Ablation of ghrelin receptor in leptin-deficient ob/ob mice has paradoxical effects on glucose homeostasis when compared with ablation of ghrelin in ob/ob mice

Ablation of ghrelin receptor in leptin-deficient ob/ob mice has paradoxicaleffects on glucose homeostasis when compared with ablation of ghrelinin ob/ob mice

Xiaojun Ma,1,2* Yuezhen Lin,3* Ligen Lin,1 Guijun Qin,2 Fred A. Pereira,4 Morey W. Haymond,1,3

Nancy F. Butte,1 and Yuxiang Sun1,4

1US Department of Agriculture/Agricultural Research Service Children’s Nutrition Research Center, Department ofPediatrics, Baylor College of Medicine, Houston, Texas; 2Division of Endocrinology, Department of Internal Medicine,The First Affiliated Hospital of Zhengzhou University, Zhengzhou, Henan, China; 3Department of Pediatric Endocrinologyand Metabolism, Texas Children’s Hospital, Baylor College of Medicine, Houston, Texas; and 4Huffington Center on Aging,Department of Molecular and Cellular Biology, Baylor College of Medicine, Houston, Texas

Submitted 15 November 2011; accepted in final form 4 June 2012

Ma X, Lin Y, Lin L, Qin G, Pereira FA, Haymond MW, Butte NF,Sun Y. Ablation of ghrelin receptor in leptin-deficient ob/ob mice hasparadoxical effects on glucose homeostasis when compared with ablation ofghrelin in ob/ob mice. Am J Physiol Endocrinol Metab 303: E422–E431,2012. First published June 5, 2012; doi:10.1152/ajpendo.00576.2011.—Theorexigenic hormone ghrelin is important in diabetes because it has aninhibitory effect on insulin secretion. Ghrelin ablation in leptin-deficient ob/ob (Ghrelin�/�:ob/ob) mice increases insulin secretionand improves hyperglycemia. The physiologically relevant ghrelinreceptor is the growth hormone secretagogue receptor (GHS-R), andGHS-R antagonists are thought to be an effective strategy for treatingdiabetes. However, since some of ghrelin’s effects are independent ofGHS-R, we have utilized genetic approaches to determine whetherghrelin’s effect on insulin secretion is mediated through GHS-R andwhether GHS-R antagonism indeed inhibits insulin secretion. Weinvestigated the effects of GHS-R on glucose homeostasis in Ghsr-ablated ob/ob mice (Ghsr�/�:ob/ob). Ghsr ablation did not rescue thehyperphagia, obesity, or insulin resistance of ob/ob mice. Surpris-ingly, Ghsr ablation worsened the hyperglycemia, decreased insulin,and impaired glucose tolerance. Consistently, Ghsr ablation in ob/obmice upregulated negative �-cell regulators (such as UCP-2, SREBP-1c, ChREBP, and MIF-1) and downregulated positive �-cell regula-tors (such as HIF-1�, FGF-21, and PDX-1) in whole pancreas; thissuggests that Ghsr ablation impairs pancreatic �-cell function in leptindeficiency. Of note, Ghsr ablation in ob/ob mice did not affect the isletsize; the average islet size of Ghsr�/�:ob/ob mice is similar to that ofob/ob mice. In summary, because Ghsr ablation in leptin deficiencyimpairs insulin secretion and worsens hyperglycemia, this suggeststhat GHS-R antagonists may actually aggravate diabetes under certainconditions. The paradoxical effects of ghrelin ablation and Ghsrablation in ob/ob mice highlight the complexity of the ghrelin-signaling pathway.

growth hormone secretagogue receptor; insulin secretion; type 2 diabetes

OBESITY IS ONE OF THE MOST ALARMING HEALTH CONCERNS in Westernand developing countries. Obesity causes insulin resistance, oftenleading to type 2 diabetes. Ghrelin is a multifaceted hormone bestknown for its orexigenic action; growth hormone secretagoguereceptor (GHS-R) is recognized as a physiologically relevantreceptor for ghrelin, mediating ghrelin’s effects on growth hor-

mone release, food intake, and adiposity (6, 12, 46). Both ghrelinand GHS-R are expressed in pancreatic islets (51, 53). Wedemonstrated previously that whereas ghrelin gene ablation inwild-type mice has no effect on hyperphagia or obesity, ghrelinablation in leptin-deficient ob/ob mice ameliorates the diabeticcondition (44). Studies have demonstrated that exogenous ghrelinadministration inhibits insulin secretion both in vivo and in vitro(4, 16, 41, 44), confirming ghrelin as a negative regulator ofinsulin secretion and implying that ghrelin has an important role inglucose homeostasis.

We reported previously that circulating ghrelin increases duringfasting and that glucose concentrations decrease in calorie-restricted ghrelin- and Ghsr-ablated mice, which suggests thatboth ghrelin and GHS-R are involved in glucose sensing (45, 46).We have shown that ghrelin’s stimulatory effects on growthhormone (GH) release and feeding are mediated through GHS-R(46). However, it is unknown whether ghrelin’s effect on insulinsecretion is mediated through GHS-R. A widely used GHS-Rantagonist, [D-Lys3]-GHRP-6, has been shown to block ghrelin’sinhibitory effect on insulin secretion (16). It was thus inferred thatghrelin’s effect on insulin secretion is mediated through theGHS-R and that GHS-R antagonists would be a viable approachfor treating diabetic hyperglycemia (1, 2, 16). However, a recentreport showed that [D-Lys3]-GHRP-6 blocks receptors other thanGHS-R; for example, the CCR5 and CXCR4 receptors, which areused by HIV to enter cells, can also be blocked by [D-Lys3]-GHRP-6 (34). In addition, GHS-R is known to have constitutiveactivity, which is capable of producing its biological response inthe absence of a bound ligand (21). Some GHS-R-associatedeffects may be due to the constitutive activity of GHS-R ratherthan the activation of ghrelin. We have shown recently thatghrelin- and Ghsr-null mice have differential phenotypes in ther-mogenic regulation and sleep; others have shown that ghrelindirectly stimulates liver glucose output and adipogenesis by mech-anisms independent from GHS-R (18, 30, 47, 48, 50). Theevidence collectively suggests that some ghrelin functions may bemediated by subtype receptor(s) other than GHS-R. It is unknownwhether ghrelin’s effect on pancreatic �-cells is mediated exclu-sively by GHS-R or whether GHS-R antagonists can be used asantidiabetic agents.

The objectives of this study were to 1) assess the effect ofGHS-R ablation on glucose homeostasis under obese and diabeticstates, 2) determine whether ghrelin deletion and GHS-R deletionhave similar effects on glycemic control in leptin-deficient mice,

* X. Ma and Y. Lin contributed equally to this work.Address for reprint requests and other correspondence: Y. Sun, Children’s

Nutrition Research Center, Dept. of Pediatrics, Baylor College of Medicine,1100 Bates Ave., Rm. 5024, Houston, TX 77030 (e-mail: [email protected]).

Am J Physiol Endocrinol Metab 303: E422–E431, 2012.First published June 5, 2012; doi:10.1152/ajpendo.00576.2011.

