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Departments of
* Internal Medicine and
Physiology and Biophysics, State University of Campinas, Campinas-SP, Brazil
1Correspondence: Departamento de Clínica Médica, Faculdade de Ciências Médicas, Universidade Estadual de Campinas, SP, Brazil. E-mail: lavelloso{at}fcm.unicamp.br
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Key Words: short-term inhibition of UCP2 • antisense oligonucleotide • ATP synthase • anti-UCP2 antibody
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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2
3)
. In contrast to the prototypic UCP1, which has been well characterized as a modulator of brown adipose tissue thermogenesis by promoting dissipation of the electrochemical gradient that activates ATP synthase (4
, 5)
, the function of UCP2 is still a matter of intense investigation (6
7
8
9
10)
. Recent studies have implicated UCP2 in the regulation of free fatty acid (FFA) metabolism and transport (11)
, control of reactive oxygen species (ROS) formation (10
, 12
13
14)
, and inhibition of insulin secretion (3
, 15)
.
Both genetic and pharmacological approaches have been used as tools to modulate UCP2 expression and/or activity, providing evidence for the role of this protein in pancreatic islet function (1
, 3
, 15
, 16)
. It was initially shown that UCP2 is expressed in normal pancreatic islets and that the induction of its overexpression leads to decreased glucose-stimulated insulin secretion (GSIS) (17)
. Similarly, overexpression of UCP2 in an insulin-producing cell line increased mitochondrial respiration while decreasing the coupling to oxidative phosphorylation; this resulted in low intracellular ATP level and reduced GSIS (18)
. In addition, knockout of the UCP2 gene in mice produced a phenotype of hyperinsulinemia and increased whole-body sensitivity to insulin. Introducing this defect in the diabetes-prone ob/ob mouse partially reverted glucose intolerance (19)
.
In a recent study, we used an antisense oligonucleotide to UCP2, which inhibits the expression of this protein by up to 60% in isolated pancreatic islets and leads to a significant increase in GSIS (3)
. Since the development of diabetes in primarily insulin-resistant subjects is thought to be the result of a failure of the pancreatic ß-cell to compensate for the peripheral demand for insulin (20)
, we decided to evaluate the outcome of a short-term in vivo treatment with the UCP2 antisense oligonucleotide in two animal models of diabetes and insulin resistance. To our surprise, not only was a significant improvement in insulin secretion achieved, but enhanced whole-body sensitivity to insulin was also obtained.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Sense and antisense oligonucleotide treatment protocols
Phosphorthioate-modified sense and antisense oligonucleotides (produced by Invitrogen Corp., Carlsbad, CA, USA) were diluted to a final concentration of 10 nmol/ml in dilution buffer containing 10 mmol/L Tris-HCl and 1.0 mmol/L EDTA. The mice were injected i.p. with one daily dose of 200 µl of dilution buffer containing, or not, sense (UCP2/S) or antisense oligonucleotides (UCP2/AS). The oligonucleotides were designed according to the Mus musculus UCP2 sequence deposited at the NIH-NCBI (http://www.ncbi.nlm.nih.gov/entrez) under the designation NM 011671 and were composed of 5'-TGC ATT GCA GAT CTC A-3' (sense) and 5'-TGA GAT CTG CAA TGC A-3' (antisense).
