Resveratrol Inhibits Neointimal Growth after Arterial Injury in High-Fat-Fed Rodents: The Roles of SIRT1 and AMPK.

We have shown that both insulin and resveratrol (RSV) decrease neointimal hyperplasia in chow-fed rodents via mechanisms that are in part overlapping and involve the activation of endothelial nitric oxide synthase (eNOS). However, this vasculoprotective effect of insulin is abolished in high-fat-fed insulin-resistant rats. Since RSV, in addition to increasing insulin sensitivity, can activate eNOS via pathways that are independent of insulin signaling, such as the activation of sirtuin 1 (SIRT1) and AMP-activated kinase (AMPK), we speculated that unlike insulin, the vasculoprotective effect of RSV would be retained in high-fat-fed rats. We found that high-fat feeding decreased insulin sensitivity and increased neointimal area and that RSV improved insulin sensitivity (p < 0.05) and decreased neointimal area in high-fat-fed rats (p < 0.05). We investigated the role of SIRT1 in the effect of RSV using two genetic mouse models. We found that RSV decreased neointimal area in high-fat-fed wild-type mice (p < 0.05), an effect that was retained in mice with catalytically inactive SIRT1 (p < 0.05) and in heterozygous SIRT1-null mice. In contrast, the effect of RSV was abolished in AMKPα2-null mice. Thus, RSV decreased neointimal hyperplasia after arterial injury in both high-fat-fed rats and mice, an effect likely not mediated by SIRT1 but by AMPKα2.

A histone deacetylase gene, SlHDA3, acts as a negative regulator of fruit ripening and carotenoid accumulation.

KEY MESSAGE: SlHDA3 functions as an inhibitor and regulates tomato fruit ripening and carotenoid accumulation. Post-translational modifications, including histones acetylation, play a pivotal role in the changes of chromatin structure dynamic modulation and gene activity. The regulation of histone acetylation is achieved by the action of histone acetyltransferases and deacetylases, which play crucial roles in the regulation of transcription activation. There is an increasing research focus on histone deacetylation in crops, but the role of histone deacetylase genes (HDACs) in tomato has not been elucidated. With the aim of characterizing the tomato RPD3/HDA1 family histone deacetylase genes, SlHDA3 was isolated and its RNA interference (RNAi) lines was obtained. The fruit of SlHDA3 RNAi lines exhibited accelerated ripening process along with short shelf life characteristics. The accumulation of carotenoid was increased due to the alteration of the carotenoid pathway flux. Climacteric ethylene production also stimulated along with significantly up-regulated expression of ethylene biosynthetic genes (ACS2, ACS4, ACO1 and ACO3) and fruit ripening-associated genes (RIN, E4, E8, PG, Pti4, LOXB, Cnr and TAGL1) in SlHDA3 RNAi lines. Besides, fruit cell wall metabolism-associated genes (HEX, MAN, TBG4, XTH5 and XYL) were enhanced in transgenic lines. Relative to wild type (WT) plants, SlHDA3 RNAi seedlings displayed shorter hypocotyls and more sensitivity to ACC (1-aminocyclopropane-1-carboxylate). These results indicated that SlHDA3 is involved in the regulation of fruit ripening by affecting ethylene biosynthesis and carotenoid accumulation.

In vivo effect of insulin to decrease matrix metalloproteinase-2 and -9 activity after arterial injury.

UNLABELLED: In vitro, insulin has both growth-promoting and vasculoprotective effects. In vivo, the effect of insulin is mainly protective. Insulin treatment (3 U/day) decreases smooth muscle cell (SMC) migration and neointimal growth after carotid angioplasty in normal rats maintained at normoglycemia by oral glucose. SMC migration requires limited proteolysis of the extracellular matrix, which is mediated by matrix metalloproteinases (MMPs). In this study, we investigated the effects of normoglycemic hyperinsulinemia on MMP activity after balloon angioplasty. Rats were divided into three groups: (1) control implants and tap water; (2) control implants and oral glucose, and (3) insulin implants (3 U/day) and oral glucose. RESULTS: Gelatin zymography revealed that insulin reduced the gelatinolytic activity of pro-MMP-2 by 46% (p < 0.05), MMP-2 by 44% (p < 0.05) and MMP-9 by 51% (p < 0.05) compared to controls after arterial injury. Insulin also reduced mRNA levels of MMP-2 (p < 0.05) and MMP-9 (p < 0.05) and protein levels of MMP-2 (p < 0.05). In contrast, there were no significant changes in membrane-type 1 MMP protein and tissue inhibitors of MMP activity after insulin treatment. Thus, these results suggest a mechanism by which insulin inhibits SMC migration and supports a vasculoprotective role for insulin in vivo.

