Role of the Duodenum and Macronutrient Type in Ghrelin Regulation

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Nov 4, 2004 - The orexigenic hormone ghrelin is implicated in preprandial hunger and meal initiation in part because circulating levels increase before meals ...
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Endocrinology 146(2):845– 850 Copyright © 2005 by The Endocrine Society doi: 10.1210/en.2004-0609

Role of the Duodenum and Macronutrient Type in Ghrelin Regulation Joost Overduin, R. Scott Frayo, Harvey J. Grill, Joel M. Kaplan, and David E. Cummings Department of Medicine (J.O., R.S.F., D.E.C.), Division of Metabolism, Endocrinology and Nutrition, University of Washington, Veterans Affairs Puget Sound Health Care System, Seattle, Washington 98108; and Department of Psychology (H.J.G., J.M.K.), University of Pennsylvania, Philadelphia, Pennsylvania 19104 The orexigenic hormone ghrelin is implicated in preprandial hunger and meal initiation in part because circulating levels increase before meals and decrease after food intake. The mechanisms underlying postprandial ghrelin suppression are unknown. Although most ghrelin is produced by the stomach, we have shown that neither gastric nutrients nor gastric distension affect ghrelin levels. We hypothesized that the nutrient-sensing mechanism regulating ghrelin is in the duodenum, the second richest source of ghrelin. To test whether duodenal nutrient exposure is required for ghrelin suppression, we infused nutrients into either the proximal duodenum or proximal jejunum in rats bearing chronic intestinal cannulas. At 0, 30, 60, 90, 120, 180, 240, and 300 min after infusions, blood was sampled via jugular-vein catheters for ghrelin, insulin, and glucose measurements. To elucidate further the mechanisms governing nutrient-related ghrelin suppression,

we also assessed the ghrelin responses to isocaloric (3 kcal) infusions of glucose, amino acids, or lipids delivered into the stomach or small intestine of chronically catheterized rats. Regardless of macronutrient type, the depth and duration of ghrelin suppression were equivalent after gastric, duodenal, and jejunal infusions. Glucose and amino acids suppressed ghrelin more rapidly and strongly (by ⬃70%) than did lipids (by ⬃50%). Because jejunal nutrient infusions suppressed ghrelin levels as well as either gastric or duodenal infusions, we conclude that the inhibitory signals mediating postprandial ghrelin suppression are not derived discretely from either the stomach or duodenum. The relatively weak suppression of ghrelin by lipids compared with glucose or amino acids could represent one mechanism promoting high-fat dietary weight gain. (Endocrinology 146: 845– 850, 2005)

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HRELIN IS A RECENTLY discovered hormone that is secreted primarily by the stomach and duodenum (1– 4). Exogenous administration potently stimulates food intake (5–9), and endogenous ghrelin is implicated in mealtime hunger and meal initiation (10). Evidence favoring this hypothesis includes the observation that circulating levels of ghrelin increase before meals and are suppressed by food intake (5, 11, 12). It is not known whether the signals mediating meal-related ghrelin suppression originate from the gastrointestinal tract or from postabsorptive sites. Although parenteral nutrient and/or insulin infusions can suppress ghrelin levels when administered for prolonged periods or at supraphysiological doses in rats and humans (13–18), physiological doses that mimic postprandial fluctuations do not affect ghrelin in humans (19, 20). In contrast, enteral nutrients consistently suppress ghrelin levels in both species, even at low doses (5, 11, 17, 19, 21–23). These observations suggest that gastrointestinal signals contribute to prandial ghrelin regulation, but the location of these signals is unknown. Although most ghrelin is produced by the stomach, this organ does not appear to contain the putative nutrientsensing mechanism that suppresses ghrelin after meals because neither gastric distension nor the presence of nutrients

in the stomach lumen is required for this response (5, 23, 24). Therefore, it is likely that nutrient-related ghrelin suppression results from signals originating distal to the stomach. The first goal of this study was to examine the contribution of nutrients within the duodenal lumen to ghrelin suppression. We hypothesized that the duodenum is necessary for the prandial ghrelin response based, in part, on observations that ghrelin regulation is disrupted after Roux-en-Y gastric bypass (RYGB) surgery (12, 25, 26). Ingested nutrients strongly regulate ghrelin, and RYGB excludes the majority of ghrelin-producing tissue (i.e. the stomach and duodenum) from contact with enteral nutrients. Because nutrients constrained within the stomach do not affect ghrelin levels (24), disordered ghrelin regulation after RYGB suggests a role for duodenal nutrients in ghrelin regulation. Moreover, nutrients within the duodenum regulate levels of other gut hormones involved in appetite control as well as endocrine and exocrine pancreatic output, gastric motility, and satiety (27– 30). If the nutrient-sensing mechanism mediating prandial ghrelin suppression is located discretely in the duodenum, then nutrients delivered into the stomach or duodenum should suppress ghrelin levels, whereas equivalent intestinal infusions delivered distal to the duodenum should not suppress ghrelin levels. To determine whether the duodenum is required for nutrient-related ghrelin suppression, we infused nutrients into the stomach, duodenum, or jejunum in conscious, unanesthetized rats fitted with chronic, indwelling intestinal catheters. The second goal of this study was to compare the magnitude of ghrelin suppression after intestinal infusions of different macronutrient types. Absorption of food from the

First Published Online November 4, 2004 Abbreviations: D-AUC, Decremental area under the curve; GN, ghrelin nadir; RYGB, Roux-en-Y gastric bypass; TGN, time to ghrelin nadir. Endocrinology is published monthly by The Endocrine Society (http:// www.endo-society.org), the foremost professional society serving the endocrine community.

