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Endocrinology, doi:10.1210/en.2006-1403
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Endocrinology Vol. 148, No. 3 1089-1098
Copyright © 2007 by The Endocrine Society

Protein Kinase C{zeta} Is Required for Oleic Acid-Induced Secretion of Glucagon-Like Peptide-1 by Intestinal Endocrine L Cells

Roman Iakoubov, Angelo Izzo, Andrea Yeung, Catharine I. Whiteside and Patricia L. Brubaker

Departments of Physiology (R.I., A.I., A.Y., P.L.B.) and Medicine (C.I.W., P.L.B.), University of Toronto, Toronto, Ontario, Canada M5S 1A8; and Department of Internal Medicine I (R.I.), Merheim Medical Center, D-51109 Cologne, Germany

Address all correspondence and requests for reprints to: Dr. P. L. Brubaker, Room 3366 Medical Sciences Building, University of Toronto, 1 King’s College Circle, Toronto, Ontario, Canada M5S 1A8. E-mail: p.brubaker{at}utoronto.ca.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Long-chain, monounsaturated fatty acids (FAs) stimulate secretion of the incretin hormone, glucagon-like peptide-1 (GLP-1) from the intestinal L cell. Because the atypical protein kinase C (PKC), PKC{zeta}, is involved in FA signaling in many cells, the role of PKC{zeta} in FA-induced GLP-1 secretion was investigated, using the murine GLUTag L cell line and primary rat intestinal L cells. GLUTag cells expressed mRNA for several PKC isoforms, including PKC{zeta}, and PKC{zeta} protein was localized throughout the cytoplasm in GLUTag and primary L cells as well as normal mouse and rat L cells. Treatment with oleic acid (150–1000 µM) for 2 h increased GLP-1 secretion (P < 0.001), and this was abrogated by the PKC{zeta} inhibitor ZI (P < 0.05) and PKC{zeta} small interfering RNA transfection (P < 0.05) but not inhibition of classical/novel PKC isoforms. Although most PKC{zeta} was localized in the particulate compartment of GLUTag cells, oleate treatment did not alter PKC{zeta} levels or activity in this cell fraction. GLUTag cells expressed mRNA for the Gq-coupled FA receptor GPR120; however, oleic acid did not induce any changes in Akt, MAPK, or calcium, and pretreatment with LY294002 and PD98059 to inhibit phosphatidylinositol 3-kinase and MAPK, respectively, did not prevent the effects of oleic acid. Finally, GLUTag cells also released GLP-1 in response to arachidonic acid (P < 0.001) but were not affected by other long-chain FAs. These findings demonstrate that PKC{zeta} is required for oleic acid-induced GLP-1 secretion. This enzyme may therefore serve as a therapeutic target to enhance GLP-1 release in type 2 diabetes.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
GLUCAGON-LIKE PEPTIDE (GLP)-1 is an intestinal hormone involved in the regulation of nutrient homeostasis through insulinotropic effects on the ß-cell as well as inhibition of glucagon release and gastric emptying and induction of satiety (1, 2). Together, these biological actions contribute to the ability of GLP-1 to reduce glycemia and hemoglobin A1c levels as well as prevent body weight gain in patients with type 2 diabetes (3, 4). Furthermore, GLP-1 exerts multiple effects to increase ß-cell mass in rodents through enhancement of islet neogenesis and ß-cell proliferation and inhibition of ß-cell apoptosis (5, 6). These antidiabetic actions of GLP-1 have resulted in intense interest in the development of GLP-1 and its long-acting analogs as a therapy for type 2 diabetes (2, 7, 8). An alternative approach to the exogenous administration of GLP-1 is stimulation of endogenous release, possibly in combination with approaches to reduce GLP-1 degradation (9). However, circulating GLP-1 levels are actually reduced in patients with type 2 diabetes (10, 11). Because GLP-1 clearance is normal in these individuals (12), these findings suggested that GLP-1 secretion is attenuated in type 2 diabetes. It is therefore important that the mechanisms underlying GLP-1 release from the intestinal L cell be elucidated.

The majority of the GLP-1-secreting L cells are located in the distal small intestine and colon (13). GLP-1 release from the L cell is regulated by nutrient ingestion, demonstrating a biphasic pattern of secretion in response to either mixed meals or carbohydrate or fat alone (14, 15). The first phase of GLP-1 secretion begins within minutes after a meal and is regulated by neural signals originating in the proximal intestine (16, 17, 18). In contrast, the second phase is induced by direct nutrient stimulation of the L cells, resulting in prolonged secretion of GLP-1 (19, 20, 21). Importantly, the increased GLP-1 secretion in response to fat appears to be physiologically relevant because only fats reach the distal intestine in sufficient concentrations to be able to stimulate the L cell directly (22). Furthermore, in vitro studies using primary rat L cells in culture have shown that the GLP-1 response to fat is highly specific, requiring monounsaturated fatty acids (MUFAs) with a chain length of 16 or more carbons (e.g. palmitoleic acid, 16:1; or oleic acid, 18:1) (19). Similarly, the murine GLUTag L cell line has been demonstrated to respond preferentially to long-chain MUFAs (20, 23). Consistent with these findings, GLP-1 secretion is increased by diets enriched in long-chain MUFAs, compared with saturated fat in both rats and humans (24, 25). One recent study implicated a novel G protein-coupled (GPR), long-chain fatty acid (FA) receptor, GPR120, in the regulation of GLP-1 secretion from a secretin tumor cell (STC-1) line (26, 27). Similar studies demonstrated a role for the related FA receptor, GPR40, in the regulation of insulin secretion (28). Both GPR120 and GPR40 are Gq-coupled receptors linked to the activation of Akt, MAPK, and/or calcium signaling. However, roles for these pathways in FA-induced GLP-1 secretion have not been demonstrated (26, 27).

