Dysregulation of micro-RNA 143-3p as a Biomarker of Carotid Atherosclerosis and the Associated Immune Reactions During Disease Progression.

Atherosclerosis commonly remains undiagnosed until disease manifestations occur. The disease is associated with dysregulated micro(mi)RNAs, but how this is linked to atherosclerosis-related immune reactions is largely unknown. A mouse model of carotid atherosclerosis, human APOB100-transgenic Ldlr-/- (HuBL), was used to study the spatiotemporal dysregulation of a set of miRNAs. Middle-aged HuBL mice with established atherosclerosis had decreased levels of miR-143-3p in their carotid arteries. In young HuBL mice, early atherosclerosis was observed in the carotid bifurcation, which had lower levels of miR-15a-5p, miR-143-3p, and miR-199a-3p, and higher levels of miR-155-5p. The dysregulation of these miRNAs was reflected by specific immune responses during atheroprogression. Finally, levels of miR-143-3p were 70.6% lower in extracellular vesicles isolated from the plasma of patients with carotid stenosis compared to healthy controls. Since miR-143-3p levels progressively decrease when transitioning between early and late experimental carotid atherosclerosis, we propose it as a biomarker for atherosclerosis.

Role of miR-15a-5p and miR-199a-3p in the inflammatory pathway regulated by NF-κB in experimental and human atherosclerosis.

BACKGROUND: Cardiovascular diseases (CVDs) prevalence has significantly increased in the last decade and atherosclerosis development is the main trigger. MicroRNAs (miRNAs) are non-coding RNAs that negatively regulate gene expression of their target and their levels are frequently altered in CVDs. METHODS: By RT-qPCR, we analysed miR-9-5p, miR-15a-5p, miR-16-5p and miR-199a-3p levels in aorta from apolipoprotein knockout (ApoE-/- ) mice, an experimental model of hyperlipidemia-induced atherosclerosis, and in human aortic and carotid atherosclerotic samples. By in silico studies, Western blot analysis and immunofluorescence studies, we detected the targets of the altered miRNAs. RESULTS: Our results show that miR-15a-5p and miR-199a-3p are significantly decreased in carotid and aortic samples from patients and mice with atherosclerosis. In addition, we found an increased expression in targets of both miRNAs that participate in the inflammatory pathway of nuclear factor kappa B (NF-κB), such as IKKα, IKKß and p65. In human vein endothelial cells (HUVECs) and vascular smooth muscle cells (VSMCs), the overexpression of miR-15a-5p or miR-199a-3p decreased IKKα, IKKß and p65 protein levels as well as NF-κB activation. On the other hand, miR-15a-5p and miR-199a-3p overexpression reduced ox-LDL uptake and the inflammation regulated by NF-κB in VSMCs. Moreover, although miR-15a-5p and miR-199a-3p were significantly increased in exosomes from patients with advanced carotid atherosclerosis, only in the ROC analyses for miR-15a-5p, the area under the curve was 0.8951 with a p value of .0028. CONCLUSIONS: Our results suggest that the decrease of miR-199a-3p and miR-15a-5p in vascular samples from human and experimental atherosclerosis could be involved in the NF-κB activation pathway, as well as in ox-LDL uptake by VSMCs, contributing to inflammation and progression atherosclerosis. Finally, miR-15a-5p could be used as a novel diagnostic biomarker for advanced atherosclerosis.

Increased let-7d-5p in non-alcoholic fatty liver promotes insulin resistance and is a potential blood biomarker for diagnosis.

