Goran Koricanac

Role of PI3K/AKT, cPLA2 and ERK1/2 Signaling Pathways in Insulin Regulation of Vascular Smooth Muscle Cells Proliferation

Vascular smooth muscle cells (VSMCs) respond to arterial wall injury by intimal proliferation and play a key role in atherogenesis by proliferating and migrating excessively in response to repeated injury, such as hypertension and atherosclerosis. In contrast, fully differentiated, quiescent VSMCs allow arterial vasodilatation and vasoconstriction. Exaggerated and uncontrolled VSMCs proliferation appears therefore to be a common feature of both atherosclerosis and hypertension. Phosphorylation/dephosphorylation reactions of enzymes belonging to the family of mitogen-activated protein kinases (MAPKs), phosphatidylinositol 3-kinase (PI3K) and protein kinase B (Akt) play an important role in the transduction of mitogenic signal. We have previously shown that among extracellular signal-regulated protein kinases (ERKs), the 42 and 44 kDa isoforms (ERK1/2) as well as Akt and cytosolic phospholipase 2 (cPLA2) participate in the cellular mitogenic machinery triggered by several VSMCs activators, including insulin (INS). The ability of INS to significantly increase VSMCs proliferation has been demonstrated in several systems, but understanding of the intracellular signal transduction pathways involved is incomplete. Signal transduction pathways involved in regulation of the VSMCs proliferation by INS remains poorly understood. Thus, this review examines recent findings in signaling mechanisms employed by INS in modulating the regulation of proliferation of VSMCs with particular emphasis on PI3K/Akt, cPLA2 and ERK1/2 signaling pathways that have been identified as important mediators of VSMCs hypertrophy and vascular diseases. These findings are critical for understanding the role of INS in vascular biology and hyperinsulinemia.

Insulin regulation of proliferation involves activation of AKT and ERK 1/2 signaling pathways in vascular smooth muscle cells.

This investigation used primary cultured rat vascular smooth muscle cells (VSMCs) to examine the effect of insulin (INS) on proliferation of VSMCs. In this study, we investigated the role of protein kinase B (Akt) and p42/44 mitogen-activated protein kinase (ERK 1/2) signaling pathways in mediating the mitogenic action of INS in VSMCs. Incubation of rat VSMCs with INS (100 nM) for 10 min resulted in an increase of Akt phosphorylation by 6-fold (p<0.001) and ERK 1/2 phosphorylation by 3-fold (p<0.001). Pretreatment for 15 min with 10 muM of PI3K/Akt inhibitor LY294002 or with 20 muM PD98059, inhibitor of ERK 1/2, significantly reduced INS-stimulated Akt and ERK 1/2 phosphorylation by 76 and 75%, respectively. Prolonged treatment of VSMCs with INS for 24 h did not have an effect on either Akt or ERK1/2 phosphorylation. Incubation of rat VSMCs with INS resulted in an increase of VSMCs proliferation by 87% (p<0.001.) The effect of INS on VSMCs proliferation was significantly reduced by 68% by pretreatment with LY294002 (p>0.01) and by 71% (p>0.01) by pretreatment with PD98059. These results indicate that INS acts through Akt and ERK 1/2 signaling pathways to up-regulate proliferation of VSMCs.

Interaction Between Insulin and Estradiol in Regulation of Cardiac Glucose and Free Fatty Acid Transporters

