Objective: The aim of this study was to investigate a possible link between high-fat diet (HFD)–induced obesity and the expression of protein phosphatase 2A (PP2A) and Cdc42-interacting protein 4 (CIP4) proteins, potential downstream components of the IRS/PI3K/AKT and CAP/Cbl/TC10 pathway, respectively, in the visceral adipose tissue.
Methods and Procedures: Twenty male Sprague-Dawley rats were randomly divided into two groups and were given either HFD or the normal diet (ND) for 8 weeks. The HFD-induced changes in the expression of the epididymal adipose tissue genes involved in the insulin-signaling pathways were evaluated using real-time reverse-transcription PCR and western blot analysis.
Results: The exposure of rats to HFD for 8 weeks resulted in a significant increase in the expression of PP2A at both the transcriptional and translational levels, along with a marked reduction in the levels of phosphorylated AKT and insulin receptor substrate-1 (IRS-1) in the cytosol of visceral adipocytes, compared with the ND rats. Besides, there were significant HFD-induced decreases in the mRNA and protein levels of CIP4 and TC10 in the adipose tissue of rats.
Discussion: These data suggest that HFD might have a relevance to insulin resistance by increasing the expression of PP2A, an inhibitor of AKT activity in the phosphatidylinositol 3-kinase (PI3K)/AKT pathway, and also by suppressing the expression of TC10 and CIP4, downstream effectors of the Cbl/CAP/TC10 insulin-signaling cascade in the visceral adipose tissue.
High-fat diet (HFD) is a single risk factor leading to whole-body and muscle insulin resistance, hyperinsulinemia, and the accumulation of fat in insulin target organs (1). Abdominal adiposity is well accepted as a predisposing factor for insulin resistance (2), and body fat distribution, especially the accumulation of visceral adipose tissue rather than subcutaneous adipose tissue, is thought to constitute impaired glucose tolerance (3). Insulin-stimulated glucose uptake is primarily mediated by the glucose transporter 4 (GLUT4), which is predominantly expressed in the skeletal muscle and fat tissues (4). Most of GLUT4 (>90%) is in the basal state sequestered in an intracellular pool, and insulin stimulates glucose uptake mainly by recruiting this intracellular GLUT4 pool to the plasma membrane (5). The two independent signaling pathways—the phosphatidylinositol 3-kinase (PI3K)/v-akt murine thymoma viral oncogene homolog (AKT) pathway and the Cbl-associated protein (CAP)/casitas b-lineage lymphoma (Cbl)/TC10 pathway—are known to be involved in the insulin-stimulated GLUT4 translocation (6). In most animal and cellular models of insulin resistance, insulin-stimulated GLUT4 translocation to the plasma membrane is reduced (7). Although the relationship between the HFD-induced visceral adiposity and insulin resistance is compelling, its precise mechanism has not yet been fully elucidated.
We observed from the cDNA microarray analysis of the epididymal adipose tissue gene expression that protein phosphatase 2A (PP2A) and cell division cycle 42 (Cdc42)-interacting protein 4 (CIP4) were upregulated and downregulated, respectively, in rats fed the HFD (data not shown). PP2A is one of the most abundant phosphatases regulating the activities of signal transduction proteins (8), and it is known to regulate the insulin-signaling pathway negatively by inhibiting AKT activity in 3T3L1 adipocytes (9). Meanwhile, the activation of TC10, a Rho family GTPase that is highly expressed in muscle and adipose tissues, is essential for insulin-stimulated GLUT4 translocation via the alternative insulin-signaling pathway, CAP/Cbl/TC10 (ref. 10). CIP4 is known to be involved in the regulation of the actin cytoskeleton and membrane trafficking through the interaction with the GTP-bound Cdc42 (ref. 11). A possible link between CIP4 protein and insulin resistance has been raised from the in vitro study using 3T3 adipocytes, suggesting that CIP4 protein is required for insulin-stimulated GLUT4 translocation via its interaction with TC10 (ref. 12).
In this study, we have addressed the following questions. Is PP2A, which has been reported to inhibit AKT activity in 3T3L1 adipocytes, affected by the HFD in the visceral adipose tissue of rats? Is HFD-induced insulin resistance, manifested in the visceral adipose tissue of rats, associated with the suppression of CIP4 protein and with the subsequent suppression of the CAP/Cbl/TC10 insulin-signaling pathway?
