Diseases caused by nematodes and protozoa have been reported to be associated with nutritional deficiencies, wasting and diabetes. An association between human T. cruzi infection and obesity and diabetes has been suspected and there has been general belief, although not proven, that the incidence of diabetes may be increased in the chagasic population.
There may be a relationship between Chagas disease and both obesity and underweightedness; the common factor appears to be poverty (Barreto et al. 2003
). Previous studies from our laboratory demonstrated that when mice with chemical-induced diabetes were infected with T. cruzi
, they had a higher parasitemia and mortality (Tanowitz et al. 1988
). The same observation was noted in infected diabetic db/db
mice (Tanowitz et al. 1988
). The underlying pathophysiological mechanisms of these observations remain unknown. A personal communication from a physician in Argentina (MM Aranda) stated that there are groups of “aboriginal people” where Chagas disease afflicts over 50% of the population and that there is a strong co-existence with the features of the metabolic syndrome in the same population. These observations need to be examined more systematically.
We determined the metabolic consequences of T. cruzi
infection on basal glucose levels and insulin sensitivity. Acute infection of CD-1 mice with the Brazil-strain of T. cruzi
is usually associated with severe hypoglycemia and generally correlated with mortality (Combs et al. 2005
). Interestingly, the metabolic response to bacterial sepsis is often associated with hyperglycemia, insulin resistance, profound negative nitrogen balance and the diversion of protein from skeletal muscle to splanchnic tissues. Thus, the response to this infection differs from that generally observed in bacterial sepsis. It is possible that there is an effect on glucose metabolism due to invasion of the liver by the parasite. During acute infection, glucose levels in all of the T. cruzi
-infected mice were below those measured in the control mice. Even though the baseline glucose levels in the infected animals were lower, the oral glucose tolerance test indicated a relatively normal ability to clear ingested glucose despite the high degree of inflammation associated with this infection.
The level of adiponectin was decreased during T. cruzi
infection of CD-1 mice (Combs et al. 2005
) and FVB mice (unpublished observations). Reduced levels of adiponectin are often associated with insulin resistance, hyperglycemia and obesity, i.e., the metabolic syndrome. Decreased levels of adiponectin are observed in some conditions of inflammation and cardiovascular disease, as noted above. Importantly, acute inflammation induced by endotoxemia does not affect adiponectin levels (Keller et al. 2003
). The infection-induced hypoglycemia cannot be readily explained by changes in adiponectin. This is an example of a physiologically relevant condition that combines hypoglycemia and normal glucose tolerance with significantly reduced adiponectin levels. The decreased insulin levels observed 30 days post-infection in the mouse model of T. cruzi
infection are consistent with a physiological response to very low glucose levels during that time. In addition, leptin levels were significantly reduced in infected mice compared to controls. Resistin levels, another fat cell-specific secretory factor with insulin-desensitizing properties, were not affected by infection. Levels of plasminogen activator inhibitor-1, which is also prominently expressed in adipocytes, were also completely unaffected by infection. However, proinflammatory markers such as cytokines (TNF-α, IL-1β, IFN-γ) and chemokines were markedly elevated in the adipose tissue of acutely infected mice (unpublished observations). This elevation often persisted into the chronic phase.
The significant decrease in leptin levels was initially surprising since the infected mice gained more weight than the control mice. Magnetic resonance imaging studies, as well as body composition studies using an ECHO magnetic resonance spectrometry body composition instrument, revealed a decrease in abdominal adipose tissue. Mice that had marked right ventricular dilation had a greater loss of fat deposits. The weight gain in infected mice appeared to be related to edema, which may have been a consequence of right-sided heart failure (Combs et al. 2005
CD-1 and FVB mice infected with the Brazil strain of T. cruzi displayed a reduction in plasma levels of adiponectin, suggesting that the infection of adipocytes may also have consequences on other proteins synthesized in adipose tissue. Consistent with the reduction of plasma adiponectin, the level of adiponectin in adipose tissue was reduced during acute infection in a number of fat pads known to be important sources of adiponectin. At 30 days post-infection, the acute-phase reactants α-1 acid glycoprotein and SAA3, which are expressed in adipocytes, were upregulated. Consistent with the infection-induced increase in macrophages and inflammatory mediators (cytokines and chemokines) (, ), there was a concomitant reduction in adiponectin and peroxisome proliferator-activated receptor-γ (PPAR-γ). Both of these proteins are negative regulators of the inflammatory pathway (unpublished observations).
