Defective Glucose-Dependent Insulinotropic Polypeptide Receptor Expression in Diabetic Fatty Zucker Rats Francis C. Lynn,1 Nathalie Pamir,1 Eddy H.C. Ng,1 Christopher H.S. McIntosh,1 Timothy J. Kieffer,2 and Raymond A. Pederson1

Glucose-dependent insulinotropic polypeptide (GIP) is a that is released postprandially from the small intestine and acts in concert with - large proportion of postprandial secre- like peptide (GLP)-1 to potentiate glucose-induced in- tion is stimulated by secreted from sulin secretion from the pancreatic ␤-cell. In type 2 the small intestine. Glucose-dependent insuli- diabetes, there is a decreased responsiveness of the notropic polypeptide (GIP) (gastric inhibitory to GIP; however, the insulin response to GLP-1 A polypeptide), the proglucagon –derived glucagon-like remains intact. The literature suggests that the ineffec- tiveness of GIP in type 2 diabetes may be a result of peptide (GLP)-1(7-37), and the COOH-terminal truncated chronic homologous desensitization of the GIP recep- form GLP-1(7-36) amide are the major that act via tor. Yet, there has been no conclusive evidence suggest- this endocrine system to potentiate glucose-induced insu- ing that GIP levels are elevated in diabetes. The lin secretion (1). GIP and GLP-1 both signal via serpentine hypothesis of the present study is that one cause of seven-transmembrane G-–coupled receptors of the decreased responsiveness to GIP in type 2 diabetes is an /VIP superfamily. Binding of the incretins to their inappropriate expression of the GIP receptor in the respective receptors on the ␤-cell surface activates ade- pancreatic islet. This hypothesis was tested using a strain nylyl cyclase, increases cAMP, and stimulates insulin se- of diabetic fatty Zucker rats. The obese rats displayed basal GIP levels similar to the control animals; however, cretion (2–6). Recent studies have demonstrated that the they were unresponsive to a GIP infusion (4 pmol ⅐ min–1 ⅐ GIP receptor displays characteristics similar to other G- kg–1), whereas the lean animals displayed a significant protein–coupled receptors in terms of ligand binding, de- reduction in blood glucose (GIP levels, 50% control sensitization, and subsequent internalization (7,8). after 60 min, P < 0.05) as well as a significant increase A wide range of experimental techniques have been in circulating insulin. GIP also potently stimulated first- used to demonstrate the physiological importance of GIP phase insulin secretion from isolated perifused islets and GLP-1. In vivo administration of exendin(9-39) and ؋ ؎ (10.3 3.0 basal), and GIP and GLP-1 potentiated GIP(7-30), GLP-1, and GIP receptor antagonists resulted in -insulin secretion from the perfused pancreas (6 ؋ con trol area under the curve [AUC]) from lean animals. GIP decreased insulin responses to oral glucose (9–11). Fur- yielded no significant effect in the Vancouver diabetic thermore, both GIP and GLP-1 receptor knockout mice fatty Zucker (VDF) rat , whereas GLP-1 elic- display compromised insulin release and, therefore, al- ited an eightfold increase of insulin secretion from the tered glucose tolerance to an oral load (12,13). From these perfused VDF pancreas. Islets from lean animals sub- studies, it has been concluded that secretion of the incre- jected to static incubations with GIP showed a 2.2-fold tins could account for up to 70% of the postprandial insulin increase in cAMP, whereas GIP failed to increase islet response to glucose (14). GIP and GLP-1 both require cAMP in the VDF islets. Finally, the expression of both elevated levels of ambient glucose to stimulate pancreatic GIP receptor mRNA and protein was decreased in islets ␤ from VDF rats. These data suggest that the decreased -cell insulin secretion; hence, there is considerable inter- effectiveness of GIP in the VDF rat and in type 2 est in using analogs of these in the diabetes may be a result of a decreased receptor expres- treatment of type 2 diabetes (15–20). sion in the islet. Diabetes 50:1004–1011, 2001 One shortfall of using GIP in therapy is the controversy over its effectiveness as an incretin in type 2 diabetes (21,22). Human studies have shown that there is a de- From the 1Department of Physiology, University of British Columbia, Vancou- ver; and the 2Departments of Medicine and Physiology, University of Alberta, creased incretin effect in type 2 diabetes, and this has been Edmonton, Canada. attributed mainly to an attenuation of GIP-stimulated in- Address correspondence and reprint requests to R.A. Pederson, Department of Physiology, Faculty of Medicine, University of British Columbia, 2146 sulin secretion either via a change in GIP receptor expres- Health Sciences Mall, Vancouver, BC, Canada V6T 1Z3. E-mail: pederson@ sion or a change in circulating GIP levels, although altered interchange.ubc.ca. signal transduction pathways could play a role (23). Cur- Received for publication 28 July 2000 and accepted in revised form 17 January 2001. rently, there is no consensus regarding the circulating AUC, area under the curve; BSA, bovine serum albumin; DPIV, dipepti- levels of GIP in type 2 diabetic subjects; studies have dylpeptidase IV; DTT, dithiothreitol; GIP, glucose-dependent insulinotropic polypeptide; GLP, glucagon-like peptide; HBSSϩ, Hank’s balanced salt solu- demonstrated increased, decreased, and unchanged GIP tion supplemented with 10 mmol/l HEPES, 2 mmol/l L-glutamine, and 0.2% levels (24–27). Thus, it cannot be concluded that chronic BSA; IBMX, 3-isobutyl-1-methylxanthine; IP, intraperitoneal; KRBH, HEPES- homologous desensitization of the GIP receptor in type 2 buffered Krebs-Ringer bicarbonate buffer; PCR, polymerase chain reaction; Rf, relative mobility; RIPA, radioimmunoprecipitation assay; RT, reverse tran- diabetes causes an ineffective incretin response (28,29). scription; TBST, Tris-buffered saline with 0.5% Tween 20. Furthermore, studies have shown point mutations in the

