Catherine Cerutti

The microRNA Signature in Response to Insulin Reveals Its Implication in the Transcriptional Action of Insulin in Human Skeletal Muscle and the Role of a Sterol Regulatory Element–Binding Protein-1c/Myocyte Enhancer Factor 2C Pathway

OBJECTIVE Factors governing microRNA expressions in response to changes of cellular environment are still largely unknown. Our aim was to determine whether insulin, the major hormone controlling whole-body energy homeostasis, is involved in the regulation of microRNA expressions in human skeletal muscle. RESEARCH DESIGN AND METHODS We carried out comparative microRNA (miRNA) expression profiles in human skeletal muscle biopsies before and after a 3-h euglycemic-hyperinsulinemic clamp, with TaqMan low-density arrays. Then, using DNA microarrays, we determined the response to insulin of the miRNA putative target genes in order to determine their role in the transcriptional action of insulin. We further characterized the mechanism of action of insulin on two representative miRNAs, miR-1 and miR-133a, in human muscle cells. RESULTS Insulin downregulated the expressions of 39 distinct miRNAs in human skeletal muscle. Their potential target mRNAs coded for proteins that were mainly involved in insulin signaling and ubiquitination-mediated proteolysis. Bioinformatic analysis suggested that combinations of different downregulated miRNAs worked in concert to regulate gene expressions in response to insulin. We further demonstrated that sterol regulatory element–binding protein (SREBP)-1c and myocyte enhancer factor 2C were involved in the effect of insulin on miR-1 and miR-133a expression. Interestingly, we found an impaired regulation of miRNAs by insulin in the skeletal muscle of type 2 diabetic patients, likely as consequences of altered SREBP-1c activation. CONCLUSIONS This work demonstrates a new role of insulin in the regulation of miRNAs in human skeletal muscle and suggests a possible implication of these new modulators in insulin resistance.

Time versus frequency domain techniques for assessing baroreflex sensitivity.

Background Newer techniques to evaluate baroreflex sensitivity (BRS) are based on the analysis of blood pressure (BP) and heart rate (HR) time series in the time or frequency domain. These novel approaches are steadily gaining popularity, since they do not require injection of vasoactive substances, nor do they rely on a complex experimental set-up. Aim This review outlines and compares some basic features of the latest methods to assess spontaneous baroreflex function. Results Modern techniques for the estimation of spontaneous BRS are based on a variety of signal processing schemes and derive information on the baroreflex function from different perspectives. Thus factors such as respiration and other non-stationary agents may have different influences on the estimates provided by each of these approaches. Notwithstanding such individual specificity, however, it has been observed that in several physiological and pathophysiological conditions these techniques often provide comparable information on BRS changes over time, particularly when the estimates are averaged over time windows of a few minutes. Conclusions Due to the general agreement in the pattern of BRS among most modern methods, it seems reasonable to employ the most validated of these techniques, for which data obtained in several studies are already available.

Hemodynamic analysis of arterial pressure oscillations in conscious rats

This study examined the contribution of rhythmic fluctuations of regional blood flow and vascular conductance to the genesis of low- (LF, 0.27-0.74 Hz) and high- (HF, 0.76-5 Hz) frequency oscillations of arterial pressure. In conscious 15-week-old male intact (n = 11), guanethidine-sympathectomized (n = 8) and chronically sinoaortic denervated (n = 7) rats, arterial pressure and regional blood flow velocities (pulsed Doppler probes) were simultaneously recorded. Indices of subdiaphragmatic aortic, hindquarters and superior mesenteric conductances were calculated on a beat-to-beat basis over a 60-min period. Spectral power was calculated in the LF and HF bands using a fast Fourier transform algorithm. Transfer function analysis was also performed to calculate coherence and phase between arterial pressure and regional flows and conductances. In the LF band, spectral power of arterial pressure was decreased by approx. 85% in sympathectomized and approx. 54% in sinoaortic denervated rats. In the HF band, spectral power did not differ between the groups. In the three groups of rats, relations between arterial pressure and blood flow were characterized by a significant coherence in the HF band with little or no phase delay (synchronous oscillations). Relations between arterial pressure and vascular conductance were characterized in intact rats by a significant coherence in the LF band and a phase delay tending to pi radians (opposite oscillations), whereas in both sympathectomized and sinoaortic denervated rats, coherence did not reach significance. It is concluded that LF oscillations of arterial pressure are mostly secondary to rhythmic fluctuations in the vasomotor sympathetic tone in several regional circulations. Part of these oscillations originate from the synchronizing influence of the baroreceptor reflex. The study also suggests that the respiratory (HF) oscillations of arterial pressure involve fluctuations in cardiac output of purely mechanical origin.