BACKGROUND AND AIMS: The molecular mechanisms driving non-alcoholic fatty liver disease (NAFLD) are poorly understood; however, microRNAs might play a key role in these processes. We hypothesize that let-7d-5p could contribute to the pathophysiology of NAFLD and serve as a potential diagnostic biomarker. METHODS: We evaluated let-7d-5p levels and its targets in liver biopsies from a cross-sectional study including patients with NAFLD and healthy donors, and from a mouse model of NAFLD. Moreover, the induction of let-7d-5p expression by fatty acids was evaluated in vitro. Further, we overexpressed let-7d-5p in vitro to corroborate the results observed in vivo. Circulating let-7d-5p and its potential as a NAFLD biomarker was determined in isolated extracellular vesicles from human plasma by RT-qPCR. RESULTS: Our results demonstrate that hepatic let-7d-5p was significantly up-regulated in patients with steatosis, and this increase correlated with obesity and a decreased expression of AKT serine/threonine kinase (AKT), insulin-like growth factor 1 (IGF1), IGF-I receptor (IGF1R) and insulin receptor (INSR). These alterations were corroborated in a NAFLD mouse model. In vitro, fatty acids increased let-7d-5p expression, and its overexpression decreased AKT, IGF-IR and IR protein expression. Furthermore, let-7d-5p hindered AKT phosphorylation in vitro after insulin stimulation. Finally, circulating let-7d-5p significantly decreased in steatosis patients and receiver operating characteristic (ROC) analyses confirmed its utility as a diagnostic biomarker. CONCLUSIONS: Our results highlight the emerging role of let-7d-5p as a potential therapeutic target for NAFLD since its overexpression impairs hepatic insulin signalling, and also, as a novel non-invasive biomarker for NAFLD diagnosis.

Oxidative Stress Modulation by ncRNAs and Their Emerging Role as Therapeutic Targets in Atherosclerosis and Non-Alcoholic Fatty Liver Disease.

Atherosclerosis and non-alcoholic fatty liver disease (NAFLD) are pathologies related to ectopic fat accumulation, both of which are continuously increasing in prevalence. These threats are prompting researchers to develop effective therapies for their clinical management. One of the common pathophysiological alterations that underlies both diseases is oxidative stress (OxS), which appears as a result of lipid deposition in affected tissues. However, the molecular mechanisms that lead to OxS generation are different in each disease. Non-coding RNAs (ncRNAs) are RNA transcripts that do not encode proteins and function by regulating gene expression. In recent years, the involvement of ncRNAs in OxS modulation has become more recognized. This review summarizes the most recent advances regarding ncRNA-mediated regulation of OxS in atherosclerosis and NAFLD. In both diseases, ncRNAs can exert pro-oxidant or antioxidant functions by regulating gene targets and even other ncRNAs, positioning them as potential therapeutic targets. Interestingly, both diseases have common altered ncRNAs, suggesting that the same molecule can be targeted simultaneously when both diseases coexist. Finally, since some ncRNAs have already been used as therapeutic agents, their roles as potential drugs for the clinical management of atherosclerosis and NAFLD are analyzed.

Implication of miR-155-5p and miR-143-3p in the Vascular Insulin Resistance and Instability of Human and Experimental Atherosclerotic Plaque.

(1) Background: Cardiovascular diseases (CVDs) are the main cause of death in developed countries, being atherosclerosis, a recurring process underlying their apparition. MicroRNAs (miRNAs) modulate the expression of their targets and have emerged as key players in CVDs; (2) Methods: 18 miRNAs were selected (Pubmed and GEO database) for their possible role in promoting atherosclerosis and were analysed by RT-qPCR in the aorta from apolipoprotein E-deficient (ApoE-/-) mice. Afterwards, the altered miRNAs in the aorta from 18 weeks-ApoE-/- mice were studied in human aortic and carotid samples; (3) Results: miR-155-5p was overexpressed and miR-143-3p was downregulated in mouse and human atherosclerotic lesions. In addition, a significant decrease in protein kinase B (AKT), target of miR-155-5p, and an increase in insulin-like growth factor type II receptor (IGF-IIR), target of miR-143-3p, were noted in aortic roots from ApoE-/- mice and in carotid plaques from patients with advanced carotid atherosclerosis (ACA). Finally, the overexpression of miR-155-5p reduced AKT levels and its phosphorylation in vascular smooth muscle cells, while miR-143-3p overexpression decreased IGF-IIR reducing apoptosis in vascular cells; (4) Conclusions: Our results suggest that miR-155-5p and miR-143-3p may be implicated in insulin resistance and plaque instability by the modulation of their targets AKT and IGF-IIR, contributing to the progression of atherosclerosis.