1004 DIABETES, VOL. 50, MAY 2001 F.C. LYNN AND ASSOCIATES

GIP receptor gene in human populations that affect GIP from exocrine tissue via centrifugation (1,000g at 4°C) through a discontinu- signaling in cell models; however, it has not been possible ous dextran gradient. Finally, islets were picked under a dissecting micro- scope, washed in HBSSϩ, and used for mRNA isolation or cultured in RPMI to associate these mutations with type 2 diabetes (30,31). 1640 with 8.8 mmol/l glucose, 10% fetal calf serum (Cansera, Rexdale, ON, In the current study, we set out to test the hypothesis Canada), antibiotics (50 U/ml each penicillin G and streptomycin), 0.07% that the ineffective GIP responses observed in type 2 human serum albumin, 0.0025% human apotransferrin, 25 pmol/l sodium diabetes can result from long-term downregulation of GIP selenite, and 20 ␮mol/l ethanolamine hydrochloride for 20–24 h in 10-cm plastic culture dishes (Falcon; Becton Dickinson) in a humidified 5% CO receptor expression in the ␤-cell plasma membrane. To 2 test this hypothesis, we used the Vancouver diabetic fatty environment. Perifusion of pancreatic islets. After the culture period, 40 healthy islets Zucker (VDF) rat as a model of type 2 diabetes. We were selected and sandwiched between two layers of Cytodex-3 beads demonstrated that there is a marked decrease in the GIP (Amersham Pharmacia, Baie d’Urfe´, PQ, Canada) in 0.2-ml polyethylene receptor mRNA expression in the pancreatic islets of these perifusion chambers (Endotronics, Coon Rapids, MN). The chambers were animals, which results in a decrease in GIP-stimulated then perifused in an Acusyst-s perifusion apparatus (Endotronics) under a humid 37°C 5% CO environment at a flow rate of 0.5 ml/min with 10 mmol/l insulin release from the pancreatic ␤-cells despite normal 2 HEPES-buffered Krebs-Ringer bicarbonate buffer (KRBH) supplemented with sensitivity to GLP-1. This provides evidence that defective 0.2% BSA. Perifusion experiments were carried out for 80 min after a 60-min GIP receptor expression may be a cause of the inappro- equilibration period in 2.8 mmol/l glucose (low glucose) perifusate. After 20 priate incretin response observed in many type 2 diabetic min, the perifusate glucose concentration was switched to 16 mmol/l with or subjects. without GIP. Samples were collected every 2 min, and insulin levels were determined by radioimmunoassay as previously described (32). Measurement of cAMP production. After an overnight culture, 40 healthy RESEARCH DESIGN AND METHODS islets were selected, washed twice with 0.5 ml of KRBH supplemented with Chemicals. Synthetic human GIP and GLP-1 were purchased from Bachem 0.2% BSA, and allowed to equilibrate for 30 min in a humidified 5% CO2 California (Torrance, CA), and 3-isobutyl-1-methylxanthine (IBMX) was pur- environment. The islets were then stimulated with either vehicle, 10 ␮mol/l chased from Research Biochemicals International (Natick, MA). All chemicals forskolin, or 10 nmol/l GIP for 30 min in the presence of 0.5 mmol/l IBMX. The of reagent or molecular biology grade were from Sigma (Oakville, ON, islets were then lysed by boiling for 5 min in 0.05N hydrochloric acid. Samples Canada) or Fisher Scientific International (Pittsburgh, PA). were then dried by vacuum centrifugation (Speed-Vac; Sorvall, Farmingdale, Animals. VDF rats spontaneously developed from a Zucker strain kept in our NY) and stored at –20°C for cAMP radioimmunoassay (Biomedical Technol- laboratory. These diabetic rats were homozygous recessive for a mutation in ogies, Stoughton, MA).

