The Epigenetic Legacy of Maternal Protein Restriction: Renal Ptger1 DNA Methylation Changes in Hypertensive Rat Offspring.

Nutrient imbalances during gestation are a risk factor for hypertension in offspring. Although the effects of prenatal nutritional deficiency on the development of hypertension and cardiovascular diseases in adulthood have been extensively documented, its underlying mechanisms remain poorly understood. In this study, we aimed to elucidate the precise role and functional significance of epigenetic modifications in the pathogenesis of hypertension. To this end, we integrated methylome and transcriptome data to identify potential salt-sensitive hypertension genes using the kidneys of stroke-prone spontaneously hypertensive rat (SHRSP) pups exposed to a low-protein diet throughout their fetal life. Maternal protein restriction during gestation led to a positive correlation between DNA hypermethylation of the renal prostaglandin E receptor 1 (Ptger1) CpG island and high mRNA expression of Ptger1 in offspring, which is consistently conserved. Furthermore, post-weaning low-protein or high-protein diets modified the Ptger1 DNA hypermethylation caused by fetal malnutrition. Here, we show that this epigenetic variation in Ptger1 is linked to disease susceptibility established during fetal stages and could be reprogrammed by manipulating the postnatal diet. Thus, our findings clarify the developmental origins connecting the maternal nutritional environment and potential epigenetic biomarkers for offspring hypertension. These findings shed light on hypertension prevention and prospective therapeutic strategies.

Optimized method for determining free L-cysteine in rat plasma by high-performance liquid chromatography with the 4-aminosulfonyl-7-fluoro-2,1,3-benzoxadiazole conversion reagent.

The analytical method was optimized for L-cysteine (Cys) in rat plasma with co-existing L-cystine (Cyss). We observed that more than 100% Cyss in rat plasma was converted to Cys under typical conditions for the conversion with 7-fluoro-2,1,3-benzoxadiazole-4-sulfonate (SBD-F). Another conversion reagent, 4-aminosulfonyl-7-fluoro-2,1,3-benzoxadiazole (ABD-F), was then employed, with which the reaction could be carried out at a low temperature without the use of a reducing reagent. Under the optimized conditions of 4 °C and pH 8.3, the conversion ratio of Cyss to Cys in rat plasma was as low as 5-7%. We determined the Cys concentration in plasma of the portal vein of rats that had been orally administered with Cys and Cyss by applying this method. The result indicated that Cys administration and also Cyss administration effectively increased the plasma Cys level. The method developed in this study is well suited for determining the thiol compounds in biological samples.

Maternal protein restriction induces renal AT2R promoter hypomethylation in salt-sensitive, hypertensive rats.

SCOPE: We previously demonstrated that protein restriction in utero induced salt-sensitive hypertension and changed renal levels of angiotensin type 2 receptor (AT2R) in Stroke-Prone Spontaneously Hypertensive Rat (SHRSP). Here, we investigated if this characteristic alteration of AT2R is related to AT2R DNA methylation profiles. METHODS AND RESULTS: First, we examined the relation between AT2R DNA methylation and its promoter activity in vitro. Luciferase assays revealed a negative correlation between these two variables. Next, we fed SHRSP dams and grand-dams a control 20% casein diet or a 9% casein diet during pregnancy. Adult offspring and grand-offspring were supplied either water or 1% saline solution for 2 weeks. Renal AT2R promoter DNA near the TATA-box was hypomethylated, mRNA expression was suppressed, and protein expression tended to be higher, in adult offspring of mothers fed a low casein diet. Moreover, adult grand-offspring exhibited high blood pressure after salt loading, along with suppressed transcription of AT2R mRNA and elevated translated protein. CONCLUSIONS: Under a fetal environment of protein restriction, the increase in protein expression due to hypomethylation of the AT2R promoter region occurs as a response to increased salt sensitivity, and controlling this mechanism may be important for the prevention of hypertension.

Integrated omics profiling of dextran sodium sulfate-induced colitic mice supplemented with Wolfberry (Lycium barbarum).

