M. Pilar Ramos

Perivascular Adipose Tissue and Mesenteric Vascular Function in Spontaneously Hypertensive Rats

Objective—Perivascular adipose tissue of normotensive rats releases a transferable factor that induces relaxation by opening voltage-dependent K+ (Kv) channels. The relevance of these observations to hypertension is unknown. Methods and Results—We characterized mesenteric perivascular adipose tissue from 3-month-old Wistar Kyoto rats (WKY) and aged-matched spontaneously hypertensive rats (SHR). Mesenteric bed (MB) weight and MB total lipid content were lower in SHR than in WKY. Freshly isolated MB adipocytes were smaller in SHR. Plasma triglycerides, glycerol, nonesterified free-fatty acids, and cholesterol were also lower in SHR. Plasma and mesenteric leptin were correlated with the quantity of mesenteric fat. To study vascular function, the MB was cannulated and perfused at a constant 2 mL/min flow. The Kv channel blocker 4-aminopyridine (4-AP; 2 mmol/L) increased perfusion pressure less in SHR MB than WKY and was directly correlated with the mesenteric fat amount. In isolated mesenteric artery rings, 4-AP (2 mmol/L) induced a contractile effect that was attenuated in SHR compared with WKY. The anticontractile effects of perivascular fat were reduced in SHR mesenteric artery rings compared with WKY. Conclusions—Differences in visceral perivascular adipose tissue mass and function may contribute to the increased vascular resistance observed in SHR.

Hyperinsulinemia Induces Insulin Resistance on Glucose and Lipid Metabolism in a Human Adipocytic Cell Line: Paracrine Interaction with Myocytes

CONTEXT Adipocytes release a variety of factors which deregulation could provide the basis for complications such as insulin resistance, an early defect on the onset of type 2 diabetes. Such insulin resistance can initially be overcome by compensatory hyperinsulinemia, but the prolonged presence of the hormone can be detrimental for insulin sensitivity. OBJECTIVE The objective of the study was to dissect the molecular mechanisms that may regulate hyperinsulinemia-induced insulin resistance in a human liposarcoma cell line and its paracrine interactions with a human rhabdomyosarcoma cell line. DESIGNS We studied glucose uptake, lipolysis, insulin signaling, and secretion pattern at different days of adipocyte differentiation in the presence of insulin. RESULTS Adipocytes differentiated for 14 d gain insulin sensitivity on glucose uptake and inhibition of lipolysis, but prolonged cultures develop an insulin-resistant state characterized by an increase in phosphatase and tensin homolog-deleted on chromosome 10 expression and defects in insulin signaling at the insulin receptor substrate-1/AKT level. The secretion pattern of nonesterified fatty acids, IL-6, adiponectin, leptin, and monocyte chemotactic protein-1 was in keeping with the changes in insulin sensitivity during differentiation. An inverse biphasic response was also observed in human myocytes when they were cultured with various adipocyte-conditioned media, although insulin resistance was detected earlier than in adipocytes. This behavior mimics hyperinsulinemia because insulin action was restored when adipocytes were cultured in the absence of the hormone. Pharmacological treatment of adipocytes with a liver X receptor agonist reestablishes insulin-stimulated glucose uptake, whereas treatment with a peroxisome proliferator-activated receptor-gamma agonist restored the antilipolytic action of insulin. CONCLUSIONS Hyperinsulinemia deregulates adipocyte secretion pattern, producing insulin resistance in adipocytes and myocytes, a situation that can be ameliorated with nuclear receptor agonists.

Vitamin E reduces adipose tissue fibrosis, inflammation, and oxidative stress and improves metabolic profile in obesity.

To test whether enhancing the capability of adipose tissue to store lipids using antioxidant supplementation may prevent the lipotoxic effects and improve the metabolic profile of long‐term obesity.

Leptin resistance develops spontaneously in mice during adult life in a tissue-specific manner. Consequences for hepatic steatosis.