Maternal taurine supplementation in rats partially prevents the adverse effects of early-life protein deprivation on ß-cell function and insulin sensitivity.

Dietary protein restriction during pregnancy and lactation in rats impairs ß-cell function and mass in neonates and leads to glucose intolerance in adult offspring. Maternal taurine (Tau) supplementation during pregnancy in rats restores ß-cell function and mass in neonates, but its long-term effects are unclear. The prevention of postnatal catch-up growth has been suggested to improve glucose tolerance in adult offspring of low-protein (LP)-fed mothers. The objective of this study was to examine the relative contribution of ß-cell dysfunction and insulin resistance to impaired glucose tolerance in 130-day-old rat offspring of LP-fed mothers and the effects of maternal Tau supplementation on ß-cell function and insulin resistance in these offspring. Pregnant rats were fed i) control, ii) LP, and iii) LP+Tau diets during gestation and lactation. Offspring were given a control diet following weaning. A fourth group consisting of offspring of LP-fed mothers, maintained on a LP diet following weaning, was also studied (LP-all life). Insulin sensitivity in the offspring of LP-fed mothers was reduced in females but not in males. In both genders, LP exposure decreased ß-cell function. Tau supplementation improved insulin sensitivity in females and ß-cell function in males. The LP-all life diet improved ß-cell function in males. We conclude that i) maternal Tau supplementation has persistent effects on improving glucose metabolism (ß-cell function and insulin sensitivity) in adult rat offspring of LP-fed mothers and ii) increasing the amount of protein in the diet of offspring adapted to a LP diet after weaning may impair glucose metabolism (ß-cell function) in a gender-specific manner.

Local insulin application on the carotid artery inhibits neointima formation.

Anti-mitogenic agents currently used to prevent restenosis in drug-eluting stents delay re-endothelialization. Delayed re-endothelialization is now considered as the main cause of late stent thrombosis with drug-eluting stents, which emphasizes the need for new treatments. We have shown that systemic insulin treatment decreases neointimal growth and accelerates re-endothelialization after arterial injury in a rat model of restenosis. However, systemic insulin treatment cannot be given to non-diabetic individuals because of the risk of hypoglycemia. Thus, we investigated whether local insulin treatment is also effective in reducing neointimal growth after arterial injury. Rats were given local vehicle or local insulin delivered via Pluronic gel applied around the carotid artery immediately following balloon injury. Plasma glucose and systemic insulin levels were not affected by local insulin treatment. Insulin decreased intimal area at 28 days (P < 0.05) and also inhibited vascular smooth muscle cell migration by 60% at 4 days (P < 0.05). NPH (a longer-lasting insulin) also decreased neointimal area. These results indicate that local insulin treatment can lead to decreased restenosis, suggesting a protective vascular effect of insulin in vivo and that local insulin treatment, possibly via insulin-eluting stents, may be clinically relevant.

Histone deacetylase gene SlHDT1 regulates tomato fruit ripening by affecting carotenoid accumulation and ethylene biosynthesis.

Fruit development and ripening is a complicated biological process, that is not only regulated by plant hormones and transcription factors, but also affected by epigenetic modifications. Histone deacetylation is an important way of epigenetic modification, and little information about it is available. In this study, an RNAi vector was constructed and transferred successfully into wild-type tomato for further research on the detailed functions of the histone deacetylation gene SlHDT1. The expression level of PSY1 was upregulated, and the transcription levels of LCY-B, LCY-E and CYC-B were downregulated, which was consistent with the increased accumulation of carotenoids. In addition, the expression levels of ethylene biosynthetic genes (ACS2, ACS4 and ACO1, ACO3), ripening-associated genes (RIN, E4, E8, PG, Pti4 and LOXB) and fruit cell wall metabolism genes (HEX, MAN, TBG4, XTH5 and XYL) were significantly upregulated further strengthening the results, including an increased ethylene content, advanced fruit ripening time and a shortened shelf life of tomato fruits. In addition, the increased total histone H3 acetylation level also provides evidence of a connection between epigenetic regulation by histone deacetylation and fruit development and ripening. Hence, SlHDT1 is a negative regulator and plays an essential role in regulating ethylene and carotenoid biosynthesis during fruit ripening through influences on the acetylation level.

Roles of vascular endothelial and smooth muscle cells in the vasculoprotective effect of insulin in a mouse model of restenosis.