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gut is accompanied by a characteristic pattern of humoral and neural responses for each class of macronutrient (28, 31). Therefore, differences in the dynamics of ghrelin suppression after isocaloric infusions of various macronutrients may shed light on the identity of signals mediating ghrelin suppression. Furthermore, such findings might have practical relevance for the design of diets intended to lower ghrelin levels. We examined the temporal profile of ghrelin suppression in rats infused with isocaloric solutions of glucose, lipids, or amino acids via indwelling intestinal catheters. Materials and Methods Animals Experiments were performed on 14- to 20-wk-old male Sprague Dawley rats (ATL, Kent, WA) weighing 350 – 450 g. Animals were housed individually at the Veterans Affairs Puget Sound Health Care System, Seattle Division (an American Association for Accreditation of Laboratory Animal Care-accredited facility). Rats had ad libitum access to pelleted chow between experiments and were provided water at all times. They were maintained on a 12-h light, 12-h dark cycle, with lights on at 0600 h. All procedures were approved by the Veterans Affairs Puget Sound Health Care System Institutional Animal Care and Use Committee.

Rat surgery For surgery, animals were anesthetized with a mixture of 60 mg/kg ketamine and 7 mg/kg xylazine (Phoenix, St. Joseph, MO) administered ip. Duodenal and jejunal feeding catheter placement and testing. Rats received either a proximal duodenal catheter (entry point at 2 cm distal to the pylorus) or a proximal jejunal catheter (entry point at 25 cm distal to the pylorus, i.e. ⬃15 cm distal to the duodenojejunal junction). Silastic tubing (outer diameter, 0.047 in; inner diameter, 0.025 in; Braintree Scientific, Braintree, MA) was fitted with a collar consisting of a 4-cm2 piece of Bard surgical mesh (Davol, Cranston, RI), which was used to tether the catheter to the intestine. After visualization of the stomach and small intestine, a puncture hole was made in the intestinal wall using an 18gauge needle. The end of the SILASTIC brand silicon tubing (Dow Corning, Midland, MI) distal to the mesh was inserted approximately 2 cm into the intestinal lumen, pointing caudally. The mesh was tethered to the outer intestinal wall with a three-loop purse string of 6 – 0 silk suture (U.S. Surgical, Norwalk, CT). In pilot studies designed to verify that jejunal infusions did not flow in a retrograde direction into the duodenum, we infused barium sulfate into jejunal catheters, using the same flow rate and volume as were used for nutrient infusions in our experiments. Fluoroscopic visualization revealed only a minute amount of retrograde flow, extending less than 2 cm from the catheter tip, which was located approximately 17 cm distal to the duodenum. Similarly, visual inspection of slow infusions of methylene blue into the jejunal catheters revealed no detectable retrograde flow (data not shown). Jugular-vein catheter placement. A 2-cm incision was made through the skin in the right jugular area, and the jugular vein was cannulated with SILASTIC brand silicon tubing (outer diameter, 0.037 in; inner diameter, 0.02 in; VWR Scientific, West Chester, PA). The other end of the tubing was tunneled sc to the head, onto which it was secured, along with the intestinal catheter, with an acrylic skull cap (Lang Dental, Wheeling, IL) (32).

Experimental protocol Rats recovered from surgery for at least 2 wk before nutrient infusions began; by this time, body weights of all animals had returned to presurgical levels. Experiments were conducted in custom-built, Plexiglas test cages (30 ⫻ 25 ⫻ 35 cm) that facilitated intestinal infusions and repeated blood sampling without disturbance of the animals. Several days before the start of each experiment, rats were habituated overnight to the experimental cages. At 1700 h on the day before infusions, each animal was placed individually in an experimental cage and was food deprived for 18 h to establish high baseline ghrelin levels (33). One hour

