1 Division of Molecular Metabolism and Diabetes, Tohoku University Graduate School of Medicine, Sendai, Japan
2 Division of Advanced Therapeutics for Metabolic Diseases, Center for Translational and Advanced Animal Research, Tohoku University Graduate School of Medicine, Sendai, Japan
3 Division of Gastroenterology, Tohoku University Graduate School of Medicine, Sendai, Japan
4 Department of Internal Medicine, Faculty of Medicine, University of Tokyo, Tokyo, Japan
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ABSTRACT |
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An explosive increase in the number of diabetic patients, which has become a major public health concern in most industrialized countries in recent decades (1), is mainly the result of excess energy intake and physical inactivity. When food intake chronically exceeds metabolic needs, efficient metabolism causes excess energy storage and results in obesity, a common condition associated with diabetes, hyperlipidemia, and premature heart disease. Excess energy in cells lowers the response to insulin, namely insulin resistance. However, the major treatment modalities for diabetes, including insulin injection and oral sulfonylureas, aim at lowering blood glucose levels by driving glucose into cells in peripheral tissues such as muscle and fat. This further exacerbates insulin resistance when energy intake is in excess, resulting in a vicious cycle. Therefore, novel therapies that promote increased energy expenditure are needed.
Inefficient metabolism, such as the generation of heat instead of ATP, is a potential treatment strategy for type 2 diabetes associated with obesity. Uncoupling proteins (UCPs) were discovered members of the mitochondrial inner membrane carrier family. These proteins leak protons into the mitochondrial matrix, dissipating energy as heat rather than allowing it to be captured in ATP (2). UCP1 (thermogenin) was originally identified in brown adipose tissue and demonstrated to mediate nonshivering thermogenesis. UCP1 plays an important role in mediating cold exposureinduced thermogenesis (3) and is also a likely regulator of diet-induced thermogenesis (4).
Several laboratories have reported overexpression of UCPs, using the transgenic approach, in mice (5,6,7,8). These reports indicate that overexpression of UCPs in white adipose tissue and skeletal muscle has preventive effects on development of genetic and dietary obesity and the resultant insulin resistance. However, it is still unclear whether ectopic UCP1 expression exerts therapeutic effects after the development of diabetes associated with obesity.
The liver is one of the major metabolic organs involved in glucose and lipid metabolism and insulin action. In addition, the liver can store and release abundant fat dynamically, in response to the energy balance. We reported that hepatic AKT activation resulted in marked alterations in glucose and lipid metabolism (9), suggesting that the liver is a potential site of ectopic expression. We herein expressed UCP1 protein in the liver, before or after diabetes associated with dietary obesity had developed. We found that hepatic UCP1 expression improved diabetes and obesity under high-fat diet conditions through local effects in the liver as well as remote effects in adipose tissues, muscle, and the hypothalamus. However, in standard dietfed lean mice, effects on glucose and lipid metabolism were minimal. Using gene transduction after disease development, as in this study, provides useful information allowing analysis of therapeutic, rather than preventive, effects that would be difficult to examine using congenitally gene-engineered animal models.
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RESEARCH DESIGN AND METHODS |
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Animals.
Animal studies were conducted under protocols in accordance with the institutional guidelines for animal experiments at Tohoku University. Male C57BL/6N mice were housed individually and divided into high-fat diet (32% safflower oil, 33.1% casein, 17.6% sucrose, and 5.6% cellulose [14]) and standard diet (65% carbohydrate, 4% fat, and 24% protein) groups at 5 weeks of age, when body weights were 21.2 ± 0.25 g (means ± SE). Four weeks after separation, body weightmatched mice for each group received an injection of adenovirus via the tail vein. Viruses were administered intravenously at a dose of 2 x 108 plaque-forming units. For pair-feeding experiments, after 4 weeks of high-fat diet, mice were allotted into three groups. Two groups of mice received an injection of UCP1 or LacZ adenovirus. After 24 h, mice in the third group received an injection of LacZ adenovirus. The latter LacZ mice were given their daily food allotments on the basis of the previous days consumption by UCP1 mice.
