Tungstate Decreases Weight Gain and Adiposity in Obese Rats through Increased Thermogenesis and Lipid Oxidation
http://www.100md.com
《内分泌学杂志》
Endocrinology and Nutrition Unit (M.C., H.C., I.C., S.B.-B., R.G.), Hospital Clínic Universitari, Institut d’Investigacions Biomèdiques August Pi i Sunyer, Faculty of Medicine, and Institut de Recerca Biomèdica de Barcelona (J.J.G.), Parc Científic de Barcelona, University of Barcelona
Pharmacology and Toxicology Department (J.S.), Institut d’Investigacions Biomèdiques de Barcelona, 08036 Barcelona, Spain
Abstract
The increasing worldwide incidence of obesity and the limitations of current treatments raise the need for finding novel therapeutic approaches to treat this disease. The purpose of the current study was first to investigate the effects of tungstate on body weight and insulin sensitivity in a rat model of diet-induced obesity. Second, we aimed to gain insight into the molecular mechanisms underlying its action. Oral administration of tungstate significantly decreased body weight gain and adiposity without modifying caloric intake, intestinal fat absorption, or growth rate in obese rats. Moreover, the treatment ameliorated dislipemia and insulin resistance of obese rats. These effects were mediated by an increase in whole-body energy dissipation and by changes in the expression of genes involved in the oxidation of fatty acids and mitochondrial uncoupling in adipose tissue. Furthermore, treatment increased the number of small adipocytes with a concomitant induction of apoptosis. Our results indicate that tungstate treatment may provide the basis for a promising novel therapy for obesity.
Introduction
OBESITY IS A complex metabolic disorder characterized by an excess of body fat and associated with an increased risk of suffering diabetes, hypertension, cardiovascular disease, and cancer. Obesity appearance is determined by interactions between genetic, environmental, and psychosocial factors, which alter long-term body energy homeostasis. Thus, obesity develops when energy intake chronically exceeds total body expenditure (1).
The prevalence of obesity and associated comorbid diseases is increasing at an alarming rate. However, currently available antiobesity drugs have limited efficacy or safety concerns (2). Consequently, the development of safer and more effective antiobesity therapeutic approaches is becoming more of an imperative. Recent advances in the understanding of the mechanisms and molecules involved in the regulation of body weight (3) have provided potential strategies for the treatment of obesity. These strategies aimed to modify, centrally and peripherally, food intake regulation, nutrient absorption, thermogenesis, and fat metabolism or storage (4).
Oral administration of tungstate reverts the diabetic phenotype in several induced and genetic animal models. Tungstate up-regulates glucose transporter expression and translocation in muscle (5), restores hepatic glucose metabolism (6, 7), and increases -cell mass (8, 9) in a number of diabetic animal models. Furthermore, administration of tungstate attenuates body weight gain in healthy rats (6, 7, 8, 9). This observation led us to examine the efficacy of tungstate for the treatment of obesity. We studied the effects of this compound on body weight and insulin sensitivity in a rat model of diet-induced obesity. In addition, we aimed to gain insight into the molecular mechanisms underlying the action of tungstate.
Our results show that tungstate, by increasing thermogenesis and lipid oxidation in adipose tissue, prevents body weight gain of obese rats on a high-fat diet. This experimental evidence raises tungstate as an effective novel agent to consider for the treatment of obesity.
Materials and Methods
Diet-induced obesity
Male Wistar rats (IFFA CREDO, L’Arbresse, France), weighing 220–240 g, were caged individually in a 12-h light, 12-h dark cycle in a temperature- and humidity-controlled environment. Animals were divided into two dietary sets for 30 d. One group was fed with standard chow diet (supplying 8% of calories as fat; type AO4 from Panlab, Barcelona, Spain). To induce obesity, the second group was fed with a cafeteria diet as previously described (10), with minor modifications. This diet consisted of standard chow and a daily intake of cookies, liver pate, bacon, and whole-milk supplemented with 333 g/liter of sucrose and 10 g/liter of a mineral and vitamin complex (Gevral; Cynamid Ibérica, Madrid, Spain). Sixty-five percent of the energy of this diet derived from lipids. All the food items were weighed daily and presented in excess. Daily caloric intake was calculated by multiplying the consumption of each item in the diet by its caloric density provided by the manufacturer.
Tungstate treatment
After 30 d of diet, lean and obese rats were divided in treated and untreated groups. Treatment was carried out by giving a solution of 0.3, 0.7, or 2 mg/ml sodium tungstate (Na2WO4x 2H2O; Carlo Erba, Milano, Italy) in distilled water for 30 d ad libitum. After treatment, we tested the reversibility of tungstate effects by withdrawing treatment under the same dietary conditions for 35 additional days (recovery period). During all the experimental period, fluid, food consumption (corrected by the amount of water lost for each item), and body weight of all rats were recorded daily.
At the end of the treatment period, rats from each group were killed. Gastrocnemius muscle (gM), interscapular brown adipose tissue (iBAT), epididymal white adipose tissue (eWAT), and liver were excised, weighed, and rapidly frozen in liquid nitrogen or fixed in 10% buffered formalin. All procedures were conducted in accordance with principles of laboratory animal care (European Community and local government guidelines) and approved by the Animal Research Committee of the University of Barcelona.
Metabolic measurements
All rats were fasted 6 h before metabolic measurements. Blood samples were collected from the tail using a capillary blood collection system with EDTA (Sarstedt, Nümbrecht, Germany). Blood glucose measurements were performed with the Roche (Mannheim, Germany) AccuTrend glucose sensor. Plasma insulin levels were measured by ELISA kits from Mercodia (Uppsala, Sweden). Plasma leptin levels were assessed by RIA (Linco Research, Inc., St. Charles, MO). Plasma triglycerides (TG) and nonesterified fatty acids (NEFA) levels were measured using colorimetric kits from Sigma (St. Louis, MO) and Roche, respectively.