The mammalian protein kinase C (PKC) family consists of 12 isozymes grouped into several classes: classical (cPKC: {alpha}, -ßI, -ßII, and -{gamma}), novel (nPKC: {delta}, -{epsilon}, -{theta}, and -{eta}), atypical (aPKC: -{zeta} and -{lambda}/{iota}), and PKC-related (µ and {nu}) (29, 30). Importantly, a number of studies implicated unsaturated fatty acids as regulators of aPKC activity and translocation (31, 32, 33), and aPKCs have been demonstrated to play a role in the regulation of insulin secretion by long-chain FAs (34). Furthermore, we have previously shown that prolonged treatment of primary rat intestinal L cells with phorbol-12-myristate-13-acetate (PMA) to down-regulate cPKC and nPKC isoforms, or treatment with inhibitors of these PKC isozymes, does not affect oleate-stimulated GLP-1 release (19). We therefore hypothesized that an aPKC isoform, specifically PKC{zeta}, may be involved in the regulation of GLP-1 secretion by oleic acid.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Cell models
The murine GLUTag L cell line was cultured in DMEM supplemented with 10% (vol/vol) fetal bovine serum (FBS), as previously described (20, 35). Medium was changed every 2–3 d, and cells were passaged by trypsinization and reseeding at a 1:3 dilution. Fetal rat intestinal cell (FRIC) cultures were prepared as previously described (19, 36, 37, 38, 39). In brief, fetal Wistar rat intestines were collected on d 19–20 of gestation, and the cells were dispersed and plated in 60-mm culture dishes for 24 h in DMEM containing 5% (vol/vol) FBS, 50 IU/ml penicillin, and 50 µg/ml streptomycin. All animal protocols were approved by the Animal Care Committee of the University of Toronto. Both the GLUTag and FRIC models used in this study have been validated with respect to the regulation of GLP-1 secretion such that, consistent with in vivo findings, GLP-1 release is stimulated by a wide variety of secretagogues, including gastrin-releasing peptide, glucose-dependent insulinotropic peptide, leptin, muscarinic agonists, and oleic but not palmitic acid (18, 19, 20, 35, 36, 37, 38, 39, 40, 41).

GLP-1 secretion experiments
Palmitoleic (16:1), oleic (18:1), {alpha}-linolenic (18:3), and {gamma}-linolenic (18:3) acids (Sigma Chemical Co., St. Louis, MO) were dissolved in 0.5 N NaOH at 80–90 C, whereas palmitic (16:0), stearic (18:0), linoleic (18:2), and arachidonic (20:4) acids (Sigma) were dissolved in absolute ethanol. The resulting stock solutions were added to calcium-free DMEM containing 0.5% free FA-free BSA (Sigma). CaCl2 was then added to a final concentration of 1.8 mM (42). GLUTag cells treated with an equivalent volume of either NaOH or ethanol alone did not show any changes in GLP-1 secretion (data not shown).

For secretion experiments, GLUTag cells were plated in 24-well culture plates and allowed to recover for a minimum of 2 d (~70% confluence). On the day of the experiment, GLUTag cells or FRIC cultures were washed once with DMEM containing 5.6 mmol/liter glucose and 0.5% FA-free BSA and incubated with media alone (control), PMA (positive control for classical and novel PKCs), or different FAs (150–1000 µM) for 2 h. A similar time course has previously been used for studies on GLP-1 release in response to FAs at concentrations of up to 1500 µM in the absence of BSA (19, 20, 23). Some cells were preincubated for 24 h with 25 µM ZI [a cell-permeant myristoylated peptide (RRGARRWRK) that inhibits aPKCs, including PKC{zeta} (43, 44); Centre for Applied Genomics, Hospital for Sick Children, Toronto, Ontario, Canada], for 45 min with 1 µM UCN-01 [7-hydroxystaurosporine, a specific inhibitor of classical and novel isoforms of PKC (45, 46); a generous gift from Kyowa Hakko Kogyo Co. Ltd., Tokyo, Japan] or for 30 min with either 100 µM LY294002 (Calbiochem, San Diego, CA) or 50 µM PD98059 (Sigma) to inhibit phosphatidylinositol 3-kinase (PI3-K) or p44/p42 MAPK, respectively. At the end of the incubation period, medium was collected, centrifuged at 1300 x g for 10 min to remove any floating cells, and 200 µl of 1% (vol/vol) trifluoroacetic acid was added. Attached cells were collected by scraping and homogenized in extraction medium [1 N HCl containing 5% (vol/vol) HCOOH, 1% NaCl (wt/vol), and 1% (vol/vol) trifluoroacetic acid]. Peptides were extracted from each fraction by passage through a cartridge of C18 silica (Sep-Pak; Waters Associates, Mississauga, Ontario, Canada). Samples were subjected to RIA for GLP-1, as previously reported (20, 35), and GLP-1 secretion was calculated as the total amount of GLP-1 in the medium, normalized for the total content of GLP-1 in the cells plus medium; total content did not differ during the 2-h treatment period with oleic acid (data not shown). We previously demonstrated that this method effectively extracts GLP-1 from cells and media and that bioactive GLP-17–36NH2 is the predominant form of GLP-1 synthesized and secreted by GLUTag cells and FRIC cultures (35, 47).