Overduin et al. • Nutrient Regulation of Ghrelin

before infusions, each animal’s head cap was connected with respective lines (PE-100; VWR) for blood sampling and nutrient administration. Infusions were initiated at 1100 h (5 h into the light cycle). Gastrointestinal infusions. Infusion conditions were run in random order and spaced at least 1 wk apart from one another. The carbohydrate solution consisted of 25% glucose, which is known to suppress ghrelin levels when delivered into the stomach (24). Three milliliters were delivered into the small intestine over 10 min at a mean rate of 0.3 ml/min. This rate of delivery approximates that of normal gastric emptying in rats voluntarily ingesting 25% glucose (34). The lipid and amino acid infusions, also administered over 10 min, were made isocaloric to the glucose infusion. The lipid infusion consisted of Intralipid (Baxter Healthcare, Deerfield, IL), a broad-spectrum mixture of long-chain triglycerides and phospholipids, that was diluted with distilled water to obtain a volume of 3 ml. The amino acid solution was Prosol 20% Amino Acids, which is a water-soluble, broad-spectrum mixture of essential and nonessential amino acids (a generous gift from Baxter Healthcare). To render the amino acid infusion isocaloric to the glucose and lipid infusions, we used a slightly larger volume of this solution (3.75 ml). Two control conditions were also examined. First, 3 ml of 25% glucose was infused into the stomach by gavage, as a positive control in which nutrients entered the small intestine more physiologically than by direct infusion (i.e. mixed and diluted with gastric secretions, then passed though the pylorus). Second, we infused 3 ml of physiologic saline (0.9% NaCl) intestinally over 10 min to control for the volume-related effects of intestinal infusions on ghrelin levels. Blood sampling and analysis. Five minutes before the start of nutrient infusions, a baseline 250-␮l blood sample was drawn from the jugularvein catheter, and additional 250-␮l samples were taken at 30, 60, 90, 120, 180, 240, and 300 min after the onset of infusions. Blood glucose levels were analyzed in a small drop from each sample using a portable glucose meter (Accu-Check; Roche, Indianapolis, IN). The remaining blood was transferred to 0.5-ml microcentrifuge tubes containing 10 ␮l of 7.5% EDTA and then placed immediately on ice. As soon as possible, blood samples were centrifuged, and the plasma was withdrawn and stored at ⫺80 C. Plasma levels of total ghrelin were measured in duplicate samples of 25 ␮l each by RIA using a primary antibody against rat ghrelin and 131I-labeled ghrelin as the tracer (kit RK-031-31; Phoenix Pharmaceuticals, Belmont, CA) (11). This assay detects both acylated and des-acyl ghrelin. Although only acylated ghrelin is bioactive, levels of the total and acylated forms correlate closely with one another over a wide variety of physiological manipulations that affect ghrelin (35–37). Plasma insulin was measured in duplicate samples of 25 ␮l each using a commercial RIA (kit SRI-13K; Linco Research Inc., St. Charles, MO). The intra- and interassay coefficients of variance were 5.5 and 9.6%, respectively, for ghrelin, and 3.7 and 5.7%, respectively, for insulin.

Data analysis Data were analyzed using Unistat software (Unistat Ltd., London, UK) running under Excel (Microsoft, Redmond, WA). For most purposes, ghrelin levels were expressed as percentages of baseline values. Three parameters were calculated to describe the dynamics of ghrelin response to gastrointestinal infusions. Ghrelin nadir (GN) was the lowest ghrelin level (expressed as a percentage of baseline) reached within the 5-h test period after infusions. Time to ghrelin nadir (TGN) was the time required for ghrelin to reach its lowest postinfusion level. Decremental area under the curve (D-AUC) was calculated to obtain an overall index of the ghrelin response, reflecting both the depth and duration of suppression. The D-AUC was defined as 100% minus the area under the ghrelin curve, which was determined using the trapezoidal rule. Graphically, D-AUC represents the area enclosed by the preinfusion baseline level (100%) and the postinfusion ghrelin curve. Two-tailed paired t tests with a significance threshold of P ⫽ 0.05 were used to test differences between pre- and postinfusion ghrelin levels. The effect of duodenal vs. jejunal infusion site on ghrelin regulation was tested with two-sided unpaired t tests for each macronutrient, with a significance threshold of P ⫽ 0.05. Effects of macronutrient type on ghrelin suppression were tested by one-way ANOVA, followed by unpaired two-sided t tests. To account for multiple post hoc comparisons, we used the Bonferroni correction, setting the significance threshold at P ⫽ 0.05/3 ⫽ 0.0167. Results are expressed as mean ⫾ sem, unless otherwise stated.

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between duodenal and jejunal effects on GN, TGN, and DAUC parameters; Fig. 1 and Table 1). Effect of macronutrient type on plasma ghrelin levels

Given that there was no effect of infusion site (duodenal vs. jejunal) on ghrelin responses, data derived from both sites were pooled for subsequent tests of the effect of various macronutrient types on ghrelin levels. All of the following results pertaining to these pooled data were also statistically significant for both the duodenal and jejunal infusions considered separately, except as noted otherwise in one case for TGN. GN. A one-way ANOVA revealed a significant effect of macronutrient type on GN (F(2,38) ⫽ 26.4, P ⬍ 0.001). GN was significantly lower after both glucose and amino acids than after lipids (P ⬍ 0.001 for both; Fig. 2). No differences were found between glucose and amino acid infusions. FIG. 1. Plasma ghrelin profiles after gastric, duodenal, or jejunal infusions of glucose in chronically catheterized rats. Immediately after time 0, all animals received isocaloric 3-ml infusions of 25% glucose, and the ghrelin response was measured using blood samples drawn serially from indwelling jugular-vein catheters. All rats (n ⫽ 9) receiving gastric infusions had indwelling duodenal or jejunal catheters. Seven rats received duodenal infusions, and nine rats received jejunal infusions. Mean baseline ghrelin levels were equivalent among all groups, and for each group, ghrelin levels decreased significantly after glucose infusion (P ⬍ 0.001).

