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Originally published In Press as doi:10.1074/jbc.M408268200 on September 16, 2004

J. Biol. Chem., Vol. 279, Issue 48, 49747-49754, November 26, 2004
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Ligand-independent Activation of Peroxisome Proliferator-activated Receptor-{gamma} by Insulin and C-peptide in Kidney Proximal Tubular Cells

DEPENDENT ON PHOSPHATIDYLINOSITOL 3-KINASE ACTIVITY*

Nawal M. Al-Rasheed{ddagger}§, Ravinder S. Chana{ddagger}, Richard J. Baines{ddagger}, Gary B. Willars{ddagger}, and Nigel J. Brunskill{ddagger}||

From the {ddagger}Department of Cell Physiology and Pharmacology, University of Leicester, Leicester LE1 9HN, United Kingdom, the Department of Infection Immunity and Inflammation, University of Leicester, Leicester LE1 9HN, United Kingdom, and the §Department of Pharmacology, King Saud University, Riyadh, Saudi Arabia

Received for publication, July 21, 2004 , and in revised form, September 15, 2004.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Peroxisome proliferator-activated receptor {gamma} (PPAR{gamma}) 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 PPAR{gamma} transcriptional activity in opossum kidney proximal tubular cells. Both insulin and C-peptide induced a concentration-dependent stimulation of PPAR{gamma} transcriptional activity. Both agents substantially augmented thiazolidinedione-stimulated PPAR{gamma} transcriptional activity. Neither insulin nor C-peptide had any effect on the expression levels of PPAR{gamma}. GW9662, a PPAR{gamma} antagonist, blocked PPAR{gamma} activation by thiazolidinediones but had no effect on either insulin- or C-peptide-stimulated PPAR{gamma} transcriptional activity. Co-transfection of opossum kidney cells with dominant negative mitogen-activated protein kinase kinase significantly depressed basal PPAR{gamma} transcriptional activity but had no effect on that induced by either insulin or C-peptide. Both insulin- and C-peptide-stimulated PPAR{gamma} 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 PPAR{gamma} was observed after stimulation by C-peptide or insulin. C-peptide effects but not insulin on PPAR{gamma} transcriptional activity were abolished by pertussis toxin pretreatment. Finally both C-peptide and insulin positively control the expression of the PPAR{gamma}-regulated CD36 scavenger receptor in human THP-1 monocytes. We concluded that insulin and C-peptide can stimulate PPAR{gamma} 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 PPAR{gamma}-related cell functions.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
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 (1). Thus C-peptide has been shown to improve glucose utilization both in vivo and in vitro. Specifically, in streptozotocin-induced diabetic rats C-peptide stimulates whole body glucose utilization (2) and stimulates glucose transport in healthy human skeletal muscle (3, 4). In the BB/Wor rat model of type 1 diabetes chronic C-peptide infusion prevents both acute and chronic metabolic, functional, and structural changes (5, 6).

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)1 kinase, phosphatidylinositol (PI) 3-kinase, and protein kinase C activity in kidney proximal tubular (PT) cells (7). 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 (8). C-peptide also enhances DNA-cAMP-response element-binding protein/ATF1 interactions via the p38 mitogen-activated protein kinase pathway in the mouse lung capillary endothelial cell (9). The majority of these effects are pertussis toxin (PTX)-sensitive indicating that C-peptide binds to a G{alpha}i/o-coupled receptor (1, 10). Enhancement by C-peptide of the insulin-stimulated insulin receptor autophosphorylation and tyrosine kinase activity (11) 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, PPAR{alpha}, PPAR{beta} (also known as {delta}), and PPAR{gamma} have been identified, and all isoforms are present in the kidneys of humans and other species (12, 13). 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 (14). Each PPAR isoform exerts distinct actions on cellular function and exhibits different specificity in ligand-binding properties (15–17).

Glitazones or thiazolidinediones (TZDs) are synthetic high affinity PPAR{gamma} ligands that act as insulin-sensitizing agents and are currently used for the treatment of type 2 diabetes mellitus (18). They improve lipid profiles together with insulin sensitivity and glucose tolerance in patients with type 2 diabetes and thus control hyperglycemia (19, 20). These metabolic effects of TZDs are mediated via PPAR{gamma}-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 (21–23). Several lines of evidence indicate convergence between insulin and PPAR{gamma} signaling pathways. Firstly the TZD, pioglitazone, augments insulin-stimulated tyrosine phosphorylation of IR/IRS-1 (19), secondly insulin participates in the control of PPAR{gamma} expression (23, 24), and finally insulin mediates ligand-independent activation of PPAR{gamma} in a phosphorylation-dependent manner (25).