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and 3) shed light on whether ghrelin’s inhibitory effect on insulinsecretion is mediated through GHS-R. To enable these studies, webred our Ghsr�/� mice with leptin-deficient ob/ob mice to gen-erate a mouse model lacking both GHS-R and leptin (Ghsr�/�:ob/ob).

METHODS

Generation of Ghsr�/�:ob/ob mice. Our studies were approved bythe Institutional Animal Care and Use Committee at Baylor College ofMedicine. The generation of ghrelin�/�, Ghsr�/�, and ghrelin�/�:ob/ob mice has been described by us previously (43, 44, 46). All miceused in the study were on the C57BL/6J background. To generateGhsr�/�:ob/ob mice, N12 Ghsr�/� mice were bred to ob/� micefrom The Jackson Laboratory, creating compound heterozygotes, i.e.,Ghsr�/�:ob/OB mice. In the second cross, these mice were interbredto generate Ghsr�/�:ob/ob mice and Ghsr�/�:OB/OB (Ghsr�/�)mice. In parallel, Ghsr�/�:ob/OB mice were bred to each other toproduce Ghsr�/�:ob/ob (ob/ob) and Ghsr�/�:OB/OB [wild-type(WT)] mice. To minimize animal-to-animal variations, only littermatemale mice were used. Mice were maintained under controlled tem-perature (�75°F) and illumination (12:12-h light-dark cycle, 6 AM to6 PM), with free access to water and regular chow.

General phenotypical characterization. Body weight and foodintake were measured weekly, and blood glucose was measuredbiweekly. Measurements were taken at the same time each day(between 9 and 10 AM) from 8 to 16 wk of age. Blood glucoseconcentrations were determined by a One-Touch Ultra glucometer(Lifescan, Milpitas, CA). Plasma triglycerides, total cholesterol, HDL,LDL, and free fatty acids (FFA) were measured by the lipid core ofHormone Assay & Analytical Services at Vanderbilt University.

Body composition and indirect calorimetry. Whole body composi-tion (fat and lean mass) of mice was measured by an Echo MRI-100whole body composition analyzer (Echo Medical Systems, Houston,TX). Metabolic parameters were obtained using the Oxymax (Colum-bus Instruments, Columbus, OH) open-circuit indirect calorimetrysystem. To minimize the confounding effects of stress, mice werecaged individually in metabolic chambers and given free access toregular chow and water for 1 wk. They were then placed in metaboliccages for �4 days before the indirect calorimetry testing. Indirectcalorimetry studies were carried out for 72 h. The first 24 h wereconsidered the acclimation phase, and average data of the final 48 hwere analyzed. Oxygen consumption (V̇O2; ml·kg�1·h�1), carbondioxide production (V̇CO2; ml·kg�1·h�1), and locomotor activity(beam break counts) were measured. Respiratory exchange ratio(RER) and energy expenditure (EE; or heat generation) were calcu-lated as we described previously (27, 33). Locomotor activity (onx-axis) was measured using infrared beams, and the number of beambreaks during the recording period was defined as locomotor activity.

Glucose and insulin tolerance test. Mice were fasted for 18 h (from3 PM to 9 AM) prior to testing and then given an intraperitoneal (ip)injection of D-glucose (0.625 g/kg for obese mice, or 2.0 g/kg for leanmice). Blood glucose was measured by tail bleeds at different timepoints. Fifty micoliters of blood from tails was collected in EDTA-coated tubes, and plasma samples were obtained by low-speed cen-trifugation. Insulin was analyzed by Hormone Assay & AnalyticalServices Core at Vanderbilt University using RIA assays. The insulintolerance test (ITT) was done similarly, except the mice were fastedfor only 6 h after lights-on, and Humulin (Eli Lilly, Indianapolis, IN)was administered by ip injection. 2.5 U/kg Humulin was used forobese mice.

Quantitative gene expression. All mice were euthanized in the morn-ing between 9 and 11 AM. Total RNA from whole pancreas wasextracted using TRIzol (Invitrogen, Carlsbad, CA) according to themanufacturer’s instructions. Relative quantitative RT-PCR was per-formed in triplicates, as described previously (44). Primer information isavailable upon request.

Histological analysis. Entire pancreases were fixed overnight in10% formaldehyde solution at room temperature and then dehydratedand embedded in paraffin. Tissue blocks of whole pancreas (showinghead, body, and tail of the pancreas) were then sectioned at 5 �m andstained with hematoxylin and eosin (H & E) for morphometricanalysis. The H & E staining was carried out according to the standardprotocols. The average area of pancreatic islets was measured usingAxiophot microscope, and the image was analyzed using the ScionImage for Windows analysis software [National Institutes of Health(NIH), Bethesda, MD]. The sectional area of islets was measured at amagnification of �10. All islets on each randomly selected sectionwere counted, and �120 islets were counted for each mouse.

Statistical analyses. All data are expressed as means � SE. Weused a two-tailed Student t-test, or one- or two-way ANOVA, todetermine significance of differences between genotypes or treat-ments. P 0.05 is defined as statistical significance.

RESULTS

GHS-R deficiency affects neither the body weight nor foodintake of ob/ob mice. No differences were observed in food intakeor body weight between WT and Ghsr�/� mice (Fig. 1, A and B).Ghsr�/�:ob/ob mice were hyperphagic compared with the leanWT and Ghsr�/� mice, but food intake was similar to that ofob/ob mice (Fig. 1A). Similar to Ghrelin�/�:ob/ob mice, the bodyweight of Ghsr�/�:ob/ob mice was significantly higher than thatof WT and Ghsr�/� mice but similar to that of ob/ob mice (Fig.1B). These results suggest that ablation of GHS-R in ob/ob micedoes not protect mice from hyperphagia or obesity resulting fromleptin deficiency.

GHS-R ablation affects neither body composition nor lipidprofile of ob/ob mice. The body fat contents of ob/ob andGhsr�/�:ob/ob mice were significantly higher than that of WTand Ghsr�/� mice, but there was no difference between ob/oband Ghsr�/�:ob/ob mice (Fig. 1C). Furthermore, comparedwith ob/ob mice, the Ghsr�/�:ob/ob mice showed similarplasma lipid profiles in triglyceride, cholesterol, HDL, andLDL (Fig. 1D) as well as FFA (Fig. 1E). Thus, GHS-R ablationaffects neither body composition nor lipid profiles of ob/obmice.

Metabolic profile of Ghsr�/�:ob/ob mice during indirectcalorimetry. Under the ad libitum-fed condition, there was nodifference in total food intake (Fig. 2A) or locomotor activity(Fig. 2B) between ob/ob and Ghsr�/�:ob/ob mice during cal-orimetry study. The characteristics of light and dark circadianrhythm were totally lost in mice on ob/ob the background (Fig.2, C and D). No differences were observed in total V̇O2, V̇CO2,or energy expenditure between ob/ob and Ghsr�/�:ob/ob micewhether normalized by either body weight or lean mass (Fig.2C). The Ghsr�/�:ob/ob mice showed a lower RER comparedwith ob/ob mice, and the difference persisted throughout thelight and dark phases (Fig. 2D). There were no differences inenergy expenditure or RER between ob/ob and Ghrelin�/�:ob/ob mice (data not shown).

