Discussion
58
Discussion
59
4. DISCUSSION
The present thesis project has allowed us both to investigate the metabolic consequences of Haptoglobin deficiency in the white adipose tissue and to better characterize the biological role of this inflammatory / adiposity molecule in the WAT. The results shown in this project will be extensively discussed in the following sections.
Inflammation / obesity is becoming an inseparable binomial association that has yet to be fully unraveled in terms of clinical consequences and underlying cytological, biochemical and molecular mechanisms. Haptoglobin (Hp), an inflammatory molecule that is up-regulated in the WAT of obese subjects, constitutes an interesting tool to further explore this relationship. In this study we demonstrated that murine WAT not only expresses but also releases Hp at levels that equal, if not overcome, the hepatic release in terms of contribution to Hp circulating concentration, in agreement with what observed in humans by Fain et al. (Fain JN et al., 2004). Furthermore, we proved that in obese mice, which show increased serum Hp levels, Hp expression is augmented in the WAT, but not in the liver, the main source of this protein, thus suggesting that obesity induces a specific WAT Hp up-regulation. These premises should be considered when analyzing the relationship between Hp expression, metabolism and WAT inflammatory profile.
For the first time, this study shows that in the HFD-treated mouse Hp deficiency results in an attenuation of obesity associated complications including hepatomegaly / steatosis and reduced glucose tolerance. In this regard, we proved the relevance of WAT for this phenotype through our insulin signaling data that indicate this organ as the one with the highest preservation (with respect to HFD WT mice) of insulin sensitivity, among metabolically active tissues. Moreover, we showed that the lack of Hp leads to a less activated machinery for hepatic triglyceride
Discussion
60 accumulation, allowing to attenuate the hepatomegaly / steatosis often associated with the
obesity state. In addition, microarray analysis showed changes in HFD Hp−/− liver expression
profile that are consistent with lower activation of the apoptotic pathways (Lisi S, Gamucci O et al.,2011). Apoptosis is a classical feature of hepatic steatosis progressing towards hepatitis and irreversible liver damage (Mahli H et al., 2010; Yin XM and Ding WX, 2003) and what we observe upon Hp deficiency is therefore consistent with a phenotype that is more protected against the onset of this pathology. Overall our data could be interpreted within two possible scenarios respectively based on a local or systemic action of Hp. Whether changes in liver
expression observed by us in HFD Hp−/− mice are due specifically to one of them or both Hp
actions cannot be fully established yet. The local scenario would imply an Hp autocrine effect, possibly modulating deleterious gene expression programs. According to the systemic hypothesis, the liver would be healthier because of surrounding conditions of improved glucose homeostasis and insulin sensitivity determined by other organs. In this regard, the benign consequences of Hp deficiency are associated to and may derive from reduced macrophages infiltration into WAT and higher adiponectin levels. Both aspects imply a direct involvement of WAT, this being in line with the specific WAT Hp up-regulation induced by obesity in the WT mouse. Interestingly, in a recent study WAT dysfunction was associated with hepatosteatosis and
plasma Hp defined as a prognostic marker for non-alcoholic fatty liver disease (NAFLD) and
non-alcoholic steatohepatitis in animals exposed to HFD (Duval C et al., 2010).
We recently reported that Hp is a chemotactic molecule (Maffei M et al., 2009), which is
consistent with the reduced ATM infiltration observed in the obese Hp−/− mice. Macrophage
infiltration in the WAT of obese individuals is related to the low chronic inflammatory state that often characterizes the obesity status. In particular, the release of inflammatory factors by macrophages, such as interleukin-6 and tumor necrosis factor-α, contributes to the onset of insulin resistance (Bourlier V and Bouloumie A, 2009).