Viability of the GLUTag cells after FA treatment was determined using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, as previously reported (48). In brief, cells were plated in 96-well dishes for 24 h and then treated with medium alone (control), 5 mM H2O2 (positive control), or 500 µM oleic acid for 2 h, after which the MTT reaction was carried out and the resulting absorbance was determined at 570 nm. Increasing absorbance correlates with increased cell viability in this assay.

RT-PCR
Total RNA was extracted from 10-cm plates of GLUTag cells using an RNeasy kit as instructed by the manufacturer (QIAGEN Inc., Mississauga, Ontario, Canada). RNA (1 µg) was reverse transcribed and the PCR was performed using a OneStep RT-PCR kit (QIAGEN). The primers used for amplification are listed in Table 1Go and were obtained from Invitrogen Canada, Inc. (Burlington, Ontario, Canada). PKCß, -{gamma}, -{delta}, -{lambda}, and -µ primers were based on those published elsewhere (49), whereas other primers were designed using PrimerQuest (http://scitools.idtdna.com/Primerquest). Negative controls included omission of the template as well as PCR without reverse transcription. Products were analyzed by agarose gel electrophoresis and visualized with ethidium bromide.


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  		TABLE 1. PCR primers

 
Immunofluorescence
GLUTag cells were plated on glass coverslips in 6-well dishes, allowed to grow for 2 d, and then washed with ice-cold PBS and fixed in methanol at –20 C for 10 min, followed by fixation in formalin overnight. FRIC cultures were plated in multiwell chamber slides, allowed to grow overnight, and washed with PBS and fixed in acetone at –20 C for 5 min. Formalin-fixed, paraffin-embedded intestinal sections from 129Sv mice and Wistar rats were deparaffinized in xylene and then rehydrated through a graded series of ethanol. Cells and tissue sections were washed with PBS or TBST [0.1 M Tris-HCl and 0.15 M NaCl (pH 7.4) with 0.1% Triton X-100], respectively, pretreated with blocking solution (4–10% donkey serum in PBS/TBST) for 30 min and incubated overnight with blocking solution containing mouse antihuman GLP-1 (1:100, a kind gift from Dr. D. D’Alessio, University of Cincinnati, Cincinnati, OH) and/or rabbit antirat PKC{zeta} [1:500; no. sc-216, Santa Cruz Biotechnologies, Inc. (Santa Cruz, CA); this antiserum also detects PKC{lambda} (see Fig. 1Go); in a humid chamber]. Slides were then washed three times with PBS/TBST and treated with donkey antimouse or donkey antirabbit IgG secondary antibody, as appropriate, labeled with either Cy2 or Cy3 (1:200 to 1:500; Jackson ImmunoLaboratories Ltd., West Grove, PA) for 1–3 h. Slides were washed again with PBS/TBST (three times for 30 min) and then mounted with mounting medium for fluorescence containing DAPI (VectaShield; Vector Laboratories, Inc., Burlingame, CA). Pictures were taken using an Axioplan deconvolution microscope (Carl Zeiss Canada, Ltd., Don Mills, Ontario, Canada). The specificity of the staining was shown by a lack of detectable immunofluorescence when primary antisera were omitted (data not shown).


Figure 1
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  		FIG. 1. Expression of PKC isoforms in GLUTag cells. A, Total RNA from GLUTag cells was analyzed by RT-PCR for the PKC isoforms {alpha}, -ß, -{gamma}, -{delta}, -{lambda} -µ, and -{zeta}, and the products separated on agarose gels and visualized with ethidium bromide. A representative gel is shown from n = 3 independent experiments, with the molecular weight ladder on the left. B, Total protein from two preparations of GLUTag cells was analyzed by Western blot for aPKC isoforms, using a PKC{zeta} antiserum that also recognizes PKC{lambda}. The molecular weight (MW) ladder is shown on the left.