Given this evidence of cross-talk between signaling pathways we postulated that C-peptide, insulin signaling, and PPAR{gamma} functions could be linked. Therefore we investigated the effects of C-peptide and insulin on PPAR{gamma} transcriptional activity in opossum kidney (OK) PT cells. In this report we demonstrate that both insulin and C-peptide activate PPAR{gamma} 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 PPAR{gamma}-regulated macrophage differentiation marker CD36. These findings for the first time implicate C-peptide in the activation of PPAR{gamma} with significant functional consequences.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
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-PPAR{alpha}, anti-PPAR{beta}, anti-PPAR{gamma}, and anti-PI 3-kinase p85-{alpha} were from Santa Cruz Biotechnology Inc. The plasmid, pCMX-PPAR{gamma} encoding mouse PPAR-{gamma}1 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). {beta}-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 ({Delta}p85) or the wild-type p85 under the control of the LacSwitch have been described previously (26). 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. {Delta}p85 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-{gamma}, pMEK1-CA, pMEK1-KD. and pSV{beta}gal. 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-PPAR{alpha}, anti-PPAR{beta}, anti-PPAR{gamma}, anti-PI 3-kinase p85-{alpha}, anti-CD36, or anti-{beta}-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 PPAR{gamma} 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 {beta}-galactosidase assay, and luciferase activity was normalized to {beta}-galactosidase content.

[32P]Orthophosphate Labeling of OK Cells and Immunoprecipitation of Phospho-PPAR{gamma}—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-PPAR{gamma} 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 (27, 28) was examined in whole cell lysates by Western blotting using anti-{beta}-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.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
Western blotting was used to determine PPAR isoform expression in OK cells whole cell lysates. Our previous PPRE-luciferase reporter studies in OK cells (29) indicated transcriptional activation only by PPAR{gamma} or PPAR{beta} ligands. In agreement with this observation both PPAR{gamma} and PPAR{beta} but not PPAR{alpha} 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).



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  		FIG. 1.
Expression of PPAR subtypes in OK cells. PPAR subtypes were detected by immunoblotting of cell lysates. Blots are representative of three experiments.

 



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  		FIG. 2.
C-peptide- and insulin-stimulated PPAR{gamma} activity in OK cells. OK cells transiently transfected with pPPRE-TK-luc and pSV{beta}gal were treated for 24 h with the indicated concentrations of C-peptide, insulin, or ciglitazone. Cells were lysed, and luciferase activity in lysates was determined and normalized for transfection efficiency using {beta}-galactosidase activity. Normalized luciferase activity is expressed as the percentage compared with control in non-stimulated cells. Results are expressed as means ± S.E. of three or four experiments

 
To confirm that the observed effects of C-peptide or insulin were mediated via activation of PPAR{gamma} cells were transfected with pPPRE-TK-luc ± pCMX-PPAR{gamma} (Fig. 3A). Overexpression of PPAR{gamma} 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 PPAR{gamma} protein in pCMX-PPAR{gamma}-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 PPAR{gamma}-mediated activation of PPRE were not related to changes in expression of PPAR{gamma} itself (Fig. 5).



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  		FIG. 3.
C-peptide- and insulin-stimulated PPAR activity is mediated though PPAR{gamma}. A, OK cells were transiently transfected with pPPRE-TK-luc and pSV{beta}gal co-transfected with pCMX-PPAR{gamma} or empty vector. Transiently transfected cells were treated with the indicated concentrations of C-peptide, insulin, or ciglitazone. Cells were lysed, and luciferase activity was determined and normalized for transfection efficiency using {beta}-galactosidase activity. Normalized luciferase activity is expressed as a percentage compared with that measured in non-stimulated pPPRE-TK-luc- and pSV{beta}gal-transfected wild-type cells. Results are expressed as means ± S.E. of three experiments. B, overexpression of PPAR{gamma} in transfected cells. Whole cell lysates of wild-type and pCMX-PPAR{gamma}-transfected cells were immunoblotted using monoclonal anti-PPAR{gamma}. This blot is representative of three independent experiments. *, p < 0.05; **, p < 0.01 versus control.

