Differentiation Sensitivity Research Articles

The potential insulin sensitizing and glucose lowering effects of a novel indole derivative in vitro and in vivo

Thiazolidinediones (TZDs) such as rosiglitazone are antidiabetic peroxisome proliferator-activated receptor γ (PPARγ) agonists. PPARγ agents improve diabetes by increasing insulin sensitivity and enhancing the differentiation of preadipocytes into adipocytes. The present study aimed to identify if 1-(4-chlorobenzoyl)-5-hydroxy-2-methyl-3-indoleacetitic acid (GY3), a newly synthesized indole compound, could enhance adipocytes differentiation and insulin sensitivity. The results showed that both GY3 and rosiglitazone significantly increased the lipid accumulating of 3T3-L1 adipocytes induced by isobutylmethylxanthine, dexamethasone and insulin mixture, but GY3 (not rosiglitazone) failed to increase the lipid accumulation when induced by insulin alone. In addition, GY3- or rosiglitaozne-induced protein expression of GLUT4 and adiponectin was determined by Western blot analysis. GY3 activated PPARα weakly but did not affect PPARγ, while rosiglitazone activated PPARγ significantly, suggesting different mechanisms between GY3 and rosiglitazone on adipocyte differentiation. Furthermore, both GY3 and rosiglitazone enhanced the adiponectin and insulin pathway proteins expression and adiponectin secretion in mature adipocytes, but only GY3 not rosiglitazone elevated gene expression of leptin and resistin. Both GY3 and rosiglitazone enhanced glucose consumption in HepG2 cells especially in the presence of insulin. In the in vivo study, GY3 decreased serum glucose and insulin in db/ db mice, indicating the insulin sensitizing effect might contribute to its antidiabetic mechanism. Altogether, these results suggest that GY3 could improve insulin resistance and lower glucose level, GY3 and its derivatives might be developed as a substitution therapy for diseases with insulin resistance.

Developmental aspects of adipose tissue in GH receptor and prolactin receptor gene disrupted mice: site-specific effects upon proliferation, differentiation and hormone sensitivity

Direct metabolic effects of GH on adipose tissue are well established, but effects of prolactin (PRL) have been more controversial. Recent studies have demonstrated PRL receptors on adipocytes and effects of PRL on adipose tissue in vitro. The role of GH in adipocyte proliferation and differentiation is also controversial, since GH stimulates adipocyte differentiation in cell lines, whereas it stimulates proliferation but inhibits differentiation of adipocytes in primary cell culture. Using female gene disrupted (ko) mice, we showed that absence of PRL receptors (PRLRko) impaired development of both internal and s.c. adipose tissue, due to reduced numbers of adipocytes, an effect differing from that of reduced food intake, where cell volume is decreased. In contrast, GHRko mice exhibited major decreases in the number of internal adipocytes, whereas s.c. adipocyte numbers were increased, even though body weight was decreased by 40-50%. The changes in adipose tissue in PRLRko mice appeared to be entirely due to extrinsic factors since preadipocytes proliferated and differentiated in similar fashion to wild-type animals in vitro and their response to insulin and isoproterenol was similar to wild-type animals. This contrasted with GHRko mice, where s.c. adipocytes proliferated, differentiated, and responded to hormones in identical fashion to controls, whereas parametrial adipocytes exhibited markedly depressed proliferation and differentiation potential and failed to respond to insulin or noradrenaline. Our results provide in vivo evidence that both GH and PRL stimulate differentiation of adipocytes but that the effects of GH are site specific and induce intrinsic changes in the precursor population, which are retained in vitro.

Expression and Potential Role of Peroxisome Proliferator-Activated Receptor γ in the Placenta of Diabetic Pregnancy

