Augmentation of angiotensinogen expression in the proximal tubule by intracellular angiotensin II via AT1a/MAPK/NF-кB signaling pathways.

Long-term angiotensin II (ANG II) infusion significantly increases ANG II levels in the kidney through two major mechanisms: AT1 receptor-mediated augmentation of angiotensinogen (AGT) expression and uptake of circulating ANG II by the proximal tubules. However, it is not known whether intracellular ANG II stimulates AGT expression in the proximal tubule. In the present study, we overexpressed an intracellular cyan fluorescent ANG II fusion protein (Ad-sglt2-ECFP/ANG II) selectively in the proximal tubule of rats and mice using the sodium and glucose cotransporter 2 (sglt2) promoter. AGT mRNA and protein expression in the renal cortex and 24-h urinary AGT excretion were determined 4 wk following overexpression of ECFP/ANG II in the proximal tubule. Systolic blood pressure was significantly increased with a small antinatriuretic effect in rats and mice with proximal tubule-selective expression of ECFP/ANG II (P < 0.01). AGT mRNA and protein expression in the cortex were increased by >1.5-fold and 61 ± 16% (P < 0.05), whereas urinary AGT excretion was increased from 48.7 ± 5.7 (n = 13) to 102 ± 13.5 (n = 13) ng/24 h (P < 0.05). However, plasma AGT, renin activity, and ANG II levels remained unaltered by ECFP/ANG II. The increased AGT mRNA and protein expressions in the cortex by ECFP/ANG II were blocked in AT1a-knockout (KO) mice. Studies in cultured mouse proximal tubule cells demonstrated involvement of AT1a receptor/MAP kinases/NF-кB signaling pathways. These results indicate that intracellular ANG II stimulates AGT expression in the proximal tubules, leading to increased AGT formation and secretion into the tubular fluid, which contributes to ANG II-dependent hypertension.

[Evaluation of the number of clones and the degree of nucleotide diversity of the HCV genome by FSSA (fluorescence single strand conformation polymorphism and sequence analysis) in patients with chronic active hepatitis C receiving interferon therapy].

We evaluated the number of clones and the degree of nucleotide diversity in the HVR of the HCV genome in patients with chronic active hepatitis C who were treated with IFN (interferon). We could not obtain the nucleotide sequence per se of the HCV genome but were able to evaluate the degree of diversity in the nucleotide sequence of the HCV genome using FSSA. Nonresponders to IFN therapy showed significant diversity in the nucleotide sequence, even if their number of HCV clones are small. Responders showed little diversity in the nucleotide sequence, which suggests that their viral clones were composed of populations with less variability in the HVR. IFN therapy had more impact on diversity in the nucleotide sequence than on the number of HCV clones.

Insulin/glucose-, pyruvate- and polyunsaturated fatty acid-responsive region(s) of rat fatty acid synthase gene promoter.

To investigate the regulatory DNA sequences required for polyunsaturated fatty acid (PUFA)-suppression of fatty acid synthase (FAS) gene as well as for insulin and/or carbohydrate-stimulation of this gene, primary hepatocytes were transfected with plasmids containing the 5'-flanking sequence of the rat FAS gene fused to the CAT gene. Sequences from -1604, -88 or -57 to +79 of the FAS gene directed an increase in CAT activity in the hepatocytes when insulin/glucose was added to the medium, in accordance with the responses on the endogenous FAS gene expression. The CAT activities were reduced by the addition of PUFA. Further deletion to -34, however, resulted in loss of the responses. The results suggest that the region from -57 to -34 of the FAS gene may be responsible for regulation due to insulin/glucose and PUFAs. Moreover, the region was also responsible for stimulation due to pyruvate alone.

Insulin- and polyunsaturated fatty acid-responsive region(s) of rat ATP citrate lyase gene promoter.

