Emma Barroso

An overview of the crosstalk between inflammatory processes and metabolic dysregulation during diabetic cardiomyopathy

Metabolic disorders such as obesity, insulin resistance and type 2 diabetes mellitus are all linked to cardiovascular diseases such as cardiac hypertrophy and heart failure. Diabetic cardiomyopathy in particular, is characterized by structural and functional alterations in the heart muscle of people with diabetes that finally lead to heart failure, and which is not directly attributable to coronary artery disease or hypertension. Several mechanisms have been involved in the pathogenesis of diabetic cardiomyopathy, such as alterations in myocardial energy metabolism and calcium signaling. Metabolic disturbances during diabetic cardiomyopathy are characterized by increased lipid oxidation, intramyocardial triglyceride accumulation, and reduced glucose utilization. Overall changes result in enhanced oxidative stress, mitochondrial dysfunction and apoptosis of the cardiomyocytes. On the other hand, the progression of heart failure and cardiac hypertrophy usually entails a local rise in cytokines in cardiac cells and the activation of the proinflammatory transcription factor nuclear factor (NF)-κB. Interestingly, increasing evidences are arising in the recent years that point to a potential link between chronic low-grade inflammation in the heart and metabolic dysregulation. Therefore, in this review we summarize recent new insights into the crosstalk between inflammatory processes and metabolic dysregulation in the failing heart during diabetes, paying special attention to the role of NF-κB and peroxisome proliferator activated receptors (PPARs). In addition, we briefly describe the role of the AMP-activated protein kinase (AMPK), sirtuin 1 (SIRT1) and other pathways regulating cardiac energy metabolism, as well as their relationship with diabetic cardiomyopathy.

Oleate prevents saturated-fatty-acid-induced ER stress, inflammation and insulin resistance in skeletal muscle cells through an AMPK-dependent mechanism

Aims/hypothesisAlthough the substitution of saturated fatty acids with oleate has been recommended in the management of type 2 diabetes mellitus, the mechanisms by which oleate improves insulin resistance in skeletal muscle cells are not completely known. Here, we examined whether oleate, through activation of AMP-activated protein kinase (AMPK), prevented palmitate-induced endoplasmic reticulum (ER) stress, which is involved in the link between lipid-induced inflammation and insulin resistance.MethodsStudies were conducted in mouse C2C12 myotubes and in the human myogenic cell line LHCN-M2. To analyse the involvement of AMPK, activators and inhibitors of this kinase and overexpression of a dominant negative AMPK construct (K45R) were used.ResultsPalmitate increased the levels of ER stress markers, whereas oleate did not. In palmitate-exposed cells incubated with a lower concentration of oleate, the effects of palmitate were prevented. The induction of ER stress markers by palmitate was prevented by the presence of the AMPK activators AICAR and A-769662. Moreover, the ability of oleate to prevent palmitate-induced ER stress and inflammation (nuclear factor-kappa B [NF-κB] DNA-binding activity and expression and secretion of IL6) as well as insulin-stimulated Akt phosphorylation and 2-deoxyglucose uptake was reversed in the presence of the AMPK inhibitor compound C or by overexpression of a dominant negative AMPK construct. Finally, palmitate reduced phospho-AMPK levels, whereas this was not observed in oleate-exposed cells or in palmitate-exposed cells supplemented with oleate.Conclusions/interpretationOverall, these findings indicate that oleate prevents ER stress, inflammation and insulin resistance in palmitate-exposed skeletal muscle cells by activating AMPK.

The PPARβ/δ Activator GW501516 Prevents the Down-Regulation of AMPK Caused by a High-Fat Diet in Liver and Amplifies the PGC-1α-Lipin 1-PPARα Pathway Leading to Increased Fatty Acid Oxidation

