Student-Newman-Keuls

923

Hypothyroidism and skeletal muscle UCP3 gene expression Hypercaloric cafeteria-like diet induced

UCP3 gene expression in skeletal

muscle is impaired by hypothyroidism

Departamentos de 1Fisiologia e Biofísica and

2Histologia e Embriologia, Instituto de Ciências Biomédicas,Universidade de S?o Paulo, S?o Paulo, SP, Brasil

M.A. Christoffolete 1and A.S. Moriscot 2

Abstract

The uncoupling protein UCP3 belongs to a family of mitochondrial carriers located in the inner mitochondrial membrane of certain cell types. It is expressed almost exclusively at high levels in skeletal muscle and its physiological role has not been fully determined in this tissue. In the present study we have addressed the possible interaction between a hypercaloric diet and thyroid hormone (T 3), which are strong stimulators of UCP3 gene expression in skeletal muscle. Male Wistar rats weighing 180 ± 20 g were rendered hypothyroid by thyroidectomy and the addition of methimazole (0.05%; w/v) to drinking water after surgery. The rats were fed a hypercaloric cafeteria diet (68% carbohydrates, 13% protein and 18% lipids) for 10 days and sacrificed by decapitation. Subsequently, the gastrocnemius muscle was dissected, total RNA was isolated with Trizol? and UCP3 gene expression was determined by Northern blotting using a specific probe. Statistical analysis was performed by one-way analysis of variance (ANOVA) followed by the Student-Newman-Keuls post-test. Skeletal muscle UCP3 gene expression was decreased by 60% in hypothyroid rats and UCP3 mRNA expression was increased 70% in euthyroid cafeteria-fed rats compared to euthyroid chow-fed animals,confirming previous studies. Interestingly, the cafeteria diet was unable to stimulate UCP3 gene expression in hypothyroid animals (40% lower as compared to euthyroid cafeteria-fed animals). The results show that a hypercaloric diet is a strong stimulator of UCP3gene expression in skeletal muscle and requires T 3 for an adequate action.

Correspondence

A.S. Moriscot

Departamento de Histologia e Embriologia, ICB, USP Av. Lineu Prestes, 152405508-900 S?o Paulo, SP Brasil

E-mail: moriscot@usp.br Research partially supported by FAPESP (Nos. 97/0650-0 and 98/10498-4.

Received May 6, 2003Accepted February 10, 2004

Key words

?Thyroid hormone ?Hypothyroidism ?Hypercaloric diet ?Uncoupling protein ?

UCP3

It is well recognized that food processing induces a basal heat production, which com-bined with other physiological processes,results in the so-called obligatory thermo-genesis. In addition, food intake also triggers a regulated form of thermogenesis which takes place mainly in the brown adipose tissue (BAT) of small rodents and plays a key role in the control of energy balance, a pro-cess called diet-induced thermogenesis (DIT)(1,2). Several studies have shown that BAT is capable of buffering excessive caloric in-take by uncoupling mitochondria from ATP synthesis. This is possible due to an inner

Brazilian Journal of Medical and Biological Research (2004) 37: 923-927ISSN 0100-879X Short Communication

924M.A. Christoffolete and A.S. Moriscot

mitochondrial membrane protein called UCP1, which bypasses the mitochondrial proton gradient without ATP synthesis. The role of UCP1 (the first UCP cloned and identified) in energy dissipation has been confirmed in animals during starvation (3), in transgenic mice overexpressing UCP1 in white adipose tissue (4), and in animals with deficient UCP1 expression in BAT (4).

Other members of the UCP family are also considered to be possibly involved in DIT. Indeed, some studies have reported a correlation between UCP3 expression and increased oxygen consumption in skeletal muscle (5-8). Several studies have shown that UCP3 mRNA in muscle, heart and adi-pose tissue is up-regulated by increased plasma free fatty acid levels due to starva-tion in humans (9,10), by a high lipid content diet (11) and by genetic/experimental diabe-tes in rodents (12). However, a rise in fatty acids is not required for UCP3 up-regulation in gastrocnemius muscle during starvation (13). In addition, hypothyroid animals on a high calorie/high lipid content diet were found to present increased oxygen consump-tion compared to hypothyroid animals on a regular diet (14). Since increased oxygen consumption cannot be achieved in BAT of hypothyroid animals, alternative sites have been suggested to be involved in energy balance control, such as skeletal muscle. In contrast, during fasting, a condition in which energy dissipation tends to decrease, UCP3 expression is up-regulated (10,15) and acute physical activity (15), which promotes en-ergy depletion, also appears to increase UCP3 gene expression.

