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Originally published In Press as doi:10.1074/jbc.M412802200 on April 18, 2005

J. Biol. Chem., Vol. 280, Issue 25, 24085-24094, June 24, 2005
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The Adipocyte as an Important Target Cell for Trypanosoma cruzi Infection*

Terry P. Combs{ddagger}§, Nagajyothi¶, Shankar Mukherjee¶, Cecilia J. G. de Almeida||, Linda A. Jelicks**, William Schubert||, Ying Lin{ddagger}, David S. Jayabalan{ddagger}, Dazhi Zhao¶, Vicki L. Braunstein¶, Shira Landskroner-Eiger{ddagger}, Aisha Cordero{ddagger}, Stephen M. Factor¶, Louis M. Weiss¶{ddagger}{ddagger}, Michael P. Lisanti||, Herbert B. Tanowitz¶{ddagger}{ddagger}, and Philipp E. Scherer{ddagger}{ddagger}{ddagger}§§

From the Departments of {ddagger}Cell Biology, Pathology, ||Molecular Pharmacology, **Physiology and Biophysics, and {ddagger}{ddagger}Medicine, Albert Einstein College of Medicine, Bronx, New York 10461

Received for publication, November 12, 2004 , and in revised form, March 31, 2005.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Adipose tissue plays an active role in normal metabolic homeostasis as well as in the development of human disease. Beyond its obvious role as a depot for triglycerides, adipose tissue controls energy expenditure through secretion of several factors. Little attention has been given to the role of adipocytes in the pathogenesis of Chagas disease and the associated metabolic alterations. Our previous studies have indicated that hyperglycemia significantly increases parasitemia and mortality in mice infected with Trypanosoma cruzi. We determined the consequences of adipocyte infection in vitro and in vivo. Cultured 3T3-L1 adipocytes can be infected with high efficiency. Electron micrographs of infected cells revealed a large number of intracellular parasites that cluster around lipid droplets. Furthermore, infected adipocytes exhibited changes in expression levels of a number of different adipocyte-specific or adipocyte-enriched proteins. The adipocyte is therefore an important target cell during acute Chagas disease. Infection of adipocytes by T. cruzi profoundly influences the pattern of adipokines. During chronic infection, adipocytes may represent an important long-term reservoir for parasites from which relapse of infection can occur. We have demonstrated that acute infection has a unique metabolic profile with a high degree of local inflammation in adipose tissue, hypoadiponectinemia, hypoglycemia, and hypoinsulinemia but with relatively normal glucose disposal during an oral glucose tolerance test.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Chagas disease caused by the protozoan parasite Trypanosoma cruzi is endemic in Mexico and Central and South America, where it causes significant morbidity and mortality (1). In fact, it is a major cause of heart disease in endemic areas. Based on seroepidemiologic studies, it is estimated that there are at least 100,000 immigrants in the United States chronically infected with this parasite (2). In addition, Chagas disease is now appreciated as an opportunistic infection in immune-compromised individuals, including those with HIV1/AIDS (3). Some investigators have concluded that the cause of death in acute Chagas disease is a septic shock-like picture accompanied by hypoglycemia (4, 5), but a more detailed metabolic characterization has not yet been reported.

For many years, the association between human T. cruzi infection and diabetes has received little formal evaluation. Anecdotally, there is a general belief that the incidence of diabetes is greater in the chagasic population. In recent years, there have been several reports suggesting that diabetes is indeed more common in the setting of increased T. cruzi infection (6, 7). One study demonstrated a significant reduction in insulin among chronically infected individuals (8). Interestingly, our previous studies indicated that when mice with chemically induced diabetes are infected with T. cruzi, they have a higher parasitemia and mortality (9). The same observation is seen when diabetic db/db mice are infected (9, 10). The underlying reasons for these phenomena are unknown.

The adipocyte and its relationship to the pathogenesis of infection has only recently been explored (11, 12). The fat-laden cells of the skin are among the cells that are initially encountered by trypomastigotes of T. cruzi and may be one of the initial targets. The adipose tissue in the acute and chronic state may serve as one of the reservoirs for the parasite from which recrudescence may occur during immune suppression.

As no systematic approach has been undertaken to more precisely define the role of the adipocyte in the normal and diabetic state during infection with T. cruzi, we examined the direct effects of T. cruzi infection on adipocytes in vitro and in vivo. Our investigations indicate that the adipocyte is an important target for infection and profoundly changes its cellular homeostasis, both in vitro as well as in vivo, as a consequence of infection. Such changes permit the adipocyte to function as an important reservoir host cell for chronic Chagas disease. Because the adipocyte has found increasing appreciation in recent years as a highly active endocrine cell (11), the observation that these cells represent a target tissue for T. cruzi significantly changes our perspectives of the role of the adipocyte in the progression and reactivation of this disease.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Animals—Mice were maintained on a 12-hour light/dark cycle and standard chow diet. All animal experimental protocols were approved by the Institute for Animal Studies of the Albert Einstein College of Medicine. The Brazil strain of T. cruzi was maintained by passage in C3H/Hej mice (Jackson Laboratories, Bar Harbor, ME). Male CD1 mice (Jackson Laboratories) were infected at 8-10 weeks of age with 5 x 104 trypomastigotes, and the various experiments were performed on the indicated days post-infection. Parasitemia was evaluated by counting in a hemocytometer as described previously (9).

