Thyroid Hormone Receptor Alpha Deletion in ApoE-/- Mice Alters the Arterial Renin-Angiotensin System and Vascular Smooth Muscular Cell Cholesterol Metabolism.

Thyroid hormone (TH) regulates gene transcription by binding to TH receptors (TRs). TRs regulate the genes of lipid metabolism and the renin-angiotensin system (RAS). We examined the effect of TRα deletion in ApoE-/- mice (DKO mice) on the following: (i) the expression of genes controlling cholesterol metabolism and tissue (t)RAS in the liver and aorta and (ii) the expression of these genes and the regulation of cholesterol content in cultured vascular smooth muscle cells (VSMCs). TRα deletion in ApoE-/- mice led to the repression of genes involved in the synthesis and influx of cholesterol in the liver. However, TRα deletion in the arterial wall suppressed the expression of genes involved in the esterification and excretion of cholesterol and enhanced the expression of angiotensinogen (AGT). The VSMCs of the ApoE-/- and DKO mice increased their cholesterol content during cholesterol loading, but failed to increase the expression of ATP-binding cassette transporter A1 (ABCA1). T3 addition partially corrected these abnormalities in the cells of the ApoE-/- mice but not those of the DKO mice. In conclusion, TRα deletion in ApoE-/- mice slightly increases the expression of tRAS in the aorta and aggravates the dysregulation of cholesterol content in the VSMCs.

Changes in Brain Monoamines Underlie Behavioural Disruptions after Zebrafish Diet Exposure to Polycyclic Aromatic Hydrocarbons Environmental Mixtures.

Zebrafish were exposed through diet to two environmentally relevant polycyclic aromatic hydrocarbons (PAHs) mixtures of contrasted compositions, one of pyrolytic (PY) origin and one from light crude oil (LO). Monoamine concentrations were quantified in the brains of the fish after six month of exposure. A significant decrease in noradrenaline (NA) was observed in fish exposed to both mixtures, while a decrease in serotonin (5HT) and dopamine (DA) was observed only in LO-exposed fish. A decrease in metabolites of 5HT and DA was observed in fish exposed to both mixtures. Several behavioural disruptions were observed that depended on mixtures, and parallels were made with changes in monoamine concentrations. Indeed, we observed an increase in anxiety in fish exposed to both mixtures, which could be related to the decrease in 5HT and/or NA, while disruptions of daily activity rhythms were observed in LO fish, which could be related to the decrease in DA. Taken together, these results showed that (i) chronic exposures to PAHs mixtures disrupted brain monoamine contents, which could underlie behavioural disruptions, and that (ii) the biological responses depended on mixture compositions.

[Vascular calcifications, the hidden side effects of vitamin K antagonists]. / Les calcifications vasculaires sous anti-vitamines K : un effet indésirable méconnu.

Despite the availability of new oral anticoagulants, vitamin K antagonists (VKA, such as fluindione, acenocoumarol or warfarin) remain currently the goal standard medicines for oral prevention or treatment of thromboembolic disorders. They inhibit the cycle of the vitamin K and its participation in the enzymatic gamma-carboxylation of many proteins. The VKA prevent the activation of the vitamin K-dependent blood clotting factors limiting thus the initiation of the coagulation cascade. But other proteins are vitamin K-dependent and also remain inactive in the presence of VKA. This is the case of matrix Gla-protein (MGP), a protein that plays a major inhibitory role in the development of vascular calcifications. Several experimental and epidemiological results suggest that the use of the VKA could promote the development of vascular calcifications increasing thus the cardiovascular risk. This risk seems to be higher in patients with chronic kidney disease or mellitus diabetes who are more likely to develop vascular calcifications, and may be due to a decrease of the MGP activity. This review aims at summarizing the data currently available making vascular calcifications the probably underestimated side effects of VKA.

Computational identification of potential transcriptional regulators of TGF-ß1 in human atherosclerotic arteries.