Peroxisome proliferator-activated receptor γ (PPARγ) is expressed predominantly in adipose tissue and is known to be involved in adipocyte differentiation and insulin sensitivity. Recent reports indicated that PPARγ-deficient mice were embryonic lethal due to abnormal placental development, suggesting that PPARγ plays an important role in normal development of placenta. On the other hand, expression of vascular endothelial growth factor (VEGF), the other important factor in placental development, has been demonstrated to be regulated by PPARγ in vascular smooth muscle cells. Also, diabetic pregnancy is often associated with defective placental functions. In order to investigate physiological roles of PPARγ and VEGF in placental development during diabetic pregnancy, we examined the expressions of PPARγ and VEGF in placentas, which were obtained from normal and streptozotocin-induced diabetic pregnant mouse, and studied in vitro effects of hyperglycemic condition and PPARγ ligands (rosiglitazone and 15-deoxy-delta(12,14)prostaglandin J 2) on trophoblasts using human choriocarcinoma cell lines. In diabetic mouse placentas ( n = 5), expressions of PPARγ and VEGF proteins significantly increased as compared with these in normal placenta ( n = 3 or 4). In vitro studies indicated that hyperglycemic condition (42 mM) significantly enhanced the PPARγ expression and hCG production, and significantly suppressed cell proliferation, however these effects were attenuated by PPARγ ligands that accompanied with increased VEGF production. These data suggest that the PPARγ pathway might be involved in the impairment of placental development induced by high glucose conditions, and that VEGF might play some roles in this pathway.

Energy balance and food intake: The role of PPARγ gene polymorphisms

Mechanisms regulating energy balance involve complex interactions between genetic, environmental and behavioural (learnt and intrinsic) factors. Genotype may drive the partitioning of energy metabolism and predispose to site-specific adiposity, culminating in a state of energy imbalance. One candidate gene with a direct link to adiposity is the peroxisome proliferator-activated receptor γ ( PPARG) gene. PPARG is a cell nuclear receptor expressed almost exclusively in adipose tissue that regulates adipocyte differentiation, lipid metabolism and insulin sensitivity. PPARγ appears to be a key regulator of energy balance, with polymorphisms on the PPARG gene linked to obesity and effects on body composition. Our research has confirmed an association between the pro12ala allele and reduced incidence of obesity in pre-pubertal children and there are strong associations between genetic variation at the PPARG locus and percentage body fat. Moreover, our evidence suggests that PPARG C-681G and pro12ala polymorphisms display opposing effects in terms of growth phenotype, with pro12Ala associated with deficient energy utilisation, leading to reduced growth and the G-681 variant associated with accelerated growth compared with wildtypes. Common differences in this gene have also been associated with variations in body weight in response to dietary macronutrients. Preliminary evidence suggests that PPARG variants may even be involved in the control of short term energy compensation. Taken together these data suggest that the role of PPARG is varied and complex, influencing fat deposition and growth velocity early in life, with potential impact in the control of energy intake and appetite regulation, and could provide a key target for future research and anti-obesity agents.

Les lipodystrophies secondaires aux traitements antirétroviraux de l’infection par le VIH

HIV infection requires the continuous administration of antiretroviral molecules. Individual molecules belonging to the two main classes, protease inhibitors (PIs) and nucleoside analogues inhibitors of the viral reverse transcriptase (NRTIs) have been shown to be involved in deleterious side effects collectively called the lipodystrophy syndrome. This syndrome associates altered body fat repartition (peripheral lipoatrophy and visceral fat hypertrophy) and metabolic alterations (dyslipidemia, insulin resistance and diabetes). The pathophysiology of these alterations is complex but different studies argue for adipose tissue being a target of some PIs and NRTIs acting through different mechanisms. NRTIs are able to induce mitochondrial dysfonction and to modify adipocyte phenotype and adipose tissue pattern of secretion of cytokines (TNFalpha, IL-6) and other adipokines (adiponectin, leptin) probably through the production of reactive oxygen species. Some PIs also act on adipocyte, alter its differentiation and insulin sensitivity and also the pattern of secretion of adipokines by adipose tissue. These hypotheses could explain the loss of adipose tissue, while the mechanisms of visceral fat hypertrophy remain speculative. Since some adipokines and the free fatty acids released by adipocytes play a major role in the control of liver and muscles insulin sensitivity, these alterations are probably involved in the metabolic alterations seen in the patients. In addition, lipodystrophic adipose tissue could be involved in the increased lesions of atherogenesis and steatohepatitis presented by these patients. The treatment of lipodystrophy remains difficult and, at present, privileges the switch of the more deleterious drugs towards new molecules less aggressive for adipose tissue.

