Uncoupling protein-2 (UCP2) is a mitochondrial membrane transporter expressed in white adipose tissue. We observed that circulating adiponectin levels and adiponectin gene expression in adipose tissue are reduced in UCP2-null mice. We studied whether mitochondrial activity and its control by UCP2 may regulate adiponectin gene expression. In 3T3-L1 cells, increasing UCP2 mitochondrial levels by adenoviral-mediated gene transfer induced adiponectin gene expression, whereas oligomycin and antimycin A, inhibitors of ATP synthesis and mitochondrial respiration, led to a downregulation. Reactive oxygen species (ROS) scavengers alleviated the repression of adiponectin gene expression caused by oligomycin or antimycin A. The action of ROS involves the transcription factor CHOP-10, the abundance of which was reduced in response to UCP2 and was induced by oligomycin. CHOP-10 inhibited adiponectin gene expression by interfering with the −117/−73 CCAAT/enhancer binding protein–binding region in the adiponectin gene promoter. Moreover, CHOP-10 levels were increased in adipose tissue from UCP2-null mice. Results indicate that the modulation of ROS levels by mitochondrial activity, and specifically as a consequence of the action of UCP2, controls adiponectin gene expression. This provides a physiological mechanism by which the adipose tissue energetic status may determine the extent of adiponectin release and influence systemic insulin sensitivity.

In the last decade, new endocrine functions have been discovered for adipose tissue (13), indicating that adipocytes are important regulators of systemic metabolism via the production of multiple proteins (adipokines) and metabolites with powerful signaling capacities in systemic metabolism. Adiponectin is a 30-kDa adipokine produced by adipose tissue (4,5). It contributes to the regulation of glucose and lipid metabolism and is involved in metabolic energy homeostasis. Adiponectin increases glucose uptake and free fatty acid oxidation in muscle and enhances hepatic insulin action. Plasma levels of adiponectin have been found to be decreased in obesity, cardiovascular diseases, hypertension, and metabolic syndrome. Furthermore, adiponectin gene expression and circulating levels of adiponectin are modulated by insulin, tumor necrosis factor-α, β-adrenergic agonists, and interleukin-6 (68). Because of its insulin-sensitizing action, strategies aimed to increase adiponectin levels are among the potential therapeutic approaches for type 2 diabetes, and for instance, part of the insulin-sensitizing action of thiazolidinediones is known to occur via increased adiponectin release by adipose tissue (8). Transcription of the adiponectin gene is under the positive control of peroxisome proliferator–activated receptor-γ (PPARγ) ligands, such as thiazolidinediones, and of transcription factors of the CCAAT/enhancer binding protein (C/EBP) family, which are considered to mediate preferential expression of adiponectin in adipose tissue (911).

Mitochondria are the main cellular sites involved in energy metabolism. ATP is synthesized in mitochondria thanks to the protomotive gradient generated by the oxidation of reduced metabolites and the subsequent pumping of protons across the mitochondrial inner membrane. The gradient is sustained by the intrinsically low permeability of the mitochondrial inner membrane. However, this low permeability and the resultant efficiency in the transduction of the energy released because of oxidation into ATP synthesis can be modulated by the activity of uncoupling proteins. Members of this family of mitochondrial proteins are known to permeabilize the inner mitochondrial membrane to protons, thus lowering the protomotive gradient and uncoupling ATP synthesis from respiration. Uncoupling protein-2 (UCP2) is expressed to differing extents in several mammalian tissues, including white adipose tissue (12). The function of UCP2 was initially thought to be related to energy metabolism. The UCP2 gene is located within a region where quantitative trait loci for obesity and type 2 diabetes are present (13), and several UCP2 gene polymorphisms were linked to increased BMI in Pima Indians (14) and other populations (15), although this has not been confirmed by other studies (16). The association of UCP2 gene polymorphisms with insulin resistance and type 2 diabetes has also been reported in several studies (15,1719).

Experimental studies have demonstrated the involvement of UCP2 in physiological processes other than the control of energy balance. UCP2-null mice did not show major changes in body weight, thermogenesis, or resistance to increased body weight in response to a high-fat diet (20,21), and the main phenotype of these mice was related to increased insulin production in response to glucose (21) as well as resistance to infections and inflammatory processes (20). Enhanced insulin secretion in UCP2-null mice appears to be due to a reduced ATP-to-ADP ratio in β-cells, whereas the effects on inflammation and responsiveness to infections have been associated with enhanced reactive oxygen species (ROS) production in macrophage mitochondria due to the lack of UCP2. This is in agreement with the control of ROS production by mitochondria as a major biochemical consequence of the mildly uncoupled status of mitochondria elicited by UCP2 (2224). The potential activity of UCP2 in controlling ROS production may have far-reaching functional consequences in cells because ROS are gaining recognition as intracellular signaling molecules mediating cross-talk between mitochondrial activity and overall gene expression (25). Moreover, changes in mitochondrial activity and UCP2 levels in adipose tissue may have consequences beyond intracellular processes and may influence systemic metabolism via modifications in the release of adipokines by adipose tissue. In the present study, we show that UCP2 and mitochondrial function regulate adiponectin release and adiponectin gene expression via modifications in intracellular ROS production.

The care and use of mice were in accordance with the European Community Council Directive 86/609/EEC and approved by the Experimental Animal Ethics Committee, University of Barcelona. Seven- to 10-week-old UCP2-null male mice on the C57BL/6J genetic background (26) and control littermates were used. Fed mice were killed in the morning, at the beginning of the light cycle, by cervical dislocation, and inguinal adipose tissue and blood were collected. Serum was prepared, and adipose tissue was frozen in liquid nitrogen. Serum metabolites were quantified using a reflection photometry-based automatic system (Accutrend; Roche).

Cell culture and treatment.

Medium, antibiotics, and serum were from GIBCO. The 3T3-L1 cells were grown in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% FCS and 5% penicillin/streptomycin (10,000 units/ml), at 37°C in 5% CO2. Adipogenic differentiation of 3T3-L1 cells was achieved by the use of DMEM containing 10% FCS, 5 μg/ml insulin, and 125 μmol/l indomethacin, a method described to enhance endocrine capacities of 3T3-L1 cells (27). After 48 h, cells were washed with PBS, and insulin treatment was maintained for an additional 48 h to achieve differentiation. Oligomycin, antimycin A, parthenolide, AICAR (5-aminoimidazole-4-carboxamide-1-b-riboside) (Sigma), H89, and Trolox (Calbiochem) were dissolved in ethanol. The ROS-sensitive fluorescent probe 6-carboxy-2′,7′-dichlorodihydrofluorescein diacetate, diacetoxymethyl-ester (H2-DCFDA) (Molecular Probes) was prepared in DMSO. The final concentration of ethanol or DMSO was kept <0.1%, and incubation with vehicle was performed as control.