Insulin tolerance tests were performed on ad libitum-fed rats by the administration of an ip injection of insulin (1 IU/kg body weight; Eli Lilly & Co., Indianapolis, IN). Blood samples collected at 0, 15, 30, 60, and 120 min after injection were used for glycemia determinations.
Histologic and morphometric analysis of adipose tissue
Formalin-fixed eWAT specimens were dehydrated, embedded in paraffin, and cut into 8-μm-thick sections. Adipose tissue sections were stained with hematoxylin and eosin, following standard protocols. For the quantitation of the number and size of adipocytes, the sectional areas of eWAT were manually analyzed in at least four fields from four animals per experimental group using an Olympus Corp. microscope (Tokyo, Japan) and MicroImage software (Hamburg, Germany).
Apoptosis determination
The presence of apoptotic nuclei in eWAT was assessed by terminal deoxynucleotidyl transferase-mediated deoxyuridine 5-triphosphate nick-end-labeling using the Apoalert DNA fragmentation assay kit (CLONTECH Laboratories, Inc., Palo Alto, CA), following the manufacturer’s instructions.
Growth rate and feces lipid content assessment
Growth rate was assessed by femur length measurement. At the end of treatment, feces were collected over 24 h and dried at 60 C until achieving constant mass. Total feces lipid content was determined by the Folch method (11).
Indirect calorimetry
Volume of oxygen consumption (VO2) was measured using an open-circuit indirect calorimetry system (MM-100 Metabolic Monitor System; CWE Inc., Ardmore, PA). Age and weight-matched rats were housed overnight in the measurement chamber to become acclimatized. Next, either vehicle (NaCl, 154 mmol/liter) or 150 mg/kg body weight of tungstate was administered to rats by gavage. This dose was previously verified as effective in reducing body weight gain. In vivo VO2 was then measured at 15-min intervals at baseline (vehicle) and for 8 h after administration of tungstate. Metabolic measurements included VO2, volume of carbon dioxide produced (VCO2), and respiratory exchange ratio (RER = VCO2/VO2).
RNA isolation and Northern blot analysis
Total RNA was purified from tissues by either Trizol (Invitrogen, Carlsbad, CA) or guanidinium isothiocyanate-phenol chloroform extraction (Invitrogen), following the manufacturer’s instructions. Total RNA derived from gM, eWAT, and iBAT tissues was electrophoresed on 1.2% formaldehyde-agarose gels and transferred to Nytran-plus membranes (Schleicher & Schuell, Inc., Keene, NH). Membranes were hybridized with specific (-32P) deoxycytidine triphosphate (Amersham Pharmacia Biotech, Buckinghamshire, UK) random primed-labeled cDNA probes generated from total RNA by RT-PCR using specific primers for peroxisome proliferators-activated receptor (PPAR), PPAR, uncoupling protein (UCP)1, UCP2, UCP3, lipoprotein lipase (LPL), muscle-carnitine palmitoyltransferase-1 (mCPT1), adipocyte protein 2 (aP2), fatty acid translocase (FAT/CD36), and acyl-CoA oxidase (ACO). Band intensities obtained by Northern blots were normalized to the signal obtained after stripping and reprobing the same membranes with 32P-labeled 28S cDNA probe. We quantified the amount of both of the signals, target gene and 28S, by densitometry using a Personal FX Phosphorimager and QuantityOne software (Bio-Rad Laboratories Inc., Hercules, CA), divided the signal of target mRNA by that of 28S, and then converted the results to a percentage. The results were expressed as mean ± SEM.
Statistical analysis
All results are expressed as mean ± SEM. Differences between the experimental groups were evaluated by the Student t test or by the nonparametric Mann-Whitney U test. P < 0.05 was considered significant.
Results
Diet-induced obesity
Administration of a cafeteria diet for 30 d led to obesity, increasing body weight of rats by 24% in relation to standard chow-fed rats (449 ± 7 vs. 362 ± 9 g; P < 0.05). As expected, the estimated cumulative energy intake was significantly higher in cafeteria-fed rats than in those fed on a standard chow diet (15,604 ± 181 vs. 7704 ± 147 kcal/kg·30 d; P < 0.001). Obese rats displayed higher plasma levels of TG (310 ± 33 vs. 159 ± 25 mg/dl; P < 0.05), NEFA (0.86 ± 0.07 vs. 0.52 ± 0.06 nmol/μl; P < 0.01), insulin (3.06 ± 0.50 vs. 0.73 ± 0.11 ng/ml; P < 0.05), and leptin (31.6 ± 2.6 vs. 7.2 ± 0.5 ng/ml; P < 0.01) than lean ones. Moreover, obese rats became glucose intolerant as assessed by a glucose tolerance test (data not shown). No differences in femur length were observed between rats fed the cafeteria and standard chow diet [51 ± 1 vs. 48 ± 1 mm; not significant (NS)].
Tungstate decreases body weight gain and adiposity of obese rats fed with a cafeteria diet
To determine the optimal dose at which oral tungstate exerts its effect, obese rats were treated with a range of doses of this compound (0.3, 0.7, and 2 mg/ml in the drinking solution) in addition to the cafeteria diet for another 30-d period (Fig. 1A). Administration of these doses led to an averaged tungstate ingestion of 33 ± 1, 77 ± 2, and 227 ± 9 mg/kg·d for the 0.3, 0.7, and 2 mg/ml, respectively. At all the doses tested, rats did not show the side effects, such as diarrhea or other toxic effects, that have been associated with the use of drugs that inhibit absorption of dietary fat. At 0.3 mg/ml of tungstate, no significant effects on body weight were observed. However, obese rats treated with either 0.7 or 2 mg/ml decreased their weight gain by 26 and 88%, respectively, compared with sex- and age-matched untreated obese (UO) rats on the cafeteria diet (Fig. 1A). Thus, we decided to retain only the dose of 2 mg/ml, which was the most effective in decreasing body weight gain.