Experimental protocols
Male 3-wk-old Swiss (Sw/Uni) inbred strain mice, originally imported from the Jackson Laboratory (Bar Harbor, ME, USA) and currently bred at the State University of Campinas Breeding Center (CEMIB), were used in the first part of the study. The investigation followed university guidelines for the use of animals in experimental studies and conforms to the Guide for the Care and Use of Laboratory Animals published by the U.S. National Institutes of Health (NIH publication no. 85–23, revised 1996). The animals were maintained on a 12:12 h artificial light:dark cycle and housed in individual cages. We set 8 wk of fat-rich diet feeding (Table 1
) as the time when all Sw/Uni mice have developed diabetes; Sw/Uni were treated with a daily dose of either oligonucleotide dilution vehicle (control), UCP2/S oligonucleotides (Sw/Uni/S), or UCP2/AS oligonucleotides (Sw/Uni/AS) from 8 wk of the diet onward. In this part of the study, hormonal and biochemical parameters were evaluated every second day. On day 16 after the onset of oligonucleotide treatment, glucose and insulin tolerance tests were performed. Some animals were anesthetized and used for tissue extraction to determine UCP2 expression, insulin-induced activation of IR, IRS1, Akt, and FOXO1 signaling. Islets were isolated for static and dynamic insulin secretion studies, measurement of SOD1 and catalase, and determination of ATP content. In the second part of the study we used male 10-wk-old ob/ob mice. The ob/ob mice were purchased from the Jackson Laboratory and currently are established as a colony at the University of Campinas Central Animal Breeding Center. The mice were allowed ad libitum access to standard rodent chow and water. Similar to the Sw/Uni mice, ob/ob mice were treated with either a daily dose of oligonucleotide dilution vehicle (control), UCP2/S oligonucleotides (ob/ob/S), or UCP2/AS oligonucleotides (ob/ob/AS). At day 16 after the start of oligonucleotide treatment, glucose and insulin tolerance tests were performed.
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Hormone and biochemical measurements
Plasma insulin was determined by RIA according to a described method (21)
. Serum glucose was determined by the glucose oxidase method (22)
. Serum triglycerides were determined by colorimetric assays (Wako Chemicals, Neuss, Germany).
Intraperitoneal (i.p.) glucose tolerance test
After an overnight fast, mice were anesthetized; after collection of an unchallenged sample (time 0), a solution of 25% glucose (11.1 mmol/kg body wt) was administered by i.p. injection. Blood samples were collected from the tail at 15, 30, 60, and 120 min to determine glucose and insulin concentrations.
Intraperitoneal insulin tolerance test (ITT)
Insulin (1.5 U/kg) was administered by i.p. injection and blood samples were collected at 0, 5, 10, 15, 20, 25, and 30 min to determine serum glucose. The constant rate for glucose disappearance (Kitt) was calculated using the formula 0.693/t1/2. Glucose t1/2 was calculated from the slope of the least-squares analysis of plasma glucose concentrations during the linear decay phase (23)
.
Islet isolation and static and dynamic insulin secretion studies
Islets were isolated by handpicking following collagenase digestion (24)
. To measure insulin secretion, groups of five islets were preincubated for 45 min at 37°C in Krebs bicarbonate buffer. The solution was then replaced by fresh buffer containing low (2.8 mmol/L) or supraphysiological (16.7 mmol/L) concentrations of glucose and islets were incubated for 1.0 h. The insulin content of the medium at the end of the incubation period was determined by RIA. For dynamic insulin secretion studies, groups of 50 freshly isolated islets were placed on a Millipore SW 1300 filter (8.0 µm pore) in a perfusion chamber. Islets were continuously perfused at a flow rate of 0.8 ml/min. During the initial 20 min of perfusion, the buffer consisted of Krebs bicarbonate solution containing 2.8 mmol/L glucose. Finally, perfusion buffer containing 16.7 mmol/L glucose was introduced. Samples of perfusate for quantification of insulin were collected every other minute starting 10 min after the onset of perfusion. The insulin content of the perfusion period was determined by RIA.
ATP content
Before chromatographic analysis, ATP, ADP, AMP, and ADO were extracted from snap-frozen islets (200 islets/group) according to a published method, with minor modifications (25
, 26)
. Then 1.5 ml of a solution containing 50 mmol/L KH2PO4 and 25 mmol/L citric acid (pH 4.5) was used to resuspend the islets. The mixture was kept in a water bath (85°C) for 3 min and the pellet of islets was lysed by mechanical stress. Afterward, samples were vortex-mixed for 1 min and transferred to Eppendorf tubes for centrifugation (8000 rpm, 5 min). One milliliter of supernatant was mixed with 100 µl of 2-chloroacetaldehyde solution and heated at 80°C for 20 min; 25 µl of the reaction solution was then resolved by liquid chromatography.
