Bombesin and nutrients independently and additively regulate hormone release from GIP/Ins cells
Lin Li and
Burton M. Wice
Department of Internal Medicine, Division of Endocrinology, Diabetes and Metabolism, Washington University School of Medicine, Saint Louis, Missouri
Submitted 30 July 2004
; accepted in final form 17 September 2004
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ABSTRACT
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Glucose-dependent insulinotropic polypeptide (GIP) regulates glucose homeostasis and high-fat diet-induced obesity and insulin resistance. Therefore, elucidating the mechanisms that regulate GIP release is important. GIP is produced by K cells, a specific subtype of small intestinal enteroendocrine (EE) cell. Bombesin-like peptides produced by enteric neurons and luminal nutrients stimulate GIP release in vivo. We previously showed that PMA, bombesin, meat hydrolysate, glyceraldehyde, and methylpyruvate increase hormone release from a GIP-producing EE cell line (GIP/Ins cells). Here we demonstrate that bombesin and nutrients additively stimulate hormone release from GIP/Ins cells. In various cell systems, bombesin and PMA regulate cell physiology by activating PKD signaling in a PKC-dependent fashion, whereas nutrients regulate cell physiology by inhibiting AMPK signaling. Western blot analyses of GIP/Ins cells using antibodies specific for activated and/or phosphorylated forms of PKD and AMPK and one substrate for each kinase revealed that bombesin and PMA, but not nutrients, activated PKC, but not PKD. Conversely, nutrients, but not bombesin or PMA, inhibited AMPK activity. Pharmacological studies showed that PKC inhibition blocked bombesin- and PMA-stimulated hormone release, but AMPK activation failed to suppress nutrient-stimulated hormone secretion. Forced expression of constitutively active vs. dominant negative PKDs or AMPKs failed to perturb bombesin- or nutrient-stimulated hormone release. Thus, in GIP/Ins cells, PKC regulates bombesin-stimulated hormone release, whereas nutrients may control hormone release by regulating the activity of AMPK-related kinases, rather than AMPK itself. These results strongly suggest that K cells in vivo independently respond to neuronal vs. nutritional stimuli via two distinct signaling pathways.
K cells; protein kinase C; protein kinase D; adenosine 5'-monophosphate-activated protein kinase; adenosine 5'-monophosphate-activated protein kinase-related kinase; enteric neurons; nutrient sensing
ENTEROENDOCRINE (EE) CELLS are hormone-producing intestinal epithelial cells. Although these singly dispersed cells comprise <1% of the intestinal epithelium, as a whole they represent the largest endocrine organ in the body. There are at least 16 different subpopulations of EE cells, based on the major product(s) synthesized and secreted by individual cells (1). These hormones play important roles in regulating gastrointestinal secretion, motility, and blood flow and also regulate whole animal physiology (1, 35, 43, 48, 50, 59). For example, glucagon-like peptides-1 and -2 are important growth and trophic factors for islet
-cells and the intestine, respectively (13). Glucose-dependent insuloinotropic polypeptide (GIP), ghrelin, CCK, and peptide tyrosine regulate food intake and/or adiposity (3, 5, 40, 49, 57). GIP and glucagon-like peptide-1 potentiate glucose-stimulated insulin release and thus play important roles in maintaining blood glucose homeostasis (18). Surprisingly, little is known about the molecular mechanisms that regulate hormone release from different subtypes of EE cells. Because GIP promotes both obesity and glucose-stimulated insulin release, we have been particularly interested in understanding the molecular mechanisms that regulate release of this hormone.
GIP is secreted in response to nutrients present in the lumen of the gut but not those circulating in the blood (12, 18, 48). GIP release is also regulated by molecules produced by enteric neurons [e.g., bombesin-like peptides (29)], other enteroendocrine cells [e.g., somatostatin inhibits GIP release (59)], and possibly enterocytes (42, 54, 60). Therefore, K cells integrate input from numerous sources to release appropriate amounts of GIP. Using GIP/Ins cells (42), we have begun to study the regulation of K cell physiology using the well-characterized islet
-cell as a model.
In
-cells, glucose metabolism increases the intracellular ATP-to-ADP (ATP/ADP) ratio, which, in turn, inhibits ATP-sensitive K+ (KATP) channels. This causes cell depolarization, influx of calcium via voltage-dependent calcium channels, and finally exocytosis of insulin from secretory granules. Islet
-cells also exhibit KATP channel-independent mechanisms of secretion that involve mobilization of calcium from endoplasmic reticulum (ER)-derived stores (44). Release of ER calcium stores can be regulated by ryanodine receptors (27) and/or inositol 1,4,5-trisphosphate receptors (IP3Rs) (7, 19, 55, 56). Surprisingly, hormone release from EE cells that produce GIP, glucagon-like peptide-1, CCK, or somatostatin, but not chromogranin A or serotonin, appears to be mostly independent from KATP channels and IP3Rs (42, 61). EE cells that produce secretin or substance P express a heterogeneous phenotype with respect to expression of KATP channels and IP3Rs. Hormone release from GIP/Ins cells is not regulated by ryanodine receptors (unpublished observation). Therefore, different subtypes of EE cells exhibit unexpected complexity, heterogeneity, and novelty concerning the molecules that regulate hormone release.
Bombesin, PMA, protein hydrolysates, glyceraldehyde, and methylpyruvate are secretagogues for GIP/Ins cells (42). However, it is unknown which signaling pathways are activated by these secretagogues. Protein kinase D (PKD) is a serine/threonine protein kinase regulated by diacylglycerol signaling (58). Bombesin and phorbol esters stimulate proliferation of 3T3 cells by activating PKD via a PKC-dependent, bisindolylmaleimide I (GF-1)-inhibitable signaling pathway (67, 68). Thus bombesin and PMA could potentially stimulate hormone release from GIP/Ins cells by activating PKC/PKD signaling. In many systems, AMP-activated protein kinase (AMPK) activity is inhibited by nutrients, which coordinately turns on ATP-consuming pathways and inhibits ATP-generating pathways (20, 21, 45). Hydrolyzed proteins, glyceraldehyde, and methylpyruvate are nutritionally rich compounds. Thus these secretagogues could potentially regulate hormone release from GIP/Ins cells by inhibiting AMPK activity. Results presented in this paper indicate that, in GIP/Ins cells, PKC signaling regulates hormone release stimulated by PMA and bombesin but not nutrients. Conversely, nutrients but not PMA or bombesin regulate AMPK signaling, but AMPK-related kinases rather than AMPK itself may regulate nutrient-stimulated hormone release. Furthermore, signaling via bombesin and nutrients is independent and additive, which would allow K cells to independently respond to neural vs. nutritional stimuli.