Results Effects of different gastrointestinal sites of nutrient infusion on plasma ghrelin levels

Consistent with previous reports (5, 24), glucose delivered into the stomach suppressed plasma ghrelin. Levels fell from 4017 ⫾ 436 pg/ml at baseline to 1495 ⫾ 113 pg/ml at the nadir (P ⬍ 0.001), and this response was observed in all animals (Fig. 1). This finding demonstrates that the indwelling intestinal catheters did not impair normal nutrientrelated ghrelin regulation. To assess whether duodenal exposure to nutrients is required for prandial ghrelin regulation, nutrients were infused either into the proximal duodenum or more distally into the jejunum. For each of the three macronutrient types, duodenal and jejunal infusions suppressed ghrelin levels equally well, with similar magnitude and timing of suppression. This can been seen in Fig. 1, as well as in Table 1, which quantifies the depth, speed, and overall magnitude of ghrelin suppression after duodenal or jejunal isocaloric infusions of glucose, amino acids, or lipids. For each macronutrient type, duodenal and jejunal infusions yielded equivalent results in terms of the postinfusion GN, TGN, and integrated D-AUC (P ⬎ 0.2 for all comparisons

D-AUC. There was a main effect of macronutrient type on D-AUC (one-way ANOVA, F(2,38) ⫽ 9.2, P ⬍ 0.001; Fig. 2). D-AUC after glucose infusions was larger than after either amino acids or lipids (P ⬍ 0.001). No significant difference in D-AUC was found between amino acids and lipids. TGN. A one-way ANOVA showed a significant effect of macronutrient type on TGN (F(2,38) ⫽ 16.9, P ⬍ 0.001), and results for TGN largely mirrored those for GN (Fig. 2). TGN was similar after glucose (58 ⫾ 5 min) and amino acid infusions (64 ⫾ 4 min), and each of these times was shorter than that after lipid infusions (133 ⫾ 17 min, P ⬍ 0.001). As with GN and D-AUC, the pattern of results was identical when data for duodenal and jejunal infusions were considered separately, except that the difference in TGN for jejunal infusions of amino acids vs. lipids did not quite reach statistical significance (P ⫽ 0.06). Blood glucose levels. Compared with baseline values, blood glucose levels increased by 49.5 ⫾ 4.2 mg/dl after glucose infusions (P ⬍ 0.001) and by 16.2 ⫾ 3.0 mg/dl after amino acid infusions (P ⬍ 0.001). These levels returned to baseline by 120 min after the start of both infusions (Fig. 3A). Lipids did not affect blood glucose levels. The modest rise in blood glucose observed after amino acid infusions presumably resulted from gluconeogenesis after conversion of absorbed amino acids in these 18-h fasted rats (31). Insulin levels. Plasma insulin levels increased from baseline by 374 ⫾ 78 pmol/liter after glucose infusions (P ⫽ 0.003), by 162 ⫾ 13 pmol/liter after amino acids (P ⬍ 0.001), and

TABLE 1. Measures of ghrelin suppression during the 5 h after isocaloric duodenal or jejunal infusions of three different macronutrient types Duodenal infusions

Glucose Amino acids Lipids

Jejunal infusions

No.

GN (% baseline)

Time to nadir (min)

D-AUC (min%)

No.

GN (% baseline)

Time to nadir (min)

D-AUC (min%)

7 6 6

29 ⫾ 8 30 ⫾ 8 48 ⫾ 16

60 ⫾ 25 60 ⫾ 0 160 ⫾ 62

15,508 ⫾ 2,404 10,352 ⫾ 4,134 8,446 ⫾ 2,796

9 8 6

27 ⫾ 7 32 ⫾ 9 55 ⫾ 11

57 ⫾ 18 68 ⫾ 21 105 ⫾ 45

14,194 ⫾ 3,339 9,617 ⫾ 3,308 8,178 ⫾ 4,036

None of the nine types of measurements displayed here differed significantly between duodenal and jejunal infusions. Values are means ⫾ SD.

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FIG. 2. Plasma ghrelin profiles after intestinal infusions of different macronutrients. Because ghrelin profiles after duodenal and jejunal infusions were equivalent for each macronutrient, combined data from these infusions are displayed. Rats received either saline (open diamonds, n ⫽ 12) or isocaloric (3 kcal) infusions of glucose (closed circles, n ⫽ 16), amino acids (open triangles, n ⫽ 14), or lipids (closed squares, n ⫽ 12). Ghrelin levels were measured in blood samples drawn serially from indwelling jugular-vein catheters. Mean baseline ghrelin levels were equivalent among all groups.