 
In vitro PKC{zeta} kinase activity
PKC{zeta} activity assay was performed on GLUTag immunoprecipitates by 32P-phosphorylation of Ser159-PKC{epsilon} pseudosubstrate peptide (44, 50), with some modifications. In brief, GLUTag cells were grown on 10-cm culture dishes and starved for 48 h in DMEM containing 0.5% FA-free BSA. Cells were then treated with media alone (control) or with 500 µM oleic acid for 2 h, as above, washed with ice-cold PBS, scraped into 200 µl of lysis buffer [1 mM NaHCO3, 5 mM MgCl2 · 6H2O, 50 mM Tris HCl, 10 mM EGTA, 2 mM EDTA (pH 7.5) and 1x EDTA-free protease inhibitor tab (Roche Diagnostics, Laval, Québec, Canada)] and passed 15 times through a 25-gauge needle. After incubation on ice for 30 min, samples were centrifuged at 100,000 x g for 1 h, and the supernatants were collected and used as the cytosol fraction. The pellets were resuspended in lysis buffer containing 1% Triton X-100 and centrifuged at 100,000 x g for 1 h, and the supernatant was used as the membrane fraction. The particulate fraction was not examined in this assay due to the requirement for solubilization using sodium dodecyl sulfate. Protein content of the cytosolic and membrane fractions was determined by Bradford assay (Bio-Rad Laboratories Canada Ltd., Mississauga, Ontario, Canada), and 500 µg of protein were added to 500 µl immunoprecipitation buffer [25 mM HEPES, 150 mM NaCl, 1 mM EGTA, 2 mM EDTA, 10 mM NaF, 50 mM ß-glycerophosphate, 1 mM Na3VO4, 1% Triton X-100, and 100 nM okadeic acid (pH 7.5)] containing 2 µg polyclonal rabbit PKC{zeta} antiserum (no. sc-216; Santa Cruz Biotechnologies) or 2 µg rabbit IgG (for determination of nonspecific enzyme activity; Santa Cruz Biotechnologies). Samples were rocked overnight at 4 C, followed by immunoprecipitation with 65 µl Protein G Plus-Agarose (Santa Cruz Biotechnologies) over 1 h. The samples were centrifuged at 15,000 x g for 1 min and washed three times with immunoprecipitation buffer and then three times with kinase buffer [50 mM Tris HCl, 5 mM Mg-acetate, 1 mM NaF, 0.1 mM Na3VO4, and 0.1 mM Na2P2O7 · 10 H2O (pH 7.5)]. Samples were recollected in 40 µl kinase buffer containing 40 µM ATP, 50 µCi [{gamma}-P32]ATP (Amersham Biosciences Inc., Baie d’Urfe, Québec, Canada), 2.5 µM okadeic acid, 5 µg PKC{epsilon} substrate (44), and 1:20 of an EDTA-free protease inhibitor tab. After an 8-min incubation at 32 C, the reaction was terminated by the addition of 25 µl quench solution [0.1 mM ATP and 100 mM EDTA (pH 8.0)], and 45 µl of the product were spotted on P-81 cellulose disks (Whatman Inc., Clifton, NJ), washed three times with 75 mM phosphoric acid, and then washed once with 80% ethanol. The disks were then air dried overnight, and the counts per minute determined by liquid scintillation counting.

Western blot
The effect of oleic acid on Akt and/or MAPK phosphorylation was examined in 10-cm plates of GLUTag cells treated with media alone (control) or oleic acid (500 µM) or insulin (100 nM; positive control) for 5 min. PKC{zeta} protein levels were determined in 10-cm plates of GLUTag cells treated with media alone (control) or 500 µM oleic acid, as above. Some cells were fractionated into cytosolic and membrane fractions, as described above, with the final pellet being dissolved in 10% sodium dodecyl sulfate to make the particulate fraction (44). Protein was determined by Bradford assay, and 10–50 µg of protein from whole-cell extracts or 6, 30, and 10 µg of protein from the cytoplasmic, membrane, and particulate fractions, respectively, were analyzed by Western blot using rabbit antiphosphoSer473-Akt (1:1000), rabbit antitotal Akt (1:1000), rabbit antiphospho-p44/p42 MAPK (1:1000), rabbit antitotal MAPK (1:1000; all from Cell Signaling Technology Inc., Beverly, MA), rabbit antirat PKC{zeta} (1:1000; no. sc-216, Santa Cruz Biotechnologies), and rabbit antipanactin (1:5000; Sigma) primary antisera, followed by detection using horseradish peroxidase (HRP)-linked goat antirabbit IgG (1:2000) with an enhanced chemiluminescence detection kit (both from Amersham Biosciences), as previously described (5, 18, 44).

Calcium imaging
GLUTag cells were seeded on glass coverslips and allowed to recover for 2 d and were then washed, preincubated in calcium-free medium with 0.5% FA-free BSA for 2 h, loaded with 5 µM Fluo-3 (Invitrogen Canada, Inc.) in the same media containing 0.04% pluronic acid for 30 min, and allowed to recover for 30 min. Cells were then transferred to media containing calcium, and single cells were photographed under a fluorescence microscope (Carl Zeiss Canada) every 20 sec for 7 min, with addition of 500 µM oleic acid at t = 1 min and 10 µM ionomycin (Sigma) at t = 3 min. Relative fluorescence intensity was analyzed using ImageJ software (National Institutes of Health, Bethesda, MD).

Small interfering (si) RNA transfection
GLUTag cells were plated in poly-D-lysine-coated 24-well dishes and allowed to recover for 48 h. Scrambled siRNA (control) and two siRNAs targeting PKC{zeta} coding sequences were custom designed by Ambion (Austin, TX). Transfection was performed in Opti-MEM medium using siRNAs (20 pM) and 1 µl Lipofectamine 2000 (Invitrogen) as instructed by the manufacturer. The cells were incubated for 4 h, washed twice with DMEM containing 5.6 mmol/liter glucose and 10% (vol/vol) FBS and allowed to recover for 48 h. GLP-1 secretion experiments and determination of PKC{zeta} protein levels via Western blot were performed as described above.

Statistical analyses
All results are expressed as mean ± SEM. Statistical analysis was performed with SAS software (SAS Institute, Cary, NC) using one- and two-way ANOVA followed by post hoc testing, as appropriate. Some data were log10 transformed before analysis to normalize variances. Significance was assumed at P < 0.05.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
RT-PCR for PKC{alpha}, -ß, -{gamma}, -{delta}, -{lambda}, -µ, and -{zeta} mRNA transcripts was performed on total RNA extracted from GLUTag cells to determine the expression of specific isoforms of PKC. As shown in Fig. 1AGo, all of the tested PKC isozymes, except for the PKC-related enzyme, PKCµ, were detected in the GLUTag cells. To determine whether the mRNA transcript for PKC{zeta} is translated into protein in the L cell, Western blot analysis of GLUTag whole-cell extracts was performed (Fig. 1BGo). The cells were found to express a major band at 78 kDa, corresponding to the molecular mass of PKC{zeta} (31). After longer exposures, a minor band at 67 kDa was detected in some cell preparations, consistent with the presence of very small amounts of the other atypical PKC, PKC{lambda} (51). When detectable, this band represented less than 12% of the total immunoreactivity detected by the PKC{zeta} antiserum.