The estrogen binding to specific extranuclear receptors (ER) activates several intracellular pathways that are activated by insulin as well. Moreover, insulin and estradiol (E2) influence cardiac energy substrates, blood glucose and free fatty acids (FFAs), and both hormones exert cardio-beneficial effects. In view of these facts, we suggest that cross-talk between their signaling pathways might have an important role in regulation of cardiac energy substrate transport. Ovariectomized rats were treated with insulin, estradiol (E2), or their combination 20, 30, or 40 min before analysis of blood glucose and FFA level, as well as cardiac plasma membranes (PM) and low density microsomes (LDM) content of glucose (GLUT4 and GLUT1) and FFA (CD36) transporters. Insulin, given alone, or in combination with E2, decreased plasma glucose level at all time points, but did not influence FFA level, while E2 treatment itself did not change glucose and FFA concentration. Insulin increased PM GLUT4 and GLUT1 content 30 and 40 min after treatment and the increases were partially accompanied by decrease in transporter LDM content. E2 increased PM content and decreased LDM content only of GLUT4 at 30 min. Insulin generally, and E2 at 20 min increased CD36 content in PM fraction. Both hormones decreased CD36 LDM content 20 min after administration. Effect of combined treatment mostly did not differ from single hormone treatment, but occasionally, particularly in distribution of GLUT4, combined treatment emphasized single hormone effect, suggesting that insulin and E2 act synergistically in regulation of energy substrate transporters in cardiac tissue.

Estradiol enhances effects of fructose rich diet on cardiac fatty acid transporter CD36 and triglycerides accumulation

Fructose rich diet increases hepatic triglycerides production and has deleterious cardiac effects. Estrogens are involved in regulation of lipid metabolism as well, but their effects are cardio beneficial. In order to study effects of fructose rich diet on the main heart fatty acid transporter CD36 and the role of estrogens, we subjected ovariectomized female rats to the standard diet or fructose rich diet, with or without estradiol (E2) replacement. The following parameters were analyzed: feeding behavior, visceral adipose tissue mass, plasma lipids, cardiac CD36 expression, localization and insulin regulation, as well as the profile of cardiac lipids. Results show that fructose rich diet significantly increased plasma triglycerides and decreased plasma free fatty acid (FFA) concentration, while E2 additionally emphasized FFA decrease. The fructose diet increased cardiac plasma membrane content of CD36 in the basal and insulin-stimulated states, and decreased its low density microsomes content. The E2 in fructose-fed rats raised the total cardiac protein content of CD36, its presence in plasma membranes and low density microsomes, and cardiac deposition of triglycerides, as well. Although E2 counteracts fructose in some aspects of lipid metabolism, and separately they have opposite cardiac effects, in combination with fructose rich diet, E2 additionally enhances CD36 presence in plasma membranes of cardiac cells and triglycerides accumulation, which paradoxically might promote deleterious effects of fructose diet on cardiac lipid metabolism. Taken together, the results presented in this work are of high importance for clinical administration of estrogens in females with a history of type 2 diabetes.

Impairment of cardiac insulin signaling in fructose-fed ovariectomized female Wistar rats.

BackgroundFructose consumption produces deleterious metabolic effects in animal models. The sites of fructose-induced insulin resistance are documented to be the liver, skeletal muscle, and adipose tissue, but effects of fructose-rich diet on cardiac insulin signaling and action were not investigated.Purpose and methodsIn order to study the potential fructose effects on development of cardiac insulin resistance, we analyzed biochemical parameters relevant for insulin action and phosphorylation of insulin signaling molecules, plasma membrane glucose transporter type 4 (GLUT4) content, and phosphorylation of endothelial nitric oxide synthase (eNOS), in ovariectomized female rats on fructose-enriched diet, in basal and insulin-stimulated conditions.ResultsFructose-fed rats (FFR) had increased content of visceral adipose tissue, but not body weight. Food intake was decreased, while fluid and caloric intake were increased in FFR. Additionally, fructose diet increased plasma insulin, blood triglycerides level, and HOMA index. Stimulation of protein kinase B (Akt) signaling pathway by insulin was reduced in rats on fructose-enriched diet, but effect of fructose on extracellular signal-regulated kinase (Erk 1/2) phosphorylation was not observed. Furthermore, insulin-induced GLUT4 presence in plasma membranes of cardiac cells was decreased by fructose diet, as well as insulin stimulation of eNOS phosphorylation at Ser1177.ConclusionIn summary, these results strongly support our hypothesis that fructose diet-induced changes of plasma lipid profile and insulin sensitivity are accompanied with decrease in cardiac insulin action in ovariectomized female rats.

