Antagonistic effect of TNF-alpha and insulin on uncoupling protein 2 (UCP-2) expression and vascular damage
© Gómez-Hernández et al.; licensee BioMed Central Ltd. 2014
Received: 23 April 2014
Accepted: 27 June 2014
Published: 31 July 2014
It has been reported that increased expression of UCP-2 in the vasculature may prevent the development of atherosclerosis in patients with increased production of reactive oxygen species, as in the diabetes, obesity or hypertension. Thus, a greater understanding in the modulation of UCP-2 could improve the atherosclerotic process. However, the effect of TNF-α or insulin modulating UCP-2 in the vascular wall is completely unknown. In this context, we propose to study new molecular mechanisms that help to explain whether the moderate hyperinsulinemia or lowering TNF-α levels might have a protective role against vascular damage mediated by UCP-2 expression levels.
We analyzed the effect of insulin or oleic acid in presence or not of TNF-α on UCP-2 expression in murine endothelial and vascular smooth muscle cells. At this step, we wondered if some mechanisms studied in vitro could be of any relevance in vivo. We used the following experimental models: ApoE−/− mice under Western type diet for 2, 6, 12 or 18 weeks, BATIRKO mice under high-fat diet for 16 weeks and 52-week-old BATIRKO mice with o without anti-TNF-α antibody pre-treatment.
Firstly, we found that TNF-α pre-treatment reduced UCP-2 expression induced by insulin in vascular cells. Secondly, we observed a progressive reduction of UCP-2 levels together with an increase of lipid depots and lesion area in aorta from ApoE−/− mice. In vivo, we also observed that moderate hyperinsulinemic obese BATIRKO mice have lower TNF-α and ROS levels and increased UCP-2 expression levels within the aorta, lower lipid accumulation, vascular dysfunction and macrovascular damage. We also observed that the anti-TNF-α antibody pre-treatment impaired the loss of UCP-2 expression within the aorta and relieved vascular damage observed in 52-week-old BATIRKO mice. Finally, we observed that the pretreatment with iNOS inhibitor prevented UCP-2 reduction induced by TNF-α in vascular cells. Moreover, iNOS levels are augmented in aorta from mice with lower UCP-2 levels and higher TNF-α levels.
Our data suggest that moderate hyperinsulinemia in response to insulin resistance or lowering of TNF-α levels within the aorta attenuates vascular damage, this protective effect being mediated by UCP-2 expression levels through iNOS.
KeywordsTNF-α Insulin UCP-2 Atherogenesis
Uncoupling proteins (UCPs) belong to the family of mitochondrial transporter proteins and are important for lowering mitochondrial membrane potential and dissipating metabolic energy as heat, maintenance of respiration, glucose disposal rate, insulin secretion, prevention of reactive oxygen species (ROS) production ,. UCP-1 was the first member identified, expressed primarily in brown adipose tissue and the major contributor to energy expenditure . Other four members of UCP (−2 to −5) family have been indentified. In contrast to UCP-4 and −5, human UCP-2 and −3 are both more closely related to UCP-1 ,. UCP-2 is expressed widely and in human is highly expressed in white adipose tissue. Other tissues as skeletal muscle, heart, cell of immune system and vascular cells express considerable amounts of UCP-2 . Recent studies from UCP-2 and −3 knockout mice suggest that both UCPs have uncoupling activity and decreased ROS production in macrophages and skeletal muscle, respectively –. More recently, a direct role for UCP-2 in the regulation of atherogenesis has been suggested by the observation that bone marrow transplantation from UCP-2-deficient mice to LDLR−/− mice markedly increased atherosclerotic lesion size . Moreover, it has been described that UCP-2 overexpression in the vasculature may prevent the development of atherosclerosis in patients with increased ROS, such as in diabetes, obesity or hypertension  and ameliorate hyperglycemia-induced endothelial dysfunction . Furthermore, UCP-2 might be playing an important role in the regulation of energy expenditure and are likely to contribute to obesity and type 2 diabetes mellitus (T2DM). In this regard, several UCP-2 gene polymorphisms were linked to an increased body weight index or obesity in Pima Indians , and in Balinese population  or with insulin resistance or T2DM –. Thus, reduced UCP gene expression has been found in adipose tissue of obese subjects and in the first-degree relatives of T2DM patients. On the other hand, both obese and diabetic patients have associated vascular complications such as atherosclerosis ,, insulin resistance with hyperinsulinemia and elevated circulating TNF-α levels . To get a new insight on that UCP-2 protective effect on the vasculature, we have studied new molecular mechanisms that help to explain whether the moderate hyperinsulinemia or reduction TNF-α levels might have a protective role against vascular damage mediated by UCP-2 modulation. Firstly, we have analyzed the effect of insulin and/or TNF-α on UCP-2 levels in endothelial and vascular smooth muscle cells. After that, we wondered if some mechanisms studied in vitro could be of any relevance in vivo. We used the following experimental models: ApoE−/− mice at 8, 12, 18 or 24 weeks of age, BATIRKO mice under high-fat diet for 16 weeks and 52-wk-old BATIRKO mice with o without anti-TNF-α treatment to address the relationship between UCP-2 expression, or lipid accumulation, or vascular damage, or oxidative stress, or insulin or TNF- α plasma levels. Finally, we searched the role of iNOS in the inhibition of UCP-2 expression by TNF-α.
