Gender differences in the expression and cellular localization of lipin 1 in the hearts of fructose-fed rats.

To give new insight to alterations of cardiac lipid metabolism accompanied by a fructose-rich diet (FRD), rats of both sexes were exposed to 10 % fructose in drinking water during 9 weeks. The protein level and subcellular localization of the main regulators of cardiac lipid metabolism, such as lipin 1, peroxisome proliferator-activated receptor α (PPARα), peroxisome proliferator-activated receptor γ coactivator-1 α (PGC-1α), carnitine palmitoyltransferase I (CPTI), and CD36 were studied. Caloric intake in fructose-fed rats (FFR) of both sexes was increased. Circulating triacylglyceroles (TAG) and non-esterified fatty acids were increased in male FFR, while females increased visceral adiposity and blood TAG. Total expression of lipin 1 in cardiac cell lysate and its cytosolic and microsomal level were increased in the hearts of male FFR. PPARα and PGC-1α content were decreased in the nuclear extract. In addition, cardiac deposition of TAG in male FFR was elevated, as well as inhibitory phosphorylation of insulin receptor substrate 1 (IRS-1). In contrast, in female FFR, lipin 1 level was increased in nuclear extract only, while overall CPTI expression and phosphorylation of IRS-1 at serine 307 were decreased. The results of our study suggest that fructose diet causes gender-dependent alterations in cardiac lipid metabolism. Potentially detrimental effects of FRD seem to be limited to male rats. Most of the observed changes might be a consequence of elevated expression and altered localization of lipin 1. Increased inhibitory phosphorylation of IRS-1 is possible link between cardiac lipid metabolism and insulin resistance in FFR.

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.

Does oestradiol attenuate the damaging effects of a fructose-rich diet on cardiac Akt/endothelial nitric oxide synthase signalling?

Fructose-rich diets (FRD) cause cardiac insulin resistance manifested by impairment of Akt/endothelial NO synthase (eNOS) signalling. In contrast, oestradiol (E2) activates this signalling pathway in the heart. To study the ability of E2 to revert the detrimental effect of fructose on cardiac Akt/eNOS, female rats were subjected to a FRD and ovariectomy followed with or without E2 replacement. We also analysed the effects of the FRD and E2 on cardiac extracellular signal-regulated kinase (Erk 1/2) signalling related to their role in cardiac hypertrophy development. Expression of Akt, eNOS and Erk 1/2, as well as regulatory phosphorylations of these molecules were determined. The protein expression of cardiac Akt and eNOS was not affected by the diet or E2 treatment. However, the FRD was accompanied by a decrease in Akt phosphorylation at Ser(473) and Thr(308), and eNOS at Ser(1177), while the phosphorylation of eNOS at Thr(495) was increased. E2 replacement in ovariectomised fructose-fed rats caused a reversion of the diet effect on Akt and eNOS serine phosphorylation, but mostly had no effect on threonine phosphorylation of the molecules. The FRD and E2 treatment did not influence Erk 1/2 expression and phosphorylation and heart mass as well. The data show that E2 selectively suppress the negative effects of a FRD on Akt/eNOS signalling and probably point to the different effects of E2 on kinase/phosphatase pathways responsible for phosphorylation/dephosphorylation of Akt and eNOS. Furthermore, the results suggest that the heart of females in the reproductive period is partially protected against the damaging effects of increasedfructose intake.

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.

Regulation of inducible nitric oxide synthase activity/expression in rat hearts from ghrelin-treated rats

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The purpose of this study was to examine the effects of ghrelin on protein kinase B (Akt) and mitogen-activated protein kinase p42/44 (ERK1/2) activation as well as ghrelin effects on inducible nitric oxide (NO) synthase (iNOS; for gene Nos2) activity/expression in rat hearts. Male Wistar rats were treated with ghrelin (0.3 nmol/5 ìl) or an equal volume of phosphate-buffered saline, injected every 24 h into the lateral cerebral ventricle for 5 days and 2 h after the last treatment the animals were sacrificed. Serum NO, L-arginine (L-Arg), and arginase activity were measured spectrophotometrically. For phosphorylation of Akt, ERK1/2, and iNOS protein expression, Western blot method was used. The expression of Nos2 mRNA was measured by the quantitative real-time polymerase chain reaction (qRT-PCR). Treatment with ghrelin significantly increased NO production in serum by 1.4-fold compared with control. The concentration of L-Arg was significantly higher in ghrelin-treated rats than in control while arginase activity was significantly lower in ghrelin-treated than in control hearts. Ghrelin treatment increased phosphorylation of Akt by 1.9-fold and ERK1/2 by 1.6-fold and increased iNOS expression by 2.5-fold compared with control. In addition, ghrelin treatment increased Nos2 gene expression by 2.2-fold as determined by qRT-PCR. These results indicate that ghrelin regulation of iNOS expression/activity is mediated via Akt/ERK1/2 signaling pathway. These results may be relevant to understanding molecular mechanisms underlying direct cardiovascular actions of ghrelin (AU)