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EXPERIMENTAL PROCEDURES
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Cells and culture conditions.
GIP/Ins Clone 10 cells are GIP-producing EE cells that were engineered to express the human insulin gene. These cells secrete both insulin and GIP in response to secretagogues (42, 60). Thus insulin release is a surrogate marker for GIP secretion in these cells. Cells were cultured in an atmosphere of 5% CO2-95% air and 100% humidity in DMEM containing 10% FBS as previously described (42).
Insulin secretion.
Insulin secretion was measured essentially as described (47). Briefly, cells were plated at
105 cells/well in 12-well plates. When
80% confluent, cells were washed twice with secretion buffer [Krebs-Ringer bicarbonate buffer containing HEPES plus 0.1% BSA (KRBH-Alb)] (47) and then preincubated at 37°C in secretion buffer. One hour later, buffer was replaced with fresh KRBH-Alb containing the appropriate secretagogue(s). When more than one secretagogue was added to cells, both compounds were added at the same time and not sequentially. Sixty minutes later, assay media were collected, centrifuged to remove any detached cells, and assayed for human insulin by RIA. Bombesin (Calbiochem, San Diego, CA) was added to the cells from a 100x stock solution prepared in KRBH-Alb. Meat hydrolysate (Sigma, St. Louis, MO) was prepared as a 20% (wt/vol) stock solution in PBS. Methylpyruvate, glyceraldehyde (Sigma), and 5-aminoimidazole-4-carboxamide-1-
-D-ribofuranoside (AICAR; Calbiochem) were prepared as 100x stock solutions in water. GF-1 (Sigma) was prepared as a 100x stock in DMSO. GF-1 and AICAR were included during the preincubation and also added along with the indicated secretagogue. All values are the average of quadruplicate samples.
Western blot analysis.
Cells were treated exactly as described for insulin secretion assays. After collection of the KRBH-Alb, cells were washed twice with ice-cold PBS and lysed directly in 1x SDS-PAGE gel loading buffer. Cell proteins were then separated by SDS-PAGE and transferred to nitrocellulose membranes. Membranes were stained with Ponceau S to confirm that samples were equally loaded and transferred. The names and catalog numbers for antibodies obtained from Cell Signaling Technology (Beverly, MA) are as follows: PKD/PKCµ antibody, no. 2052; phospho-PKD/PKCµ (Ser744/748) antibody, no. 2054; phospho-PKD/PKCµ (Ser916) antibody, no. 2051;
-AMPK antibody, no. 2532; and phospho-
-AMPK (Thr172) antibody, no. 2535. The names and catalog numbers for antibodies obtained from Upstate USA (Charlottesville, VA) are as follows: anti-acetyl-CoA carboxylase, no. 07-439, and anti-phospho-acetyl- CoA carboxylase (Ser79), no. 07-303. Antibodies to Kidins220 phosphorylated at Ser919 (no. K1725-01) were obtained from United States Biological (Swampscott, MA). Western blots were performed according to protocols provided with each antibody.
Transient transfection and human growth hormone secretion assays.
These assays are similar to those described in which MIN6 insulinoma cells were cotransfected with a human growth hormone (hGH) reporter construct plus a cDNA that encodes a putative regulatory protein (11, 31). The transiently produced hGH is stored by the endocrine cells and is released along with insulin after stimulation with appropriate secretagogues. Like MIN6, GIP/Ins cells store the transiently produced hGH and release it with appropriate GIP secretagogues. Thus, by transfecting cDNAs that encode hGH along with cDNAs that encode constitutively active or dominant negative AMPKs or PKDs, we can determine the effects of these protein kinases on secretagogue-stimulated hormone release from GIP/Ins cells. GIP/Ins cells were plated at 105 cells/well in 12-well dishes. The next day, quadruplicate wells of cells were transfected with the indicated cDNAs (see below) using the LT-1 transfection reagent (Panvera, Madison, WI) according to the manufacturer's protocol. The following day, cells were washed twice with fresh media, refed, incubated for an additional 24 h, and then assayed for hGH release using the insulin secretion assay protocol. hGH levels were determined with a high-sensitivity ELISA (Abazyme, Needham, MA). Cells in each well were transfected with 0.25 µg of the pTKGH hGH reporter construct (Nichols Diagnostic, San Clemente, CA) plus 0.25 µg of the indicated control or test plasmid(s). The constitutively active
1-AMPK, dominant negative
1-AMPK, and dominant negative
2-AMPK constructs in pCDNA3 were generously provided by Dr. David Carling (Imperial College School of Medicine, University of London, London, UK; see Refs. 52, 63). Constitutively active (
PH) and kinase dead (K612W; dominant negative) PKD1 constructs in pEGFP-N1 were generously provided by Dr. Angelika Hausser (Institute for Cell Biology and Immunology, University of Stuttgart, Stuttgart, Germany; see Refs. 23, 28).
Isolation of pools of stably transfected GIP/Ins cells.
GIP/Ins cells were plated in 100-mm dishes. The next day, cells were transfected with 1 µg of pIRES/hygro (BD Biosciences Clontech, Palo Alto, CA) plus 9 µg of pCDNA3, constitutively active AMPK, or dominant negative
1- plus
2-AMPKs (4.5 µg of each). Forty-eight hours later, cells were refed complete media containing hygromycin (400 µg/ml). The next day, cells were trypsinized and replated at different densities. Cells plated at low densities were allowed to grow as individual colonies, which allowed estimation of the number and size of clones from cells transfected with each set of plasmids. Cells replated at higher dilutions were trypsinized and maintained as pools until selection was complete (4 wk). Hygromycin treatment killed 100% of nontransfected cells with the use of this protocol.
RESULTS
PMA and bombesin but not nutrients activate PKC signaling in GIP/Ins cells.