Chromatography
Chromatographic analyses were carried out on a Waters Alliance equipment series 2695 (Milford, MA, USA) equipped with a quaternary pump, an autosampler, a degasser, and a Waters 2475 fluorescence detector model. The fluorescence of derivatized compounds (ATP, ADP, AMP, and ADO) was monitored with excitation and emission wavelengths set at 280 and 420 nm, respectively. Chromatographic separations of the compounds were achieved at room temperature, using a reversed-phase Cosmosil 5C18-MS column (150x4.6 mm i.d.; 5 µm particle size) with a Cosmosil guard column (5C18-MS 10x4.6 mm) purchased from Phenomenex (Torrance, CA, USA). The mobile phase composition was 50 mmol/L KH2PO4, 25 mmol/L citric acid (pH 4.5), and methanol (90:10, v/v), which was prepared immediately before use and filtered through a 0.45 µm filter (Millipore, Milford, MA, USA). The column was equilibrated and eluted under isocratic conditions using a flow rate of 1.0 ml/min. The chromatographic run time for each analysis was 20 min. Aliquots of 25 µl were injected into the HPLC system. System control, data acquisition, and processing were performed with a PC-Pentium IV Processor personal computer from Dell, operated with Microsoft Windows XP version 2003 and Waters Empower 2002 chromatography software. A validation chromatographic run included a set of calibration samples assayed in duplicate and quality control samples at four levels in triplicate. The standard calibration curves for known amounts of ATP, ranging from 0.025 to 10.0 µmol/L, were linear (R>0,999) and could be described by the linear regression equation: y = 0.4992*x – 0.0463 (n=4, P<0.0001, r=0.9997), in which y is the ATP concentration in micromoles and x is the chromatogram peak area.
Immunoprecipitation and immunoblotting
To evaluate insulin signal transduction, the abdominal cavities of anesthetized mice were opened and the animals received an injection of insulin (100 µl, 10–6 mol/L) or saline (100 µl) through the cava vein. After different intervals (see Results), fragments (3.0x3.0x3.0 mm) of white adipose tissue (WAT) and gastrocnemius muscle were excised and immediately homogenized in solubilization buffer at 4°C [1% Triton X-100, 100 mmol/L Tris-HCl (pH 7.4), 100 mmol/L sodium pyrophosphate, 100 mmol/L sodium fluoride, 10 mmol/L EDTA, 10 mmol/L sodium orthovanadate, 2.0 mmol/L PMSF, and 0.1 mg aprotinin/ml] with a Polytron PTA 20S generator (model PT 10/35; Brinkmann Instruments, Westbury, NY, USA). Insoluble material was removed by centrifugation for 40 min at 11,000 rpm in a 70.Ti rotor (Beckman, Fullerton, CA, USA) at 4°C. The protein concentration of the supernatants was determined by the Bradford dye binding method. Aliquots of the resulting supernatants containing 2.0 mg of total protein were used for immunoprecipitation with antibodies against IR and IRS1 at 4°C overnight, followed by SDS/PAGE, transfer to nitrocellulose membranes, and blotting with antiphosphotyrosine (pY) antibodies. In direct immunoblot experiments, 0.2 mg of protein extracts obtained from each tissue (muscle and WAT) were separated by SDS-PAGE, transferred to nitrocellulose membranes, and blotted with anti-IR, anti-IRS1, anti-Akt, antiphospho [Ser-473] Akt, anti-FOXO1, and antiphospho [Ser-256] FOXO1. To determine UCP2, SOD1, and catalase expression, fragments (3.0x3.0x3.0 mm) of spleen, WAT, gastrocnemius muscle, heart, brain, or isolated pancreatic islets (1500 per sample) were obtained from anesthetized mice, homogenized in solubilization buffer as above, and samples containing 0.2 mg were separated by SDS-PAGE, transferred to nitrocellulose membranes, and blotted with respective antibodies. Specific bands were labeled with 125I-protein A and visualization was performed by exposure of the membranes to RX films.