Concerted regulation of non-alcoholic fatty liver disease progression by microRNAs in apolipoprotein E-deficient mice.

The prevalence of non-alcoholic fatty liver disease (NAFLD) is constantly increasing, and altered expression of microRNAs (miRNAs) fosters the development and progression of many pathologies, including NAFLD. Therefore, we explored the role of new miRNAs involved in the molecular mechanisms that trigger NAFLD progression and evaluated them as biomarkers for diagnosis. As a NAFLD model, we used apolipoprotein E-deficient mice administered a high-fat diet for 8 or 18 weeks. We demonstrated that insulin resistance and decreased lipogenesis and autophagy observed after 18 weeks on the diet are related to a concerted regulation carried out by miR-26b-5p, miR-34a-5p, miR-149-5p and miR-375-3p. We also propose circulating let-7d-5p and miR-146b-5p as potential biomarkers of early stages of NAFLD. Finally, we confirmed that circulating miR-34a-5p and miR-375-3p are elevated in the late stages of NAFLD and that miR-27b-3p and miR-122-5p are increased with disease progression. Our results reveal a synergistic regulation of key processes in NAFLD development and progression by miRNAs. Further investigation is needed to unravel the roles of these miRNAs for developing new strategies for NAFLD treatment. This article has an associated First Person interview with the joint first authors of the paper.

Severe Hepatic Insulin Resistance Induces Vascular Dysfunction: Improvement by Liver-Specific Insulin Receptor Isoform A Gene Therapy in a Murine Diabetic Model.

BACKGROUND: Cardiovascular dysfunction is linked to insulin-resistant states. In this paper, we analyzed whether the severe hepatic insulin resistance of an inducible liver-specific insulin receptor knockout (iLIRKO) might generate vascular insulin resistance and dysfunction, and whether insulin receptor (IR) isoforms gene therapy might revert it. METHODS: We studied in vivo insulin signaling in aorta artery and heart from iLIRKO. Vascular reactivity and the mRNA levels of genes involved in vascular dysfunction were analyzed in thoracic aorta rings by qRT-PCR. Finally, iLIRKO mice were treated with hepatic-specific gene therapy to analyze vascular dysfunction improvement. RESULTS: Our results suggest that severe hepatic insulin resistance was expanded to cardiovascular tissues. This vascular insulin resistance observed in aorta artery from iLIRKO mice correlated with a reduction in both PI3K/AKT/eNOS and p42/44 MAPK pathways, and it might be implicated in their vascular alterations characterized by endothelial dysfunction, hypercontractility and eNOS/iNOS levels' imbalance. Finally, regarding long-term hepatic expression of IR isoforms, IRA was more efficient than IRB in the improvement of vascular dysfunction observed in iLIRKO mice. CONCLUSION: Severe hepatic insulin resistance is sufficient to produce cardiovascular insulin resistance and dysfunction. Long-term hepatic expression of IRA restored the vascular damage observed in iLIRKO mice.

miRNA Dysregulation in the Development of Non-Alcoholic Fatty Liver Disease and the Related Disorders Type 2 Diabetes Mellitus and Cardiovascular Disease.