GHS-R ablation aggravates hyperglycemia of ob/ob mice.As expected, under the ad libitum-fed condition, the lean micewere euglycemic, and the ob/ob mice developed hyperglyce-mia when compared with lean mice. Surprisingly, in contrast tothe improved glycemic condition observed in Ghrelin�/�:ob/ob mice, the Ghsr�/�:ob/ob mice exhibited worsened hy-perglycemia (Fig. 3A) and reduced plasma insulin and C-peptide(Fig. 3B) compared with ob/ob mice. Whereas plasma glucagonlevels of Ghsr�/� mice were significantly lower than those of WT

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mice, the glucagon levels in Ghsr�/�:ob/ob mice showed a trendof decrease compared with that of ob/ob mice, but this trend failedto reach statistical significance (Fig. 3C). These results suggestthat ablation of GHS-R further exacerbates hyperglycemia ofob/ob mice by inhibiting insulin secretion.

GHS-R ablation inhibits insulin secretion of ob/ob mice buthas no effect on insulin sensitivity. A low-dose (0.625 g/kg) ipglucose tolerance test (GTT) was selected to study the glucoseand insulin responses of Ghsr�/�:ob/ob mice, because mice onob/ob background are glucose intolerant (44). Compared withob/ob mice, Ghsr�/�:ob/ob mice displayed increased bloodglucose excursions at 30, 60, and 120 min following an ipglucose bolus (Fig. 3D); first-phase (15 min) plasma insulinconcentrations were decreased in Ghsr�/�:ob/ob mice com-pared with ob/ob mice (Fig. 3E). To elucidate whether theworsened hyperglycemia of Ghsr�/�:ob/ob mice was alsoattributable to insulin sensitivity, ITTs were performed. Nostatistically significant differences were observed in bloodglucose concentrations between ob/ob and Ghsr�/�:ob/obmice (Fig. 3F). These data suggest that GHS-R ablation inleptin-deficient mice further impairs �-cell insulin secretoryfunction but has no effect on insulin sensitivity.

Morphology of pancreatic islets in Ghsr�/�:ob/ob mice.Although obese mice (ob/ob, Ghrelin�/�:ob/ob, and Ghsr�/�:ob/ob) had significantly enlarged islets compared with lean WTmice (Fig. 4, A–D), there was no difference in islet sizebetween ob/ob and Ghsr�/�:ob/ob mice. On the other hand,Ghrelin�/�:ob/ob mice appeared to have larger islets than ob/oband Ghsr�/�:ob/ob mice, but the difference failed to reach statis-tical significance (P 0.08) due to the wide variation in islet size

(Fig. 4E). We noted that ob/ob and Ghsr�/�:ob/ob mice appearedto have more islet vascularization than WT and Ghrelin�/�:ob/obmice (Fig. 4, A–D). Ghsr�/�:ob/ob mice had pancreatic isletmorphology similar to that of ob/ob mice but were different fromthat of Ghrelin�/�:ob/ob mice.

Expression profiles of �-cell regulatory genes in whole pan-creas of ob/ob, Ghrelin�/�:ob/ob, and Ghsr�/�:ob/ob mice. Westudied the expression of regulators involved in insulin secretionand pancreatic �-cell mass. We showed previously that pancreaticuncoupling protein 2 (UCP2) mRNA expression is decreased inwhole pancreas of Ghrelin�/�:ob/ob mice (44). In contrast, theGhsr�/�:ob/ob mice have increased UCP2 expression in wholepancreas compared with ob/ob mice (Fig. 5A). The sterol regula-tory element-binding protein-1c (SREBP-1c) and peroxisomeproliferator-activated receptor-� coactivator-1� (PGC-1�) are posi-tive transcriptional regulators of UCP2, targeting the E-box and TREregions of UCP2 promoter, respectively (32). Indeed, SREBP-1clevels in whole pancreas were decreased in Ghrelin�/�:ob/ob micebut increased in that of Ghsr�/�:ob/ob mice (Fig. 5B), which is inline with UCP2 levels shown in these mouse models; however,there is no difference in PGC-1� levels (Fig. 5C). Whereas UCP2decreases ATP levels in �-cells, hypoxia-inducible factor-1�(HIF-1�) exerts its effect on �-cell function by stimulating ATP(9). Fibroblast growth factor-21 (FGF-21) has been shown toimprove �-cell function and survival by activation of extracellularsignal-regulated kinase 1/2 and the Akt signaling pathway (52).Remarkably, although we detected significant increases in theexpression of both HIF-1� and FGF-21 in whole pancreases ofGhrelin�/�:ob/ob mice, we found much lower levels of HIF-1�

Fig. 1. General characterization of growth hor-mone secretagogue receptor (Ghsr)�/�:ob/obmice. A and B: food intake and body weight.C: body composition of fat content. D: fedplasma lipid concentrations: triglycerides(TG), cholesterol, HDL, and LDL. E: fed freefatty acid (FFA). The data are presented asmeans � SE (n 8). P 0.05, obese vs. leanmice. P � 0.05, ob/ob vs. Ghsr�/�:ob/obmice. WT, wild type.

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and FGF-21 expression in those of Ghsr�/�:ob/ob mice (Fig. 5,D and E). Carbohydrate response element-binding protein(ChREBP) is a key regulator of glucose metabolism and fatstorage (15). Pancreatic and duodenal homeobox-1 (PDX-1) is atranscription factor necessary for pancreatic development and�-cell maturation. It has been shown that PDX-1 is negativelyregulated by ChREBP (13). Our results showed that ChREBPexpression was decreased in whole pancreases of Ghrelin�/�:ob/ob mice but increased in thos of Ghsr�/�:ob/ob mice (Fig. 5F).In contrast, PDX-1 showed an opposite expression pattern inwhole pancreas of both double-null mice compared with ChREBP(Fig. 5G). Deficiency of macrophage migration inhibitory factor-1(MIF-1) has been shown to protect pancreatic islets from palmiticacid-induced apoptosis (40). Yet whereas we detected lowerlevels of MIF-1 in whole pancreases of Ghrelin�/�:ob/ob mice,we observed higher levels of ChREBP in Ghsr�/�:ob/ob mice(Fig. 5H). In addition, we have studied expression of forkhead boxprotein O1 (FOXO1). FOXO1 has been shown to protect pancre-atic �-cells from fatty acid insult (31), but FOXO1 levels did notchange in our mouse models (Fig. 5I). Collectively, these data

exemplify the dramatic differences between the pancreatic geneexpression profiles of Ghrelin�/�:ob/ob and Ghsr�/�:ob/ob mice,suggesting that whereas ablation of ghrelin in ob/ob mice im-proves �-cell function, ablation of Ghsr in ob/ob mice mayworsen �-cell function.