The deceleration in body weight gain of 2 mg/ml tungstate-treated obese (TO) rats was accompanied by a significant decrease in adiposity when compared with UO rats. No significant changes in iBAT, gM, or liver mass were recorded at the end of treatment (Table 1). Moreover, no significant differences in femur length were observed between TO and UO rats (65 ± 2 for TO vs. 68 ± 2 mm for UO; NS), ruling out possible effects of tungstate on growth rate. Treatment withdrawal (recovery period) led to progressive and sustained body weight gain, indicating that tungstate does not induce a persistent state of weight loss (Fig. 1B).
Tungstate ameliorates insulin resistance and metabolic parameters
Tungstate effects on body weight and adiposity were accompanied by improved lipid profile and insulin sensitivity. Treatment decreased the circulating levels of TG, NEFA, and insulin (Fig. 2, A, B, and C, respectively). Moreover, ip insulin tolerance tests after 30 d of treatment showed an improvement of insulin sensitivity in TO when compared with UO rats (Fig. 2D). Noteworthy, amelioration of insulin sensitivity appeared as early as d 5 of treatment (data not shown).
Antiobesity effects of tungstate are not mediated by fat malabsorption, reduced energy intake, or changes in plasma leptin
Deficient intestinal fat absorption might account for the effects of tungstate on body weight and adiposity in obese rats. However, the feces lipid content in the total fecal mass was unaffected by tungstate treatment (12.1 ± 0.7 for TO vs. 12.8 ± 0.7% for UO; NS), demonstrating that this compound did not alter intestinal lipid absorption. No differences were observed in daily food, fluid (Fig. 3, A and B), or cumulative energy intake during the treatment period, between TO and UO rats (10,452 ± 229 for TO vs. 10,112 ± 246 kcal/kg·30 d for UO; NS). Indeed, regression analysis showed no correlation between food consumption and tungstate intake in TO rats fed with a cafeteria diet (Fig. 3C).
We next studied whether treatment altered the expression of leptin, an adipocyte-derived hormone involved in the regulation of energy homeostasis and plasma concentrations of this hormone are highly correlated with adipose tissue mass and body weight (12). However, plasma leptin levels at the end of the treatment were unaffected in TO rats when compared with UO counterparts (Fig. 3D).
Tungstate prevents adipocyte hypertrophy
Histological analysis of eWAT from TO rats showed an increased number of small (4500 μm2) adipocytes (40.3 ± 2.8 for TO vs. 17.3 ± 1.9% for UO; P < 0.05) and a trend to reduce the proportion of large (9000 μm2) adipocytes (35.1 ± 8.3 for TO vs. 43.5 ± 4.8% for UO; NS), (Fig. 4, A and B). In addition, treatment induced apoptosis in eWAT [7.8 ± 2.7 for TO vs. 3.2 ± 1.2% for UO (percentage of apoptotic nuclei referred to total nuclei); P < 0.05]. Collectively, these data indicate that tungstate remodels adipocyte population, thereby reducing white adipose tissue mass and alleviating insulin resistance.
Metabolic and morphometric results described above were similar to those reported for the modulation of PPARs, key transcription factors in the control of adipogenesis and lipid metabolism (13, 14). However, we did not observe changes in the gene expression of PPAR (data not shown) or PPAR in either iBAT or eWAT due to treatment (Fig. 4C).
Tungstate enhances energy dissipation and expression of proteins involved in lipid metabolism
To test whether the effects of tungstate on body weight were mediated by increased thermogenesis, we monitored metabolic rate by VO2 measurements and analyzed the gene expression of the UCPs, molecules involved in energy dissipation, oxidative phosphorylation, and lipid metabolism (15). In treated obese rats, VO2 averaged 732 ± 20 compared with 607 ± 15 ml/h·kg0.75 for the UO group, which represented an overall increase of 21% (Fig. 5A; P < 0.0001). Moreover, the average RER was lower for treated obese rats (0.74 ± 0.012) than for the UO rats (0.77 ± 0.019; Fig. 5B; P < 0.0001), indicating increased fatty acid oxidation by tungstate administration. Tungstate administration also increased UCP1 expression in iBAT (Fig. 5C). Taken together, these data indicate that tungstate treatment increased thermogenesis as fatty acid oxidation. In eWAT, this compound failed to induce ectopic expression of UCP1 (data not shown) but increased the expression of UCP2 (Fig. 5D). UCP2 and UCP3 mRNA levels were also up-regulated by treatment in gM (Fig. 5E).
To study a possible link between increased energy dissipation and enhanced fatty acid oxidation, we analyzed the expression of genes involved in fatty acid transport (LPL, FAT/CD36, and aP2) and oxidation (ACO and mCPT1) in several tissues. Tungstate did not alter the expression of these genes in gM, indicating that this compound has no effect on fatty acid uptake or oxidation in this tissue (Fig. 6A). In contrast, treatment resulted in the up-regulation of LPL, aP2, and mCPT1 but did not alter the mRNA levels of FAT/CD36 or ACO in eWAT (Fig. 6B).
Taken together, these results indicate that tungstate treatment increases fatty acid oxidation in adipose tissue but limits an excessive production of reactive oxidative species.
Discussion
In recent years, numerous studies have reported the antidiabetic properties of chemically resembled inorganic compounds such as tungstate vanadate, molybdate, and selenate (6, 7, 8, 9, 16, 17, 18). In the present study, we investigated the effectiveness of tungstate in obesity treatment and demonstrated that chronic administration of tungstate prevents body weight gain of high-fat diet-induced obese rats, ameliorates the metabolic profile associated with the obese phenotype, and leads to reduced adiposity and improved insulin sensitivity. Antiobesity effects of tungstate occur without any adverse effects such as gastrointestinal discomfort, the main side effect described for vanadate (19). Indeed, tungstate has a rather low toxicity, as demonstrated by its oral LD50 value, high bioavailability, rapid total plasma clearance, and elimination half-life in rats (19, 20, 21, 22). Moreover, short- or long-term administration of tungstate in rodents neither causes diarrhea nor exerts hepatotoxic or nephrotoxic effects (6, 7, 8, 9, 19, 20, 21, 22). Thereby, from the results presented here and the toxicity data, tungstate emerges as a promising new therapy for the treatment of obesity.
