Protein Kinase C␨ Activation Mediates Glucagon-Like Peptide-1–Induced Pancreatic ␤-Cell Proliferation Jean Buteau,1 Sylvain Foisy,1 Christopher J. Rhodes,2 Lee Carpenter,3 Trevor J. Biden,3 and Marc Prentki1 Glucagon-like peptide-1 (GLP-1), an insulinotropic and effects (DNA synthesis, metabolic enzymes, and insu- glucoincretin hormone, is a potentially important ther- lin gene expression) of the glucoincretin. Diabetes 50: apeutic agent in the treatment of diabetes. We previ- 2237–2243, 2001 ously provided evidence that GLP-1 induces pancreatic ␤-cell growth nonadditively with glucose in a phospha- tidylinositol-3 kinase (PI-3K)–dependent manner. In the present study, we investigated the downstream effec- lucagon-like peptide-1 (GLP-1)-(7-36) amide, a tors of PI-3K to determine the precise signal transduc- potent glucoincretin hormone (1,2), is secreted ␤ tion pathways that mediate the action of GLP-1 on -cell by the intestinal L-cells in response to fat meals proliferation. GLP-1 increased extracellular signal-re- and carbohydrates (3,4). It is a potentially lated kinase 1/2, p38 mitogen-activated protein kinase G (MAPK), and protein kinase B activities nonadditively important drug in the treatment of diabetes in view of its with glucose in pancreatic ␤(INS 832/13) cells. GLP-1 ability to improve insulin secretion in both patients with also caused nuclear translocation of the atypical pro- impaired glucose tolerance and type 2 diabetes (5,6). tein kinase C (aPKC) ␨ isoform in INS as well as in GLP-1 is also an insulinotropic agent through its ability to dissociated normal rat ␤-cells as shown by immunolo- stimulate insulin gene expression and proinsulin biosyn- calization and Western immunoblotting analysis. Triti- thesis (7) and acts as a potent ␤-cell growth factor (8). ated thymidine incorporation measurements showed that GLP-1 increases the expression level of the ␤-cell specific the p38 MAPK inhibitor SB203580 suppressed GLP-1– transcription factor pancreatic and duodenal homeobox ␤ induced -cell proliferation. Further investigation was gene-1 (PDX-1) (8,9). In addition, the glucoincretin in- performed using isoform-specific pseudosubstrates of creases ␤-cell proliferation nonadditively with glucose in a classical (␣, ␤, and ␥)or␨ aPKC isoforms. The PKC␨ pseudosubstrate suppressed the proliferative action of phosphatidylinositol-3 kinase (PI-3K)–dependent manner GLP-1, whereas the inhibitor of classical PKC isoforms in ␤(INS-1) cells (8) as well as islet mass in mouse pan- had no effect. Overexpression of a kinase-dead PKC␨ creas (9). However, the precise signal transduction path- acting as a dominant negative protein suppressed GLP- way that mediates the proliferative action of GLP-1 is not 1–induced proliferation. In addition, ectopic expression completely elucidated. of a constitutively active PKC␨ mutant stimulated tri- PI-3K is a family of proteins known to be activated in tiated thymidine incorporation to the same extent as response to various growth factors in different cell types GLP-1, and the glucoincretin had no growth-promoting (10). Many downstream effectors of PI-3K mediate prolif- action under this condition. The data indicate that GLP- ␨ ␤ erative signals. Extracellular signal-related kinases (ERK) 1–induced activation of PKC is implicated in the -cell 1/2 and p38 mitogen-activated protein kinase (MAPK) are proliferative signal of the insulinotropic hormone. The results are consistent with a model in which GLP-1– in some instances downstream targets of PI-3K (11), induced PI-3K activation results in PKC␨ translocation mediating the proliferative response of a variety of exter- to the nucleus, which may play a role in the pleiotropic nal signals (12). ERK 1/2 and p38 MAPK also promote cell growth by being involved in antiapoptotic processes (13). Glucose activates p38 MAPK in pancreatic ␤-cell, an action From the 1Molecular Nutrition Unit, Department of Nutrition, University of that may be causally implicated in insulin gene induction Montreal, the Centre de Recherche du CHUM and Institut du Cancer, by the sugar via phosphorylation of the transcription Montreal, Quebec, Canada; the 2Pacific Northwest Research Institute & Department of Pharmacology, University of Washington, Seattle, Washington; factor PDX-1 (14). However, the involvement of p38 in and the 3Garvan Institute of Medical Research, Darlinghurst, New South PDX-1 activation was challenged recently (15). Among Wales, Australia. other downstream effectors of PI-3K are phosphoinositide- Address correspondence and reprint requests to Dr. Marc Prentki, CR- CHUM, Pavillon de Se`ve, 4e, 1560 Sherbrooke Est, Montreal, PQ H2L 4M1, dependent kinases (PDK), which in turn activate protein Canada. E-mail: [email protected]. kinase B (PKB) (also