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Department of Cell Physiology and Pharmacology, University of Leicester, Leicester LE1 9HN, United Kingdom, theDepartment of Infection Immunity and Inflammation, University of Leicester, Leicester LE1 9HN, United Kingdom, and the
To whom correspondence should be addressed: Dept. of Cell Physiology and Pharmacology, Medical Sciences Bldg., University of Leicester, University Rd., Leicester, LE1 9HN, United Kingdom. Tel.: 44-116-258-8043; Fax: 44-116-258-4764;
Department of Cell Physiology and Pharmacology, University of Leicester, Leicester LE1 9HN, United Kingdom, theDepartment of Infection Immunity and Inflammation, University of Leicester, Leicester LE1 9HN, United Kingdom, and the
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Peroxisome proliferator-activated receptor γ (PPARγ) has key roles in the regulation of adipogenesis, inflammation, and lipid and glucose metabolism. C-peptide is believed to be inert and without appreciable biological functions. Recent studies suggest that C-peptide possesses multiple functions. The present study investigated the effects of insulin and C-peptide on PPARγ transcriptional activity in opossum kidney proximal tubular cells. Both insulin and C-peptide induced a concentration-dependent stimulation of PPARγ transcriptional activity. Both agents substantially augmented thiazolidinedione-stimulated PPARγ transcriptional activity. Neither insulin nor C-peptide had any effect on the expression levels of PPARγ. GW9662, a PPARγ antagonist, blocked PPARγ activation by thiazolidinediones but had no effect on either insulin- or C-peptide-stimulated PPARγ transcriptional activity. Co-transfection of opossum kidney cells with dominant negative mitogen-activated protein kinase kinase significantly depressed basal PPARγ transcriptional activity but had no effect on that induced by either insulin or C-peptide. Both insulin- and C-peptide-stimulated PPARγ transcriptional activity were attenuated by wortmannin and by expression of a dominant negative phosphatidylinositol (PI) 3-kinase p85 regulatory subunit. In addition PI 3-kinase-dependent phosphorylation of PPARγ was observed after stimulation by C-peptide or insulin. C-peptide effects but not insulin on PPARγ transcriptional activity were abolished by pertussis toxin pretreatment. Finally both C-peptide and insulin positively control the expression of the PPARγ-regulated CD36 scavenger receptor in human THP-1 monocytes. We concluded that insulin and C-peptide can stimulate PPARγ activity in a ligand-independent fashion and that this effect is mediated by PI 3-kinase. These results support a new and potentially important physiological role for C-peptide in regulation of PPARγ-related cell functions.
Connecting peptide (C-peptide) is a 31-amino-acid enzymatic cleavage product derived from proinsulin during the biosynthesis and release of insulin from pancreatic beta cells. Although insulin and C-peptide are released into the circulation in equimolar amounts, it has become generally accepted that C-peptide does not possess biological activity of its own. This view has been challenged, however, with recent studies suggesting that C-peptide may possess multiple and significant functions (
This discovery of the physiological effects of C-peptide has been accompanied by the elucidation of various cell-signaling actions. Recently we have shown that C-peptide stimulates extracellular signal-regulated mitogen-activated protein (ERK MAP)
). Other workers have shown in addition that C-peptide increases nitric oxide production by increasing endothelial nitric oxide synthase protein via ERK MAP kinase-dependent up-regulation of endothelial nitric oxide synthase gene transcription (
) supports the notion of commonality between insulin and C-peptide signaling pathways.
The peroxisome proliferator-activated receptors (PPARs) are members of the nuclear receptor superfamily of ligand-activated transcription factors. Three PPAR isoforms, PPARα, PPARβ (also known as δ), and PPARγ have been identified, and all isoforms are present in the kidneys of humans and other species (
). PPARs regulate gene expression by binding as heterodimers with one of three retinoid X receptor proteins to cis-acting peroxisome proliferator response elements (PPRE) within the promoter regions of target genes (
). These metabolic effects of TZDs are mediated via PPARγ-regulated transcription of genes involved in glucose and lipid homeostasis. Similarly, insulin also promotes an altered expression of important genes regulating glucose and fatty acids metabolism (
Given this evidence of cross-talk between signaling pathways we postulated that C-peptide, insulin signaling, and PPARγ functions could be linked. Therefore we investigated the effects of C-peptide and insulin on PPARγ transcriptional activity in opossum kidney (OK) PT cells. In this report we demonstrate that both insulin and C-peptide activate PPARγ in a ligand-independent manner in OK cells. This phenomenon is pertussis toxin-sensitive and reliant upon PI 3-kinase but not ERK MAP kinase activity. Further, when applied to monocytes both C-peptide and insulin induce the expression of the PPARγ-regulated macrophage differentiation marker CD36. These findings for the first time implicate C-peptide in the activation of PPARγ with significant functional consequences.