Bombesin and bombesin-like peptides stimulate hormone release from gut endocrine cells in the absence of additional agonists (29, 32, 38). In many cell systems, bombesin and PMA activate PKD signaling via a PKC-dependent, GF-1-inhibitable signaling pathway. Phosphorylation of PKD at Ser744/748 or Ser916 profoundly increases PKD activity (26, 67, 68). Therefore, we determined whether secretagogues increased phosphorylation of these residues in GIP/Ins cells. Cells were preincubated for 60 min in the absence of secretagogues, treated for the indicated time with secretagogues, and then analyzed by Western blots with the use of antibodies against total or specifically phosphorylated forms of PKD. As shown in Fig. 1, bombesin profoundly increased phosphorylation of PKD on Ser744/748. Maximal stimulation was observed within 15 min, and increased phosphorylation was maintained for at least 90 min (Fig. 1). Throughout this period, total PKD levels or PKD phosphorylated at Ser916 remained relatively constant. Addition of PMA to GIP/Ins cells resulted in similar patterns of PKD expression and phosphorylation in a dose-dependent fashion (not shown). Meat hydrolysate is a nutrient-rich mixture of peptides and amino acids that would be present in the lumen of the gut after ingestion of a meal. Stimulation of GIP/Ins cells with meat hydrolysate did not alter total PKD levels or PKD phosphorylation at Ser744/748 or Ser916. GIP/Ins cells do not secrete hormones in response to glucose (42, 60). However, glyceraldehyde and methylpyruvate are nutrients that bypass glycolysis and stimulate hormone release from these cells (42, 60). As with meat hydrolysate, glyceraldehyde and methylpyruvate had no affect on PKD phosphorylation (Fig. 2). Therefore, PKD is phosphorylated in response to PMA and bombesin, but not nutrients.

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Fig. 1. Bombesin and meat hydrolysate regulate PKC and AMP-activated protein kinase (AMPK) signaling, respectively, in GIP/Ins cells. Cells were cultured in standard growth media until 80% confluent and preincubated for 60 min in KRBH-Alb (see EXPERIMENTAL PROCEDURES for details of buffer) without secretagogues. Fresh KRBH-Alb containing no secretagogue (None), 107 M bombesin (Bomb), or 1% meat hydrolysate (MH) was then added to cells. Cells were harvested for Western blots at the indicated time after addition of secretagogues. From top to bottom: Western blots were probed with antibodies specific for protein kinase D (PKD) phosphorylated at Ser744/748, PKD phosphorylated at Ser916, total PKD, Kidins220 phosphorylated at Ser919, AMPK phosphorylated at Thr172, total AMPK, and acetyl-CoA carboxylase (ACC) phosphorylated at Ser79. Bombesin but not meat hydrolysate increases PKC activity, since PKD phosphorylation is increased at Ser744/748. Conversely, meat hydrolysate but not bombesin decreases AMPK signaling, since this nutrient reduces AMPK-P and ACC-P levels.
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Fig. 2. PKC regulates PKD phosphorylation in GIP/Ins cells. Cells were treated as described in Fig. 1 and harvested just before (0 min) or 60 min after addition of secretagogues. Cells were analyzed by Western blots as described in Fig. 1. When added, bisindolylmaleimide I (GF-1) (3.5 µM) and 5-aminoimidazole-4-carboxamide-1- -D-ribofuranoside (AICAR; 1 mM) were present during preincubation and treatment with secretagogues. Note that 1) GF-1 inhibits bombesin-stimulated phosphorylation of PKD; 2) AICAR, which activates AMPK in many systems, partially activated AMPK in GIP/Ins cells treated with meat hydrolysate; and 3) meat hydrolysate, glyceraldehyde (Glycerald), and methylpyruvate (Me-Pyr) but not bombesin inhibited AMPK activation and signaling.
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PKC regulates bombesin- but not nutrient-stimulated hormone release.
GF-1 is a highly selective inhibitor of phorbol ester-activated PKCs but has no direct effect on PKD activity (58). Addition of GF-1 to 3T3 cells prevents bombesin-stimulated PKD activation (58). As shown in Fig. 2, GF-1 completely inhibited bombesin-stimulated PKD phosphorylation at Ser744/748 but had no effect on phosphorylation at Ser916. GF-1 had no apparent affect on PKD phosphorylation in cells treated with meat hydrolysate. GF-1 completely prevented bombesin- and PMA-stimulated hormone release but had no affect on hormone release from cells treated with meat hydrolysate (Fig. 3 and not shown). Therefore, PKC increases PKD phosphorylation and also stimulates bombesin- or PMA- but not nutrient-stimulated hormone release.

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Fig. 3. Inhibition of PKC prevents bombesin- but not meat hydrolysate-stimulated hormone release from GIP/Ins cells. Cells were treated with or without GF-1 plus bombesin (A) or meat hydrolysate (B) as described in Figs. 1 and 2. Secretion buffer was collected after 60 min and assayed for human insulin. Note that GF-1 inhibits bombesin-stimulated insulin release and has no effect on meat hydrolysate-stimulated insulin release. GF-1 also completely inhibited PMA-stimulated hormone release (not shown).
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Kidins220 is selectively expressed in the brain and in neuroendocrine cells and is phosphorylated on Ser919 by activated PKD (24). Thus Western blots were probed with antibodies specific for Kidins220 phosphorylated on Ser919. Compared with unstimulated cells at every time point examined, addition of bombesin, meat hydrolysate, glyceraldehyde, or methylpyruvate did not alter phosphorylation of Kidins220 at Ser919 (Fig. 1 and not shown), suggesting that although PKD phosphorylation is increased, PKD activity may not regulate hormone release from GIP/Ins cells. To directly test this hypothesis, a transient hGH secretion assay was utilized to determine whether constitutively active and dominant negative (kinase dead) PKDs (in pEGPF-N1) perturb secretagogue-stimulated hormone release from GIP/Ins cells (see EXPERIMENTAL PROCEDURES). Deletion of the pleckstrin homology domain of PKD (PKD-
PH) has been shown to produce a constitutively active form of PKD (23, 25, 28). Conversely, transient overexpression of a K612W kinase dead PKD (PKD-KD) has been shown to suppress PKD-mediated NF-
B-dependent gene expression (28). Empty pEGPF-N1 and pCDNA3 vectors served as controls. As shown in Fig. 4A, GIP/Ins cells transiently expressing the hGH plus control vectors secrete hGH. As noted for insulin release from nontransfected cells (Fig. 3), hGH secretion was increased after addition of bombesin. Importantly, the kinase dead PKD did not suppress bombesin-stimulated hGH release, and the constitutively active PKD did not increase basal hGH release to levels observed after addition of bombesin. Thus PKD activity does not regulate hormone release from GIP/Ins cells. However, because basal hGH release was similarly increased in cells expressing either PKD construct, it is possible that the transiently expressed PKDs can interact with and sequester proteins that inhibit hormone release from GIP/Ins cells. Taken together, these results indicate that bombesin- and PMA-stimulated hormone release from GIP/Ins cells is PKC dependent and does not require PKD activity.