According to the World Health Organization, the continuing surge in obesity pandemic creates a substantial increase in incidences of metabolic disorders, such as non-alcoholic fatty liver disease (NAFLD), type 2 diabetes mellitus, and cardiovascular disease. MicroRNAs (miRNAs) belong to an evolutionarily conserved class of short (20-22 nucleotides in length) and single-stranded non-coding RNAs. In mammals, miRNAs function as critical post-transcriptional negative regulators involved not only in many biological processes but also in the development of many diseases such as NAFLD and comorbidities. More recently, it has been described that cells can secrete miRNAs in extracellular vesicles, transported by body fluids, and uptaken by other tissues regulating gene expression. Therefore, this could be a mechanism of signaling involved not only in physiological pathways but also in the development of diseases. The association of some miRNA expression profiles with certain disorders has made them very interesting molecules for diagnosis, prognosis, and disease management. The finding of specific miRNA signatures to diagnose NAFLD and related diseases could anticipate the risk of development of related complications and, actually, it is the driving force of present health strategies worldwide. In this review, we have included latest advances in knowledge about the miRNAs involved in the development of NAFLD and related diseases and examined how this knowledge could be used to identify new non-invasive biomarkers and new pharmacological interventions.

The Interplay between Oxidative Stress and miRNAs in Obesity-Associated Hepatic and Vascular Complications.

Nowadays, the obesity pandemic is one of the most relevant health issues worldwide. This condition is tightly related to comorbidities such as non-alcoholic fatty liver disease (NAFLD) and cardiovascular diseases (CVDs), namely atherosclerosis. Dysregulated lipid metabolism and inflammation link these three diseases, leading to a subsequent increase of oxidative stress (OS) causing severe cellular damage. On the other hand, microRNAs (miRNAs) are short, single-stranded, non-coding RNAs that act as post-transcriptional negative regulators of gene expression, thus being involved in the molecular mechanisms that promote the development of many pathologies including obesity and its comorbidities. The involvement of miRNAs in promoting or opposing OS in disease progression is becoming more evident. Some miRNAs, such as miR-200a and miR.421, seem to play important roles in OS control in NAFLD. On the other hand, miR-92a and miR-133, among others, are important in the development of atherosclerosis. Moreover, since both diseases are linked to obesity, they share common altered miRNAs, being miR-34a and miR-21 related to OS. This review summarizes the latest advances in the knowledge about the mechanisms of oxidative stress (OS) generation in obesity-associated NAFLD and atherosclerosis, as well as the role played by miRNAs in the regulation of such mechanisms.

Specific knockout of p85α in brown adipose tissue induces resistance to high-fat diet-induced obesity and its metabolic complications in male mice.

OBJECTIVE: An increase in mass and/or brown adipose tissue (BAT) functionality leads to an increase in energy expenditure, which may be beneficial for the prevention and treatment of obesity. Moreover, distinct class I PI3K isoforms can participate in metabolic control as well as in systemic dysfunctions associated with obesity. In this regard, we analyzed in vivo whether the lack of p85α in BAT (BATp85αKO) could modulate the activity and insulin signaling of this tissue, thereby improving diet-induced obesity and its associated metabolic complications. METHODS: We generated BATp85αKO mice using Cre-LoxP technology, specifically deleting p85α in a conditional manner. To characterize this new mouse model, we used mice of 6 and 12 months of age. In addition, BATp85αKO mice were submitted to a high-fat diet (HFD) to challenge BAT functionality. RESULTS: Our results suggest that the loss of p85α in BAT improves its thermogenic functionality, high-fat diet-induced adiposity and body weight, insulin resistance, and liver steatosis. The potential mechanisms involved in the improvement of obesity include (1) increased insulin signaling and lower activation of JNK in BAT, (2) enhanced insulin receptor isoform B (IRB) expression and association with IRS-1 in BAT, (3) lower production of proinflammatory cytokines by the adipose organ, (4) increased iWAT browning, and (5) improved liver steatosis. CONCLUSIONS: Our results provide new mechanisms involved in the resistance to obesity development, supporting the hypothesis that the gain of BAT activity induced by the lack of p85α has a direct impact on the prevention of diet-induced obesity and its associated metabolic complications.

Liver-specific insulin receptor isoform A expression enhances hepatic glucose uptake and ameliorates liver steatosis in a mouse model of diet-induced obesity.