To investigate the regulatory DNA sequences required for insulin-stimulation of the ATP citrate-lyase (ACL) gene as well as for polyunsaturated fatty acid (PUFA)-suppression of this gene, primary cultured hepatocytes were transfected with plasmids containing the 5'-flanking sequence of the rat ACL gene fused to the chloramphenicol acetyltransferase (CAT) gene. Sequences from -861, -194 or -104 to +128 of the ACl gene directed an increase in CAT activity in hepatocytes when insulin was added to the medium containing either glucose or pyruvate. The CAT activities stimulated by insulin were reduced by the addition of PUFA, in accordance with the responses on the endogenous ACL gene expression. Further deletion to -20, however, resulted in loss of the responses. The results suggest that the region from -104 to -20 of the ACL gene is responsible for regulation due to insulin and PUFAs. In particular, the region from -61 to -49 of the ACL has sequence similarity to the insulin-responsive regions of fatty acid synthase and acetyl-CoA carboxylase.

Effects of nutrients and hormones on gene expression of ATP citrate-lyase in rat liver.

Northern-blot analyses demonstrated a strong gene expression of ATp citrate-lyase in liver and adipose tissue of rat and a weak expression in brain, heart, small intestine and muscle. After refeeding a carbohydrate/protein diet to fasted rats, the transcriptional rate had already increased within 2 h, the mRNA concentration reached a maximal level of approximately 30-fold increased in 16 h, and the enzyme induction increased sixfold in 48 h. By feeding only carbohydrate without protein, the transcriptional rate was increased threefold, and the mRNA concentration and enzyme induction comparably, to the levels in the carbohydrate/protein diet. It appears that protein feeding is not necessary to induce ATP citrate-lyase. In diabetic rats fed on a glucose diet, the transcriptional rate, mRNA concentration and enzyme level were very low in comparison with the normal. By fructose feeding, however, the transcriptional rate was more greatly increased and the mRNA concentration increased comparably to the levels reached by insulin treatment, while the enzyme induction was not so increased. Thus, it is suggested that insulin is important in regulated translation in addition to transcription. However, triiodothyronine treatment did not have much effect on the gene expression. As a result of the present experiment, it is noted that ATP citrate-lyase-gene expression was greatly dependent on carbohydrate.

Nutritional and hormonal regulation of mRNA levels of lipogenic enzymes in primary cultures of rat hepatocytes.

The effects of nutrients and hormones on the mRNA levels of acetyl-CoA carboxylase, fatty acid synthase, malic enzyme, and glucose 6-phosphate dehydrogenase were examined in primary cultures of rat hepatocytes during the process of induction. The addition of both glucose and insulin to the culture medium markedly enhanced the lipogenic enzyme mRNA induction due to either of them, in 16 h. Fructose or glycerol proved to be an effective substitute for glucose, suggesting that glycolytic metabolites were involved in the mRNA induction. It is remarkable that mRNA induction of acetyl-CoA carboxylase was the most sensitive to glucose and also to insulin among the lipogenic enzymes. Polyunsaturated fatty acids markedly reduced the mRNA induction of lipogenic enzymes. Dexamethasone enhanced all the lipogenic enzyme mRNA induction by insulin. On the other hand, triiodothyronine addition greatly increased the mRNA concentrations of lipogenic enzymes, but dexamethasone decreased rather than increased the mRNA induction by triiodothyronine. The effects of insulin on the induction of the lipogenic enzyme mRNAs were similar, but those of triiodothyronine were not. Triiodothyronine markedly enhanced malic enzyme mRNA induction by insulin with dexamethasone, and tended to enhance the induction of the acetyl-CoA carboxylase and fatty acid synthase mRNAs, but not that of glucose 6-phosphate dehydrogenase mRNA. It appeared that insulin and triiodothyronine synergistically enhanced lipogenic enzyme mRNA induction by glucose, but the mechanisms were different.

Regulation of hepatic lipogenic enzyme gene expression by diet quantity in rats fed a fat-free, high carbohydrate diet.