Mitochondria preparation and use in immunoblot experiments
Brain, spleen, heart, and WAT mitochondria were isolated by homogenization in ice-cold medium containing 100 mmol/L sucrose, 100 mmol/L KCl, 50 mmol/L Tris-HCl, 1.0 mmol/L K2HPO4, 0.1 mmol/L EGTA, and 0.2% BSA, pH 7.4, followed by differential centrifugation as described (27)
. After the last 10,000 g centrifugation step, the supernatant was recovered and used in parallel with mitochondria (pellet) protein extracts to determine UCP2 expression by immunoblot as described above. Samples containing 30 µg mitochondria protein and 200 µg cytosolic, mitochondria-free (supernatant) protein were separated by SDS-PAGE, transferred to nitrocellulose membranes, and blotted with anti-UCP2 antibodies.
Immunodepletion
In immunodepletion experiments, aliquots of spleen, skeletal muscle, and WAT total protein extracts containing 1.0 mg protein were used in four consecutive rounds of immunoprecipitation with the anti-UCP2 antibody. At each immunoprecipitation round, a sample was obtained from the supernatant and used in SDS-PAGE and immunoblotting analysis with the UCP2 antibody.
Statistical analysis
Specific protein bands present in the blots were quantified by digital densitometry (ScionCorp, Inc., Frederick, MD, USA). Mean values ± SE obtained from densitometric scans, ATP contents, static and dynamic insulin secretion amounts, blood hormone and biochemical parameters, body weight, and food intake were compared utilizing the Mann-Whitney test. A P value of <0.05 was accepted as statistically significant.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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. Mitochondria was prepared from tissues known to express high levels of the protein, such as spleen and WAT, and from tissues known to express undetectable levels of UCP2, such as brain and heart. As depicted in Fig. 1
A, UCP2 was detected in mitochondria but not in the supernatants of spleen and WAT, whereas no expression of the protein was seen in mitochondria or supernatants of brain and heart. Using 0.2 mg protein samples from total protein extracts, we detected bands of 
30 kDa in spleen, WAT, skeletal muscle, and pancreatic islets (Fig. 1B
). To evaluate whether these bands corresponded to UCP2, samples containing 1.0 mg total protein were submitted to an immunodepletion protocol using the UCP2 antibody. Samples recovered from the supernatants after each round of immunoprecipitation were used to detect the target protein by immunoblot. As shown in Fig. 1C
, UCP2 was depleted in samples of spleen and WAT but not in skeletal muscle, suggesting that the 30 kDa band present in this tissue did not correspond to UCP2. Due to the large amounts of pancreatic islets required to obtain enough protein for immunodepletion experiments, we did not include this tissue in the evaluation. Nevertheless, we performed long-run SDS-PAGEs with 0.2 mg protein samples from pancreatic islets separated in parallel with samples from crude (nonimmunodepleted) and completely immunodepleted protein extracts from spleen. As shown in Fig. 1D
, the 30 kDa band detected in pancreatic islet total protein extract migrates in the same position as that of the UCP2 expressed in spleen.