slightly after lipids (by 55 ⫾ 9 pmol/liter, P ⫽ 0.007; Fig. 3B). As with blood glucose, insulin levels returned to baseline by 120 min after the start of all infusions. Maximum increases of insulin were larger after glucose and amino acid infusions than after lipids (P ⫽ 0.002 and P ⬍ 0.001, respectively). Maximum increases were larger after glucose than after amino acid infusions (P ⬍ 0.02). Correlations among ghrelin, insulin, and blood glucose responses to gastrointestinal nutrient infusions. The especially strong suppression of ghrelin after intestinal infusions of either glucose or amino acids, both of which stimulate insulin secretion, suggests a possible dominant role for insulin and/or glucose in the postprandial ghrelin response. If this were true, the depth and total magnitude of nutrient-related ghrelin suppression should correlate with the height and total magnitude of increases in insulin and/or glucose. Thus, we analyzed these correlations, as shown in Table 2. For all macronutrient infusions analyzed together, GN correlated inversely with the maximum rise in both insulin and glucose levels (Spearman rank correlations, P ⬍ 0.001 for both; Table 2). Similarly, the D-AUC of ghrelin correlated significantly with the AUCs of insulin and glucose (P ⬍ 0.001 for both; Table 2). Discussion

We found that glucose infusions delivered into the stomach, duodenum, or jejunum all suppressed plasma ghrelin equally well. Ghrelin levels were also suppressed equivalently by duodenal and jejunal nutrient infusions when lipids or amino acids were administered. Using both barium and methylene blue, we confirmed that there was no detectable retrograde flow into the duodenum or stomach from our jejunal catheters. Because jejunal nutrient infusions suppressed ghrelin levels equally well as did either gastric or duodenal infusions, we conclude that neither gastric nor duodenal exposure to nutri-

FIG. 3. Blood glucose (A) and insulin (B) responses to intestinal infusions of saline (open diamonds) or isocaloric solutions of glucose (closed circles), amino acids (open triangles), or lipids (closed squares). Data are derived from the experiments displayed in Fig. 2.

ents is required for nutrient-related suppression of ghrelin. Our results, together with our prior observation that nutrients constrained to the stomach do not affect ghrelin (24), indicate that inhibitory signals arising either from intestinal sites distal to the stomach and duodenum or from postabsorptive events are sufficient to mediate nutrient-related ghrelin suppression. Our data do not rule out the possibility that the duodenum, acting in concert with more distal sites, participates in ghrelin regulation. It is conceivable that ghrelin suppression can be achieved equally by various different parts of the intestine coming in contact with nutrients. This pluripotent model is suggested by our observation that gastric and duodenal infusions suppressed ghrelin levels equally well as jejunal fusions, even though in the latter two conditions, much of the small caloric load we used was probably absorbed in the duodenum, without reaching the jejunum. Presuming that ghrelin is regulated similarly in rats and humans, as has generally been found in studies to date (14, 15, 38), one implication of these findings is that the perturbation of ghrelin regulation that typically follows RYGB surgery (12, 25, 26) is unlikely to result from lack of nutrient flux through the stomach and duodenum. We had initially hy-

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TABLE 2. Spearman rank correlation coefficients between nadir or D-AUC for plasma ghrelin vs. maximum rise or AUC of insulin and glucose levels during 5 h after macronutrient infusions Correlation coefficient

GN Insulin peak Glucose peak Ghrelin D-AUC Insulin AUC Glucose AUC

⫺0.69 ⫺0.61 0.69 0.55

All correlations are significant with P ⬍ 0.001 and are based on data for all macronutrient infusions combined.

pothesized that prandial ghrelin suppression results from direct contact between ingested nutrients within the gastrointestinal lumen and ghrelin-producing cells, most of which are in the stomach and duodenum (3). Accordingly, we had hypothesized that the condition of a permanently empty stomach and duodenum after RYGB would cause unrelenting ghrelin stimulation, leading ultimately to paradoxical ghrelin suppression via override inhibition (12). This phenomenon would be analogous to the paradoxical suppression of gonadotropins or GH by continuous infusions of GnRH or GHRH, respectively (39, 40). In view of our current findings, however, showing that nutrient-related ghrelin regulation does not require nutrients within the lumen of the stomach or duodenum, it now seems unlikely that this override inhibition hypothesis is valid. Our second principal finding was that isocaloric intestinal infusions of either glucose or amino acids suppressed ghrelin levels more rapidly and effectively than did lipid infusions. These observations are consistent with and extend those of a recent report showing that intragastric infusions of glucose, peptone, casein, or corn oil all suppressed plasma ghrelin in anesthetized rats (41). In that work, however, infusions across the different macronutrient classes were not isocaloric, and the ghrelin responses were only assessed at a single postinfusion time point. Thus, quantitative comparisons of the ghrelin responses to various macronutrients are not possible from the prior study. Our finding that ghrelin was suppressed less effectively by lipids than by glucose or amino acids could have clinical implications. Theoretically, weak suppression of an orexigenic hormone by ingested lipids could be one of the mechanisms underlying high-fat diet-induced weight gain (42). For all infusions combined, the depth of ghrelin suppression correlated significantly with the magnitude of increase in both glucose and insulin levels; similarly, the D-AUC for ghrelin correlated with the AUCs for glucose and insulin. Although we did not directly test whether increases in blood glucose and/or insulin cause ghrelin suppression, studies of rats made insulin deficient with the ␤-cell toxin, streptozotocin, suggest that insulin participates in postprandial ghrelin suppression in this species but that other factors are also involved (43). Two observations that support this assertion can be made from our current data. First, lipid infusions suppressed ghrelin levels by approximately 50% (Table 1), without any increase in glucose levels and with only a marginal increase in insulin (Fig. 3). Second, regardless of the macronutrient type infused, ghrelin levels remained suppressed long