the receptor gene, fa (Gln269Pro). Rats carrying one normal Fa allele Isolation and measurement of islet GIP receptor mRNA by real-time were phenotypically lean and displayed normal glucose tolerance. Only male reverse transcriptase–polymerase chain reaction. Rat islet RNA was animals aged 14–16 weeks were used in this study. All animals tested isolated immediately after islet isolation using Trizol and the standard displayed severe glucose intolerance and decreased first-phase insulin re- protocol supplied by the manufacturer (Life Technologies). Specifically, 1 ml

sponse characteristic of VDF rats. Lean littermates were used as control Trizol reagent was used per 100 islets, and all A260/A280 ratios of isolated RNA animals in these experiments. were Ն1.80. After RNA isolation, 1 ␮g islet RNA was subjected to reverse Intraperitoneal glucose tolerance test. Zucker rats were anesthetized with transcription (RT). Total RNA was reverse transcribed in a volume of 10 ␮l an intraperitoneal (IP) injection of sodium pentobarbital (65 mg/kg) (Somno- containing 0.5 mmol/l deoxynucleotide triphosphate, 15 pmol gene-specific tol; MTC Pharmaceuticals, Cambridge, ON, Canada). The right jugular vein primer targeted at the COOH-terminus of the rat GIP receptor (GTT CTG GAG was then exposed and cannulated with heparinized polyethylene tubing TAG AGG TCC GTG TA), 75 pmol random hexamers (Amersham Pharmacia), (PE50; Becton Dickinson, Sparks, MD). GIP (4 pmol ⅐ min–1 ⅐ kg–1)or 100 U Superscript II RNase HϪ Reverse Transcriptase (Life Technologies), 10 physiological saline was infused (30 ␮l/min) via the cannula using an infusion U RNase inhibitor (RNA Guard; Amersham Pharmacia), 1 mmol/l dithiothre-

pump (Harvard Apparatus, South Natick, MA) for 5 min before an IP glucose itol, 50 mmol/l Tris-HCl, pH 8.3, 75 mmol/l KCl, and 3 mmol/l MgCl2. After RT, injection (40%, 1 g/kg). Blood samples (0.5 ml) were collected from the tail 100 ng rat islet tissue cDNA was used in the real-time polymerase chain vein in heparinized Caraway/Natelson collecting tubes (Fisher Scientific reaction (PCR) to measure GIP receptor expression, whereas 10 ng cDNA was International) 5 min before (basal) and 10, 20, 30, and 60 min after glucose used in the GAPDH control PCR. The PCR mix consisted of 1ϫ TaqMan Buffer ␮ injection. Concomitantly, blood glucose measurements were taken before the A (PE Applied Biosystems, Foster City, CA), 10 mmol/l MgCl2; 200 mol/l infusion started (basal) and then every 10 min after glucose administration dATP, dCTP, and dGTP and 400 ␮mol/l dUTP; 200 nmol/l rat GIP receptor 5Ј using a handheld blood glucose meter (SureStep; LifeScan, Burnaby, BC, forward primer (5Ј-CCG CGC TTT TCG TCA TCC-3Ј), 200 nmol/l rat GIP Canada). Plasma was then separated from red cells by centrifugation at receptor 3Ј reverse primer (5Ј-CCA CCA AAT GGC TTT GAC TT-3Ј), 200 10,000g for 20 min at 4°C and then stored at –20°C until GIP and insulin nmol/l GIP receptor probe colabeled with the fluorescent dyes FAM and radioimmunoassays could be carried out (32). TAMRA (5Ј-CCC AGC ACT GCG TGT TCT CGT ACA GG-3Ј), 0.01 U/␮l In vitro pancreatic perfusion. Anesthesia was established using Somnotol, AmpErase uracil-DNA glycosylase (UNG) (PE Applied Biosystems), and 0.025 and pancreases were isolated as previously described (16). The perfusate U/␮l of AmpliTaq Gold (PE Applied Biosystems). The GAPDH reactions consisted of a modified Krebs-Ringer bicarbonate buffer containing 3% dex- included the above reaction conditions with the exception of the primers and tran and 0.2% bovine serum albumin (BSA) (Fraction V, RIA grade; Sigma) probe, which were purchased from PE Applied Biosystems and were directed