To gain insight into the mechanisms of action of tungstate on body weight, we studied its putative anorectic and thermogenic effects. This compound significantly reduced body weight gain and adiposity in diet-induced obese rats, without alterations in fat absorption or growth rate. However, calorimetry and gene expression studies showed that tungstate increased energy expenditure and UCP1 expression in iBAT, the most important thermogenic protein in rodents (15). UCP1 plays a key role in adaptive thermogenesis by uncoupling ATP production from oxidative phosphorylation, a process that has long been recognized as a protective mechanism against the development of obesity (23). Moreover, tungstate treatment also increased mRNA levels of UCP2 and UCP3; and, although their physiological functions are still under debate, experimental evidence suggests a putative role for these UCPs in thermogenesis, fuel metabolism, and reactive oxidative species production (15, 23, 24, 25, 26).
Tungstate also induced a coordinate increase in the expression of genes involved in fatty acid transport (LPL and aP2) and oxidation (mCPT1) in adipose tissue. We observed a 4-fold up-regulation of the LPL gene, which might contribute to the improvement of plasma lipid profile and, hence, insulin sensitivity of treated obese rats. LPL catalyzes the key step of TG hydrolysis and is an important determinant of TG metabolism and its circulating concentrations (27). Tungstate treatment also increased the mRNA levels of aP2, a cytosolic protein that facilitates the NEFA transport to oxidative organules (28), and mCPT1, the rate-limiting enzyme of mitochondrial -oxidation (29). These changes suggest that tungstate promotes the shunting of fatty acids to oxidation and their entry into the mitochondria.
Therefore, variations in UCP expression, as well as in genes involved in lipid transport and oxidation, might lead to increased energy dissipation and reduced storage of TG in adipose tissue and, consequently, contribute to reduce body weight gain and adiposity and to improve insulin sensitivity. Indeed, it is well known that the capacity to increase fat oxidation or to reduce TG storage may affect whole-body energy homeostasis and prevent obesity (30, 31, 32).
UCPs also play a role when energy partitioning is oriented toward NEFA oxidation, reducing the toxic effects of excessive lipid metabolism in the cell (33). Therefore, the up-regulation of UCP2 and UCP3 by tungstate could also prevent excessive free radical generation and lipid accumulation in the mitochondria during fatty acid oxidation, limiting oxidative damage.
To induce the effects described above, tungstate might also modulate the transcriptional activity of PPARs. These transcription factors play a central role in the coordination of rates of fatty acid oxidation and storage by transactivating proteins with direct links to adipocyte and lipid metabolism (13, 14). PPAR predominantly regulates pathways of fatty acid oxidation, whereas PPAR, the subtype preferentially expressed in adipose tissue, is involved in insulin sensitivity. Therefore, its modulators are efficacious as insulin-sensitizing agents (13, 14), probably by increasing the number of small insulin-sensitive adipocytes, which finally leads to alleviation of insulin resistance (34). Although we did not observe changes in the mRNA levels of PPARs due to treatment, tungstate acted as an insulin sensitizer, reducing both high-fat diet-induced adipocyte hypertrophy and insulin resistance. Moreover, tungstate regulated the expression of genes that are targets for these nuclear factors, such as UCPs, and enzymes involved in lipid homeostasis (13, 14). The mechanisms for PPAR regulation include activation by several naturally occurring compounds and by synthetic molecules (13, 14, 35, 36, 37, 38) but also by kinases/phosphatases pathways that involve MAPK activation (39, 40, 41). Because activation of MAPK by tungstate has been recently observed in various cell types, including 3T3-L1 adipocytes (Ref.42 ; and H. Corominola, unpublished results), it is reasonable to speculate that tungstate probably mediates MAPK activation and subsequent modulation of PPAR transcriptional activity by phosphorylation. Interestingly, activation of the MAPK pathway, which, in turn, phosphorylates PPAR, has also been described for insulin (41).
In summary, tungstate increases whole-body energy expenditure and favors oxidative catabolism of fatty acids over anabolic pathways, without excessive free radical generation. These actions improve lipid profile and insulin sensitivity and reduce weight gain and adiposity in obese rats fed with a high-fat palatable diet. These findings strongly support tungstate as an attractive therapeutic approach for the treatment of obesity.
Acknowledgments
We thank R. Gasa and B. Nadal for their helpful suggestions and comments. We also thank J. Moreno and M. Parés for excellent technical assistance with animals and R. Casamitjana for leptin determinations.
Footnotes
This work was supported by Grant SAF2003-06018 from the Ministerio de Ciencia y Tecnologia (Spain) and grants from Fundació la Marató de TV3 and Fundación Lilly. The research group was supported by Redes C03/08, G03/212, and PIO20483 from the Ministerio de Sanidad y Consumo (Spain). M.C. was supported by Grants SAF2000-0053 and SAF2003–06018 from the Ministerio de Ciencia y Tecnología (Spain).
1 M.C. and H.C. contributed equally to the work.
Abbreviations: ACO, Acyl-CoA oxidase; aP2, adipocyte protein 2; eWAT, epididymal white adipose tissue; FAT/CD36, fatty acid translocase; gM, gastrocnemius muscle; iBAT, interscapular brown adipose tissue; LPL, lipoprotein lipase; mCPT1, muscle-carnitine palmitoyltransferase-1; NEFA, nonesterified fatty acids; NS, not significant; PPAR, peroxisome proliferator-activated receptor; RER, respiratory exchange ratio; TG, triglyceride(s); TO, 2 mg/ml tungstate-treated obese; UCP, uncoupling protein; UO, untreated obese; VO2, oxygen consumption volume.