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Inhibition of UCP2 expression reverses diet-induced diabetes
To evaluate the effect of inhibition of UCP2 expression on glucose homeostasis and insulinemia of Sw/Uni mice, a phosphorthioate-modified antisense oligonucleotide to UCP2 (UCP2/AS) was used. Since glucose and insulin levels became significantly higher from 8 wk onward, we started UCP2/AS treatment after the introduction of a fat-rich diet to Sw/Uni mice. First, the capacity of UCP2/AS to inhibit UCP2 expression was tested in a dose-response experiment. As shown in Fig. 2
A, a daily dose of 0.5 nmol UCP2/AS was sufficient to inhibit UCP2 expression by 40 ± 8%, whereas 2.0 nmol/day resulted in 87 ± 13% inhibition of UCP2 expression in pancreatic islets. One daily dose of 1.0 nmol UCP2/AS was sufficient to inhibit UCP2 expression by 85 ± 10% and 84 ± 15% in islet and WAT of Sw/Uni mice, respectively (Fig. 2B
), and so was used in the remaining experiments. UCP2/AS was specific since no significant change in the expression of the structural protein actin was detected in islets and adipose tissue (Fig. 2B
). In addition, treatment with UCP2/sense oligonucleotide resulted in no modulation of UCP2 or actin expression in the tissues evaluated (Fig. 2B
). To determine the effect of the inhibition of UCP2 expression on blood glucose and insulin levels, mice were treated with one daily dose of UCP2/AS and blood samples were collected every second day. As shown in Fig. 2C
, a significant fall in glucose level was observed from 8 days onward after treatment started. In addition, 4 days after treatment began, blood insulin levels rose higher than controls (Fig. 2D
). During an i.p. glucose tolerance test, mice treated with UCP2/AS for 16 days presented a reduced area under the glucose curve and an increased area under the insulin curve (Fig. 3
A, B); during an insulin tolerance test, a higher glucose decay constant (kitt) was observed in Sw/Uni/AS mice (Fig. 3C
). None of these effects of UCP2/AS was accompanied by any significant changes in body weight, food intake, serum triglycerides, or epidydimal fat (Table 2
).
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The in vivo inhibition of UCP2 expression improves insulin secretion by isolated pancreatic islets
To determine the role of UCP2 expression inhibition in insulin secretion, mice were treated for 16 days with 1.0 nmol/day of UCP2/AS; at the end of the experimental period, pancreatic islets were isolated and used to determine static and dynamic insulin secretion. Figure 4
A shows that with a low glucose level (2.8 mmol/L glucose), the reduction of UCP2 expression did not affect insulin secretion. However, during incubation with high glucose levels (16.7 mmol/L glucose), inhibition of UCP2 expression led to a significant (1.5-fold) increase in insulin secretion. Similarly, during the low glucose phase of the dynamic insulin secretion evaluation, UCP2 expression inhibition promoted no change in insulin secretion whereas during the high glucose phase, inhibition of UCP2 expression produced a remarkable improvement in insulin secretion (Fig. 4B
).
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Inhibition of UCP2 expression increases islet ATP content and does not affect the expression of SOD1 and catalase
The capacity of UCP2 to modulate intracellular ATP content is thought to be the main mechanism involved in the UCP2-dependent control of insulin secretion. Therefore, we determined the content of ATP in islets from UCP2/AS-treated mice. As shown in Fig. 5
A, inhibition of UCP2 expression did not affect the ATP content in pancreatic islets exposed to 5.6 mmol/L glucose but significantly increased the ATP content in islets exposed to high glucose levels (16.7 mmol/L). In addition, since the expression of uncoupling proteins is postulated to play a role in the control of oxidative stress, we evaluated the expression of two key enzymes involved in the cell’s response to this sort of insult. As shown in Fig. 5B
, treating mice with the UCP2/AS did not promote significant modulation of the expression of SOD1 and catalase.
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Improved insulin signal transduction in WAT but not in muscle of UCP2/AS-treated mice
To evaluate the effect of UCP2/AS on the activity of the insulin signal transduction pathway, Sw/Uni mice, treated or not with UCP2/AS for 16 days, were anesthetized and acutely treated with a single dose of saline (200 µl) or insulin (200 µl, 10–6 mol/L) through the cava vein. After 2 or 5 min, fragments of WAT and muscle were obtained and used in typical immunoprecipitation and immunoblotting experiments to assess tyrosine phosphorylation of the IR and IRS1 and serine phosphorylation of Akt and FOXO1. The inhibition of UCP2 expression was associated with a significant increase in insulin-induced tyrosine phosphorylation of the IR (Fig. 6
A) and IRS1 (Fig. 6B
), and a significant increase in serine phosphorylation of Akt (Fig. 6C
) and FOXO1 (Fig. 6D
) in adipose tissue. Conversely, in muscle the inhibition of UCP2 expression promoted a significant fall only in insulin-induced tyrosine phosphorylation of the IR (Fig. 6E
), with no modifications detected in the more distal steps of the insulin signaling pathway (Fig. 6F-H
). These phenomena were not accompanied by the modulation of expression of any of the proteins studied (Fig. 6A-H
).