gassed with 95% O2/5% CO2 to achieve a pH of 7.4. The abdominal aorta was toward rodent GAPDH. PCRs were carried out in triplicate in the PE Applied perfused at a rate of 4 ml/min, and portal venous outflow was collected at Biosystems 7700 sequence detection system. The reaction profile included a 1-min intervals. After a 10-min equilibration period, the pancreatic perfusion 10-min preincubation at 50°C to allow the UNG to degrade any uracil- continued with 4.4 mmol/l glucose for 4 min followed by 8.8 mmol/l glucose containing nucleic acids and a further 10 min incubation at 94°C to activate for the remainder of the experiment. GIP (10 pmol/l), GLP-1 (50 pmol/l), or the AmpliTaq Gold. After these preincubations, a two-step PCR protocol was saline was introduced into the perfusion system from 20 to 40 min. The carried out, which included a denaturation step at 94°C for 15 s followed by different doses of GIP and GLP-1 were chosen because they had equal a 1-min annealing/extension step at 60°C. Fluorescence was measured during insulinotropic activities in the lean Zucker perfused pancreas. the annealing/extension steps over 40 cycles and used to calculate a cycle Isolation and culture of rat pancreatic islets. Rat pancreatic islets were threshold, i.e., the point at which the reaction is in the exponential phase and isolated as previously described (33). Briefly, the rat was anesthetized, and a is detectable by the hardware. All reactions followed the typical sigmoidal midline incision was made. The common bile duct was cannulated, and the reaction profile, and cycle threshold was used as a measure of amplicon pancreas was inflated with collagenase (320 mg/l, Type XI; Sigma) in Hank’s abundance (34). balanced salt solution supplemented with 10 mmol/l HEPES, 2 mmol/l Western analysis of islet GIP receptor protein. Islets were isolated as L-glutamine, and 0.2% BSA (HBSSϩ) (Life Technologies, Burlington, ON, previously described. After isolation, islet GIP receptor protein was analyzed Canada). The pancreas was then removed from the rat and macerated with as previously described (35). Briefly, islets were lysed in ice-cold radioimmu- scissors before collagenase . The pancreatic tissue was initially noprecipitation assay buffer (150 mmol/l NaCl, 20 mmol/l Tris-Cl, pH 7.5, 1 digested in a shaking 37°C water bath for 20 min and 10 min for pancreases mmol/l EDTA, 1% Nonidet P-40, 1% deoxycholate, 0.1% SDS, 5 mmol/l NaF, 1 from lean and fat rats, respectively; a second digestion was then carried out mmol/l phenymethylsulfonyl fluoride, 1 mmol/l dithiothreitol [DTT], 10 ␮g/ml for 10 and 7 min for the lean and fat rats, respectively. After the collagenase , 10 ␮g/ml pepstatin A, 10 ␮g/ml bestatin, and 1% Trasylol [Bayer digestions, the islets were passed through a 1-mm nylon screen and separated Pharmaceuticals, Etobicoke, ON, Canada]) for 30 min on ice. Protein concen-

DIABETES, VOL. 50, MAY 2001 1005 DEFECTIVE GIP RECEPTOR EXPRESSION IN DIABETIC RATS tration was determined using a Bicinchoninic Acid kit (Pierce, Rockford, IL). Then, 50 ␮g total islet protein was denatured under reducing conditions (100 mmol/l DTT) at 100°C for 5 min and run by SDS-PAGE. were then transferred to nitrocellulose membrane, blocked with 5% skim milk (in Tris-buffered saline with 0.5% Tween 20 [TBST]), and incubated with a well-characterized polyclonal anti-GIP receptor antibody (35). Membranes were then washed three times in TBST and incubated with horseradish peroxidase–conjugated goat anti-rabbit IgG secondary antibody (Jackson Immunoresearch Laboratories, West Grove, PA). After further washing, the immunoreactive bands were visualized using enhanced chemiluminescence (Amersham Pharmacia). Finally, bands were subjected to densitometry using Eagle Eye II software (Stratagene, La Jolla, CA), and molecular weight was determined using relative mobility (Rf) analysis. Data analysis. All data are expressed as mean Ϯ SE, with the sample size indicated in the appropriate figure legend. Unpaired two-tailed t tests were carried out to compare groups of animals. P values Յ0.05 were considered statistically significant. Area under the curve was determined using curve analysis software (Graphpad; Prism, San Diego, CA).