In addition to the discrepant reports about the physiological role of UCP3 in skeletal muscle, the interactions of known UCP3 regulators are also poorly understood. For instance, thyroid hormone (T3) and hyperca-loric/lipidic diets are known to be strong stimulators of UCP3 gene expression (5,7,8, 16,17), yet it is not clear how such activators interact. Therefore, in the present study, the effect of T3 and a hypercaloric diet on UCP3 gene expression in skeletal muscle was ad-dressed by feeding rats a cafeteria diet.

Experiments were performed on male Wistar rats weighing 180 ± 20 g, obtained from our breeding colony. Animals were maintained on a 12:12-h dark-light cycle at room temperature and, unless specified, had free access to standard chow and water. Surgical thyroidectomy was performed un-der light ether anesthesia and was followed by administration of 0.05% methimazole (Sigma, St. Louis, MO, USA) in drinking water. Each experimental group consisted of 2-3 animals per experiment.

The cafeteria diet is based on the prod-ucts found in a typical American cafeteria, which are industrialized products with high caloric values. The macronutrient composi-tion of the cafeteria diet was 68% carbohy-drates, 13% protein and 18% lipids while the standard chow diet is composed of 80% carbohydrates, 19% protein and less than 1% lipids. The animals were placed in indi-vidual cages and food intake was monitored for 10 days. The weights of the diet compo-nents were determined individually the day before and the day after they were offered to the rats (prior to the beginning of the dark cycle), with the difference between weights corresponding to the amount of food eaten. Four groups were studied: I) euthyroid rats on a standard chow diet; II) hypothyroid rats on a standard chow diet; III) euthyroid rats on a cafeteria diet, and IV) hypothyroid rats on a cafeteria diet. At the end of each experi-ment the animals were sacrificed by decapi-tation and the gastrocnemius muscle was dissected, immediately frozen in liquid ni-trogen and stored at -70oC. Total RNA was isolated by the Trizol? method (Invitrogen) and transferred to a nylon membrane, and a 20-μg amount was pre-hybridized with 0.5 M sodium phosphate, pH 6.8, 7% SDS, 1% BSA, 1 mM EDTA, and 200 μg/ml salmon DNA for 4 h at 65oC. Subsequently, the membrane was incubated with a specific

925 Hypothyroidism and skeletal muscle UCP3 gene expression

probe (Genbank #U92069) for UCP3 for 18 h and 18S rRNA was used as internal con-trol. The membrane was washed twice for 5 min in SSC 0.1X and 0.1% SDS at 50oC, and for 15 min in SSC 2X and 1% SDS 1 at 55oC. Images were obtained with a STORM 840 instrument (Molecular Dynamics, Sunnyvale, CA, USA) and band densitometry was deter-mined using the ImageQuant software (Mo-lecular Dynamics). Data are reported as means ± SEM. One-way analysis of variance (ANOVA) was used,followed by the Stu-dent-Newman-Keuls post-test. For all tests, P < 0.05 was considered statistically signifi-cant. For statistical analysis we used the GraphPad Instat Software (GraphPad Soft-ware Inc., San Diego, CA, USA).

Euthyroid animals submitted to the caf-eteria diet increased their caloric intake by ~1.6-fold compared to euthyroid chow-fed animals. An increase in caloric intake by cafeteria-fed hypothyroid rats was also noted as compared to chow-fed hypothyroid ani-mals (Figure 1). Despite the clear increase in caloric intake in the cafeteria-fed groups, the ?body weight did not differ significantly between the chow and cafeteria groups (23 ±5.7 g euthyroid chow-fed, 31.8 ± 2.8 g euthy-roid cafeteria-fed, -2.3 ± 4 g hypothyroid chow-fed, and -11 ± 6.1 g hypothyroid caf-eteria-fed rats).