BACKGROUND: Insulin exerts vasculoprotective effects on endothelial cells (ECs) and growth-promoting effects on vascular smooth muscle cells (SMCs) in vitro, and suppresses neointimal growth in vivo. Here we determined the role of ECs and SMCs in the effect of insulin on neointimal growth. METHODS: Mice with transgene CreERT2 under the control of EC-specific Tie2 (Tie2-Cre) or SMC-specific smooth muscle myosin heavy chain promoter/enhancer (SMMHC-Cre) or littermate controls were crossbred with mice carrying a loxP-flanked insulin receptor (IR) gene. After CreERT2-loxP-mediated recombination was induced by tamoxifen injection, mice received insulin pellet or sham (control) implantation, and underwent femoral artery wire injury. Femoral arteries were collected for morphological analysis 28 days after wire injury. RESULTS: Tamoxifen-treated Tie2-Cre+ mice showed lower IR expression in ECs, but not in SMCs, than Tie2-Cre- mice. Insulin treatment reduced neointimal area after arterial injury in Tie2-Cre- mice, but had no effect in Tie2-Cre+ mice. Tamoxifen-treated SMMHC-Cre+ mice showed lower IR expression in SMCs, but not in ECs, than SMMHC-Cre- mice. Insulin treatment reduced neointimal area in SMMHC-Cre- mice, whereas unexpectedly, it failed to inhibit neointima formation in SMMHC-Cre+ mice. CONCLUSION: Insulin action in both ECs and SMCs is required for the "anti-restenotic" effect of insulin in vivo.

Insulin inhibits and oral sucrose increases neointimal growth after arterial injury in rats.

BACKGROUND/AIMS: In our previous studies, rats on insulin treatment (5 U/day) and oral glucose to avoid hypoglycemia had reduced neointimal growth after arterial injury. However, plasma glucose in the insulin-treated rats was lower than normal and the effect of oral glucose remained undetermined. In this study, the effects of normoglycemic hyperinsulinemia and oral glucose or sucrose were investigated in the same model. METHODS: Rats were divided into 6 groups: (1) control implants and tap water; (2) insulin implants (5 U/day) and oral glucose + i.p. glucose to avoid any glucose lowering; (3) insulin implants (4 U/day) and oral glucose; (4) insulin implants (4 U/day) and oral sucrose; (5) control implants and oral glucose, and (6) control implants and oral sucrose. RESULTS: Insulin treatment at both doses reduced neointimal area (p < 0.001) 14 days after injury in rats receiving oral glucose but not in those receiving oral sucrose. Oral glucose, without insulin, had no effect on neointimal formation, whereas oral sucrose increased neointimal growth (p < 0.05). Oral sucrose (p < 0.05) but not oral glucose decreased insulin sensitivity measured with hyperinsulinemic clamps. CONCLUSIONS: (1) Insulin decreases neointimal growth after arterial injury independent of glucose-lowering or oral glucose administration and (2) oral sucrose per se affects neointimal growth.

Silencing SlMED18, tomato Mediator subunit 18 gene, restricts internode elongation and leaf expansion.

Mediator complex, a conserved multi-protein, is necessary for controlling RNA polymerase II (Pol II) transcription in eukaryotes. Given little is known about them in tomato, a tomato Mediator subunit 18 gene was isolated and named SlMED18. To further explore the function of SlMED18, the transgenic tomato plants targeting SlMED18 by RNAi-mediated gene silencing were generated. The SlMED18-RNAi lines exhibited multiple developmental defects, including smaller size and slower growth rate of plant and significantly smaller compound leaves. The contents of endogenous bioactive GA3 in SlMED18 silenced lines were slightly less than that in wild type. Furthermore, qRT-PCR analysis indicated that expression of gibberellins biosynthesis genes such as SlGACPS and SlGA20x2, auxin transport genes (PIN1, PIN4, LAX1 and LAX2) and several key regulators, KNOX1, KNOX2, PHAN and LANCEOLATE(LA), which involved in the leaf morphogenesis were significantly down-regulated in SlMED18-RNAi lines. These results illustrated that SlMED18 plays an essential role in regulating plant internode elongation and leaf expansion in tomato plants and it acts as a key positive regulator of gibberellins biosynthesis and signal transduction as well as auxin proper transport signalling. These findings are the basis for understanding the function of the individual Mediator subunits in tomato.

Combined Hyperglycemia- and Hyperinsulinemia-Induced Insulin Resistance in Adipocytes Is Associated With Dual Signaling Defects Mediated by PKC-ζ.