RESULTS Effect of GIP on glucose tolerance in the VDF rat. The optimal GIP dose was determined by carrying out the bioassay with various GIP concentrations ranging from 2 pmol ⅐ min–1 ⅐ kg–1 to2nmol⅐ min–1 ⅐ kg–1 (data not shown). The optimum dose determined from this study (4 pmol ⅐ min–1 ⅐ kg–1) produced a submaximal glucose-lowering response in the lean animals. Thus, this dose was used for the remainder of the experiments. Figure 1A shows the blood glucose response to an IP glucose tolerance test with and without GIP in the lean control animals. This figure clearly shows that GIP was able to significantly improve glucose tolerance in the lean animals as early as 30 min after IP glucose injection. Furthermore, this im- provement in glucose tolerance was maintained as long as the GIP infusion was continued. Figure 1B shows that the integrated glucose response (over 60 min) for the lean animals receiving GIP was significantly smaller than that in animals receiving saline. The fat animals displayed basal hyperglycemia, with an average of 8.3 Ϯ 0.4 mmol/l, compared with 5.5 Ϯ 0.2 FIG. 1. Glucose response to infused GIP (‚) and saline (f) in control mmol/l for the lean animals (Fig. 1A and C). The fat Fa/? (A and B) and VDF fa/fa (C and D) rats. Basal blood glucose animals also had much higher peak glucose levels in samples were taken, and then a 4 pmol ⅐ min–1 ⅐ kg–1 dose of GIP was response to the glucose challenge, with the control (sa- infused into the jugular vein. After 5 min of infusion, glucose (1 g/kg) was administered via an IP injection. Blood glucose measurements line) values peaking at 15 mmol/l (compared with 11 were made on a handheld glucose analyzer at 10-min intervals. B and D mmol/l in lean animals); thus, the fatty animals were were obtained by integrating the AUC in A and C. *Statistical signifi- .P < 0.05). Values are expressed as means ؎ SE ,12 ؍ glucose intolerant and hyperglycemic. The GIP infusion cance (n did not result in a decrease in circulating glucose levels in the fat animals, as no difference (P Ͼ 0.05) was observed levels were much higher at all times during the infusion between GIP and saline infusions at any time after IP protocol in this group (Fig. 2C). Furthermore, there was glucose (Fig. 1C). Furthermore, the integrated glucose neither a significant increase in insulin secretion elicited response for the fatty animals that received GIP was not by GIP infusion (Fig. 2C) nor an increase in the integrated different from the integrated response of saline-infused insulin response in these animals (Fig. 2D). animals (Fig. 1D). Thus, GIP yielded no improvement in Effect of GIP on insulin release from the pancreas of glucose tolerance in the diabetic fatty animals. the VDF rat. Pancreatic perfusions were carried out to Effect of GIP on insulin secretion in the VDF rat. determine if the release of insulin was a direct response to Plasma was collected at –5, 10, 20, 30, and 60 min after IP GIP in the intraperitoneal glucose tolerance test. As indi- glucose and assayed for insulin and GIP content. There cated in Fig. 3A and C, both 10 pmol/l GIP and 50 pmol/l was no difference observed in basal circulating GIP levels GLP-1 in the presence of 8.8 mmol/l glucose evoked between the fat (16.7 Ϯ 1.8 pmol/l) and lean (15.5 Ϯ 1.6 approximately equal peak 2.8-fold (6.1 AUC) and 3.4-fold pmol/l) animals. GIP yielded a significant increase in (5.3 AUC) increases, respectively, in insulin secretion from circulating insulin levels in the lean animals with a peak of the lean pancreas compared with control conditions. How- 400 pmol/l at 20 min, which was before the glucose peak, ever, this augmentation in insulin secretion was not ob- in the same test group (Figs. 1A and 2A). Additionally, the served in the fatty Zucker pancreas when stimulated with integrated insulin response was significantly greater in the GIP (Fig. 3B and D) where insulin secretion decreased lean animals that received GIP (Fig. 2B). Because of the from ϳ8.5 nmol/l to around 3.7 nmol/l during the high insulin-resistant state of the fatty animals, the insulin glucose and GIP infusion period. In contrast, GLP-1 caused

1006 DIABETES, VOL. 50, MAY 2001 F.C. LYNN AND ASSOCIATES

response to glucose alone seen in the lean rat islets (Fig. 4A and B). Effect of GIP on cAMP production in VDF rat islets. Islet cAMP studies were carried out to elucidate where the defect in the GIP signaling pathway in the fatty animals occurred. Figure 5 illustrates the effect of GIP and forsko- lin on cAMP production in islets from fat and lean rats. GIP (10 nmol/l) produced a significant (2.2-fold basal) re- sponse in the lean control islets; however, there was no observable cAMP response to GIP in the islets from obese animals. The basal values of cAMP production did not differ significantly between the two phenotypes. Addition- ally, the forskolin responses of the fat and lean animals did not differ significantly, indicating that the cAMP produc- tion machinery was not defective in the VDF rats.