Among the main complications associated with obesity are insulin resistance and altered glucose and lipid metabolism within the liver. It has previously been described that insulin receptor isoform A (IRA) favors glucose uptake and glycogen storage in hepatocytes compared with isoform B (IRB), improving glucose homeostasis in mice lacking liver insulin receptor. Thus, we hypothesized that IRA could also improve glucose and lipid metabolism in a mouse model of high-fat-diet-induced obesity. We addressed the role of insulin receptor isoforms in glucose and lipid metabolism in vivo We expressed IRA or IRB specifically in the liver by using adeno-associated viruses (AAVs) in a mouse model of diet-induced insulin resistance and obesity. IRA, but not IRB, expression induced increased glucose uptake in the liver and muscle, improving insulin tolerance. Regarding lipid metabolism, we found that AAV-mediated IRA expression also ameliorated hepatic steatosis by decreasing the expression of Fasn, Pgc1a, Acaca and Dgat2 and increasing Scd-1 expression. Taken together, our results further unravel the role of insulin receptor isoforms in hepatic glucose and lipid metabolism in an insulin-resistant scenario. Our data strongly suggest that IRA is more efficient than IRB at favoring hepatic glucose uptake, improving insulin tolerance and ameliorating hepatic steatosis. Therefore, we conclude that a gene therapy approach for hepatic IRA expression could be a safe and promising tool for the regulation of hepatic glucose consumption and lipid metabolism, two key processes in the development of non-alcoholic fatty liver disease associated with obesity.This article has an associated First Person interview with the first author of the paper.

Chronic Exercise Improves Mitochondrial Function and Insulin Sensitivity in Brown Adipose Tissue.

The aim of the present work was to study the consequences of chronic exercise training on factors involved in the regulation of mitochondrial remodeling and biogenesis, as well as the ability to produce energy and improve insulin sensitivity and glucose uptake in rat brown adipose tissue (BAT). Male Wistar rats were divided into two groups: (1) control group (C; n = 10) and (2) exercise-trained rats (ET; n = 10) for 8 weeks on a motor treadmill (five times per week for 50 min). Exercise training reduced body weight, plasma insulin, and oxidized LDL concentrations. Protein expression of ATP-independent metalloprotease (OMA1), short optic atrophy 1 (S-OPA1), and dynamin-related protein 1 (DRP1) in BAT increased in trained rats, and long optic atrophy 1 (L-OPA1) and mitofusin 1 (MFN1) expression decreased. BAT expression of nuclear respiratory factor type 1 (NRF1) and mitochondrial transcription factor A (TFAM), the main factors involved in mitochondrial biogenesis, was higher in trained rats compared to controls. Exercise training increased protein expression of sirtuin 1 (SIRT1), peroxisome proliferator-activated receptor γ coactivator 1α (PGC1α) and AMP-activated protein kinase (pAMPK/AMPK ratio) in BAT. In addition, training increased carnitine palmitoyltransferase II (CPT II), mitochondrial F1 ATP synthase α-chain, mitochondrial malate dehydrogenase 2 (mMDH) and uncoupling protein (UCP) 1,2,3 expression in BAT. Moreover, exercise increased insulin receptor (IR) ratio (IRA/IRB ratio), IRA-insulin-like growth factor 1 receptor (IGF-1R) hybrids and p42/44 activation, and decreased IGF-1R expression and IR substrate 1 (p-IRS-1) (S307) indicating higher insulin sensitivity and favoring glucose uptake in BAT in response to chronic exercise training. In summary, the present study indicates that chronic exercise is able to improve the energetic profile of BAT in terms of increased mitochondrial function and insulin sensitivity.

Potential role of insulin receptor isoforms and IGF receptors in plaque instability of human and experimental atherosclerosis.