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Pharmacology and Toxicology Department (J.S.), Institut d’Investigacions Biomèdiques de Barcelona, 08036 Barcelona, Spain
Abstract
The increasing worldwide incidence of obesity and the limitations of current treatments raise the need for finding novel therapeutic approaches to treat this disease. The purpose of the current study was first to investigate the effects of tungstate on body weight and insulin sensitivity in a rat model of diet-induced obesity. Second, we aimed to gain insight into the molecular mechanisms underlying its action. Oral administration of tungstate significantly decreased body weight gain and adiposity without modifying caloric intake, intestinal fat absorption, or growth rate in obese rats. Moreover, the treatment ameliorated dislipemia and insulin resistance of obese rats. These effects were mediated by an increase in whole-body energy dissipation and by changes in the expression of genes involved in the oxidation of fatty acids and mitochondrial uncoupling in adipose tissue. Furthermore, treatment increased the number of small adipocytes with a concomitant induction of apoptosis. Our results indicate that tungstate treatment may provide the basis for a promising novel therapy for obesity.
Introduction
OBESITY IS A complex metabolic disorder characterized by an excess of body fat and associated with an increased risk of suffering diabetes, hypertension, cardiovascular disease, and cancer. Obesity appearance is determined by interactions between genetic, environmental, and psychosocial factors, which alter long-term body energy homeostasis. Thus, obesity develops when energy intake chronically exceeds total body expenditure (1).
The prevalence of obesity and associated comorbid diseases is increasing at an alarming rate. However, currently available antiobesity drugs have limited efficacy or safety concerns (2). Consequently, the development of safer and more effective antiobesity therapeutic approaches is becoming more of an imperative. Recent advances in the understanding of the mechanisms and molecules involved in the regulation of body weight (3) have provided potential strategies for the treatment of obesity. These strategies aimed to modify, centrally and peripherally, food intake regulation, nutrient absorption, thermogenesis, and fat metabolism or storage (4).
Oral administration of tungstate reverts the diabetic phenotype in several induced and genetic animal models. Tungstate up-regulates glucose transporter expression and translocation in muscle (5), restores hepatic glucose metabolism (6, 7), and increases -cell mass (8, 9) in a number of diabetic animal models. Furthermore, administration of tungstate attenuates body weight gain in healthy rats (6, 7, 8, 9). This observation led us to examine the efficacy of tungstate for the treatment of obesity. We studied the effects of this compound on body weight and insulin sensitivity in a rat model of diet-induced obesity. In addition, we aimed to gain insight into the molecular mechanisms underlying the action of tungstate.
Our results show that tungstate, by increasing thermogenesis and lipid oxidation in adipose tissue, prevents body weight gain of obese rats on a high-fat diet. This experimental evidence raises tungstate as an effective novel agent to consider for the treatment of obesity.
Materials and Methods
Diet-induced obesity
Male Wistar rats (IFFA CREDO, L’Arbresse, France), weighing 220–240 g, were caged individually in a 12-h light, 12-h dark cycle in a temperature- and humidity-controlled environment. Animals were divided into two dietary sets for 30 d. One group was fed with standard chow diet (supplying 8% of calories as fat; type AO4 from Panlab, Barcelona, Spain). To induce obesity, the second group was fed with a cafeteria diet as previously described (10), with minor modifications. This diet consisted of standard chow and a daily intake of cookies, liver pate, bacon, and whole-milk supplemented with 333 g/liter of sucrose and 10 g/liter of a mineral and vitamin complex (Gevral; Cynamid Ibérica, Madrid, Spain). Sixty-five percent of the energy of this diet derived from lipids. All the food items were weighed daily and presented in excess. Daily caloric intake was calculated by multiplying the consumption of each item in the diet by its caloric density provided by the manufacturer.
Tungstate treatment
After 30 d of diet, lean and obese rats were divided in treated and untreated groups. Treatment was carried out by giving a solution of 0.3, 0.7, or 2 mg/ml sodium tungstate (Na2WO4x 2H2O; Carlo Erba, Milano, Italy) in distilled water for 30 d ad libitum. After treatment, we tested the reversibility of tungstate effects by withdrawing treatment under the same dietary conditions for 35 additional days (recovery period). During all the experimental period, fluid, food consumption (corrected by the amount of water lost for each item), and body weight of all rats were recorded daily.
At the end of the treatment period, rats from each group were killed. Gastrocnemius muscle (gM), interscapular brown adipose tissue (iBAT), epididymal white adipose tissue (eWAT), and liver were excised, weighed, and rapidly frozen in liquid nitrogen or fixed in 10% buffered formalin. All procedures were conducted in accordance with principles of laboratory animal care (European Community and local government guidelines) and approved by the Animal Research Committee of the University of Barcelona.
Metabolic measurements
All rats were fasted 6 h before metabolic measurements. Blood samples were collected from the tail using a capillary blood collection system with EDTA (Sarstedt, Nümbrecht, Germany). Blood glucose measurements were performed with the Roche (Mannheim, Germany) AccuTrend glucose sensor. Plasma insulin levels were measured by ELISA kits from Mercodia (Uppsala, Sweden). Plasma leptin levels were assessed by RIA (Linco Research, Inc., St. Charles, MO). Plasma triglycerides (TG) and nonesterified fatty acids (NEFA) levels were measured using colorimetric kits from Sigma (St. Louis, MO) and Roche, respectively.
Insulin tolerance tests were performed on ad libitum-fed rats by the administration of an ip injection of insulin (1 IU/kg body weight; Eli Lilly & Co., Indianapolis, IN). Blood samples collected at 0, 15, 30, 60, and 120 min after injection were used for glycemia determinations.