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after blood glucose and insulin levels had returned to baseline (Figs. 2 and 3). Thus, it is unlikely that nutrient-related ghrelin suppression is driven solely by circulating glucose and insulin, although these factors may contribute to the response, as suggested by several studies (43, 44). Two additional factors, rate of nutrient absorption and increases in osmolarity within the intestinal lumen, may partly explain the dynamics of ghrelin responses in our experiments. Glucose and amino acids, which are quickly absorbed from the gut, suppressed ghrelin rapidly and deeply. In contrast, lipids, which require intestinal digestion before absorption (45), suppressed ghrelin more gradually and to a lesser extent. This difference could imply that nutrientrelated ghrelin suppression results from signals generated during or after nutrient absorption. Moreover, although our infusions of various macronutrients were isocaloric with one another, they varied significantly in osmolarity, and the strength of ghrelin suppression followed a pattern consistent with a possible contribution from changes in intestinal osmolarity. Specifically, infusions of glucose and amino acids (with osmolarities of 1450 and 1800 mOsm/liter, respectively) suppressed ghrelin more strongly than did the comparatively less osmotic lipid infusions (ⱕ450 mOsm/liter). Other potential pathways governing meal-related ghrelin suppression could include signaling from factors secreted by intestinal enteroendocrine cells (e.g. serotonin, glucose-dependent insulinotropic peptide, cholecystokinin, glucagon-like peptide-1, etc.). These cells respond to all macronutrients in vitro and in vivo (28). Prandial ghrelin suppression could also involve neural pathways, specifically the myenteric plexus but probably not the vagus nerve. Although vagal afferent pathways are involved in other nutrient-related signaling from the gut (28, 29), the vagus is not required for prandial ghrelin regulation, as judged from experiments in vagotomized rats (33). In summary, we find that nutrient-related ghrelin suppression does not require the presence of nutrients in either the stomach or duodenum, which are the principal sites of ghrelin production. Thus, prandial ghrelin regulation is probably mediated by intestinal signals located downstream of the ligament of Treitz (or at least distributed throughout the intestine) and/or by postabsorptive mechanisms. Among the latter, circulating glucose and insulin probably contribute to nutrient-related ghrelin suppression. However, they are unlikely to explain the entire response because ghrelin remained suppressed in our study long after normalization of glucose and insulin levels and because lipids suppressed ghrelin in the absence of substantial increases in glucose or insulin levels. Our findings pertain only to the regulation of circulating ghrelin, which is produced primarily by the stomach and proximal small intestine. The data do not address regulation of the very small amount of ghrelin that has been reported to be produced in the hypothalamus (46). The weaker suppression of peripheral ghrelin, an orexigenic hormone, by lipids than by glucose or amino acids could constitute one of many reasons why highfat diets promote weight gain. Acknowledgments We are very grateful to Drs. Karen E. Foster-Schubert and Diana L. Williams for their insightful intellectual input into these studies and the manuscript.

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Received May 12, 2004. Accepted October 13, 2004. Address all correspondence and requests for reprints to: David E. Cummings, M.D., Associate Professor of Medicine, University of Washington, Veterans Affairs Puget Sound Health Care System, 1660 South Columbian Way, S-111-Endo, Seattle, Washington 98108. E-mail: davidec@ u.washington.edu. This work was supported by National Institutes of Health Grant R01 DK61516 (to D.E.C.) and by the Veterans Affairs Puget Sound Health Care System.