BACKGROUND: Clinical complications associated with atherosclerotic plaques arise from luminal obstruction due to plaque growth or destabilization leading to rupture. We previously demonstrated that overexpression of insulin receptor isoform A (IRA) and insulin-like growth factor-I receptor (IGF-IR) confers a proliferative and migratory advantage to vascular smooth muscle cells (VSMCs) promoting plaque growth in early stages of atherosclerosis. However, the role of insulin receptor (IR) isoforms, IGF-IR or insulin-like growth factor-II receptor (IGF-IIR) in VSMCs apoptosis during advanced atherosclerosis remains unclear. METHODS: We evaluated IR isoforms expression in human carotid atherosclerotic plaques by consecutive immunoprecipitations of insulin receptor isoform B (IRB) and IRA. Western blot analysis was performed to measure IGF-IR, IGF-IIR, and α-smooth muscle actin (α-SMA) expression in human plaques. The expression of those proteins, as well as the presence of apoptotic cells, was analyzed by immunohistochemistry in experimental atherosclerosis using BATIRKO; ApoE-/- mice, a model showing more aggravated vascular damage than ApoE-/- mice. Finally, apoptosis of VSMCs bearing IR (IRLoxP+/+ VSMCs), or not (IR-/- VSMCs), expressing IRA (IRA VSMCs) or expressing IRB (IRB VSMCs), was assessed by Western blot against cleaved caspase 3. RESULTS: We observed a significant decrease of IRA/IRB ratio in human complicated plaques as compared to non-complicated regions. Moreover, complicated plaques showed a reduced IGF-IR expression, an increased IGF-IIR expression, and lower levels of α-SMA indicating a loss of VSMCs. In experimental atherosclerosis, we found a significant decrease of IRA with an increased IRB expression in aorta from 24-week-old BATIRKO; ApoE-/- mice. Furthermore, atherosclerotic plaques from BATIRKO; ApoE-/- mice had less VSMCs content and higher number of apoptotic cells. In vitro experiments showed that IGF-IR inhibition by picropodophyllin induced apoptosis in VSMCs. Apoptosis induced by thapsigargin was lower in IR-/- VSMCs expressing higher IGF-IR levels as compared to IRLoxP+/+ VSMCs. Finally, IRB VSMCs are more prone to thapsigargin-induced apoptosis than IRA or IRLoxP+/+ VSMCs. CONCLUSIONS: In advanced human atherosclerosis, a reduction of IRA/IRB ratio, decreased IGF-IR expression, or increased IGF-IIR may contribute to VSMCs apoptosis, promoting plaque instability and increasing the risk of plaque rupture and its clinical consequences.

Differential Role of Adipose Tissues in Obesity and Related Metabolic and Vascular Complications.

This review focuses on the contribution of white, brown, and perivascular adipose tissues to the pathophysiology of obesity and its associated metabolic and vascular complications. Weight gain in obesity generates excess of fat, usually visceral fat, and activates the inflammatory response in the adipocytes and then in other tissues such as liver. Therefore, low systemic inflammation responsible for insulin resistance contributes to atherosclerotic process. Furthermore, an inverse relationship between body mass index and brown adipose tissue activity has been described. For these reasons, in recent years, in order to combat obesity and its related complications, as a complement to conventional treatments, a new insight is focusing on the role of the thermogenic function of brown and perivascular adipose tissues as a promising therapy in humans. These lines of knowledge are focused on the design of new drugs, or other approaches, in order to increase the mass and/or activity of brown adipose tissue or the browning process of beige cells from white adipose tissue. These new treatments may contribute not only to reduce obesity but also to prevent highly prevalent complications such as type 2 diabetes and other vascular alterations, such as hypertension or atherosclerosis.

Prevalent role of the insulin receptor isoform A in the regulation of hepatic glycogen metabolism in hepatocytes and in mice.