Methods and Procedures
Animals and experimental diets
Twenty 5-week-old male Sprague-Dawley rats (Orient, Gyeonggi-do, Korea) were housed in a temperature- (21 ± 2.0 °C) and humidity- (50 ± 5%) controlled room with a ratio of 12-h light/12-h darkness, and were fed a commercial diet (Purina, St. Louis, MO) for 1 week. Rats were randomly divided into two groups: the normal-diet (ND) group and the HFD group. The HFD contained 200 g fat/kg (170 g lard and 30 g corn oil) and 1% cholesterol by weight. The HFD was formulated to provide 40% of the total energy generated by the diet from fat by replacing carbohydrate energy with lard and corn oil, and it had the same amount of vitamins and minerals per kilojoule as the ND did.
At the end of the 8-week feeding period, the rats were anesthetized with diethyl ether following overnight fasting. Blood was drawn from the abdominal aorta into a vacuum tube and the epididymal fat pads were removed, weighed, and frozen with liquid nitrogen. This study adhered to the Guide for the Care and Use of Laboratory Animals developed by the Institute of Laboratory Animal Resources of the National Research Council, and was approved by the Institutional Animal Care and Use Committee of Yonsei University in Seoul, Korea.
The serum glucose concentration was measured using an automatic analyzer (Express Plus; Chiron Diagnostics, East Walpole, MA). The serum insulin level was measured by radioimmunoassay (Linco Research, St. Charles, MO), and the total cholesterol level was determined using a commercial kit (Sigma, St. Louis, MO).
RNA isolation and real-time reverse-transcription PCR analysis
Total RNA was isolated from the epididymal fat tissue of each rat using Trizol (Invitrogen, Carlsbad, CA) and was reverse-transcribed using the Superscript II kit (Invitrogen), according to the manufacturer's recommendations. The primers for real-time PCR analysis were synthesized at Bioneer (Daejun, Korea). Forward (F) and reverse (R) primer sequences for the genes involved in the insulin-signaling pathway are shown in Table 2. Real-time PCRs were then carried out in a 20-μl reaction mixture (2 μl cDNA, 16 μl SYBR green PCR master mix, which includes 2 μl 1 × LightCycler, 2.4 μl 1.5 mmol/l MgCl2 and 11.6 μl H2O, and a 1 μl 0.5 μmol/l specific gene primer pair) in a LightCycler instrument (Roche Diagnostics, Indianapolis, IN). The PCR program was initiated for 10 min at 95 °C before 40 thermal cycles consisting of 10 s at 95 °C, 5 s at 55 °C, and 30 s at 70 °C. The data obtained were analyzed using the comparative cycle threshold method, and these were normalized by the β-actin expression value. Melting curves for each PCR were generated to ensure the purity of the amplification product.
Table 2. The mRNA level of epididymal adipose tissue genes as determined by real-time RT-PCR analyses
Western blot analysis
Epididymal fat pads were homogenized at 4 °C in an extraction buffer (100 mmol/l Tris-HCl, pH 7.4, 5 mmol/l EDTA, 50 mmol/l NaCl, 50 mmol/l sodium pyrophosphate, 50 mmol/l NaF, 100 mmol/l orthovanadate, 1% Triton X-100, 1 mmol/l phenylmethylsulphonylfluoride, 2 μg/ml aprotinin, 1 μg/ml pepstatin A, and 1 μg/ml leupeptin). Tissue homogenates were centrifuged (13,000g, 20 min, 4 °C) and the resulting supernatants (whole-tissue extract) were used for western blot analyses. The plasma membrane protein was extracted from the epididymal adipose tissue using a commercially available protein extraction kit (ReadyPrep-Signal; Bio-Rad, Hercules, CA) according to the manufacturer's instruction. The total protein concentrations of whole-tissue and plasma membrane extracts were determined by the Lowry assay (Bio-Rad).
Protein samples (80 μg/lane) were separated by 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis and were transferred onto the polyvinylidene difluoride membrane (Millipore, Billerica, MA). After incubation in 5% skim milk in Tris-buffered saline-Tween 20 for 1 h, the membrane was incubated overnight with the specific antibodies against AKT (Cell Signaling, Beverly, MA); phospho-AKT at Ser 473 (Cell Signaling); CIP4 (BD Transduction, Lexington, KY); PP2A (Upstate, Lake Placid, NY); TC10 (Santa Cruz, Santa Cruz, CA); Cbl (Santa Cruz); phospho-Cbl at Tyr 700 (Santa Cruz); PI3K (p85) (Santa Cruz); PI3K (p110) (Santa Cruz); insulin receptor substrate-1 (IRS-1, Santa Cruz); phosphor-IRS-1 at Tyr 941 (Santa Cruz); β-actin (Santa Cruz); and caveolin (BD Transduction). After incubation with the relative second antibody, immunoreactive signals were then detected using the chemiluminescent detection system (Amersham, Uppsala, Sweden), and were quantified using the Quantity One analysis software (Bio-Rad).