Examination of the GLUTag cells by immunofluorescence demonstrated the presence of PKC{zeta} throughout the cytoplasm, with some protein also found in a punctate pattern in the nucleus (Fig. 2AGo). Immunoreactive PKC{zeta} also colocalized with GLP-1 in the cytoplasm of L cells in the normal mouse intestine (Fig. 2BGo). Similarly, PKC{zeta} was distributed throughout the cytoplasm of GLP-1-expressing cells in primary FRIC cultures as well as in L cells in the normal rat intestine (Fig. 2Go, C and D).


Figure 2
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  		FIG. 2. Distribution of PKC{zeta} in GLUTag, FRIC, and rodent. A, PKC{zeta} (red) was visualized in GLUTag cells with a rabbit anti-PKC{zeta} antiserum followed by Cy3-coupled donkey antirabbit IgG. Nuclei were stained with DAPI (blue) (x1000 magnification). B–D, Double-label immunofluorescence for GLP-1 (green) and PKC{zeta} (red) in mouse intestinal sections (x1000) (B), FRIC cultures (x400) (C), and rat intestinal sections (x400) (D). GLP-1 was detected using a mouse anti-GLP-1 antiserum followed by Cy2-coupled donkey antimouse IgG, and PKC{zeta} was visualized as above.

 
To determine the dose-dependent effects of long-chain FA on GLP-1 secretion, GLUTag cells were incubated in DMEM containing 0.5% FA-free BSA for 2 h in the absence or presence of oleic acid. Basal secretion was 13.1 ± 2.1% of total cell content, whereas PMA (positive control for cPKCs and nPKCs) significantly increased secretion to 27.6 ± 2.1% (P < 0.001; Fig. 3AGo). Oleic acid had no effect on GLP-1 secretion at 150 µM but significantly stimulated secretion at both 500 and 1000 µM, to 24.2 ± 1.9 and 26.6 ± 1.2% of total cell content (P < 0.001), respectively. Because no difference between the two higher doses was observed, 500 µM was selected for use in all further experiments. Cell viability studies demonstrated that the oleic acid treatment did not cause any cell death and, in fact, slightly increased cell viability (by 33 ± 12%, P < 0.05), compared with control cells (Fig. 3BGo).


Figure 3
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  		FIG. 3. Effects of oleic acid on GLP-1 secretion and cell viability. A, GLUTag cells were treated for 2 h with media alone (with 0.5% FA-free BSA; control), PMA (1 µM; positive control), or oleic acid (150–1000 µM), and the media and cell content of GLP-1 were determined by RIA (n = 6–10). ***, P < 0.001 vs. control. B, GLUTag cells were treated for 2 h with media alone (control), H2O2 (5 mM; positive control), or oleic acid (500 µM), followed by determination of cell viability by MTT assay. *, P < 0.05 vs. control. C and D, GLUTag cells (C) and primary FRIC cells (D) were pretreated for 24 h with media alone or 25 µM ZI before treatment for 2 h with media alone (control), PMA (1 µM; positive control), or 500 µM oleic acid without or with continued ZI (n = 6–10). *, P < 0.05; **, P < 0.01; ***, P < 0.001 vs. control; #, P < 0.05 vs. oleate alone. E, GLUTag cells were pretreated for 2 h with media alone or with 1 µM UCN-01, before treatment for 2 h with media alone (control), PMA (1 µM; positive control), or 500 µM oleic acid in the absence or presence of UCN-01 (n = 5–6). *, P < 0.05 vs. paired control; #, P < 0.05 vs. PMA alone.

 
To establish whether PKC{zeta} is involved in oleate-induced GLP-1 secretion, GLUTag cells were pretreated for 24 h with the PKC{zeta} inhibitor, ZI (25 µM; Fig. 3CGo). Whereas ZI alone did not affect GLP-1 secretion, preincubation with ZI significantly reduced the stimulatory effect of 500 µM oleate on the GLUTag cells to control levels (from 22.8 ± 2.8 to 14.5 ± 1.3% of total cell content, P < 0.05 vs. oleate; P > 0.05 vs. controls). Similar results were obtained with the primary FRIC cultures, in which ZI also completely abrogated oleic acid-induced GLP-1 secretion (Fig. 3DGo). Finally, to test the possible involvement of other PKC isoforms in oleate-induced GLP-1 secretion, GLUTag cells were pretreated for 45 min with 1 µM UCN-01, a staurosporine analog that inhibits classical and novel PKC isoforms without affecting PKC{zeta} (45, 46). UCN-01 pretreatment alone had no effect on GLP-1 secretion but significantly decreased PMA-induced GLP-1 secretion (from 21.6 ± 3.7 to 13.0 ± 1.0% of total cell content, P < 0.05; Fig. 3CGo). In contrast, oleic acid-induced GLP-1 secretion was not affected by pretreatment with UCN-01 (P > 0.05 vs. oleate alone, P < 0.05 vs. UCN-01 alone). We similarly reported that staurosporine or H7 (nonspecific c/nPKC inhibitors) or 24 h preincubation with PMA (to down-regulate c/nPKCs) does not prevent oleic acid-induced GLP-1 release by FRIC cultures (19).