AIMS/HYPOTHESIS: In the postprandial state, the liver regulates glucose homeostasis by glucose uptake and conversion to glycogen and lipids. Glucose and insulin signalling finely regulate glycogen synthesis through several mechanisms. Glucose uptake in hepatocytes is favoured by the insulin receptor isoform A (IRA), rather than isoform B (IRB). Thus, we hypothesised that, in hepatocytes, IRA would increase glycogen synthesis by promoting glucose uptake and glycogen storage. METHODS: We addressed the role of insulin receptor isoforms on glycogen metabolism in vitro in immortalised neonatal hepatocytes. In vivo, IRA or IRB were specifically expressed in the liver using adeno-associated virus vectors in inducible liver insulin receptor knockout (iLIRKO) mice, a model of type 2 diabetes. The role of IR isoforms in glycogen synthesis and storage in iLIRKO was subsequently investigated. RESULTS: In immortalised hepatocytes, IRA, but not IRB expression induced an increase in insulin signalling that was associated with elevated glycogen synthesis, glycogen synthase activity and glycogen storage. Similarly, elevated IRA, but not IRB expression in the livers of iLIRKO mice induced an increase in glycogen content. CONCLUSIONS/INTERPRETATION: We provide new insight into the role of IRA in the regulation of glycogen metabolism in cultured hepatocytes and in the livers of a mouse model of type 2 diabetes. Our data strongly suggest that IRA is more efficient than IRB at promoting glycogen synthesis and storage. Therefore, we suggest that IRA expression in the liver could provide an interesting therapeutic approach for the regulation of hepatic glucose content and glycogen storage.

Insulin receptor isoform A ameliorates long-term glucose intolerance in diabetic mice.

Type 2 diabetes mellitus is a complex metabolic disease and its pathogenesis involves abnormalities in both peripheral insulin action and insulin secretion. Previous in vitro data showed that insulin receptor isoform A, but not B, favours basal glucose uptake through its specific association with endogenous GLUT1/2 in murine hepatocytes and beta cells. With this background, we hypothesized that hepatic expression of insulin receptor isoform A in a mouse model of type 2 diabetes could potentially increase the glucose uptake of these cells, decreasing the hyperglycaemia and therefore ameliorating the diabetic phenotype. To assure this hypothesis, we have developed recombinant adeno-associated viral vectors expressing insulin receptor isoform A (IRA) or isoform B (IRB) under the control of a hepatocyte--specific promoter. Our results demonstrate that in the long term, hepatic expression of IRA in diabetic mice is more efficient than IRB in ameliorating glucose intolerance. Consequently, it impairs the induction of compensatory mechanisms through beta cell hyperplasia and/or hypertrophy that finally lead to beta cell failure, reverting the diabetic phenotype in about 8 weeks. Our data suggest that long-term hepatic expression of IRA could be a promising therapeutic approach for the treatment of type 2 diabetes mellitus.

Severe Brown Fat Lipoatrophy Aggravates Atherosclerotic Process in Male Mice.

Obesity is one of the major risk factors for the development of cardiovascular diseases and is characterized by abnormal accumulation of adipose tissue, including perivascular adipose tissue (PVAT). However, brown adipose tissue (BAT) activation reduces visceral adiposity. To demonstrate that severe brown fat lipoatrophy might accelerate atherosclerotic process, we generated a new mouse model without insulin receptor (IR) in BAT and without apolipoprotein (Apo)E (BAT-specific IR knockout [BATIRKO];ApoE(-/-) mice) and assessed vascular and metabolic alterations associated to obesity. In addition, we analyzed the contribution of the adipose organ to vascular inflammation. Brown fat lipoatrophy induces visceral adiposity, mainly in gonadal depot (gonadal white adipose tissue [gWAT]), severe glucose intolerance, high postprandial glucose levels, and a severe defect in acute insulin secretion. BATIRKO;ApoE(-/-) mice showed greater hypertriglyceridemia than the obtained in ApoE(-/-) and hypercholesterolemia similar to ApoE(-/-) mice. BATIRKO;ApoE(-/-) mice, in addition to primary insulin resistance in BAT, also showed a significant decrease in insulin signaling in liver, gWAT, heart, aorta artery, and thoracic PVAT. More importantly, our results suggest that severe brown fat lipoatrophy aggravates the atherosclerotic process, characterized by a significant increase of lipid depots, atherosclerotic coverage, lesion size and complexity, increased macrophage infiltration, and proinflammatory markers expression. Finally, an increase of TNF-α and leptin as well as a decrease of adiponectin by BAT, gWAT, and thoracic PVAT might also be responsible of vascular damage. Our results suggest that severe brown lipoatrophy aggravates atherosclerotic process. Thus, BAT activation might protect against obesity and its associated metabolic alterations.