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Inhibition of UCP2 expression reverses diet-induced diabetes in ob/ob mice
To evaluate the effect of inhibition of UCP2 expression on glucose homeostasis and insulinemia of ob/ob mice, a phosphorthioate-modified antisense oligonucleotide to UCP2 (UCP2/AS) was used. In the University of Campinas colony, the leptin-deficient ob/ob mice become diabetic at 8 wk of age (28)
. Therefore, we began treating mice at 10 wk of age when basal glucose levels were 312 ± 28 mg/dl (at this point, Ob/? mice presented basal blood glucose levels of 94±11 mg/dl). As for Sw/Uni mice, treatment with UCP2/AS for 16 days promoted no significant changes in body mass, food intake, serum triglycerides, or epidydimal fat mass in ob/ob mice (data not shown). However, during an i.p. glucose tolerance test (GTT), mice treated with UCP2/AS presented a reduced area under the glucose curve and an increased area under the insulin curve (Fig. 7
A, B). In addition, during an ITT, a significantly higher constant of glucose decay (Kitt) was determined in Sw/Uni/AS mice (Fig. 7C
).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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and, after 6–8 wk exposure to the experimental diet, develops hyperglycemia, obesity, and hepatic steatosis (30)
. This model develops, through a combination of genetic and environmental factors, a phenotype that resembles human metabolic syndrome and therefore is an interesting tool for studying aspects related to the pathophysiology and therapeutics of this condition. The ob/ob mouse is a model of monogenic obesity that secondarily develops insulin resistance, diabetes, and hypertension (31)
, and is one of the most widely used models for studying mechanisms involved in developing the components of the metabolic syndrome.
Initially, we evaluated the method to detect UCP2 protein expression in different tissues. Similar to a previous study, UCP2 was detected in mitochondria of spleen and WAT; using an immunodepletion protocol, we demonstrated that an UCP2-specific 30 kDa band could be determined in total protein extracts from spleen and WAT. In skeletal muscle, a 30 kDa band could not be depleted by the UCP2 antibody, suggesting it does not correspond to UCP2, which is in accordance with previous reports (32)
.
To achieve UCP2 inhibition, we used a phosphorthioate-modified antisense oligonucleotide specific for the mouse mRNA sequence of UCP2. Modified antisense oligonucleotides bind, by the Watson-Crick hybridization mechanism, to specific sequences of the mRNA. The resulting heteroduplex is a substrate for catalytic degradation by endogenous RNase H (33)
. In recent years, an increasing number of studies have used antisense oligonucleotidic compounds either as tools for investigating effects of the reduced expression of any given protein or as experimental/clinical therapeutic approaches for different diseases (34
35
36
37
38
39
40)
. In the present work, in vivo treatment with the UCP2/AS oligonucleotide promoted an 85% reduction in UCP2 expression in pancreatic islets and adipose tissue. The fact that the same treatment led to no modulation of the expression of the 30 kDa band in skeletal muscle (not shown) reinforces the findings of other groups that suggest that UCP2 is absent or present at undetectable levels in this tissue. Nevertheless, the treatment of Sw/Uni mice fed a hyperlipidic diet with the UCP2/AS led to a decrease in blood glucose level and an increase in blood insulin level without changing food intake, body mass, body fat distribution, or blood triglyceride levels. These findings match the effects of UCP2 knockout in lean and ob/ob mice, since in both models the most important phenotypic outcomes were related to insulin secretion and blood glucose levels without affecting body mass (15)
. In addition, since in the present model the effect of UCP2 inhibition on glucose and insulin was already detected after 4–8 days of UCP2/AS treatment, we can assume that it results specifically from the abrogation of UCP2 and not as a consequence of complex developmental modulations, as could be argued for the knockout models (41
, 42)
.