TGF-ß is protective in atherosclerosis but deleterious in metastatic cancers. Our aim was to determine whether TGF-ß transcriptional regulation is tissue-specific in early atherosclerosis. The computational methods included 5 steps: (i) from microarray data of human atherosclerotic carotid tissue, to identify the 10 best co-expressed genes with TGFB1 (TGFB1 gene cluster), (ii) to choose the 11 proximal promoters, (iii) to predict the TFBS shared by the promoters, (iv) to identify the common TFs co-expressed with the TGFB1 gene cluster, and (v) to compare the common TFs in the early lesions to those identified in advanced atherosclerotic lesions and in various cancers. Our results show that EGR1, SP1 and KLF6 could be responsible for TGFB1 basal expression, KLF6 appearing specific to atherosclerotic lesions. Among the TFs co-expressed with the gene cluster, transcriptional activators (SLC2A4RG, MAZ) and repressors (ZBTB7A, PATZ1, ZNF263) could be involved in the fine-tuning of TGFB1 expression in atherosclerosis.

Early adverse changes in liver microvascular circulation during experimental septic shock are not linked to an absolute nitric oxide deficit.

Nitric oxide (NO) is believed to play a key role in adverse microvascular changes during sepsis. A deficit in NO has been evoked as a potential mechanism of microcirculatory deterioration in the early phase of septic shock. The aim of this study was to evaluate simultaneously and continuously both hepatic microcirculation and local NO production during early experimental sepsis. Wistar male rats were divided into two groups: a sepsis group undergoing cecal ligation and puncture (CLP) peritonitis and a control group undergoing sham surgery. Hepatic microcirculation was continuously monitored using a laser Doppler probe and local nitric oxide (NO) production by means of a specific electrode. Constitutive and inducible NO synthase production was assessed 2h after surgery, at onset of shock, and at 2 and 3h after shock. In control animals, hepatic microcirculatory perfusion and NO production remained stable throughout the experiment. In septic animals, whereas a fall in microcirculatory perfusion was noted as early as 2h after CLP, NO concentration remained stable and further increased after the onset of shock. At this time, inducible NO synthase was the only isoform significantly elevated. In this non-resuscitated experimental model of sepsis, an absolute liver deficit of NO could not explain the early adverse changes in the local microvascular system.

Physiological regulation of pro-inflammatory cytokines expression in rat cardiovascular tissues by sympathetic nervous system and angiotensin II.

Pro-inflammatory cytokines regulation by sympathetic nervous system (SNS) and angiotensin II (ANG II) was widely described in cardiovascular system, but the role of such neuro-humoral interaction needs further investigation in this context. We tested SNS-ANG II interaction on IL-6 and TNF-α mRNA expression in left ventricle and aorta from normotensive rats by sympathectomy with guanethidine and blockade of the ANG II AT1 receptors (AT1R) antagonist with losartan. mRNA synthesis of IL-6 and TNF-α were performed by Q-RT-PCR. In the left ventricle, IL-6 mRNA increased by 63% (p < 0.01) after sympathectomy, still unchanged after losartan treatment and decreased by 38% (p < 0.05) after combined treatment. TNF-α mRNA decreased by 44% (p < 0.01), only after combined treatment. In the aorta, IL-6 mRNA increased equally by 65% (p < 0.05) after sympathectomy or losartan treatment. TNF-α mRNA decreased by 28, 41, and 42% (p < 0.05) after sympathectomy, losartan and combined treatments, respectively. Our data suggest that ANG II stimulates directly (via AT1R) and indirectly (via SNS) IL-6 mRNA synthesis in left ventricle and aorta and TNF-α mRNA in left ventricle. ANG II seems unable to influence directly TNF-α mRNA synthesis in the aorta but can stimulate this cytokine via SNS. The results are relevant to prevent or reduce proinflammatory cytokines overexpression seen in cardiovascular diseases.

The IDEAL study : towards personalized drug treatment of hypertension.