Antibodies.
UCP1, acetyl-CoA carboxylase 1 (ACC 1), and insulin receptor antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). The -subunit of AMP-activated protein kinase (AMPK), phospho-AMPK (Thr172), and phospho-ACC (Ser79) antibodies were purchased from Cell Signaling Technology (Beverly, MA). Affinity-purified antibody against insulin receptor substrate 1 (IRS1) was prepared as described previously (15).
Immunoblotting.
Tissue samples were prepared as previously described (9), and tissue protein extracts (250 µg of total protein) were boiled in Laemmli buffer that contained 10 mmol/l dithiothreitol and subjected to SDS-PAGE. The immunoblots were visualized with an enhanced chemiluminescence detection kit (Amersham, Buckinghamshire, U.K.).
Triglyceride content of the liver.
Frozen livers were homogenized, and triglycerides were extracted with CHCl3:CH3OH (2:1, vol:vol), dried, and resuspended in 2-propanol (16). Triglyceride contents were measured using Lipidos liquid (TOYOBO, Osaka, Japan).
Oxygen consumption.
Oxygen consumption was measured with an O2/CO2 metabolism measuring system (model MK-5000RQ; Muromachikikai, Tokyo, Japan). Each mouse was kept unrestrained in a sealed chamber with an air flow of 0.5 l/min for 5 h at 25°C without food or water during the light cycle. Air was sampled every 3 min, and the consumed oxygen concentration (VO2) was calculated.
Histological analysis.
Livers as well as epididymal fat (white adipose tissue) and brown adipose tissues were removed and fixed with 10% formalin and embedded in paraffin. Tissue sections were stained with hematoxylin and eosin. Total adipocyte areas were traced manually and analyzed. Brown and white adipocyte areas were measured in 100 or more cells per mouse in each group.
Measurement of body temperature.
Rectal temperature was measured with a Thermalert TH-5 (Physitemp, Clifton, NJ).
Measurement of ATP.
The ATP levels in liver homogenates were measured with a luciferase-luciferin system (17) by using an ATP determination kit (Molecular Probes, Eugene, OR).
Measurement of AMPK activity.
Livers were homogenized, and aliquots of supernatant were incubated with anti-AMPK -subunit antibody. AMPK activity in the immunoprecipitates was assessed as a function of SAMS peptide phosphorylation, as previously described (18).
Tyrosine phosphorylation of insulin receptor and IRS1.
Mice that were fasted for 16 h received an injection of 100 µl of normal saline (0.9% NaCl), with or without 10 units/kg body wt insulin, via the tail vein. Hindlimb muscles were removed 300 s later and immediately homogenated. After centrifugation, the resultant supernatants were used for immunoprecipitation with antiinsulin receptor or anti-IRS1 antibody. Immunoprecipitates were subjected to SDS-PAGE and then immunoblotted using anti-phosphotyrosine antibody (4G10) or individual antibodies as described previously (15).
Blood analysis.
Blood glucose was assayed with Antsense II (Horiba Industry, Kyoto, Japan). Serum insulin and leptin were determined with ELISA kits (Morinaga Institute of Biological Science, Yokohama, Japan). Serum adiponectin and tumor necrosis factor- (TNF-
) concentrations were measured with an ELISA kit (Ohtsuka Pharmaceutical, Tokyo, Japan) and a TNF-
assay kit (Amersham Biosciences, Uppsala, Sweden), respectively. Serum total cholesterol, triglyceride, and free fatty acid concentrations were determined with a Cholescolor liquid, Lipidos liquid (TOYOBO), and NEFA C (Wako Pure Chemical, Osaka, Japan) kits, respectively.
Glucose, insulin, and leptin tolerance tests.