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Fig. 4. PKD and AMPK do not regulate hormone release from GIP/Ins cells. GIP/Ins cells were transiently cotransfected with a human growth hormone (hGH) reporter construct plus the indicated control, PKD, or AMPK expression vector(s). Cells were then treated with no secretagogue or with bombesin or meat hydrolysate and assayed for hGH released into the medium. pCDNA3, pCDNA3 vector with no insert; EGFP, pEGFP-N1 vector with no insert; PKD- PH, constitutively active PKD in pEGFP-N1; PKD-KD, kinase dead PKD in pEGFP-N1; CA-AMPK, constitutively active AMPK in pCDNA3; DN-AMPK, equal amounts of dominant negative 1- plus 2-AMPKs in pCDNA3. Each condition was tested in quadruplicate, and results are means ± SE. Similar results were obtained in a second independent experiment. The pCDNA3 and pEGFP controls for A and B are the same samples. Note that cells expressing the constitutively active vs. kinase dead or dominant negative constructs respond similarly to all test conditions, indicating that PKD or AMPK activity does not regulate hormone release from GIP/Ins cells.
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Nutrients but not bombesin or PMA inhibit AMPK activity in GIP/Ins cells.
Next, we investigated whether nutrients regulate AMPK rather than PKC activity in GIP/Ins cells. AMPK activity is profoundly increased by phosphorylation at Thr172 (20, 45). Typically, AMPK phosphorylation and activity are high under nutrient-poor conditions. GIP/Ins cells were treated as described above and then analyzed for total AMPK or AMPK phosphorylated at Thr172. In the absence of secretagogues, AMPK is highly phosphorylated at Thr172 (Fig. 1). Addition of meat hydrolysate caused a rapid and profound decrease in AMPK phosphorylation that was maintained for at least 90 min. Glyceraldehyde and methylpyruvate also inhibited phosphorylation of AMPK at Thr172 (Fig. 2). Conversely, treatment of GIP/Ins cells with bombesin (Figs. 1 and 2) or PMA (not shown) did not reduce AMPK phosphorylation.
Activated AMPK phosphorylates acetyl-CoA carboxylase (ACC) at Ser79 (2, 11). As shown in Fig. 1, addition of meat hydrolysate to GIP/Ins cells caused a rapid and profound decrease in phosphorylation of ACC at Ser79 that paralleled the decrease in AMPK phosphorylation at Thr172. Similar decreases were noted in AMPK and ACC phosphorylation when GIP/Ins cells were treated with glyceraldehyde or methylpyruvate (Fig. 2). Conversely, treatment of cells with bombesin (Figs. 1 and 2) or PMA (not shown) had no affect on the phosphorylation states of AMPK or ACC. Therefore, nutritional secretagogues but not bombesin or PMA rapidly inhibit AMPK activity and signaling in GIP/Ins cells.
AMPK does not regulate nutrient-stimulated hormone release from GIP/Ins cells.
AICAR is a cell-permeable analog of adenosine that can activate AMPK in vivo (53). AICAR (0.13 mM) did not inhibit meat hydrolysate-stimulated hormone release from GIP/Ins cells (not shown). However, addition of 1 mM AICAR reversed much of the meat hydrolysate-stimulated decrease in AMPK phosphorylation (Fig. 2) but did not prevent the meat hydrolysate-regulated decrease in ACC phosphorylation (Fig. 2). These observations suggest that AMPK may not be the major regulator of ACC phosphorylation. Two sets of experiments were conducted to directly determine whether AMPK regulates nutrient-stimulated hormone release from GIP/Ins cells. First, cells were transiently transfected with the hGH reporter construct along with cDNA vectors that encode a constitutively active AMPK vs. dominant negative
1- plus
2-AMPKs, as described for PKD. Both dominant negative forms of AMPK were included, since inhibition of one AMPK isoform can increase expression of the other (36). Control cells were transfected with empty pCDNA3 or enhanced green fluorescent protein (EGFP) vectors. Similar to insulin release from nontransfected cells, GIP/Ins cells transfected with the hGH reporter plus either control vector secreted increased amounts of hGH in response to bombesin and meat hydrolysate (Fig. 4B). However, the response to meat hydrolysate was somewhat blunted compared with that of nontransfected cells (Fig. 3). Basal hGH release was increased to a similar extent in cells transfected with the dominant negative or constitutively active AMPKs. Furthermore, expression of the constitutively active AMPK failed to inhibit meat hydrolysate-stimulated hGH release. As expected, expression of the AMPK plasmids had no effect on bombesin-stimulated hGH release. Next, separate dishes of GIP/Ins cells were transfected with an empty pCDNA vector or with constitutively active or
1- plus
2-dominant negative AMPK plasmids. Pools of stably transfected cells were then isolated and assayed for insulin release as described in Fig. 3. Each pool was composed of
1,500 similarly sized clones, suggesting that AMPK activity did not alter cell growth or viability (not shown). Addition of meat hydrolysate or glyceraldehyde increased insulin release from each of the three different pools of transfected cells threefold and fourfold, respectively (not shown). Therefore, nutrients but not PMA or bombesin inhibit phosphorylation of AMPK in GIP/Ins cells. However, AMPK does not regulate hormone release.
Bombesin- and nutrient-signaling pathways independently and additively regulate hormone release from GIP/Ins cells.
The previous results indicate that bombesin and nutrients regulate different signaling pathways. Nutrient- and bombesin-signaling pathways could converge at a common downstream point or else represent parallel independent signaling pathways. To distinguish between these two possibilities, GIP/Ins cells were treated with varying concentrations of bombesin and/or meat hydrolysate. As shown in Fig. 5, hormone release was maximally stimulated by 109 M bombesin alone (3-fold) or 0.3% meat hydrolysate alone (2.5-fold). Addition of 109 M bombesin plus 0.3% meat hydrolysate resulted in a sixfold increase in hormone release. Therefore, bombesin- and nutrient-regulated signaling pathways are working in an independent and additive manner to stimulate hormone release from GIP/Ins cells.

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Fig. 5. Bombesin and nutrients independently and additively regulate hormone release from GIP/Ins cells. Secretion assays were conducted with GIP/Ins cells treated with the indicated concentrations of bombesin and meat hydrolysate. Both secretagogues were added to cells at the same time. Note that insulin release is additive in cells treated with maximal concentrations of bombesin plus a maximal concentration of meat hydrolysate.
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DISCUSSION
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Hormones produced by EE cells regulate a variety of important gastrointestinal and whole animal physiological events. Therefore, it is critical for EE cells to be able to tightly control production and release of specific EE cell products. To accomplish this, each specific subtype of EE cell must be able to sense and respond not only to changes in specific luminal contents but also to neurotransmitters released from adjacent neurons as well as to other regulatory molecules produced by additional intestinal and nonintestinal cell types (35). Previous studies from this laboratory have shown that different subtypes of EE cells utilize remarkably distinct and heterogeneous sets of proteins to regulate hormone release (42, 60, 61).