FIG. 2. Insulin responses to infused GIP (‚) and saline (f) in control Fa/? (A and B) and VDF fa/fa (C and D) rats. Basal blood glucose samples were taken, and then a 4 pmol ⅐ min–1 ⅐ kg–1 dose of GIP was infused into the jugular vein. After 5 min of infusion, glucose (1 g/kg) was administered via an IP injection. Blood samples (500 ␮l) were collected from the tip of the nicked tail, and plasma insulin was assayed using radioimmunoassay. B and D were obtained by taking the AUC ؍ from the time course studies in A and C. *Statistical significance (n .P < 0.05). Values are expressed as means ؎ SE ,4 a significant insulin secretion from the pancreas of the fat animals that reached a plateau at 10 times the control perfusion level of secretion (Fig. 3B and D). Pancreatic islet perifusion was carried out to determine whether the defect in GIP stimulation of insulin secretion was confined to the islet. As seen in Fig. 4A, 10 nmol/l GIP in the presence of 16 mmol/l glucose was able to stimulate insulin secretion from the lean Zucker islet, with a peak level 10 times the basal level of 62 pmol/l. High glucose (16 mmol/l) alone only produced a fourfold increase in insulin release from the islets. Figure 4B illustrates the effects of GIP on the islets from the fatty Zucker rat. As illustrated, FIG. 3. Insulin responses from perfused pancreas of control (A and C) 10 nmol/l GIP had little effect on insulin release from these and VDF (B and D) rats. Pancreases were perfused at a rate of 4 ml/min after equilibration, the preparations (4 ؍ with Krebs buffer. At 4 min (t islets in the presence of 16 mmol/l glucose. The peak were subjected to 8.8 mmol/l glucose (छ, glucose alone). GIP (F,10 GIP-stimulated insulin release from the fat islets in the pmol/l) or GLP-1 (Ⅺ, 50 pmol/l) was then added via an infusion pump perifusion system was 4.3 Ϯ 1.5 ϫ basal, whereas glucose and a side arm at 20 min. Samples were collected every minute and Ϯ assayed for insulin content using radioimmunoassay. The AUCs (C and alone produced about a 3.2 1.5-fold increase from a D) were determined using Graphpad software. Values are expressed as .6–3 ؍ basal level of 95 pmol/l; this response was similar to the means ؎ SE. n

DIABETES, VOL. 50, MAY 2001 1007 DEFECTIVE GIP RECEPTOR EXPRESSION IN DIABETIC RATS

marked decrease in GIP receptor protein level in islets from VDF rats. This decrease is in accordance with that seen with mRNA levels as well as insulin release and cAMP stimulation of islets with GIP.

DISCUSSION Human type 2 diabetic subjects have been characterized by a decreased incretin response (21,26). This response has been attributed to dysfunction in the GIP portion of the enteroinsular axis because GLP-1 continues to have both insulinotropic and blood glucose–lowering actions in these individuals (22,26,36). The current study examined the expression and function of the GIP receptor on the pancreatic ␤-cell of the VDF rat—an animal model for type 2 diabetes. It was shown for the first time that there is a decreased level of GIP receptor mRNA and protein in the pancreatic islets of animals exhibiting characteristics of type 2 diabetes: hyperinsulinemia and insulin resistance. This decrease in GIP receptor resulted in a decreased insulin response to physiological levels of GIP. The VDF rats used in this study were both glucose intolerant (Fig. 1) and hyperinsulinemic (Figs. 2 and 3). Additionally, these animals did not exhibit a second-phase insulin response to glucose (Fig. 3) and, therefore, have developed a syndrome similar to that observed in human type 2 diabetes. The pancreas perfusions (Fig. 3) demon- strated that there is a blunted first-phase insulin secretion in the fat animals: 125% increase compared with a 600% increase in lean animals in response to the introduction of 8.8 mmol/l glucose. Furthermore, in the normal lean animal, 10 pmol/l GIP stimulated a characteristic biphasic insulin response, whereas this hormone had no effect on second-phase insulin secretion in the fat animal (Fig. 3A and B). Interestingly, 10 pmol/l GIP and 50 pmol/l GLP-1 had similar insulinotropic effects in the perfused pancreas from the lean animals (Fig. 3C); however, only GLP-1 was able to potentiate glucose-induced insulin secretion from FIG. 4. Insulin release from perifused islets isolated from control (A) and VDF (B) rats. Islets were isolated, cultured, and perifused as the VDF pancreas (Fig. 3D). The perifusion data demon- described in RESEARCH DESIGN AND METHODS. After a 70-min equilibration strate that GIP was able to significantly increase first- at 2.8 mmol/l glucose, 10 nmol/l GIP in 16.6 mmol/l glucose (‚) or 16.6 mmol/l alone (f) was applied and continued for the remainder of the experiment. Insets depict AUCs that were determined using the trap- ezoid rule (Graphpad). Fractions were collected every 2 min, and perifusate was assayed for insulin using radioimmunoassay. Data are .3 ؍ expressed as means ؎ SE. n

GIP receptor mRNA expression in the VDF rat islets. The cAMP data suggested that a decrease of GIP receptor expression could be responsible for the decreased effective- ness of GIP in the fatty Zucker rat. This hypothesis was tested by carrying out real-time RT-PCR on RNA isolated from islets of lean and fat animals. We observed a signif- icant (75 Ϯ 5%) decrease in GIP receptor mRNA in the islets from the fatty Zucker rats, as seen in Fig. 6A. Additionally, the reduction of GIP receptor mRNA was obtained when measured with RT-competitive PCR—an alternate means of measuring RNA expression (data not FIG. 5. Islet cAMP responses to GIP and forskolin from control and shown). VDF rat islets. Islets were isolated and cultured as described in GIP receptor protein expression in the Zucker rat RESEARCH DESIGN AND METHODS. Forty islets were allowed to equilibrate in 8.8 mmol/l glucose KRBH at 37°C for 30 min before GIP stimulation (10 islets. The Western blot in Fig. 6B is representative of nmol/l). GIP, forskolin (10 ␮mol/l), or glucose alone was then added to those obtained when comparing fat and lean islet GIP the islets in KRBH supplemented with 0.5 mmol/l IBMX. Islets were receptor protein content. The posttranslationally modified stimulated for 30 min before cAMP extraction and measurement using -Sta* .(4 ؍ ϳ radioimmunoassay. Data are expressed as means ؎ SE (n GIP receptor appears to run at 65 kDa, which is in tistical significance between control and GIP-stimulated conditions agreement with previous work (35). Figure 6B illustrates a (P < 0.05).