DISCUSSION

The best-known functions of ghrelin are its roles in stimu-lating GH release and promoting appetite and adiposity. Weand others have shown that ghrelin is a negative regulator ofinsulin secretion (16, 44, 45). We reported that ablation ofghrelin expression augments insulin secretion and improvesinsulin sensitivity, resulting in the improvement of hypergly-cemia and glucose intolerance in diabetic ob/ob mice (44).Blockade of ghrelin has also been shown to increase insulinsecretion and prevent glucose intolerance induced by a high-fatdiet (16). These findings suggest that suppressing ghrelinsignaling prevents both genetically and environmentally in-duced �-cell impairments. It has thus been speculated that

Fig. 2. Metabolic profiles of Ghsr�/�:ob/ob mice.Food intake (A), activity (B), energy expenditure(EE) normalized by body weight (BW) or leanmass (C), and respiratory exchange ratio (RER; D)were analyzed using the Comprehensive Labora-tory Animal Monitoring System. The data arepresented as means � SE (n 8). *P 0.05,ob/ob vs. Ghsr�/�:ob/ob mice.

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GHS-R antagonists may serve as antidiabetic agents. Althoughthe effects of deletion and/or pharmacological blockade ofGHS-R on glucose homeostasis have been examined in normallean mice (11, 29, 60), the effect of GHS-R in obese anddiabetic subjects is unknown. Most diabetic patients are obese,so it is important to understand the role of GHS-R on glucosehomoeostasis under an obese diabetic state.

Leptin and ghrelin have opposite effects on food intake andbody weight regulation. It is thus important to understand theinterplay between leptin signaling and ghrelin signaling. Boththe leptin receptor and the ghrelin receptor are expressedwithin the same nuclei of the hypothalamic arcuate nucleus,which regulate appetite and satiety (35). One report showedthat the effects of ghrelin on GH secretion and food intake aresuppressed in leptin receptor-knockout db/db mice, suggestingthat cross-talk exists between the ghrelin- and leptin-signalingpathways (24). Another report showed that ghrelin receptordeficiency does not impact the anorexigenic effect of leptin,suggesting that ghrelin and leptin may act on separate anddistinct neuronal populations (35). In diet-induced obese mice,the circulating ghrelin level is lower, and the role of ghrelin onfood intake is suppressed (5, 24). In obese leptin-resistantZucker rats, GHRP-6-induced Fos response is increased, butcentral infusion of leptin suppresses this GHRP-6-induced Fos

response (22). These studies suggest that ghrelin-regulatorycircuits in the hypothalamus are dynamically regulated, and theregulation may vary based on nutritional states.

To study the role of GHS-R on glucose homeostasis underobese and diabetic conditions, we bred the Ghsr�/� mice withleptin-deficient ob/ob mice to generate Ghsr�/�:ob/ob mice. Sim-ilarly to Ghrelin�/�:ob/ob mice, we observed that GHS-R abla-tion in ob/ob mice failed to rescue the hyperphagic or obesephenotypes of ob/ob mice (Fig. 1, A–C). Plasma lipids, such asplasma levels of triglyceride, cholesterol, HDL, LDL, and FFA,play important roles in insulin resistance and glucose homeostasis.Here, we found that Ghsr�/�:ob/ob mice had lipid profiles similarto those of ob/ob mice (Fig. 1, D and E). These findings indicatethat unopposed action of ghrelin or GHS-R in ob/ob mice is notthe underlying cause of leptin-dependent obesity and that leptinhas a dominant effect on energy homeostasis.

We reported recently that ghrelin ablation and Ghsr ablationhave distinct effects on energy expenditure in older mice; olderGhsr�/� mice (but not older Ghrelin�/� mice) have an ele-vated energy expenditure (30) due to increased thermogenesisin brown adipose tissue (27). Characterization of the metabolicstate of Ghsr�/�:ob/ob mice revealed no differences in energyintake or locomotor activity (Fig. 2, A and B). Regardless ofnormalizing by body weight or lean mass, the energy expen-

Fig. 3. Ghsr�/�:ob/ob mice have more severe hyperglycemia than ob/ob mice. A: fed blood glucose concentrations at various ages. B and C: plasma insulin andC-peptide and glucagon concentrations at 16 wk of age. D and E: blood glucose and plasma insulin concentrations during glucose tolerance tests after 18 h offasting. F: blood glucose concentrations during insulin tolerance tests after 6 h of fasting. The data are presented as means � SE (n 8). *P 0.05, ob/ob vs.Ghsr�/�:ob/ob mice.

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diture of Ghsr�/�:ob/ob mice was no different from that ofob/ob mice (Fig. 2C). It is known that ob/ob mice have almostno brown adipose tissue, which makes them severely thermo-genetically impaired (23). The lack of brown adipose tissue inGhsr�/�:ob/ob may thus obscure the effect of the GHS-R onenergy expenditure. Interestingly, Ghsr�/�:ob/ob mice had asignificant reduction in RER during both the light and the darkperiods (Fig. 2D), indicating that the Ghsr�/�:ob/ob micepreferentially utilize fat as an energy source under the leptin-deficient background. Our data are consistent with previousreports that Ghrelin�/� and Ghsr�/� mice have decreasedRER in a diet-induced obese state (54, 60). However, the bodycomposition analysis (Fig. 1C) showed that the preferential fatconsumption associated with GHS-R ablation is insufficient torescue the obesity of ob/ob mice. This again supports the

conclusion that leptin plays a dominant role in regulatingenergy metabolism and body composition.

In surprising contrast to Ghrelin�/�:ob/ob mice, we foundthat Ghsr�/�:ob/ob mice exhibited higher basal glucose andlower insulin and C-peptide levels than ob/ob mice (Fig. 3,A and B). C-peptide, generated during proinsulin processingand secreted along with insulin, is a more accurate measure-ment for insulin secretion than plasma insulin itself, becauseplasma insulin concentrations can also be affected by degra-dation (55). The decreased C-peptide levels along with lowerinsulin concentrations confirm that insulin secretion is indeedreduced in Ghsr�/�:ob/ob mice. GHS-R is reported to beexpressed in both �- and �-cells of pancreatic islets (14, 25).Chuang et al. (11) showed that ghrelin injections increaseblood glucose and plasma glucagon in wild-type mice; consis-

Fig. 4. Histological analysis of pancreatic is-lets. Pancreas sections from age-matched WT,ob/ob, Ghrelin�/�:ob/ob, and Ghsr�/�:ob/obmice were stained with hematoxylin and eosinfor morphological characterization. The dataare presented as means � SE (n 3). *P 0.05, WT vs. ob/ob, Ghrelin�/�:ob/ob, andGhsr�/�:ob/ob mice. P 0.08, ob/ob vs.Ghsr�/�:ob/ob mice.

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tently, they showed that GHS-R knockout mice have lowerglucagon and fasting blood glucose. This suggests that ghre-lin’s regulation of blood glucose may also involve stimulationof glucagon secretion from �-cells. Similar to the observationmade by Chuang et al. (11), we detected lower glucagon levelsin Ghsr�/� mice compared with those of WT mice (Fig. 3C).As expected, both obese ob/ob and Ghsr�/�:ob/ob mice werehyperglucagonemic compared with lean WT and Ghsr�/�

mice. Although there was a trend toward decreasing gluca-

gon in Ghsr�/�:ob/ob mice compared with that of ob/obmice, it did not reach statistical significance (Fig. 3C). Thediscrepancy between the glucagon phenotype observed inGhsr�/�:ob/ob mice vs. that of Ghsr�/� mice might be dueto the nutritional state of the double-null mice and/or theleptin-deficient background. Since we did not detect signif-icant elevation of glucagon in Ghsr�/�:ob/ob mice, theworsened hyperglycemia of the double-mutant mice cannotbe explained by elevated glucagon secreted by �-cells.