Materials—Human 31-amino-acid C-peptide and a 31-amino-acid scrambled C-peptide were generously provided by Dr. John Wahren, Karolinska Institute (Stockholm, Sweden). Ciglitazone was from Biomol (Mamhead, UK). GW9662 was purchased from Cayman Chemical Co (Nottingham, UK). Bovine insulin was obtained from Sigma. Wortmannin and PTX were obtained from Calbiochem. Anti-PPARα, anti-PPARβ, anti-PPARγ, and anti-PI 3-kinase p85-α were from Santa Cruz Biotechnology Inc. The plasmid, pCMX-PPARγ encoding mouse PPAR-γ1 was kindly provided by Dr. R. Evans (Salk Institute, San Diego, CA). The reporter plasmid pPPRE-TK-luc was kindly provided by Dr. M. Lazar (University of Pennsylvania, Philadelphia, PA). The plasmid pFC-MEK1 encoding constitutively active MAP kinase kinase (MEK-1) (pMEK1-CA), and the kinase-dead mutant of MEK-1 in pCMV5 (pMEK1-KD) were provided by Dr. J. Blank (University of Leicester, Leicester, UK). Luciferase assays were performed using the LucLite kit (Packard, Pangbourne, England). β-Galactosidase assay kits were obtained from Promega. General laboratory chemicals were from Sigma unless otherwise stated.
Cell Culture and Transfection Studies—Wild-type OK cells were obtained from Dr. J. Caverzasio (University Hospital, Geneva, Switzerland). Mutant OK cells expressing a dominant negative form of the p85 regulatory subunit of the class 1A p85/p110 PI 3-kinase (Δp85) or the wild-type p85 under the control of the LacSwitch have been described previously (
). Wild-type OK cells were maintained in Dulbecco's modified Eagle's medium-Ham's F12 mix supplemented with 10% fetal calf serum, 2 mm l-glutamine, 100 units/ml penicillin, and 0.1 mg/ml streptomycin. Δp85 cells were maintained in the above medium with the addition of 200 μg/ml hygromycin and 300 μg/ml G418. The human monocytic cell line THP-1 was obtained from ATCC (Manssas, VA) and cultured in RPMI 1640 medium containing 10% fetal calf serum, 100 units/ml penicillin, and 0.1 mg/ml streptomycin. All cells were incubated at 37 °C in a humidified atmosphere of 5% CO2, 95% air. In all experiments cells were serum-starved overnight before being subjected to agonist stimulation to ensure as far as possible that cells were not exposed to mitogenic agents or C-peptide derived from fetal calf serum.
All transient transfection studies were performed in fully supplemented medium using FuGENE 6 transfection reagent (Roche Diagnostics) according to the manufacturer's instructions with OK cells growing in 24-well plates at 50% confluence. OK cells were transfected with pPPPE-TK-luc and various combinations of pCMX-PPAR-γ, pMEK1-CA, pMEK1-KD. and pSVβgal. For 24-well plates 0.25 μg of each plasmid DNA was used in each well, and in experiments involving the transfection of multiple plasmids, equivalent concentrations of DNA were maintained by addition the appropriate empty vector.
Where appropriate native and recombinantly expressed proteins were detected in cell lysates by Western blotting using anti-PPARα, anti-PPARβ, anti-PPARγ, anti-PI 3-kinase p85-α, anti-CD36, or anti-β-actin primary antisera. In all cases, primary antibodies were visualized using peroxidase-conjugated secondary antibodies and enhanced chemiluminescence (Amersham Biosciences).
Luciferase Reporter Assays—24 h after transient transfection with pPPTE-TK-luc and/or other plasmids, cells were incubated in serum-free medium for 18 h before stimulation with various potential PPARγ agonists. Where applicable 100 ng/ml PTX was added at this stage to the serum-free medium. In some experiments inhibitors were applied to cells 30 min prior to the addition of agonists, and the cells were subsequently incubated with agonists with or without inhibitors for a further 24 h. Medium was then removed, and cells were lysed in a buffer containing of 500 mm HEPES, 2% Triton N101, 1 mm CaCl2, 1 mm MgCl2, pH 7.8. Cell lysis was allowed to proceed for 10 min, and luciferase activity was measured using the LucLite assay kit in a LumiCount luminometer (Packard, Pangbourne, England). In all experiments a 50-μl aliquot of lysate was removed for β-galactosidase assay, and luciferase activity was normalized to β-galactosidase content.