 



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  		FIG. 4.
Synergistic stimulation of PPAR{gamma} activity by both C-peptide and insulin with TZD. Cells were transiently transfected with pPPRE-TK-luc and pSV{beta}gal to normalize transfection efficiency to {beta}-galactosidase activity. Transiently transfected OK cells were treated for 24 h with the indicated concentrations of C-peptide, insulin, ciglitazone, or combination. Cells were lysed, and luciferase activity in the lysates was determined. Normalized luciferase activity is expressed as a percentage compared with control in non-stimulated cells. Results are expressed as means ± S.E. of three experiments. *, p < 0.05; **, p < 0.01 versus ciglitazone alone.

 



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  		FIG. 5.
Effects of C-peptide and insulin on protein levels of PPAR{gamma} in OK cells. Cells were treated with indicated concentrations of C-peptide or insulin for 24 h, and PPAR{gamma} was detected by immunoblotting. Membranes were stripped and reprobed for {beta}-actin as a loading control. This is a representative blot of four independent experiments.

 
To determine whether C-peptide or insulin per se are PPAR{gamma} ligands, or whether treatment with these agents caused the release of intracellular PPAR{gamma} ligands, cells were pretreated with 0.5 µM GW9662, a selective irreversible PPAR{gamma} 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).



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  		FIG. 6.
PPAR{gamma} antagonist GW9662 has no effect on C-peptide- or insulin-induced PPAR{gamma} activity. Cells were transiently transfected with pPPRE-TK-luc and pSV{beta}gal to normalize transfection efficiency. Transiently transfected cells were treated with GW9662 for 30 min before stimulation and during treatment for 24 h with the indicated concentrations of C-peptide, insulin, and ciglitazone. Cells were lysed, and the luciferase activity in lysates was determined. Normalized luciferase activity is expressed as a percentage compared with the control in non-stimulated, non-GW9662-treated cells. Results are expressed as the means ± S.E. of three experiments. *, p < 0.05 relative to ciglitazone alone.

 
Insulin has been shown previously to activate PPAR{gamma} via a ligand-independent mechanism involving PPAR{gamma} 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).



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  		FIG. 7.
ERK MAP kinase is not required for C-peptide- or insulin-induced PPAR{gamma} activity. Cells were transiently transfected with pPPRE-TK-luc and pSV{beta}gal to normalize for transfection efficiency. A, transiently transfected cells were treated with 5 µM PD98059 for 30 min prior to stimulation and during treatment for 24 h with the indicated concentrations of C-peptide and insulin. B, cells were co-transfected with either constitutive active MEK1, dominant negative MEK1, or empty vector. Transiently transfected cells were treated for 24 h with the indicated concentrations of C-peptide or insulin. Cells were lysed, and the luciferase activity in lysates was determined. Normalized luciferase activity is expressed as a percentage compared with control in non-stimulated cells. Results are expressed as the means ± S.E. of three experiments.

 
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 {Delta}p85 the stimulatory effects of insulin and C-peptide on PPRE-mediated luciferase activity were significantly attenuated compared with non-IPTG-induced {Delta}p85-transfected controls (Fig. 8B). Induced expression of wild-type p85 had no effect on C-peptide or insulin-induced activation of PPAR{gamma} (Fig. 8C). Successful induction of recombinant p85/{Delta}p85 was confirmed by Western blotting (Fig. 8D).