Fig. 5. The mRNA expression of negative and positive �-cell regulatory genes in pancreata of obese mice. Uncoupling protein 2 (UCP2; A), sterol regulatoryelement-binding protein-1c (SREBP-1c; B), perxisome proliferator-activated receptor-� coactivator-1� (PGC-1�; C), hypoxia-inducible factor-1� (HIF-1�; D),fibroblast growth factor-21 (FGF21; E), carbohydrate response element-binding protein (ChREBP; F), pancreatic and duodenal homeobox-1 (PDX-1; G),macrophage migration inhibitory factor-1 (MIF-1; H), and forkhead box protein O1 (FOXO1; I). The data are presented as means � SE (n 9–12). *P 0.05and **P 0.001, ob/ob vs. Ghrelin�/�:ob/ob mice or ob/ob vs. Ghsr�/�:ob/ob mice. #P 0.05, Ghrelin �/�:ob/ob vs. Ghsr�/�:ob/ob mice.

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Our hormonal analysis data suggest that the Ghsr ablation inob/ob mice decreases insulin secretion. In agreement, glucosetolerance tests revealed that Ghsr�/�:ob/ob mice have wors-ened glucose tolerance, showing increased glucose but reducedinsulin when compared with ob/ob mice (Fig. 3, D and E).First-phase insulin secretion is the earliest detectable sign ofprediabetes (36). In line with the attenuated glucose responsein Ghsr�/�:ob/ob mice, first-phase (15 min) plasma insulinconcentrations were decreased in Ghsr�/�:ob/ob mice com-pared with ob/ob mice, supporting that Ghsr ablation attenu-ates insulin secretion in the leptin-deficient background (Fig.3E). GHS-R ablation in ob/ob mice does not have a significantimpact on insulin sensitivity (Fig. 3F), suggesting that insulinsensitivity does not contribute to the worsened hyperglycemiaand glucose intolerance of Ghsr�/�:ob/ob mice. Collectively,our data suggest that Ghsr ablation in ob/ob mice does notaffect glucose counterregulation or insulin sensitivity but fur-ther diminishes insulin secretion and aggravates hyperglyce-mia. This surprising outcome reveals that ghrelin and GHS-Rhave distinct effects on insulin secretion in ob/ob mice.

Pancreatic function can be affected by islet function or isletcell mass. The ob/ob mice have increased islet cell mass, dueprimarily to islet cell hyperplasia and hypertrophy (26, 49). Toexclude the possibility that the reduced pancreatic function ofGhsr�/�:ob/ob mice was due to reduced islet cell mass, weperformed histological examinations by H & E staining toevaluate islet size. As expected, we found that islet size ofobese (Ghrelin�/�:ob/ob, Ghsr�/�:ob/ob, and ob/ob) mice wassignificantly greater than that of lean WT mice, but there wasno difference in islet size between ob/ob and Ghsr�/�:ob/obmice (Fig. 4E). This suggests that the islet impairment inGhsr�/�:ob/ob mice is likely due to the effect of GHS-R on�-cell function but not on islet cell mass. It is intriguing thatthe islet blood vessel distribution of Ghrelin�/�:ob/ob mice issimilar to normal WT lean mice, whereas the islet vasculaturemorphology of Ghsr�/�:ob/ob mice closely resembles that ofob/ob mice (Fig. 4, A–D). The ob/ob mouse islets are moresensitive to sympathetic inhibition of catecholamines in thecirculation (39) and have reduced capacity for blood flow (28).This vascular feature of ob/ob mice increases �-cell stress andcauses more islet cell damage. These morphology data suggestthat ghrelin ablation may improve islet vasculature of ob/obmice, but Ghsr ablation does not. Again, this is in line with ourobservation that ghrelin�/�:ob/ob and Ghsr�/�:ob/ob micehave differential diabetic phenotypes. Ghsr ablation-associatedpancreatic impairment is likely attributable to the effect ofGHS-R on pancreatic function but not islet cell mass.

Mitochondrial UCP2 is a negative regulator of pancreatic�-cell function. UCP2 decreases ATP production and results ina reduced ATP/ADP ratio, thereby inhibiting insulin secretionin pancreatic �-cells (17, 56). Overexpression of UCP2 leads toreduced insulin secretion (7); in contrast, UCP2-deficient micehave increased insulin secretion (56). UCP2-ablated ob/obmice have restored first-phase insulin secretion and improvedglycemia (56). Previously, we showed that Ghrelin�/�:ob/obmice have attenuated hyperglycemia and improved �-cell func-tion, compared with that of ob/ob mice, resulting from down-regulation of UCP2 (44). Our current study shows that pancre-atic UCP2 mRNA was increased significantly in whole pan-creas of Ghsr�/�:ob/ob mice compared with that of ob/ob mice(Fig. 5A). Similarly, Ghsr ablation in ob/ob mice upregulated

negative �-cell regulators (such as SREBP-1c, ChREBP, andMIF-1) and downregulated positive �-cell regulators (such asHIF-1�, FGF-21, and PDX-1) (Fig. 5, B–I) in whole pancreas.The differential gene expression profiles are in line with theworsened hyperglycemia in Ghsr�/�:ob/ob mice and the im-proved glycemic control in Ghrelin�/�:ob/ob mice. This sup-ports our conclusion that ghrelin ablation in ob/ob mice im-proves pancreatic �-cell function, whereas Ghsr ablation inob/ob mice impairs pancreatic �-cell function. It is worthnoting that the pancreatic phenotypes may not be a reflection ofchanges solely in �-cells, since the gene expression studieswere carried out in whole pancreas. Pancreatic cell types otherthan �-cells may contribute to the pancreatic phenotypes viaendocrine and/or exocrine mechanisms.

It is important to emphasize that the glycemic phenotypes ofthe double-null mice were observed on the leptin-deficientobese background, which are different from that observed ineither ghrelin ablation or Ghsr ablation on the wild-type leanbackground. The glycemic phenotypes of the double-null micelikely result from the interplay between leptin signaling andghrelin/GHS-R signaling. Our data showed that ghrelin andGHS-R in a leptin-deficient background have differential rolesin glucose homeostasis. It is possible that ghrelin’s inhibitoryeffect on insulin secretion is mediated through subtype recep-tor(s) other than GHS-R. Culture studies of the direct effect ofghrelin in GHS-R knockdown pancreatic �-cells or GHS-R-null islets may help to further address this question.

