insulin-like growth factor-binding protein-1 (IGFBP-1) and phosphoenolpyruvate carboxykinase (PEPCK) gene expression. These results indicate that PQ and H2O2 impair insulin action effectively and are suitable for examining crosstalk between oxidative stress and insulin signaling in liver-cell culture systems.

Insulin-like growth factor-I messenger RNA content in the oviduct of Japanese quail (Coturnix coturnix japonica): changes during growth and development or after estrogen administration

Complementary DNA (cDNA) of insulin-like growth factor-I (IGF-I) of Japanese quail was cloned. The nucleotide sequence analysis of the cDNA showed that only seven bases differed from those of chicken IGF-I cDNA in the 440 bases of the cloned region. This difference in nucleotide sequence did not cause changes in the amino acid sequence. Using this cloned cDNA, the changes in IGF-I mRNA content in the tissues of female quail during growth and development were investigated. In the oviduct, IGF-I mRNA was high about 5 weeks after hatching, concomitant with the rapid increase in total DNA content in this tissue (and the increases in total RNA content and RNA/DNA ratio). It decreased after 6 weeks, in accordance with the appearance of ovalbumin mRNA. When immature quails (6-day-old) were injected with diethylstilbestrol (DES), induction of IGF-I mRNA was observed after 24 hr. A few days later, there was a strong induction of ovalbumin mRNA. These two inductions were dependent on the dose of DES. The sequential inductions of these two mRNAs were also noted when DES was re-administered to the immature quail to which it had been first administered and from which then withdrawn. The present results showed that IGF-I gene is expressed extensively during development of the oviduct, probably in accordance with the activity of DNA replication, because the highest IGF-I mRNA content was observed when the total DNA content of the tissues increased extensively. The results suggest that IGF-I in the oviduct of Japanese quail works in an autocrinal or paracrinal mode during the development of this tissue.

Soy Protein Suppresses Gene Expression of Acetyl-CoA Carboxylase Alpha from Promoter PI in Rat Liver

Dietary soy protein isolate (SPI) reduces hepatic lipogenesis by suppressing gene expression of lipogenic enzymes, including acetyl-CoA carboxylase (ACC). In order to elucidate the mechanism of this regulation, the effect of dietary SPI on promoter (PI and PII) specific gene expression of ACC alpha was investigated. Rats were fed experimental diets containing SPI or casein as a nitrogen source. SPI feeding decreased the hepatic contents of total ACC mRNA as well as triglyceride (TG) content, but dietary SPI affected the amount of sterol-regulatory element binding protein (SREBP)-1 mRNA and protein very little. The amount of ACC mRNA transcribed from PII promoter containing SRE was not significantly affected by dietary protein, while a significant decrease in PI-generated ACC mRNA content was observed in rats fed the SPI diet. These data suggest that SPI feeding decreased the hepatic contents of ACC alpha mRNA mainly by regulating PI promoter via a nuclear factor(s) other than SREBP-1.

Effect of protein restriction on messenger RNA of insulin-like growth factor-I and insulin-like growth factor-binding proteins in liver of ovariectomized rats.

Effects of dietary protein restriction and ovariectomy on plasma concentrations and hepatic messenger RNA (mRNA) of insulin-like growth factor-I (IGF-I) and insulin-like growth factor binding proteins (IGFBP) were investigated in young female rats. Ovariectomy increased plasma IGF-I concentration in rats fed on either a 50 g casein/kg diet (protein-restricted diet) or a 200 g casein/kg diet (control diet), but it increased IGF-I mRNA in liver only in the rats fed on the control diet. On the other hand, by Western ligand blot analysis, we observed that ovariectomy increased plasma IGFBP-3 concentration, and decreased plasma IGFBP-4 concentration. Ovariectomy did not affect IGFBP-1 and IGFBP-2 mRNA in liver, but dietary protein restriction significantly increased them, which may correspond to their plasma concentrations. The present results show that ovarian hormones and dietary protein content affect the plasma concentrations of IGF-I, IGFBP-3 and IGFBP-4 and hepatic mRNA of IGF-I, IGFBP-1, IGFBP-2, IGFBP-3 and IGFBP-4 in different manners.