Daidzein stimulates glucose uptake through activation of PPARγ in 3T3‐L1 adipocytes

Recent studies have shown that soy isoflavones favorably alter metabolic phenotypes associated with type 2 diabetes. However, the insulin-sensitizing effects of soy isoflavones are unclear. We have previously reported that daidzein, one of major isoflavones, enhanced differentiation, glucose uptake and translocation of GLUT4 protein in 3T3-L1 adipocytes. Given the role of PPARγ in adipocyte differentiation and insulin sensitivity, we investigated the effects of daidzein on the differentiation, PPARγ activity, and PPARγ-dependent downstream responses. A PPAR-responsive reporter gene was transiently cotransfected with PPARγ expression plasmids in 3T3-L1 cells. Compared to control, daidzein significantly increased transcriptional activity of PPARγ in a dose-dependent manner (3 and 4.2-fold at 10 and 20 μM, respectively). Furthermore, the daidzein-enhanced adipocyte differentiation was markedly inhibited by a PPARγ antagonist (BADGE). We also examined whether the potentiating effects of daidzein on glucose uptake in adipocytes were mediated via PPARγ. The daidzein-enhanced glucose uptake correlated with an increase in total cellular and plasma membrane expression of GLUT4 protein as well as GLUT4 mRNA. Combination of daidzein and BADGE (100 μM) completely abrogated daidzein-induced glucose uptake as well as GLUT4 protein abundance. These results indicate that the mechanism by which daidzein increases glucose uptake may be accounted for upregulation of GLUT4 expression through PPARγ activation.

223 COMPARISON OF NEURAL DIFFERENTIATION BETWEEN RAT AND CAT MESENCHYMAL STEM CELLS UNDER CHALLENGE BY THE SAME NEUROINDUCTION AGENT

Bone marrow-derived mesenchymal stem cells (MSCs) have the potential to differentiate into a variety of cell types, including osteocytes, chondrocytes, and adipocytes. Moreover, MSCs have the capacity to differentiate into neural lineages. The present study aimed to investigate the differences between the expression profiles during neural differentiation of rat and cat MSCs. MSCs in the 4th passage, which were isolated from the femurs and tibias by standard methodology, were used in our study. Culture media was divided into pre-induction medium, which consisted of DMEM-HG, 10% fetal bovine serum, 10 ng mL-1 of basic fibroblast growth factor (bFGF), and 1 mM �-mercaptoethanol, for 24 h, and neuron induction medium, which was composed of DMEM-HG, 2% DMSO, 200 �M BHA, 25 mMKCl, 10 �M forskolin, 5 �g mL-1 insulin-transferrin-sodium selenite, and 20 ng mL-1 bFGF, for 24 h. Thereafter, cell morphology and growth traits were determined by light microscopy imaging and by examining the cell-surface antigen profile and differentiation repertoire by immunocytochemistry, respectively. Regarding the expression of 3 MSC-related surface antigens, rat cells were positive for CD18 and CD44, whereas cat cells expressed CD9 and CD44. Under proneurogenic conditions, rat neuron-like cells progressively increased at 30 min post-induction, peaked at 1 h, and gradually declined after 12 h. At 24 h after neural induction, there were still some neuron-like cells. Meanwhile, cat cells were expressed increasingly during the first hour, peaked at 2 to 3 h, were sustained for 8 h after neural induction, and then gradually declined.A few neuron-like cells remained until 24 h post-induction. In neural differentiation, rat MSCs were positive for �III-tubulin, NF-L, NF-M, NF-H, trkA, and vimentin; cat MSCs were negative for �III-tubulin and NF-H but positive for NF-L, NF-M, trkA, and vimentin. Both MSCs were negative for oligodendrocyte markers. Our results revealed that there is variation in neural differentiation sensitivity due to species type. Rat cells were more sensitive in response to neuroinduction agents, maintained their morphology for a longer time, and expressed relatively mature neuronal markers. On the contrary, the sensitivity of cat cells was weaker, and survival time was shorter compared to that of the rat cells. The cat cells expressed immature neuronal markers. The present data suggest a significant aspect of the culture of MSCs from higher-grade species. This work was supported by KOSEF (Grant ? M10525010001-05N2501-00110).