Metabolic syndrome-associated dyslipidemia is mainly initiated by hepatic overproduction of the plasma lipoproteins carrying triglycerides. Here we examined the effects of the peroxisome proliferator-activated receptors (PPAR)-β/δ activator GW501516 on high-fat diet (HFD)-induced hypertriglyceridemia and hepatic fatty acid oxidation. Exposure to the HFD caused hypertriglyceridemia that was accompanied by reduced hepatic mRNA levels of PPAR-γ coactivator 1 (PGC-1)-α and lipin 1, and these effects were prevented by GW501516 treatment. GW501516 treatment also increased nuclear lipin 1 protein levels, leading to amplification in the PGC-1α-PPARα signaling system, as demonstrated by the increase in PPARα levels and PPARα-DNA binding activity and the increased expression of PPARα-target genes involved in fatty acid oxidation. These effects of GW501516 were accompanied by an increase in plasma β-hydroxybutyrate levels, demonstrating enhanced hepatic fatty acid oxidation. Moreover, GW501516 increased the levels of the hepatic endogenous ligand for PPARα, 16:0/18:1-phosphatidilcholine and markedly enhanced the expression of the hepatic Vldl receptor. Interestingly, GW501516 prevented the reduction in AMP-activated protein kinase (AMPK) phosphorylation and the increase in phosphorylated levels of ERK1/2 caused by HFD. In addition, our data indicate that the activation of AMPK after GW501516 treatment in mice fed HFD might be the result of an increase in the AMP to ATP ratio in hepatocytes. These findings indicate that the hypotriglyceridemic effect of GW501516 in HFD-fed mice is accompanied by an increase in phospho-AMPK levels and the amplification of the PGC-1α-lipin 1-PPARα pathway.

Early alterations in energy metabolism in the hippocampus of APPswe/PS1dE9 mouse model of Alzheimer's disease

The present study had focused on the behavioral phenotype and gene expression profile of molecules related to insulin receptor signaling in the hippocampus of 3 and 6 month-old APPswe/PS1dE9 (APP/PS1) transgenic mouse model of Alzheimers disease (AD). Elevated levels of the insoluble Aβ (1-42) were detected in the brain extracts of the transgenic animals as early as 3 months of age, prior to the Aβ plaque formation (pre-plaque stage). By the early plaque stage (6 months) both the soluble and insoluble Aβ (1-40) and Aβ (1-42) peptides were detectable. We studied the expression of genes related to memory function (Arc, Fos), insulin signaling, including insulin receptor (Insr), Irs1 and Irs2, as well as genes involved in insulin growth factor pathways, such as Igf1, Igf2, Igfr and Igfbp2. We also examined the expression and protein levels of key molecules related to energy metabolism (PGC1-α, and AMPK) and mitochondrial functionality (OXPHOS, TFAM, NRF1 and NRF2). 6 month-old APP/PS1 mice demonstrated impaired cognitive ability, were glucose intolerant and showed a significant reduction in hippocampal Insr and Irs2 transcripts. Further observations also suggest alterations in key cellular energy sensors that regulate the activities of a number of metabolic enzymes through phosphorylation, such as a decrease in the Prkaa2 mRNA levels and in the pAMPK (Thr172)/Total APMK ratio. Moreover, mRNA and protein analysis reveals a significant downregulation of genes essential for mitochondrial replication and respiratory function, including PGC-1α in hippocampal extracts of APP/PS1 mice, compared to age-matched wild-type controls at 3 and 6 months of age. Overall, the findings of this study show early alterations in genes involved in insulin and energy metabolism pathways in an APP/PS1 model of AD. These changes affect the activity of key molecules like NRF1 and PGC-1α, which are involved in mitochondrial biogenesis. Our results reinforce the hypothesis that the impairments in both insulin signaling and energy metabolism precede the development of AD amyloidogenesis.

Peroxisome Proliferator-Activated Receptor (PPAR)β /δ: A New Potential Therapeutic Target for the Treatment of Metabolic Syndrome

Metabolic syndrome is defined as the clustering of multiple metabolic abnormalities, including abdominal obesity, dyslipidemia (high serum triglycerides and low serum HDL-cholesterol levels), glucose intolerance and hypertension. The pathophysiology underlying metabolic syndrome involves a complex interaction of crucial factors, but two of these, insulin resistance and obesity (especially visceral obesity), play a major role. The nuclear receptors Peroxisome Proliferator-Activated Receptors (PPAR)α and PPARγ are therapeutic targets for hypertriglyceridemia and insulin resistance, respectively. Evidence is now emerging that the PPARβ/δ isotype is a potential pharmacological target for the treatment of disorders associated with metabolic syndrome. PPARβ/δ activation increases lipid catabolism in skeletal muscle, heart and adipose tissue and improves the serum lipid profile and insulin sensitivity in several animal models. In addition, PPARβ/δ ligands prevent weight gain and suppress macrophage-derived inflammation. These data are promising and indicate that PPARβ/δ ligands may become a therapeutic option for the treatment of metabolic syndrome. However, clinical trials in humans assessing the efficacy and safety of these drugs should confirm these promising perspectives in the treatment of the metabolic syndrome.