Reagents—Dulbecco's modified Eagle's medium was purchased from Mediatech Inc. (Herndon, VA). All other chemicals were purchased from Fisher and were of the highest purity.

Magnetic Resonance Imaging—Mice were anesthetized with ketamine/xylazine injection (intraperitoneal) and were placed in a custom built 35-mm inner diameter radiofrequency coil. Body temperature was maintained by keeping the NESLAB gradient water cooling system set at 30 °C. The 1H NMR spectra of the mice were acquired using a routine 1-pulse experiment with a 5-s relaxation delay, signal averaging of four transients, and a GE Omega 9.4-tesla vertical bore NMR system (Fremont, CA). The spectra were integrated using the standard vendor NMR software. All procedures were approved by our institutional animal care and use committee and are in accordance with accepted institutional and governmental policies.

Adipocyte Differentiation in Cell Culture—3T3-L1 murine fibroblasts (a generous gift of Dr. Charles Rubin, Department of Molecular Pharmacology, Albert Einstein College of Medicine) were propagated and differentiated to adipocytes as described previously (13). In brief, the cells were propagated in FCS (Dulbecco's modified Eagle's medium containing 10% fetal calf serum (JRH Biosciences) supplemented with penicillin/streptomycin (100 units/ml each) and allowed to reach confluence (day-2). After 2 days (day 0), the medium was changed to DM1 (containing FCS, 160 nM insulin, 250 nM dexamethasone, and 0.5 mM 3-isobutyl-1-methylxanthine). Two days later (day 2), the medium was switched to DM2 (FCS containing 160 nM insulin). After another 2 days, the cells were switched back to FCS. Cells were used between days 8 and 12 post-induction of differentiation. Cells were infected at a multiplicity of infection of 2:1.

Assay Protocols—Serum values for glucose were measured by Fast-Blue B glucose assay (Sigma-Aldrich), and values for adiponectin with a mouse adiponectin radioimmunoassay kit (LINCO Research, St. Charles, MO). Other adipokines were measured using LINCOplex reagents (LINCO Research). All metabolic parameters were measured in the fasted state; animals were assayed at 10 a.m. following a 16-h fast. Oral glucose tolerance tests of 2.5 mg/g body weight of glucose load by oral gavage were performed on animals without access to food for 2 h prior to administration and during the course of the study.

Quantitative Determination of Parasite Load in Tissue—Heart tissue, spleen tissue, brown adipose tissue (BAT), and white adipose tissue (WAT) were collected from mice 15, 30, 60, and 300 days post-infection and stored at -80 °C. DNA was isolated from these tissues, as were T. cruzi epimastigotes using the Qiagen DNeasy tissue kit following the manufacturer's protocol.

A standard curve in the range of 50 pg to 50 ng for the quantification of T. cruzi DNA by real time PCR was developed using the T. cruzi 195-bp repeat DNA-specific primers TCZ-F (5'-GCTCTTGCCCACAAGGGTGC-3') and TCZ-R (5'-CCAAGCAGCGGATAGTTCAGG-3') (14) and genomic DNA purified from T. cruzi epimastigotes.

Quantitative PCR was performed using samples containing 50 ng of genomic DNA, 0.5 µM TCZ-F and TCZ-R primes (that amplify a 182-bp product), 1.6 µl of MgCl2 (25 mM), LightCycler FastStart Master SYBER Green 1 (Roche Applied Science), and PCR grade water (Roche Applied Science) to a final total volume of 20 µl. A parallel reaction was done for each sample using 50 ng of genomic DNA and 0.2 µM murine-specific microglobulin primers {beta}2F2 (5'-TGGGAAGCCGAACATACTG-3') and {beta}2R2 (5'-GCAGGCGTATGTATCAGTCTCA-3'), designed by TIB Molbiol LLC, which amplify the 190-bp product. These reaction mixes were loaded into Roche LightCycler Capillaries, capped, centrifuged for 10 s at 2000 rpm, and placed in the LightCycler. In the denaturation phase, the capillary was heated to 95 °C at a 20 °C/s ramp and held for 10 min. During the 45 cycles of the amplification phase, there were three steps: 95 °C for 2 s, 57 °C for 5 s, and 72 °C for 10 s, all at a 20 °C/s ramp. During extension, the fluorescence intensity was acquired as single color detection (SYBER Green 1). The third phase was a hold at 95 °C at a 20 °C/s ramp for 0 s, 65 °C at a 20 °C/s ramp for 15 s, and finally 95 °C at a 0.1 °C/s ramp for 0 s. During the melting phase, the acquisition setting was set at "continuous." The final phase was the cooling phase, which lasted 30 s at 40 °C at a 20 °C/s ramp. Data were acquired and analyzed with LightCycler version 3.0 software. Each run contained a negative control lacking template DNA.