Fig. 1 adipose tissue of CD-1 mice infected with the Brazil strain of Trypanosoma cruzi (for 30 days). Upper panel are immunoblots demonstrating a reduction in adiponectin expression in perirenal and visceral adipose tissue. Lower panel are immunoblots demonstrating (more ...)
Fig. 2 adipose tissue of CD-1 mice infected with the Brazil strain of Trypanosoma cruzi. For 30 days. F4/80 staining of brown fat (A: uninfected; B: infected). Note the intense staining of macrophages in infected adipose tissue [from Combs et al. (2005) with (more ...)
We have previously demonstrated by qPCR that T. cruzi
DNA in adipose tissue 300 days post-infection was present at the same levels as seen in the heart (Combs et al. 2005
) (). This observation suggests that the adipocyte proper may serve as an important target for T. cruzi
and in chronic Chagas disease adipocytes may represent an important long-term reservoir for parasites from which relapse of infection can occur.
Fig. 3 quantitative PCR demonstrating presence of Trypanosoma cruzi in various tissues 60 and 300 days post-infection in CD-1 mice infected with the Brazil strain. BAT: brown adipose tissue; EWAT: epididymal white adipose tissue [from Combs et al. (2005) with (more ...)
In vitro studies were subsequently performed to evaluate the role of adipocytes in T. cruzi
infection in a model system devoid of many of the other cell types ordinarily found in adipose tissue. Although we were not the first group to observe T. cruzi
in fat cells (Andrade & Silva 1995
), we published the first systematic analysis of the detailed events occurring during acute and chronic stages of T. cruzi
infection in fat (Combs et al. 2005
, Nagajyothi et al. 2008
). Intracellular amastigotes were monitored by electron microscopy (), revealing that adipocytes infected for 96 h maintained their integrity. T. cruzi
infection of cultured adipocytes induced an inflammatory phenotype. For example, there was increased expression of chemokines, such as CCL2, CCL3, CCL5 and CXCL10, as well as the cytokines TNF-α, IL-10 and interferon-γ. The expression of STAT3, an important downstream mediator of cytokine signaling, was also increased. Toll-like receptors, which have been reported to be activated during T. cruzi
infection of other cell types, were upregulated in cultured adipocytes (TLR-2 and -9) and there was also activation of components of the Mitogen-Activated Protein Kinase (MAPK pathway). Specifically extracellular signal-regulated kinase (ERK) and p38 MAPK were activated, whereas Jun N-terminal Kinase was not (Nagajyothi et al. 2008
Fig. 4 A: an uninfected cell; B–D: representative transmission electron micrographs of 3T3-L1 adipocytes 48 h post-infection. Note the close proximity of parasites to lipid droplets indicated by arrowheads; E–G: scanning electron micrographs (more ...) T. cruzi
infection of cultured adipocytes also resulted in the increased expression of cyclin D1. Cyclin D1 is generally associated with cell proliferation; however, cultured adipocytes are usually terminally differentiated. The increased expression of cyclin D1 is of interest because it is upregulated by ERK and inversely regulated by caveolin-1 (Hulit et al. 2000
). Indeed, we have demonstrated that infection resulted in a reduction in the expression of caveolin-1 and the activation of ERK. Both of these events increase the expression of cyclin D1. A reduction in caveolin-1 expression has also been demonstrated to be associated with an increased proinflammatory cytokine response (Cohen et al. 2003
). Interestingly, infection activates the Notch pathway, which regulates, in part, the expression of cyclin D1 (Stahl et al. 2006
PPAR-γ is expressed in adipose tissue and reduces the inflammatory process, similar to adiponectin (Nawrocki et al. 2006
, Kim et al. 2007
). As noted, a reduction in the level of adiponectin is associated with an increase in inflammation (Desruisseaux et al. 2007
). In addition, there is an inverse relationship between PPAR-γ and inflammation, as well as between PPAR-γ and cyclin D1 (Wang et al. 2003
). It has been demonstrated that increased expression of cyclin D1 is associated with a reduction in PPAR-γ. Recent evidence suggests a similar relationship between adiponectin and PPAR-γ (Nawrocki et al. 2006
, Kim et al. 2007
). Our observations clearly demonstrated that T. cruzi
infection resulted in a reduction in the expression of adiponectin and PPAR-γ () and an increase in the expression of cyclin D1 and inflammatory mediators.