Primary vascular smooth muscle cells (VSMC) were obtained from thoracic aorta arteries, immortalized and cultured as described previously . Endothelial cell line, SVEC4-10EE2 (clone 2167) was purchased from ATCC and was cultured in DMEM medium supplemented with 10% of horse bovine serum, respectively. Both cell lines were growth-arrested by incubation in medium without serum for 5 h, and then incubated with the corresponding stimuli. For experiments in vitro, we have used TNF-α (10 ng/mL), insulin (10 nmol/L), oleate (1 mmol/L) and L-NAME (1 mmol/L).
Male mice were maintained in the Animal Care Facility under the standard conditions of temperature and 12 h light/dark cycle. All animals from three experimental models used are under C57BL/6 genetic background. Male ApoE−/− knockout mice and their control mice were fed a Western type diet (A04 + 21% kcal from fat) at six week-age for 2, 6, 12 or 18 weeks respectively. Male BATIRKO mice  were fed on high-fat diet (A04 + 61% kcal from fat) for 16 weeks or standard diet (3% calories from fat, A04) for 52 weeks. Moreover, one group of 52-wk-old BATIRKO mice were treated with LEAF purified anti-TNF-α (MP6-XT22, Bio-Legend, San Diego, CA) (50 μg/mouse ip.) every 3 days for 6 weeks as previously described . All animal experimentation described in this manuscript was conducted according with accepted standards of human animal care, as approved by the corresponding institutional committee. The investigation also conforms to the Guide for the Care and Use of Laboratory Animals published by the National Institutes of Health (NIH Publication No. 85–23, revised 1996) and in accordance with The ARRIVE Guideline for Reporting Animal research .
Western blot analyses were performed on protein extracts from VSMCs, ECs or aorta artery as previously described . The antibodies used were anti-phospho-AKT (T308), AKT, p-p70S6K (T389), p70S6K, p-p44/42 (S202/T204) and p44/42 from Cell Signalling, anti-UCP-2 was from Calbiochem and anti-β-actin or α-tubulin was from Sigma-Aldrich Corp.
RNA extraction and real-time quantitative PCR
Total RNA was extracted from ECs, VSMCs or aorta artery from mice by TRIzol method (Invitrogen, Carlsbad, CA). The gene expression was analyzed by real-time quantitative PCR (qRT-PCR) as described .
Plasma levels of insulin and TNF-α were analyzed using ELISA kits (Millipore and SABioSciences, Frederick, MD, respectively).
Aortic roots were OCT-embedded and sections of 7 μm interval were Oil-Red-O/hematoxylin stained was done to measure lipid depot. The lesion size on aortic root was also measured as described . Macrophages and nitrotyrosine levels were detected by immunoperoxidase with rat anti-mouse F4/80 antigen (MCA497GA, AbD serotec) and rabbit anti-nitrotyrosine polyclonal Ab (06–284, Upstate), respectively.