The primer set TCZ-F::TCZ-R has been used previously (14) for routine PCR-based detection of T. cruzi (16). Primer set TCZ amplifies a 195-bp genomic sequence repeated in tandem, e.g. satellite DNA, present in ~120,000 copies in the Y strain of T. cruzi (15). As an internal control, {beta}2-microglobulin, a double copy mouse gene, was used to normalize the amount of mouse DNA present, e.g. number of cells, in each analysis. A standard for murine {beta}2-microglobulin concentration was developed from a serial dilution of DNA (PCR-produced murine genomic DNA). The number of parasites per cell was calculated by dividing the number of parasites (copies of T. cruzi DNA obtained by real time PCR) by number of cells (copies of {beta}2-microglobulin obtained by real time PCR).

Immunoblot Analysis—After infection, plates of differentiated 3T3-L1 cells were washed twice with phosphate-buffered saline and lysed in 1 ml of SDS-PAGE sample buffer (0.75% SDS, 0.5 M Tris-HCl, pH 6.8, 16 mM EDTA) plus 1 mM phenylmethylsulfonyl fluoride, and lysates were boiled for 5 min followed by brief sonication. Total protein (30 µg) was resolved by SDS-PAGE on 12% acrylamide gels and transferred to BA83 nitrocellulose (Schleicher & Schüll). Blots were probed with various antibodies as indicated. The rabbit polyclonal antibodies to the guanine nucleotide dissociation inhibitor (GDI) were a generous gift from Dr. Perry Bickel (Washington University, St. Louis, MO). Primary and secondary antibodies were diluted in phosphate-buffered saline or Tris-buffered saline with 0.1% Tween 20 and 1% bovine serum albumin. Bound antibodies were detected by enhanced chemiluminescence according to the manufacturer's instructions (Amersham Biosciences). Immunoblots from a minimum of three independent experiments were scanned, and the background-corrected signal from each band was quantitated by densitometry using an Alpha Innotech Multiimage Light Cabinet with Chemiimager 4400 software. Signal for each sample lane was normalized to the signals obtained for either GDI or {beta}-actin. The normalized relative levels for each experimental group are represented as mean ± S.E.

Immunohistochemistry—Freshly isolated tissues were fixed with phosphate-buffered formalin overnight and then embedded in paraffin wax. 5-µm sections were incubated overnight with a monoclonal anti-F4/80 antibody. After washing in phosphate-buffered saline, slides were incubated with biotinylated goat anti-rat or anti-rat IgG at 5 µg/ml (Vector Laboratories, Burlingame, CA) for 1 h at room temperature. Slides were developed using a peroxidase detection kit (Vector Laboratories) and counterstained with hematoxylin (Sigma-Aldrich).

Transmission Electron Microscopy—3T3-L1 adipocytes were fixed with 2.5% glutaraldehyde in 0.1 M sodium cacodylate buffer, post-fixed with 1% osmium tetroxide followed by 1% uranyl acetate, dehydrated through a graded series of ethanol solution, and embedded in LX112 resin (LADD Research Industries, Burlington, VT). Ultrathin sections (80 nm) were cut on a Reichert Ultracut UCT, stained with uranyl acetate followed by lead citrate, and viewed on a JEOL 1200EX transmission electron microscope at 80 kV. For electron microscopy on primary adipocytes, mice were anesthetized, and both brown and white adipose tissue (epididymal) were carefully dissected. These samples were then fixed with 2% paraformaldehyde and 2.5% glutaraldehyde in 0.1 M sodium cacodylate buffer. They were then post-fixed with 1% osmium tetroxide followed by 1% uranyl acetate, dehydrated through a graded series of ethanol solution, embedded in LX112 resin, and sectioned and imaged as indicated above.

Scanning Electron Microscopy—Fat cells were quick fixed in buffer (1% osmium tetroxide, 0.1 M sodium cacodylate, 0.2 M sucrose, 5 mM MgCl2 pH 4) for 5 s followed by two changes of 2.5% glutaraldehyde in SEM buffer (250 mM sucrose, 1 mM EDTA, 20 mM MOPS, pH 7.4). Cells were then fixed with 1% osmium tetroxide in SEM buffer and dehydrated through a graded series of ethanol solution. Critical point drying was accomplished using liquid carbon dioxide in a Tousimis Samdri 795 Critical Point Drier (Rockville, MD). Cells were then sputter-coated with gold palladium in a Denton Vacuum Desk-2 Sputter Coater (Cherry Hill, NJ) and examined in a JEOL JSM6400 scanning electron microscope (Peabody, MA) using an accelerating voltage of 10 kV.