Dihydrotestosterone deteriorates cardiac insulin signaling and glucose transport in the rat model of polycystic ovary syndrome.

It is supposed that women with polycystic ovary syndrome (PCOS) are prone to develop cardiovascular disease as a consequence of multiple risk factors that are mostly related to the state of insulin resistance and consequent hyperinsulinemia. In the present study, we evaluated insulin signaling and glucose transporters (GLUT) in cardiac cells of dihydrotestosterone (DHT) treated female rats as an animal model of PCOS. Expression of proteins involved in cardiac insulin signaling pathways and glucose transporters, as well as their phosphorylation or intracellular localization were studied by Western blot analysis in DHT-treated and control rats. Treatment with DHT resulted in increased body mass, absolute mass of the heart, elevated plasma insulin concentration, dyslipidemia and insulin resistance. At the molecular level, DHT treatment did not change protein expression of cardiac insulin receptor and insulin receptor substrate 1, while phosphorylation of the substrate at serine 307 was increased. Unexpectedly, although expression of downstream Akt kinase and its phosphorylation at threonine 308 were not altered, phosphorylation of Akt at serine 473 was increased in the heart of DHT-treated rats. In contrast, expression and phosphorylation of extracellular signal regulated kinases 1/2 were decreased. Plasma membrane contents of GLUT1 and GLUT4 were decreased, as well as the expression of GLUT4 in cardiac cells at the end of androgen treatment. The obtained results provide evidence for alterations in expression and especially in functional characteristics of insulin signaling molecules and glucose transporters in the heart of DHT-treated rats with PCOS, indicating impaired cardiac insulin action.

Interference between insulin and estradiol signaling pathways in the regulation of cardiac eNOS and Na+/K+-ATPase

Insulin and estradiol share some of signaling pathways and regulate same target molecules exerting mostly beneficial cardiac effects. In order to study their cardiac interaction, ovariectomized female rats were treated with hormones, separately or simultaneously (20, 30 or 40min before analysis), and the phosphorylations of protein kinase B (Akt), extracellular signal-regulated kinases 1 and 2 (ERK 1/2), endothelial nitric oxide synthase (eNOS) were analyzed, as well as the plasma membrane content of α2 subunit of Na(+)/K(+)-ATPase. Insulin, particularly, and estradiol stimulate Ser(473) Akt phosphorylation. The combined treatment was stimulatory, but less than insulin alone was. The general increase of Thr(308) Akt phosphorylation by insulin was stronger than at Ser(473) and reduced in the presence of estradiol, which also stimulated this phosphorylation given alone. The estradiol induction of ERK 1/2 phosphorylation was inverted to the decrease by the combined treatment, while insulin had no effect. Only insulin increased the plasma membrane content of α2. Estradiol did increase the phosphorylation of eNOS, whereas the insulin effect was controversial. The effect of the combined treatment on target molecules was generally opposite to single hormone treatment. In summary, both hormones exerted an effect on Akt phosphorylation, but only estradiol stimulated ERK 1/2 phosphorylation. The α2 plasma membrane content was increased only by insulin, while estradiol increased eNOS phosphorylation more consistently. Finally, if these hormones were administered together, it seems that they disturb each other in having a full effect on cardiac Akt, ERK 1/2, and downstream effectors, eNOS and Na(+)/K(+)-ATPase.

Basal and glucocorticoid induced changes of hepatic glucocorticoid receptor during aging: relation to activities of tyrosine aminotransferase and tryptophan oxygenase.

