Discussion and Perspectives

Adipose tissue integrity is crucial for whole body energy homeostasis. Its functional deregulation, as observed in obesity or lipodystrophy, is linked to diseases, such as type 2 diabetes, usually found associated with the metabolic syndrome. PPARγ occupies key functions in WAT, where it participates in adipocyte differentiation and maintenance, including promotion of lipid storage (369). In the absence of PPARγ, preadipocytes do not differentiate into adipocytes, and its deletion in mature adipocytes leads to their death (162). The prime role of PPARγ in the metabolic balance is also underscored by the fact that it is the molecular target of type 2 diabetes drugs, which directly bind to PPARγ. Interestingly, PPARγ +/- mice were shown to be resistant to HFD-induced obesity and more sensitive to insulin then their wt counterparts (189,247). Paradoxically, untreated PPARγ +/- mice on a HFD were as sensitive to insulin as wt mice on the same diet, but treated with a PPARγ agonist, the insulin sensitizer Rosiglitazone (246).

We aimed to understand the reasons for this paradox. We suspected that lipid storage is by far not the sole metabolic function of PPARγ. However, in previous studies, challenging the PPARγ +/- mice with a HFD most likely exacerbated the lipid storage function of PPARγ and therefore other key functions went unnoticed. For this reason, we chose to study the metabolic role of PPARγ in the absence of any specific nutritional challenge by investigating young adult PPARγ +/- male mice fed on a SD.

The plasmatic analyses showed that if insulin and glucose concentrations were normal in basal feeding conditions, there was an alteration of the glycaemic control in the mutated mice during fasting. Interestingly, the plasmatic lipid profile of these animals, especially FFA and glycerol, suggested the possibility of an abnormal lipolysis. This presumption was strengthened by the fact that the plasmatic concentrations of ketone bodies were also low. Although this would designate the adipose tissue as the main culprit for these deregulations, we nevertheless extended our study to the liver and skeletal muscle. All three organs are either important consumers (skeletal muscle) or producers (WAT and liver) of energy. Furthermore, all three express PPARγ, although at different levels, WAT presenting by far the highest levels of this receptor. Based on their gene expression

profiles, we concluded that liver and skeletal muscle in PPARγ +/- mice are most likely not involved in the metabolic deregulation of these animals when fed a SD. Of all the genes tested, which are involved in glycolysis and FA oxidation in both tissues, as well as in gluconeogenesis in the liver, none presented a modified expression in the PPARγ +/- mice. These results are in agreement with liver and muscle gene expression profiles in fatless mice or mice with an organ-specific deletion of PPARγ in skeletal muscle. In these studies, no modification was observed in the expression of genes involved in β-oxidation and gluconeogenesis (248,253,255,370).

On the contrary, deletion of one PPARγ allele had unexpectedly profound effects on the expression of genes involved in WAT growth and energy production. The present study shows that genes involved in the IGF-1 signaling pathway, known to participate in adipocyte differentiation and growth (245,371-374) were down-regulated in PPARγ +/- WAT. This explains the resistance to growth hormone action and the smaller adipocyte size already observed by us and others in the PPARγ +/- WAT (189,245,289). In addition to the IGF-1 pathway, genes belonging to the insulin signaling pathway, which is an important activator of the glycolytic and lipogenic pathways in WAT, were also down-regulated.

Unexpected at first sight, but in agreement with a down-regulation of the insulin pathway, basal glucose uptake and glycolysis were decreased in the mutant WAT. Glycolysis is thought to play three majors roles in WAT by (i) participating in the de novo synthesis of fatty acids, (ii) promoting their storage as fat and, finally, (iii) producing ATP for adipocyte survival. Firstly, the acetyl CoA and NADPH synthesized by the glycolytic and pentose phosphate pathways, respectively, were decreased in the PPARγ +/- WAT. As an immediate consequence, de novo fatty acid synthesis was decreased, an effect reinforced by the down-regulation of enzymes involved in this process. Secondly, storage was altered since glyceroneogenesis was affected in the PPARγ +/- WAT. In fact, the activated glycerol produced by glyceroneogenesis is used for fatty acid esterification into TG, the main form of lipid storage. Thirdly, glucose is required for adipocyte survival, and ATP production is an important feature of this process, deficiency of which affects the integrity of the cell. In this respect, we observed a significant decrease of the ATP levels in the WAT of the mutant mice. This result is in agreement with previous data from cell culture experiments where decreased ATP concentration in 3T3L1 or primary adipocytes interfered with

RESULTS-PPARγ and adipose tissue integrity

insulin signaling and lipolysis. Glycolysis and FA oxidation are the major ATP producers, but no increase of β-oxidation was observed in the mutant WAT to compensate for the energy deficit.