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.

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

BACKGROUND: Fructose 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 METHODS: In 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. RESULTS: Fructose-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 Ser(1177). CONCLUSION: In 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.

Regulation of inducible nitric oxide synthase activity/expression in rat hearts from ghrelin-treated rats.

The purpose of this study was to examine the effects of ghrelin on protein kinase B (Akt) and mitogen-activated protein kinase p42/44 (ERK1/2) activation as well as ghrelin effects on inducible nitric oxide (NO) synthase (iNOS; for gene Nos2) activity/expression in rat hearts. Male Wistar rats were treated with ghrelin (0.3 nmol/5 µl) or an equal volume of phosphate-buffered saline, injected every 24 h into the lateral cerebral ventricle for 5 days and 2 h after the last treatment the animals were sacrificed. Serum NO, L-arginine (L-Arg), and arginase activity were measured spectrophotometrically. For phosphorylation of Akt, ERK1/2, and iNOS protein expression, Western blot method was used. The expression of Nos2 mRNA was measured by the quantitative real-time polymerase chain reaction (qRT-PCR). Treatment with ghrelin significantly increased NO production in serum by 1.4-fold compared with control. The concentration of L-Arg was significantly higher in ghrelin-treated rats than in control while arginase activity was significantly lower in ghrelin-treated than in control hearts. Ghrelin treatment increased phosphorylation of Akt by 1.9-fold and ERK1/2 by 1.6-fold and increased iNOS expression by 2.5-fold compared with control. In addition, ghrelin treatment increased Nos2 gene expression by 2.2-fold as determined by qRT-PCR. These results indicate that ghrelin regulation of iNOS expression/activity is mediated via Akt/ERK1/2 signaling pathway. These results may be relevant to understanding molecular mechanisms underlying direct cardiovascular actions of ghrelin.

Diabetes and antioxidants: myth or reality?

Numerous studies have shown that increased oxidative stress (OxS) is present in diabetic patients. There is evidence that this OxS can be increased before complications associated with diabetes mellitus (DM) occur. However, the role and influence of OxS in the initiation and progression of DM remains the subject of debate. It has been suggested that in DM, OxS is caused by increased production of reactive oxygen species (ROS), and associated with reduction in antioxidant defenses and altered cellular redox status. Acute and chronic OxS which could enhance the development of complications associated with DM. This review considers recent findings on the role of antioxidants in controlling OxS and the incidence of DM with emphasis on animal and human studies.

Impact of estradiol on insulin signaling in the rat heart.

It is well known that variation in the concentration of estrogens affects insulin action. In this study we examine the impact of estradiol (E2) on insulin signaling in the rat heart. Ovariectomized female rats were treated with E2 6 h prior to analysis of basal protein and mRNA content of insulin signaling molecules, and additionally with insulin 30 min before the experiment to delineate E2 effects on phosphorylations and molecular associations relevant for insulin signaling. The results show that E2 decreased insulin receptor (IR) tyrosine phosphorylation, while it did not alter IR protein and mRNA content. E2 administration did not change IR substrate 1 (IRS-1) protein content and tyrosine phosphorylation, while decreased mRNA content and increased its association with the p85 subunit of phosphatidylinositol 3-kinase (PI3K). E2 decreased protein and mRNA content of IR substrate 2 (IRS-2), while did not change IRS-2 tyrosine phosphorylation and IRS-2 association with p85. The increase of IRS-1/p85 is accompanied by increase of p85 protein and mRNA levels, and by stimulation of protein kinase B (Akt) Ser(473) phosphorylation. In contrast, Akt protein and mRNA content were not changed. In summary, although in some aspects cardiac insulin signaling is obviously improved by E2 treatment (increase of p85 mRNA and protein levels, enhancement of IRS-1/p85 association and Ser(473)Akt phosphorylation), the observed decrease of IR tyrosine phosphorylation, IRS-2 protein content, and IRSs mRNA contents, suggest very complex interplay of beneficial and suppressive effects of E2, both genomic and non-genomic, in regulation of heart insulin signaling.