The characteristics of glucocorticoid receptors, their sensitivity to glucocorticoid as well as the basal and glucocorticoid induced thyrosine aminotranferase (TAT) and tryptophan oxygenase (TO) activities were studied in rat liver during aging. The concentration (N) and dissociation constant (Kd) of glucocorticoid receptor (GR) significantly change during the aging both in untreated and dexamethasone treated animals. The level of receptors was lower in dexamethasone treated rats of all analyzed aged groups compared to untreated animals. In comparison to untreated groups, there was no correlation between the changes of N and Kd during the lifespan. According to immunochemical analysis, the decline of receptor protein content occurs during lifespan. Dexamethasone treatment reduced the level of receptor protein compare to respective age group of untreated rats. The glucocorticoid-receptor (G-R) complexes from both untreated and treated animals underwent thermal activation, although the extent of activation was more pronounced in the case of untreated groups compared to treated animals. The magnitude of heat activation of receptor complexes was more pronounced in the liver of the youngest untreated rats compared to elderly ones, while the receptor activation between treated groups of studied ages has shown less significant differences. Besides, basal as well as induced TAT and TO activities after dexamethasone injection also showed age-related alterations. The observed alterations in GR might play a role in the changes of the cell responses to glucocorticoid during the age. This presumption is supported by detected changes in basal and dexamethasone induced TAT and TO activities during aging.

Age-related changes of insulin receptors, plasma insulin and glucose level

The effects of aging on hepatic and erythrocyte insulin receptors have been investigated in 6, 12, 18 and 21-months-old compare to 3-months-old rats. Plasma insulin was elevated in 6, 12 and 18-months-old rats. Specific binding of insulin in liver was increased at the age of 8 months and accompanied with increase in concentration of low affinity binding sites, while specific binding to erythrocytes as well as concentration of both classes of binding sites was increased in 6-months-old rats. The protein and mRNA content of hepatic receptor were decreased only in the oldest animals. Plasma glucose was elevated starting from 12-months-old rats, while, after decrease in 6-months-old animals, citrulline was raised in the oldest group. The results demonstrating that specific binding of insulin in liver and erythrocytes and the concentration of binding sites in both tissues were not decreased during aging, as well as the absence of changes in affinity of insulin binding sites do not point out to occurrence of insulin resistance. However, the increase in insulinemia in the middle of lifespan, elevated plasma glucose and citrulline as well as decrease of hepatic receptor protein and mRNA content in the oldest animals indicate some age-related changes in insulin signaling.

Low intensity exercise prevents disturbances in rat cardiac insulin signaling and endothelial nitric oxide synthase induced by high fructose diet.

Increase in fructose consumption together with decrease in physical activity contributes to the development of metabolic syndrome and consequently cardiovascular diseases. The current study examined the preventive role of exercise on defects in cardiac insulin signaling and function of endothelial nitric oxide synthase (eNOS) in fructose fed rats. Male Wistar rats were divided into control, sedentary fructose (received 10% fructose for 9 weeks) and exercise fructose (additionally exposed to low intensity exercise) groups. Concentration of triglycerides, glucose, insulin and visceral adipose tissue weight were determined to estimate metabolic syndrome development. Expression and/or phosphorylation of cardiac insulin receptor (IR), insulin receptor substrate 1 (IRS1), tyrosine-specific protein phosphatase 1B (PTP1B), Akt, extracellular signal-regulated protein kinases 1 and 2 (ERK1/2) and eNOS were evaluated. Fructose overload increased visceral adipose tissue, insulin concentration and homeostasis model assessment index. Exercise managed to decrease visceral adiposity and insulin level and to increase insulin sensitivity. Fructose diet increased level of cardiac PTP1B and pIRS1 (Ser307), while levels of IR and ERK1/2, as well as pIRS1 (Tyr 632), pAkt (Ser473, Thr308) and pERK1/2 were decreased. These disturbances were accompanied by reduced phosphorylation of eNOS at Ser1177. Exercise managed to prevent most of the disturbances in insulin signaling caused by fructose diet (except phosphorylation of IRS1 at Tyr 632 and phosphorylation and protein expression of ERK1/2) and consequently restored function of eNOS. Low intensity exercise could be considered as efficient treatment of cardiac insulin resistance induced by fructose diet.