named Akt) (16). PKB participates in Received for publication 16 November 2000 and accepted in revised form 29 proliferative signals in response to many stimuli in differ- June 2001. aPKC, atypical protein kinase C; BSA, bovine serum albumin; CA, constitu- ent cell types possibly via the activation of the mammalian tively active; cPKC, classical protein kinase C; DN, dominant-negative; DTT, target of rapamycin and p70s6 kinase (17). Other targets of dithiothreitol; ERK, extracellular signal-related kinases; GLP-1, glucagon-like peptide-1; MAPK, mitogen-activated protein kinase; MEK, mitogenic-extracel- PDK that could play a role in cell growth regulation lular signal-regulated kinase; MOI, multiplicity of infection; NF␬B, nuclear- include the atypical isoform ␨ of protein kinase C (PKC) factor ␬B; PBS, phosphate-buffered saline; PDK, phosphoinositide-dependent (18,19). PKC is a multigene family divided into three kinases; PDX-1, pancreatic and duodenal homeobox gene-1; PI-3K, phospha- tidylinositol-3 kinase; PKB, protein kinase B; PKC, protein kinase C; PMSF, classes depending on their cofactor requirements: classi- phenylmethylsulfonyl fluoride; WT, wild-type. cal PKCs (cPKCs), which are sensitive to calcium/diacyl- DIABETES, VOL. 50, OCTOBER 2001 2237 PKC␨ AND GLP-1–INDUCED ␤-CELL GROWTH glycerol and tumor-promoting phorbol esters; novel PKCs, were then washed with PBS, incubated in the absence and presence of GLP-1, which are sensitive to diacylglycerol and tumor-promoting and subsequently fixed as described above for INS cells. For immunofluores- cence, cells were blocked with 1% BSA/PBS for 10 min, incubated for 1 h with phorbol esters only; and atypical PKCs (aPKCs), which are both a polyclonal PKC␨ (10 ␮g/ml) and a monoclonal mouse anti-insulin (10 insensitive to all three regulators (20). ␮g/ml) primary antibody, washed three times with PBS, incubated for 1 h with We report here that GLP-1 increases the PI-3K down- both a goat anti-rabbit fluorescein secondary antibody (Pierce) and a rhoda- stream targets ERK 1/2, p38 MAPK, and PKB activities mine-conjugated donkey anti-mouse secondary antibody (Jackson Immuno- nonadditively with glucose in INS(832/13) cells. GLP-1 also research, West Grove, PA), and finally washed three times with PBS. Image causes PKC␨ nuclear translocation. However, only PKC␨ acquisition was performed using an LSM-410 confocal microscope (Carl Zeiss). and p38 MAPK are likely to be involved in GLP-1–induced Preparation of nuclear extracts and immunoblot analysis of PKC␨. proliferation as revealed by tritiated thymidine incorpora- Nuclear extracts were isolated using a published procedure (25). Briefly, cells tion measurements in the presence of specific inhibitors. (40 ϫ 106 per condition) previously grown in 225 cm2 Petri dishes were The implication of the aPKC isoform ␨ in the proliferative harvested with a rubber policeman in cold PBS, sedimented at 3,500 g for 4 min, and lysed in 1 ml of ice-cold buffer A (15 mmol/l KCl, 2 mmol/l MgCl ,10 action of GLP-1 is demonstrated using recombinant adeno- 2 ␨ mmol/l HEPES [pH 7.4], 0.1% phenylmethylsulfonyl fluoride [PMSF], and 0.5% viruses, which allow expression of various PKC con- Nonidet P-40). After a 10-min incubation on ice, nuclei were collected by structs. centrifugation (1,000g for 5 min) and washed with buffer A without Nonidet P-40. Nuclei were lysed in a buffer containing 2 mmol/l KCl, 25 mmol/l HEPES (pH 7.4), 0.1% EDTA, and 1 mmol/l DTT. After a 15-min incubation period on RESEARCH DESIGN AND METHODS ice, a dialysis buffer (25 mmol/l HEPES [pH 7.4], 1 mmol/l DTT, 0.1% PMSF, 2 ␮ Reagents. Pharmacological inhibitors (SB203580, PD98059, LY294002, KN-93, g/ml aprotinin, 0.1 mmol/l EDTA, and 11% glycerol) was added to the nuclei myristoylated PKC␨, and cPKC [20–28] peptide inhibitors) were purchased preparations. Samples were centrifuged (16,000g for 20 min), and the super- from Biomol (Plymouth Meeting, PA). Human GLP-1 fragment 7–36 amide was natants containing the nuclear proteins were used for protein determinations, obtained from Sigma (St. Louis, MO). The anti-PKC␨ antibody was purchased subsequently aliquoted (50 ␮l), and kept frozen at Ϫ70°C for subsequent from Upstate Biotechnology (Lake Placid, NY). The anti-insulin antibody was immunoblot analysis. Lysates were subjected to electrophoresis on SDS- from Sigma. RPMI-1640 and the supplements, including fetal calf serum, were polyacrylamide gels and transferred onto nitrocellulose membranes (Schlei- purchased from Gibco BRL (Burlington, Ontario, Canada). Methyl [3H]- cher & Schuell, Keene, NH). Membranes were probed with a PKC␨ primary thymidine was from ICN (Costa Mesa, CA). antibody and subsequently with peroxidase-conjugated goat anti-rabbit IgG. Cell culture and incubation.