All values were expressed as means +/−sem. Data were analyzed using a one-way analysis of variance, followed by a Bonferroni test if differences were noted (SPSS 15.0 program). Spearman’s correlation coefficient analysis was used to assess associations between several parameters of experimental model. The null hypothesis was rejected when the p value was less than 0.05.
Differential effect of TNF-alpha and Insulin on UCP-2 expression in vascular cells
Protective role of UCP-2 against lipid accumulation and vascular damage
Relationship between TNF-α and UCP-2 expression levels in vivo
Effect of insulin on UCP-2 expression levels in vivo
At this step, we wondered if UCP-2 overexpression induced by insulin in vitro could be of any relevance in vivo. For this purpose, we observed that obese BATIRKO mice with moderate hyperinsulinemia had higher UCP-2 levels in aorta and lesser vascular damage than normoinsulinemic obese BATIRKO mice (Figure 3A, B and E and Additional file 1: Figure S1B). Moreover, we established a positive and significant correlation between circulating insulin levels and UCP-2 levels in aorta (Figure 3F). In the third experimental model, we also observed this correlationship between insulin and UCP-2 expression levels (Figure 4C). Thus, 52-week-old control group displaying moderate hyperinsulinemia showed a significant increase in UCP-2 expression levels in the aorta (Figure 3A and Additional file 1: Figure S1D). However, 52-week-old BATIRKO mice showing a lower insulinemia manifested a significant reduction of UCP-2 expression levels and higher vascular alterations (Figure 3A and Additional file 1: Figure S1C and D). On the other hand, UCP-2 might modify atherosclerotic process due to the fact that elevated levels of this protein reduce ROS levels . Thus, we observed a significant decrease of superoxide anion and nitrotyrosine levels in aortic roots from moderate hyperinsulinemic as compared with normoinsulinemic obese BATIRKO mice (Additional file 2: Figure S2C).
Role of iNOS in the UCP-2 downregulation induced by TNF-α
Protector role of UCP-2 against lipid depot and vascular damage
Atherosclerosis is a multi-factorial chronic vascular inflammatory disease characterized by endothelial dysfunction and accumulation of lipids, inflammatory cells, smooth muscle cells and extracellular matrix in the arterial neointima . Several studies suggest that ROS are involved in plaque formation  and all plaque cellular components may respond to and be damaged by ROS, contribute to plaque progression and finally, to plaque rupture . Thus, several approaches to stop ROS production and to alter disease progression have been used ,. In addition, it has been previously published that UCP-2 overexpression in macrophages decreases intracellular ROS levels and reduces their immune activity ,. Moreover, UCP-2 might function as an adaptive antioxidant defense to protect against the development of atherosclerosis in response to high fat and cholesterol diet  and improve hyperglycemia-induced endothelial dysfunction . Under this scenario, our results demonstrate that high-fat diet BATIRKO mice showing lower UCP-2 expression levels manifested higher oxidative stress in the aorta. Moreover, the decrease in UCP-2 levels in the aorta is strongly inversely correlated with lipid accumulation and lesion area from 24-week-old ApoE−/− mice or normoinsulinemic BATIRKO mice in the aorta. Previous results have also suggested a protective role of UCP-2 against atherosclerosis  showing an antiatherogenic effect in macrophages, ECs and VSMCs . Thus, UCP-2 higher expression reduced proliferation, migration and plasminogen activator 1 expression in human VSMCs .
Insulin induces UCP-2 overexpression in aorta protecting against vascular damage
A better knowledge of UCP-2 expression levels regulation in the vasculature may improve the management of the atherosclerotic process. Thus, we explored the association between insulin and UCP-2 in vivo and in vitro. Our results suggest that insulin or moderate hyperinsulinemia in response to insulin resistance induces UCP-2 expression in ECs and VSMCs or in the aorta from BATIRKO MH mice respectively. On this regard, we previously demonstrated that insulin or IGF-1 induce UCP-1 expression through IRS-1 or AP-1 activity in a PI3K/Akt dependent manner ,. Others authors had also described similar effects of insulin on UCP-2 expression levels in bovine retinal microvascular endothelial cells  or in skeletal muscle . Moreover, it have been described that intensive insulin therapy suppressed iNOS gene expression in liver and skeletal muscle, possibly in part via reduced NF-κB activation, and lowered the elevated circulating NO levels . So, insulin might also reduced NF-κB activation and iNOS levels in aorta and in consequence favours UCP-2 overexpression and protect against vascular damage.