Statistical Analysis—The results are shown as means ± S.E. Statistical analysis was performed by one- or two-way analysis of variance. Significance was accepted at p < 0.05.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Parasitology—CD1 mice had a 60% mortality by day 35-40 post-infection. The peak parasitemia at day 35-40 post-infection ranged from 5 x 105 to 1 x 106 trypomastigotes/ml of blood, and the parasitemia waned so that by day 60 postinfection, parasites were not observed on routine blood examinations.



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  		FIG. 1.
T. cruzi infection causes severe hypoglycemia but does not have an effect on glucose clearance during an oral glucose tolerance test. A, fasting plasma glucose levels before and during infection of CD1 mice with 104 T. cruzi (Brazil strain). Plasma glucose levels were determined in the fasted state prior to infection and on days 15, 30, 60, and 90 post-infection. Note the marked hypoglycemia on day 30. B, oral glucose tolerance tests at various stages of infection. Note that despite hypoglycemia, the rate of glucose clearance remains unaltered compared with control animals. Uninfected mice, infected mice that survived, and infected mice that died during infection are plotted separately. Data for days 15 and 60 are not shown but look similar to days 0 and 90. C, hyperglycemia is associated with increased mortality. Plasma glucose levels have been plotted for individual mice at various stages of infection. , uninfected mice; {circ}, infected mice that survived until day 90; x, mice that died during the infection. n = 10 for each group. In all cases, significant differences (p < 0.05) are indicated with an asterisk.

 
Infection of Mice with T. cruzi Caused Transient Hypoglycemia during the Acute Phase of the Infection but DidNot Impair Glucose Tolerance—To determine the metabolic consequences of T. cruzi infection, we monitored basal glucose levels as well as insulin sensitivity by means of an oral glucose tolerance test prior to infection, during the acute phase of infection (day 30) as well as during the chronic stage (~90 days post-infection), and compared it with a cohort of mice that were injected with vehicle only. The acute phase of the infection was associated with severe hypoglycemia in mice (Fig. 1A), in agreement with data reported previously (4, 5). Clinically, similar observations have been reported for patients. Conventionally, the metabolic response to sepsis entails hyperglycemia with insulin resistance, profound negative nitrogen balance, and diversion of protein from skeletal muscle to splanchnic tissues. The underlying causes of the hypoglycemia during acute T. cruzi infections, which may be a major reason for mortality, are not known. The oral glucose tolerance tests between the two groups were overlapping before infection (day 0) and in the chronic stage of infection (day 90) when the mice had fully recovered. Surprisingly, during the acute phase of infection (day 30), glucose levels in the infected animals remained at all stages below the levels measured for the control animals. Even though the base-line glucose levels in the infected animals were lower, the oral glucose tolerance tests indicate relatively normal ability to clear the ingested glucose despite the high degree of inflammation associated with this state (Fig. 1B).



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  		FIG. 2.
Infection with T. cruzi is associated with transient changes in plasma adipokine levels. A, adiponectin levels during the indicated stages of infection. B, plasma insulin, leptin, and resistin levels on days 15 and 30 post-infection. C, IL-6 and monocyte chemoattractant protein 1 (MCP-1) levels on days 15 and 30 post-infection. All values were obtained using LINCOplex assays. D, weight of the mouse cohorts at different stages of infection.

 
Hypoglycemia Is a Predictor of Mortality in T. cruzi-infected Mice—To test whether the hypoglycemia may directly or indirectly be linked to mortality, we compared the fasting glucose levels and survival rate of infected mice (Fig. 1C). There was a strong correlation between the relative reduction in glucose levels and mortality. The average normal glucose levels of CD1 mice were 150 mg/dl. During acute infection, glucose levels were reduced as low as 20 mg/dl, a level that can be fatal over a sustained period of time, although there are rare examples of mice that spontaneously recovered from such a severe hypoglycemic condition. Even though the basal glucose levels correlate well, we failed to detect significant differences in glucose clearance during oral glucose tolerance tests between survivors and mice that died in the course of infection (Fig. 1B).