PPARβ/δ prevents endoplasmic reticulum stress-associated inflammation and insulin resistance in skeletal muscle cells through an AMPK-dependent mechanism

Aim/hypothesisEndoplasmic reticulum (ER) stress, which is involved in the link between inflammation and insulin resistance, contributes to the development of type 2 diabetes mellitus. In this study, we assessed whether peroxisome proliferator-activated receptor (PPAR)β/δ prevented ER stress-associated inflammation and insulin resistance in skeletal muscle cells.MethodsStudies were conducted in mouse C2C12 myotubes, in the human myogenic cell line LHCN-M2 and in skeletal muscle from wild-type and PPARβ/δ-deficient mice and mice exposed to a high-fat diet.ResultsThe PPARβ/δ agonist GW501516 prevented lipid-induced ER stress in mouse and human myotubes and in skeletal muscle of mice fed a high-fat diet. PPARβ/δ activation also prevented thapsigargin- and tunicamycin-induced ER stress in human and murine skeletal muscle cells. In agreement with this, PPARβ/δ activation prevented ER stress-associated inflammation and insulin resistance, and glucose-intolerant PPARβ/δ-deficient mice showed increased phosphorylated levels of inositol-requiring 1 transmembrane kinase/endonuclease-1α in skeletal muscle. Our findings demonstrate that PPARβ/δ activation prevents ER stress through the activation of AMP-activated protein kinase (AMPK), and the subsequent inhibition of extracellular-signal-regulated kinase (ERK)1/2 due to the inhibitory crosstalk between AMPK and ERK1/2, since overexpression of a dominant negative AMPK construct (K45R) reversed the effects attained by PPARβ/δ activation.Conclusions/interpretationOverall, these findings indicate that PPARβ/δ prevents ER stress, inflammation and insulin resistance in skeletal muscle cells by activating AMPK.

Targeting endoplasmic reticulum stress in insulin resistance

The endoplasmic reticulum (ER) is involved in the development of insulin resistance and progression to type 2 diabetes mellitus (T2DM). Disruption of ER homeostasis leads to ER stress, which activates the unfolded protein response (UPR). This response is linked to different processes involved in the development of insulin resistance (IR) and T2DM, including inflammation, lipid accumulation, insulin biosynthesis, and β-cell apoptosis. Understanding the mechanisms by which disruption of ER homeostasis leads to IR and its progression to T2DM may offer new pharmacological targets for the treatment and prevention of these diseases. Here, we examine ER stress, the UPR, and downstream pathways in insulin sensitive tissues, and in IR, and offer insights towards therapeutic strategies.

High-fat diet-induced deregulation of hippocampal insulin signaling and mitochondrial homeostasis deficiences contribute to Alzheimer disease pathology in rodents

Global obesity is a pandemic status, estimated to affect over 2 billion people, that has resulted in an enormous strain on healthcare systems worldwide. The situation is compounded by the fact that apart from the direct costs associated with overweight pathology, obesity presents itself with a number of comorbidities, including an increased risk for the development of neurodegenerative disorders. Alzheimer disease (AD), the main cause of senile dementia, is no exception. Spectacular failure of the pharmaceutical industry to come up with effective AD treatment strategies is forcing the broader scientific community to rethink the underlying molecular mechanisms leading to cognitive decline. To this end, the emphasis is once again placed on the experimental animal models of the disease. In the current study, we have focused on the effects of a high-fat diet (HFD) on hippocampal-dependent memory in C57/Bl6 Wild-type (WT) and APPswe/PS1dE9 (APP/PS1) mice, a well-established mouse model of familial AD. Our results indicate that the continuous HFD administration starting at the time of weaning is sufficient to produce β-amyloid-independent, hippocampal-dependent memory deficits measured by a 2-object novel-object recognition test (NOR) in mice as early as 6months of age. Furthermore, the resulting metabolic syndrome appears to have direct effects on brain insulin regulation and mitochondrial function. We have observed pathological changes related to both the proximal and distal insulin signaling pathway in the brains of HFD-fed WT and APP/PS1 mice. These changes are accompanied by a significantly reduced OXPHOS metabolism, suggesting that mitochondria play an important role in hippocampus-dependent memory formation and retention in both the HFD-treated and AD-like rodents at a relatively young age.