To confirm the obligatory role of PKC{zeta} in oleate-induced GLP-1 secretion, PKC{zeta} expression levels were decreased in GLUTag cells using an siRNA approach, as confirmed by Western blot (Fig. 4AGo). Although oleic acid increased GLP-1 release in both control and siRNA-transfected cells (P < 0.05), the GLP-1 response to stimulation with oleic acid was significantly reduced in the siRNA-transfected cells, compared with control cells (125.7 ± 4.3 vs. 192.0 ± 35.0%, respectively, P < 0.05; Fig. 4BGo). When taken together with the inhibitor studies, these findings indicate that PKC{zeta}, but not the cPKC or nPKC isozymes, plays an essential role in oleate-stimulated release of GLP-1 from the GLUTag cells.


Figure 4
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  		FIG. 4. Effect of PKC{zeta} siRNA in GLUTag cells. GLUTag cells were treated with lipofectamine or transfected with scrambled siRNA or two different PKC{zeta} siRNAs, followed by a 48-h recovery and then stimulation with media alone (control) or 500 µM oleic acid (OA) for 2 h. A, GLUTag cells were examined by Western blot for PKC{zeta} (78 kDa) and ß-actin (42 kDa) using rabbit anti-PKC{zeta} and rabbit antipanactin antisera, respectively, followed by detection using HRP-linked goat antirabbit IgG and an enhanced chemiluminescence detection kit. –, Control cells; +, oleic acid-treated cells. B, GLUTag cells were treated for 2 h with media alone (control; –OA) or 500 µM oleic acid (+ OA). Data from cells treated with lipofectamine alone or scrambled siRNA were combined to make the control group, whereas that from cells treated with either of the two PKC{zeta} siRNAs were combined to form the PKC{zeta} siRNA group (n = 7–8). *, P < 0.05 vs. paired control; #, P < 0.05.

 
Whether oleic acid treatment altered the intracellular distribution of PKC{zeta} was examined by Western blot analysis on subcellular fractions of GLUTag cells treated for 2 h with media alone (control) or 500 µM oleic acid (Fig. 5AGo). Immunoreactive PKC{zeta} was detected in the cytosolic, membrane and particulate fractions; however, the majority of the protein (>90%) was found in the particulate fraction (10 µg of particulate fraction protein required only a 1 sec exposure, whereas 6 and 30 µg of cytosolic and membrane protein, respectively, required 30 sec exposures). Membrane levels of PKC{zeta} were increased by 2-fold (P < 0.05) in response to treatment with 500 µM oleic acid but did not change in either the cytoplasmic or particulate fraction. In contrast to these findings, PKC{zeta} enzyme activity in the membrane fraction was found to decrease by 39% (P < 0.05) in response to treatment of the GLUTag cells with oleic acid, with no changes observed in the cytosolic compartment (Fig. 5BGo).


Figure 5
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  		FIG. 5. PKC{zeta} protein and enzyme activity in subcellular fractions of GLUTag cells. GLUTag cells were treated for 2 h with media alone [control (C)] or 500 µM oleic acid (O) and then fractionated into cytosolic, membrane, and particulate fractions. A, PKC{zeta} (78 kDa) and ß-actin (42 kDa) were detected by Western blot using rabbit anti-PKC{zeta} and rabbit antipanactin antisera, respectively, followed by detection using HRP-linked goat antirabbit IgG. Representative blots and the results of quantification by densitometry are shown (n = 3–4). B, PKC{zeta} activity was determined in GLUTag cytosolic and membrane fractions using an in vitro assay (n = 4–7). *, P < 0.05 vs. control.

 
The nature of the cellular machinery involved in FA uptake and transport has not been previously explored in GLUTag cells. The cells were therefore examined for expression of mRNA transcripts for transport and binding proteins with selectivity for long-chain FAs, including the G protein-coupled membrane receptors, GPR40 (28) and GPR120 (26), the long-chain FA membrane transport proteins (FATPs), FATP1 and FATP4 (52, 53), and the intracellular intestinal FA binding and transport protein (iFABP) (54). All of these different transcripts were detected in the GLUTag cells (Fig. 6Go), consistent with the known capacity of these cells to respond to long-chain FA (present study and Refs. 20 and 23).


Figure 6
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  		FIG. 6. Expression of mRNA transcripts for FA uptake, translocation, and signaling proteins in GLUTag cells. Total RNA from GLUTag cells was analyzed by RT-PCR and the products separated on agarose gels and visualized with ethidium bromide. A, Detection of the long-chain FA membrane receptor (GPR40), iFABP, and FATP-1 and -4. Representative gels are shown from n = 3 independent experiments. B, Detection of the G protein-coupled fatty acid transporter, GPR120, in two separate extracts of GLUTag cells as well as mouse colon (positive control), using two separate primer pairs.