A hyperglycemic and hyperinsulinemic environment characteristic of type 2 diabetes causes insulin resistance. In adipocytes, defects in both insulin sensitivity and maximum response of glucose transport have been demonstrated. To investigate the molecular mechanisms, freshly isolated rat adipocytes were incubated in control (5.6 mM glucose, no insulin) and high glucose (20 mM)/high insulin (100 nM) (HG/HI) for 18 hours to induce insulin resistance. Insulin-resistant adipocytes manifested decreased sensitivity of glucose uptake associated with defects in insulin receptor substrate (IRS)-1 Tyr phosphorylation, association of p85 subunit of phosphatidylinositol-3-kinase, Akt Ser473 and Thr308 phosphorylation, accompanied by impaired glucose transporter 4 translocation. In contrast, protein kinase C (PKC)-ζ activity was augmented by chronic HG/HI. Inhibition of PKC-ζ with a specific cell-permeable peptide reversed the signaling defects and insulin sensitivity of glucose uptake. Transfection of dominant-negative, kinase-inactive PKC-ζ blocked insulin resistance, whereas constitutively active PKC-ζ recapitulated the defects. The HG/HI incubation was associated with stimulation of IRS-1 Ser318 and Akt Thr34 phosphorylation, targets of PKC-ζ. Transfection of IRS-1 S318A and Akt T34A each partially corrected insulin signaling, whereas combined transfection of both completely normalized insulin signaling. In vivo hyperglycemia/hyperinsulinemia in rats for 48 hours similarly resulted in activation of PKC-ζ and increased phosphorylation of IRS-1 Ser318 and Akt Thr34. These data indicate that impairment of insulin signaling by chronic HG/HI is mediated by dual defects at IRS-1 and Akt mediated by PKC-ζ.

The tomato histone deacetylase SlHDA1 contributes to the repression of fruit ripening and carotenoid accumulation.

Histone deacetylation is one of the well characterized post-translational modifications related to transcriptional repression in eukaryotes. The process of histone deacetylation is achieved by histone deacetylases (HDACs). Over the last decade, substantial advances in our understanding of the mechanism of fruit ripening have been achieved, but the role of HDACs in this process has not been elucidated. In our study, an RNA interference (RNAi) expression vector targeting SlHDA1 was constructed and transformed into tomato plants. Shorter fruit ripening time and decreased storability were observed in SlHDA1 RNAi lines. The accumulation of carotenoid was increased through an alteration of the carotenoid pathway flux. Ethylene content, ethylene biosynthesis genes (ACS2, ACS4 and ACO1, ACO3) and ripening-associated genes (RIN, E4, E8, Cnr, TAGL1, PG, Pti4 and LOXB) were significantly up-regulated in SlHDA1 RNAi lines. In addition, the expression of fruit cell wall metabolism genes (HEX, MAN, TBG4, XTH5 and XYL) was enhanced compared with wild type. Furthermore, SlHDA1 RNAi seedlings displayed shorter hypocotyls and were more sensitive to ACC (1-aminocyclopropane-1-carboxylate) than the wild type. The results of our study indicate that SlHDA1 functions as a negative regulator of fruit ripening by affecting ethylene synthesis and carotenoid accumulation.

Silencing of histone deacetylase SlHDT3 delays fruit ripening and suppresses carotenoid accumulation in tomato.

The acetylation levels of histones on lysine residues are regulated by histone acetyltransferases and histone deacetylases, which play an important but understudied role in the control of gene expression in plants. There is an increasing research focus on histone deacetylation in crops, but to date, there is little information regarding tomato. With the aim of characterizing the tomato HD2 family of histone deacetylases, an RNA interference (RNAi) expression vector of SlHDT3 was constructed and transformed into tomato plants. The time of fruit ripening was delayed and the shelf life of the fruit was prolonged in SlHDT3 RNAi lines. The accumulation of carotenoid was decreased by altering of the carotenoid pathway flux. Ethylene content was also reduced and expression of ethylene biosynthetic genes (ACS2, ACS4 and ACO1, ACO3) and ripening-associated genes (RIN, E4, E8, PG, Pti4 and LOXB) was significantly down-regulated in SlHDT3 RNAi lines. The expression of genes involved in fruit cell wall metabolism (HEX, MAN, TBG4, XTH5 and XYL) was inhibited compared with wild type. These results indicate that SlHDT3 functions as a positive regulator of fruit ripening by affecting ethylene synthesis and carotenoid accumulation and that SlHDT3 lies upstream of SlMADS-RIN in the fruit ripening regulatory network.