We used a multi-omics profiling approach to investigate the suppressive effects of 2% Wolfberry (WOL)-enriched diets on dextran sodium sulfate (DSS)-induced colitis in mice. It was observed that in mice fed the WOL diet, the disease activity index, colon shortening, plasma concentrations of matrix metalloproteinase-3 and relative mesenteric fat weight were significantly improved as compared to the DSS group. Results from colon transcriptome and proteome profiles showed that WOL supplementation significantly ameliorated the expression of genes and proteins associated with the integrity of the colonic mucosal wall and colonic inflammation. Based on the hepatic transcriptome, proteome and metabolome data, genes involved in fatty acid metabolism, proteins involved in inflammation and metabolites related to glycolysis were downregulated in WOL mice, leading to lowered inflammation and changes in these molecules may have led to improvement in body weight loss. The integrated nutrigenomic approach thus revealed the molecular mechanisms underlying the ameliorative effect of whole WOL fruit consumption on inflammatory bowel disease.

Low-arginine and low-protein diets induce hepatic lipid accumulation through different mechanisms in growing rats.

BACKGROUND: Dietary protein deficiency and amino acid imbalance cause hepatic fat accumulation. We previously demonstrated that only arginine deficiency or total amino acid deficiency in a diet caused significant hepatic triglyceride (TG) accumulation in young Wistar rats. In this study, we explored the mechanisms of fatty liver formation in these models. METHODS: We fed 6-week-old male Wistar rats a control diet (containing an amino acid mixture equivalent to 15% protein), a low-total-amino acid diet (equivalent to 5% protein; 5PAA), and a low-arginine diet (only the arginine content is as low as that of the 5PAA diet) for 2 weeks. RESULTS: Much greater hepatic TG accumulation was observed in the low-arginine group than in the low-total-amino acid group. The lipid consumption rate and fatty acid uptake in the liver did not significantly differ between the groups. In contrast, the low-total-amino acid diet potentiated insulin sensitivity and related signaling in the liver and enhanced de novo lipogenesis. The low-arginine diet also inhibited hepatic very-low-density lipoprotein secretion without affecting hepatic insulin signaling and lipogenesis. CONCLUSIONS: Although the arginine content of the low-arginine diet was as low as that of the low-total-amino acid diet, the two diets caused fatty liver via completely different mechanisms. Enhanced lipogenesis was the primary cause of a low-protein diet-induced fatty liver, whereas lower very-low-density lipoprotein secretion caused low-arginine diet-induced fatty liver.

Sardine peptide with angiotensin I-converting enzyme inhibitory activity improves glucose tolerance in stroke-prone spontaneously hypertensive rats.

An enzymatic hydrolysate of sardine protein (sardine peptide, SP) derived from sardine muscle possesses angiotensin I-converting enzyme (ACE) inhibitory activity. In the present study, we investigated the effect of SP on the blood glucose levels in stroke-prone spontaneously hypertensive rats (SHRSPs). Ten-week-old SHRSPs were assigned to three groups. The control group was given tap water for 4 weeks, while the experimental groups were given water containing SP (1 g/kg/d) or an ACE inhibitor, captopril (8 mg/kg/d). Treatment with SP and captopril decreased ACE activity in the kidney, aorta, and mesentery. There were no differences in fasting blood glucose levels among the three groups, whereas SP and captopril administration significantly suppressed the increase in blood glucose after glucose loading in the control SHRSPs. No difference was observed in plasma insulin levels among the three groups. Thus treatment with captopril and ACE-inhibitory sardine peptides ameliorated the glucose tolerance of this rat strain.

Maternal Protein Restriction Alters the Renal Ptger1 DNA Methylation State in SHRSP Offspring.