Response of human rhabdomyosarcoma cell lines to retinoic acid: Relationship with induction of differentiation and retinoic acid sensitivity

The ability of retinoids to induce growth inhibition associated with differentiation of diverse cell types makes them potent anti-cancer agents. We examined the effect of retinoic acid (RA) in cell lines derived from rhabdomyosarcoma (RMS), a malignant soft-tissue tumor committed to the myogenic lineage, but arrested prior to terminal differentiation. We showed that several RMS derived cell lines, including RD human rhabdomyosarcoma cells, are resistant to the growth-inhibitory and differentiation effects of RA. We established that this RA-resistance correlates with reduced expression and activity of RA-receptors in RD cells. We stably expressed either RARalpha, RARbeta, RARgamma, or RXRalpha expression vector into RD cells and found that only RARbeta or RARgamma induced a significant RA growth arrest without promoting differentiation indicating that changes in the amounts of RARs and RXRs are not sufficient to determine the RA myogenic response of rhabdomyosarcoma cells. Activation of RD cell differentiation by ectopic MRF4 expression enhanced RA-receptor activity and led to RA induction of differentiation. These studies demonstrate that RA-resistance of RD cells is linked to their lack of differentiation and suggest that the differentiation-promoting activity of RA requires factors other than RAR-RXR heterodimers.

Ligands for the peroxisome proliferator-activated receptor-γ have inhibitory effects on growth of human neuroblastoma cells in vitro

The thiazolidinedione (TZD) or glitazone class of peroxisome proliferator-activated-γ (PPAR-γ) ligands not only induce adipocyte differentiation and increase insulin sensitivity, but also exert growth inhibitory effects on several carcinoma cell lines in vitro as well as in vivo. In the current study the in vitro effect of four PPAR-γ agonists (ciglitazone, pioglitazone, troglitazone, rosiglitazone) on the cell growth of seven human neuroblastoma cell lines (Kelly, LAN-1, LAN-5, LS, IMR-32, SK-N-SH, SH-SY5Y) was investigated. Growth rates were assessed by a colorimetric XTT-based assay kit. Expression of PPAR-γ protein was examined by immunohistochemistry and Western blot analysis. All glitazones inhibited in vitro growth and viability of the human neuroblastoma cell lines in a dose-dependent manner showing considerable effects only at high concentrations (10 μM and 100 μM). Effectiveness of the glitazones on neuroblastoma cell growth differed depending on the cell line and the agent. The presence of PPAR-γ protein was demonstrated in all cell lines. Our findings indicate that ligands for PPAR-γ may be useful therapeutic agents for the treatment of neuroblastoma. Thus the effect of glitazones on the growth of neuroblastoma should now be investigated in an in vivo animal model.

More Evidence of Cardiorenal Protective Effects of Peroxisome Proliferator-Activated Receptor Activation

Peroxisome proliferator-activated receptors (PPARs) are nuclear receptors that heterodimerize with the retinoid X receptor and modulate functions of many target genes. Three PPARs, α, β/δ, and γ, have been demonstrated. PPARα is involved in fatty acid oxidation and expressed in liver, kidney, and skeletal muscle; PPARβ/δ is ubiquitous and regulates lipid metabolism; and PPARγ plays a role in fat cell differentiation, lipid storage, and insulin sensitivity.1 PPARs are present in cardiovascular tissues, including the endothelium, smooth muscle cells, macrophages, and the heart.2–4 Fibrates (hypolipemic agents) and fatty acids activate PPARα, and thiazolidinediones or glitazones (antidiabetic drugs) stimulate PPARγ.5 However, the endogenous ligands of PPARs remain unknown. PPARα and PPARγ have increasingly been demonstrated to exert cardiovascular protective effects, independent of their metabolic actions.3,4 Whereas metabolic effects of PPARs are mediated by activation of a PPAR-responsive element present in the promoter region of different genes, the cardiovascular protective actions may result from anti-inflammatory and antioxidant actions mediated by transrepression of proinflammatory and pro-oxidant …

Modelling and process optimization for functionally graded materials

AbstractWe optimize continuous quench process parameters to produce functionally graded aluminium alloy extrudates. To perform this task, an optimization problem is defined and solved using a standard non‐linear programming algorithm. Ingredients of this algorithm include (1) the process parameters to be optimized, (2) a cost function: the weighted average of the precipitate number density distribution, (3) constraint functions to limit the temperature gradient (and hence distortion and residual stress) and exit temperature, and (4) their sensitivities with respect to the process parameters. The cost and constraint functions are dependent on the temperature and precipitate size which are obtained by balancing energy to determine the temperature distribution and by using a reaction‐rate theory to determine the precipitate particle sizes and their distributions. Both the temperature and the precipitate models are solved via the discontinuous Galerkin finite element method. The energy balance incorporates non‐linear boundary conditions and material properties. The temperature field is then used in the reaction rate model which has as many as 105 degrees‐of‐freedom per finite element node. After computing the temperature and precipitate size distributions we must compute their sensitivities. This seemingly intractable computational task is resolved thanks to the discontinuous Galerkin finite element formulation and the direct differentiation sensitivity method. A three‐dimension example is provided to demonstrate the algorithm. Copyright © 2004 John Wiley & Sons, Ltd.