T. cruzi Infection Has a Profound Impact on Systemic Inflammation and Adipokine Levels—Prompted by the very low glucose levels, we decided to examine the plasma levels of two key adipokines released by adipocytes. Adiponectin has potent insulin-sensitizing properties, and decreased levels of this hormone tend to be associated with insulin resistance, hyperglycemia, and obesity. In addition, decreased levels of adiponectin are observed with increased systemic inflammation associated with cardiovascular disease. However, acute inflammation triggered by endotoxemia does not have an effect on adiponectin levels (12). The hypoglycemia coupled with the potent proinflammatory profile associated with the acute phase of T. cruzi infection makes it difficult to predict how adiponectin levels would be affected. We therefore monitored the levels throughout the experimental period. Adiponectin levels were significantly reduced during acute infection (Fig. 2A), suggesting that the proinflammatory component of the infection is the main driving force for the expression of adiponectin. The hypoglycemia observed during that period cannot be explained by excess adiponectin levels. To our knowledge, this is the first example of a physiologically relevant condition that combines hypoglycemia and normal glucose tolerance with significantly reduced serum adiponectin levels. In contrast to the situation during infection of mice with rodent malaria, which is also accompanied by hypoglycemia triggered by a severe hyperinsulinemia (16), insulin levels were not significantly affected on days 15 and 30 post-infection. In fact, there was a trend toward lower levels of insulin on day 30 in the infected group, but it failed to reach statistical significance (Fig. 2B, left panel). The decreased insulin levels are consistent with a physiological response to the very low glucose levels during that time. It is unlikely that this is a reflection of pathological changes at the level of the pancreatic {beta} cells, because the {beta} cell morphology is normal on day 30 of infection, and insulin levels revert to normal levels at later stages (data not shown). Leptin levels were significantly decreased in infected mice compared with control mice (Fig. 2B, middle panel). Resistin, another fat cell-specific secretory factor with insulin-desensitizing properties, was unaffected by infection (Fig. 2B, right panel). Similarly, levels of plasminogen activator inhibitor type 1, which is also prominently expressed in adipocytes, are completely unaffected by infection (not shown). In contrast, proinflammatory markers, such as IL-6 and monocyte chemoattractant protein 1 (Fig. 2C) and TNF{alpha} (not shown), were markedly elevated in the infected mice in response to the high parasite load on days 15 and 30. The significant decrease in leptin levels was surprising because the infected mice gained more weight than the control mice (Fig. 2C). To test whether the weight gain was because of an increase in fat mass or because of other reasons such as edema formation, we performed a detailed body composition analysis by magnetic resonance imaging. Consistent with the decreased leptin levels, we found significantly reduced levels of adipose tissue during acute infection (Table I). Interestingly, the decrease in adipose tissue persisted even at later stages during chronic infection and was at that stage primarily because of a decrease in abdominal adipose tissue. Mice that suffered from cardiac dilation (used here as a surrogate marker for the increased severity of infection at the earlier stages) had an even more dramatic loss of both total and abdominal fat depots. The weight gain in infected animals appeared to be related to edema, which is a consequence of cardiac dysfunction in these animals.


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  		TABLE I
Levels of adipose tissue during acute and chronic infection

 
Adipose Tissue Is an Important Target of T. cruzi—Because there was an alteration in systemic levels of one of the adipokines, we wanted to determine whether the parasite directly invades adipose tissue. A real time PCR approach was utilized to evaluate the number of T. cruzi parasite host cells (using genomic copies of {beta}2-microglobulin as a normalization standard). As shown in Fig. 3A, adipose tissue is a significant target tissue, harboring parasites in larger numbers than has been observed in more conventionally studied tissues, such as the heart and the spleen, with an average of >7 parasites/cell (day 15 post-infection) and 4 parasites/cell (day 30 post-infection) during acute infection.

Particularly noteworthy is that in the chronic stage, on day 60 post-infection, adipose tissue remained a prominent reservoir of parasites. Even 300 days post-infection, a comparable number of parasites were present in adipose and heart tissue when normalized on a per cell basis (Fig. 3B). This indicates that adipose tissue is a likely reservoir tissue for these parasites. This could be even more physiologically important in the setting of significant obesity.

Adipose tissue contains a number of different cell types. The stromal vascular fraction contains pre-adipocytes and macrophages, as well as other cell types. An important role for local adipose tissue macrophages has recently been described (17). In the obese state, additional macrophages are recruited into adipose tissue and may significantly contribute to local inflammation. The two cell types combined may be important contributors to systemic inflammation. Because there were numerous parasites in adipose tissue, we suspected that the massive local parasite count should be responsible for a considerable influx of macrophages. We wanted to test whether the high propensity to harbor and propagate T. cruzi in adipocytes also had an effect on macrophage levels. We therefore isolated white adipose tissue from two different fat depots of infected mice. As judged by an immunohistological stain for the macrophage-specific marker F4/80, in WAT there was a massive increase of F4/80 positive cells during the acute infection on day 18 (Fig. 3C). Even more pronounced changes were observed in BAT at the same time period (not shown), and these could still be seen on day 30 post-infection (Fig. 3D). The small inset in Fig. 3D demonstrates a higher magnification and highlights the intracellular location of the parasites within the adipocytes that was characterized by multiple smaller intracellular lipid droplets in the case of brown adipocytes, as opposed to the densely packed white adipocytes that contained a single large lipid droplet. Despite these dramatic morphological changes in WAT and BAT during the acute infection, which demonstrate the huge impact of a large number of parasites, the effects are at least morphologically fully reversible. In BAT (not shown) and epididymal WAT (Fig. 3E) during chronic infection (60 and 90 days post-infection), there was a full recovery with reduced levels of macrophages that did not differ in number between infected and non-infected mice.