Adenoviral UCP2 gene transfer.

AdCMV-UCP2 adenovirus driving human UCP2 expression (28) was provided by Dr. I.K. Lee (Keimyung University, Daegu, Korea). AdCMV-βGal expressing the Escherichia coli β-galactosidase gene was used as control. 3T3-L1 confluent cells were infected with adenovirus (either AdCMV-UCP2 or the control AdCMV-βGal) at a multiplicity of infection (MOI) of 50, for 2 h in the presence of polylysine at 0.5 μg/ml. This treatment led to an efficiency of transduction of ∼90%, as assessed in previous experiments analyzing fluorescence driven by AdCMV-GFP transduction in these same conditions. Cells were studied 3 days after transduction.

Quantification of adiponectin protein levels.

Adiponectin protein levels were determined in 3T3-L1 culture medium and in mice serum. After overnight serum deprivation, 3T3-L1 cells were incubated in fresh serum-free medium. At the end of the experiment, adiponectin release in cell culture medium was measured. Assays were performed using a Mouse/Rat Adiponectin ELISA Kit (B-Bridge International). Absorbance was detected at 450 nm using an automatic ELISA plate reader (Bio-Rad Benchmark Plus).

Nuclear and mitochondrial extract preparation.

Nuclear extracts were prepared as reported elsewhere (29). The 3T3-L1 cells were washed twice with ice-cold PBS and scraped off the plates in buffer containing 10 mmol/l HEPES, pH 7.9, 10 mmol/l KCl, 0.1 mmol/l EDTA, 0.1 mmol/l EGTA, 1 mmol/l dithiothreitol, 0.5 mmol/l phenylmethylsulfonyl fluoride (PMSF), and protease inhibitors (5 mg/ml each of aprotinin, leupeptin, and pepstatin). Cells were allowed to swell on ice for 15 min; then 25 μl Nonidet P-40 (0.5%) was added, and the suspension was mixed for 10 s. The homogenate was centrifuged (16,000 × g, 4°C, 60 s), and the nuclear pellet was resuspended in 50 μl ice-cold buffer containing 20 mmol/l HEPES, pH 7.9, 0.4 mol/l NaCl, 1 mmol/l EDTA, 1 mmol/l EGTA, 1 mmol/l dithiothreitol, 1 mmol/l PMSF, and protease inhibitors. The nuclear extract was cleared (16,000 × g, 4°C, 5 min).

Mitochondrial proteins were isolated from 3T3-L1 cells as described previously (30). Cells were homogenized in a buffer containing 220 mmol/l mannitol, 70 mmol/l sucrose, 5 mmol/l MOPS, 2 mmol/l EGTA, and 0.2% BSA, pH 7.4. The homogenates were centrifuged for 10 min at 500 × g (4°C). The pellet was homogenized and centrifuged as above. Supernatants were mixed and then centrifuged at 10,000 × g (10 min, 4°C). The resulting pellet was washed twice in the same buffer and centrifuged at 5,000 × g (10 min, 4°C). Finally, the pellet was resuspended, and protein concentrations were determined by the Bradford assay (Bio-Rad).

CHOP-10 and UCP2 immunoblot analysis.

Samples containing 40 μg cellular or mitochondrial protein or nuclear proteins after immunoprecipitation with C/EBPβ antibody (2-h incubation; SC-150; Santa Cruz) or 100 μg protein from adipose tissue were mixed with equal volumes of 2× SDS loading buffer, incubated at 90°C for 5 min, and electrophoresed on SDS/12% polyacrylamide gels. Proteins were transferred to Immobilon-P membranes (Millipore), and immunological detection was performed using rabbit or goat affinity-pure IgG against CHOP-10 or UCP2, respectively (SC-575 and SC-6526; Santa Cruz). Detection was achieved with horseradish peroxidase–coupled anti-rabbit and anti-goat (SC-2004 and SC-2354; Santa Cruz) antibodies and an enhanced chemiluminescence detection kit (Amersham). Mitochondrial extracts from HeLa cells previously transfected with a plasmid expression vector driving UCP2 cDNA were used as positive control for UCP2 detection. The sizes of proteins were estimated using protein molecular-mass standards (Bio-Rad).

RNA isolation and quantitative real-time PCR analyses.

RNA was prepared from frozen aliquots of adipose tissue or 3T3-L1 cells (RNeasy; Qiagen). Quantitative mRNA expression analysis was performed using TaqMan real-time RT-PCR (Applied Biosystems). The reverse transcriptase reaction was performed on 0.5 μg RNA using TaqMan standardized reagents, and the real-time PCR reaction was performed using TaqMan Universal PCR Master Mix and standardized gene expression primer probes (“Assay-on-Demand”): adiponectin (Mm00456425), glutathione peroxidase-1 (Mm00656767), superoxide dismutase-2 (Mm00449726), and PPARγ (Mm00440945). The amount of mRNA for the gene of interest in each sample was normalized to that of the housekeeping reference 18S rRNA (Hs99999901). Samples were run in duplicate on the ABI-PRISM 7700HT sequence detection system (Applied Biosystems).

Determination of intracellular ROS.

The H2-DCFDA probe was used to estimate intracellular ROS (31). After incubation with 10 mmol/l H2-DCFDA for 30 min, 3T3-L1 cells were washed twice with ice-cold PBS and scraped on ice into water. After sonication, fluorescence was measured at 527 nm after excitation at 493 nm. Fluorescence data were normalized relative to protein concentrations in the cell lysates.

Electrophoretic mobility shift assay.

A double-stranded oligonucleotide containing the −117/−73 fragment of the mouse adiponectin promoter (5′-GCCCACTCATTGGCTATTGGCCTTGACTGGGTT GGCCAATGGTAA G-3′; Roche) was end-labeled using [α-32P]dCTP and Klenow enzyme. The DNA probe (30,000 cpm) was added to 10 μg nuclear protein extract mixture and incubated for 20 min at room temperature in binding buffer (25 mmol/l HEPES, pH 7.6, 0.5 mmol/l dithiothreitol, 12.5 mmol/l ZnSO4, 50 mmol/l KCl, 1 mg/ml BSA, 5% glycerol, 0.1% Nonidet P-40, and 2.5 μg poly[dI-dC]). Specific binding was assessed by competition tests; preincubating samples with a 100-fold molar excess of unlabeled doubled-stranded oligonucleotide. Samples were run on 5% nondenaturing polyacrylamide gels in 0.5× Tris-borate-EDTA at 130 V and 4°C for 60 min. Specificity of the C/EBPβ binding was further assessed by inducing a super-shifted band after incubating nuclear extracts with anti-C/EBPβ antibody (SC-150; Santa Cruz) for 30 min at room temperature.