It is also noteworthy that three peptides, ghrelin (acylatedghrelin), des-acyl ghrelin, and obestatin, are derived from thepreproghrelin gene, and all are expressed in the pancreas (8,42). Whereas ghrelin is known to activate GHS-R, des-acylghrelin and obestatin do not, and their receptors are eitherunknown or debatable. Des-acyl ghrelin is shown to increasefood intake and promote obesity (38, 48, 50, 58), whereasobestatin inhibits food intake and enhances energy expenditure(3, 57). More intriguingly, des-acyl ghrelin and obestatin havebeen shown to have opposing glucoregulatory effects com-pared with ghrelin; des-acyl ghrelin functions as a potentinsulin secretagogue (19, 58), and des-acyl ghrelin and obesta-tin increase islet cell mass and prevent streptozocin-induceddiabetes (10, 20, 37). In Ghrelin�/� mice, the signaling ofghrelin, des-acyl ghrelin, and obestatin are abolished. InGhsr�/� mice, the signaling pathway of ghrelin is abolished,but the signaling pathways of des-acyl ghrelin and obestatinremain intact. The glycemic phenotype of Ghsr�/� mice maybe a result of unopposed des-acyl ghrelin signaling and/orobestatin signaling.

Ghrelin O-acyltransferase (GOAT) is an acyltransferase thatcatalyzes ghrelin octanoylation. Ablation of GOAT in mice hasno phenotype under a regular diet or high-fat diet. Intriguingly,at 60% calorie restriction, the GOAT-null mice show severehypoglycemia and low circulating GH (59). This result indi-cates that ghrelin is essential for maintaining GH levels duringsevere calorie restriction to prevent hypoglycemia and death.In contrast, our studies were in overnourished obese mice,wherein ablation of ghrelin in ob/ob mice appeared to bebeneficial, but ablation of GHS-R in ob/ob mice appeared to bedetrimental. The differential effects of ghrelin deficiency inleptin-deficient background mice vs. the GOAT-ablated micemay be due to the difference in nutritional states of the miceand/or the effects of des-acyl ghrelin and obestatin present in

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GOAT-null mice. Our findings in obese and diabetic leptin-deficient mice suggest that GHS-R antagonists may be harmfulwhen used in treating diabetes in certain obese conditions.However, since most obese humans are leptin resistant ratherthan leptin deficient (having a metabolic state more like leptinreceptor-deficient db/db mice), further studies of the phenotypeof GHS-R ablation in db/db mice may provide additionalinsight.

In conclusion, our data show that ghrelin ablation and GHS-Rablation have opposite effects on glycemic control of leptin-deficient ob/ob mice; ghrelin ablation improves it, and GHS-Rablation worsens it. Ghrelin and Ghsr ablation have differentialeffects on glucose-induced pancreatic insulin secretion in theleptin-deficient background. In contrast to ghrelin neutralization,GHS-R antagonism may inhibit pancreatic insulin secretion andhas deleterious effects on pancreatic function. Ghrelin’s inhibitoryeffect on insulin secretion may be mediated by receptor(s) otherthan GHS-R. Our new findings highlight the extreme complexityof the ghrelin-signaling pathway in the pancreas and demonstratethat it is critically important to distinguish the effects of ghrelinneutralization from that of GHS-R antagonism. Further studies areneeded to fully understand the molecular mechanisms by whichghrelin and GHS-R regulate pancreatic �-cell function and todetermine the proper therapeutic applications for ghrelin neutral-ization and GHS-R antagonists.

ACKNOWLEDGMENTS

We thank Drs. Monique Rijnkels and Marta L. Fiorotto at the Children’sNutrition Research Center and the Department of Pediatrics at Baylor Collegeof Medicine for their insightful advice and input in real-time PCR analysis andcalorimetry studies, respectively. We thank Geetali Pradhan and Michael R.Honig for their editorial assistance.

GRANTS

This work is a publication of the US Department of Agriculture/Agricul-tural Research Service (USDA/ARS) Children’s Nutrition Research Center,Department of Pediatrics, Baylor College of Medicine, Houston, TX, and hasbeen funded in part with federal funds from the USDA/ARS under CooperativeAgreement No. 58-6250-0-008. The contents of this publication do not nec-essarily reflect the views or policies of the USDA, nor does mention of tradenames, commercial products, or organizations imply endorsement from the USgovernment. This work was also supported by NIH/NIA Grant 1-R03-AG-029641-01 (Y. Sun), the American Heart Association 12IRG9230004 (Y.Sun), an NIH-Diabetes and Endocrinology Research Center grant at BaylorCollege of Medicine (P30-DK-079638), and the Lipid Core of Mouse Meta-bolic Phenotyping Center at Vanderbilt University (U24-DK-59637).

DISCLOSURES

No conflicts of interest, financial or otherwise, are declared by the authors.

AUTHOR CONTRIBUTIONS

X.M., Y.L., and Y.S. did the conception and design of the research; X.M.,Y.L., and Y.S. performed the experiments; X.M. and Y.L. analyzed the data;X.M., L.L., N.F.B., and Y.S. interpreted the results of the experiments; X.M.and Y.L. prepared the figures; X.M. drafted the manuscript; X.M., Y.L., L.L.,G.Q., F.A.P., M.W.H., N.F.B., and Y.S. edited and revised the manuscript;X.M., G.Q., F.A.P., M.W.H., N.F.B., and Y.S. approved the final version of themanuscript.

REFERENCES

1. Ahima RS. Antagonism of ghrelin for glycemic control in type 2 diabetesmellitus? Endocrinology 148: 5173–5174, 2007.

2. Asakawa A, Inui A, Kaga T, Katsuura G, Fujimiya M, Fujino MA,Kasuga M. Antagonism of ghrelin receptor reduces food intake and bodyweight gain in mice. Gut 52: 947–952, 2003.

3. Bresciani E, Rapetti D, Dona F, Bulgarelli I, Tamiazzo L, Locatelli V,Torsello A. Obestatin inhibits feeding but does not modulate GH andcorticosterone secretion in the rat. J Endocrinol Invest 29: RC16–RC18,2006.

4. Briggs DI, Andrews ZB. A recent update on the role of ghrelin in glucosehomeostasis. Curr Diabetes Rev 7: 201–207, 2011.

5. Briggs DI, Enriori PJ, Lemus MB, Cowley MA, Andrews ZB. Diet-induced obesity causes ghrelin resistance in arcuate NPY/AgRP neurons.Endocrinology 151: 4745–4755, 2010.

6. Castañeda TR, Tong J, Datta R, Culler M, Tschöp MH. Ghrelin in theregulation of body weight and metabolism. Front Neuroendocrinol 31:44–60, 2010.

7. Chan CB, MacDonald PE, Saleh MC, Johns DC, Marban E, WheelerMB. Overexpression of uncoupling protein 2 inhibits glucose-stimulatedinsulin secretion from rat islets. Diabetes 48: 1482–1486, 1999.

8. Chanoine JP, Wong AC, Barrios V. Obestatin, acylated and total ghrelinconcentrations in the perinatal rat pancreas. Horm Res 66: 81–88, 2006.

9. Cheng K, Ho K, Stokes R, Scott C, Lau SM, Hawthorne WJ,O’Connell PJ, Loudovaris T, Kay TW, Kulkarni RN, Okada T, WangXL, Yim SH, Shah Y, Grey ST, Biankin AV, Kench JG, Laybutt DR,Gonzalez FJ, Kahn CR, Gunton JE. Hypoxia-inducible factor-1alpharegulates beta cell function in mouse and human islets. J Clin Invest 120:2171–2183, 2010.