Fig. 5 mRNA levels of peroxisome proliferator-activated receptor γ (PPAR-γ) and adiponectin in Trypanosoma cruzi infected cultured differentiated 3T3-L1 adipocytes. Note that infection reduces the expression of both adiponectin and PPAR-γ. (more ...)
Interestingly, T. cruzi infection also results in the increased expression of PI3kinase and the activation of AKT/PKB (thymoma viral protoco-oncogene), suggesting that this infection may induce components of the insulin/IGF-1 receptor cascade. This is surprising since the upregulation of proinflammatory pathways is usually associated with a downregulation of the insulin signal transduction pathway. It is not clear what is responsible for this phenomenon. T. cruzi is likely to have an impact on lipid pathways in vivo, yet these issues have not been examined to date. These observations are significant because there is usually a correlation between inflammation and insulin resistance. However, the infection of adipocytes with a parasite that resides intracellularly can be viewed as different from exposing adipocytes to an endotoxin. The continued intracellular presence of the parasites clearly has a differential effect on insulin sensitivity, perhaps by lowering the levels of one of the critical lipid mediators of insulin resistance. Although IRS-1 levels are not necessarily indicative of activity, it is generally accepted that pAkt levels downstream are an excellent reflection of the cellular insulin signaling activity in 3T3-L1 adipocytes.
Infection also results in increased expression of PI3kinase and the activation of AKT, suggesting that this infection may induce components of the insulin/IGF-1 receptor cascade. This would be surprising as the upregulation of proinflammatory pathways is generally associated with a downregulation of the insulin signal transduction pathway (Hotamisligil 2006
, Ferrante 2007
). It is not clear what is responsible for this phenomenon, but it can be observed with a high degree of reproducibility in these cells. Despite the upregulation of some of the components of the pathway, there were no differences with respect to a dose-response to insulin in infected cells (unpublished observations). It remains to be determined whether other pathways influenced by insulin, such as events leading to differences in the rate of lipid accumulation or lipolysis, may be affected. T. cruzi
is likely to have an impact on lipid pathways in vivo, yet these issues have not been examined to date.
In summary, fat and glucose metabolism are interrelated and dysregulated in T. cruzi
infection. We have clearly demonstrated that adipocytes and adipose tissue represent an important target of and reservoir for infection. This is a reservoir from which parasites can be reactivated during periods of immunosuppression. In addition, the infection of adipocytes and adipose tissue create an inflammatory phenotype that affects a variety of metabolic processes. The reduction in the expression of adiponectin and PPAR-γ perpetuate this inflammatory phenotype. Since adiponectin null mice have a cardiomyopathic phenotype, it is tempting to suggest that the reduction in adiponectin and PPAR-γ contributes to the cardiomyopathy of Chagas disease. Recently Coura (2007)
has commented on the need for new approaches to the study of Chagas disease. Although the study of adipose tissue was not specifically mentioned, we believe that this is a new and fruitful area of research.