[32P]Orthophosphate Labeling of OK Cells and Immunoprecipitation of Phospho-PPARγ—Confluent wild-type OK cells in 6-well plates were serum-starved overnight, incubated with or without 100 nm wortmannin for 30 min, and then washed three times with phosphate-free Dulbecco's modified Eagle's medium-Ham's F12 mix. Cells were then incubated with serum-free medium containing 200 μCi/ml [32P]orthophosphate and 5 nm C-peptide or 100 nm insulin for 4 h at 37 °C. Cell monolayers were then washed three times with ice-cold phosphate-buffered saline, pH 7.4, and incubated for 30 min with ice-cold lysis buffer composed of 1% Nonidet P-40, 0.5% sodium deoxycholate, 0.1% sodium dodecyl sulfate, and 200 μm sodium vanadate made up in phosphate-buffered saline, pH 7.4. The lysates were clarified by 5 min of centrifugation, and the supernatant was precleared with protein A-Sepharose for 45 min at 4 °C and then incubated with 10 μl of anti-PPARγ overnight at 4 °C. Immune complexes were collected using protein A-Sepharose beads. The beads were then washed three times with lysis buffer and then 35 μl of Laemmli buffer (60 mm Tris, pH 6.8, 10% glycerol, 2% SDS, 100 mm dithiothreitol, and 0.01% bromphenol blue) was added. Immunoprecipitated proteins were then separated on 12% polyacrylamide gels, and radiolabeled proteins were detected by autoradiography. Densitometry of phosphorylated protein bands was performed using Scion Image Beta 4.0.2 image analysis software, Scion, Frederick, MD.
THP-1 Cell Differentiation Experiments—THP-1 cells were plated in 6-well plates in serum-free medium containing potential stimulators of differentiation, including as a positive control 1 μm phorbol myristate acetate, for either 24 or 48 h. Cells were collected by centrifugation, washed twice with serum-free medium, and then lysed in polyacrylamide gel electrophoresis loading buffer. Expression of the differentiation marker CD 36 (
) was examined in whole cell lysates by Western blotting using anti-β-actin as a loading control.
Statistical Analyses—Data are presented as the mean ± S.E. Data were analyzed using unpaired Student's t test for comparison between two experimental groups. For multiple comparisons, one-way analysis of variance with Tukey's correction was used. Differences were considered significant at p < 0.05.
Western blotting was used to determine PPAR isoform expression in OK cells whole cell lysates. Our previous PPRE-luciferase reporter studies in OK cells (
) indicated transcriptional activation only by PPARγ or PPARβ ligands. In agreement with this observation both PPARγ and PPARβ but not PPARα proteins were detected in OK cells by Western blotting in the current study (Fig. 1). A concentration-dependent increase in luciferase expression in pPPRE-TK-luc-transfected OK cells was observed after stimulation with the TZD ciglitazone (maximum 207 ± 2.8% of control., EC50 80 nm), C-peptide (maximum 170 ± 6.5% of control, EC50 4 nm), and insulin (maximum 250 ± 4.5% of control, EC50 10 nm) (Fig. 2).
To confirm that the observed effects of C-peptide or insulin were mediated via activation of PPARγ cells were transfected with pPPRE-TK-luc ± pCMX-PPARγ (Fig. 3A). Overexpression of PPARγ enhanced both basal PPRE activity and that induced by 5 nm C-peptide, 100 nm insulin, or 5 μm ciglitazone by 11, 20, and 14-fold, respectively, above that observed in non-stimulated, wild-type pPPRE-TK-luc-transfected OK cells. Increased levels of PPARγ protein in pCMX-PPARγ-transfected cells were confirmed by Western blotting (Fig. 3B). Combining either 5 nm C-peptide or 100 nm insulin with 5 μm ciglitazone resulted in an augmented luciferase response (Fig. 4) compared with that seen in cells treated with each agonist alone (270 ± 6 and 400% ± 8, respectively, compared with 190 ± 3% for ciglitazone alone). However the combination of insulin and C-peptide was not synergistic in terms of luciferase activity (Fig. 4). The effects of C-peptide and insulin on the PPARγ-mediated activation of PPRE were not related to changes in expression of PPARγ itself (Fig. 5).