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  		FIG. 3.
Quantitative assessment of the parasite load at different stages of infection. A, assessment of the parasite load by quantitative reverse transcription-PCR in heart, spleen, brown adipose tissue, and epididymal white adipose tissue (EWAT) 15, 30, 60, and 300 days post-infection. B, data for days 60 and 300 are shown again on a different scale. C, immunohistochemical analysis for the presence of macrophages in white adipose tissue on day 18 post-infection using antibodies against the macrophage marker F4/80. Perirenal WAT is shown in the top panels, epididymal WAT is shown in the bottom panels. A monoclonal control antibody of the same isotype was used at the same concentration in the left panels. D, immunohistochemical analysis for the presence of macrophages in brown adipose tissue on day 30 post-infection using antibodies against F4/80. The inset (middle) highlights the intracellular presence of the parasites in adipocytes. BAT was isolated from infected (top two panels) or uninfected (bottom two panels) mice. A monoclonal control antibody of the same isotype was used at the same concentration in the left panels. E, analysis of macrophage infiltration in epididymal WAT on days 60 (top four panels) and 90 (bottom four panels) post-infection using F4/80 stains. F, electron microscopy analysis of brown adipocytes at different magnifications. LD, lipid droplet. Arrows indicate parasites. Parasites are ~4-5 µm in diameter. G, electron microscopy analysis of white adipocytes (epididymal) at different magnifications. Arrows indicate parasites.

 
Primary Adipocytes Display Vast Numbers of Intracellular Parasites—To address the issue of whether the parasites found in adipose tissue indeed have an intracellular location in adipocytes, we have systemically examined tissue pads of brown and white primary adipose tissue from infected mice. Fig. 3F shows several micrographs from infected brown adipocytes. Note the characteristic multilocular small lipid droplet distribution and the abundant mitochondria. Similarly, a large number of parasites can be found in adipocytes isolated from white adipose tissue (Fig. 3G). It is interesting to note that the parasites are frequently found in close juxtaposition to the lipid droplet, suggesting that they may take advantage of local lipolysis at the surface of the lipid droplet to cover their energetic needs through the uptake and {beta}-oxidation of free fatty acids.

Infected Adipocytes Exhibited Changes in the Expression Levels of a Number of Different Adipocyte-specific or Adipocyte-enriched Proteins—The altered serum levels of adiponectin suggest that the invasion of adipocytes by parasites may also have direct consequences on additional proteins produced in adipose tissue. To directly address this issue, we performed immunoblot analysis for a number of proteins that are expressed in adipose tissue. Consistent with the reduction of plasma adiponectin, the levels of adiponectin in adipose tissue were reduced during acute infection in a number of different fat pads. Particularly perirenal and visceral adipose pads (both of which are important systemic sources of adiponectin) were significantly reduced on day 30 post-infection, whereas the levels in brown adipose tissue were unaffected. (Fig. 4A). We extended our analysis of protein expression on brown and perirenal adipose tissue 30 days post-infection to a number of additional inflammatory markers. The acute phase reactants {alpha}1 acid glycoprotein and serum amyloid A3, both of which we characterized previously to be expressed at high levels in adipocytes, are up-regulated. Even more dramatic effects were observed for TNF{alpha}, interferon-{gamma}, and IL-1{beta}, which were upregulated at least 10-fold 30 days post-infection (Fig. 4B). At day 90 post-infection, we could not detect any measurable metabolic or immunohistochemical differences in white adipose tissue, but noticed that significant differences with respect to inflammatory markers such as TNF{alpha}, interferon-{gamma}, and IL1-{beta} persisted. These differences suggest that parasites in adipose tissue during chronic infection continued to affect the local inflammatory state (Fig. 4C). Under all conditions, levels of control protein GDI, a marker that we have used extensively for normalization in the past, were unchanged.

Infection by Trypomastigotes of 3T3-L1 Adipocytes in Vitro—To determine whether the in vivo observations extend to an isolated cell system, 3T3-L1 adipocytes, a common cell line model for adipocytes, was used. These cells are propagated as fibroblasts and can conveniently be differentiated into adipocytes over a period of 8 days (13). Isolated adipocytes cultured in vitro display a similar expression pattern with respect to the induction of specific proinflammatory markers upon infection as the primary adipocytes. Forty-eight hours post-infection, adiponectin production was reduced, whereas the levels of Toll-like receptor 2, an inflammatory marker, were up-regulated compared with {beta}-actin. Similarly, levels of TNF{alpha} were increased 48 h post-infection. Interferon-{gamma} and IL1-{beta} were similarly up-regulated under those conditions, albeit to a lesser extent than observed in vivo (Fig. 5A, quantitation provided in Fig. 5B).