The UCP3 gene expression was increased 1.7-fold by the cafeteria diet in euthyroid animals compared to the euthyroid chow-fed group. Hypothyroidism induced a ~60% re-duction in UCP3 gene expression in chow-fed animals. Interestingly, in cafeteria-fed animals, hypothyroidism also induced a ~40% decrease in UCP3 gene expression (Figure 2A,B).

Our results confirm previous data in the literature showing that hypothyroidism de-creases (16) while high lipid content diet increases (11) UCP3 mRNA levels in skel-etal muscle. In the present study, the in-crease in UCP3 expression observed in eu-thyroid animals on the cafeteria diet was

Figure 1. Daily energy intake (kcal/kg body weight) by euthyroid chow-fed (Eu-chow), hypothyroid chow-fed (Hypo-chow), euthyroid hypercaloric diet-fed (Eu-hypercal) and hypo-thyroid hypercaloric diet-fed (Hypo-hypercal) rats during the 10 days of the experiment. *P < 0.01 vs Eu-chow, +P < 0.01 vs Hypo-chow, #P < 0.01 vs Eu-hypercal (ANOVA followed by the Student-Newman-Keuls post-test).

Eu-chow Hypo-chow Eu-hypercal Hypo-hypercal

UCP3

18S

Figure 2. Abundance of mRNA/UCP3 in euthyroid chow-fed (Eu-chow), hypothyroid chow-fed (Hypo-chow), euthyroid hypercaloric diet-fed (Eu-hypercal) and hypothyroid hypercaloric diet-fed (H ypo-hypercal) rats. A, Northern blot analysis of UCP3 mRNA levels and 18S rRNA in gastrocnemius muscle. B, Histogram representing the densitometry of the bands. Data are reported as arbitrary units of mRNA for UCP3/rRNA 18S. *P < 0.05 vs Eu-chow, +P < 0.05 vs H ypo-chow, #P < 0.05 vs Eu-hypercal (ANOVA followed by the Student-Newman-Keuls post-test).

926M.A. Christoffolete and A.S. Moriscot

minimized in hypothyroid animals receiving the same diet, showing that T3 plays a key role in UCP3 gene responsiveness to a hypercaloric stimulus.

BAT, the major site of facultative ther-mogenesis in rodents, is not functional in hypothyroids rats (18). A previous study has shown that hypothyroid rats on a cafeteria diet increase their oxygen consumption com-pared to hypothyroid rats on a chow diet, probably due to DIT, suggesting that in the absence of functional BAT other unknown adaptive thermogenic mechanisms might be recruited in hypothyroid rats (14). It is rea-sonable to assume that skeletal muscle may be a natural alternative candidate for mediat-ing DIT, possibly due to increased UCP3 expression. Our findings show that UCP3 gene expression was unchanged in hypothy-roid rats submitted to a hypercaloric diet for 10 days. These data support the idea that alternative thermogenic mechanisms such as uncoupling by UCP2 may be activated, al-though the UCP2 gene has been shown to function similarly to the UCP3 gene under certain physiological conditions (10). In agreement with our observations (17), it has been suggested that an increase in UCP3 expression may not be the only factor re-sponsible for the T3-induced uncoupling ef-fect. In addition, UCP3 knock-out mice do not develop obesity, with their weights being similar to those of wild-type mice (19). The role of UCP2 and UCP3 in adaptive thermo-genesis, in addition to their ability to un-couple mitochondrial respiration, remain unclear. In fact, recent studies have pro-posed that the primary function of UCP2 and UCP3 is to act as regulators of reactive oxy-gen species production in the mitochondria, having a secondary effect on energy expen-diture and, consequently, on body weight (20).

In vitro studies involving different skel-etal muscle cell lines should be conducted in the future in order to provide further insight into the possible interactions between ener-getic substrate loading and T3 upon UCP3 gene expression and provide a better under-standing of the physiological role of UCP3 in skeletal muscle.

Acknowledgments

The authors would like to acknowledge José Luiz dos Santos and Alessandra Cres-cenzi for excellent technical assistance.

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