To determine whether C-peptide or insulin per se are PPARγ ligands, or whether treatment with these agents caused the release of intracellular PPARγ ligands, cells were pretreated with 0.5 μm GW9662, a selective irreversible PPARγ antagonist, for 30 min before the application of 100 nm insulin, 5 nm C-peptide, or 5 μm ciglitazone. GW9662 treatment significantly inhibited the effects of ciglitazone on PPRE activity to ∼60% of that observed compared with non-GW9662-treated cells. However, neither C-peptide nor insulin-induced PPRE activity was affected by GW9662 treatment (Fig. 6).
Insulin has been shown previously to activate PPARγ via a ligand-independent mechanism involving PPARγ phosphorylation by ERK MAP kinase. To determine whether this was the operative mechanism in the current experiments OK cells were pretreated with or without 5 μm PD98059, a specific inhibitor of MEK1, for 30 min before stimulation with 5 nm C-peptide or 100 nm insulin, and the antagonist was kept during the entire stimulation period. The effect of C-peptide or insulin on PPRE activation was not attenuated to any significant level by the PD98059 treatment (Fig. 7A). In cells transfected with pMEK1-CA basal unstimulated PPRE-driven luciferase expression was increased by 270 ± 10%, and no further significant stimulation by agonists was observed (Fig. 7B). Transfection with pMEK1-KD markedly inhibited basal transactivation to ∼55% ± 8, but the effects of C-peptide or insulin were not attenuated compared with the new basal luciferase expression (Fig. 7B).
Pretreatment with 100 nm wortmannin for 30 min prior to stimulation with 5 nm C-peptide or 100 nm insulin attenuated the effects of both insulin and C-peptide on PPRE activation (Fig. 8A). Furthermore in cells induced to express Δp85 the stimulatory effects of insulin and C-peptide on PPRE-mediated luciferase activity were significantly attenuated compared with non-IPTG-induced Δp85-transfected controls (Fig. 8B). Induced expression of wild-type p85 had no effect on C-peptide or insulin-induced activation of PPARγ (Fig. 8C). Successful induction of recombinant p85/Δp85 was confirmed by Western blotting (Fig. 8D).
Immunoprecipitation of PPARγ from [32P]-labeled cells revealed that both C-peptide and insulin treatment resulted in phosphorylation of PPARγ (Fig. 9). Inhibition of PI 3-kinase activity with wortmannin had no effect on basal PPARγ phosphorylation but abolished that stimulated by both insulin and C-peptide. Exposure of cells to 100 ng/ml PTX for 18 h prior to stimulation completely abolished the effect of C-peptide on PPRE activity but did not attenuate the effect of insulin to any significant level (Fig. 10).
The expression of the monocyte/macrophage differentiation marker CD36 in THP-1 cells was examined as an example of a protein product of a well established PPARγ-regulated gene. Stimulation of the cells with 5 nm C-peptide, 100 nm insulin, or 1 μm phorbol myristate acetate for 48 h increased CD36 protein levels ∼2-, 2.2-, and 2.5-fold over the basal level, respectively (Fig. 11).
A growing body of evidence challenges the generally accepted dogma that C-peptide is devoid of biological function (
) we now provide for the first time unequivocal confirmation that treatment of OK PT cells with C-peptide at physiologically relevant concentrations results in significant transactivation of PPRE. Likewise we also demonstrated a similar, but less potent, dose-dependent effect of insulin itself. With respect to the PPAR isoform responsible for this effect, PPARα is not expressed in these cells and accordingly shows no response to PPARα agonists (
), and thus we reasoned that PPARs α and β were unlikely to be involved. We therefore hypothesized that PPARγ was the subtype mediating PPRE transactivation and confirmed this by demonstrating augmentation of C-peptide- and insulin-evoked PPRE activity in cells overexpressing recombinant PPARγ. In adipocytes insulin treatment leads to increased expression of both PPARγ mRNA and protein (
). Enhanced expression of the PPARγ protein was not induced by insulin or C-peptide in OK cells in the current study, and thus changes in PPARγ expression levels cannot explain the demonstrated increase in PPRE transactivation by insulin and C-peptide in wild-type OK cells.