OBJECTIVE: To identify markers (phenotypic, genetic, or environmental) of blood pressure (BP) response profiles to angiotensin converting enzyme inhibitors (ACEIs) and diuretics. METHODS: IDEAL was a crossover (two active and two wash out phases), double-blind, placebo-controlled trial. Eligible patients were untreated hypertensive, aged 25 to 70. After two visits, patients were randomized to one of four sequences. The main outcome was BP differences between the active treatment and placebo. RESULTS: One hundred and twenty-four patients were randomised: mean age 53, men 65%, family history of hypertension 60%. Average BP fall at each visit before randomisation was about 2% of the initial level reflecting both a regression to the mean and a placebo effect. CONCLUSION: The results are expected to improve knowledge in drug's mechanisms of action and pathophysiology of hypertension, and to help in personalizing treatment. The estimation of BP responses to each drug in standardized conditions provided a benefit to each participant.

Regulation of aortic extracellular matrix synthesis via noradrenergic system and angiotensin II in juvenile rats.

CONTEXT: Extracellular matrix (ECM) synthesis regulation by sympathetic nervous system (SNS) or angiotensin II (ANG II) was widely reported, but interaction between the two systems on ECM synthesis needs further investigation. OBJECTIVE: We tested implication of SNS and ANG II on ECM synthesis in juvenile rat aorta. MATERIALS AND METHODS: Sympathectomy with guanethidine (50 mg/kg, subcutaneous) and blockade of the ANG II AT1 receptors (AT1R) blocker with losartan (20 mg/kg/day in drinking water) were performed alone or in combination in rats. mRNA and protein synthesis of collagen and elastin were examined by Q-RT-PCR and immunoblotting. RESULTS: Collagen type I and III mRNA were increased respectively by 62 and 43% after sympathectomy and decreased respectively by 31 and 60% after AT1R blockade. Combined treatment increased collagen type III by 36% but not collagen type I. The same tendency of collagen expression was observed at mRNA and protein levels after the three treatments. mRNA and protein level of elastin was decreased respectively by 63 and 39% and increased by 158 and 15% after losartan treatment. Combined treatment abrogates changes induced by single treatments. DISCUSSION AND CONCLUSION: The two systems act as antagonists on ECM expression in the aorta and combined inhibition of the two systems prevents imbalance of mRNA and protein level of collagen I and elastin induced by single treatment. Combined inhibition of the two systems prevents deposit or excessive reduction of ECM and can more prevent cardiovascular disorders.

Interaction between sympathetic nervous system and renin angiotensin system on MMPs expression in juvenile rat aorta.

The aim of our present study is to investigate the interaction between angiotensin II (ANG II) and sympathetic nervous system (SNS) on matrix metalloproteinase MMP-2 and MMP-9 expression and activity in juvenile rat aorta under normal conditions. Sympathectomy with guanethidine and blockade of the ANG II receptors (AT1R) by losartan were performed alone or in combination on new-born rats. mRNA, protein expression and activity of MMP-2 and MMP-9 were examined by Q-RT-PCR, immunoblotting and zymography, respectively. MMP-2 mRNA and protein amount were decreased after sympathectomy or AT1R blockade and an additive effect was observed after combined treatment. However, MMP-9 expression was reduced to the same level in the three treated groups. There were some detectable gelatinolytic activity of the MMPs in both control and treated rats. We concluded that ANG II stimulates directly and indirectly (via sympathostimulator pathway) the MMP-2 expression but seems unable to affect MMP-9 expression through direct pathway. Combined inhibition of SNS and ANG II were more efficient than a single inhibition in reducing MMP amounts in rat vessels.

Evaluation of oxidative stress among coronary diabetics patients.

OBJECTIVES: Determination of the superoxide dismutase (SOD), glutathione peroxidase (GPX) and the total antioxidant status (TAS) and evaluation of inflammation by the use of high sensitivity C reactive protein (hs-CRP) among Tunisian coronary diabetic patients. MATERIALS AND METHODS: We measured the erythrocyte GPX activity and the plasmatic TAS concentration by colorimetric methods, the apolipoproteins [ApoA1, ApoB], hs-CRP and the fibrinogen by immunonephelometry assays. RESULTS: TAS and GPX were significantly decreased among patients compared to the controls [TAS: 1,14 +/- 0,28 mmol/l vs 1,55 +/- 0,35 mmol/l, GPX: 59,32 +/- 10,72 U/gHb vs 149,19 +/- 30,95 U/gHb]. For the coronary diabetic patients, the TAS is correlated positively with hs-CRP [r= 0,01, p<10(-3)]. Pearson's correlation shows a significantly positive correlation between GPX and TAS among all patients. CONCLUSIONS: Determination of antioxidative defense markers contributes to understanding the effect of stress oxidative on the development, prevention and therapy of cardiovascular disease. (www.actabiomedica.it).

