Giovanna R. Degasperi

Saturated Fatty Acids Produce an Inflammatory Response Predominantly through the Activation of TLR4 Signaling in Hypothalamus: Implications for the Pathogenesis of Obesity

In animal models of diet-induced obesity, the activation of an inflammatory response in the hypothalamus produces molecular and functional resistance to the anorexigenic hormones insulin and leptin. The primary events triggered by dietary fats that ultimately lead to hypothalamic cytokine expression and inflammatory signaling are unknown. Here, we test the hypothesis that dietary fats act through the activation of toll-like receptors 2/4 and endoplasmic reticulum stress to induce cytokine expression in the hypothalamus of rodents. According to our results, long-chain saturated fatty acids activate predominantly toll-like receptor 4 signaling, which determines not only the induction of local cytokine expression but also promotes endoplasmic reticulum stress. Rats fed on a monounsaturated fat-rich diet do not develop hypothalamic leptin resistance, whereas toll-like receptor 4 loss-of-function mutation and immunopharmacological inhibition of toll-like receptor 4 protects mice from diet-induced obesity. Thus, toll-like receptor 4 acts as a predominant molecular target for saturated fatty acids in the hypothalamus, triggering the intracellular signaling network that induces an inflammatory response, and determines the resistance to anorexigenic signals.

Deletion of tumor necrosis factor-alpha-receptor 1 (TNFR1) protects against diet-induced obesity by means of increased thermogenesis

In diet-induced obesity, hypothalamic and systemic inflammatory factors trigger intracellular mechanisms that lead to resistance to the main adipostatic hormones, leptin and insulin. Tumor necrosis factor-α (TNF-α) is one of the main inflammatory factors produced during this process and its mechanistic role as an inducer of leptin and insulin resistance has been widely investigated. Most of TNF-α inflammatory signals are delivered by TNF receptor 1 (R1); however, the role played by this receptor in the context of obesity-associated inflammation is not completely known. Here, we show that TNFR1 knock-out (TNFR1 KO) mice are protected from diet-induced obesity due to increased thermogenesis. Under standard rodent chow or a high-fat diet, TNFR1 KO gain significantly less body mass despite increased caloric intake. Visceral adiposity and mean adipocyte diameter are reduced and blood concentrations of insulin and leptin are lower. Protection from hypothalamic leptin resistance is evidenced by increased leptin-induced suppression of food intake and preserved activation of leptin signal transduction through JAK2, STAT3, and FOXO1. Under the high-fat diet, TNFR1 KO mice present a significantly increased expression of the thermogenesis-related neurotransmitter, TRH. Further evidence of increased thermogenesis includes increased O2 consumption in respirometry measurements, increased expressions of UCP1 and UCP3 in brown adipose tissue and skeletal muscle, respectively, and increased O2 consumption by isolated skeletal muscle fiber mitochondria. This demonstrates that TNF-α signaling through TNFR1 is an important mechanism involved in obesity-associated defective thermogenesis.

Reactive oxygen species generation in peripheral blood monocytes and oxidized LDL are increased in hyperlipidemic patients

OBJECTIVES Experimental and in vitro evidences have established that reactive oxygen species (ROS) generated by vascular wall cells play a key role in atherogenesis. Here, we evaluated the rate of ROS generation by resting peripheral monocytes in naive hyperlipidemic subjects. DESIGN AND METHODS Primary hypercholesterolemic, combined hyperlipidemic, and normolipidemic individuals were studied. ROS generation and the mitochondrial electrical transmembrane potential were estimated by flow cytometry. Plasma oxidized (ox) LDL levels and lipid profile were measured by ELISA and enzymatic colorimetric methods. RESULTS Both hyperlipidemic groups presented significantly higher rates of monocyte ROS generation and elevated plasma levels of ox-LDL. Combined hyperlipidemic subjects presented increased levels of small dense LDL and insulin. Significant positive correlations between monocyte ROS generation and ox-LDL concentrations were found in pooled data. CONCLUSIONS These data provide evidence that ROS production by circulating monocytes from hyperlipidemic subjects may contribute to the systemic oxidative stress and possibly to atherogenesis.