Histologic and morphometric analysis of adipose tissue
Formalin-fixed eWAT specimens were dehydrated, embedded in paraffin, and cut into 8-μm-thick sections. Adipose tissue sections were stained with hematoxylin and eosin, following standard protocols. For the quantitation of the number and size of adipocytes, the sectional areas of eWAT were manually analyzed in at least four fields from four animals per experimental group using an Olympus Corp. microscope (Tokyo, Japan) and MicroImage software (Hamburg, Germany).
Apoptosis determination
The presence of apoptotic nuclei in eWAT was assessed by terminal deoxynucleotidyl transferase-mediated deoxyuridine 5-triphosphate nick-end-labeling using the Apoalert DNA fragmentation assay kit (CLONTECH Laboratories, Inc., Palo Alto, CA), following the manufacturer’s instructions.
Growth rate and feces lipid content assessment
Growth rate was assessed by femur length measurement. At the end of treatment, feces were collected over 24 h and dried at 60 C until achieving constant mass. Total feces lipid content was determined by the Folch method (11).
Indirect calorimetry
Volume of oxygen consumption (VO2) was measured using an open-circuit indirect calorimetry system (MM-100 Metabolic Monitor System; CWE Inc., Ardmore, PA). Age and weight-matched rats were housed overnight in the measurement chamber to become acclimatized. Next, either vehicle (NaCl, 154 mmol/liter) or 150 mg/kg body weight of tungstate was administered to rats by gavage. This dose was previously verified as effective in reducing body weight gain. In vivo VO2 was then measured at 15-min intervals at baseline (vehicle) and for 8 h after administration of tungstate. Metabolic measurements included VO2, volume of carbon dioxide produced (VCO2), and respiratory exchange ratio (RER = VCO2/VO2).
RNA isolation and Northern blot analysis
Total RNA was purified from tissues by either Trizol (Invitrogen, Carlsbad, CA) or guanidinium isothiocyanate-phenol chloroform extraction (Invitrogen), following the manufacturer’s instructions. Total RNA derived from gM, eWAT, and iBAT tissues was electrophoresed on 1.2% formaldehyde-agarose gels and transferred to Nytran-plus membranes (Schleicher & Schuell, Inc., Keene, NH). Membranes were hybridized with specific (-32P) deoxycytidine triphosphate (Amersham Pharmacia Biotech, Buckinghamshire, UK) random primed-labeled cDNA probes generated from total RNA by RT-PCR using specific primers for peroxisome proliferators-activated receptor (PPAR), PPAR, uncoupling protein (UCP)1, UCP2, UCP3, lipoprotein lipase (LPL), muscle-carnitine palmitoyltransferase-1 (mCPT1), adipocyte protein 2 (aP2), fatty acid translocase (FAT/CD36), and acyl-CoA oxidase (ACO). Band intensities obtained by Northern blots were normalized to the signal obtained after stripping and reprobing the same membranes with 32P-labeled 28S cDNA probe. We quantified the amount of both of the signals, target gene and 28S, by densitometry using a Personal FX Phosphorimager and QuantityOne software (Bio-Rad Laboratories Inc., Hercules, CA), divided the signal of target mRNA by that of 28S, and then converted the results to a percentage. The results were expressed as mean ± SEM.
Statistical analysis
All results are expressed as mean ± SEM. Differences between the experimental groups were evaluated by the Student t test or by the nonparametric Mann-Whitney U test. P < 0.05 was considered significant.
Results
Diet-induced obesity
Administration of a cafeteria diet for 30 d led to obesity, increasing body weight of rats by 24% in relation to standard chow-fed rats (449 ± 7 vs. 362 ± 9 g; P < 0.05). As expected, the estimated cumulative energy intake was significantly higher in cafeteria-fed rats than in those fed on a standard chow diet (15,604 ± 181 vs. 7704 ± 147 kcal/kg·30 d; P < 0.001). Obese rats displayed higher plasma levels of TG (310 ± 33 vs. 159 ± 25 mg/dl; P < 0.05), NEFA (0.86 ± 0.07 vs. 0.52 ± 0.06 nmol/μl; P < 0.01), insulin (3.06 ± 0.50 vs. 0.73 ± 0.11 ng/ml; P < 0.05), and leptin (31.6 ± 2.6 vs. 7.2 ± 0.5 ng/ml; P < 0.01) than lean ones. Moreover, obese rats became glucose intolerant as assessed by a glucose tolerance test (data not shown). No differences in femur length were observed between rats fed the cafeteria and standard chow diet [51 ± 1 vs. 48 ± 1 mm; not significant (NS)].
Tungstate decreases body weight gain and adiposity of obese rats fed with a cafeteria diet
To determine the optimal dose at which oral tungstate exerts its effect, obese rats were treated with a range of doses of this compound (0.3, 0.7, and 2 mg/ml in the drinking solution) in addition to the cafeteria diet for another 30-d period (Fig. 1A). Administration of these doses led to an averaged tungstate ingestion of 33 ± 1, 77 ± 2, and 227 ± 9 mg/kg·d for the 0.3, 0.7, and 2 mg/ml, respectively. At all the doses tested, rats did not show the side effects, such as diarrhea or other toxic effects, that have been associated with the use of drugs that inhibit absorption of dietary fat. At 0.3 mg/ml of tungstate, no significant effects on body weight were observed. However, obese rats treated with either 0.7 or 2 mg/ml decreased their weight gain by 26 and 88%, respectively, compared with sex- and age-matched untreated obese (UO) rats on the cafeteria diet (Fig. 1A). Thus, we decided to retain only the dose of 2 mg/ml, which was the most effective in decreasing body weight gain.