Effects of dexamethasone on insulin receptor in aging.

The aim of this study was to examine the effects of dexamethasone (Dex) on functional properties of the rat insulin receptor (IR). Male Mill Hill hooded rats, 3, 6, 12, 18 and 21 months old, were injected with Dex (4 mg/kg) and rat liver and erythrocytes were used for experiments 18 h after Dex administration. Treatment with Dex lowered the specific binding (SB) of insulin (INS) in the liver of 3- and 18-month-old rats and concentration of INS binding sites (N1, N2) and the dissociation constant of low-affinity binding sites (Kd2) in the liver of 6- and 18-month-old rats. In addition, Dex treatment lowered the liver IR protein level in all analyzed groups, except 21-month-old rats where it remained unchanged, but raised the IR mRNA level in 18-month-old rats. In erythrocytes, treatment with Dex decreased SB and Kd2 (in animals 3 and 6 months old) and N1 (in ones 3 and 18 months old). Following Dex treatment, the INS plasma level increased (in rats 3, 18 and 21 months old), while glucose (Glu) concentration increased in 3 and 12 months old, but decreased in 6- and 21-month-old rats. In summary, Dex exerts the strongest effect on the erythrocyte IR of 3- and 6-month-old rats and the hepatic IR of 18-month-old rats. IR in both tissues is almost insensitive to Dex in 12- and 21-month-old rats. The pattern of age-related changes of IR induced by Dex does not correlate with changes of plasma Glu and INS.

Hypothetical mechanism of sodium pump regulation by estradiol under primary hypertension.

Causal relationship between sodium and hypertension has been proposed and various changes in Na+,K+-ATPase (sodium pump) activity have been described in established primary hypertension. A number of direct vascular effects of estradiol have been reported, including its impact on the regulation of sodium pump activity and vasomotor tone. The effects of estradiol involve the activation of multiple signaling cascades, including phosphatydil inositol-3 kinase (PI3K) and p42/44 mitogen-activated protein kinase (p42/44(MAPK)). In addition, some of the effects of estradiol have been linked to activity of cytosolic phospholipase A(2) (cPLA(2)). One possible cardioprotective mechanism of estradiol involves of the interaction between estradiol and the rennin-angiotensin system (RAS). Elevated circulating and tissue levels of angiotensin II (Ang II) have been implicated in the development of hypertension and heart failure. The aim of our investigation was to elucidate the signaling mechanisms employed by estradiol and Ang II in mediating sodium pump, in vascular smooth muscle cells (VSMC). The aim of our investigation was to elucidate the signaling mechanisms employed by estradiol and Ang II in mediating sodium pump activity/expression in VSMC, with particular emphasis on PI3K/cPLA(2)/p42/44(MAPK) signaling pathways. Our primary hypothesis is that estradiol stimulates sodium pump activity/expression in VSMC via PI3K/cPLA(2)/p42/44(MAPK) dependent mechanism and, that impaired estradiol-stimulated sodium pump activity/expression in hypertensive rodent models (i.e. SHR), Ang II-mediated vascular impairment of estradiol is related to a decrease ability of estradiol to stimulate the PI3K/cPLA(2)/p42/44(MAPK) signaling pathways. An important corollary to this hypothesis is that in hypertensive state (i.e. SHR rats) the decreasing in ACE enzyme activity and/or AT1 receptor expression caused by administration of estradiol is accompanying with abrogated ability of Ang II to decrease IRS-1/PI3K association, and consequent PI3K/cPLA(2)/p42/44(MAPK) activity and associated sodium pump activity/expression. A clear characterization of how Ang II attenuates estradiol signaling may lead to a better understanding of the molecular mechanism(s) underlying pathophysiological conditions such as hypertension and to understanding how certain pathophysiological situations affect sodium pump activity/expression in VSMC.

Regulation of the inducible nitric oxide synthase and sodium pump in type 1 diabetes.