Real time PCR for fast detection of the angiotensinogen polymorphisms.

OBJECTIVE: to develop a rapid and reliable real-time PCR to detect polymorphisms of angiotensinogen (AGT), to compare the two methods of MS-PCR (Mutagenically Separated PCR) and real-time PCR to determine three polymorphisms of the angiotensinogen gene M235T, the A (-6) G and A (-20) C. METHODS: the method of real-time PCR was developed on the PLC Roche LightCycler1 with SYBR Green I. We used two sense primers and a primer nonsense. Detection of polymorphisms of angiotensinogen gene was performed by comparing the melting curves. RESULTS: the DNA samples were analyzed by two methods: real-time PCR and MS-PCR. In our study, no differences were found between the two techniques. DISCUSSION: The real-time PCR is a rapid and reliable method for detecting gene polymorphisms on the AGT M235T, the A (-6) G and A (-20) C. CONCLUSION: this method of real-time PCR is a reliable genetic test, which is fast and cheap and can be used in practice to study particular polymorphisms of AGT gene associated with cardiovascular disease.

Conditional inactivation of TGF-ß type II receptor in smooth muscle cells and epicardium causes lethal aortic and cardiac defects.

To understand the role of TGF-ß signaling in cardiovascular development, we generated mice with conditional deletion of the TGF-ß type II receptor (TßRII) gene (Tgfbr2) in cells expressing the smooth muscle cell-specific protein SM22α. The SM22α promoter was active in tissues involved in cardiovascular development: vascular smooth muscle cells (VSMCs), epicardium and myocardium. All SM22-Cre(+/-)/Tgfbr2 (flox/flox) embryos died during the last third of gestation. About half the mutant embryos exhibited heart defects (ventricular myocardium hypoplasia and septal defects). All mutant embryos displayed profound vascular abnormalities in the descending thoracic aorta (irregular outline and thickness, occasional aneurysms and elastic fiber disarray). Restriction of these defects to the descending thoracic aorta occurred despite similar levels of Tgfbr2 invalidation in the other portions of the aorta, the ductus arteriosus and the pulmonary trunk. Immunocytochemistry identified impairment of VSMC differentiation in the coronary vessels and the descending thoracic aorta as crucial for the defects. Ventricular myocardial hypoplasia, when present, was associated to impaired α-SMA differentiation of the epicardium-derived coronary VSMCs. Tgfbr2 deletion in the VSMCs of the descending thoracic aorta diminished the number of α-SMA-positive VSMC progenitors in the media at E11.5 and drastically decreased tropoelastin (from E11.5) and fibulin-5 (from E.12.5) synthesis and/or deposition. Defective elastogenesis observed in all mutant embryos and the resulting dilatation and probable rupture of the descending thoracic aorta might explain the late embryonic lethality. To conclude, during mouse development, TGF-ß plays an irreplaceable role on the differentiation of the VSMCs in the coronary vessels and the descending thoracic aorta.

Lipogenesis in arterial wall and vascular smooth muscular cells: regulation and abnormalities in insulin-resistance.