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  		FIG. 8.
PI 3-kinase is necessary for C-peptide- and insulin-induced PPAR{gamma} activity. Cells were transiently transfected with pPPRE-TK-luc and pSV{beta}gal to normalize for transfection efficiency. A, transiently transfected wild-type cells were treated with or without 100 nM wortmannin for 30 min before stimulation for 24 h with the indicated concentrations of C-peptide or insulin. B, transiently transfected {Delta}p85-transfected OK cells were treated with or without 5 mM IPTG for 24 h prior to stimulation for a further 24 h with the indicated concentrations of C-peptide or insulin. C, transiently transfected wild-type p85-transfected OK cells were treated with or without 5 mM IPTG for 24 h prior to stimulation for a further 24 h with the indicated concentrations of C-peptide or insulin. Cells were lysed, and the luciferase activity in the lysates was determined. Normalized luciferase activity is expressed as a percentage compared with control in non-stimulated, non-wortmannin-treated, or non-IPTG-treated cells. Results are expressed as means ± S.E. of three or four experiments. D, induction of {Delta}p85 and p85 expression by IPTG in OK cells. {Delta}p85-transfected OK cells and p85-transfected OK cells were treated with 5 mM IPTG for 24 h, and lysates were immunoblotted with anti-PI 3-kinase p85{alpha}. Blots are representative of three independent experiments.

 
Immunoprecipitation of PPAR{gamma} from [32P]-labeled cells revealed that both C-peptide and insulin treatment resulted in phosphorylation of PPAR{gamma} (Fig. 9). Inhibition of PI 3-kinase activity with wortmannin had no effect on basal PPAR{gamma} 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).



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  		FIG. 9.
Phosphorylation of PPAR{gamma} in response to C-peptide and insulin. Wild-type OK cells were labeled with [32P]orthophosphate, treated with the indicated concentrations of C-peptide and insulin with or without wortmannin pretreatment, and then immunoprecipitated with anti-PPAR{gamma}. Immunoprecipitates were collected on Protein A-Sepharose and then subjected to PAGE followed by autoradiography. A representative gel is shown in the upper panel where the labeled band represents phosphorylated PPAR{gamma}. The lower panel depicts the combined results of densitometric analysis from three identical experiments. *, p < 0.05 relative to non-stimulated, non-wortmannin-inhibited control condition.

 



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  		FIG. 10.
PTX inhibits C-peptide but not insulin-induced activation of PPAR{gamma}. Cells were transiently transfected with pPPRE-TK-luc and pSV{beta}gal to normalize for transfection efficiency. A, transiently transfected wild-type OK cells were treated with or without 100 ng/ml PTX for 18 h prior to stimulation for 24 h with the indicated concentrations of C-peptide or insulin. Cells were lysed, and the luciferase activity in lysates was determined. Normalized luciferase activity is expressed as a percentage compared with control in non-stimulated cells. Results are expressed as means ± S.E. of three experiments. *, p < 0.05 relative to non-PTX-treated, C-peptide-stimulated cells.

 
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 PPAR{gamma}-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).



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  		FIG. 11.
C-peptide and insulin increase CD36 protein levels in THP-1 cells. Cells were treated with the indicated concentrations of C-peptide, insulin, or phorbol myristate acetate (PMA) in serum-free medium for the indicated times. CD36 protein was detected by immunoblotting. A representative blot is shown (upper panel). Bands were quantified by densitometry and values expressed graphically (lower panel) as the -fold increase over the basal. Results are expressed as means ±S.E., n = 3. *, p < 0.01 relative to control at the same time point.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
A growing body of evidence challenges the generally accepted dogma that C-peptide is devoid of biological function (1, 30). Particularly interesting are the insulinomimetic effects of C-peptide whereby cross-talk is observed between C-peptide and insulin signaling pathways (11), C-peptide stimulates glucose uptake and glycogen synthesis in muscle cells (3), and C-peptide increases whole body glucose utilization in streptozotocin diabetic rats (2). In view of our developing interests in the function of the insulin sensitizer PPAR{gamma} in the kidney (29, 31), and primarily using OK PT cells as a model, we elected to determine whether C-peptide and/or insulin were able to activate the transcriptional activity of PPAR{gamma}.