We previously reported that maternal protein restriction (LP) during pregnancy increases salt sensitivity in offspring using the Stroke-Prone Spontaneously Hypertensive Rat (SHRSP). In the present study, we focus on DNA methylation profiles of prostaglandin E receptor 1 gene (ptger1), which is known to be associated with hypertension. We evaluated the ptger1 DNA methylation status via bisulfite sequencing, and analyzed the expression of ptger1-related genes. The results of these analyses showed that, compared to controls, the LP-S offspring exhibited both marked ptger1 hypermethylation, and significantly increased ptger1 expression. Moreover, they also exhibited significantly decreased expression of the downstream gene epithelial Na⁺ channel alpha (enacα). Interestingly, LP offspring that were provided with a standard water drinking supply (W) also exhibited increased ptger1 methylation and expression. Together, these results suggest that maternal protein restriction during pregnancy modulates the renal ptger1 DNA methylation state in SHRSP offspring, and thereby likely mediates ptger1 and enacα gene expression to induce salt sensitivity.

Branched-chain amino acid supplementation restores reduced insulinotropic activity of a low-protein diet through the vagus nerve in rats.

BACKGROUND: Previously, we reported that a low-protein diet significantly reduced insulin secretion in response to feeding within 1 h in rats, suggesting that the insulinotropic effect of dietary protein plays an important role in maintaining normal insulin release. The current study aimed to elucidate whether deficiency of certain amino acids could diminish the insulinotropic activity and to investigate whether reduced insulin secretion in response to a low-protein diet is restored by supplementation with certain amino acids. METHODS: First, we fed male Wistar rats (5-6 rats per group) with diets deficient in every single amino acid or three branched-chain amino acids (BCAAs); within 1-2 h after the onset of feeding, we measured the plasma insulin levels by using an enzyme-linked immunosorbent assay (ELISA). As insulin secretion was reduced in BCAA-deficient groups, we fed low-protein diets supplemented with BCAAs to assess whether the reduced insulin secretion was restored. In addition, we treated the pancreatic beta cell line MIN6 with BCAAs to investigate the direct insulinotropic activity on beta cells. Lastly, we investigated the effect of the three BCAAs on sham-operated or vagotomized rats to assess involvement of the vagus nerve in restoration of the insulinotropic activity. RESULTS: Feeding a low-protein diet reduced essential amino acid concentrations in the plasma during an absorptive state, suggesting that reduced plasma amino acid levels can be an initial signal of protein deficiency. In normal rats, insulin secretion was reduced when leucine, valine, or three BCAAs were deficient. Insulin secretion was restored to normal levels by supplementation of the low-protein diet with three BCAAs, but not by supplementation with any single BCAA. In MIN6 cells, each BCAA alone stimulated insulin secretion but the three BCAAs did not show a synergistic stimulatory effect. The three BCAAs showed a synergistic stimulatory effect in sham-operated rats but failed to stimulate insulin secretion in vagotomized rats. CONCLUSIONS: Leucine and valine play a role in maintaining normal insulin release by directly stimulating beta cells, and supplementation with the three BCAAs is sufficient to compensate for the reduced insulinotropic activity of the low-protein diet, through the vagus nerve.

Reduction of salt sensitivity in stroke-prone spontaneously hypertensive rats administered an AT1 receptor antagonist during suckling.

BACKGROUND: In this study, antihypertensive therapy was started during suckling and the effect on blood pressure (BP) and salt sensitivity of stroke-prone spontaneously hypertensive rats (SHRSP) was determined. METHODS: The SHRSP were treated with an AT1 receptor antagonist (losartan: 100 mg/L in drinking water) from 2 to 4 weeks of age. After stopping treatment at 4 weeks of age, the control group and the losartan group were fed a commercial diet with tap water ad libitum until 10 weeks of age. Both the control and losartan groups were switched to 1% saline at the age of 10 weeks. RESULTS: Salt loading was started at 10 weeks of age, with BP levels of 203+/-3 and 199+/-6 mm Hg for the control group and the losartan group, respectively, at that age. After 4 weeks of salt loading, BP levels were 253+/-7 mm Hg in the control group and 242+/-5 mm Hg in the losartan group, showing a mild elevation in the losartan group. The life span of the losartan group (104+/-78 days) was significantly greater than that of the control group (37+/-17 days). Plasma aldosterone concentrations of the losartan group were lower than those of the control group at 4 and 15 weeks of age. CONCLUSIONS: We have demonstrated the renin-angiotensin-aldosterone system may play a key role in the establishment of end-organ salt sensitivity, and the period of lactation in critical for salt sensitivity in later life.