The TZD ciglitazone is a prototypic PPARγ ligand that also transactivates PPRE in OK cells. Combining ciglitazone with C-peptide or insulin resulted in synergistic PPARγ-mediated transactivation of PPRE whereas combining C-peptide with insulin did not, suggesting distinct means of PPARγ activation. Although it seemed unlikely that either insulin or C-peptide themselves could be ligands for PPARγ we considered the possibility that an endogenous intracellular PPARγ ligand may be produced as a result of their respective signaling capabilities. GW9662 binds to the ligand-binding domain of PPARγ resulting in the covalent modification of Cys285 thus conferring upon GW9662 properties of a full PPARγ antagonist because of irreversible loss of ligand binding (
). Interestingly in the current study GW9662 was able to inhibit ciglitazone but not C-peptide- or insulin-mediated PPRE transactivation. Therefore unlike the situation with the prototypic TZD PPARγ ligands, activation of PPARγ by insulin and C-peptide appears to be independent of ligand binding. Overall these results are consistent with a model whereby insulin and C-peptide stimulate PPARγ-mediated PPRE activation via signaling cascades downstream of their cell surface receptors. Indeed, for C-peptide at least, the pertussis toxin sensitivity of PPRE activation is coherent with the evolving concept of C-peptide-mediated signaling via a Gαi- or Gαo-coupled receptor (
Post-translational modification of PPARγ by N-terminal phosphorylation may be linked with positive or negative regulation of the ligand-independent transcriptional activity. Several groups of workers have demonstrated that ERK MAP kinase-mediated phosphorylation of the serine 82 of PPARγ1 and the serine 112 of PPARγ2 is accompanied by a reduction in transcriptional activity (
) demonstrated that activators of protein kinase A evoked a ligand-independent activation and phosphorylation of PPARγ. Ligand-independent activation of PPARγ by insulin has previously been reported by Zhang et al. (
) who showed that insulin and TZDs combined synergistically to stimulate PPARγ-mediated transactivation of an aP2 promoter-luciferase reporter gene in association with the stimulation of PPARγ phosphorylation by activation of the ERK MAP kinase pathway. An insulin-regulated, ligand-independent activation domain in the N-terminal region of PPARγ has also been delineated by Werman et al. (
Despite the previous reports that ERK MAP kinase is a key regulator of ligand-independent PPARγ activity, our results provided several lines of evidence to suggest that the ERK MAP kinase pathway is not involved in C-peptide- or insulin-mediated alterations in PPARγ activity in OK cells. Firstly, although we have demonstrated previously a robust ERK MAP kinase activation by C-peptide in OK cells (
), the stimulatory effects of C-peptide and insulin on PPRE transactivation were not affected by a chemical ERK MAP kinase inhibitor. Secondly, although overexpression of a dominant negative MEK1 significantly depressed basal transactivation of PPARγ, activity the effects of C-peptide and insulin on PPRE transactivation were preserved. In OK cells, rather than inhibiting PPARγ transcriptional activity as demonstrated by others, expression of constitutively active MEK1 leads to a maximally enhanced basal activation of endogenous PPARγ activity such that ligand-dependent and -independent activation can no longer be detected.
In view of our earlier observations that C-peptide activated PI 3-kinase in OK cells (
), we studied the reliance of ligand-independent PPARγ activation by these agents on the activity of PI 3-kinase. Wortmannin-induced attenuation of the PPRE transactivation stimulated by both C-peptide and insulin strongly implicates PI 3-kinase in this effect, and the concordant results of the experiments using Δp85 confirm the involvement of type 1A PI 3-kinase. Furthermore, ligand-independent activation of PPARγ by C-peptide and insulin is clearly associated with PI 3-kinase-dependent phosphorylation of PPARγ, although the precise kinase mediating the phosphorylation is not established by these studies.
These observations raise a major question as to whether the PPRE activation induced by C-peptide and insulin observed using luciferase reporter constructs is mirrored by transcriptional activation of established PPARγ-regulated genes in native cells. Although data regarding PPARγ function in PT cells is beginning to accrue, little information is available regarding specific PPARγ-regulated genes in these cells. A variety of PPARγ regulated genes have been identified in macrophages, and co-localization of C-peptide with macrophages in atherosclerotic lesions described in diabetic patients suggests a potential role for C-peptide in regulation of function in this cell type (
). By treating the human monocyte cell line THP-1 with insulin and C-peptide, and comparing with a phorbol ester inducer of differentiation, we provided proof of the principle that C-peptide has the ability to induce the expression of PPARγ-regulated genes.
In summary our results demonstrated C-peptide- and insulin-evoked, phosphorylation-associated, ligand-independent activation of PPARγ with the requirement for PI 3-kinase activity in OK cells. These effects measured using reporter constructs are mirrored by C-peptide- and insulin-stimulated expression of PPARγ-regulated genes in a monocyte cell line. The findings indicate a potentially major new role for C-peptide in the regulation of cell biology, insulin sensitivity, glucose homeostasis, and PPARγ function.