This investigation concerns the effects of the level of intake of a high carbohydrate diet on transcriptional rate, mRNA concentration and enzyme induction for lipogenic enzymes in rat liver. Six hours after refeeding fasted rats, the transcriptional rates in livers reached low maximum levels with small quantities of diet, but the mRNA concentrations continued to increase as diet intake increased. Greater diet intake primarily increased transcriptional rates and mRNA concentrations of lipogenic enzymes. After refeeding for 16 h, the mRNA concentrations were sigmoidly increased relative to the diet quantity and reached maximum levels of 20-, 110-, 22- and 16-fold above each fasted level for acetyl-CoA carboxylase, fatty acid synthase, malic enzyme and glucose-6-phosphate dehydrogenase, respectively. After 3 d of refeeding (in a steady state of lipogenic enzyme activities), however, the transcriptional rates, mRNA concentrations and activity inductions of all the enzymes were sigmoidly increased relative to diet quantity, but were not different among the enzymes. Consequently, fatty acid synthesis and triglyceride levels in the liver were not increased by feeding less than 70% of ad libitum intake but were greatly increased by feeding greater than 70% of ad libitum intake.

Effects of nutrients and hormones on transcriptional and post-transcriptional regulation of fatty acid synthase in rat liver.

The effects of nutrients and hormones on transcriptional and post-transcriptional regulation of fatty acid synthase in rat liver were investigated following cDNA cloning. When fasted rats were fed a carbohydrate/protein diet, the transcriptional rate was greatly increased even in 1 h. The transcriptional rate, mRNA concentration and enzyme induction reached maximum levels in 4 h, 8-16 h and 48 h, respectively. Although dietary carbohydrate increased each level more than protein did, both carbohydrate and protein were required to reach a high level. Corn oil feeding markedly decreased the transcriptional rate. In diabetic rats, the transcriptional rate, mRNA concentration and enzyme induction were very low in comparison with the normal. By treating the diabetic rats with insulin, however, the transcriptional rate was increased 5-fold in 1 h and 15-fold in 6 h, preceding a great increase in the mRNA and enzyme levels. On the other hand, fructose feeding or triiodothyronine treatment of diabetic rats abundantly increased the mRNA concentration and somewhat increased the transcriptional rate. Thus, it is suggested that insulin mainly stimulates the transcription of the fatty acid synthase gene, whereas triiodothyronine and fructose mainly increase the mRNA stability.

Effects of nutrients and hormones on transcriptional and post-transcriptional regulation of acetyl-CoA carboxylase in rat liver.

The effects of nutrients and hormones on transcriptional and post-transcriptional regulation of acetyl-CoA carboxylase in rat liver were investigated following a cDNA cloning. After refeeding a carbohydrate/protein diet to fasted rats, the transcriptional rate was increased 2.5-fold in only 1 h. The mRNA concentration reached a maximal level of 9-12-fold increase in 8-16 h, and the enzyme induction increased 10-fold in 48 h. By a carbohydrate diet without protein, the transcriptional rate, mRNA concentration and enzyme induction were similarly increased to the levels in the carbohydrate/protein diet. It appears that protein feeding is not necessary to induce acetyl-CoA carboxylase. Corn oil feeding decreased the transcriptional rate. In diabetic rats, the transcriptional rate, mRNA concentration and enzyme induction were very low in comparison with the normal. After insulin treatment, the transcriptional rate was increased 2-fold (the normal level) in 2 h in diabetic rats. By fructose feeding to diabetic rats, the transcriptional rate and mRNA concentration were increased similarly to the levels reached by insulin treatment, while the enzyme induction was increased by only 60%. Thus, it is suggested that insulin is importantly involved in the transcription and also translation of acetyl-CoA carboxylase. On the other hand, triiodothyronine treatment increased the mRNA and enzyme levels in diabetic and normal rats, and somewhat increased the transcriptional rate only in diabetic rats. Triiodothyronine appears to stabilize the mRNA besides having an insulin-like action in acetyl-CoA carboxylase transcription.