Reactive oxygen species production is increased in the peripheral blood monocytes of obese patients

Infiltrating macrophages play an important role in the production of inflammatory mediators by the adipose tissue of obese subjects. To reach the adipose tissue, peripheral monocytes are recruited by locally produced chemoattractants. However, little is known about the activation of monocytes in the peripheral blood of obese subjects. The objective of this study was to determine reactive oxygen species and endoplasmic reticulum stress as early markers of monocytic commitment with an inflammatory phenotype in the peripheral blood of nondiabetic obese patients. Patients were recruited from an academic general hospital; controls were voluntary students. Seven lean controls and 6 nondiabetic obese patients were included in the study. Monocytes were prepared from peripheral blood. Immunoblot, flow cytometry, and polymerase chain reaction were used to determine reactive oxygen species and endoplasmic reticulum stress. Increased reactive oxygen species and activation of endoplasmic reticulum stress were detected in the monocytes from obese patients. Reducing endoplasmic reticulum stress with a chemical chaperone reversed monocytic activation, as determined by the reduction of reactive oxygen species production. Thus, monocytes from nondiabetic obese patients are already committed with an inflammatory phenotype in peripheral blood; and reducing endoplasmic reticulum stress negatively modulates their activation.

Fyn Mediates Leptin Actions in the Thymus of Rodents

Background Several effects of leptin in the immune system rely on its capacity to modulate cytokine expression and apoptosis in the thymus. Surprisingly, some of these effects are dependent on signal transduction through the IRS1/PI3-kinase, but not on the activation of JAK2. Since all the well known effects of leptin in different cell types and tissues seem to be dependent on JAK2 activation, we hypothesized that, at least for the control of thymic function, another, unknown kinase could mediate the transduction of the leptin signal from the ObR towards the IRS1/PI3-kinase signaling cascade. Methodology/Principal Findings Here, by employing immunoblot, real-time PCR and flow citometry we show that the tyrosine kinase, Fyn, is constitutively associated with the ObR in thymic cells. Following a leptin stimulus, Fyn undergoes an activating tyrosine phosphorylation and a transient association with IRS1. All these effects are independent of JAK2 activation and, upon Fyn inhibition, the signal transduction towards IRS1/PI3-kinase is abolished. In addition, the inhibition of Fyn significantly modifies the effects of leptin on thymic cytokine expression. Conclusion/Significance Therefore, in the thymus, Fyn acts as a tyrosine kinase that transduces the leptin signal independently of JAK2 activation, and mediates some of the immunomodulatory effects of leptin in this tissue.

High susceptibility of activated lymphocytes to oxidative stress-induced cell death

The present study provides evidence that activated spleen lymphocytes from Walker 256 tumor bearing rats are more susceptible than controls to tert-butyl hydroperoxide (t-BOOH)-induced necrotic cell death in vitro. The iron chelator and antioxidant deferoxamine, the intracellular Ca2+ chelator BAPTA, the L-type Ca2+ channel antagonist nifedipine or the mitochondrial permeability transition inhibitor cyclosporin A, but not the calcineurin inhibitor FK-506, render control and activated lymphocytes equally resistant to the toxic effects of t-BOOH. Incubation of activated lymphocytes in the presence of t-BOOH resulted in a cyclosporin A-sensitive decrease in mitochondrial membrane potential. These results indicate that the higher cytosolic Ca2+ level in activated lymphocytes increases their susceptibility to oxidative stress-induced cell death in a mechanism involving the participation of mitochondrial permeability transition.

METHODS FOR ASSESSING AND MODULATING UCP2 EXPRESSION AND FUNCTION

Uncoupling protein 2 (UCP2) is a member of the uncoupling protein family. It is expressed in the inner mitochondrial membrane and plays a role in the control of free radical production, oxidative damage, insulin secretion, and fatty-acid peroxide exportation. Although UCP2 expression occurs in several tissues, some of its most remarkable functions are exerted in organs of difficult experimental access, such as the central nervous system, particularly the hypothalamus and the pancreatic islets. In addition, due to its low levels of expression in the mitochondrial membrane, studying UCP2 expression and function depends on specific- and well-established methods. This chapter describes methods for directly assessing UCP2 expression and function in different tissues. Purified mitochondria preparations are used for enhancing the capacity of detection of UCP2 protein or for evaluating the role of UCP2 in mitochondria respiration. Exposure of experimental animals to cold environment leads to increased UCP2 expression, while reduction of its expression can be achieved directly by targeting its mRNA with antisense oligonucleotides, or indirectly by targeting PGC-1alpha expression with antisense oligonucleotides.