Di Guida, Francesca (2017) Pharmacological effects of palmitoylethanolamide on hypertension, insulin-resistance and obesity in murine models. [Tesi di dottorato]

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N-Palmitoylethanolamide (PEA) is an endogenous N-acylethanolamine, first identified in lipid extracts from brain, liver, and muscle of rat and guinea pig. PEA is formed “on demand” from membrane phospholipids and it is gaining ever-increasing interest not only for its anti-inflammatory and analgesic effects mediated by peroxisome-proliferator activated receptor (PPAR)-α, but also for its novel metabolic effects. Overweight and obesity are defined as abnormal or excessive fat accumulation that may impair health. Main consequence of obesity is cardiovascular disease (CVD). The sum of the risk factors that predisposes to CVD goes by the name of “metabolic syndrome” (MetS). Hypertension is an important hallmark of MetS and a common cause of kidney disease. In the first part of this thesis, we investigated the mechanisms underpinning PEA blood pressure lowering effect, exploring the contribution of epoxyeicosatrienoic acids (EETs), CYP-dependent arachidonic acid (AA) metabolites, as endothelium derived hyperpolarizing factors (EDHF), and renin-angiotensin system (RAS) modulation. To achieve this aim, SHR and Wistar-Kyoto normotensive (WKY) rats were treated with PEA (30 mg/kg/day, s.c.) for five weeks. Functional evaluations on mesenteric bed were performed to analyze EDHF mediated vasodilation. Moreover, mesenteric bed and carotid were harvested to measure the soluble epoxide hydrolase (sEH), which is the enzyme responsible for EETs degradation in their corresponding inactive diols. Effect of PEA on RAS modulation was investigated by analyzing angiotensin converting enzyme (ACE) and angiotensin receptor (AT)1 expression. We showed that EDHF-mediated dilation in response to acetylcholine (Ach) was increased in mesenteric beds of PEA-treated SHR. Interestingly, in both vascular tissues, PEA significantly decreased the sEH protein level, accompanied by a reduced serum concentration of its metabolite 14-15 dihydroxyeicosatrienoic acid (DHET), implying a reduction in EET hydrolysis. Moreover, PEA treatment down-regulated AT1 and ACE expression, indicating a reduction in Ang II-mediated effects. Our data clearly demonstrate the involvement of EETs and RAS in the blood pressure lowering effect of PEA. The relationship between obesity, insulin-resistance (IR) type 2 diabetes mellitus (T2DM) and MetS is well known. IR is defined as an inefficient glucose uptake and utilization in peripheral tissues in response to insulin stimulation. IR in the prediabetes stage is a feature of glucose intolerance, which includes impaired fasting glucose and/or impaired glucose tolerance. When insulin binds to its transmembrane receptor (InsR), promotes its autophosphorylation (pInsR). Activated pInsR recruits insulin receptor substrate (IRS), leading to insulin signaling cascade. A potential link between inflammation and IR has been shown. Indeed, obesity is characterized by chronic low grade inflammation, where the release of adipose tissue-derived cytokines can block insulin action and cause systemic IR. In fact, serum tumor necrosis factor (TNF)-α and interleukin (IL)-6 are significantly increased in serum from obese patients. All cytokines induce IRS1 protein degradation, which suppresses insulin signaling pathway and subsequently suppresses glucose transporter (GLUT) translocation and glycogen synthesis, contributing to IR and hyperglycemia. Our study was focused on the pharmacological effect of PEA in an animal model of diet-induced obesity (DIO), feeding mice with a high-fat diet (HFD), and on the mechanisms by which this lipid mediator could modulate the storage and availability of energy sources, restoring lipid/glucose homeostasis. To achieve this aim, mice were fed a standard chow diet (STD group) or HFD (DIO group). After twelve weeks, both STD or HFD mice were treated with PEA (30 mg/kg/day, o.s.) for ten weeks. At the end of the experimental period, body parameters were determined, and serum and tissues collected for following determinations. Interestingly, PEA caused a reduction in body weight and fat mass, improved glucose tolerance and prevented IR, induced by HFD feeding. Moreover, PEA restored the alterations of serum biochemical and inflammatory parameters, inducing a marked reduction of ALT, AST, cholesterol, and pro-inflammatory cytokines, such as TNF-α, IL-1 and monocyte chemoattractant protein (MCP)-1. PEA also normalized metabolic hormone levels and restored insulin sensitivity. At hepatic level, PEA treatment significantly induced an increase in the activation AMPK/ACC pathway, stimulating fatty acid oxidation, compromised in obese mice. To evaluate tissue insulin-sensitivity, we determined the hepatic expression of the InsR, whose expression decreased in liver of DIO mice compared to that of STD animals, and increased in PEA-treated mice. Then, we evaluated the effectiveness of hepatic insulin signaling through the evaluation of InsR and Akt phosphorylated state and the expression of GLUT-2. PEA treatment restored insulin signaling. The protective effect of PEA was strengthened by the evaluation of hepatic IL-6 and TNF-α, whose transcription, upregulated by HFD feeding, was reduced. To address the direct effect of PEA on hepatic insulin-sensitivity, we evaluated the restoration of insulin signaling, altered by the induction of IR, in HepG2 cells, a human hepatocarcinoma cell line. Therefore, we demonstrated in vitro that PEA increased the phosphorylation of Akt in insulin resistant cells, following insulin stimulation. PEA was also able to modulate glucose homeostasis at hypothalamic level. Therefore, we examined neuronal activation at the arcuate (ARC) and ventromedial (VMH) nuclei, evaluating c-fos immunostaining. In the ARC nucleus of DIO mice, a decrease in c-fos labeling was found. Interestingly, in the PEA-treated DIO group, a trend of c-fos labeling increase was evidenced. Consistently, in the VHM of DIO mice a significant decrease in the neuronal activation was shown compared to STD mice, although, no differences were found between DIO and PEA-treated DIO mice. The involvement of the hypothalamic control of glucose homeostasis by PEA was confirmed in in vitro experiments, using human SH-SY5Y neuroblastoma cell line. When insulin-resistant cells were treated with PEA, the re-stimulation with insulin showed a restoration of Akt phosphorylation, and therefore of insulin-sensitivity. These findings show that this acylethanolamide also displays a central effect on glucose homeostasis, reducing neuronal IR. Our data strengthened evidence on the metabolic activity of PEA, through the involvement of central and peripheral mechanisms. PEA clearly ameliorates glucose-tolerance and insulin-sensitivity, indicating its therapeutic potential for the treatment of metabolic dysfunctions associated to obesity, such as IR and T2DM.

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