Mental Stress–Induced Increase in Blood Pressure Is Not Related to Baroreflex Sensitivity in Middle-Aged Healthy Men

The baroreflex that acts to blunt blood pressure (BP) variations through opposite variations in heart rate should limit the BP increase produced by an emotional challenge. However, relations between baroreflex sensitivity and BP reactivity induced by a psychological stress in a large group of adults have never been firmly established. In 280 healthy men, rest (10 minutes) and stress (5 minutes) BP and heart rate were recorded beat to beat by a blood pressure monitor. The mental stress was elicited by a well-standardized computerized version of a word color conflict stress test (Stroop Color Test). Rest and stress baroreflex sensitivity was assessed by the cross-spectral analysis of BP and heart rate and by the sequence method. The stress-induced increase in systolic BP (22.4+/-0.1 mm Hg) was not correlated with resting baroreflex sensitivity but was slightly correlated (r=0.2, P<0.001) with BP variability assessed either by standard deviation or by mid-frequency band spectral power. Our results suggested that a centrally mediated sympathetic stimulation overcame cardiac autonomic regulation and emphasized the role of the sympathetic vasoconstriction in the pressure response at the onset of the stressing stimulation. During the sustained sympathoexcitatory phase, the cardiac baroreflex blunts BP variations but at a lower sensitivity.

ARTERIAL BAROREFLEX CONTROL OF HEART PERIOD IS NOT RELATED TO BLOOD PRESSURE VARIABILITY IN CONSCIOUS HYPERTENSIVE AND NORMOTENSIVE RATS

1. The short‐term (within 30 min periods) and the long‐term (among 30 min periods) variabilities, expressed as variation coefficients, of blood pressure (BP) and heart period (HP) were studied using a computer analysis of BP recordings in freely moving genetically hypertensive (LH), normotensive (LN) and low BP (LL) rats of Lyon strains at ages 5, 9, 21 and 40 weeks. The baroreflex control of HP was estimated with the slope of the linear relationship between systolic BP (SBP) and HP (SBP‐HP slope) computed after phenylephrine and nitroglycerin injections.

Cathepsin G is associated with atheroma formation in human carotid artery.

Objective To elucidate the organization of the tissue angiotensin system, we investigated the expression and cellular localization of angiotensin system components and cathepsins D and G, potentially involved in intraparietal angiotensin II formation and atheroma. Methods Total RNA was extracted from atheroma plaque, fatty streaks and macroscopically intact tissue obtained during carotid endarterectomy in 21 hypertensive patients. mRNA levels were compared between these tissues using a semi-quantitative reverse transcriptase-polymerase chain reaction (RT-PCR). In situ hybridization and immunohistochemistry were used to define the cellular localization of the transcripts and their respective proteins. Results Apart from renin and angiotensin type 2 (AT2) receptors, which were never detected, the studied mRNAs could be measured in all patients. Angiotensin-converting enzyme (ACE) mRNA was increased five-fold in atheroma, and angiotensin type 1 receptor (AT1) mRNA decreased 2.5-fold in atheroma and 1.4-fold in fatty streaks compared to intact tissue. A two-fold increase in cathepsin G mRNA was observed in atheroma plaque. In atheroma and intact tissue, significant positive correlations were found between cathepsin G and angiotensinogen, AT1 receptor and ACE mRNAs. Angiotensinogen and cathepsin mRNAs and proteins were detected in both arterial layers. AT1 immunoreactivity was mainly associated with α-actin-positive cells. Conclusion All components required for angiotensin II formation are expressed locally in the arterial wall, where, in the absence of renin, cathepsin G could be a major angiotensin-generating enzyme. Overexpression of ACE and cathepsin G may lead to angiotensin II overproduction and contribute, with decreased number of differentiated smooth muscle cells, to the lower amount of AT1 receptor in atheroma.

High blood pressure and metabolic disorders are associated in the Lyon hypertensive rat.