Plasmid constructs and transfection assays.

A double-stranded oligonucleotide corresponding to the −117/−73 region of the mouse adiponectin promoter known to contain the C/EBPβ-response element (10) was inserted, using the HindIII and XhoI sites, upstream of the herpes simplex virus thymidine kinase basal promoter driving firefly luciferase expression (Tk109-Luc vector). This construct was transfected into confluent 3T3-L1 cells using Fugene reagent (Roche). PRL-TK (Promega), an expression vector of Renilla luciferase, was co-transfected. Luciferase activities were measured in a Turner-Designs luminometer (TD20/20) using the Dual-Luciferase Reporter kit (Promega). Luciferase activity elicited by the −117/−73 TK-Luc fragment construct was normalized for variation in transfection efficiency using Renilla luciferase. The CHOP-10 expression vector SH-GADD153 was provided by S. Ambrosio (University of Barcelona, Barcelona, Spain).

Statistical analysis.

Data, shown as means ± SE, were analyzed with Student's t test, and differences were considered to be significant at a P value <0.05.

Circulating adiponectin levels and adipose tissue adiponectin gene expression are downregulated in UCP2-null mice.

In a first attempt to determine whether changes in respiratory chain regulation elicited by mitochondrial uncoupling could be involved in the biological signals produced by adipose tissue, we measured the levels of adiponectin and metabolites in serum from control and UCP2-null mice. Table 1 shows that glucose and lactate levels were significantly lowered in UCP2-null mice, whereas triglyceride levels were not significantly altered. Overall, this pattern of circulating metabolites is in accordance with previous reports (20,21,32). Table 1 also shows that adiponectin levels in the serum of UCP2-null mice are significantly reduced with respect to control mice. Adiponectin mRNA levels were dramatically downregulated in adipose tissue from UCP2-null mice (a 65% reduction vs. controls) (Table 2). This suggested that the reduced serum adiponectin levels were caused by impaired adiponectin gene expression in adipose tissue due to the lack of UCP2. The reduction in adiponectin mRNA levels was not shared by other genes, and for instance, SOD2 mRNA levels were increased in UCP2-null mice (Table 2). Upregulation of the SOD2 gene suggests the occurrence of altered oxidative status in adipose tissues from UCP2-null mice. PPARγ mRNA levels were not significantly affected in UCP2-null mice.

Effects of UCP2, oligomycin, and antimycin A treatments on adiponectin production by 3T3-L1 cells.

To determine whether mitochondrial respiratory function and specifically UCP2 influence adiponectin production, adipogenic 3T3-L1 cells were used. Cells were transduced with an adenoviral vector driving UCP2 expression (AdCMV-UCP2) in conditions leading to an approximate twofold increase (2.16 ± 0.6–fold) in UCP2 protein levels in mitochondrial protein extracts, as assessed by immunoblot assays (Fig. 1A). The increase in UCP2 levels led to a rise in adiponectin mRNA levels in 3T3-L1 cells with respect to controls (Fig. 1B) and a significant increase in the release of adiponectin protein to the culture medium (1.44 ± 0.16–fold induction, P < 0.05). To ascertain the biochemical basis of the UCP2 effects, parallel assays were performed exposing 3T3-L1 cells to oligomycin or antimycin A, inhibitors of ATP synthase and respiratory chain complex III, respectively. These drugs are known to reduce ATP synthesis, as UCP2 does in 3T3-L1 adipocytes (33), but in this case, this is associated with an inhibition of the respiratory chain activity, in contrast with the action of mitochondrial uncoupling that increases respiration. Oligomycin and antimycin A produced effects opposite those of UCP2, leading to a significant reduction in adiponectin mRNA levels (Fig. 1B). Adiponectin protein release was downregulated in parallel (0.62 ± 0.05–fold decrease in oligomycin-treated cells, P < 0.05). These results indicated that a blockage of mitochondrial respiratory function represses adiponectin gene expression, whereas the mitochondrial modifications elicited by UCP2 enhance this expression. To ascertain the potential mechanisms underlying the observed effects, 3T3-L1 cells were incubated in the presence of inhibitors or activators of different signaling pathways potentially related to mitochondrial activity. Oligomycin or antimycin A treatment are expected to increase AMP levels, and therefore it could be hypothesized that AMP kinase activation could be involved in the regulation of adiponectin gene expression. However, Fig. 1C shows that AICAR, a drug that mimics AMP kinase, did not affect adiponectin mRNA levels, thus indicating that disturbances in the ATP/ADP/AMP relationship were not the main determinants of changes in adiponectin gene expression in response to altered mitochondrial respiratory function. Furthermore, when protein kinase A was inhibited by H89, no significant changes in the oligomycin effects on adiponectin mRNA levels were observed (data not shown), suggesting that changes in cAMP levels are not involved in the regulation of adiponectin mRNA expression. Treatment of 3T3-L1 cells with Trolox, an ROS scavenging drug, did not modify basal levels of adiponectin mRNA but alleviated oligomycin-dependent and antimycin A-dependent reduction in adiponectin mRNA expression (Fig. 1C). This suggests that a rise in ROS elicited by oligomycin treatment could mediate the downregulation of adiponectin gene expression. This would explain the upregulation of adiponectin mRNA levels due to UCP2, considering its known capacity to reduce mitochondrial ROS levels. Thus, a major role for mitochondrial ROS production in the control of adiponectin gene expression would explain the opposite effects of UCP2 and oligomycin.

Involvement of ROS and CHOP-10 in the control of adiponectin gene expression.