The combined evaluation of the results of the GTT and the ITT suggested that short-term in vivo inhibition of UCP2 expression promoted not only an improvement of GSIS, but also an improvement of peripheral insulin action. The effect of UCP2 in insulin secretion has been evaluated in earlier studies, and in general shown to be intimately associated with control of the ATP content of the islet (15
, 16)
. In the present study, we demonstrate that after short-term inhibition of UCP2 expression, pancreatic islet ATP levels are increased and the GSIS, measured by two distinct methods, is increased. Thus, it becomes clear that UCP2 acts as a switch for insulin secretion by providing an effective means to control the flux of electrochemical energy toward ATP synthase in the inner membrane of the mitochondrial wall.
It has been a matter of intense debate as to whether UCP2 activity provides a mechanism for regulating ROS accumulation in certain cell types (10
, 12
13
14)
. Since ROS accumulation in pancreatic islets is known to participate in the mechanisms involved in autoimmune destruction of insulin-producing cells (43
, 44)
and also in the defective GSIS observed in type 2 diabetes (44
, 45)
, we determined the effect of UCP2 inhibition on the expression of two enzymes that participate in the response to oxidative stress: SOD1 and catalase. Since no modulation of the expression of either protein was detected in islets of mice treated with UCP2/AS, we believe that, at least on a short-term basis, the negative modulation of UCP2 expression does not impose an adverse stimulus on insulin-producing cells.
With regard to the improvement in insulin action in UCP2-inhibited mice, our finding, as determined by the ITT, is unique since it provides evidence that short-term in vivo inhibition of UCP2 expression is capable of affecting glucose homeostasis not only because of an effect on insulin production, but also by improving insulin action on glucose clearance. One possibility would be that the increased GSIS could act initially by reducing glucotoxicity, which would indirectly improve insulin signaling in peripheral tissues. However, by evaluating key elements of the initial, intermediary, and final steps of the insulin signaling pathway, we observed that the effect of UCP2 inhibition on insulin action coincides with a significant improvement in insulin signaling in adipose tissue, but not in muscle. Since treatment with UCP2/AS resulted in specific inhibition of the target protein in WAT, and considering that no significant amounts of the protein could be detected in muscle, we believe that the effect on insulin signal transduction results from a direct intracellular mechanism and is not due to intertissue cross-talk. Thus, we provide an important piece of evidence for the participation of UCP2 in insulin action at the molecular level, enhancing the importance of this mitochondrial protein as an active regulator of metabolic function.
In the final part of the study, we treated ob/ob mice with the same compound to evaluate the effect of short-term inhibition of UCP2 in a model that develops obesity due to a monogenic defect. Similar to the Sw/Uni mouse, the ob/ob mouse treated with UCP2/AS presented a rapid improvement in glucose homeostasis due to a combined effect on insulin secretion and action.
In conclusion, by promoting a rapid restoration of glucose levels in two distinct animal models, we provide further support for the role for UCP2 as a potential target for the therapeutics of diabetes mellitus and related disorders. These results were obtained after only a few days of treatment with a single dose of the compound, and the animals presented no signs of drug intolerance such as mortality, irritability, major changes in feeding behavior, or gross appearance of sickness. Although oligonucleotidic compounds have been approved for therapeutics in human diseases (36
, 37)
, the general costs for its production and potential unspecific interactions with the mRNA processing machinery have hindered the development of drugs of this chemical class. A recent study reported finding a natural compound from Gardenia sp. genipin that acts as an inhibitor of UCP2 activity and is able to increase islet ATP content and improve GSIS (16)
. Certainly, it will be of major interest to evaluate the outcomes of the therapeutic use of this drug in animal models of metabolic syndrome.
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ACKNOWLEDGMENTS |
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Received for publication August 17, 2006. Accepted for publication November 23, 2006.
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