The deceleration in body weight gain of 2 mg/ml tungstate-treated obese (TO) rats was accompanied by a significant decrease in adiposity when compared with UO rats. No significant changes in iBAT, gM, or liver mass were recorded at the end of treatment (Table 1). Moreover, no significant differences in femur length were observed between TO and UO rats (65 ± 2 for TO vs. 68 ± 2 mm for UO; NS), ruling out possible effects of tungstate on growth rate. Treatment withdrawal (recovery period) led to progressive and sustained body weight gain, indicating that tungstate does not induce a persistent state of weight loss (Fig. 1B).
Tungstate ameliorates insulin resistance and metabolic parameters
Tungstate effects on body weight and adiposity were accompanied by improved lipid profile and insulin sensitivity. Treatment decreased the circulating levels of TG, NEFA, and insulin (Fig. 2, A, B, and C, respectively). Moreover, ip insulin tolerance tests after 30 d of treatment showed an improvement of insulin sensitivity in TO when compared with UO rats (Fig. 2D). Noteworthy, amelioration of insulin sensitivity appeared as early as d 5 of treatment (data not shown).
Antiobesity effects of tungstate are not mediated by fat malabsorption, reduced energy intake, or changes in plasma leptin
Deficient intestinal fat absorption might account for the effects of tungstate on body weight and adiposity in obese rats. However, the feces lipid content in the total fecal mass was unaffected by tungstate treatment (12.1 ± 0.7 for TO vs. 12.8 ± 0.7% for UO; NS), demonstrating that this compound did not alter intestinal lipid absorption. No differences were observed in daily food, fluid (Fig. 3, A and B), or cumulative energy intake during the treatment period, between TO and UO rats (10,452 ± 229 for TO vs. 10,112 ± 246 kcal/kg·30 d for UO; NS). Indeed, regression analysis showed no correlation between food consumption and tungstate intake in TO rats fed with a cafeteria diet (Fig. 3C).
We next studied whether treatment altered the expression of leptin, an adipocyte-derived hormone involved in the regulation of energy homeostasis and plasma concentrations of this hormone are highly correlated with adipose tissue mass and body weight (12). However, plasma leptin levels at the end of the treatment were unaffected in TO rats when compared with UO counterparts (Fig. 3D).
Tungstate prevents adipocyte hypertrophy
Histological analysis of eWAT from TO rats showed an increased number of small (4500 μm2) adipocytes (40.3 ± 2.8 for TO vs. 17.3 ± 1.9% for UO; P < 0.05) and a trend to reduce the proportion of large (9000 μm2) adipocytes (35.1 ± 8.3 for TO vs. 43.5 ± 4.8% for UO; NS), (Fig. 4, A and B). In addition, treatment induced apoptosis in eWAT [7.8 ± 2.7 for TO vs. 3.2 ± 1.2% for UO (percentage of apoptotic nuclei referred to total nuclei); P < 0.05]. Collectively, these data indicate that tungstate remodels adipocyte population, thereby reducing white adipose tissue mass and alleviating insulin resistance.
Metabolic and morphometric results described above were similar to those reported for the modulation of PPARs, key transcription factors in the control of adipogenesis and lipid metabolism (13, 14). However, we did not observe changes in the gene expression of PPAR (data not shown) or PPAR in either iBAT or eWAT due to treatment (Fig. 4C).
Tungstate enhances energy dissipation and expression of proteins involved in lipid metabolism
To test whether the effects of tungstate on body weight were mediated by increased thermogenesis, we monitored metabolic rate by VO2 measurements and analyzed the gene expression of the UCPs, molecules involved in energy dissipation, oxidative phosphorylation, and lipid metabolism (15). In treated obese rats, VO2 averaged 732 ± 20 compared with 607 ± 15 ml/h·kg0.75 for the UO group, which represented an overall increase of 21% (Fig. 5A; P < 0.0001). Moreover, the average RER was lower for treated obese rats (0.74 ± 0.012) than for the UO rats (0.77 ± 0.019; Fig. 5B; P < 0.0001), indicating increased fatty acid oxidation by tungstate administration. Tungstate administration also increased UCP1 expression in iBAT (Fig. 5C). Taken together, these data indicate that tungstate treatment increased thermogenesis as fatty acid oxidation. In eWAT, this compound failed to induce ectopic expression of UCP1 (data not shown) but increased the expression of UCP2 (Fig. 5D). UCP2 and UCP3 mRNA levels were also up-regulated by treatment in gM (Fig. 5E).
To study a possible link between increased energy dissipation and enhanced fatty acid oxidation, we analyzed the expression of genes involved in fatty acid transport (LPL, FAT/CD36, and aP2) and oxidation (ACO and mCPT1) in several tissues. Tungstate did not alter the expression of these genes in gM, indicating that this compound has no effect on fatty acid uptake or oxidation in this tissue (Fig. 6A). In contrast, treatment resulted in the up-regulation of LPL, aP2, and mCPT1 but did not alter the mRNA levels of FAT/CD36 or ACO in eWAT (Fig. 6B).
Taken together, these results indicate that tungstate treatment increases fatty acid oxidation in adipose tissue but limits an excessive production of reactive oxidative species.
Discussion
In recent years, numerous studies have reported the antidiabetic properties of chemically resembled inorganic compounds such as tungstate vanadate, molybdate, and selenate (6, 7, 8, 9, 16, 17, 18). In the present study, we investigated the effectiveness of tungstate in obesity treatment and demonstrated that chronic administration of tungstate prevents body weight gain of high-fat diet-induced obese rats, ameliorates the metabolic profile associated with the obese phenotype, and leads to reduced adiposity and improved insulin sensitivity. Antiobesity effects of tungstate occur without any adverse effects such as gastrointestinal discomfort, the main side effect described for vanadate (19). Indeed, tungstate has a rather low toxicity, as demonstrated by its oral LD50 value, high bioavailability, rapid total plasma clearance, and elimination half-life in rats (19, 20, 21, 22). Moreover, short- or long-term administration of tungstate in rodents neither causes diarrhea nor exerts hepatotoxic or nephrotoxic effects (6, 7, 8, 9, 19, 20, 21, 22). Thereby, from the results presented here and the toxicity data, tungstate emerges as a promising new therapy for the treatment of obesity.