Discussion
61 explain this association include a secondary effect of the altered availability of lipids generated by the insulin resistance state and a direct consequence of the altered WAT expression profile (Marra F and Bertolani C., 2009). Kolak and colleagues interestingly reported (Kolak M et al., 2007) that increased fat content in the liver is independent of the degree of obesity, but significantly associated with adipose tissue inflammation and, specifically, with the abundance of markers including among others, CD68 and MCP1. MCP-1 was initially described as a potent chemoattractant factor (Yoshimura T et al., 1989) and has been recently implicated in the recruitment of macrophages in WAT. Supporting evidence in this direction derives from the increased infiltration of macrophages observed by Kanda and coworkers in lean mice over-expressing MCP-1 in WAT and, consistently, from the significantly lower content of these cells in obese mice deficient for this factor (Kanda H et al., 2006) or for its receptor (CCR2) (Weisberg SP et al., 2006), as compared to controls. These data were not always confirmed, since Inouye et al. (Inouye KE et al., 2007) reported that macrophage content in the WAT of
MCP 1 −/− obese mice is, if nothing higher, than that of obese controls. In no case anyway
genetic obese models for MCP-1 or CCR2 exhibited a level of ATM normalized to that observed in lean mice, thus implying the presence of other factors in the modulation of this phenomenon, many of which yet to be identified. According to our published and currently reported evidence (Maffei M et al., 2009), Hp presents all the features to be considered one of them. Tissue distribution differentiates MCP-1 from Hp, because abundance in WAT of the former is prevalently due (80%) to its expression in the stromal vascular fraction (Di Gregorio GB et al., 2010; Christiansen et al., 2005), whereas Hp gene expression and release are almost totally confined to the adipocyte fraction (do Nascimento CO et al., 2004). This strengthens the notion that the adipocyte itself is able to produce chemotactic molecules and attract macrophages both by active release and by simple spill-over in case of cell death (Cinti S et al., 2005). We previously demonstrated that Hp is able to internalize CCR2 in cultured monocytes and that pharmacologic inhibition of this chemokine receptor results in the abolition of Hp chemotactic
Discussion
62 activity (Maffei M et al., 2009), thus suggesting that Hp interacts with CCR2 to recruit
macrophages. As we found for the obese Hp−/− mice, CCR2−/− animals exposed to a HFD
showed reduced ATM content, improved insulin sensitivity, reduced hepatic steatosis, and
increased adiponectin (Weisberg SP et al., 2006). Obese Hp−/− mice show higher adiponectin
expression compared to controls (Lisi S, Gamucci O et al., 2011). This adipokine is considered an important player in the determination of insulin sensitivity. A large amount of evidence points to the lack of a sufficient amount of this factor as a key signal for the onset of insulin resistance (Cook JR and Semple RK, 2010) and for the development of hepatic steatosis. In HFD-treated mice, decreased adiponectin often precedes steatohepatitis (Larter CZ et al., 2009) and
adiponectin reverses steatosis in ob/ob mice (Xu A et al., 2003). Obese Hp−/− mice exhibit higher
circulating and local levels of adiponectin compared with matched WT controls, which may partly explain their improved glucose tolerance and reduced hepatosteatosis. Our observation
regarding adiponectin in HFD-treated Hp−/− mice was independently proved by the inhibitory
effect exerted by Hp on adiponectin expression and secretion in cultured adipocytes (Lisi S, Gamucci O et al., 2011). To our knowledge, this is the first report demonstrating that adiponectin is significantly downregulated by Hp, thus introducing a new player in the regulation of this adipokine. Accordingly, similar inhibitory capacities were previously reported for other inflammatory factors, including tumor necrosis factor-α and interleukin-6 (Fasshauer M et al., 2003; Hajer GR et al., 2004, Maeda N et al., 2001), also upregulated in the WAT of obese subjects.
An increased insulin sensitivity and a higher expression of adiponectin (van Tienen FH et al., 2005; Roberts R et al., 2009) have also been positively related to small adipocytes and to a protection against the excessive size of adipocytes upon obesity. Findings presented in this study
indicate that obese Hp−/− mice have a smaller adipocyte size as compared to WT (Gamucci O et
Discussion
63 insulin-dependent AKT activation, that define WAT as a key element in the protection against
the obesity associated onset of systemic insulin resistance in the Hp−/− model (Lisi S, Gamucci O
et al., 2011). The herein found higher expression of hormone sensitive lipase and perilipin suggests an increased activation of the TG hydrolysis machinery and may at least partly explain this phenotype. It is of interest to compare our findings on obese mice with those from Jo and colleagues (Jo J et al., 2009), who employed a sophisticated computational approach to the study of hypertrophy and hyperplasia of adipocytes in obesity resistant FVB and obesity susceptible C57BL/6J mice during regular CFD and HFD. Their results indicate that hypertrophy and not hyperplasia is a major determinant in the rapid enlargement of fat mass typical of HFD. Cell size growth moves the mode of the cell size distribution to larger sizes and increases the spread of the distribution around the mode. This effect is more pronounced in the C57BL/6J mice that become insulin resistant in response to HFD, as compared to the FVB that remain more insulin sensitive.