High-dose metformin (420mg/kg daily p.o.) increases insulin sensitivity but does not affect neointimal thickness in the rat carotid balloon injury model of restenosis.

OBJECTIVE: Our laboratory has shown that insulin's effect to decrease neointimal thickness after arterial injury is greatly diminished in insulin resistant conditions. Thus, in these conditions, a better alternative to insulin could be to use an insulin sensitizing agent. Metformin, the most commonly prescribed insulin sensitizer, has a cardiovascular protective role. Therefore, the objective of this study was to investigate the potential benefit of metformin on neointimal area after arterial injury in a rat model of restenosis. METHODS: Rats fed with either normal or high fat diet and treated with or without oral metformin (420mg/kg daily) underwent carotid balloon injury. Effects of metformin on clamp-determined insulin sensitivity, vessel AMPK (AMP-activated protein kinase) phosphorylation (activation marker) and neointimal area were evaluated. RESULTS: Metformin increased insulin sensitivity, but did not affect neointimal thickness in either the normal fat or high fat diet-fed rats. Furthermore, metformin activated AMPK in uninjured but not in injured vessels. Similarly, 10mmol/L metformin inhibited proliferation and activated AMPK in smooth muscle cells of uninjured but not injured vessels, whereas 2mmol/L metformin did not have any effect. CONCLUSION: In rats, metformin does not decrease neointimal growth after arterial injury, despite increasing whole body insulin sensitivity.

The effect of insulin to decrease neointimal growth after arterial injury is endothelial nitric oxide synthase-dependent.

In vitro, insulin has mitogenic effects on vascular smooth muscle cells (VSMC) but also has protective effects on endothelial cells by stimulating nitric oxide (NO) production and endothelial nitric oxide synthase (eNOS) expression. Furthermore, NOS inhibition attenuates the effect of insulin to inhibit VSMC migration in vitro. Using an in vivo model, we have previously shown that insulin decreases neointimal growth and cell migration and increases re-endothelialization after arterial injury in normal rats. Since insulin can stimulate NOS, and NO can decrease neointimal growth, we hypothesized that NOS, and more specifically eNOS was required for the effects of insulin in vivo. Rats were given subcutaneous insulin implants (3 U/day) alone or with the NOS inhibitor l-NAME (2 mg kg(-1) day(-1)) 3 days before arterial (carotid or aortic) balloon catheter injury. Insulin decreased both neointimal area (P < 0.01) and cell migration (P < 0.01), and increased re-endothelialization (P < 0.05). All of these effects were prevented by the co-administration of l-NAME. Insulin was found to decrease inducible NOS expression (P < 0.05) but increase eNOS phosphorylation (P < 0.05). These changes were also translated at the functional level where insulin improved endothelial-dependent vasorelaxation. To further study the NOS isoform involved in insulin action, s.c. insulin (0.1 U/day) was given to wild-type and eNOS knockout mice. We found that insulin was effective at decreasing neointimal formation in wild-type mice after wire injury of the femoral artery, whereas this effect of insulin was absent in eNOS knockout mice. These results show that the vasculoprotective effect of insulin after arterial injury is mediated by an eNOS-dependent mechanism.

Resveratrol inhibits neointimal formation after arterial injury through an endothelial nitric oxide synthase-dependent mechanism.

Revascularization procedures used for treatment of atherosclerosis often result in restenosis. Resveratrol (RSV), an antioxidant with cardiovascular benefits, decreases neointimal formation after arterial injury by a mechanism that is still not fully clarified. Our main objective was to address the role of nitric oxide synthases (NOSes) and more specifically the endothelial-NOS (eNOS) isoform as a mediator of this effect. RSV (4 mg/kg/day, s.c.) alone or in combination with the NOS inhibitor N-nitro-L-arginine methyl ester (L-NAME) (2 mg/kg/day, s.c.) was given to Sprague-Dawley rats beginning at 3 days before arterial (carotid or aortic) injury. RSV reduced neointimal formation by 50% (P<0.01), decreased intimal cell proliferation by 37% (P<0.01) and reduced inflammatory markers such as PECAM and MMP-9 mRNA. These effects of RSV were all abolished by coadministration of l-NAME. Oral RSV (beginning at 5 days before arterial injury) reduced neointimal thickness after femoral wire injury in mice, however this effect was not observed in eNOS knockout mice. This is the first report of RSV decreasing neointimal cell proliferation and neointimal growth through an eNOS-dependent mechanism.