References 1. Kojima M, Hosoda H, Date Y, Nakazato M, Matsuo H, Kangawa K 1999 Ghrelin is a growth-hormone-releasing acylated peptide from stomach. Nature 402:656 – 660 2. Date Y, Kojima M, Hosoda H, Sawaguchi A, Mondal MS, Suganuma T, Matsukura S, Kangawa K, Nakazato M 2000 Ghrelin, a novel growth hormonereleasing acylated peptide, is synthesized in a distinct endocrine cell type in the gastrointestinal tracts of rats and humans. Endocrinology 141:4255– 4261 3. Ariyasu H, Takaya K, Tagami T, Ogawa Y, Hosoda K, Akamizu T, Suda M, Koh T, Natsui K, Toyooka S, Shirakami G, Usui T, Shimatsu A, Doi K, Hosoda H, Kojima M, Kangawa K, Nakao K 2001 Stomach is a major source of circulating ghrelin, and feeding state determines plasma ghrelin-like immunoreactivity levels in humans. J Clin Endocrinol Metab 86:4753– 4758 4. Gnanapavan S, Kola B, Bustin SA, Morris DG, McGee P, Fairclough P, Bhattacharya S, Carpenter R, Grossman AB, Korbonits M 2002 The tissue distribution of the mRNA of ghrelin and subtypes of its receptor, GHS-R, in humans. J Clin Endocrinol Metab 87:2988 –2991 5. Tschop M, Smiley DL, Heiman ML 2000 Ghrelin induces adiposity in rodents. Nature 407:908 –913 6. Nakazato M, Murakami N, Date Y, Kojima M, Matsuo H, Kangawa K, Matsukura S 2001 A role for ghrelin in the central regulation of feeding. Nature 409:194 –198 7. Wren AM, Small CJ, Ward HL, Murphy KG, Dakin CL, Taheri S, Kennedy AR, Roberts GH, Morgan DG, Ghatei MA, Bloom SR 2000 The novel hypothalamic peptide ghrelin stimulates food intake and growth hormone secretion. Endocrinology 141:4325– 4328 8. Asakawa A, Inui A, Kaga T, Yuzuriha H, Nagata T, Ueno N, Makino S, Fujimiya M, Niijima A, Fujino MA, Kasuga M 2001 Ghrelin is an appetitestimulatory signal from stomach with structural resemblance to motilin. Gastroenterology 120:337–345 9. Wren AM, Seal LJ, Cohen MA, Brynes AE, Frost GS, Murphy KG, Dhillo WS, Ghatei MA, Bloom SR 2001 Ghrelin enhances appetite and increases food intake in humans. J Clin Endocrinol Metab 86:5992–5995 10. Cummings DE, Shannon MH 2003 Roles for ghrelin in the regulation of appetite and body weight. Arch Surg 138:389 –396 11. Cummings DE, Purnell JQ, Frayo RS, Schmidova K, Wisse BE, Weigle DS 2001 A preprandial rise in plasma ghrelin levels suggests a role in meal initiation in humans. Diabetes 50:1714 –1719 12. Cummings DE, Weigle DS, Frayo RS, Breen PA, Ma MK, Dellinger EP, Purnell JQ 2002 Human plasma ghrelin levels after diet-induced weight loss and gastric bypass surgery. N Engl J Med 346:1623–1630 13. Lucidi R, Murdolo G, DiLoreto C, DeCicco A, Parlanti N, Fanelli C, Santeusanio F, Bolli GB, DeFeo P 2002 Ghrelin is not necessary for adequate hormonal counterregulation of insulin-induced hypoglycemia. Diabetes 51:2911–2914 14. Saad MF, Bernaba B, Hwu CM, Jinagouda S, Fahmi S, Kogosov E, Boyadjian R 2002 Insulin regulates plasma ghrelin concentration. J Clin Endocrinol Metab 87:3997– 4000 15. McCowen KC, Maykel JA, Bistrian BR, Ling PR 2002 Circulating ghrelin concentrations are lowered by intravenous glucose or hyperinsulinemic euglycemic conditions in rodents. J Endocrinol 175:R7–R11 16. Mohlig M, Spranger J, Otto B, Ristow M, Tschop M, Pfeiffer ARH 2002 Euglycemic hyperinsulinemia, but not lipid infusion, decreases circulating ghrelin levels in humans. J Endocrinol Invest 25:RC36 –RC38 17. Nakagawa E, Nagaya N, Okumura H, Enomoto M, Oya H, Ono F, Hosoda H, Kojima M, Kangawa K 2002 Hyperglycaemia suppresses the secretion of ghrelin, a novel growth-hormone-releasing peptide: responses to the intravenous and oral administration of glucose. Clin Sci (Lond) 103:325–328 18. Flanagan DE, Evans ML, Monsod TP, Rife F, Heptulla RA, Tamborlane WV, Sherwin RS 2003 The influence of insulin on circulating ghrelin. Am J Physiol Endocrinol Metab 284:E313–E316 19. Caixas A, Bashore C, Nash W, Pi-Sunyer FX, Laferrere B 2002 Insulin, unlike food intake, does not suppress ghrelin in human subjects. J Clin Endocrinol Metab 87:1902–1906