Moreover, genes involved in the lipolytic activity as well as in the glycerol release were down-regulated in the PPARγ +/- WAT. Thus, PPARγ +/- WAT suffers from a generalized energy shortage probably due to the ATP crisis in the adipocytes, which we link to a deregulation of the glycolytic pathway.

To our knowledge and for the first time, the findings reported herein link PPARγ expression levels with the regulation of the systemic metabolic rate and physical activity. Although deletion of just one PPARγ allele decreased the metabolic rate and physical activity of the mice, we recorded no effect on the relative amounts of carbohydrates versus lipids used as fuel molecules. Evidence that this systemic effect is PPARγ-specific comes from the Pioglitazone treatment of the PPARγ +/- mice, which alleviated the deleterious effect of heterozygocity.

We speculate that the reduced level of both metabolic rate and physical activity are part of a protective survival strategy in conditions of energy shortage. When faced with an energy crisis, animals employ various behavioural and physiological responses to reduce metabolism, which prolongs the period of time during which energy reserves can cover metabolic needs. Such behavioural responses can include a reduction in metabolic rate and spontaneous activity.

Collectively, the data obtained by taking advantage of PPARγ +/- animals fed a SD, showed that PPARγ activity in WAT is not only important for fat storage, as previously showed. In fact, in the absence of any nutritional challenge, PPARγ controls IGF-1 and insulin signaling with effects on energy production via the regulation of the glycolytic and lipolytic pathways. Most importantly, deregulation of these functions in WAT most likely influences the whole-body energetic balance with its impacts on metabolic rate and physical activity, which appear to adapt to the available energy.

Furthermore, our results showed that deletion of one PPARγ allele affects the integrity of the white adipose tissue. Beside the delay in the adipocyte differentiation, several key proteins involved in the detoxification pathway were deregulated in the mutated WAT. This deregulation was accompanied by an increased inflammatory reaction. Proteins involved in the inflammatory reaction

as well as in macrophage activation were up-regulated in the PPARγ +/- WAT. Surprisingly the expression of some of these adipokines and chemokines was shown to be stimulated by fasting. This is an interesting observation since it makes the link between the inflammatory status of the mutant WAT and its energetic deficiency that we explored above.

Measurements of the ROS and lipid peroxide concentrations in WAT but also in other organs such as the liver, should tell us more about the deregulation of the detoxification program.

Moreover, extensive measurements of the expression of inflammatory molecules as well as histological analysis of inflammatory cells infiltration in WAT should clarify these observations.

Thus, in normal conditions, PPARγ plays an important role not only in lipid storage but also participates in the metabolic regulation of the WAT as well as to its integrity. Deletion of only one PPARγ allele deregulates the WAT equilibrium and has a whole body systemic effect.

These results shows for the first time that PPARγ is not only involved in the lipid uptake and storage as it was suggested by studies in models of diet induced obesity, but it also controls the general metabolism of the WAT, regulating the glycolytic and lipolytic status of the tissue as well as its energy production. Besides metabolism and energy production, we showed for the first time that normal PPARγ expression levels are required for the integrity of adipocytes where its expression levels are important. The link between inflammation and energetic status of the tissue argue against the utilization of PPARγ antagonists as drug replacement therapy for the treatment of type 2 diabetes. Moreover, it shows again that the metabolic integrity of the WAT is a prerequisite for an uninflamed tissue, deregulation of its metabolism either by obesity or lipodystrophy will stimulate the inflammatory reactions.

In the line of our results, selective modulators of PPARγ are from far a better alternative since they are not ablating PPARγ activity, rather they seem to preferentially activate genes involved in insulin signaling without affecting genes involved in the lipid metabolism (195,375,376).

II. Setup of the small interference RNA (siRNA) technique for the knock-down of different