Effects of ageing on transcriptional and post-transcriptional regulation of malic enzyme and glucose-6-phosphate dehydrogenase in rat liver.

We previously found an age-dependent impairment of induction of lipogenic enzymes in rat liver [Iritani et al. (1981) Biochim. Biophys. Acta 665, 636-639]. Further, we have found that after refeeding a fat-free diet to fasted rats, increases in transcriptional rate, mRNA concentration and enzyme induction of hepatic malic enzyme and glucose-6-phosphate dehydrogenase were always lower in 18-month-old rats than in 1.5-month-old rats. In the young rats, the transcriptional rates reached the maximum level in 4 h and the mRNA reached maximum levels in 16 h. The peaks tended to delay in the older rats. The half-lives of the mRNAs were not significantly longer in the old than in the young animals. The incorporation of [3H]leucine into the enzyme proteins was also decreased roughly in proportion to the enzyme induction. The mRNA concentrations in the liver polysomes were roughly proportional to the total mRNA. Thus, no effects of ageing on mRNA stability or on the translational activity of the enzymes could be found. It is suggested that the age-dependent decreases of malic enzyme and glucose-6-phosphate dehydrogenase induction can be mainly ascribed to the transcriptional steps. Moreover, the transcriptional rate, mRNA concentration and induction of malic enzyme were increased by triiodothyronine treatment at a similar rate in both the young and old rats, but the absolute increments were lower in the old animals. The triiodothyronine response to malic enzyme induction also appeared to be primarily decreased at the transcription level.

Effects of nutrients and insulin on transcriptional and post-transcriptional regulation of glucose-6-phosphate dehydrogenase synthesis in rat liver.

The transcriptional and post-transcriptional regulation of glucose-6-phosphate dehydrogenase induction of rat liver was investigated using a cDNA cloned in our laboratory. By feeding a carbohydrate/protein diet to fasted rats, the mRNA concentration and enzyme induction of glucose-6-phosphate dehydrogenase (EC 1.1.1.49) reached maximal levels about 10-fold those in the fasted rats at 16 h and 72 h, respectively, whereas the transcriptional rate was increased about 3-fold in 6 h. In the protein fed (without carbohydrate) group, both the mRNA concentration and enzyme induction were increased to about 60% of the levels in the carbohydrate/protein fed group and in the group fed on a carbohydrate diet (without protein) to 30-40%. Further, dietary fat significantly reduced the transcriptional rate, mRNA concentration and enzyme induction to less than half, suggesting that dietary fat primarily reduced transcription. Thus, dietary nutrients appear to be involved in the steps preceding the translation. On the other hand, in diabetic rats, the transcriptional rate was significantly decreased as compared to the normal level and restored by insulin-treatment in 4 h. The mRNA concentration was very low in diabetic rats, and was restored to the normal level by insulin treatment in 8 h, and was half restored by fructose feeding. However, the enzyme induction of glucose-6-phosphate dehydrogenase was scarcely restored by fructose, unless accompanied by insulin treatment. Thus, it is suggested that insulin is involved in translation as well as in transcription. Further, the insulin-dependent increase of glucose-6-phosphate dehydrogenase mRNA was blocked by cycloheximide, suggesting that synthesis of a peptide is required.

Effects of insulin and fructose on transcriptional and post-transcriptional regulation of malic enzyme synthesis in diabetic rat liver.