In many cells, bombesin and PMA activate PKD signaling in a PKC-dependent, GF-1-inhibitable manner (9, 34, 64, 67, 68). Bombesin and PMA but not nutrients increased phosphorylation of PKD on Ser744/748 but not on Ser916. Because GF-1 inhibited bombesin and PMA stimulated phosphorylation of PKD at Ser744/748, these two secretagogues activate PKC in GIP/Ins cells. Site-directed mutagenesis of positions 744/748 of PKD has demonstrated that phosphorylation of these two residues alone is sufficient to regulate PKD activity (26, 65, 66). However, addition of bombesin or PMA did not increase phosphorylation of Kidins220, the only known in vivo substrate for activated PKD, and overexpression of constitutively active or dominant negative PKDs did not perturb secretagogue-stimulated hormone release. Because GF-1 inhibited phosphorylation of PKD and also prevented bombesin- and PMA-stimulated hormone release, the effects of bombesin and PMA are mediated by PKC and are mostly independent of PKD activity. There have been two other reports that examined the potential role for PKD in regulating hormone release from gut endocrine cells. In agreement with our results, Moore et al. (38) presented evidence that PKC but not PKD regulates hormone release from purified human antral gastrin-producing EE cells. The authors also showed that gastrin-producing EE cells express at least six different isoforms of PKC. Thus it will be important to determine which PKC isoforms are expressed by GIP/Ins cells and to evaluate the physiological role for each isozyme. Conversely, Li et al. (32) concluded that PKD plays a central role in regulating neurotensin release from BON cells. However, in these latter studies, expression of a constitutively active PKD in BON cells increased basal neurotensin release only
2-fold, whereas PMA increased neurotensin release
10-fold. In addition, BON cells transfected with the gastrin-releasing peptide receptor exhibit a 10-fold increase in neurotensin release after addition of bombesin. However, after transfection with PKD small interfering RNA, these same cells exhibited only a twofold reduction in bombesin-stimulated neurotensin release even though PKD expression and activation appeared to be completely blocked. Thus PKC may also be much more important than PKD for regulating neurotensin release.
Phosphorylation of AMPK at Thr172 greatly increases its enzyme activity (20). Addition of meat hydrolysate, glyceraldehyde, or methylpyruvate, but not PMA or bombesin, to GIP/Ins cells resulted in decreased phosphorylation of AMPK at Thr172. AMPK signaling also appeared to be inhibited, since these same nutrients suppressed phosphorylation of ACC. However, forced expression of constitutively active vs. dominant negative AMPKs did not perturb secretagogue-stimulated hormone release from GIP/Ins cells. These results suggest that AMPK signaling does not regulate hormone release but may be important for regulating other aspects of K cell physiology. In contrast to our results, forced expression of a dominant negative AMPK in MIN6 insulinoma cells led to unregulated insulin release, whereas forced expression of a constitutively active AMPK blocked glucose-stimulated insulin release (11). These results further illustrate the remarkable differences in the proteins that regulate hormone release from islet
-cells vs. GIP-producing EE cells.
It was initially unclear why AICAR, which can activate AMPK in other systems, failed to inhibit insulin release from GIP/Ins cells. One possible explanation is that GIP/Ins cells do not efficiently metabolize AICAR. Alternatively, there is some evidence that, in islet
-cells, AICAR can be phosphorylated to ZMP, which can then be phosphorylated further to ZTP, an ATP mimetic. This, in turn, could stimulate depolarization and subsequent hormone release from
-cells by inhibiting KATP channel activity without inhibiting AMPK activity (45). However, KATP channels do not regulate hormone release from GIP/Ins cells (42, 60). Therefore, it is possible that additional intermediates derived from metabolism of AICAR could inhibit rather than activate AMPK. A much more intriguing and unifying explanation for our results is that nutrients control hormone release from GIP/Ins cells by regulating activity of AMPK-related kinases, rather than AMPK itself. Twelve human AMPK-related kinases have recently been identified and partially characterized (33). Like AMPK, 11 of these AMPK-related kinases are phosphorylated in their T-loops by LKB1, an AMPK kinase. Phosphorylation at these sites increased enzyme activity >50-fold. In contrast to AMPK, the activity of LKB1 or the AMPK-related kinases is not increased by AICAR. Although there were marked differences in the relative rates, all of the AMPK-related kinases also phosphorylated each of three peptides that are substrates for AMPK. Therefore, our results suggest that AMPK-related kinases regulate hormone release from GIP/Ins cells, since 1) AICAR activated AMPK but did not inhibit phosphorylation of ACC, a substrate for AMPK; 2) AICAR did not inhibit nutrient-stimulated hormone release; and 3) forced expression of AMPKs did not perturb hormone release from GIP/Ins cells.
It was predicted that PMA and bombesin vs. nutrients would control hormone release by regulating different signaling pathways. However, it was unexpected to see that there was no apparent cross talk between the PKC and AMPK/AMPK-related kinase signaling pathways and that hormone release regulated by these two pathways was additive. Because bombesin-like peptides are produced by enteric neurons, these results imply that K cells independently sense and respond to nutritional and neuronal stimuli.
As already discussed, K cells do not utilize many of the same regulatory proteins to control hormone release that are used by other endocrine, excitatory, and some EE cells (42, 60, 61). However, evidence presented in this paper indicates that GIP secretagogues regulate two conserved signaling pathways in GIP/Ins cells. This implies that steps leading to the activation of PKC and/or inactivation of AMPK/AMPK-related kinases may represent critical steps in regulating K cell physiology and GIP release in vivo. This also implies that PKC and APMK/AMPK-related kinase signaling can regulate hormone release from other subtypes of EE cells and that cell-specific differences in the regulatory cascades that activate or inactivate these kinases control release of other hormones. For example, cell-specific molecules that regulate bombesin receptor signaling could potentially confer differential responses to bombesin-like peptides in distinct subtypes of EE cells. With respect to nutrients, GIP is released in response to a set of amino acids different from those that stimulate gastrin and CCK release (59). Therefore, amino acid transporters present on one type of EE cell but not on another could potentially initiate cell-specific cascades that inhibit AMPK/AMPK-related kinase signaling and thus stimulate release of hormones from only a specific type of EE cell. However, it is also possible that different subtypes of EE cells express distinct AMPK-related kinases, and each of these kinases can be regulated by metabolites derived from different nutrients.