Our intra-genotype analyses, although not as accurate, revealed that in Hp−/− mice the cell size
distribution spread due to HFD is attenuated as compared to WT, thus confirming a resistance to the effect of HFD, somewhat reminiscent of what takes place in the FVB strain. It is interesting to analyse these findings also through the innovative model proposed by Muller (Muller G, 2011), who speculates about the existence of signals sent by large adipocytes to order small adipocytes to “take-over” the excess of lipid loading. This should lead to the up-regulation of lipid storage in small adipocytes and thus blunt the spread (small / large) of cell size within the tissue. If any, Hp action within this dialogue has to be negative, given that both during obesity, Hp deficiency attenuated the spread effect, implying that lipid storage is more homogeneously distributed among existing cells.
In the lean Hp−/− mouse we did not observe any metabolic alteration and, in contrast to what
Discussion
64
expression of the main genes of adipogenesis LPL, FAS, aP2 and PPAR
γ
characterizes the leanadult Hp−/− WAT. Accordingly, data obtained in MEFs in terms of both TG accumulation and
gene expression show a reduced capacity to be converted to adipocytes in the Hp−/− as compared
to WT cells, thus suggesting a less active adipogenesis in the WAT of the Hp−/− mouse (Gamucci
O et al., 2012).
In the effort to reconcile data obtained on obese Hp deficient animals and on Hp−/− MEFs, we
could speculate that in the absence of the antioxidant inflammatory-related factor Hp the adipocyte size distribution tends to be more static upon the HFD induced hypertrophy. While the
impaired adipogenic potential observed in Hp−/− MEFs might account for a more static
hyperplastic phase that characterizes the first stages of adipogenesis, the relationship with the observation about hypertrophy is somewhat controversial. Adipogenesis role in the onset / protection against obesity and associated comorbidities is being debated as exemplified by the following studies: Transgenic mice that over express The groucho family member
transducin-like enhancer of split 3 (TLE3), a potent facilitator of PPARγ activity and an adipogenic factor,
in WAT show an ameliorated insulin resistance upon HFD (Villanueva CJ et al., 2011). By contrast, an ameliorated glucose and fat metabolism is observed in mice that are deficient for
Phospholipase C (PLC) δ1, which also promotes adipogenesis (Hirata M et al., 2011). De novo
adipogenesis is not the only determinant of adipocyte size distribution, that is also importantly controlled by cell loss. Excessive size triggers a necrotic cell death phenomenon in obesity (Cinti S et al., 2005; Jo J et al., 2010). Further, enhanced oxidative stress (OS) has been implied in HFD-dependent cell death in adipose tissue (DeFuria J et al., 2009). This effect could be amplified by the lack of Hp, the main carrier of the potent oxidant free haemoglobin. Although
pure speculative, a possibility is that in the obese Hp−/− mouse very large cells, more susceptible
to die in normal conditions, are further selected negatively by high OS and become less represented in the population.
Discussion
65 In conclusion, we have established that Hp not only marks the intersection between obesity and inflammation, but also actively contributes to the WAT inflammatory profile often found in obese subjects and to the downregulation of adiponectin. Our data are therefore consistent with an Hp deficient phenotype being partially protected from the onset of the metabolic derangements, including severe insulin resistance and hepatosteatosis / hepatomegaly (Lisi S, Gamucci O et al., 2011), that obesity implies and to which the increased expression of the
inflammatory molecule Hp seems to concur. Moreover, adipocytes from Hp−/− mice seem to be
less susceptible to become oversized during obesity (Gamucci O et al., 2012): this is in line with the concept of a WAT that becomes a key element in the protection against the obesity-associated onset of systemic insulin resistance, due to higher adiponectin production and
insulin-dependent AKT activation in the Hp−/− model.
Further, Hp deficiency impairs adipose conversion of mouse embryonic fibroblasts, this being