 
As the response of GPR40 and GPR120 to specific long-chain FA has recently been reported (26, 27, 28), GLUTag cells were treated with a variety of different saturated and unsaturated long-chain FA, ranging from 16 to 20 carbons in length (each at 500 µM in 0.5% BSA; Fig. 7Go). Only oleic acid and arachidonic acid were found to significantly stimulate GLP-1 secretion, by 36 ± 8 and 152 ± 32%, respectively (P < 0.05–0.001). Furthermore, the GPRs have been demonstrated to signal through a Gq-linked mechanism, increasing both calcium flux and phosphorylation of Akt as well as p44/p42 MAPK in a variety of different endocrine cell types (26, 27, 28). However, under the identical conditions in which oleic acid significantly stimulated GLP-1 secretion from the GLUTag cells, no changes in intracellular calcium concentrations were observed over either 2 (Fig. 8AGo) or 7 min (data not shown). Furthermore, no changes in Akt or MAPK phosphorylation were observed in response to oleate treatment (Fig. 8BGo). Positive controls in each experiment (e.g. ionomycin to enhance intracellular calcium levels and insulin treatment to increase Akt and p44/p42 MAPK phosphorylation) demonstrated that this lack of responsiveness to oleic acid was not due to an inability to induce or detect such changes in the GLUTag cells. Finally, pretreatment of the cells with 100 µM LY294002 or 50 µM PD98059 to inhibit PI3-K and MAPK signaling, respectively, did not prevent the effects of oleic acid on GLP-1 release (Fig. 8CGo).


Figure 7
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  		FIG. 7. Effects of different long-chain FA on GLP-1 secretion by GLUTag cells. GLUTag cells were treated for 2 h with media alone (control), PMA (1 µM; positive control), or different FA (palmitic 16:0; palmitoleic 16:1; stearic 18:0; oleic 18:1; linoleic 18:2; {alpha}-linolenic 18:3; {gamma}-linolenic 18:3; arachidonic 20:4; each at 500 µM), and the media and cell content of GLP-1 were determined by RIA (n = 6–10). *, P < 0.05; **, P < 0.01; and ***, P < 0.001 vs. control.

 

Figure 8
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  		FIG. 8. Effects of oleic acid on calcium, Akt, and MAPK signaling in GLUTag cells. A, GLUTag cells were loaded with fluo3 and single cells were analyzed for changes in fluorescence every 20 sec for 1 min (baseline), every 20 sec for 2 min after addition of 500 µM oleic acid, and every 20 sec for 4 min after addition of 10 µM ionomycin (calcium ionophore; positive control) (n = 4–5 cells from each of five different cell preparations). B, GLUTag cells were treated for 5 min with media alone (control) or 500 µM oleic acid or 100 nM insulin, followed by Western blot analysis of total protein for phosphorylated and total Akt and p44/p42 MAPK. Data are expressed as the ratio of phosphorylated to total protein (n = 3). C, GLUTag cells were pretreated for 30 min with media alone (control) or 100 µM LY294002 or 50 µM PD98059, followed by treatment for 2 h with oleic acid alone or LY294002 or PD98059, and the media and cell content of GLP-1 were determined by RIA (n = 6–10). *, P < 0.05; **, P < 0.01; ***, P < 0.001 vs. paired control.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Previous studies have established that long-chain MUFA stimulate release of GLP-1 from the intestinal L cell, both in vitro and in vivo (19, 20, 23, 24, 25). The intracellular mechanisms underlying FA-induced GLP-1 secretion have remained unclear. However, the demonstration that oleic acid-induced GLP-1 secretion in primary FRIC cultures is not prevented by either prolonged treatment with PMA to down-regulate cPKC and nPKC isoforms or pretreatment with staurosporine or H7 to inhibit these isozymes led to the hypothesis that an aPKC isoform, specifically PKC{zeta}, may be involved in the regulation of GLP-1 secretion by oleic acid (19).

The results of the present study demonstrated that, despite the presence of multiple PKC isoforms in the GLUTag cells, the GLP-1 response to oleic acid was prevented by pretreatment with an aPKC inhibitor and by siRNA-induced reductions in PKC{zeta} levels, but was not affected by inhibition of both classical and novel PKC isozymes. As the major aPKC expressed by GLUTag cells is PKC{zeta}, this isoform of PKC appears to play an essential role in oleic acid-induced GLP-1 secretion from the intestinal L cell. Similar findings were observed in primary rat intestinal cells, such that the aPKC inhibition abolished oleic-acid-induced GLP-1 secretion, consistent with our previous demonstration that inhibition of the diacylglycerol-sensitive c/nPKCs does not prevent long-chain MUFA-induced stimulation of GLP-1 release from the primary rat L cell (19). Nonetheless, PMA does stimulate GLP-1 secretion from the intestinal L cell, through the c/nPKCs. Interestingly, several PKC isozymes have also been detected in other gut endocrine cell populations (55, 56). Furthermore, bombesin- but not nutrient-induced hormone secretion by the GIP/Ins cell line has been reported to be dependent on the c/nPKCs (57). When taken together, these findings therefore indicate that PKC-mediated regulation of GLP-1 secretion from the intestinal L cell is complex, involving multiple isoforms of PKC that likely respond to different extracellular secretagogues but that PKC{zeta} appears to be the major mediator of oleic acid-stimulated GLP-1 release. Further experiments will be required to determinate whether the effects of PKC{zeta} are specific to oleic acid or include other secretagogues, such as arachidonic acid, acetylcholine, or gastrin-releasing peptide (17, 18).