1008 DIABETES, VOL. 50, MAY 2001 F.C. LYNN AND ASSOCIATES

half-life of the incretins (39). Currently, there is contro- versy as to whether GIP levels are elevated, normal, or lowered in type 2 diabetes and animal models for the disease (24–27,40). One possible explanation for the inef- fectiveness of GIP in the VDF rat is an increase in DPIV in these animals, which could inactivate GIP prior to its actions on the ␤-cell. Because it was impossible to mea- sure differences in intact versus NH2-terminally truncated GIP with our radioimmunoassay, it was not possible to determine the role of DPIV in our findings, nor was it possible to determine the concentration of bioactive GIP in these animals. However, it has been demonstrated by Pederson et al. (41) that the levels of circulating DPIV in VDF rats and their lean littermates are similar; therefore, this explanation for GIP ineffectiveness can be ruled out. Exposure of the rat islet to GIP leads to desensitization of the islet to GIP (29). Currently, it is not clear whether this is due to a reversible phosphorylation of the GIP receptor, a decrease in GIP receptor mRNA expression, or both. However, it was demonstrated that there was a rapid homologous desensitization of GIP-stimulated insulin se- cretion in mouse ␤-cells (␤TC3) that occurred at the receptor level as well as further downstream in the signal- ing cascade. In contrast, in the current study, the cAMP response to GIP in the islets of the VDF animals suggests there is a defect in the GIP receptor–adenylyl cyclase segment of the GIP receptor signaling pathway. Further- more, we were unable to measure elevated ambient GIP levels in the VDF rat, indicating that GIP is not causing a loss of functional cell surface receptors, either by desen- sitization or downregulation, in these animals at this level of glucose intolerance. Expression of other G-protein– coupled receptors (such as the ) in the superfamily are regulated by glucose as well as the ade- nylyl cyclase activator forskolin (42). Thus, it is likely that GIP receptor downregulation and dysfunction are a result of inappropriate stimulation of the ␤-cell by abnormal levels of metabolites and hormones in this animal model. FIG. 6. A: GIP receptor mRNA levels in the islets of control (f, Fa/?) The current study demonstrates for the first time that and VDF (Ⅺ, fa/fa) rats. Islets were isolated as described in RESEARCH GIP receptor expression is downregulated at the mRNA DESIGN AND METHODS. After isolation, RNA was extracted from the islets, and 1 ␮g pancreatic islet RNA was subjected to real-time RT-PCR. GIP and protein levels in the pancreatic ␤-cell of the diabetic receptor mRNA was normalized to GAPDH mRNA content and ex- Zucker rat (Fig. 6A and B). These observations suggest pressed as a fraction of control islet content. Data are expressed as Statistical significance (P < 0.05). B: GIP that there is a decrease in receptor expression on the* .(4 ؍ means ؎ SE (n receptor protein expression in the islets of control (Fa/?) and VDF pancreatic ␤-cell and that this decrease in expression (fa/fa) rats. Islets were isolated as described in RESEARCH DESIGN AND leads to the loss of islet sensitivity to GIP. Furthermore, METHODS. After isolation, islets were lysed in ice-cold RIPA buffer. Then, 50 ␮g total cellular protein was run on a 13% polyacrylamide gel. we have recently demonstrated in clonal ␤-cells that The gel was transferred to a nitrocellulose membrane and blotted with changes in mRNA expression also produce changes in cell GIP receptor antibody, followed by horseradish peroxidase–conju- gated goat anti-rabbit secondary antibody. The immunoreactive bands surface GIP receptor expression using radioligand satura- were visualized using enhanced chemiluminescence, and GIP receptor tion binding curves (F.C.L., unpublished data). Therefore, ؍ molecular weight (65 kDa) was determined using Rf analysis. n 3. we believe that there is a decrease in cell surface GIP receptor concomitant to the decrease in total GIP receptor phase insulin secretion in islets from lean animals, but mRNA and protein in these islets. continued exposure to 10 nmol/l GIP may have resulted in Recently, Be´guin et al. (43) demonstrated that stimula- desensitization of ␤-cell surface GIP receptors, and no tion of the ␤-cell by GIP caused phosphorylation of the further significant effect of GIP on insulin secretion was Kir6.2 (KATP) channel via protein kinase A on serine 372. observed (29). Phosphorylation of this serine residue led to an increased Recently, there has been considerable interest in using open probability of the channel. This article first demon- dipeptidylpeptidase IV (DPIV) inhibitors in type 2 diabetes strates that GIP stimulation of the ␤-cell leads to protein therapy (37,38). Circulating DPIV inactivates both GLP-1 phosphorylation. However, the physiological basis for this and GIP by cleaving the NH2-terminal dipeptide from the phosphorylation event is still unclear since Be´guin et al. parent incretin polypeptide, rendering both peptides bio- believe that Kir6.2 is maximally phosphorylated in the logically inactive and thereby decreasing the circulating basal state. It is tempting to speculate that decreased