To check directly whether UCP2, oligomycin, or antimycin A treatment modulates ROS production in accordance with the above hypothesis, we directly measured ROS levels in 3T3-L1 cells infected or not with AdCMV-UCP2 and treated or not with oligomycin or antimycin A. Figure 2 shows that in our conditions, UCP2 transduction induced a significant reduction in ROS levels in 3T3-L1 cells, whereas oligomycin and antimycin A caused a significant increase in ROS levels. The treatment with Trolox described above did not modify basal ROS levels but suppressed the rise in ROS caused by oligomycin or antimycin A (Fig. 2). These results are consistent with the proposal of ROS production as a main signal of mitochondrial origin controlling adiponectin gene expression and with the action of UCP2 upregulating adiponectin gene expression via a reduction in mitochondrial ROS production. A potential mechanism of adiponectin gene regulation could be the activation of the nuclear factor-κB (NF-κB) system, because the absence of UCP2 has been reported to increase NF-κB activation and cytokine release in macrophages (34). However, treatment of 3T3-L1 cells with parthenolide, which specifically inhibits NF-κB activation by preventing inhibitor of κB degradation (35), did not prevent either the oligomycin-dependent or the antimycin-dependent repression of adiponectin gene expression (Fig. 1C), thus indicating that mitochondrial activity impairment does not mediate adiponectin gene regulation via NF-κB activation

CHOP-10 transcription factor (also known as GADD153) is an inactive analog of C/EBP proteins, known to be induced by ROS and mediating some of the ROS-dependent effects on gene transcription (3638). Because it has been demonstrated that the adiponectin gene promoter is regulated by C/EBPβ (10), we hypothesized that CHOP-10 activation could be a potential mechanism providing the signaling pathway linking ROS to adiponectin gene expression. To verify this possibility, we first determined CHOP-10 protein levels in nuclear extracts of 3T3-L1 after UCP2 transduction or oligomycin treatment. Figure 3A shows that CHOP-10 protein levels were decreased significantly in nuclear extracts from cells infected with AdCMV-UCP2 with respect to controls, whereas CHOP-10 levels were significantly increased with oligomycin treatment. This was fully consistent with variations in ROS production described above.

We next determined how the changes in CHOP-10 elicited as a consequence of mitochondrial activity influence the amounts of CHOP-10–C/EBPβ inactive heterodimers. Nuclear extracts from 3T3-L1 cells incubated or not with oligomycin were immunoprecipitated using an anti-C/EBPβ antibody, and the amount of CHOP-10 protein in the precipitate was immunodetected using an anti-CHOP-10 antibody. Results indicated that oligomycin treatment lead to a significant increase of CHOP-10–C/EBPβ inactive heterodimers in 3T3-L1 cells relative to untreated controls (Fig. 3C). This confirms that CHOP-10 protein variations result in parallel changes in the amounts of CHOP-10–C/EBPβ inactive heterodimers.

At this point, and to further establish whether the downregulation in adiponectin gene expression observed in UCP2-null mice may be associated with changes in CHOP-10 expression, immunoblot analysis was performed using adipose tissue protein extracts from wild-type and UCP2-null mice. The results indicated significantly higher levels of CHOP-10 protein in adipose tissue from UCP2-null mice (Fig. 4).

Regulation of adiponectin gene transcription.

To establish whether changes in mitochondrial activity and formation of CHOP-10/CEBPβ heterodimers have consequences on C/EBPβ binding to the adiponectin promoter, electrophoretic mobility shift assays were performed using as probe the −117/−73 fragment of the adiponectin promoter, containing the C/EBPβ binding site (10), and nuclear protein extracts from 3T3-L1 cells after UCP2 transduction or oligomycin treatment. Results indicated that C/EBPβ binding to the −117/−73 region was significantly increased (1.33 ± 0.10–fold, P < 0.05) in extracts from UCP2-transduced cells, whereas it was reduced (0.53 ± 0.15–fold, P < 0.05) using extracts from oligomycin-treated cells (Fig. 5).

Finally, to establish the involvement of CHOP-10 in the regulation of the adiponectin promoter in response to ROS production and UCP2 activity, transient transfection assays were performed using a luciferase reporter construct driven by the −117/−73 fragment of mouse adiponectin promoter. Transduction of 3T3-L1 cells with UCP2 led to a dramatic increase in luciferase activity driven by the −117/−73 adiponectin gene element, whereas it did not affect the promoter activity of the control construct (Fig. 6). Oligomycin treatment had opposite effects and led to a significant reduction in −117/−73 TK-Luc activity (0.38 ± 0.11 in oligomycin-treated cells, P < 0.05) but not in the parental TK-Luc construct. The effects of overexpressing CHOP-10 via co-transfection with a CHOP-10 expression vector were analyzed. Results showed that CHOP-10 co-transfection suppressed the capacity of UCP2 to upregulate the −117/−73 adiponectin TK-Luc construct (Fig. 6). These results indicated that UCP2 effects leading to adiponectin gene upregulation are mediated via the −117/−73 element in the adiponectin promoter region and that CHOP-10 downregulation is involved in this effect.

The influence of adipose tissue upon systemic metabolism, including the control of glycemia and insulin sensitivity, has been recognized in recent years. Adiponectin is an insulin-sensitizing molecule released by adipose tissue, and the identification of the mechanisms of control of adiponectin gene expression and release are of utmost relevance for type 2 diabetes control and prospective therapies. In the present study, we established that the action of mitochondrial activity, and specifically of UCP2, on ROS production has a pivotal influence on adiponectin gene expression. Changes in mitochondrial activity and the action of UCP2 occur via changes in ROS levels and involve changes in the levels of CHOP-10, which acts as a negative regulator of adiponectin gene transcription by interfering with the C/EBP-binding site in the promoter. These findings are consistent with reports indicating that the intracellular status of adipocytes in relation to ROS influences adiponectin gene expression (39) and with the reported capacity of CHOP-10 to convey ROS signaling into overall regulation of gene expression in adipocytes (38).

Accumulating pieces of evidence have underlined the importance of mitochondrial activity in muscle and also in adipose tissue for insulin sensitivity. For instance, adipose tissue is the main target of thiazolidinediones, and the insulin-sensitizing effects of these drugs involve the induction of gene expression of mitochondrial proteins in the tissue (40), including UCP2 (41,42), as well as the induction of adiponectin gene expression (9). Although thiazolidinediones may favor adiponectin gene expression via direct activation of PPARγ-responsive elements in the adiponectin gene, the parallel induction of adiponectin and UCP2 gene expression in adipose tissue after thiazolidinedione treatment is consistent with a role of UCP2 in inducing adiponectin gene expression, as shown here. On the other hand, several experimental or clinical insulin-resistance states associated with low adiponectin levels and reduced UCP2 expression in adipose tissue have been reported. For instance, lipodystrophy in HIV-infected, antiretroviral-treated patients is associated with systemic insulin resistance, mitochondrial impairment in adipose tissue, including UCP2 downregulation, and reduced adiponectin in plasma and adipose tissue (43,44). Abnormal downregulation of adiponectin and UCP2 gene expression has been reported in adipose tissue from first-degree relatives of type 2 diabetic patients (45,46), whereas insulin sensitization in 11β-hydroxysteroid dehydrogenase type 1–deficient mice or in Crebbp heterozygous mice is associated with parallel induction of adiponectin and UCP2 mRNA levels in adipose tissue (47,48).