BACKGROUND: Vascular smooth muscular cells (VSMC) express lipogenic genes. Therefore in situ lipogenesis could provide fatty acids for triglycerides synthesis and cholesterol esterification and contribute to lipid accumulation in arterial wall with aging and during atheroma. METHODS: We investigated expression of lipogenic genes in human and rat arterial walls, its regulation in cultured VSMC and determined if it is modified during insulin-resistance and diabetes, situations with increased risk for atheroma. RESULTS: Zucker obese (ZO) and diabetic (ZDF) rats accumulated more triglycerides in their aortas than their respective control rats, and this triglycerides content increased with age in ZDF and control rats. However the expression in aortas of lipogenic genes, or of genes involved in fatty acids uptake, was not higher in ZDF and ZO rats and did not increase with age. Expression of lipogenesis-related genes was not increased in human arterial wall (carotid endarterectomy) of diabetic compared to non-diabetic patients. In vitro, glucose and adipogenic medium (ADM) stimulated moderately the expression and activity of lipogenesis in VSMC from control rats. LXR agonists, but not PXR agonist, stimulated also lipogenesis in VSMC but not in arterial wall in vivo. Lipogenic genes expression was lower in VSMC from ZO rats and not stimulated by glucose or ADM. CONCLUSION: Lipogenic genes are expressed in arterial wall and VSMC; this expression is stimulated (VSMC) by glucose, ADM and LXR agonists. During insulin-resistance and diabetes, this expression is not increased and resists to the actions of glucose and ADM. It is unlikely that this metabolic pathway contribute to lipid accumulation of arterial wall during insulin-resistance and diabetes and thus to the increased risk of atheroma observed in these situations.

Density-dependent shift of transforming growth factor-beta-1 from inhibition to stimulation of vascular smooth muscle cell growth is based on unconventional regulation of proliferation, apoptosis and contact inhibition.

BACKGROUND: TGF-beta shifts from inhibition to stimulation of vascular smooth muscle cell (vSMC) growth when cell density increases. How proliferation and apoptosis contribute to this shift is still unknown. METHODS: In sparse and confluent V8 vSMC treated or not with TGF-beta(1) (1 ng/ml) for 3 days, cell number, mitotic activity, cell-cycle-regulatory protein levels, caspase-3 and phosphoinositide 3-kinase (PI3-K) activities were studied. RESULTS: In TGF-beta(1)-treated cells, (i) the growth curve rose constantly compared to controls, reaching post-confluent densities; (ii) mitotic activity, which was constant at all cell densities, was lower than in sparse but higher than in contact-inhibited control cells, and (iii) apoptosis occurred at sparse densities only. The mechanism of proliferation control by TGF-beta(1) was very unconventional in V8 vSMCs: (i) p15(INK4b) and cyclin D levels were similar in cells treated or not with TGF-beta(1), and (ii) p27(Kip1) levels remained very low even at high densities while cyclin E levels were not markedly decreased. TGF-beta(1)-induced apoptosis in sparse cultures and its reversal in dense cultures were inversely correlated to PI3-K activation. CONCLUSIONS: TGF-beta(1) slowed sparse V8 vSMC growth by inhibiting proliferation and inducing apoptosis. TGF-beta(1)-treated confluent vSMCs escaped contact inhibition and kept growing through unconventional regulation of p27(Kip1), cyclin E and suppression of apoptosis.

High-volume haemofiltration with a new haemofiltration membrane having enhanced adsorption properties in septic pigs.

BACKGROUND: High-volume haemofiltration (HVHF) has been suggested as an adjuvant treatment of septic shock due to its capacities to remove from blood both pro- and anti-inflammatory mediators involved in the sepsis syndrome. Adsorption properties of some haemofiltration membranes are also interesting with this indication because inflammatory mediators are caught in the membrane itself. The aim of this study was to determine the haemodynamic and immunological effects of a new haemofiltration membrane, which has enhanced adsorption properties due to a special surface treatment, allowing the adsorption of endotoxins. METHODS: We compared this membrane to a standard haemofiltration membrane both in vitro and in 20 sepsis-induced pigs, randomized in two groups. One group was haemofiltered with the treated membrane and the other with the standard haemofiltration membrane during 6-h HVHF sessions. RESULTS: At the end of the experiment, mean +/- SD crystalloids requirements (5937 +/- 1588 versus 7587 +/- 1456 ml, P = 0.026), colloids requirements (1437 +/- 320 versus 1912 +/- 538 ml, P = 0.027), lactic acidosis (pH = 7.20 +/- 0.11 versus 7.10 +/- 0.07, P = 0.026) and pulmonary arterial hypertension (MPAP = 24 +/- 7 versus 34 +/- 8 mmHg, P = 0.008) were less pronounced when HVHF was performed with the treated membrane. In addition, mean +/- SD endotoxins levels were lower in the treated membrane group after 1 hour of HVHF (1.91 +/- 1.19 versus 11.07 +/- 10.64 EU/ml, P = 0.035). Cytokines levels were not different between groups except for IL-1beta, which was slightly lower in the treated membrane group. CONCLUSIONS: The use of a membrane with enhanced adsorption properties during a 6-h HVHF session in septic pigs improves haemodynamics compared to a standard haemofiltration membrane. These results are probably due to an efficient endotoxins and cytokines adsorption. A human study using this membrane is now necessary to confirm these results.