Protective role of oleic acid against cardiovascular insulin resistance and in the early and late cellular atherosclerotic process.

BACKGROUND: Several translational studies have identified the differential role between saturated and unsaturated fatty acids at cardiovascular level. However, the molecular mechanisms that support the protective role of oleate in cardiovascular cells are poorly known. For these reasons, we studied the protective role of oleate in the insulin resistance and in the atherosclerotic process at cellular level such as in cardiomyocytes (CMs), vascular smooth muscle cells (VSMCs) and endothelial cells (ECs). METHODS: The effect of oleate in the cardiovascular insulin resistance, vascular dysfunction, inflammation, proliferation and apoptosis of VSMCs were analyzed by Western blot, qRT-PCR, BrdU incorporation and cell cycle analysis. RESULTS: Palmitate induced insulin resistance. However, oleate not only did not induce cardiovascular insulin resistance but also had a protective effect against insulin resistance induced by palmitate or TNFα. One mechanism involved might be the prevention by oleate of JNK-1/2 or NF-κB activation in response to TNF-α or palmitate. Oleate reduced MCP-1 and ICAM-1 and increased eNOS expression induced by proinflammatory cytokines in ECs. Furthermore, oleate impaired the proliferation induced by TNF-α, angiotensin II or palmitate and the apoptosis induced by TNF-α or thapsigargin in VSMCs. CONCLUSIONS: Our data suggest a differential role between oleate and palmitate and support the concept of the cardioprotector role of oleate as the main lipid component of virgin olive oil. Thus, oleate protects against cardiovascular insulin resistance, improves endothelial dysfunction in response to proinflammatory signals and finally, reduces proliferation and apoptosis in VSMCs that may contribute to an ameliorated atherosclerotic process and plaque stability.

Insulin receptor isoform A confers a higher proliferative capability to pancreatic beta cells enabling glucose availability and IGF-I signaling.

The main compensatory response to insulin resistance is the pancreatic beta cell hyperplasia to account for increased insulin secretion. In fact, in a previous work we proposed a liver-pancreas endocrine axis with IGF-I (insulin-like growth factor type I) secreted by the liver acting on IRA insulin receptor in beta cells from iLIRKO mice (inducible Liver Insulin Receptor KnockOut) that showed a high IRA/IRB ratio. However, the role of insulin receptor isoforms in the IGF-I-induced beta cell proliferation as well as the underlying molecular mechanisms remain poorly understood. For this purpose, we have used four immortalized mouse beta cell lines: bearing IR (IRLoxP), lacking IR (IRKO), expressing exclusively IRA (IRA), or alternatively expressing IRB (IRB). Pancreatic beta cell proliferation studies showed that IRA cells are more sensitive than those expressing IRB to the mitogenic response induced by IGF-I, acting through the pathway IRA/IRS-1/2/αp85/Akt/mTORC1/p70S6K. More importantly, IRA beta cells, but not IRB, showed an increased glucose uptake as compared with IRLoxP cells, this effect being likely owing to an enhanced association between Glut-1 and Glut-2 with IRA. Overall, our results strongly suggest a prevalent role of IRA in glucose availability and IGF-I-induced beta cell proliferation mainly through mTORC1. These results could explain, at least partially, the role played by the liver-secreted IGF-I in the compensatory beta cell hyperplasia observed in response to severe hepatic insulin resistance in iLIRKO mice.