To gain insight into the mechanisms of action of tungstate on body weight, we studied its putative anorectic and thermogenic effects. This compound significantly reduced body weight gain and adiposity in diet-induced obese rats, without alterations in fat absorption or growth rate. However, calorimetry and gene expression studies showed that tungstate increased energy expenditure and UCP1 expression in iBAT, the most important thermogenic protein in rodents (15). UCP1 plays a key role in adaptive thermogenesis by uncoupling ATP production from oxidative phosphorylation, a process that has long been recognized as a protective mechanism against the development of obesity (23). Moreover, tungstate treatment also increased mRNA levels of UCP2 and UCP3; and, although their physiological functions are still under debate, experimental evidence suggests a putative role for these UCPs in thermogenesis, fuel metabolism, and reactive oxidative species production (15, 23, 24, 25, 26).
Tungstate also induced a coordinate increase in the expression of genes involved in fatty acid transport (LPL and aP2) and oxidation (mCPT1) in adipose tissue. We observed a 4-fold up-regulation of the LPL gene, which might contribute to the improvement of plasma lipid profile and, hence, insulin sensitivity of treated obese rats. LPL catalyzes the key step of TG hydrolysis and is an important determinant of TG metabolism and its circulating concentrations (27). Tungstate treatment also increased the mRNA levels of aP2, a cytosolic protein that facilitates the NEFA transport to oxidative organules (28), and mCPT1, the rate-limiting enzyme of mitochondrial -oxidation (29). These changes suggest that tungstate promotes the shunting of fatty acids to oxidation and their entry into the mitochondria.
Therefore, variations in UCP expression, as well as in genes involved in lipid transport and oxidation, might lead to increased energy dissipation and reduced storage of TG in adipose tissue and, consequently, contribute to reduce body weight gain and adiposity and to improve insulin sensitivity. Indeed, it is well known that the capacity to increase fat oxidation or to reduce TG storage may affect whole-body energy homeostasis and prevent obesity (30, 31, 32).
UCPs also play a role when energy partitioning is oriented toward NEFA oxidation, reducing the toxic effects of excessive lipid metabolism in the cell (33). Therefore, the up-regulation of UCP2 and UCP3 by tungstate could also prevent excessive free radical generation and lipid accumulation in the mitochondria during fatty acid oxidation, limiting oxidative damage.
To induce the effects described above, tungstate might also modulate the transcriptional activity of PPARs. These transcription factors play a central role in the coordination of rates of fatty acid oxidation and storage by transactivating proteins with direct links to adipocyte and lipid metabolism (13, 14). PPAR predominantly regulates pathways of fatty acid oxidation, whereas PPAR, the subtype preferentially expressed in adipose tissue, is involved in insulin sensitivity. Therefore, its modulators are efficacious as insulin-sensitizing agents (13, 14), probably by increasing the number of small insulin-sensitive adipocytes, which finally leads to alleviation of insulin resistance (34). Although we did not observe changes in the mRNA levels of PPARs due to treatment, tungstate acted as an insulin sensitizer, reducing both high-fat diet-induced adipocyte hypertrophy and insulin resistance. Moreover, tungstate regulated the expression of genes that are targets for these nuclear factors, such as UCPs, and enzymes involved in lipid homeostasis (13, 14). The mechanisms for PPAR regulation include activation by several naturally occurring compounds and by synthetic molecules (13, 14, 35, 36, 37, 38) but also by kinases/phosphatases pathways that involve MAPK activation (39, 40, 41). Because activation of MAPK by tungstate has been recently observed in various cell types, including 3T3-L1 adipocytes (Ref.42 ; and H. Corominola, unpublished results), it is reasonable to speculate that tungstate probably mediates MAPK activation and subsequent modulation of PPAR transcriptional activity by phosphorylation. Interestingly, activation of the MAPK pathway, which, in turn, phosphorylates PPAR, has also been described for insulin (41).
In summary, tungstate increases whole-body energy expenditure and favors oxidative catabolism of fatty acids over anabolic pathways, without excessive free radical generation. These actions improve lipid profile and insulin sensitivity and reduce weight gain and adiposity in obese rats fed with a high-fat palatable diet. These findings strongly support tungstate as an attractive therapeutic approach for the treatment of obesity.
Acknowledgments
We thank R. Gasa and B. Nadal for their helpful suggestions and comments. We also thank J. Moreno and M. Parés for excellent technical assistance with animals and R. Casamitjana for leptin determinations.
Footnotes
This work was supported by Grant SAF2003-06018 from the Ministerio de Ciencia y Tecnologia (Spain) and grants from Fundació la Marató de TV3 and Fundación Lilly. The research group was supported by Redes C03/08, G03/212, and PIO20483 from the Ministerio de Sanidad y Consumo (Spain). M.C. was supported by Grants SAF2000-0053 and SAF2003–06018 from the Ministerio de Ciencia y Tecnología (Spain).
1 M.C. and H.C. contributed equally to the work.
Abbreviations: ACO, Acyl-CoA oxidase; aP2, adipocyte protein 2; eWAT, epididymal white adipose tissue; FAT/CD36, fatty acid translocase; gM, gastrocnemius muscle; iBAT, interscapular brown adipose tissue; LPL, lipoprotein lipase; mCPT1, muscle-carnitine palmitoyltransferase-1; NEFA, nonesterified fatty acids; NS, not significant; PPAR, peroxisome proliferator-activated receptor; RER, respiratory exchange ratio; TG, triglyceride(s); TO, 2 mg/ml tungstate-treated obese; UCP, uncoupling protein; UO, untreated obese; VO2, oxygen consumption volume.
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