The involvement of UCP2 in the control of insulin/glucose homeostasis was first recognized from studies in pancreatic β-cells from UCP2-null mice, and UCP2 was identified as a negative regulator of insulin release (21), although there have been contradictory reports based on distinct experimental approaches (49). Our present findings indicate that UCP2 levels in adipose tissue are positive inducers of adiponectin gene expression and therefore may favor insulin sensitivity. This may help to explain the mild reduction in glycemia that, despite hyperinsulinemia (21), occurs in UCP2-null mice. On the other hand, insulin has been reported to repress adiponectin gene expression (8), and therefore high insulin levels may contribute to the adiponectin downregulation in UCP2-null mice. Further research will be required to establish whether the regulatory pathways of adiponectin gene regulation described here, involving ROS-dependent pathways under the control of UCP2, are related to the intracellular mechanisms of insulin action on the adiponectin gene.

In summary, we report here a novel pathway of regulation of adiponectin gene expression mediated by mitochondrial activity and specifically by mitochondrial UCP2 levels. This finding supports the notion that mitochondrial activity and ROS production in adipose tissue are important events influencing overall insulin sensitivity and systemic metabolism, via the control of adiponectin expression and release.

FIG. 1.

Effects of UCP2 and inhibitors of mitochondrial function on adiponectin gene expression in 3T3-L1 cells. A: 3T3-L1 cells were infected with control AdCMV-βGal (MOI = 50) or AdCMV-UCP2 (MOI = 50) in the presence of 0.5 μg/ml polylysine. Representative immunoblot analysis of UCP2 content after transduction with AdCMV-UCP2 (40 μg mitochondrial protein/lane). Lane C is a positive control of 5 μg mitochondrial protein from HeLa cells transiently transfected with a plasmid vector driving UCP2 cDNA. B: 3T3-L1 cells were incubated for 6 h with 5 μg/ml oligomycin or 10 μmol/l antimycin A. C: 3T3-L1 cells were incubated with 5 μg/ml oligomycin, 10 μmol/l antimycin A, 0.5 mmol/l AICAR, 5 mmol/l Trolox, or 10 μmol/l parthenolide at the indicated combinations. Adiponectin mRNA levels were measured by TaqMan RT-PCR on total RNA. Values are means ± SE of the fold change with respect to controls from four different experiments. *P < 0.05.

FIG. 1.

Effects of UCP2 and inhibitors of mitochondrial function on adiponectin gene expression in 3T3-L1 cells. A: 3T3-L1 cells were infected with control AdCMV-βGal (MOI = 50) or AdCMV-UCP2 (MOI = 50) in the presence of 0.5 μg/ml polylysine. Representative immunoblot analysis of UCP2 content after transduction with AdCMV-UCP2 (40 μg mitochondrial protein/lane). Lane C is a positive control of 5 μg mitochondrial protein from HeLa cells transiently transfected with a plasmid vector driving UCP2 cDNA. B: 3T3-L1 cells were incubated for 6 h with 5 μg/ml oligomycin or 10 μmol/l antimycin A. C: 3T3-L1 cells were incubated with 5 μg/ml oligomycin, 10 μmol/l antimycin A, 0.5 mmol/l AICAR, 5 mmol/l Trolox, or 10 μmol/l parthenolide at the indicated combinations. Adiponectin mRNA levels were measured by TaqMan RT-PCR on total RNA. Values are means ± SE of the fold change with respect to controls from four different experiments. *P < 0.05.

FIG. 2.

Effects of UCP2, oligomycin, and antimycin A on ROS levels in 3T3-L1 cells. Cells were infected with control AdCMV-βGal (MOI = 50) or AdCMV-UCP2 (MOI = 50) or incubated with 5 μg/ml oligomycin or 10 μmol/l antimycin A for 90 min. Values are means ± SE of the fold change with respect to controls from three different experiments. *P < 0.05.

FIG. 2.

Effects of UCP2, oligomycin, and antimycin A on ROS levels in 3T3-L1 cells. Cells were infected with control AdCMV-βGal (MOI = 50) or AdCMV-UCP2 (MOI = 50) or incubated with 5 μg/ml oligomycin or 10 μmol/l antimycin A for 90 min. Values are means ± SE of the fold change with respect to controls from three different experiments. *P < 0.05.

FIG. 3.

Effects of UCP2 and oligomycin on CHOP-10 protein levels and on CHOP-10–C/EBPβ heterodimer abundance in 3T3-L1 cells. Cells were treated as in Fig. 2. CHOP-10 protein levels were measured by immunoblotting on 40 μg nuclear extract protein (A) or on 40 μg nuclear extract protein after immunoprecipitation with anti-C/EBPβ antibody (SC-150; Santa Cruz) (C). The relative intensity of the CHOP-10 band was determined by densitometric analysis. Results are means ± SE of the fold change with respect to controls from three different experiments. *P < 0.05. B and D are representative examples of immunoblot and co-immunoprecipitation assays, respectively.

FIG. 3.

Effects of UCP2 and oligomycin on CHOP-10 protein levels and on CHOP-10–C/EBPβ heterodimer abundance in 3T3-L1 cells. Cells were treated as in Fig. 2. CHOP-10 protein levels were measured by immunoblotting on 40 μg nuclear extract protein (A) or on 40 μg nuclear extract protein after immunoprecipitation with anti-C/EBPβ antibody (SC-150; Santa Cruz) (C). The relative intensity of the CHOP-10 band was determined by densitometric analysis. Results are means ± SE of the fold change with respect to controls from three different experiments. *P < 0.05. B and D are representative examples of immunoblot and co-immunoprecipitation assays, respectively.

FIG. 4.

CHOP-10 levels in adipose tissue from UCP2-null mice. Immunoblotting assays were performed on 100 μg tissue protein extracts. The relative intensity of the CHOP-10 band was determined by densitometric analysis. Results are means ± SE of the relative intensity of the immunodetected band in adipose tissue samples from five wild-type and five UCP2-null mice. *P < 0.05 (top panel). Example of immunoblot assay (bottom panel).

FIG. 4.

CHOP-10 levels in adipose tissue from UCP2-null mice. Immunoblotting assays were performed on 100 μg tissue protein extracts. The relative intensity of the CHOP-10 band was determined by densitometric analysis. Results are means ± SE of the relative intensity of the immunodetected band in adipose tissue samples from five wild-type and five UCP2-null mice. *P < 0.05 (top panel). Example of immunoblot assay (bottom panel).

FIG. 5.