Insulin action on regulation of hepatic malic enzyme has been investigated in comparison with fructose, using streptozotocin-induced diabetic rats. Insulin-treatment caused a 2.8-fold increase in the transcriptional rate of malic enzyme (EC 1.1.1.40) after 8 h, and a 5-fold increase in the mRNA concentration of the liver. In Northern blot analysis, we demonstrated that after insulin treatment, the nuclear mRNA of malic enzyme tended to increase more rapidly than the total cellular mRNA. Therefore, it is suggested that the nuclear mRNA was primarily increased by insulin. The insulin-dependent increase of malic enzyme mRNA was blocked by cycloheximide, suggesting that synthesis of a peptide is required. On the other hand, by feeding a high-fructose diet to diabetic rats, the malic enzyme mRNA concentration was considerably increased, though with a delayed peaking in comparison with the insulin-treated animals, whereas the transcriptional rate was not significantly increased. Dietary fructose may stabilize the transcripts. Fructose increased the enzyme level far less than the mRNA level. These results suggest that insulin is required in both the translational and transcriptional regulation of malic enzyme.

Transcriptional and posttranscriptional regulation of malic enzyme synthesis by insulin and triiodothyronine.

To investigate the transcriptional and posttranscriptional regulation of malate dehydrogenase (EC 1.1.1.40) induction by insulin, the transcriptional rate, mRNA concentration and enzyme induction of malic enzyme were compared in livers of normal and diabetic rats fed a high-carbohydrate diet. When rats were fed the diet for 4 days, the enzyme induction and mRNA concentration in livers of diabetic rats were only about 10% and 39%, respectively, of the values of normal rats, and the transcriptional rate was about 64%. Insulin treatment restored the transcriptional rate and mRNA concentration in 8 h and the enzyme induction in 4 days. Thus, it is suggested that insulin is involved in malic enzyme transcription of the gene and also possibly in the translation of the cytoplasm. On the other hand, by giving triiodothyronine treatment, the transcriptional rate and mRNA concentration were increased about twice and the enzyme induction, about 10-times in the diabetic animals. Triiodothyronine appears to stimulate malic enzyme transcription and possibly post-transcriptional steps even at a very low insulin level.

Effects of dietary casein and soybean protein on triglyceride turnover in rat liver.

We previously found that hepatic lipogenic enzyme induction, fatty acid synthesis, and triglyceride level were markedly lower in rats fed soybean protein than in those fed casein (Iritani et al. (1986): J. Nutr., 116: 190). After labeling triglycerides with tritiated water, the effects of dietary protein on the triglyceride degradation have been investigated. After the injection of tritiated water into rats, the radioactivities of fatty acids and triglycerides reached a plateau in 1-2 days and were markedly lower in the soybean protein group than in the casein group. The decreasing rates of triglyceride radioactivities were similar between the casein and soybean protein groups. The enzyme activities in glycerolipid synthesis were similar between the groups. Therefore, the lowering effects of soybean protein on triglyceride levels appear to be ascribed to triglyceride synthesis (due to fatty acid synthesis) rather than to the degradation.

Lipogenic enzyme induction and incorporation of exogenous fatty acid into liver and liver nuclei.

The incorporation of exogenous fatty acid into lipids of liver and liver nuclei of rats fed diets with or without fat was compared. When [3H]palmitic acid was injected into rats, more radioactivity was incorporated into triacylglycerols and phospholipids of liver and liver nuclei from rats fed the fat-free diet than from those fed the fat diet. The results were supported further by an autoradiographic study. On the other hand, the enzyme induction and quantity of malic enzyme mRNA were decreased by fat feeding. Other lipogenic enzymes were also coordinately decreased. Thus, it may be possible that exogenous fatty acid is involved in nuclear regulation in addition to cytosolic regulation of lipogenic enzyme induction.

Influence of diet on the transcriptional and post-transcriptional regulation of malic enzyme induction in the rat liver.