Insulin-like growth factor-1 (IGF-1) is a hormone and growth factor closely related to insulin. The autocrine/paracrine actions of IGF-1 involve activation of inducible nitric oxide synthase (iNOS) and the Na(+), K(+)-ATPase sodium pump in cardiovascular tissues. Data from literature indicate that iNOS is expressed in vascular smooth muscle cells (VSMC) and that IGF-1-induced release of NO is both rapid and delayed. We hypothesize that impaired IGF-1-induced sodium pump activity/expression in rats with type 1 diabetes is related to activation of phosphatidylinositol 3 kinase (PI3K)/cytosolic phospholipase 2 (cPLA(2))/protein kinase B (Akt) signaling, and that IGF-1 prevents acute and chronic dysfunction of iNOS and sodium pump activity in a chemically induced model of type 1 diabetes, the streptozotocin-treated rat heart (STZ). Understanding how iNOS and sodium pump activity are regulated by IGF-1 activation of the PI3K/cPLA(2)/Akt cascade should provide novel and fundamental knowledge regarding the regulatory actions of IGF-1 in promoting vasodilation. Since insulin resistance is currently a major focus of research, the use of IGF-1 to improve insulin resistance and glucose metabolism has opened a new arena for treatment of comorbid conditions. Future investigations should now focus on mechanisms of action of IGF-1 and its clinical applicability.

Insulin modulates rat liver glucocorticoid receptor.

This investigation used cytosol fraction of rat liver to examine the effects of insulin (INS) on functional properties of glucocorticoid receptor (GR). Male Wistar rats (220-250 g b.wt.) were injected with INS (50 microg/200 g b.wt, i.p.) and 18 h after INS administration used for experiments. INS-stimulated dissociation of G-R complexes was significantly increased by 133% compared to control level. However, INS treatment significantly stimulated stability of GR protein by 138% above control value. Furthermore, results show that INS stimulated activation of formed cytosol [3H] TA-R complexes by 143% in respect to control. [3H]TA-R complexes from INS treated animals could be activated and accumulated at higher rate in cell nuclei of control animals. The physiological relevance of the data was confirmed by INS-related stimulation of Tryptophan oxigenase (TO) activity. It was observed that INS stimulated TO activity while INS injected to adrenalectomized rats, exhibited less effects compared to control. The results indicate that a glucocorticoid hormone (CORT) enhances INS induced stimulation of TO activity, as evidenced by enhanced enzyme activity. Presented data suggest: that INS treatment leads to modifications of the GR protein and the nuclear components and that INS activates the rat liver CORT signaling pathway which mediates, in part, the activity of TO.

[Molecular basis of glucocorticoid action].

Glucocorticoid hormones are involved in regulation of cell processes and coordinate physiological response to diverse signals. These hormones, through interaction with specific intracellular receptors, coordinate components of physiological repertoires by activating the expression of gene networks. Thus hormone-receptor complexes may function as key constituent in regulation of specific cell functions as well as in provoking differentiation in already determined cells. Analysis of steroid receptors are important for understanding of molecular details of transcriptional control as well as providing the insight as to how an individual transcriptional factor such as glucocorticoid receptor, contributes to cell identity and function. The purpose of this review is to establish the general molecular mechanism of glucocorticoid action and mechanism associated hormone-receptor complexes with the control of differential patterns (i.e. "positive" and "negative") of gene expression. One of the examples of two signal pathways regulating opposite gene expression are NF-kB and GR-mediated signal pathways. These pathways have important and opposite roles in the immune function. NF-kB is transcription factor which induces the expression of many genes that participate in immune and inflamatory response, while GR is transcription factor that serves as antiinflammatory agent and immune suppressor. Their interactions within the cell, although not yet completely understood, appear to be an important, possibly even the primary mechanism of immune homeostasis. It has not been established that glucocorticoid sensitivity can be caused by mechanisms other than changes of GR number and properties, although recent studies have indicated that receptor isoforms and transcriptional factors may modulate glucocorticoid responsiveness by interacting with receptor protein or directly at the site of DNA binding. The aim of this review is also to describe the role of glucocorticoid receptors in mechanism of glucocorticoid action on cell functions, including immune responses, as well as to present emerging issues on clinical aspects of molecular mechanisms of glucocorticoid action.

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.