Effects of UCP2 and oligomycin on C/EBPβ binding to the −117/−73 adiponectin promoter region. 3T3-L1 cells were treated as in Fig. 3. C/EBPβ binding was evaluated by electrophoretic mobility shift assay using 32P-labeled double-stranded oligonucleotide corresponding to the −117/−73 region of the mouse adiponectin gene and 40 μg nuclear protein extracts from 3T3-L1 cells. A: Results are means ± SE of the fold change in the intensity (using densitometric analysis) of the C/EBPβ shifted band. Values are means of the fold change with respect to controls from seven different experiments. *P < 0.05, **P < 0.01. B: Example of electrophoretic mobility shift assay. C+ ×100, control extracts plus 100-fold excess unlabeled oligonucleotide.

FIG. 5.

Effects of UCP2 and oligomycin on C/EBPβ binding to the −117/−73 adiponectin promoter region. 3T3-L1 cells were treated as in Fig. 3. C/EBPβ binding was evaluated by electrophoretic mobility shift assay using 32P-labeled double-stranded oligonucleotide corresponding to the −117/−73 region of the mouse adiponectin gene and 40 μg nuclear protein extracts from 3T3-L1 cells. A: Results are means ± SE of the fold change in the intensity (using densitometric analysis) of the C/EBPβ shifted band. Values are means of the fold change with respect to controls from seven different experiments. *P < 0.05, **P < 0.01. B: Example of electrophoretic mobility shift assay. C+ ×100, control extracts plus 100-fold excess unlabeled oligonucleotide.

FIG. 6.

Effects of UCP2 and CHOP-10 on the transcriptional activity driven by the −117/−73 region of the mouse adiponectin gene promoter. The 3T3-L1 cells were transfected with control TK-luc or −117/−73 TK-luc and infected with AdCMV-βGal (control) or AdCMV-UCP2 adenoviral vectors as in Fig. 2. Results are means ± SE of the fold change in luciferase activity with respect to control from five different experiments. Statistically significant effect of UCP2 respect to βGal is shown as *P < 0.05.

FIG. 6.

Effects of UCP2 and CHOP-10 on the transcriptional activity driven by the −117/−73 region of the mouse adiponectin gene promoter. The 3T3-L1 cells were transfected with control TK-luc or −117/−73 TK-luc and infected with AdCMV-βGal (control) or AdCMV-UCP2 adenoviral vectors as in Fig. 2. Results are means ± SE of the fold change in luciferase activity with respect to control from five different experiments. Statistically significant effect of UCP2 respect to βGal is shown as *P < 0.05.

TABLE 1

Serum metabolite and adiponectin levels in UCP2-null mice

Wild typeUCP2 null
Glucose (mg/dl) 161.5 ± 15.4 122.0 ± 2.2* 
Triglycerides (mg/dl) 157.5 ± 24.7 129.1 ± 2.3 
Lactate (mmol/l) 4.1 ± 0.5 2.1 ± 0.4* 
Adiponectin (μg/ml) 16.7 ± 1.3 13.2 ± 0.5* 
Wild typeUCP2 null
Glucose (mg/dl) 161.5 ± 15.4 122.0 ± 2.2* 
Triglycerides (mg/dl) 157.5 ± 24.7 129.1 ± 2.3 
Lactate (mmol/l) 4.1 ± 0.5 2.1 ± 0.4* 
Adiponectin (μg/ml) 16.7 ± 1.3 13.2 ± 0.5* 

Data are means ± SE of five animals.

*

P < 0.05.

TABLE 2

Adiponectin, glutathione peroxidase-1, superoxide dismutase-2, and PPARγ mRNA levels in adipose tissue from UCP2-null mice

mRNAWild typeUCP2 null
Adiponectin 11.8 ± 3.1 4.2 ± 0.4* 
GPX1 0.5 ± 0.2 0.7 ± 0.1 
SOD2 1.8 ± 0.6 5.4 ± 1.4* 
PPARγ 6.0 ± 1.2 4.6 ± 1.7 
mRNAWild typeUCP2 null
Adiponectin 11.8 ± 3.1 4.2 ± 0.4* 
GPX1 0.5 ± 0.2 0.7 ± 0.1 
SOD2 1.8 ± 0.6 5.4 ± 1.4* 
PPARγ 6.0 ± 1.2 4.6 ± 1.7 

Data are means ± SE of five animals. mRNA levels are arbitrary units from real-time quantitative PCR normalized to 18S rRNA. GPX1, glutathione peroxidase-1; SOD2, superoxide dismutase-2.

*

P < 0.05.

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.

E.C. is the recipient of research fellowships from Nestle Foundation and Generalitat de Catalunya. This study has received support from Grants SAF2002-03648 and SAF2005-01722 from the Ministerio de Educacion y Ciencia, Spain, FISPI052336 from Ministerio de Sanidad y Consumo, Spain, and European Community 6th Framework Programme funding N LSHM-CT-2003-503041.

We thank Dr. I.K. Lee for the UCP2 adenoviral vector and S. Ambrosio and D. Haro (University of Barcelona) for SH-GADD153 and Tk109-Luc plasmid vectors, respectively.