Supplemental arginine above the requirement during suckling causes obesity and insulin resistance in rats.

Nutrition in early life is important in determining susceptibility to adult obesity, and arginine may promote growth acceleration in infants. We hypothesized that maternal arginine supplementation may promote growth in their pups and contribute to obesity and alteration of the metabolic system in later life. Dams and pups of Wistar rats were given a normal diet (15% protein) as a control (CN) or a normal diet with 2% arginine (ARG). Altered profiles of free amino acids in breast milk were observed in that the concentrations of threonine and glycine were lower in the ARG dams compared with the CN dams. The offspring of the CN and ARG dams were further subdivided into normal-diet (CN-CN and ARG-CN) groups and a high fat-diet groups (CN-HF and ARG-HF). In response to the high fat-diet feeding, the visceral fat deposits were significantly increased in the ARG-HF group (although not compared with the CN-HF group); no difference was observed between the CN-CN and ARG-CN groups. The blood glucose and insulin levels after glucose loading were significantly higher in the ARG-HF group compared with the CN-HF group. The results suggest that the offspring of dams supplemented with arginine during lactation acquired increased susceptibility to a high-fat diet, resulting in visceral obesity and insulin resistance. The lower supply of threonine and glycine to pups may be one of the contributing causes to the programming of lifelong obesity risk in offspring. Our findings also indicated that maternal arginine supplementation during suckling causes obesity and insulin resistance in rats.

Role of the renin-angiotensin-aldosterone system in the enhancement of salt sensitivity caused by prenatal protein restriction in stroke-prone spontaneously hypertensive rats.

We previously demonstrated that maternal protein restriction during pregnancy enhanced salt sensitivity and shortened life span in stroke-prone spontaneously hypertensive rats (SHRSP). The present study was conducted to investigate the participation of the renin-angiotensin-aldosterone system in the development of salt sensitivity in the offspring of dams fed a low-protein diet during pregnancy. We used SHRSP offspring from dams fed a 20% casein diet (CN) or a 9% casein diet (LP) during pregnancy. The CN and LP SHRSP offspring were further subdivided into tap-water-drinking and 1%-saline-drinking groups from the postnatal 10th week. A remarkable elevation in blood pressure in response to salt loading was observed in the LP SHRSP offspring. The protein levels of CYP11B2, an enzyme for aldosterone synthesis, were markedly elevated in response to salt loading in the kidneys of LP offspring. Treatment of the LP offspring with an aldosterone receptor antagonist prevented the blood pressure from elevating and lengthened the average life span in LP offspring in response to the drinking of 1% saline. No difference in the activity of angiotensin-converting enzyme or in the protein level of the angiotensin type 1 receptor was found between the CN and LP offspring in either the tap-water-drinking or saline-drinking conditions. In conclusion, the increment of aldosterone production in response to high-salt loading may contribute to the elevated salt sensitivity of the offspring of protein-restricted dams.

The effects of maternal mild protein restriction on stroke incidence and blood pressure in stroke-prone spontaneously hypertensive rats (SHRSP).

The effect of maternal protein restriction during pregnancy on the offspring's blood pressure was assessed in stroke-prone spontaneously hypertensive rats (SHRSP) which are genetically predisposed to hypertension and stroke. After the confirmation of pregnancy, the control group was given a 20% casein diet, and the low-protein group was fed a 9% casein diet. After the confirmation of delivery, commercial feed was given to both of the groups. No differences were seen between the control and low-protein offspring in regard to body weight, blood pressure elevation, or life span. One percent saline solution was put in the control and low-protein groups after the age of 11 weeks. Blood pressure increased markedly in the low-protein group, on the blood pressure level in the low-protein group on week 2 after salt loading (242+/-6 mmHg) was significantly higher than that in the control group (223+/-9 mmHg; p<0.05). The survival duration was significantly shorter in the low-protein group (113+/-4 days) than in the control group (135+/-22 days; p<0.05). These results suggest that maternal protein malnutrition in SHRSP exerted a high salt sensitivity and a malignant influence on stroke incidence on offspring.