The data of body weight gain, visceral fat pad weight, and serum biochemistries are presented as means ± s.e.m. of 10 rats. The real-time reverse-transcription PCR data were presented as means ± s.e.m. of the quadruplicate analyses of the RNA samples pooled from 10 rats per group. Western blot analysis was repeated at least three times. Statistical significance was defined as P < 0.05 and was determined by Student's t-test using the SPSS software.
The body weight gain and the relative weight of the epididymal fat pad of the rats fed the HFD were 113 (P < 0.001) and 84% (P < 0.01) greater than the ND rats. The rats fed the HFD showed significantly higher serum levels of cholesterol (P < 0.01), glucose (P < 0.01), and insulin (P < 0.05) than the ND rats (Table 1).
Table 1. Body weight gain, visceral fat pad weight, and serum biochemistries
The HFD-induced changes in the expression of the epididymal adipose tissue genes involved in the insulin-signaling pathways were evaluated by the real-time reverse-transcription PCR analyses. Feeding the rats the HFD for 8 weeks significantly downregulated the expressions of CIP4 (0.72-fold, P < 0.05) and TC10 (0.68-fold, P < 0.05), whereas it upregulated the PP2A gene expression (3.3-fold, P < 0.05) in the epididymal adipose tissue. The HFD, however, did not alter the mRNA levels of CAP, GLUT4, Cbl, IRS-1, IRS-2, and AKT1 in the epididymal fat tissue of the rats (Table 2).
To determine whether the HFD altered the protein level of the signaling molecules involved in the insulin-signaling pathways, western blot analysis was performed using the whole-tissue extract (Figure 1) as well as the plasma membrane fraction (Figure 2) prepared from the epididymal adipose tissue of rats. The immunoblot results of the whole-tissue extract showed that the HFD led to a 74% increase (P < 0.05) in the level of PP2A proteins in the epididymal adipose tissue compared to that in the ND rats (Figure 1). The HFD suppressed the phosphorylation of major insulin-signaling molecules such as IRS-1 at Tyr 941 (56% reduction, P < 0.05) and AKT at Ser 473 (67% reduction, P < 0.05), compared with those in the ND rats, whereas it did not affect the protein levels of the total form of IRS-1 and AKT. The protein levels of PI3K (p110) were also downregulated (25% reductions, P < 0.05) in the epididymal adipose tissue of the rats fed the HFD compared with the ND rats. The CIP4 protein level was reduced by 41% (P < 0.05) in the visceral adipose tissue when the rats were fed the HFD (Figure 1). The western blot analysis results of plasma membrane fractions indicated that the HFD decreased the pCbl at Tyr 700 (25% lower) and TC10 (60% lower) protein levels in the epididymal adipose tissue of the HFD-fed rats compared with those for the ND rats (P < 0.05) (Figure 2).
Until recently, only a few studies have demonstrated the relationship between HFD and impaired insulin-stimulated GLUT4 translocation in the skeletal muscle of rodent models. Although the activation of several components involved in the CAP/Cbl and IRS/PI3K signaling cascades has garnered some preliminary evaluation in skeletal and cardiac muscles obtained from genetic, pharmacologically induced, or HFD-induced models of insulin resistance (13,14,15,16), to the best of our knowledge, the involvement and activation of these pathways have not been evaluated in the visceral adipose tissue obtained from HFD-fed rodents. Moreover, the molecular mechanisms that may account for the insulin resistance in visceral adipose tissue induced by HFD are still not well established. In this study, the hypothesis that the HFD-induced visceral obesity may be linked to alterations of PP2A, TC10, and CIP4 expression, which are potential downstream components of the IRS/PI3K/AKT and CAP/Cbl/TC10 pathways, was tested.