Peroxisome proliferator-activated receptors and cardiovascular remodeling.

Peroxisome proliferator-activated receptors (PPARs) are nuclear receptors that heterodimerize with the retinoid X receptor and then modulate the function of many target genes. Three PPARs are known: alpha, beta/delta, and gamma. The better known are PPAR-alpha and PPAR-gamma, which may be activated by different synthetic agonists, although the endogenous ligands are unknown. PPAR-alpha is involved in fatty acid oxidation and expressed in the liver, kidney, and skeletal muscle, whereas PPAR-gamma is involved in fat cell differentiation, lipid storage, and insulin sensitivity. However, both have been shown to be present in variable amounts in cardiovascular tissues, including endothelium, smooth muscle cells, macrophages, and the heart. The activators of PPAR-alpha (fibrates) and PPAR-gamma (thiazolidinediones or glitazones) antagonized the actions of angiotensin II in vivo and in vitro and exerted cardiovascular antioxidant and anti-inflammatory effects. PPAR activators lowered blood pressure, induced favorable effects on the heart, and corrected vascular structure and endothelial dysfunction in several rodent models of hypertension. Activators of PPARs may become therapeutic agents useful in the prevention of cardiovascular disease beyond their effects on carbohydrate and lipid metabolism. Some side effects, such as weight gain, as well as documented aggravation of advanced heart failure through fluid retention by glitazones, may, however, limit their therapeutic application in prevention of cardiovascular disease.

The expression of immediate early gene X-1 (IEX-1) is differentially induced by retinoic acids in NB4 and KG1 cells: possible implication in the distinct phenotype of retinoic acid-responsive and -resistant leukemic cells.

In a cell-type- and stimulus-dependent fashion, the early response gene immediate early gene X-1 (IEX-1) is involved in growth control and modulation of apoptosis. The present study demonstrates that, in the two acute promyelocytic leukemia (APL) cell lines NB4 and KG1, exhibiting distinct responsiveness to retinoic acids (RAs), IEX-1 expression is rapidly (30-60 min) induced by all-trans- or cis-RA and independently of other signal transduction mediators, such as TNFalpha, NF-kappaB or MAP kinases. In NB4 cells (expressing PML-RARalpha), this increase is transient and completely reversible, along with a cell cycle arrest, ongoing differentiation and lower sensitivity to anti-cancer-drug-induced apoptosis. In contrast, the RA-induced IEX-1 expression in KG1 cells (expressing PLZF-RARalpha) persists over days, along with continued cell cycle progression and increased apoptotic sensitivity. Furthermore, two functional RA-response elements in the IEX-1 promoter were identified by gel shift and luciferase reporter gene assays. IEX-1 might be a rather unique transcriptional target of the two X-RARalpha fusion receptors exhibiting distinct responsiveness to RAs. Following a different time course of direct transcriptional induction by PML-RARalpha and PLZF-RARalpha in NB4 and KG1 cells, respectively, IEX-1 expression may be involved in the modified actions of these receptors and the distinct phenotypes of APL cells.

Sensitivity analysis and optimization of thermo-elasto-plastic processes with applications to welding side heater design

A computational scheme is presented to optimize quasi-static weakly coupled thermo-elasto-plastic processes in three dimensional Lagrangian reference frames. Sensitivity formulations are developed from the radial return algorithm based thermo-elasto-plastic finite element equations using the direct differentiation method. These formulations are exemplified in the optimization of the side heaters in the transient thermal tensioning welding process for minimum residual stress. The results of the direct differentiation sensitivity analysis are validated by comparing with finite difference sensitivity calculations. Optimization is performed using the BFGS line search method.

