Cell stress and detoxification

Molecular oxygen (O2) is the premier biological electron acceptor that serves vital roles in fundamental cellular functions. However, with the beneficial properties of O2 comes the inadvertent formation of reactive oxygen species (ROS) such as superoxide (O2•−

), hydrogen peroxide (H2O2),

Figure 8. A. Histological analysis of the white adipose tissue. Black arrows represent adipocyte nucleus and red arrows represent the infiltrated inflammatory cells. B. Presence of inflammatory foci in the white adipose tissue. Black arrow represent the inflammatory foci (left picture).Black arrow represent inflammatory cell found in the inflammatory foci (right picture, higher magnitude). C.

Measurement of the adipocyte size using the Image J program. D. Counting of the number of infiltrated inflammatory cells. A, B, C and D. Hematoxylin and Eosin Staining (H&E); n = 2

RESULTS-PPARγγγγ and adipose tissue integrity

and hydroxyl radical (OH^•). If unabated, ROS pose a serious threat causing cell death. To minimize the damaging effects of ROS, organisms evolved enzymatic antioxidant defences. This includes enzymes such as superoxide dismutases (Sod), catalases (Cat), glutathione peroxidases (Gpx), and glutathione S-transferases (GST). Sod, Cat and Gpx protect cell integrity by directly scavenging superoxide radicals and hydrogen peroxide, converting them to less reactive species. Sod catalyze the dismutation of O₂^•− toH₂O₂ and Cat and Gpx reduces H₂O₂ to 2H₂O (327). Thus, the Sod and Cat/Gpx serve, in tandem, as a first front-line in the antioxidant defences (328). Glutathione S transferases (GST) are enzymes acting as a second line defence against the oxidative stress. These enzymes react with various electrophiles, physiological metabolites and xenobiotics to form mercapturates which can be safely degraded by the organism (329,330).

14 % of the genes expressed in WAT are involved in cell and organism defences (303).

Deregulation of this defence has been observed in metabolic syndrome diseases such as atherosclerosis, obesity and type 2 diabetes. The increase in the oxidative stress observed in these pathologies is due to a defective antioxidant system reflected by a decrease in the expression of Gpx, Cat and Sod as well as an increase in the expression of inflammatory molecules such as IL-6, MCP1 and PAI1 (331). Observations on mouse and human adipocytes showed a correlation between increased ROS production and increased inflammatory reactions (332). Moreover, oxidative stress was shown to impair lipogenesis and glucose uptake as well as PPARγ expression in WAT (331).

Impairment of lipogenesis and glucose uptake were observed in PPARγ +/- WAT (section Results I.2.). Our microarray analysis showed a deregulation of several genes involved in the first and second line of defence in the detoxification pathways in the PPARγ +/- WAT.

As mentioned above, two of the enzymes involved in this first front-line antioxidant defence were down-regulated in the heterozygous WAT. The expression levels of Sod and Gpx1 were decreased by 1.4 and 1.7 folds respectively (Table 3). Lower Gpx activity was observed in obese and diabetic rats and decreased Gpx expression was observed in several pathologies linked to oxidative stress (191,333). Gpx knock-out mice are sensitive to an increase in H2O2 concentrations (334), whereas Sod knock-out mice suffer from an increase in lipid peroxidation (335). These results suggest that the PPARγ +/- WAT suffers from an increase in the ROS concentrations. However,

further experiments measuring the ROS concentrations as well as the Sod and Gpx activities in WAT would allow a better analysis of this situation (336).

A second line of defence in the antioxidant program is the activation of GST enzymes. These enzymes catalyze the nucleophilic attack by reduced glutathione (GSH) on nonpolar compounds containing an electrophilic carbon, nitrogen, or sulphur atom. Their substrates include metabolic derivatives such as oxidized lipids, DNA, protein, and carbohydrates as well as xenobiotics (327).

GSTs were shown to participate to the synthesis of eicosanoids and prostanoids, to reduce fatty acids, cholesteryl and phospholipid hydroperoxides and to protect DNA against oxidative damage (327,337-339). Three major families of proteins that are widely distributed in nature exhibit glutathione transferase activity. Two of these, the cytosolic and mitochondrial GST, comprise soluble enzymes that are only distantly related. The third family comprises microsomal GST and is now referred to as membrane-associated proteins in eicosanoid and glutathione (MAPEG) metabolism (327).

The glutathione S transferase mu 6 (Gstm6) is a cytosolic GST, participating to the elimination of oxidative stress molecules. Its overexpression was shown to protect against oxidative stress induced apoptosis, involving this enzyme in the adaptive response to cell stress (340,341). Its expression was up-regulated by 1.9 folds in PPARγ +/- WAT, suggesting that the heterozygous WAT needs an increased protection against the oxidative stress (Table 3). Measuring the levels of lipid peroxidation in WAT should sharpen this analysis and will definitely underline possible perturbations in the mechanism of protection against oxidative stress (336,342).

The microsomal GSTs are membrane associated proteins involved essentially in the synthesis of leukotrienes, which are lipid mediators of the inflammatory response (343). Knock-out models of microsomal GSTs were shown to be deficient in leukotriene synthesis (344). The expression of Mgst3, one of the microsomal GSTs, was down-regulated by 1.8 folds in the PPARγ +/- WAT, (Table 3). Since this class of GSTs was associated to leukotriene synthesis, it is possible that their production is reduced in the heterozygous WAT implying that the adipose tissue is deficient in the anti-inflammatory response. Moreover, the expression of Redd1, a marker of oxidative stress up-regulated by cellular stress (345), was also increased by 2.3 folds in PPARγ +/- WAT, (Table 3).

RESULTS-PPARγγγγ and adipose tissue integrity

When the cells undergo irreversible damages their apoptotic programme is activated leading to their apoptosis (346). Gadd45γ and Egr1, two inducers of apoptosis activated by DNA damage and cellular stress (347), were down-regulated by 2 folds in the PPARγ +/- WAT, (Table 3). Thus, the PPARγ +/- WAT suffers from cell stress and accumulation of undesired metabolites. In this context, down-regulation of the expression of pro-apoptotic proteins could be a protective mechanism against cellular death.

The results presented above are in agreement with our previous results showing that deletion of one PPARγ allele in WAT interfere with several metabolic pathways in the white adipose tissue leading to a deficiency in the energy production, (section Results I.2.). Deregulation of these metabolic pathways as well as the energetic deficiency could indeed be responsible for the cellular stress depicted by our microarray results.