There is a large body of biochemical, physiological, and genetic evidence indicating that GIP promotes obesity and insulin resistance (4, 6, 8, 10, 1417, 22, 30, 37, 39, 41, 46, 51, 62). Furthermore, mice lacking GIP receptors do not exhibit any serious detrimental phenotypes (37). Therefore, inhibition of GIP receptor signaling is a potential strategy to ameliorate obesity and insulin resistance. This could be accomplished by development of GIP receptor antagonists. An alternative and complementary approach would be to inhibit GIP release from K cells. One advantage of this latter approach is that potential drugs could be delivered orally to K cells, which could minimize adverse side effects due to systemic drug delivery. A second advantage is that different subtypes of EE cells utilize distinct proteins to control hormone release (42), suggesting that drugs that inhibit GIP release, but do not adversely affect other cell populations, could be rationally developed (60, 61). Therefore, as a first step in developing potential anti-obesity drugs, it is critical to elucidate the mechanisms that regulate GIP release from K cells so that K cell-specific components of those pathways can be defined. Studies are underway to determine the roles of PKC, PKD, AMPK, and AMPK-related kinase signaling pathways in regulating hormone release from K cells in vivo, as well as from other subtypes of EE cells, and to identify cell-specific components of these pathways.
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GRANTS
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This work was supported in part by a Career Development Award from the American Diabetes Association (B. M. Wice) and the Washington University Diabetes and Research Training Center (National Institute of Diabetes and Digestive and Kidney Diseases Grant 5-P60-DK-20579).
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ACKNOWLEDGMENTS
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We thank Drs. David Kipnis and Ernesto Bernal for helpful discussions.
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FOOTNOTES
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Address for reprint requests and other correspondence: B. M. Wice, Washington Univ. School of Medicine, Dept. of Internal Medicine, Division of Endocrinology, Diabetes & Metabolism, Campus Box 8127, 660 South Euclid Ave., St. Louis, MO 63110 (E-mail: bwice{at}im.wustl.edu)
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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REFERENCES
|
---|
- Aiken KD, Kisslinger JA, and Roth KA. Immunohistochemical studies indicate multiple enteroendocrine cell differentiation pathways in the mouse proximal small intestine. Dev Dyn 201: 636670, 1994.
- Andersson U, Filipsson K, Abbott CR, Woods A, Smith K, Bloom SR, Carling D, and Small CJ. AMP-activated protein kinase plays a role in the control of food intake. J Biol Chem 279: 1200512008, 2004.[Abstract/Free Full Text]
- Asakawa A, Inui A, Kaga T, Yuzuriha H, Nagata T, Ueno N, Makino S, Fujimiya M, Niijima A, Fujino MA, and Kasuga M. Ghrelin is an appetite-stimulatory signal from stomach with structural resemblance to motilin. Gastroenterology 120: 337345, 2001.[ISI][Medline]
- Baba AS, Harper JM, and Buttery PJ. Effects of gastric inhibitory polypeptide, somatostatin and epidermal growth factor on lipogenesis in ovine adipose explants. Comp Biochem Physiol B Biochem Mol Biol 127: 173182, 2000.[CrossRef][ISI][Medline]
- Batterham RL, Cowley MA, Small CJ, Herzog H, Cohen MA, Dakin CL, Wren AM, Brynes AE, Low MJ, Ghatei MA, Cone RD, and Bloom SR. Gut hormone PYY(336) physiologically inhibits food intake. Nature 418: 650654, 2002.[CrossRef][ISI][Medline]
- Beck B, Max JP, and Villaume C. Effects of somatostatin on the insulin- and gastric inhibitory polypeptide-stimulated fatty acid esterification in rat adipose tissue. Int J Obes 12: 4147, 1988.[ISI][Medline]
- Blondel O, Bell GI, and Seino S. Inositol 1,4,5-trisphosphate receptors, secretory granules and secretion in endocrine and neuroendocrine cells. Trends Neurosci 18: 157161, 1995.[CrossRef][ISI][Medline]
- Chini EN, Chini CC, Kato I, Takasawa S, and Okamoto H. CD38 is the major enzyme responsible for synthesis of nicotinic acid-adenine dinucleotide phosphate in mammalian tissues. Biochem J 362: 125130, 2002.[CrossRef][ISI][Medline]
- Chiu T and Rozengurt E. PKD in intestinal epithelial cells: rapid activation by phorbol esters, LPA, and angiotensin through PKC. Am J Physiol Cell Physiol 280: C929C942, 2001.[Abstract/Free Full Text]
- Creutzfeldt W, Ebert R, Willms B, Frerichs H, and Brown JC. Gastric inhibitory polypeptide (GIP) and insulin in obesity: increased response to stimulation and defective feedback control of serum levels. Diabetologia 14: 1524, 1978.[ISI][Medline]
- Da Silva XG, Leclerc I, Varadi A, Tsuboi T, Moule SK, and Rutter GA. Role for AMP-activated protein kinase in glucose-stimulated insulin secretion and preproinsulin gene expression. Biochem J 371: 761774, 2003.[CrossRef][ISI][Medline]
- Drucker DJ. Glucagon-like peptides. Diabetes 47: 159169, 1998.[Abstract]
- Drucker DJ. Glucagon-like peptides: regulators of cell proliferation, differentiation, and apoptosis. Mol Endocrinol 17: 161171, 2003.[Abstract/Free Full Text]
- Ebert R, Frerichs H, and Creutzfeldt W. Impaired feedback control of fat induced gastric inhibitory polypeptide (GIP) secretion by insulin in obesity and glucose intolerance. Eur J Clin Invest 9: 129135, 1979.[ISI][Medline]
- Ebert R, Nauck M, and Creutzfeldt W. Effect of exogenous or endogenous gastric inhibitory polypeptide (GIP) on plasma triglyceride responses in rats. Horm Metab Res 23: 517521, 1991.[ISI][Medline]
- Eckel RH, Fujimoto WY, and Brunzell JD. Gastric inhibitory polypeptide enhanced lipoprotein lipase activity in cultured preadipocytes. Diabetes 28: 11411142, 1979.[Abstract]
- Elahi D, Andersen DK, Muller DC, Tobin JD, Brown JC, and Andres R. The enteric enhancement of glucose-stimulated insulin release. The role of GIP in aging, obesity, and non-insulin-dependent diabetes mellitus. Diabetes 33: 950957, 1984.[Abstract]
- Fehmann H, Goke R, and Goke B. Cell and molecular biology of the incretin hormones glucagon-like peptide-1 and glucose-dependent insulin releasing polypeptide. Endocr Rev 16: 390410, 1995.[ISI][Medline]