Glucose tolerance tests were performed on fasted (10 h) mice. Mice were given oral glucose (2 g/kg body wt), and blood glucose was assayed immediately before and at 15, 30, 60, and 120 min after administration. Insulin tolerance tests were performed on fed mice. Mice received an injection of human regular insulin (0.75 units/kg body wt; Eli Lilly, Kobe, Japan) into the intraperitoneal space, and blood glucose was assayed immediately before and at 20, 40, 60, and 80 min after injection. Leptin tolerance tests were performed as reported previously (19) with slight modification. Fasted (12 h) mice received an injection of mouse leptin (7.2 mg/kg body wt; R&D Systems) into the intraperitoneal space, and food intake amounts for 12 h thereafter were determined. Ratios of food intake amounts to those of vehicle-injected mice were calculated.
Quantitative RT-PCRbased gene expression.
Total RNA was isolated from 0.1 g of mouse hepatic tissue with ISOGEN (Wako Pure Chemical), and cDNA synthesis was performed with a Cloned AMV First Strand Synthesis Kit (Invitrogen, Rockville, MD) using 5 µg of total RNA. cDNA synthesized from total RNA was evaluated in a real-time PCR quantitative system (Light Cycler Quick System 350S; Roche Diagnostics, Mannheim, Germany). The relative amount of mRNA was calculated with glyceraldehyde-3-dehydrogenase mRNA as the invariant control. The primers used are described in Table 1.
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RESULTS |
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Resting oxygen consumption on day 3 was markedly increased, by 12%, in UCP1 mice compared with controls (Fig. 1D), whereas rectal temperature did not differ between the two (Fig. 1E). Thus, ectopic UCP1 in the liver, like endogenous UCP1 in brown adipocytes, promoted inefficient metabolism, thereby enhancing energy expenditure and leading to weight reduction. This effect, however, was not sufficient to raise whole-body temperature. In addition, hepatic UCP1 expression changed food intake. Whereas without hepatic UCP1 expression, food intake amounts in high-fatfed mice were markedly increased compared with those in standard dietfed lean mice (compare Figs. 1F and 5D), hepatic UCP1 expression reversed hyperphagia in mice with high-fat dietinduced obesity and diabetes (Fig. 1F). After day 8, concomitantly with the drop in hepatic UCP1 expression, hyperphagia was restored (Fig. 1F). In contrast, mice received an intravenous injection of adenovirus encoding CPT1a, another mitochondrial protein, did not show significantly altered food consumption (data not shown), suggesting that food intake suppression induced by hepatic UCP1 expression is not a nonspecific effect of expression of any of the hepatic mitochondrial proteins.
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Hepatic UCP1 expression decreased fat contents in the liver and adipose tissues.
Hepatic and adipose fat accumulations were examined on day 7 after adenoviral gene delivery. In the high-fatfed control mice, liver weight and triglyceride content were markedly increased compared with the standard chowfed lean mice (compare Fig. 2A and B with Fig. 5E and F, respectively). Hepatic UCP1 expression significantly decreased liver weight (Fig. 2A) and triglyceride content (Fig. 2B) compared with LacZ mice, with high-fat feeding. It is interesting that hepatic UCP1 expression also decreased fat content in their adipose tissues. For example, epididymal fat weight was significantly decreased in UCP1 mice compared with that in controls (Fig. 2C). Thus, hepatic expression of UCP1 exerts not only local effects in the liver but also remote effects on metabolism in other tissues.
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Hepatic expressions of enzymes involved in lipid metabolism and glucose production.
To elucidate the underlying mechanism whereby stored fat was decreased in the liver by hepatic UCP1 expression, we examined the expressions of proteins involved in lipid metabolism by quantitative RT-PCR. Significant reductions in the expressions of the lipogenic enzymes, including stearoyl-CoA desaturase-1 and fatty acid synthase, were observed in UCP1 mice (Fig. 3A). Sterol regulatory element binding protein 1c (SREBP1c) expression in the liver tended to be diminished. In contrast, hepatic expressions of enzymes involved in fatty acid oxidation tended to be increased. In particular, expressions of fatty acid transporter and UCP2 were significantly increased (Fig. 3B).