10. Chmielewska J, Szczepankiewicz D, Skrzypski M, Kregielska D,Strowski MZ, Nowak KW. Ghrelin but not obestatin regulates insulinsecretion from INS1 beta cell line via UCP2-dependent mechanism. J BiolRegul Homeost Agents 24: 397–402, 2010.

11. Chuang JC, Sakata I, Kohno D, Perello M, Osborne-Lawrence S,Repa JJ, Zigman JM. Ghrelin directly stimulates glucagon secretionfrom pancreatic alpha-cells. Mol Endocrinol 25: 1600–1611, 2011.

12. Cummings DE. Ghrelin and the short- and long-term regulation ofappetite and body weight. Physiol Behav 89: 71–84, 2006.

13. da Silva Xavier G, Sun G, Qian Q, Rutter GA, Leclerc I. ChREBPregulates Pdx-1 and other glucose-sensitive genes in pancreatic �-cells.Biochem Biophys Res Commun 402: 252–257, 2010.

14. Date Y, Nakazato M, Hashiguchi S, Dezaki K, Mondal MS, Hosoda H,Kojima M, Kangawa K, Arima T, Matsuo H, Yada T, Matsukura S.Ghrelin is present in pancreatic alpha-cells of humans and rats andstimulates insulin secretion. Diabetes 51: 124–129, 2002.

15. Dentin R, Denechaud PD, Benhamed F, Girard J, Postic C. Hepaticgene regulation by glucose and polyunsaturated fatty acids: a role forChREBP. J Nutr 136: 1145–1149, 2006.

16. Dezaki K, Sone H, Koizumi M, Nakata M, Kakei M, Nagai H, HosodaH, Kangawa K, Yada T. Blockade of pancreatic islet-derived ghrelinenhances insulin secretion to prevent high-fat diet-induced glucose intol-erance. Diabetes 55: 3486–3493, 2006.

17. Fleury C, Neverova M, Collins S, Raimbault S, Champigny O, Levi-Meyrueis C, Bouillaud F, Seldin MF, Surwit RS, Ricquier D, WardenCH. Uncoupling protein-2: a novel gene linked to obesity and hyperinsu-linemia. Nat Genet 15: 269–272, 1997.

18. Gauna C, Delhanty PJ, Hofland LJ, Janssen JA, Broglio F, Ross RJ,Ghigo E, van der Lely AJ. Ghrelin stimulates, whereas des-octanoylghrelin inhibits, glucose output by primary hepatocytes. J Clin EndocrinolMetab 90: 1055–1060, 2005.

19. Gauna C, Kiewiet RM, Janssen JA, van de Zande B, Delhanty PJ,Ghigo E, Hofland LJ, Themmen AP, van der Lely AJ. Unacylatedghrelin acts as a potent insulin secretagogue in glucose-stimulated condi-tions. Am J Physiol Endocrinol Metab 293: E697–E704, 2007.

20. Granata R, Settanni F, Gallo D, Trovato L, Biancone L, Cantaluppi V,Nano R, Annunziata M, Campiglia P, Arnoletti E, Ghe C, Volante M,Papotti M, Muccioli G, Ghigo E. Obestatin promotes survival of pan-creatic beta-cells and human islets and induces expression of genesinvolved in the regulation of beta-cell mass and function. Diabetes 57:967–979, 2008.

21. Heppner KM, Tong J, Kirchner H, Nass R, Tschöp MH. The ghrelinO-acyltransferase-ghrelin system: a novel regulator of glucose metabo-lism. Curr Opin Endocrinol Diabetes Obes 18: 50–55, 2011.

22. Hewson AK, Tung LY, Connell DW, Tookman L, Dickson SL. The ratarcuate nucleus integrates peripheral signals provided by leptin, insulin,and a ghrelin mimetic. Diabetes 51: 3412–3419, 2002.

23. Hukshorn CJ, Saris WH. Leptin and energy expenditure. Curr Opin ClinNutr Metab Care 7: 629–633, 2004.

24. Iwakura H, Akamizu T, Ariyasu H, Irako T, Hosoda K, Nakao K,Kangawa K. Effects of ghrelin administration on decreased growth

E430 GHRELIN RECEPTOR AND GLUCOSE HOMEOSTASIS

AJP-Endocrinol Metab • doi:10.1152/ajpendo.00576.2011 • www.ajpendo.org

hormone status in obese animals. Am J Physiol Endocrinol Metab 293:E819–E825, 2007.

25. Kageyama H, Funahashi H, Hirayama M, Takenoya F, Kita T, KatoS, Sakurai J, Lee EY, Inoue S, Date Y, Nakazato M, Kangawa K,Shioda S. Morphological analysis of ghrelin and its receptor distributionin the rat pancreas. Regul Pept 126: 67–71, 2005.

26. Lavine RL, Voyles N, Perrino PV, Recant L. Functional abnormalitiesof islets of Langerhans of obese hyperglycemic mouse. Am J PhysiolEndocrinol Metab Gastrointest Physiol 233: E86–E90, 1977.

27. Lin L, Saha PK, Ma X, Henshaw IO, Shao L, Chang BH, Buras ED,Tong Q, Chan L, McGuinness OP, Sun Y. Ablation of ghrelin receptorreduces adiposity and improves insulin sensitivity during aging by regu-lating fat metabolism in white and brown adipose tissues. Aging Cell 10:996–1010, 2011.

28. Lindström P. beta-cell function in obese-hyperglycemic mice [ob/obMice]. Adv Exp Med Biol 654: 463–477, 2010.

29. Longo KA, Charoenthongtrakul S, Giuliana DJ, Govek EK, McDon-agh T, Qi Y, DiStefano PS, Geddes BJ. Improved insulin sensitivity andmetabolic flexibility in ghrelin receptor knockout mice. Regul Pept 150:55–61, 2008.

30. Ma X, Lin L, Qin G, Lu X, Fiorotto M, Dixit VD, Sun Y. Ablations ofghrelin and ghrelin receptor exhibit differential metabolic phenotypes andthermogenic capacity during aging. PLoS One 6: e16391, 2011.

31. Martinez SC, Tanabe K, Cras-Meneur C, Abumrad NA, Bernal-Mizrachi E, Permutt MA. Inhibition of Foxo1 protects pancreatic isletbeta-cells against fatty acid and endoplasmic reticulum stress-inducedapoptosis. Diabetes 57: 846–859, 2008.

32. Oberkofler H, Klein K, Felder TK, Krempler F, Patsch W. Role ofperoxisome proliferator-activated receptor-gamma coactivator-1alpha inthe transcriptional regulation of the human uncoupling protein 2 gene inINS-1E cells. Endocrinology 147: 966–976, 2006.

33. Obici S, Wang J, Chowdury R, Feng Z, Siddhanta U, Morgan K,Rossetti L. Identification of a biochemical link between energy intake andenergy expenditure. J Clin Invest 109: 1599–1605, 2002.

34. Patel K, Dixit VD, Lee JH, Kim JW, Schaffer EM, Nguyen D, TaubDD. The GHS-R blocker D-[Lys3] GHRP-6 serves as CCR5 chemokinereceptor antagonist. Int J Med Sci 9: 51–58, 2012.