TNF-α downregulates UCP-2 in aorta accelerating vascular damage
Among several proinflammatory and proatherogenic signals working on the vasculature TNF-α is relevant the most. Thus, the relationship between TNF-α and UCP-2 expression levels appears to be of importance in assessing vascular damage risk. On this regard, we have shown that insulin and TNF-α have antagonistic effect on UCP-2 expression in ECs and VSMCs. It has been previously published that proinflammatory cytokines such as TNF-α and/or IL-1β downregulated UCP-2 levels in adipocytes , INS-1 cells or rat pancreatic islets . Moreover, our data provide a strong support in vivo to the negative relationship between TNF-α and UCP-2. Thus, 52-week-old BATIRKO mice or normoinsulinemic BATIRKO mice under high-fat diet with lower UCP-2 levels showed elevated TNF-α expression levels in WAT, plasma and aorta. Moreover, TNF-α may directly downregulates adiponectin  contributing to the development of vascular insulin resistance and the decrease of UCP-2 levels in the aorta. On this regard, it has previously been described that adiponectin induces UCP-2 expression in the liver . In the two populations of BATIRKO mice, we observed a negative correlation between TNF-α and adiponectin levels in both WAT and plasma. Therefore, higher levels of adiponectin might induce UCP-2 overexpression in the aorta attenuating vascular damage. The use of the anti-TNF-α antibody pre-treatment support the concept that TNF-α downregulates UCP-2 expression levels as shown in 52-week-old BATIRKO mice.
Other mechanism involved in the inhibitory effect of TNF-α on UCP-2 expression levels is the NO-dependent pathway induction of iNOS expression in ECs and VSMCs as previously described in 3T3F442A preadipocytes . In vivo, we also demonstrated that anti-TNF-α treatment in 52-week-old BATIRKO mice is able to reduce NF-κB activation in white and brown adipose tissues and aorta, reducing iNOS levels in aorta  and increasing UCP-2 levels in aorta and as result lowering vascular damage. Moreover, LPS promoted the expression of iNOS and ROS production as well as inflammatory cytokines in UCP-2−/− macrophages ,. Our data strongly suggest an inverse correlationship between iNOS and UCP-2. Thus, 24-week-old ApoE−/− mice, normoinsulinemic BATIRKO mice under high-fat diet and 52-week-old BATIRKO mice with lower UCP-2 levels had higher iNOS levels and higher vascular damage. In addition, anti-TNF-α antibody pre-treatment reduced iNOS expression, restoring UCP-2 levels, and improving vascular alterations from 52-week-old BATIRKO mice .
In conclusion, our results suggest that insulin and TNF-α share an antagonistic effect on UCP-2 expression levels in vascular cells and also in the aorta in vivo. Thus, moderate hyperinsulinemia in response to insulin resistance or lowering of TNF-α levels within the aorta attenuates vascular damage, this protective effect being mediated by UCP-2 expression levels through iNOS.
- Ang II:
Protein kinase B (Pkb)
- ApoE−/− mice:
Apolipoprotein E knockout mice
Brown adipose tissue
BAT-specific IR knockout mice
- BATIRKO MH:
Moderate hyperinsulinemic obese BATIRKO mice
- BATIRKO N:
Normoinsulinemic obese BATIRKO mice
Endothelial cell lines
Nitro-L-arginine methyl ester hydrochloride- NOS inhibitor
Insulin-like growth factor-1
Inducible nitric oxide synthase
Nuclear factor kappa B
Reactive oxygen species
Tumor necrosis factor alpha
Uncoupling protein 2
Vascular smooth muscle cells
White adipose tissue
The authors thank Gema García-Gómez and Silvia Fernández for technical assistance. This work was supported by grants SAF2008/00031 and SAF2011/22555 from MCINN, Comunidad de Madrid (S2010/BMD-2423) and CIBERDEM ISCIII, Spain.
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