Building on our earlier work examining C-peptide signaling in kidney PT cells (7) 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, PPAR{alpha} is not expressed in these cells and accordingly shows no response to PPAR{alpha} agonists (29). Furthermore OK cells demonstrate only very modest responses to PPAR{beta} agonists (29), and thus we reasoned that PPARs {alpha} and {beta} were unlikely to be involved. We therefore hypothesized that PPAR{gamma} was the subtype mediating PPRE transactivation and confirmed this by demonstrating augmentation of C-peptide- and insulin-evoked PPRE activity in cells overexpressing recombinant PPAR{gamma}. In adipocytes insulin treatment leads to increased expression of both PPAR{gamma} mRNA and protein (23, 24). Enhanced expression of the PPAR{gamma} protein was not induced by insulin or C-peptide in OK cells in the current study, and thus changes in PPAR{gamma} 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 PPAR{gamma} ligand that also transactivates PPRE in OK cells. Combining ciglitazone with C-peptide or insulin resulted in synergistic PPAR{gamma}-mediated transactivation of PPRE whereas combining C-peptide with insulin did not, suggesting distinct means of PPAR{gamma} activation. Although it seemed unlikely that either insulin or C-peptide themselves could be ligands for PPAR{gamma} we considered the possibility that an endogenous intracellular PPAR{gamma} ligand may be produced as a result of their respective signaling capabilities. GW9662 binds to the ligand-binding domain of PPAR{gamma} resulting in the covalent modification of Cys285 thus conferring upon GW9662 properties of a full PPAR{gamma} antagonist because of irreversible loss of ligand binding (32). 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 PPAR{gamma} ligands, activation of PPAR{gamma} 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 PPAR{gamma}-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 G{alpha}i- or G{alpha}o-coupled receptor (10, 33).

Post-translational modification of PPAR{gamma} 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 PPAR{gamma}1 and the serine 112 of PPAR{gamma}2 is accompanied by a reduction in transcriptional activity (34, 35). In contrast Lazennec and co-workers (36) demonstrated that activators of protein kinase A evoked a ligand-independent activation and phosphorylation of PPAR{gamma}. Ligand-independent activation of PPAR{gamma} by insulin has previously been reported by Zhang et al. (37) who showed that insulin and TZDs combined synergistically to stimulate PPAR{gamma}-mediated transactivation of an aP2 promoter-luciferase reporter gene in association with the stimulation of PPAR{gamma} phosphorylation by activation of the ERK MAP kinase pathway. An insulin-regulated, ligand-independent activation domain in the N-terminal region of PPAR{gamma} has also been delineated by Werman et al. (25).

Despite the previous reports that ERK MAP kinase is a key regulator of ligand-independent PPAR{gamma} 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 PPAR{gamma} activity in OK cells. Firstly, although we have demonstrated previously a robust ERK MAP kinase activation by C-peptide in OK cells (7), 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 PPAR{gamma}, activity the effects of C-peptide and insulin on PPRE transactivation were preserved. In OK cells, rather than inhibiting PPAR{gamma} transcriptional activity as demonstrated by others, expression of constitutively active MEK1 leads to a maximally enhanced basal activation of endogenous PPAR{gamma} 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 (7), a capacity also shared by insulin (26), we studied the reliance of ligand-independent PPAR{gamma} 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 {Delta}p85 confirm the involvement of type 1A PI 3-kinase. Furthermore, ligand-independent activation of PPAR{gamma} by C-peptide and insulin is clearly associated with PI 3-kinase-dependent phosphorylation of PPAR{gamma}, 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 PPAR{gamma}-regulated genes in native cells. Although data regarding PPAR{gamma} function in PT cells is beginning to accrue, little information is available regarding specific PPAR{gamma}-regulated genes in these cells. A variety of PPAR{gamma} 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 (38, 39). The type B scavenger receptor CD36 is expressed during differentiation of monocytes to macrophages, and its expression is regulated by PPAR{gamma} (28, 40). CD36 is also important in the protection against insulin resistance and is essential for the actions of insulin sensitizing actions of TZDs (41–43). 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 PPAR{gamma}-regulated genes.

In summary our results demonstrated C-peptide- and insulin-evoked, phosphorylation-associated, ligand-independent activation of PPAR{gamma} 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 PPAR{gamma}-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 PPAR{gamma} function.


   FOOTNOTES
 
* 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. Back

|| 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; E-mail: njb18{at}le.ac.uk.

1 The abbreviations used are: ERK MAP, extracellular signal-regulated mitogen-activated protein; PPAR, peroxisome proliferator-activated receptor; PPRE, peroxisome proliferator response element; OK, opossum kidney; PT, proximal tubular; TZD, thiazolidinedione; PTX, pertussis toxin; PI 3-kinase, phosphatidylinositol 3-kinase; IPTG, isopropyl 1-thio-{beta}-D-galactopyranoside. Back



   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 

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