The great majority of the immunoreactive PKC{zeta} expressed by both the primary L cell and the GLUTag cells was distributed in the cytoplasm, with over 90% found in the particulate compartment after GLUTag cell fractionation. Localization in the particulate fraction is consistent with a known role for PKC{zeta} in the organization of the cytoskeleton (58, 59, 60) that plays a key role in the regulation of hormone secretion (61, 62). In addition, several studies have shown that PKC{zeta} activation is not necessarily associated with translocation to a specific subcellular compartment (60, 63). Indeed, over 90% of the total PKC{zeta} present in the intestinal Caco2 cell line also resides in the particulate fraction and is found in the constitutively active state (60). Hence, although PKC{zeta} translocation to and activity in the membrane fraction were found to be modulated by oleic acid in the present study, it seems likely that the PKC{zeta} present in the particulate fraction plays a more important role in the regulation of GLP-1 secretion from the GLUTag cells. Further studies involving disruption of the cytoskeleton will clearly be necessary to more fully characterize the function of PKC{zeta} in this subcellular fraction of the GLUTag cells.

The results of the present study clearly demonstrate that oleic acid-stimulated GLP-1 secretion by the GLUTag cells was not associated with Akt, MAPK, or calcium. Previous studies have demonstrated that the cholecystokinin release from both the GLUTag and STC-1 cell lines by medium-chain saturated FAs (e.g. 10–14 carbons) increases intracellular calcium levels (23). Furthermore, recent studies using the STC-1 cell line have demonstrated that long-chain MUFAs stimulate GLP-1 secretion via activation of GPR120 in association with increased MAPK and calcium signaling (26). However, the effects of these FAs on hormone secretion by the STC-1 cells were not prevented by pretreatment with PD98059 or nifedipine, an L-type calcium channel inhibitor. Conversely, FA treatment was found to prevent apoptosis in the STC-1 cells (consistent with the findings of the present study) through GPR120-dependent stimulation of both PI3-K/Akt and MAPK (27). Thus, the relationship among FAs, enteroendocrine hormone secretion, and MAPK/intracellular calcium appears to be complex and, at least in part, dependent on FA chain length and/or degree of saturation.

The effects of different FAs on the STC-1 cells are markedly different from the pattern observed in the GLUTag cells, with stimulation by palmitoleic and {alpha}-linolenic acid but not oleic acid (26). Similar differences arise when considering the related FA receptor GPR40, which was also detected in the STC-1 cells (26). GPR40 has been found to play a role in FA stimulation of insulin secretion from the pancreatic ß-cell in response to oleic, {alpha}-linolenic, {gamma}-linolenic, and arachidonic acid (28). Furthermore, although this effect was independent of its ability to enhance MAPK phosphorylation, nifedipine pretreatment was able to prevent the effects of FA on insulin secretion. When taken together, the results of the present study suggest that, although GPR120 and -40 are expressed by the GLUTag cells, the effects of long-chain MUFAs on GLP-1 secretion from these cells are independent of these receptors.

Another well-established model for FA uptake across the cell membrane involves FA transport via the specific FATP transport proteins (64, 65). Once inside the cell, FA diffusion through the cytoplasm is facilitated by intracellular binding proteins, most notably iFABP in intestinal cells (54, 66, 67). Intracellular FAs can then be metabolized in the mitochondria or, alternatively, may directly modulate intracellular signaling pathways through acylation of proteins, modulation of protein trafficking, and exocytosis and/or direct activation of PKC, including PKC{zeta} (31, 68). Importantly, consistent with the preferential effect of MUFAs on both GLP-1 secretion and PKC{zeta}, iFABP exhibits a significantly higher binding affinity for unsaturated compared with saturated fatty acids (69, 70). Thus, because the GLUTag cells expressed mRNA transcripts for FATP1 and FATP4 as well as iFABP, this remains a possible mechanism by which oleic acid may activate PKC{zeta} in the L cell.

In summary, the results of the current study establish for the first time that PKC{zeta} is required for oleic acid-induced GLP-1 secretion. Because diets enriched in monounsaturated fats have demonstrated benefits with respect to both GLP-1 secretion and glycemic control (24, 25), these findings implicate PKC{zeta} as a potential target to enhance GLP-1 levels in type 2 diabetes. Future studies are clearly warranted to more fully elucidate the role of PKC{zeta} in the regulation of GLP-1 secretion.


   Acknowledgments
 
The authors are grateful to Kyowa Hakko Kogyo Co. Ltd. (Tokyo, Japan) for the gift of UCN-01, Dr. D. D’Alessio for the gift of GLP-1 antiserum, Dr. J. Dlugosz and Ms. H. Cvetnic for technical assistance, and Drs. M. Leitges (Hannover, Germany) and A. Rocca (Toronto, Canada) for helpful discussions.


   Footnotes
 
This work was supported by an operating grant from the Canadian Diabetes Association. R.I. was supported by a studentship from the German National Academic Foundation and a fellowship from the Banting and Best Diabetes Centre, University of Toronto. A.Y. by a summer studentship from the Banting and Best Diabetes Centre; and P.L.B. by the Canada Research Chairs Program.

Disclosure Statement: The authors have nothing to disclose.

First Published Online November 16, 2006

Abbreviations: aPKC, Atypical PKC; cPKC, conventional PKC; FA, fatty acid; FBS, fetal bovine serum; FRIC, fetal rat intestinal cell; GLP, glucagon-like peptide; GPR, G protein-coupled receptor; MUFA, monounsaturated fatty acid; nPKC, novel PKC; PI3-K, phosphatidylinositol 3-kinase; PKC, protein kinase C; PMA, phorbol-12-myristate-13-acetate; STC, secretin tumor cell.

Received October 17, 2006.

Accepted for publication November 9, 2006.


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