PP2A is a multimeric serine/threonine phosphatase that has been highly conserved during the evolution of eukaryotes (8). Numerous evidences indicated that PP2A dephosphorylates a diversity of kinases in vitro, including AKT, PKC, mitogen-activated protein/extracellular-regulated kinase kinase (MEK), and mitogen-activated protein kinase (8). A couple of recent reports cited the possible role of PP2A in the metabolic actions of insulin: okadaic acid, an inhibitor of PP2A, activated glucose transport and GLUT4 translocation in vitro (17) and PP2A expression was increased in the skeletal muscle biopsy samples obtained from patients with type 2 diabetes (18). The results of this study provided the evidence, for the first time, that the HFD-induced visceral obesity is accompanied with increases in the expression of PP2A at both the transcriptional and translational levels in the visceral adipose tissue. A marked reduction (67% decrease) in the level of phosphorylated AKT at Ser 473 in the cytosol of visceral adipocyte, with total form of AKT being unaffected, compared with those in the ND rats, strongly supports our hypothesis that PP2A may function as an important component of the PI3K/AKT signaling pathway by mediating the dephosphorylation of AKT at serine (Figure 3).
Another important question that has yet to be answered concerns the relationship between PP2A and IRS-1 activation in the insulin-signaling pathway in the visceral adipose tissue. The data of this study indicated that HFD led to a decrease in the tyrosine phosphorylation of IRS-1 (Figure 1). Tyrosine dephosphorylation and/or the serine phosphorylation of IRS-1 has been recognized as one of the main mechanisms that lead to insulin resistance (19). IRS proteins contain many potential serine phosphorylation sites, in addition to tyrosine phosphorylation sites (20), and the serine phosphorylation of IRS-1 is capable of regulating insulin signal transduction both positively (21) and negatively (22). The serine 307 phosphorylation of IRS-1 is particularly important in modulating the interaction between IRS-1 and the insulin receptor (23), and stimulated phosphorylation of IRS-1 at serine 307 was found in insulin resistance induced by a variety of agents (24). Although several serine/threonine kinases that phosphorylate IRS-1 have been reported (21,22,23), the phosphatases that act on these sites have not yet been identified. On the basis of recent studies using 3T3L1 adipocytes (9,25), PP2A appears not to be involved in the dephosphorylation of the serine 307 residue of IRS-1. Further work is needed to determine whether serine phosphorylated IRS-1 serves as a substrate for PP2A.
It was then assessed whether HFD-induced insulin resistance is mediated by CIP4 in relation to the Cbl/CAP/TC10 pathway, the second route for insulin-signaling pathway. Although several publications have described the relationship between Cbl activation and insulin resistance in muscles (13,16), the regulation of downstream components such as TC10 and CIP4 during HFD-induced obesity and insulin resistance has not yet been determined. TC10 is a member of the Rho family small G-proteins and its activation is involved in the remodeling of the actin cytoskeleton, which is necessary for GLUT4 translocation (10). This study exhibited that HFD induced marked downregulations of CIP4 and TC10 in the visceral adipose tissue of rats, at both the transcriptional and translational levels (Figure 3). Therefore, CIP4 appears to interact with TC10, and may have a role in the regulation of actin dynamics, and eventually, in GLUT4 translocation. These results contradict the recent findings by Bernard et al. (13) that the TC10 protein level was not altered in the skeletal muscle of rats fed the HFD (13) but correspond to the reports of Gupte and Mora (16) that the TC10 protein level was suppressed in the adipose tissue of ob/ob mice.
The insulin-stimulated GLUT4 translocation in the visceral fat tissue of rats has not been determined in this study. However, ample evidences demonstrate that a HFD-fed rodent or genetic animal model of type 2 diabetes exhibit reduced insulin-stimulated GLUT4 translocation to the cell surface along with the impairment of glucose transport (26,27,28). Miura et al. (26) reported the impairment of insulin-stimulated GLUT4 translocation in skeletal muscle and adipose tissue in the Tsumura Suzuki obese diabetic mouse. Furthermore, GLUT4 translocation was completely abrogated in the muscle of insulin-stimulated high fat-fed rats (15). Taken together, these results suggest that HFD might have a relevance to insulin resistance by increasing the expression of PP2A, an inhibitor of AKT activity in the PI3K/AKT pathway, and also by suppressing the expression of TC10 and CIP4, downstream effectors of the Cbl/CAP/TC10 insulin-signaling cascade in the visceral adipose tissue.
This work was supported by the Korea Research Foundation Grant funded by the Korean Government (Ministry of Education and Human Resources Development) (KRF-2006-311-C00180) and the Brain Korea 21 Project, Yonsei University.