- Hagar RE and Ehrlich BE. Regulation of the type III InsP3 receptor and its role in beta cell function. Cell Mol Life Sci 57: 19381949, 2000.[ISI][Medline]
- Hardie DG, Carling D, and Carlson M. The AMP-activated/SNF1 protein kinase subfamily: metabolic sensors of the eukaryotic cell? Annu Rev Biochem 67: 821855, 1998.[CrossRef][ISI][Medline]
- Hardie DG, Scott JW, Pan DA, and Hudson ER. Management of cellular energy by the AMP-activated protein kinase system. FEBS Lett 546: 113120, 2003.[CrossRef][ISI][Medline]
- Hauner H, Glatting G, Kaminska D, and Pfeiffer EF. Effects of gastric inhibitory polypeptide on glucose and lipid metabolism of isolated rat adipocytes. Ann Nutr Metab 32: 282288, 1988.[ISI][Medline]
- Hausser A, Link G, Bamberg L, Burzlaff A, Lutz S, Pfizenmaier K, and Johannes FJ. Structural requirements for localization and activation of protein kinase C mu (PKC mu) at the Golgi compartment. J Cell Biol 156: 6574, 2002.[Abstract/Free Full Text]
- Iglesias T, Cabrera-Poch N, Mitchell MP, Naven TJ, Rozengurt E, and Schiavo G. Identification and cloning of Kidins220, a novel neuronal substrate of protein kinase D. J Biol Chem 275: 4004840056, 2000.[Abstract/Free Full Text]
- Iglesias T and Rozengurt E. Protein kinase D activation by mutations within its pleckstrin homology domain. J Biol Chem 273: 410416, 1998.[Abstract/Free Full Text]
- Iglesias T, Waldron RT, and Rozengurt E. Identification of in vivo phosphorylation sites required for protein kinase D activation. J Biol Chem 273: 2766227667, 1998.[Abstract/Free Full Text]
- Islam MS. The ryanodine receptor calcium channel of
-cells: molecular regulation and physiological significance. Diabetes 51: 12991309, 2002.[Abstract/Free Full Text]
- Johannes FJ, Horn J, Link G, Haas E, Siemienski K, Wajant H, and Pfizenmaier K. Protein kinase Cµ downregulation of tumor-necrosis-factor-induced apoptosis correlates with enhanced expression of nuclear-factor-
B-dependent protective genes. Eur J Biochem 257: 4754, 1998.[Abstract]
- Kieffer TJ, Buchan AM, Barker H, Brown JC, and Pederson RA. Release of gastric inhibitory polypeptide from cultured canine endocrine cells. Am J Physiol Endocrinol Metab 267: E489E496, 1994.[Abstract/Free Full Text]
- Knapper JM, Puddicombe SM, Morgan LM, Fletcher JM, and Marks V. Glucose dependent insulinotropic polypeptide and glucagon-like peptide-1(736)amide: effects on lipoprotein lipase activity (Abstract). Biochem Soc Trans 21: 135S, 1993.[Medline]
- Kotake K, Ozaki N, Mizuta M, Sekiya S, Inagaki N, and Seino S. Noc2, a putative zinc finger protein involved in exocytosis in endocrine cells. J Biol Chem 272: 2940729410, 1997.[Abstract/Free Full Text]
- Li J, O'Connor KL, Hellmich MR, Greeley GHJ, Townsend CMJ, and Evers BM. The role of protein kinase D in neurotensin secretion mediated by protein kinase C-alpha/-delta and Rho/Rho kinase. J Biol Chem 279: 2846628474, 2004.[Abstract/Free Full Text]
- Lizcano JM, Goransson O, Toth R, Deak M, Morrice NA, Boudeau J, Hawley SA, Udd L, Makela TP, Hardie DG, and Alessi DR. LKB1 is a master kinase that activates 13 kinases of the AMPK subfamily, including MARK/PAR-1. EMBO J 23: 833843, 2004.[Abstract/Free Full Text]
- Matthews SA, Rozengurt E, and Cantrell D. Protein kinase D. A selective target for antigen receptors and a downstream target for protein kinase C in lymphocytes. J Exp Med 191: 20752082, 2000.[Abstract/Free Full Text]
- Miller LJ. Gastrointestinal hormones and receptors. In: Textbook of Gastroenterology, edited by Yamada T, Alpers DH, Laine L, Owyang C, and Powell DW. Philadelphia, PA: Lippincott Williams & Wilkens, 1999, p. 3566.
- Minokoshi Y, Alquier T, Furukawa N, Kim YB, Lee A, Xue B, Mu J, Foufelle F, Ferre P, Birnbaum MJ, Stuck BJ, and Kahn BB. AMP-kinase regulates food intake by responding to hormonal and nutrient signals in the hypothalamus. Nature 428: 569574, 2004.[CrossRef][ISI][Medline]
- Miyawaki K, Yamada Y, Ban N, Ihara Y, Tsukiyama K, Zhou H, Fujimoto S, Oku A, Tsuda K, Toyokuni S, Hiai H, Mizunoya W, Fushiki T, Holst JJ, Makino M, Tashita A, Kobara Y, Tsubamoto Y, Jinnouchi T, Jomori T, and Seino Y. Inhibition of gastric inhibitory polypeptide signaling prevents obesity. Nat Med 8: 738742, 2002.[CrossRef][ISI][Medline]
- Moore ED, Ring M, Scriven DR, Smith VC, Meloche RM, and Buchan AM. The role of protein kinase C isozymes in bombesin-stimulated gastrin release from human antral gastrin cells. J Biol Chem 274: 2249322501, 1999.[Abstract/Free Full Text]
- Morgan LM, Hampton SM, Tredger JA, Cramb R, and Marks V. Modifications of gastric inhibitory polypeptide (GIP) secretion in man by a high-fat diet. Br J Nutr 59: 373380, 1988.[ISI][Medline]
- Nakazato M, Murakami N, Date Y, Kojima M, Matsuo H, Kangawa K, and Matsukura S. A role for ghrelin in the central regulation of feeding. Nature 409: 194198, 2001.[CrossRef][ISI][Medline]
- Oben J, Morgan L, Fletcher J, and Marks V. Effect of the entero-pancreatic hormones, gastric inhibitory polypeptide and glucagon-like polypeptide-1(736) amide, on fatty acid synthesis in explants of rat adipose tissue. J Endocrinol 130: 267272, 1991.[Abstract]
- Ramshur EB, Rull TR, and Wice BM. Novel insulin/GIP-producing cell lines provide unexpected insights into gut K-cell function in vivo. J Cell Physiol 192: 339350, 2002.[CrossRef][ISI][Medline]
- Roth KA and Gordon JI. Spatial differentiation of the intestinal epithelium: analysis of enteroendocrine cells containing immunoreactive serotonin, secretin, and substance P in normal and transgenic mice. Proc Natl Acad Sci USA 87: 64086412, 1990.[Abstract]
- Rutter GA. Nutrient-secretion coupling in the pancreatic islet
-cell: recent advances. Mol Aspects Med 22: 247284, 2001.[CrossRef][Medline]
- Rutter GA, Da Silva XG, and Leclerc I. Roles of 5'-AMP-activated protein kinase (AMPK) in mammalian glucose homoeostasis. Biochem J 375: 116, 2003.[CrossRef][ISI][Medline]
- Salera M, Giacomoni P, Pironi L, Cornia G, Capelli M, Marini A, Benfenati F, Miglioli M, and Barbara L. Gastric inhibitory polypeptide release after oral glucose: relationship to glucose intolerance, diabetes mellitus, and obesity. J Clin Endocrinol Metab 55: 329336, 1982.[Abstract]
- Sandler S and Eizirik DL. Culture of human pancreatic islet cells. In: Methods in Molecular Medicine: Human Cell Culture Protocols, edited by Jones GE. Totowa, NJ: Humana, 1994, p. 391407.