Leptin is an adipocyte-derived hormone which stimulates β-oxidation in peripheral tissues and prevents steatosis. Because leptin production naturally increases during adult life, we have hypothesized that leptin receptors might undergo a physiological and gradual desensitization during ageing. Therefore we have characterized in three- five- and ten-month old mice i) the weight of different white adipose pads, heart and liver, ii) lipid content in these tissues/organs, and iii) responsiveness to acute leptin, measured in terms of phosphorylation of signal transducer and activator of transcription 3 (STAT3) and protein kinase B (Akt). In this study we have detected that leptin-mediated STAT3 phosphorylation appears to be preserved in cardiac tissue even in 10-month old animals but not in adipose tissue and liver of five- and ten-month old mice, respectively. Nevertheless, leptin increased pAkt content in the liver of these mice. In a parallel study we have analyzed the functionality of leptin signalling pathways in 10-month old obese mice and we have observed that the STAT3 pathway appears to be only operative in the heart whereas the Akt pathway remains functional both in heart and liver. Nevertheless, hepatic lipids increased almost 300% compared to age-matched lean controls. Our data demonstrate that during adult life there is a lost of leptin receptor functionality which is tissue-dependent and mainly affects the STAT3 pathway. Otherwise we demonstrate that the antisteatotic effect of leptin is independent of the Akt signalling pathway.

Regulation of leptin distribution between plasma and cerebrospinal fluid by cholecystokinin receptors

Cholecystokinin (CCK) is a postprandial hormone that elicits a satiating effect and regulates feeding behaviour. CCK has been shown to enhance the effect of leptin in several experimental paradigms. The goal of this work was to characterize the effect of endogenous CCK on plasma leptin content by using CCK receptor antagonists. Therefore, we administered SR‐27,897, a selective CCK1 receptor antagonist, and L‐365,260, a selective CCK2 receptor antagonist, to fed and food‐deprived rats and determined plasma leptin concentration by enzyme immunoassay. Plasma insulin and glucose concentration as well as food intake were also determined. Under our conditions, SR‐27,897 increased plasma concentration of leptin both in fed and food‐deprived rats. It also increased food intake as well as plasma concentration of insulin in fed animals. L‐365,260 increased plasma leptin concentration only in fed rats. In animals receiving exogenous leptin, CCK‐8 increased the ratio between the concentration of leptin in cerebrospinal fluid and plasma. These results show that CCK receptor antagonists increases plasma concentration of leptin and suggest that endogenous CCK may facilitate the uptake of plasma leptin to the cerebrospinal fluid.

Characterization of the role of endogenous cholecystokinin on the activity of the paraventricular nucleus of the hypothalamus in rats.

Activation of the hypothalamic–pituitary–adrenal axis by fasting seems to involve cholecystokinin (CCK) receptors. This work aims to characterize the role of endogenous CCK in the activity of the paraventricular nucleus (PVN) of the hypothalamus during food withdrawal. We investigated, by c‐Fos immunohistochemistry, the effect of CCK1 and CCK2 receptor antagonists (SR‐27,897 and L‐365,260, respectively) on c‐Fos levels expression induced by food deprivation. Under our conditions, the number of cells expressing c‐Fos was reduced both by SR‐27,897 and L‐365,260 in food‐deprived rats. To investigate the importance of glucose availability, we studied the effect of CCK receptor antagonists on c‐Fos synthesis induced by the glucose antimetabolite 2‐deoxyglucose. In these animals, only SR‐27,897 decreased c‐Fos expression in the PVN. Our results indicate that the effect of CCK antagonists is mainly perceptible when glucose availability decreases, and suggest that CCK‐ergic inputs could drive the activity of the PVN under fasting/low glucose conditions.