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  		FIG. 4.
Effects of T. cruzi infection on protein expression in adipose tissue. A, Western blot analysis of adiponectin expression in brown, perirenal, and visceral adipose tissue in control and infected animals 30 days post-infection. B, protein expression in brown and perirenal adipose depots in control (C) and infected (I) animals 30 days post-infection for {alpha}1 acid glycoprotein, serum amyloid A3, TNF{alpha}, interferon-{gamma} (INF{gamma}), and IL-1{beta}. All signals were normalized for GDI levels and represent averages from measurements of four independent mice. C, samples are as described in B except tissue was harvested 90 days post-infection. In all cases, significant differences (p < 0.05) are indicated with an asterisk.

 
3T3-L1 adipocytes faithfully reproduce the biological properties of primary adipocytes, although they differ with respect to lipid droplet number and average lipid droplet size. Scanning electron micrographs of 3T3-L1 adipocytes revealed a large number of trypomastigotes actively invading cells (Fig. 5C). The smaller lipid droplet size makes the structural analysis of the intracellular morphology much more approachable. Comparing electron micrographs of uninfected and infected 3T3-L1 adipocytes revealed numerous intracellular amastigotes in the infected cells, clustered around the lipid droplets (Fig. 5D), similar to the observations made in primary adipocytes. These observations suggest that the parasite may have an affinity for events taking place on the surface of the lipid droplet such as lipolysis mediated by hormone-sensitive lipase. This may provide an explanation for the high affinity of parasites for this cell type. It is noteworthy that other intracellular parasites, e.g. Toxoplasma gondii, have been shown to need host cell lipids, and a similar phenomenon may explain the increased number of T. cruzi per cell in adipocytes.



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  		FIG. 5.
Effect of T. cruzi infection on 3T3-L1 adipocytes. A, 3T3-L1 adipocytes were infected at a multiplicity of infection of 2:1 with T. cruzi from the Brazil strain. 48 h later, protein extracts were prepared and analyzed by Western blot analysis for levels of adiponectin, Toll-like receptor 2 (TLR-2), TNF{alpha}, IL-1{beta}, interferon-{gamma} (INF{gamma}), and {beta}-actin. Ctrl, control. B, quantitative representation of data shown in A. Data are means ± S.D.; n = 4. In all cases, significant differences (p < 0.05) are indicated with an asterisk. Adipon, adiponectin. C, four representative scanning electron micrographs of 3T3-L1 adipocytes infected with T. cruzi. 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. The picture on the top left corresponds to an uninfected cell.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
This is, to our knowledge, the first detailed analysis of the consequences of T. cruzi infection on adipose tissue and adipocytes both in vitro and in vivo. Our data indicate that adipocytes play an important role in the pathogenesis of Chagas disease. During acute infection, there are profound changes in the transcriptional program of adipocytes and, as a result, changes at the level of adipokines released by adipose tissue. Our data suggest that adipocytes serve as a reservoir for parasites from which recrudescence may occur during immunosuppression.

Previous observations from several laboratories have indicated that adipose tissue may serve as a prominent target tissue for T. cruzi, even though more quantitative assessments were lacking in these studies. Buckner et al. (18) demonstrated a high level of T. cruzi infection of mesenteric fat pad using a genetically engineered strain of the parasite expressing {beta}-galactosidase. In addition, Lenzi et al. (19) found large numbers of parasites in adipose tissue 15 days post-infection. These observations are important and needed to be re-evaluated with our recent increased understanding of adipose tissue as both an endocrine organ and contributor to systemic inflammation.

There has been an increase in Type II diabetes in developing countries. However, there has been little attention given to the role of infectious diseases in the pathogenesis of diabetes, including the role of the adipocyte. We believe that Chagas disease lends itself particularly well to the study of the interaction of infection and the diabetic state because of the critical involvement of the adipocyte as demonstrated by the data in this study. Adipose tissue continued to harbor a significant number of parasites 300 days post-infection, as demonstrated by real time quantitative PCR. This could be in part because of the unique metabolic conditions that the parasites find inside the adipocyte. Another important aspect to this persistence is the extremely slow turnover of adipocytes. Although various methods yield different results regarding the life span of adipocytes under normal physiological conditions, there is a general consensus that adipocytes are very long-lived and do not significantly turn over in 6-12 months. The vast majority of adipocytes that are present 300 days post-infection may therefore represent cells that were already present during acute infection. Weisberg et al. (17) recently reported a significant infiltration of additional macrophages into adipose tissue in obese mice. The increased local concentration of macrophages is thought to act in concert with the adipocytes to increase local and systemic inflammatory levels. The dramatic increase in local adipose tissue macrophages during the acute phase of the infection not only highlights the large number of local parasites but may also be responsible for a significant amount of systemic inflammation in light of the high proinflammatory potential of both macrophages and adipocytes. Additional clinical studies will be required to assess the correlations of disease outcome with both the acute and chronic phases of the infection and adipose tissue mass. Depending on the level of obesity, adipose tissue can account for greater than 40-60% of total body weight. Local phenomena observed in adipose tissue can therefore have a profound systemic impact.