Overduin et al. • Nutrient Regulation of Ghrelin

20. Schaller G, Schmidt A, Pleiner J, Woloszczuk W, Wolzt M, Luger A 2003 Plasma ghrelin concentrations are not regulated by glucose or insulin. Diabetes 52:16 –20 21. Tschop M, Wawarta R, Riepl RL, Friedrich S, Bidlingmaier M, Landgraf R, Folwaczny C 2001 Post-prandial decrease of circulating human ghrelin levels. J Endocrinol Invest 24:RC19 –RC21 22. Callahan HS, Cummings DE, Pepe MS, Breen PA, Matthys CC, Weigle DS 2003 Postprandial suppression of plasma ghrelin level is proportional to ingested caloric load but does not predict intermeal interval in humans. J Clin Endocrinol Metab 89:1319 –1324 23. Shiiya T, Nakazato M, Mizuta M, Date Y, Mondal MS, Tanaka M, Nozoe S, Hosoda H, Kangawa K, Matsukura S 2002 Plasma ghrelin levels in lean and obese humans and effect of glucose on ghrelin secretion. J Clin Endocrinol Metab 87:240 –244 24. Williams DL, Cummings DE, Grill HJ, Kaplan JM 2003 Meal-related ghrelin suppression requires postgastric feedback. Endocrinology 144:2765–2767 25. Cummings DE, Shannon MH 2003 Ghrelin and gastric bypass: is there a hormonal contribution to surgical weight loss? J Clin Endocrinol Metab 88: 2999 –3002 26. Cummings DE, Overduin J, Foster-Schubert KE 2004 Gastric bypass for obesity: mechanisms of weight loss and diabetes resolution. J Clin Endocrinol Metab 89:2608 –2615 27. Rocca AS, Brubaker PL 1999 Role of the vagus nerve in mediating proximal nutrient-induced glucagon-like peptide-1 secretion. Endocrinology 140:1687–1694 28. Raybould HE 2002 Visceral perception: sensory transduction in visceral afferents and nutrients. Gut 51(Suppl 1):i11–i14 29. Buchan AM 1999 Nutrient tasting and signaling mechanisms in the gut. III. Endocrine cell recognition of luminal nutrients. Am J Physiol 277:G1103–G1107 30. Moran TH 2000 Cholecystokinin and satiety: current perspectives. Nutrition 16:858 – 865 31. Guyton AC, Hall JE 1996 Textbook of medical physiology. 9th ed. Philadelphia: WB Saunders Company 32. Remie R, van Dongen JJ, Rensema JW, van Wunnik GHJ 1990 General techniques. In: Remie R, van Dongen JJ, Rensema JW, van Wunnik GHJ, eds. Manual of microsurgery on the laboratory rat. Amsterdam: Elsevier; 81–156 33. Williams DL, Grill HJ, Cummings DE, Kaplan JM 2003 Vagotomy dissociates short- and long-term controls of circulating ghrelin. Endocrinology 144:5184 –5187 34. Kaplan JM, Spector AC, Grill HJ 1992 Dynamics of gastric emptying during and after stomach fill. Am J Physiol 263:R813–R819 35. Murakami N, Hayashida T, Kuroiwa T, Nakahara K, Ida T, Mondal MS, Nakazato M, Kojima M, Kangawa K 2002 Role for central ghrelin in food intake and secretion profile of stomach ghrelin in rats. J Endocrinol 174:283–288 36. Ariyasu H, Takaya K, Hosoda H, Iwakura H, Ebihara K, Mori K, Ogawa Y, Hosoda K, Akamizu T, Kojima M, Kangawa K, Nakao K 2002 Delayed short-term secretory regulation of ghrelin in obese animals: evidenced by a specific RIA for the active form of ghrelin. Endocrinology 143:3341–3350 37. Nakai Y, Hosoda H, Nin K, Ooya C, Hayashi H, Akamizu T, Kangawa K 2003 Plasma levels of active form of ghrelin during oral glucose tolerance test in patients with anorexia nervosa. Eur J Endocrinol 149:R1–R3 38. Cummings DE, Overduin J 2004 Circulating ghrelin levels in pathophysiological conditions. In: Ghigo E, ed. Ghrelin. Boston: Kluwer Academic Publishers; 207–223 39. Belchetz PE, Plant TM, Nakai Y, Keogh EJ, Knobil E 1978 Hypophysial responses to continuous and intermittent delivery of hypothalamic gonadotropin-releasing hormone. Science 202:631– 633 40. Rittmaster RS, Loriaux DL, Merriam GR 1987 Effect of continuous somatostatin and growth hormone-releasing hormone (GHRH) infusions on the subsequent growth hormone (GH) response to GHRH: evidence for somatotroph desensitization independent of GH pool depletion. Neuroendocrinology 45:118 –122 41. Gomez G, Englander EW, Greeley GH 2004 Nutrient inhibition of ghrelin secretion in the fasted rat. Regul Pept 117:33–36 42. Astrup A, Astrup A, Buemann B, Flint A, Raben A 2002 Low-fat diets and energy balance: how does the evidence stand in 2002. Proc Nutr Soc 61:299 –309 43. Gelling RW, Overduin J, Morrison CD, Morton GJ, Frayo RS, Cummings DE, Schwartz MW 2004 Effect of uncontrolled diabetes on plasma ghrelin concentrations and ghrelin-induced feeding. Endocrinology 145:4575– 4582 44. Cummings DE, Foster KE 2003 Ghrelin-leptin tango in body-weight regulation. Gastroenterology 124:1532–1544 45. Greenberg D, Kava RA, Lewis DR, Greenwood MR, Smith GP 1995 Time course for entry of intestinally infused lipids into blood of rats. Am J Physiol 269:R432–R436 46. Cowley MA, Smith RG, Diano S, Tschop M, Pronchuk N, Grove KL, Strasburger CM, Bidlingmaier M, Esterman M, Heiman ML, Garcia-Segura LM, Nillni EA, Mendez P, Low MJ, Sotonyi P, Friedman JM, Liu H, Pinto S, Colmers WF, Cone RD, Horvath TL 2003 The distribution and mechanism of action of ghrelin in the CNS demonstrate a novel hypothalamic circuit regulating energy homeostasis. Neuron 37:649 – 661

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