1.
Lafontan M: Fat cells: afferent and efferent messages define new approaches to treat obesity.
Annu Rev Pharmacol Toxicol
45
:
119
–146,
2005
2.
Lazar MA: The humoral side of insulin resistance.
Nat Med
12
:
43
–44,
2006
3.
Kershaw EE, Flier JS: Adipose tissue as an endocrine organ.
J Clin Endocrinol Metab
89
:
2548
–2556,
2004
4.
Scherer PE, Williams S, Fogliano M, Baldini G, Lodish HF: A novel serum protein similar to C1q, produced exclusively in adipocytes.
J Biol Chem
270
:
26746
–26749,
1995
5.
Hu E, Liang P, Spiegelman BM: AdipoQ is a novel adipose-specific gene dysregulated in obesity.
J Biol Chem
271
:
10697
–10703,
1996
6.
Kadowaki T, Yamauchi T: Adiponectin and adiponectin receptors.
Endocr Rev
26
:
439
–451,2005
7.
Berg AH, Scherer PE: Adipose tissue, inflammation, and cardiovascular disease.
Circ Res
96
:
939
–949,
2005
8.
Kadowaki T, Yamauchi T, Kubota N, Hara K, Ueki K, Tobe K: Adiponectin and adiponectin receptors in insulin resistance, diabetes, and the metabolic syndrome.
J Clin Invest
116
:
1784
–1792,
2006
9.
Maeda N, Takahashi M, Funahashi T, Kihara S, Nishizawa H, Kishida K, Nagaretani H, Matsuda M, Komuro R, Ouchi N, Kuriyama H, Hotta K, Nakamura T, Shimomura I, Matsuzawa Y: PPARγ ligands increase expression and plasma concentrations of adiponectin, an adipose-derived protein.
Diabetes
50
:
2094
–2099,
2001
10.
Park SK, Oh SY, Lee MY, Yoon S, Kim KS, Kim JW: CCAAT/enhancer binding protein and nuclear factor-Y regulate adiponectin gene expression in adipose tissue.
Diabetes
53
:
2757
–2766,
2004
11.
Qiao L, MacLean PS, Schaack J, Orlicky DJ, Darimont C, Pagliassotti M, Friedman JE, Shao J: C/EBPα regulates human adiponectin gene transcription through an intronic enhancer.
Diabetes
54
:
1744
–1754,
2005
12.
Rousset S, Alves-Guerra MC, Mozo J, Miroux B, Cassard-Doulcier AM, Bouillaud F, Ricquier D: The biology of mitochondrial uncoupling proteins.
Diabetes
53 (Suppl. 1)
:
S130
–S135,
2004
13.
Fleury C, Neverova M, Collins S, Raimbault S, Champigny O, Levi-Meyrueis C, Bouillaud F, Seldin MF, Surwit RS, Ricquier D, Warden CH: Uncoupling protein-2: a novel gene linked to obesity and hyperinsulinemia.
Nat Genet
15
:
269
–272,
1997
14.
Walder K, Norman RA, Hanson RL, Schrauwen P, Neverova M, Jenkinson CP, Easlick J, Warden CH, Pecqueur C, Raimbault S, Ricquier D, Silver MH, Shuldiner AR, Solanes G, Lowell BB, Chung WK, Leibel RL, Pratley R, Ravussin E: Association between uncoupling protein polymorphisms (UCP2-UCP3) and energy metabolism/obesity in Pima Indians.
Hum Mol Genet
7
:
1431
–1435,
1998
15.
Krempler F, Esterbauer H, Weitgasser R, Ebenbichler C, Patsch JR, Miller K, Xie M, Linnemayr V, Oberkofler H, Patsch W: A functional polymorphism in the promoter of UCP2 enhances obesity risk but reduces type 2 diabetes risk in obese middle-aged humans.
Diabetes
51
:
3331
–3335,
2002
16.
Otabe S, Clement K, Rich N, Warden C, Pecqueur C, Neverova M, Raimbault S, Guy-Grand B, Basdevant A, Ricquier D, Froguel P, Vasseur F: Mutation screening of the human UCP 2 gene in normoglycemic and NIDDM morbidly obese patients: lack of association between new UCP 2 polymorphisms and obesity in French Caucasians.
Diabetes
47
:
840
–842,
1998
17.
Yu X, Jacobs DR Jr, Schreiner PJ, Gross MD, Steffes MW, Fornage M: The uncoupling protein 2 Ala55Val polymorphism is associated with diabetes mellitus: the CARDIA study.
Clin Chem
51
:
1451
–1456,
2005
18.
Bulotta A, Ludovico O, Coco A, Di Paola R, Quattrone A, Carella M, Pellegrini F, Prudente S, Trischitta V: The common -866G/A polymorphism in the promoter region of the UCP-2 gene is associated with reduced risk of type 2 diabetes in Caucasians from Italy.
J Clin Endocrinol Metab
90
:
1176
–1180,
2005
19.
D'Adamo M, Perego L, Cardellini M, Marini MA, Frontoni S, Andreozzi F, Sciacqua A, Lauro D, Sbraccia P, Federici M, Paganelli M, Pontiroli AE, Lauro R, Perticone F, Folli F, Sesti G: The −866A/A genotype in the promoter of the human uncoupling protein 2 gene is associated with insulin resistance and increased risk of type 2 diabetes.
Diabetes
53
:
1905
–1910,
2004
20.
Arsenijevic D, Onuma H, Pecqueur C, Raimbault S, Manning BS, Miroux B, Couplan E, Alves-Guerra MC, Goubern M, Surwit R, Bouillaud F, Richard D, Collins S, Ricquier D: Disruption of the uncoupling protein-2 gene in mice reveals a role in immunity and reactive oxygen species production.
Nat Genet
26
:
435
–439,
2000
21.
Zhang CY, Baffy G, Perret P, Krauss S, Peroni O, Grujic D, Hagen T, Vidal-Puig AJ, Boss O, Kim YB, Zheng XX, Wheeler MB, Shulman GI, Chan CB, Lowell BB: Uncoupling protein-2 negatively regulates insulin secretion and is a major link between obesity, beta cell dysfunction, and type 2 diabetes.
Cell
105
:
745
–755,
2001
22.
Negre-Salvayre A, Hirtz C, Carrera G, Cazenave R, Troly M, Salvayre R, Penicaud L, Casteilla L: A role for uncoupling protein-2 as a regulator of mitochondrial hydrogen peroxide generation.
FASEB J
11
:
809
–815,
1997
23.
Skulachev VP: Uncoupling: new approaches to an old problem of bioenergetics.
Biochim Biophys Acta
1363
:
100
–124,
1998
24.
Brand MD, Esteves TC: Physiological functions of the mitochondrial uncoupling proteins UCP2 and UCP3.
Cell Metab
2
:
85
–93,
2005
25.
Poli G, Leonarduzzi G, Biasi F, Chiarpotto E: Oxidative stress and cell signalling.
Curr Med Chem
11
:
1163
–1182,
2004
26.
Blanc J, Alves-Guerra MC, Esposito B, Rousset S, Gourdy P, Ricquier D, Tedgui A, Miroux B, Mallat Z: Protective role of uncoupling protein 2 in atherosclerosis.
Circulation
107
:
388
–390,
2003
27.
Knight DM, Chapman AB, Navre M, Drinkwater L, Bruno JJ, Ringold GM: Requirements for triggering of adipocyte differentiation by glucocorticoids and indomethacin.