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UCP1 expression may activate AMPK as a result of decreased generation of ATP. AMPK activation reportedly decreases malonyl-CoA generation via inhibition of ACC (21), resulting in enhancement of fatty acid oxidation. Therefore, ATP levels and AMPK phosphorylation in the liver were examined in LacZ and UCP1 mice under ad libitum feeding conditions. Hepatic ATP concentrations in UCP1 mice were approximately half those in control mice (Fig. 3D) but still 2.3-fold those in standard dietfed control mice. Hepatic AMPK activity was increased 1.6-fold in UCP1 mice compared with LacZ mice (Fig. 3E). The phosphorylation state of the
-subunit of AMPK in the liver was enhanced in UCP1 mice (Fig. 3F). Furthermore, resultant enhancement of hepatic ACC phosphorylation was observed (Fig. 3G). These findings suggest that AMPK activation induced by UCP1 expression plays an important role in the observed marked improvement of fatty liver findings via enhanced fatty acid oxidation.
Glucose and lipid metabolism in UCP1 mice.
The results of oral glucose tolerance (Fig. 4A) and insulin tolerance (Fig. 4B) tests on day 7 after adenoviral administration clearly showed that hepatic expression of UCP1 markedly improved glucose tolerance and insulin sensitivity in obese and diabetic mice. Improved insulin sensitivity in muscle was confirmed by enhanced insulin receptor and IRS1 phosphorylation (Fig. 4C) in response to insulin administration. Thus, hepatic UCP1 expression exerts a remote beneficial effect on insulin sensitivity in muscle.
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Serum insulin levels were markedly decreased, by 57% (Fig. 4E), in UCP1 mice, despite lower blood glucose levels (Fig. 1C), indicating marked improvement of systemic insulin sensitivity. Serum adiponectin and TNF- levels were similar in these groups (Fig. 4F), suggesting that these adipocytokines are not involved in the improvement of insulin resistance in UCP1 mice. In contrast, serum leptin levels were significantly decreased, by 56%, in UCP1 mice compared with those in control mice (Fig. 4F) concomitantly with decreased food intake (Fig. 1F). In control mice that were fed a high-fat diet, marked hyperleptinemia was observed (serum leptin concentrations, standard dietfed mice versus high-fat dietfed mice: 0.48 ± 0.08 vs. 32 0.8 ± 4.6 ng/ml) despite increased food intake (compare Fig. 1F with Fig. 5D), indicating leptin resistance. The present results suggest that hepatic UCP1 expression improves hypothalamic leptin resistance in obese and diabetic mice. To directly test whether leptin sensitivity was improved, we performed leptin tolerance tests (Fig. 4G). Leptin was injected intraperitoneally into fasted mice, followed by measurement of 12-h food intakes. The food intake inhibition by leptin administration was far more profound in UCP1 mice than in LacZ mice. Thus, UCP1 mice responded strongly to leptin administration, clearly showing that hepatic UCP1 expression exerts a therapeutic effect on hypothalamic leptin resistance.
Hepatic UCP1 expression exerted minimal effects in standard dietfed lean mice.
Hepatic UCP1 expression reduced body weight and blood glucose and lipid levels in obese and diabetic mice. These are very promising results suggesting that ectopic UCP1 expression may be useful in treating diabetic individuals who are obese. However, if this were also the case in lean individuals, then these individuals would become leaner, possibly even developing malnutrition and hypoglycemia. We therefore performed experiments with a similar design but used 9-week-old standard dietfed lean mice, i.e., the same age as the high-fatfed mice.
It is intriguing that although ectopic UCP1 expression levels in the liver were similar under high-fat and standard diet conditions (Fig. 5A), the resultant phenotypes were completely different. In standard dietfed lean mice, hepatic UCP1 expression did not alter body weight (Fig. 5B), fasting blood glucose levels (Fig. 5C), or food intake amounts (Fig. 5D). In addition, hepatic weight (Fig. 5E), triglyceride content (Fig. 5F), and epididymal fat weight (Fig. 5G) were not changed. Thus, hepatic UCP1 expression did not exert significant effects on glucose metabolism or adiposity in lean mice.