Similar to the clinical disease progression reported for humans, severe hypoglycemia was observed in mice during the acute phase of the infection. Metabolically, it is not known what accounts for this dramatic drop in glucose levels, which is in sharp contrast to the situation observed in septic patients who experience insulin resistance (20, 21). Insulin resistance was not present because the mice exhibited normal glucose tolerance tests and did not have elevated insulin levels. The hypoglycemia could be a consequence of an increased glucose uptake in peripheral tissues, such as muscle or fat, or could be attributed to a massive uptake of glucose by T. cruzi (22). However, we believe a more likely explanation is a failure of the liver to adjust hepatic glucose production, resulting in decreased hepatic gluconeogenesis and the massive hypoglycemia during acute T. cruzi infection. In fact, although T. cruzi does not directly infect cultured hepatocytes, it will infect sinusoidal cells in the liver, resulting in hepatic inflammation.2 Hypoglycemia could also be attributed to insulinomimetic properties of an inositol phosphate glycan derived from a parasite glycosylphosphatidylinositol (23, 24). The precise mechanism of hypoglycemia in the setting of acute Chagas disease will be addressed in the future using techniques that will allow an assessment of glucose fluxes during the acute phase of this infection.

Hypoglycemia has also been reported in the context of malaria, both in humans (25) and in mice (16). However, in this case, the hypoglycemia is primarily attributed to either treatment with quinine that triggers hyperinsulinemia or a generalized hyperinsulinemia that is present even in the absence of treatment (26). The underlying reasons for the hypoglycemia must be fundamentally different in the case of Chagas disease, because such a hyperinsulinemia is not observed. These observations highlight a rather unique metabolic fingerprint caused by Chagas disease that has not been reported for any other infectious disease to date.

The prominent role of the adipocyte during T. cruzi infection may also have important clinical implications in the context of specific disease states, such as HIV infections. HIV represents an immune-compromised state, which can predispose an infected host to recrudescent infections. Additionally, highly active antiretroviral therapy and HIV itself are associated with lipodystrophy effects (27). This HIV-associated lipodystrophy can result in a reduction of subcutaneous tissue with fat depots redistributed toward a more central location. During this process, changes in adipocytes could lead to the release of intracellular parasites from these adipocytes into the systemic circulation, providing another mechanism (in addition to immune suppression) for the development of recrudescent Chagas disease in the setting of HIV infection (3).


   FOOTNOTES
 
* This work was supported by National Institutes of Health National Research Service Award DK61228 (to T. P. C.) and National Institutes of Health Grants AI-52739, D43TW007129 (FIC-NIH), and AI-12770 (to H. B. T.), R01-HL073163-01 (to L. A. J. and P. E. S.), AI-062730 (to L. A. J.), and R01-DK55758 and R03 EY014935-01 (to P. E. S.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

§ Present address: Dept. of Nutrition, University of North Carolina at Chapel Hill, McGavran-Greenberg Hall, CB7461, Chapel Hill, NC 27599-7461. Back

§§ Recipient of an Irma T. Hirschl Career Scientist award. To whom correspondence should be addressed: Dept. of Cell Biology, Albert Einstein College of Medicine, 1300 Morris Park Ave., Bronx, NY 10461. Tel.: 718-430-2928; Fax: 718-430-8574; E-mail: scherer{at}aecom.yu.edu.

1 The abbreviations used are: HIV, human immunodeficiency virus; BAT, brown adipose tissue; WAT, white adipose tissue; GDI, guanine nucleotide dissociation inhibitor; IL, interleukin; TNF{alpha}, tumor necrosis factor {alpha}; FCS, fetal calf serum; MOPS, 4-morpholinepropanesulfonic acid. Back

2 H. Tanowitz, unpublished results. Back


   ACKNOWLEDGMENTS
 
We thank Dr. Todd Schraw for assistance with glucose assays, Dr. Andrea Nawrocki for help in the primer design for the reverse transcription-PCR reactions, Frank Macaluso and Leslie Cummins in the Analytical Imaging Facility of the Albert Einstein College of Medicine for expert technical assistance for electron microscopy, and the Albert Einstein Diabetes Research and Training Center radioimmunoassay core laboratories for hormone and cytokine measurements.



   REFERENCES
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 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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