Objective: A large population of F2 rats, obtained from a cross between male Lyon hypertensive (LH) rats and female Lyon normotensive (LN) rats, was studied in order to assess the relationship between increased body weight, hyperlipidaemia and high blood pressure which characterize LH rats. Methods: Mean arterial pressure (MAP) was recorded in male, conscious, freely moving LH, LN, F1 and F2 rats aged 30 weeks. Plasma total cholesterol, high-density lipoprotein-, low-density lipoprotein- and very low-density lipoprotein-cholesterol, phospholipids, triglycerides, insulin and glucose were measured. Results: In the F2 cohort it was observed that high MAP was a recessive trait that depends on several genes and was unrelated to body weight. The left ventricular weight, corrected for tibia length, was correlated with MAP. Plasma total and high-density lipoprotein-cholesterol and phospholipids concentrations were lower in the F1 rats than in the LN rats, suggesting an overdominance of the LN alleles. In the F2 rats MAP was related to total, high-density lipoprotein- and low-density lipoprotein-cholesterol. Plasma triglycerides, insulin and the insulin: glucose ratio, which were higher in the LH rats than in the LN rats, were also correlated with MAP in the F2 cohort. Using stepwise multiple regression analysis, MAP remained correlated with plasma total cholesterol, insulin and the insulin: glucose ratio, but not with triglycerides. Conclusions: Hypertension in LH rats is a recessive trait that is independent of body weight. In addition, the cosegregation of blood pressure with plasma cholesterol and, to a lesser degree, with insulin levels, which was observed in the present study provides the first direct evidence that these phenotypes are associated and are not due simply to genetic drift in the Lyon model.

Influence of anaesthesia on the cardiovascular effects of rilmenidine and clonidine in spontaneously hypertensive rats

1 The acute cardiovascular effects of two α2‐adrenoceptor agonists, rilmenidine and clonidine, were studied in 15‐week‐old male spontaneously hypertensive rats (SHRs). The effects of these drugs were compared with intravenous (i.v.) and intracerebroventricular (i.c.v.) administration in conscious and pentobarbitone‐anaesthetized SHRs, in which aortic blood pressure (BP) was continuously recorded. 2 In conscious SHRs, i.v. doses of either rilmenidine (30, 100, 300 μg kg−1) or clonidine (3, 10, 30 μg kg−1) induced dose‐dependent short‐lasting increases in BP followed by moderate decreases associated with bradycardia, while the same three doses of both drugs given i.c.v. were devoid of BP and heart rate (HR) effects. 3 Pentobarbitone‐anaesthesia increased the sympathetic control of BP and suppressed the cardiac baroreflex sensitivity. 4 In anaesthetized SHRs, i.v. injections of the same 3 doses of rilmenidine and clonidine induced a slight increase in BP, rapidly followed by profound and long‐lasting BP and HR decreases. Surprisingly, when given i.c.v., these 3 doses lowered BP and HR to the same extent but in a more progressive manner. 5 The lack of efficacy of both drugs in conscious SHRs after the i.c.v. administration of i.v. active doses and the lack of more marked and rapid effects in anaesthetized SHRs, after i.c.v. than after i.v. injections, question the involvement of a major central site of action for these antihypertensive α2‐adrenoceptor agonists. Moreover, these results show that the cardiovascular effects of these drugs are profoundly influenced by baseline sympathetic nervous system activity which is enhanced by pentobarbitone‐anaesthesia.

Increased expression of IL-6 and LIF in the hypertrophied left ventricle of TGR(mRen2)27 and SHR rats

Cytokines from the interleukin-6 (IL-6) family have been reported to play an important synergistic role with angiotensin II in the development of pathological cardiac hypertrophy. Whether their expression pattern changes in vivo, in an angiotensin II-dependent hypertrophied myocardium has not been reported. In this study, we addressed that issue using two animal models of angiotensin II-dependent cardiac hypertrophy. Heterozygous transgenic TGR(mRen2)27 (TGR) with an overactive cardiac renin angiotensin system and the closely related spontaneously hypertensive rats (SHR) were compared to their respective control rats. The mRNA levels of IL-6, leukemia inhibitory factor (LIF), ciliary neurotrophic factor (CNTF) and cardiotrophin-1 (CT-1) as well as their receptor subunits, glycoprotein 130 (gp130), IL-6 receptor (IL-6R), LIFR, and CNTFR, were measured by semi-quantitative RT-PCR. The protein levels of IL-6, LIF and CT-1 were investigated by western blot. TGR and SHR both displayed significant over expression of mRNA and protein levels for IL-6 and LIF. In TGR, the increased level of LIF was accompanied by a decrease in mRNA levels for LIFR and CNTFR. In SHR, a higher level of mRNA IL-6R was observed. By contrast, the mRNA and protein levels for CT-1 and the mRNA level for gp130 did not vary in these two models. These findings suggest that IL-6 and LIF, but not CT-1, contribute to angiotensin II-dependent left ventricular hypertrophy in the two hypertensive rat models, TGR(mRen2)27 and SHR. (Mol Cell Biochem 269: 95–101, 2005)

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.