To determine why hepatic UCP1 expression in lean mice did not significantly alter metabolic conditions, we measured basal energy expenditure and hepatic ATP contents. Hepatic UCP1 expression did not significantly change basal energy expenditure (Fig. 5H) or hepatic ATP levels (Fig. 5I), suggesting that UCP1 ectopically expressed in the liver is minimally involved in mitochondrial uncoupling, when surplus energy is not stored in the liver. Thus, hepatic UCP1 is likely to dissipate excess energy while having no effect on required energy. These characteristics are favorable in terms of therapeutic strategies for the metabolic syndrome.
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DISCUSSION |
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How might a change in hepatic lipid metabolism affect the energy balance of the entire body? It is noteworthy that the weight and/or cell sizes of epididymal fat and brown adipose tissues were markedly decreased by hepatic UCP1 expression in the present study. Inhibition of fat accumulation in adipose tissues was also observed in UCP1 and in UCP3 transgenic mice under the control of muscle-specific promoters (7,8). Mice lacking ACC2, which is predominantly expressed in the heart and muscle of wild-type mice, also markedly inhibited fat accumulation in their adipose tissues (24). In reports using transgenic models, muscle is a site of increasing energy expenditure, through mitochondrial uncoupling, which prevents obesity. In the present study, hepatic expression of UCP1 reduced fat contents, rather than inhibiting fat accumulation, not only in the liver but also in adipose tissues, indicating promotion of hydrolysis of triglycerides already stored in the adipose tissues. Thus, hepatic uncoupling is likely to convey signals to peripheral adipose tissues. These signals might involve an autonomic nerve network, because the hydrolysis of triglycerides stored in adipose tissues is controlled mainly by the cAMP-mediated pathway, including sympathetic nerve activation (25). Alternatively, a decline in serum fatty acid concentrations, observed in UCP1 mice, or some unknown factors secreted by the liver might trigger lipolysis in adipose tissues. Although more work is required to elucidate the mechanism underlying this remote effect, enhancement of hepatic uncoupling is likely to exert therapeutic, rather than preventive, effects on insulin resistance associated with obesity. Thus, the liver is a potential therapeutic target for diabetes with obesity. Furthermore, unraveling the underlying mechanism may lead to development of antiobesity pharmacological agents that promote lipolysis in adipose tissues.
The present results are also interesting with respect to appetite regulation. Transgenic mice overexpressing UCP3 in skeletal muscle are reportedly hyperphagic (8), whereas UCP1 transgenic mice show no changes in food intake (7). In these transgenic mice, UCPs are continuously overexpressed throughout life, including in the fetal stage. In contrast, the UCP was expressed after development of diabetes with obesity in the present study. In obese subjects, serum leptin levels are reportedly increased with an increment in adipose tissue mass (26,27). Despite increased serum leptin levels, neither appetite nor food intake was suppressed, but instead increased, which is explained by hypothalamic leptin resistance in obese subjects. Herein, control mice on a high-fat diet were hyperphagic compared with those on a standard diet, whereas serum leptin levels were markedly elevated in high-fat dietfed mice, indicating the development of leptin resistance. It is interesting that hepatic UCP1 expression reversed hyperphagia in high-fat dietfed mice. Leptin tolerance tests show marked improvement of hypothalamic leptin resistance in UCP1 mice, another remote effect of hepatic UCP1 expression. In addition, increased fatty acid oxidation might be involved in the decreased food intake, because administration of peroxisome proliferatoractivated receptor (PPAR)- agonists reportedly reduces food intake amounts, but not in mice deficient in PPAR-
(28). Furthermore, streptozotocin-induced hyperphagia was reportedly reversed by hepatic expression of protein phosphatase-1 (29), suggesting that altering hepatic metabolism modulates appetite. Vagal pathways from the liver to the brain mediate the fat-induced changes in hypothalamic neuropeptides and feeding behavior in diabetic rats (30). Taken together with these observations, through appetite modulation, the liver also holds promise as a target for treatment of diabetes with obesity.