Mol Endocrinol
1
:
36
–43,
1987
28.
Park JY, Park KG, Kim HJ, Kang HG, Ahn JD, Kim HS, Kim YM, Son SM, Kim IJ, Kim YK, Kim CD, Lee KU, Lee IK: The effects of the overexpression of recombinant uncoupling protein 2 on proliferation, migration and plasminogen activator inhibitor 1 expression in human vascular smooth muscle cells.
Diabetologia
48
:
1022
–1028,
2005
29.
Zamora M, Merono C, Vinas O, Mampel T: Recruitment of NF-kappaB into mitochondria is involved in adenine nucleotide translocase 1 (ANT1)-induced apoptosis.
J Biol Chem
279
:
38415
–38423,
2004
30.
Dejean L, Camara Y, Sibille B, Solanes G, Villarroya F: Uncoupling protein-3 sensitizes cells to mitochondrial-dependent stimulus of apoptosis.
J Cell Physiol
201
:
294
–304,
2004
31.
Negre-Salvayre A, Auge N, Duval C, Robbesyn F, Thiers JC, Nazzal D, Benoist H, Salvayre R: Detection of intracellular reactive oxygen species in cultured cells using fluorescent probes.
Methods Enzymol
352
:
62
–71,
2002
32.
Joseph JW, Koshkin V, Zhang CY, Wang J, Lowell BB, Chan CB, Wheeler MB: Uncoupling protein 2 knockout mice have enhanced insulin secretory capacity after a high-fat diet.
Diabetes
51
:
3211
–3219,
2002
33.
Sun X, Zemel MB: Role of uncoupling protein 2 (UCP2) expression and 1alpha, 25-dihydroxyvitamin D3 in modulating adipocyte apoptosis.
FASEB J
18
:
1430
–1432,
2004
34.
Bai Y, Onuma H, Bai X, Medvedev AV, Misukonis M, Weinberg JB, Cao W, Robidoux J, Floering LM, Daniel KW, Collins S: Persistent nuclear factor-kappa B activation in Ucp2−/− mice leads to enhanced nitric oxide and inflammatory cytokine production.
J Biol Chem
280
:
19062
–19069,
2005
35.
Hehner SP, Heinrich M, Bork PM, Vogt M, Ratter F, Lehmann V, Schulze-Osthoff K, Droge W, Schmitz ML: Sesquiterpene lactones specifically inhibit activation of NF-kappa B by preventing the degradation of I kappa B-alpha and I kappa B-beta.
J Biol Chem
273
:
1288
–1297,
1998
36.
Ubeda M, Wang XZ, Zinszner H, Wu I, Habener JF, Ron D: Stress-induced binding of the transcriptional factor CHOP to a novel DNA control element.
Mol Cell Biol
16
:
1479
–1489,
1996
37.
Ron D, Habener JF: CHOP, a novel developmentally regulated nuclear protein that dimerizes with transcription factors C/EBP and LAP and functions as a dominant-negative inhibitor of gene transcription.
Genes Dev
6
:
439
–453,
1992
38.
Carriere A, Carmona MC, Fernandez Y, Rigoulet M, Wenger RH, Penicaud L, Casteilla L: Mitochondrial reactive oxygen species control the transcription factor CHOP-10/GADD153 and adipocyte differentiation: a mechanism for hypoxia-dependent effect.
J Biol Chem
279
:
40462
–40469,
2004
39.
Furukawa S, Fujita T, Shimabukuro M, Iwaki M, Yamada Y, Nakajima Y, Nakayama O, Makishima M, Matsuda M, Shimomura I: Increased oxidative stress in obesity and its impact on metabolic syndrome.
J Clin Invest
14
:
1752
–1761,
2004
40.
Wilson-Fritch L, Burkart A, Bell G, Mendelson K, Leszyk J, Nicoloro S, Czech M, Corvera S: Mitochondrial biogenesis and remodeling during adipogenesis and in response to the insulin sensitizer rosiglitazone.
Mol Cell Biol
23
:
1085
–1094,
2003
41.
Hondares E, Mora O, Yubero P, de la Concepcion MR, Iglesias R, Giralt M, Villarroya F: Thiazolidinediones and rexinoids induce peroxisome proliferator-activated receptor-coactivator (PGC)-1alpha gene transcription: an autoregulatory loop controls PGC-1alpha expression in adipocytes via peroxisome proliferator-activated receptor-gamma coactivation.
Endocrinology
147
:
2829
–2838,
2006
42.
Digby JE, Crowley VE, Sewter CP, Whitehead JP, Prins JB, O'Rahilly S: Depot-related and thiazolidinedione-responsive expression of uncoupling protein 2 (UCP2) in human adipocytes.
Int J Obes Relat Metab Disord
24
:
585
–592,
2002
43.
Tong Q, Sankale JL, Hadigan CM, Tan G, Rosenberg ES, Kanki PJ, Grinspoon SK, Hotamisligil GS: Regulation of adiponectin in human immunodeficiency virus-infected patients: relationship to body composition and metabolic indices.
J Clin Endocrinol Metab
88
:
1559
–1564,
2003
44.
Giralt M, Domingo P, Guallar JP, Rodríguez de la Concepción ML, Alegre M, Domingo JC, Villarroya F: HIV-1 infection alters gene expression in adipose tissue, contributing to HIV-1/HAART-associated lipodystrophy.
Antivir Ther
11
:
729
–740,
2006
45.
Lihn AS, Ostergard T, Nyholm B, Pedersen SB, Richelsen B, Schmitz O: Adiponectin expression in adipose tissue is reduced in first-degree relatives of type 2 diabetic patients.
Am J Physiol Endocrinol Metab
284
:
E443
–E448,
2003
46.
Pedersen SB, Nyholm B, Kristensen K, Nielsen MF, Schmitz O, Richelsen B: Increased adiposity and reduced adipose tissue mRNA expression of uncoupling protein-2 in first-degree relatives of type 2 diabetic patients: evidence for insulin stimulation of UCP-2 and UCP-3 gene expression in adipose tissue.
Diabetes Obes Metab
7
:
98
–105,
2005
47.
Morton NM, Paterson JM, Masuzaki H, Holmes MC, Staels B, Fievet C, Walker BR, Flier JS, Mullins JJ, Seckl JR: Novel adipose tissue-mediated resistance to diet-induced visceral obesity in 11 β-hydroxysteroid dehydrogenase type 1–deficient mice.
Diabetes
53
:
931
–938,
2004
48.
Yamauchi T, Oike Y, Kamon J, Waki H, Komeda K, Tsuchida A, Date Y, Li MX, Miki H, Akanuma Y, Nagai R, Kimura S, Saheki T, Nakazato M, Naitoh T, Yamamura K, Kadowaki T: Increased insulin sensitivity despite lipodystrophy in Crebbp heterozygous mice.
Nat Genet
30
:
221
–226,
2002
49.
Wang MY, Shimabukuro M, Lee Y, Trinh KY, Chen JL, Newgard CB, Unger RH: Adenovirus-mediated overexpression of uncoupling protein-2 in pancreatic islets of Zucker diabetic rats increases oxidative activity and improves β-cell function.
Diabetes
48
:
1020
–1025,
1999