Evaluation of remodeling in left and right ventricular myocytes from heterozygous (mRen2)27 transgenic rats.

Cardiac remodeling was assessed both in the pressure-overloaded left ventricle and in the normotensive right ventricle of hypertensive transgenic rats (mRen2)27 (TGR27). The present study combined histology, electrophysiology, molecular biology and biochemistry techniques. A significant increase in action potential (AP) duration was recorded both in right and left ventricular myocytes wheareas only in the latter ones were hypertrophic. The increase in AP duration is mainly supported by the reduction of the transient outward K current (I(to)) density since no significant modification was observed for the L-type calcium current (I(Ca,L)), the sodium-calcium exchange current (I(NCX)), the delayed rectifier current (I(K)) and the inward rectifier current (I(K1)). The lower amplitude of I(to) current was associated with a lower Kv4.3 protein expression both in right and left ventricles while Kv4.3 mRNA levels was decreased only in left ventricle. Thus, a differential ventricular remodeling takes place in the TGR27 model. The possible cause of electrical remodeling in right ventricular myocytes of TGR27 is discussed.

TRα inhibits arterial renin-angiotensin system expression and prevents cholesterol accumulation in vascular smooth muscle cells.

OBJECTIVES: The tissue renin-angiotensin system (tRAS) plays a key role in the maintenance of cellular homeostasis but is also implicated in atherosclerosis. Thyroid hormone (TH) contributes, via genomic effects, to control of tRAS gene expression in the arterial wall and vascular smooth muscle cells (VSMCs). We investigated the specific functions of TH receptors-α and -ß (TRα and TRß) on tRAS gene expression in the aorta and VSMCs, and the potential protective effect of TRα against atherosclerosis. MATERIAL AND METHODS: Using aorta and cultured aortic VSMCs from TRα and TRß deficient mice, tRAS gene expression was analyzed by determining mRNA levels on real-time PCR. Gene regulation under cholesterol loading mimicking atherosclerosis conditions was also examined in VSMCs in vitro. RESULTS: TRα deletion significantly increased expression of angiotensinogen (AGT) and angiotensin II receptor type 1 subtype a (AT1Ra) at transcriptional level in aorta, a tissue with high TRα expression level. TRα activity thus seems to be required for maintenance of physiological levels of AGTand AT1Raexpression in the arterial wall. In addition, during cholesterol loading, TRα deletion significantly increased cholesterol content in VSMCs, with a weaker decrease in AGTexpression. CONCLUSION: TRα seems to have an inhibitory impact on AGTand AT1Raexpression, and loss of TRα function in TRα0/0 mice increases tRAS expression in the aortic wall. More importantly, TRα deletion significantly increases VSMC cholesterol content. Our results are consistent with a protective role of TRα against atherosclerosis.

Functional meta-analysis of double connectivity in gene coexpression networks in mammals.