GC–MS based Gestational Diabetes Mellitus longitudinal study: Identification of 2-and 3-hydroxybutyrate as potential prognostic biomarkers

HIGHLIGHTSGC–MS offers high‐throughput molecular profiling to study metabolic signature of diabetes.Metabolomics may serve for early prediction of GDM and developing T2DM.2‐hydroxybutyrate and 3‐hydroxybutyrate may be potential prognostic biomarkers.Metabolomics is bringing knowledge gaps in current management of diabetes.Translation of metabolomics research for clinical practice is desired. ABSTRACT Gestational Diabetes Mellitus (GDM) causes severe short‐ and long‐term complications for the mother, fetus and neonate, including type 2‐diabetes (T2DM) later in life. In this pilot study, GC–Q/MS analysis was applied for plasma metabolomics fingerprinting of 24 healthy and 24 women with GDM at different stages of gestation (second and third trimester) and postpartum (one and three months). Multivariate (unsupervised and supervised) statistical analysis was performed to investigate variance in the data, identify outliers and for unbiased assessment of data quality. Plasma fingerprints allowed for the discrimination of GDM pregnant women from controls both in the 2nd and 3rd trimesters of gestation. However, metabolic profiles tended to be similar after delivery. Follow up of these women revealed that 4 of them developed T2DM within 2 years postpartum. Multivariate PLS‐DA models limited to women with GDM showed clear separation 3 months postpartum. In the 2nd trimester of gestation there was also a clear separation between GDM women that were normoglycemic after pregnancy and those with recognized postpartum T2DM. Metabolites that had the strongest discriminative power between these groups in the 2nd trimester of gestation were 2‐hydroxybutyrate, 3‐hydroxybutyrate, and stearic acid. We have described, that early GDM comprises metabotypes that are associated with the risk of future complications, including postpartum T2DM. In this pilot study, we provide evidence that 2‐hydroxybutyrate and 3‐hydroxybutyrate may be considered as future prognostic biomarkers to predict the onset of diabetic complications in women with gestational diabetes after delivery.

Metabolic Fingerprints of Gestational Diabetes Mellitus

Understanding the gestational diabetes pathophysiology, and the identification of potential risk factors and early diagnostic markers for both gestational diabetes and postpartum T2DM, are relevant questions. “Omics” techniques are powerful tools to shed light into the etiopathogenesis of the metabolic diseases and in the discovery of new biomarkers with high diagnostic quality. In the last years, it has been described that plasma metabolic fingerprints in gestational diabetes reveal disease-specific metabolic imbalances, indicative of low-grade inflammation and an altered redox-balance that may reflect on the specific pathophysiological context of the disease.

Englitazone Delays Fetal Growth in Late Gestation in the Rat

The mechanisms regulating hepatocyte proliferation are relevant to liver development, carcinogenesis, and regeneration. Studies of hepatocyte proliferation control during late foetal and postnatal development have been used as a model to understand such mechanisms. Since peroxisome proliferator-activated receptor gamma (PPARγ) ligands have been implicated in the inhibition of growth and differentiation of certain human cancers, in the present study, we have investigated the effect of englitazone (EG), a PPARγ ligand, on foetal and postnatal development. Our results indicate that, EG administered semi-chronically to pregnant rats, produced a body weight reduction on the progeny. This effect may be related to the diminished level of plasma IGF-I found in the neonates from treated-mothers. Surprisingly, despite receiving an anti-diabetic drug, foetus and neonates showed high levels of insulin, and were hyperglycemic. The plasma levels of leptin, other putative mitogenic factor, were not affected by the treatment. In the liver of neonates from mothers receiving EG, the expression of PPARα, IR, PI3K and IRS-1 was unchanged, as was the phosphorylation of MAPK. Nevertheless, an increase on Akt phosphorylation was observed on liver of neonates from treated-mothers, confirming a remarkable change on the mitogenic insulin/IGF-I pathway. In conclusion, the growth inhibitory effect reported herein may be associated with the ability of PPARγ ligands to reduce IGF-I concentrations and produce an insulin resistance state on foetus/neonates. These data strengthen the idea that PPARγ ligands have potential benefits on cancer treatment.