RNA interfering approach for clarifying the PPARγ pathway using lentiviral vector expressing short hairpin RNA

Peroxisome proliferator-activated receptor γ (PPARγ) plays a central role in adipocyte differentiation and insulin sensitivity. Although PPARγ also appears to regulate diverse cellular processes in other cell types such as lymphocytes, the detailed mechanisms remain unclear. In this study, we established a lentivirus-mediated short hairpin RNA expression system and identified a potent short hairpin RNA which suppresses PPARγ expression, resulting in marked inhibition of preadipocyte-to-adipocyte differentiation in 3T3-L1 cells. Our PPARγ-knockdown method will serve to clarify the PPARγ pathway in various cell types in vivo and in vitro, and will facilitate the development of therapeutic applications for a variety of diseases.

Analysis of candidate genes in Polish families with obesity.

This study analyzes the relationship between risk factors related to overweight/obesity, insulin resistance, lipid tolerance, hypertension, endothelial function and genetic polymorphisms associated with: i) appetite regulation (leptin, melanocortin-3-receptor (MCR-3), dopamine receptor 2 (D2R)); ii) adipocyte differentiation and insulin sensitivity (peroxisome proliferator-activated receptor-gamma2 (PPAR-gamma2), tumor necrosis factor-alpha (TNF-alpha)); iii) thermogenesis and free fatty acid (FFA) transport/catabolism (uncoupling protein-1 (UCP1), lipoprotein lipase (LPL), beta2- and beta3-adrenergic receptor (beta2AR, beta3AR), fatty acid transport protein-1 (FATP-1) and iv) lipoproteins (apoliprotein E (apoE), apo CIII). The 122 members of 40 obese Caucasian families from southern Poland participated in the study. The genotypes were analyzed by restriction fragment length polymorphism-polymerase chain reaction (RFLP-PCR) or by direct sequencing. Phenotypes related to obesity (body mass index (BMI), fat/lean body mass composition, waist-to-hip ratio (WHR)), fasting lipids, glucose, leptin and insulin, as well as insulin during oral glucose tolerance test (OGTT) (4 points within 2 hours) and during oral lipid tolerance test (OLTT) (5 points within 8 hours) were assessed. The insulin sensitivity indexes: homeostasis model assessment of insulin resistance, whole body insulin sensitivity index, hepatic insulin sensitivity and early secretory response to an oral glucose load (HOMA-IR, ISI-COMP, ISI-HOMA and DELTA) were calculated. The single gene mutations such as C105 T OB and Pro115 Gln PPAR-gamma2 linked to morbid obesity were not detected in our group. A weak correlation between obesity and certain gene polymorphisms was observed. Being overweight (25 < BMI > or = 30 kg/m2) significantly correlated with worse FFA tolerance in male PPAR-gamma2 12Pro, LPL-H (G) allele carriers. Insulin resistance was found in female PPAR-gamma2 Pro12, TNF-alpha (-308A) and LPL-H (G) allele carriers. Hypertension linked to the PPAR-gamma2 Pro allele carriers was characterized by high leptin output during OLTT. We conclude that the polymorphisms we investigated were weakly correlated with obesity but significantly modified the risk factors of the metabolic syndrome.

Thiazolidinediones: a new class of drugs for the therapy of ischemia-reperfusion injury.

Synthetic peroxisome proliferator-activated receptor gamma (PPAR-gamma) ligands shown to induce adipocyte differentiation and increase insulin sensitivity, thiazolidinediones have recently been implicated as regulators of cellular inflammatory and ischemic responses. Thiazolidinediones inhibit the activity of a number of transcriptional factors, including nuclear factor-gammaB, activating protein 1, signal transducers and activated T cells, and early response gene 1 by either PPAR-gamma-dependent or -independent mechanisms. Recent experimental studies demonstrated a dramatic protection by thiazolidinediones against cardiac, gastrointestinal and lung injury after ischemia-reperfusion, in addition to a significant reduction in infiltrating neutrophils. These reports have hypothesized that the inhibition of neutrophil-mediated inflammation by thiazolidinediones is involved in the reduction of ischemia-reperfusion-induced tissue injury. Altogether, recent findings suggest that thiazolidinediones could represent a novel therapeutic approach to reducing or preventing reperfusion injury.