In functional genomics, the high-throughput methods such as microarrays 1) allow analysis of the relationships between genes considering them as elements of a network and 2) lead to biological interpretations thanks to Gene Ontology. But up to now it has not been possible to find relationships between the functions and the connectivity of the genes in coexpression networks. To achieve this aim, we have defined a double connectivity for each gene by the numbers of its significant negative and positive correlations with the other genes within a given biological condition, or group. Here, based on the analysis of 1,260 DNA microarrays, we show that this double connectivity clearly separates two types of genes, those with a predominantly strong negative connectivity, hub- genes, and those with a predominantly strong positive connectivity, hub+ genes. Interestingly, the hub+ genes concerned transcription factors more often than by chance and, similarly, for the hub- genes concerning miRNA predicted targets. Furthermore, a meta-analysis of GO annotations carried out on 67 groups in humans and rats shows that these two types of genes correspond to a functional biological duality. The hub- genes were mainly involved in basic functions common to all eukaryote cells, whereas the hub+ genes were mainly involved in specialized functions related to cell differentiation and communication. The separation and the biological role of these hub- and hub+ genes provide a powerful new tool for a better understanding of the control and regulation of the key genes involved in cellular differentiation and physiopathological conditions.

Adiponectin receptors: expression in Zucker diabetic rats and effects of fenofibrate and metformin.

The insulin-sensitizing adipokine, adiponectin, acts through 2 receptors, AdipoR1 and AdipoR2. A decreased expression of these receptors could contribute to insulin resistance and diabetes. We determined if the expression of adiponectin receptors is decreased in an experimental model, the Zucker diabetic rat (ZDF), and if a peroxisome proliferator-activated receptor alpha agonist, fenofibrate, and metformin could increase these expressions. The ZDF and control (L) rats were studied at 7, 14, and 21 weeks. After initial study at 7 weeks, ZDF received no treatment (n = 10), metformin (n = 10), or fenofibrate (n = 10) until final studies at 14 or 21 weeks. The L rats received no treatment. AdipoR1 and R2 expressions were measured in liver, muscle, and white adipose tissue (WAT). As expected, ZDF rats were insulin resistant at 7 weeks, had type 2 diabetes mellitus at 14 weeks, and had diabetes with insulin deficiency at 21 weeks. Compared with L rats, AdipoRs messenger RNA was decreased only in the WAT (P < .05) of 7-week-old ZDF rats, but was unchanged in muscle and increased in liver. Metformin and fenofibrate decreased plasma triacylglycerols (P < .01) as expected. The only effect of fenofibrate on AdipoRs was a moderate increase (P < .01) of both receptors' messenger RNA in liver. Metformin increased AdipoR1 and R2 expression in muscle (P < .01) and AdipoR1 (P < .01) in WAT. These results do not support an important role for decreased AdipoRs expression in the development of insulin resistance and diabetes. Parts of the actions of fenofibrate and of metformin could be mediated by a stimulation of the expression of these receptors in liver and in insulin-sensitive, glucose-utilizing tissues (muscle, WAT), respectively.

Increased insulin-stimulated expression of arterial angiotensinogen and angiotensin type 1 receptor in patients with type 2 diabetes mellitus and atheroma.

OBJECTIVE: Because inhibition of the renin-angiotensin system (RAS) reduces the onset of type 2 diabetes (T2D) and prevents atherosclerosis, we investigated the expression of RAS in the arterial wall of T2D and nondiabetic (CTR) patients. METHODS AND RESULTS: mRNA and protein levels of angiotensinogen (AGT), angiotensin-converting enzyme (ACE) and AT1 receptor (AT1R) were determined in carotid atheroma plaque, nearby macroscopically intact tissue (MIT), and in vascular smooth muscle cells (VSMCs) before and after insulin stimulation from 21 T2D and 22 CTR patients. AGT and ACE mRNA and their protein levels were 2- to 3-fold higher in atheroma and in MIT of T2D patients. VSMCs from T2D patients had respectively 2.5- and 5-fold higher AGT and AT1R mRNA and protein contents. Insulin induced an increase in AGT and AT1R mRNA with similar ED50. These responses were blocked by PD98059, an inhibitor of MAP-kinase in the two groups whereas wortmannin, an inhibitor of PI3-kinase, partially prevented the response in CTR patients. Phosphorylated ERK1-2 was 4-fold higher in MIT from T2D than from CTR patients. CONCLUSIONS: The arterial RAS is upregulated in T2D patients, which can be partly explained by an hyperactivation of the ERK1-2 pathway by insulin.