35. Perello M, Scott MM, Sakata I, Lee CE, Chuang JC, Osborne-Lawrence S, Rovinsky SA, Elmquist JK, Zigman JM. Functionalimplications of limited leptin receptor and ghrelin receptor coexpression inthe brain. J Comp Neurol 520: 281–294, 2012.

36. Poitout V, Robertson RP. An integrated view of beta-cell dysfunction intype-II diabetes. Annu Rev Med 47: 69–83, 1996.

37. Qader SS, Hakanson R, Rehfeld JF, Lundquist I, Salehi A. Proghrelin-derived peptides influence the secretion of insulin, glucagon, pancreaticpolypeptide and somatostatin: a study on isolated islets from mouse and ratpancreas. Regul Pept 146: 230–237, 2008.

38. Rodríguez A, Gómez-Ambrosi J, Catalán V, Gil MJ, Becerril S, SáinzN, Silva C, Salvador J, Colina I, Frühbeck G. Acylated and desacylghrelin stimulate lipid accumulation in human visceral adipocytes. Int JObes (Lond) 33: 541–552, 2009.

39. Rooth P, Taljedal IB. Vital microscopy of islet blood flow: catechol-amine effects in normal and ob/ob mice. Am J Physiol Endocrinol Metab252: E130–E135, 1987.

40. Saksida T, Stosic-Grujicic S, Timotijevic G, Sandler S, Stojanovic I.Macrophage migration inhibitory factor deficiency protects pancreaticislets from palmitic acid-induced apoptosis. Immunol Cell Biol. In press.

41. Salehi A, Dornonville de la Cour C, Håkanson R, Lundquist I. Effectsof ghrelin on insulin and glucagon secretion: a study of isolated pancreaticislets and intact mice. Regul Pept 118: 143–150, 2004.

42. Soares JB, Leite-Moreira AF. Ghrelin, des-acyl ghrelin and obestatin:three pieces of the same puzzle. Peptides 29: 1255–1270, 2008.

43. Sun Y, Ahmed S, Smith RG. Deletion of ghrelin impairs neither growthnor appetite. Mol Cell Biol 23: 7973–7981, 2003.

44. Sun Y, Asnicar M, Saha PK, Chan L, Smith RG. Ablation of ghrelinimproves the diabetic but not obese phenotype of ob/ob mice. Cell Metab3: 379–386, 2006.

45. Sun Y, Butte NF, Garcia JM, Smith RG. Characterization of adultghrelin and ghrelin receptor knockout mice under positive and negativeenergy balance. Endocrinology 149: 843–850, 2008.

46. Sun Y, Wang P, Zheng H, Smith RG. Ghrelin stimulation of growthhormone release and appetite is mediated through the growth hormonesecretagogue receptor. Proc Natl Acad Sci USA 101: 4679–4684, 2004.

47. Szentirmai E, Kapas L, Sun Y, Smith RG, Krueger JM. The pre-proghrelin gene is required for the normal integration of thermoregulationand sleep in mice. Proc Natl Acad Sci USA 106: 14069–14074, 2009.

48. Thompson NM, Gill DA, Davies R, Loveridge N, Houston PA, Rob-inson IC, Wells T. Ghrelin and des-octanoyl ghrelin promote adipogen-esis directly in vivo by a mechanism independent of the type 1a growthhormone secretagogue receptor. Endocrinology 145: 234–242, 2004.

49. Tomita T, Doull V, Kimmel JR, Pollock HG. Pancreatic polypeptide andother hormones in pancreas of obese (ob/ob) mice. Diabetologia 27:454–459, 1984.

50. Toshinai K, Yamaguchi H, Sun Y, Smith RG, Yamanaka A, SakuraiT, Date Y, Mondal MS, Shimbara T, Kawagoe T, Murakami N,Miyazato M, Kangawa K, Nakazato M. Des-acyl ghrelin induces foodintake by a mechanism independent of the growth hormone secretagoguereceptor. Endocrinology 147: 2306–2314, 2006.

51. Volante M, Allia E, Gugliotta P, Funaro A, Broglio F, Deghenghi R,Muccioli G, Ghigo E, Papotti M. Expression of ghrelin and of the GHsecretagogue receptor by pancreatic islet cells and related endocrinetumors. J Clin Endocrinol Metab 87: 1300–1308, 2002.

52. Wente W, Efanov AM, Brenner M, Kharitonenkov A, Koster A,Sandusky GE, Sewing S, Treinies I, Zitzer H, Gromada J. Fibroblastgrowth factor-21 improves pancreatic beta-cell function and survival byactivation of extracellular signal-regulated kinase 1/2 and Akt signalingpathways. Diabetes 55: 2470–2478, 2006.

53. Wierup N, Yang S, McEvilly RJ, Mulder H, Sundler F. Ghrelin isexpressed in a novel endocrine cell type in developing rat islets andinhibits insulin secretion from INS-1 (832/13) cells. J Histochem Cy-tochem 52: 301–310, 2004.

54. Wortley KE, del Rincon JP, Murray JD, Garcia K, Iida K, ThornerMO, Sleeman MW. Absence of ghrelin protects against early-onsetobesity. J Clin Invest 115: 3573–3578, 2005.

55. Zavaroni I, Deferrari G, Lugari R, Bonora E, Garibotto G, Dall’AglioE, Robaudo C, Gnudi A. Renal metabolism of C-peptide in man. J ClinEndocrinol Metab 65: 494–498, 1987.

56. Zhang CY, Baffy G, Perret P, Krauss S, Peroni O, Grujic D, Hagen T,Vidal-Puig AJ, Boss O, Kim YB, Zheng XX, Wheeler MB, ShulmanGI, Chan CB, Lowell BB. Uncoupling protein-2 negatively regulatesinsulin secretion and is a major link between obesity, beta cell dysfunction,and type 2 diabetes. Cell 105: 745–755, 2001.

57. Zhang JV, Ren PG, Avsian-Kretchmer O, Luo CW, Rauch R, KleinC, Hsueh AJ. Obestatin, a peptide encoded by the ghrelin gene, opposesghrelin’s effects on food intake. Science 310: 996–999, 2005.

58. Zhang W, Chai B, Li JY, Wang H, Mulholland MW. Effect of des-acylghrelin on adiposity and glucose metabolism. Endocrinology 149: 4710–4716, 2008.

59. Zhao TJ, Liang G, Li RL, Xie X, Sleeman MW, Murphy AJ, Valen-zuela DM, Yancopoulos GD, Goldstein JL, Brown MS. Ghrelin O-acyltransferase (GOAT) is essential for growth hormone-mediated sur-vival of calorie-restricted mice. Proc Natl Acad Sci USA 107: 7467–7472,2010.

60. Zigman JM, Nakano Y, Coppari R, Balthasar N, Marcus JN, Lee CE,Jones JE, Deysher AE, Waxman AR, White RD, Williams TD, LacheyJL, Seeley RJ, Lowell BB, Elmquist JK. Mice lacking ghrelin receptorsresist the development of diet-induced obesity. J Clin Invest 115: 3564–3572, 2005.

E431GHRELIN RECEPTOR AND GLUCOSE HOMEOSTASIS

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