The peroxisome proliferator-activated receptor (PPAR) β/δ agonist GW501516 inhibits IL-6-induced signal transducer and activator of transcription 3 (STAT3) activation and insulin resistance in human liver cells.

Aim/hypothesisIL-6 induces insulin resistance by activating signal transducer and activator of transcription 3 (STAT3) and upregulating the transcription of its target gene SOCS3. Here we examined whether the peroxisome proliferator-activated receptor (PPAR)β/δ agonist GW501516 prevented activation of the IL-6–STAT3–suppressor of cytokine signalling 3 (SOCS3) pathway and insulin resistance in human hepatic HepG2 cells.MethodsStudies were conducted with human HepG2 cells and livers from mice null for Pparβ/δ (also known as Ppard) and wild-type mice.ResultsGW501516 prevented IL-6-dependent reduction in insulin-stimulated v-akt murine thymoma viral oncogene homologue 1 (AKT) phosphorylation and in IRS-1 and IRS-2 protein levels. In addition, treatment with this drug abolished IL-6-induced STAT3 phosphorylation of Tyr705 and Ser727 and prevented the increase in SOCS3 caused by this cytokine. Moreover, GW501516 prevented IL-6-dependent induction of extracellular-related kinase 1/2 (ERK1/2), a serine–threonine protein kinase involved in serine STAT3 phosphorylation; the livers of Pparβ/δ-null mice showed increased Tyr705- and Ser727-STAT3 as well as phospho-ERK1/2 levels. Furthermore, drug treatment prevented the IL-6-dependent reduction in phosphorylated AMP-activated protein kinase (AMPK), a kinase reported to inhibit STAT3 phosphorylation on Tyr705. In agreement with the recovery in phospho-AMPK levels observed following GW501516 treatment, this drug increased the AMP/ATP ratio and decreased the ATP/ADP ratio.Conclusions/interpretationOverall, our findings show that the PPARβ/δ activator GW501516 prevents IL-6-induced STAT3 activation by inhibiting ERK1/2 phosphorylation and preventing the reduction in phospho-AMPK levels. These effects of GW501516 may contribute to the prevention of cytokine-induced insulin resistance in hepatic cells.

The peroxisome proliferator-activated receptor β/δ (PPARβ/δ) agonist GW501516 prevents TNF-α-induced NF-κB activation in human HaCaT cells by reducing p65 acetylation through AMPK and SIRT1

Nuclear factor (NF)-κB is a ubiquitously expressed transcription factor controlling the expression of numerous genes involved in inflammation. The aim of this study was to evaluate whether activation of the peroxisome proliferator-activated receptor (PPAR) β/δ prevented TNF-α-induced NF-κB activation in human HaCaT keratinocytes and, if so, to determine the mechanism involved. The PPARβ/δ agonist GW501516 inhibited the increase caused by TNF-α in the mRNA levels of the NF-κB target genes interleukin 8 (IL-8), TNF-α and thymic stromal lymphopoietin (TSLP). Likewise, GW501516 prevented the increase in NF-κB DNA-binding activity observed in cells exposed to TNF-α. The reduction in NF-κB activity following GW501516 treatment in cells stimulated with TNF-α did not involve either increased IκBα protein levels or a reduction in the translocation of the p65 subunit of NF-κB. In contrast, GW501516 treatment decreased TNF-α-induced p65 acetylation. Acetylation of p65 is mainly regulated by p300, a transcriptional co-activator that binds to and acetylates p65. Of note, AMP kinase (AMPK) activation phosphorylates p300 and reduces its binding to p65. GW501516 increased AMPK phosphorylation and the subsequent p300 phosphorylation, leading to a marked reduction in the association between p65 and this transcriptional co-activator. In addition, treatment with the PPARβ/δ agonist increased SIRT1 protein levels. Finally, the reduction in IL-8 mRNA levels following GW501516 treatment in TNF-α-stimulated cells was abolished in the presence of the PPARβ/δ antagonist GSK0660, the AMPK inhibitor compound C and the SIRT1 inhibitor sirtinol, indicating that the effects of GW501516 on NF-κB activity were dependent on PPARβ/δ, AMPK and SIRT1, respectively.