Fasted rats were refed a carbohydrate/protein diet, a carbohydrate diet (without protein) or a protein diet (without carbohydrate) to investigate, using a cDNA cloned in our laboratory, the regulatory mechanisms involved in hepatic malic enzyme induction. In the carbohydrate/protein diet, although the enzyme activity and the mRNA concentration of malic enzyme were increased about 7-fold above the levels in the fasted rat, the rate of transcription was increased only 2-fold. In the carbohydrate diet group the rate of transcription and the concentration of mRNA were increased to the levels in the carbohydrate/protein diet group, whereas the enzyme activity increased only to 60% of those levels. Protein appears to contribute to an increase in the translation of malic enzyme mRNA. In the protein diet group the transcriptional rate was not low, but the mRNA concentration was about half in comparison with the level of the carbohydrate/protein diet group. Further, dietary fat did not reduce the transcriptional activity, but reduced the mRNA concentration and the enzyme activity to half of the basal levels. Therefore, it is suggested that fat stimulates the degradation of the mRNA in liver cytosol, whereas carbohydrate tends to stabilize the mRNA. On the other hand 3,5,3'-triiodothyronine treatment increased the transcriptional activity by 1.5-2-fold above the basal values on all the diets and even on fasting. Thus, it is suggested that 3,5,3'-triiodothyronine increases the transcriptional activity of malic enzyme independently from nutritional regulation, while the nutrients are predominantly involved in the post-transcriptional steps.

Effects of aging on contributions of dietary fat and triiodothyronine treatment to lipogenic enzyme induction.

Although lipogenic enzyme inductions are reduced by fat feeding, this reduction decreases with aging and is particularly detectable in the case of acetyl-CoA carboxylase and fatty acid synthetase activities. On the other hand, the fat-dependent reductions of malic enzyme and acetyl-CoA carboxylase were consistently relieved by triiodothyronine (T3) treatment. The effects of T3 treatment on these enzyme inductions were greater in 10-month-old rats than in 1-month-old rats, while the carbohydrate-dependent induction and the fat-dependent reduction of the enzymes decreased with aging. In these animals, alterations in malic enzyme mRNA translational activities were roughly in parallel to the enzyme activities. Therefore, the age-dependent alterations in effects of T3 treatment and fat on malic enzyme induction do not appear to occur in post-translation.

Effects of dietary nutrients on substrate and effector levels of lipogenic enzymes, and lipogenesis from tritiated water in rat liver.

When fasted rats were refed for 4 days with a carbohydrate and protein diet, a carbohydrate diet (without protein) or a protein diet (without carbohydrate), the effects of dietary nutrients on the fatty acid synthesis from injected tritiated water, the substrate and effector levels of lipogenic enzymes and the enzyme activities were compared in the livers. In the carbohydrate diet group, although acetyl-CoA carboxylase was much induced and citrate was much increased, the activity of acetyl-CoA carboxylase extracted with phosphatase inhibitor and activated with 0.5 mM citrate was low in comparison to the carbohydrate and protein diet group. The physiological activity of acetyl-CoA carboxylase seems to be low. In the protein diet group, the concentrations of glucose 6-phosphate, acetyl-CoA and malonyl-CoA were markedly higher than in the carbohydrate and protein group, whereas the concentrations of oxaloacetate and citrate were lower. The levels of hepatic cAMP and plasma glucagon were high. The activities of acetyl-CoA carboxylase and also fatty acid synthetase were low in the protein group. By feeding fat, the citrate level was not decreased as much as the lipogenic enzyme inductions. Comparing the substrate and effector levels with the Km and Ka values, the activities of acetyl-CoA carboxylase and fatty acid synthetase could be limited by the levels. The fatty acid synthesis from tritiated water corresponded more closely to the acetyl-CoA carboxylase activity (activated 0.5 mM citrate) than to other lipogenic enzyme activities. On the other hand, neither the activities of glucose-6-phosphate dehydrogenase and malic enzyme (even though markedly lowered by diet) nor the levels of their substrates appeared to limit fatty acid synthesis of any of the dietary groups. Thus, it is suggested that under the dietary nutrient manipulation, acetyl-CoA carboxylase activity would be the first candidate of the rate-limiting factor for fatty acid synthesis with the regulations of the enzyme quantity, the substrate and effector levels and the enzyme modification.