DIABETES, VOL. 50, MAY 2001 1009 DEFECTIVE GIP RECEPTOR EXPRESSION IN DIABETIC RATS levels of GIP receptor on the ␤-cell surface result in a stimulation of insulin release by glucose-dependent insulinotropic poly- decrease in the phosphorylation state of the K channel peptide (GIP): effect of a specific glucose-dependent insulinotropic poly- ATP peptide receptor antagonist in the rat. J Clin Invest 98:2440–2445, 1996 and decreased open probability. This in turn would lead to 12. Scrocchi LA, Brown TJ, Maclusky N, Brubaker PL, Auerbach AB, Joyner membrane depolarization and insulin secretion. If this AL, Drucker DJ: Glucose intolerance but normal satiety in mice with a null receptor deficit was great enough, there could be uncou- mutation in the glucagon-like peptide 1 receptor gene. Nat Med 2:1254– pling of glucose-stimulated insulin secretion and concom- 1258, 1996 itant ␤-cell decompensation, as observed in fatty rats. 13. Miyawaki K, Yamada Y, Yano H, Niwa H, Ban N, Ihara Y, Kubota A, In conclusion, glucose tolerance and insulin responses Fujimoto S, Kajikawa M, Kuroe A, Tsuda K, Hashimoto H, Yamashita T, Jomori T, Tashiro F, Miyazaki J, Seino Y: Glucose intolerance caused by a were studied after GIP infusion in the Vancouver Zucker defect in the entero-insular axis: a study in gastric inhibitory polypeptide diabetic fatty rat. In these animals, GIP did not potentiate receptor knockout mice. Proc Natl Acad SciUSA96:14843–14847, 1999 glucose-induced insulin secretion, either in vivo or from 14. Nauck MA, Bartels E, Orskov C, Ebert R, Creutzfeldt W: Additive insuli- the perfused rat pancreas and isolated perifused rat islets, notropic effects of exogenous synthetic human gastric inhibitory polypep- whereas the GLP-1 response remained intact. Moreover, tide and glucagon-like peptide-1-(7-36) amide infused at near-physiological insulinotropic hormone and glucose concentrations. J Clin Endocrinol GIP failed to stimulate cAMP production in isolated fa/fa Metab 76:912–917, 1993 islet static incubations. Finally, GIP receptor mRNA and 15. Jia X, Brown JC, Ma P, Pederson RA, McIntosh CH: Effects of glucose- protein levels were shown to be downregulated in the dependent insulinotropic polypeptide and glucagon-like peptide-I-(7-36) on islets of these animals, and this was hypothesized to be the insulin secretion. Am J Physiol Endocrinol Metab 268:E645–E651, 1995 basis for their insensitivity to GIP. This study suggests that 16. Pederson RA, Brown JC: The insulinotropic action of gastric inhibitory polypeptide in the perfused rat pancreas. Endocrinology 99:780–785, 1976 the decreased GIP response observed in this animal model 17. Rachman J, Turner RC: Drugs on the horizon for treatment of type 2 and in type 2 diabetes may be a result of a downregulation diabetes. Diabet Med 12:467–478, 1995 of the pancreatic GIP receptor. 18. Brown JC, Dryburgh JR, Frost JL, Otte SC, Pederson RA: Physiology and pathophysiology of GIP. Adv Exp Med Biol 106:169–171, 1978 19. Nauck MA: Glucagon-like peptide 1 (GLP-1): a potent gut hormone with a ACKNOWLEDGMENTS possible therapeutic perspective. Acta Diabetol 35:117–129, 1998 This work was supported by the Medical Research Council 20. Holst JJ: Glucagon-like peptide-1, a gastrointestinal hormone with a (MRC) of Canada (Grant 590007), the Canadian Diabetes pharmaceutical potential. Curr Med Chem 6:1005–1017, 1999 Association, the Canada Foundation for Innovation, and 21. Nauck M, Stockmann F, Ebert R, Creutzfeldt W: Reduced incretin effect in the British Columbia Health Research Foundation. F.C.L. type 2 (non-insulin-dependent) diabetes. Diabetologia 29:46–52, 1986 22. Nauck MA, Heimesaat MM, Orskov C, Holst JJ, Ebert R, Creutzfeldt W: is funded by the MRC and the Killam Trusts. 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