- Schmier WE. Gastrointestinal hormones. In: Textbook of Gastroenterology, edited by Yamada T. Philadelphia, PA: JB Lippincott, 1995, p. 2571.
- Schwartz MW, Woods SC, Porte DJ, Seeley RJ, and Baskin DG. Central nervous system control of food intake. Nature 404: 661671, 2000.[ISI][Medline]
- Sjolund K, Sanden G, Hakanson R, and Sundler F. Endocrine cells in human intestine: an immunocytochemical study. Gastroenterology 85: 11201130, 1983.[ISI][Medline]
- Starich GH, Bar RS, and Mazzaferri EL. GIP increases insulin receptor affinity and cellular sensitivity in adipocytes. Am J Physiol Endocrinol Metab 249: E603E607, 1985.[Abstract/Free Full Text]
- Stein SC, Woods A, Jones NA, Davison MD, and Carling D. The regulation of AMP-activated protein kinase by phosphorylation. Biochem J 345: 437443, 2000.[CrossRef][ISI][Medline]
- Sullivan JE, Brocklehurst KJ, Marley AE, Carey F, Carling D, and Beri RK. Inhibition of lipolysis and lipogenesis in isolated rat adipocytes with AICAR, a cell-permeable activator of AMP-activated protein kinase. FEBS Lett 353: 3336, 1994.[CrossRef][ISI][Medline]
- Sykes S, Morgan LM, English J, and Marks V. Evidence for preferential stimulation of gastric inhibitory polypeptide secretion in the rat by actively transported carbohydrates and their analogues. J Endocrinol 85: 201207, 1980.[Abstract]
- Taylor CW, Genazzani AA, and Morris SA. Expression of inositol trisphosphate receptors. Cell Calcium 26: 237251, 1999.[CrossRef][ISI][Medline]
- Thrower EC, Hagar RE, and Ehrlich BE. Regulation of Ins(1,4,5)P3 receptor isoforms by endogenous modulators. Trends Pharmacol Sci 22: 580586, 2001.[CrossRef][ISI][Medline]
- Tschop M, Smiley DL, and Heiman ML. Ghrelin induces adiposity in rodents. Nature 407: 908913, 2000.[CrossRef][ISI][Medline]
- Waldron RT, Iglesias T, and Rozengurt E. Phosphorylation-dependent protein kinase D activation. Electrophoresis 20: 382390, 1999.[CrossRef][ISI][Medline]
- Walsh JH. Gastrointestinal hormones. In: Physiology of the Gastrointestinal Tract, edited by Johnson LR. New York: Raven, 1994, p. 1128.
- Wang SY, Chi MM, Li L, Moley KH, and Wice BM. Studies with GIP/Ins cells indicate secretion by gut K-cells is KATP channel independent. Am J Physiol Endocrinol Metab 284: E988E1000, 2003.[Abstract/Free Full Text]
- Wang SY, Liu J, Li L, and Wice BM. Individual sub-types of enteroendocrine cells in the mouse small intestine exhibit unique patterns of inositol 1,4,5-trisphosphate receptor expression. J Histochem Cytochem 52: 5363, 2004.[Abstract/Free Full Text]
- Wasada T, McCorkle K, Harris V, Kawai K, Howard B, and Unger RH. Effect of gastric inhibitory polypeptide on plasma levels of chylomicron triglycerides in dogs. J Clin Invest 68: 11061107, 1981.[ISI][Medline]
- Woods A, Azzout-Marniche D, Foretz M, Stein SC, Lemarchand P, Ferre P, Foufelle F, and Carling D. Characterization of the role of AMP-activated protein kinase in the regulation of glucose-activated gene expression using constitutively active and dominant negative forms of the kinase. Mol Cell Biol 20: 67046711, 2000.[Abstract/Free Full Text]
- Yuan J, Bae D, Cantrell D, Nel AE, and Rozengurt E. Protein kinase D is a downstream target of protein kinase C
. Biochem Biophys Res Commun 291: 444452, 2002.[CrossRef][ISI][Medline]
- Yuan J, Slice L, Walsh JH, and Rozengurt E. Activation of protein kinase D by signaling through the alpha subunit of the heterotrimeric G protein G(q). J Biol Chem 275: 21572164, 2000.[Abstract/Free Full Text]
- Yuan J, Slice LW, and Rozengurt E. Activation of protein kinase D by signaling through Rho and the alpha subunit of the heterotrimeric G protein G13. J Biol Chem 276: 3861938627, 2001.[Abstract/Free Full Text]
- Zhukova E, Sinnett-Smith J, and Rozengurt E. Protein kinase D potentiates DNA synthesis and cell proliferation induced by bombesin, vasopressin, or phorbol esters in Swiss 3T3 cells. J Biol Chem 276: 4029840305, 2001.[Abstract/Free Full Text]
- Zugaza JL, Waldron RT, Sinnett-Smith J, and Rozengurt E. Bombesin, vasopressin, endothelin, bradykinin, and platelet-derived growth factor rapidly activate protein kinase D through a protein kinase C-dependent signal transduction pathway. J Biol Chem 272: 2395223960, 1997.[Abstract/Free Full Text]
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