The most intriguing finding of the present study is that, despite similar UCP1 expression levels in mice on high-fat and standard diets, the resultant phenotypes were completely different. Hepatic UCP1 expression exerted no significant effects on food intake, weight change, or blood glucose levels in standard dietfed lean mice. No alterations in energy expenditure or hepatic ATP contents were observed with hepatic UCP1 expression, indicating that, in the absence of a significant energy surplus, ectopic UCP1 has minimal effects on mitochondrial uncoupling. We performed similar experiments in a mildly obese and insulin-resistant model, 15% fat-fed mice. In these mice, hepatic UCP1 expression did not change body weight or food intake. Glucose tolerance and insulin sensitivity were significantly improved, but the effects were smaller (data not shown) than those in a more severely obese and insulin-resistant model, 32% fat-fed mice, reported here. Furthermore, under 32% high-fatfed conditions in the present study, although hepatic UCP1 expression decreased ATP levels in the liver, the reduced ATP concentrations still exceeded those in standard dietfed mice, suggesting that enhanced expression of UCPs in the liver does not itself produce an energy shortage. Taken together, hepatic UCP1 is likely to sense the metabolic state in the liver and function according to the degree of stored energy in the liver. In the reconstituted system, addition of fatty acids is indispensable for proton transport by UCP1 (31,32). Although the underlying mechanism has been widely debated (33,34), fatty acid cycling seems to be important for proton transport by UCP1 (35,36). Via such a mechanism, ectopic UCP1 activity in the liver may depend on the metabolic state, probably on the amount of stored fat in the liver. Thus, hepatic UCP1 seems to dissipate surplus energy but not to affect required energy. Therefore, the liver, in which intracellularly stored fat changes dramatically according to the energy balance, seems to be a good target tissue for enhanced expression of UCPs. This feature is of particular importance, as applied to therapeutic strategies for type 2 diabetes associated with obesity and insulin resistance.
Recently, it was reported that, using a transgenic technique, skeletal muscle expression of UCP1 in genetically obese mice lowers blood pressure (37), suggesting that uncoupling decreases the risk for atherosclerosis in patients with obesity and type 2 diabetes. In addition, uncoupling reportedly decreases the production of reactive oxygen species (38), although total oxygen consumption increases. A high mitochondrial electrochemical gradient is associated with the production of reactive oxygen species that may damage tissues, a possible cause of diabetes complications and atherosclerosis (39). Thus, the respiratory uncoupling increment in the liver may protect tissues from oxidative stress. Taken together with the results of the present study, enhancement of UCPs in the liver is a potential therapy for the metabolic syndrome via reductions in adiposity and blood glucose levels as well as possibly reactive oxygen species in obese and diabetic individuals.
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ACKNOWLEDGMENTS |
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We thank Prof. Y. Moriyama (Okayama University) for helpful suggestions for measuring ATP. We also thank I. Sato and K. Kawamura for technical support.
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FOOTNOTES |
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Address correspondence and reprint requests to Hideki Katagiri, MD, PhD, Division of Advanced and Therapeutics for Metabolic Diseases, Center for TranslationalAdvanced Animal Research, Tohoku University Graduate School of Medicine, 2-1 Seiryo-machi, Aoba-ku, Sendai 980-8575, Japan. E-mail: katagiri-tky{at}umin.ac.jp
Received for publication April 19, 2004 and accepted in revised form October 13, 2004
ACC1, acetyl-CoA carboxylase 1; AMPK, AMP-activated protein kinase; CPT1, carnitine palmitoyltransferase 1; IRS1, insulin receptor substrate 1; PPAR, peroxisome proliferatoractivated receptor; SREBP, sterol regulatory element binding protein; TNF-, tumor necrosis factor-
; UCP, uncoupling protein
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REFERENCES |
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