Ceramide Disables 3-Phosphoinositide Binding to the Pleckstrin Homology Domain of Protein Kinase B (PKB)/Akt by a PKCζ-Dependent Mechanism

Ceramide is generated in response to numerous stress-inducing stimuli and has been implicated in the regulation of diverse cellular responses, including cell death, differentiation, and insulin sensitivity. Recent evidence indicates that ceramide may regulate these responses by inhibiting the stimulus-mediated activation of protein kinase B (PKB), a key determinant of cell fate and insulin action. Here we show that inhibition of this kinase involves atypical PKCzeta, which physically interacts with PKB in unstimulated cells. Insulin reduces the PKB-PKCzeta interaction and stimulates PKB. However, dissociation of the kinase complex and the attendant hormonal activation of PKB were prevented by ceramide. Under these circumstances, ceramide activated PKCzeta, leading to phosphorylation of the PKB-PH domain on Thr(34). This phosphorylation inhibited phosphatidylinositol 3,4,5-trisphosphate (PIP(3)) binding to PKB, thereby preventing activation of the kinase by insulin. In contrast, a PKB-PH domain with a T34A mutation retained the ability to bind PIP(3) even in the presence of a ceramide-activated PKCzeta and, as such, expression of PKB T34A mutant in L6 cells was resistant to inhibition by ceramide treatment. Inhibitors of PKCzeta and a kinase-dead PKCzeta both antagonized the inhibitory effect of ceramide on PKB. Since PKB confers a prosurvival signal and regulates numerous pathways in response to insulin, suppressing its activation by a PKCzeta-dependent process may be one mechanism by which ceramide promotes cell death and induces insulin resistance.

The Orphan Nuclear Receptor Rev-Erbα Is a Peroxisome Proliferator-activated Receptor (PPAR) γ Target Gene and Promotes PPARγ-induced Adipocyte Differentiation

Rev-Erbalpha (NR1D1) is an orphan nuclear receptor encoded on the opposite strand of the thyroid receptor alpha gene. Rev-Erbalpha mRNA is induced during adipocyte differentiation of 3T3-L1 cells, and its expression is abundant in rat adipose tissue. Peroxisome proliferator-activated receptor gamma (PPARgamma) (NR1C3) is a nuclear receptor controlling adipocyte differentiation and insulin sensitivity. Here we show that Rev-Erbalpha expression is induced by PPARgamma activation with rosiglitazone in rat epididymal and perirenal adipose tissues in vivo as well as in 3T3-L1 adipocytes in vitro. Furthermore, activated PPARgamma induces Rev-Erbalpha promoter activity by binding to the direct repeat (DR)-2 response element Rev-DR2. Mutations of the 5' or 3' half-sites of the response element totally abrogated PPARgamma binding and transcriptional activation, identifying this site as a novel type of functional PPARgamma response element. Finally, ectopic expression of Rev-Erbalpha in 3T3-L1 preadipocytes potentiated adipocyte differentiation induced by the PPARgamma ligand rosiglitazone. These results identify Rev-Erbalpha as a target gene of PPARgamma in adipose tissue and demonstrate a role for this nuclear receptor as a promoter of adipocyte differentiation.

Peroxisome proliferator-activated receptors: vascular and cardiac effects in hypertension.

Peroxisome proliferator-activated receptors (PPAR) are nuclear receptors acting as transcription factors on numerous target genes after heterodimerization with the retinoid X receptor. PPAR-alpha and PPAR-gamma may be activated by different agonists, although the endogenous ligands are unknown. Although PPAR-alpha is mainly involved in fatty acid oxidation and expressed in liver, kidney, and skeletal muscle, and PPAR-gamma is mainly involved in fat cell differentiation and insulin sensitivity, both are expressed in smooth muscle cells and myocardium, although PPAR-gamma are scarce in the latter. Activators of PPAR-alpha such as fatty acids and fibrates, and PPAR-gamma such as thiazolidinediones have been shown to exert antiproliferative effects, antagonize angiotensin II actions in vivo and in vitro, and exert antioxidant actions inhibiting generation of reactive oxygen species and activation of inflammatory mediators on blood vessels and the heart. These agents lowered blood pressure in several models of hypertension and corrected endothelial dysfunction. They exerted anti-inflammatory and antifibrotic actions on blood vessels and the heart. With the development of dual alpha/gamma-PPAR activators, these newer agents may become interesting therapeutic agents for prevention of vascular and cardiac complications of hypertension as well as for preventative therapy in other forms of cardiovascular disease.