the effect on glucagon, glucagon-like peptide-1, total and acyl-ghrelin of dietary fats ingested...

The Effect on Glucagon, Glucagon-Like Peptide-1,Total and Acyl-Ghrelin of Dietary Fats Ingested withand without Potato

Angela Radulescu, Mary C. Gannon, and Frank Q. Nuttall

Endocrine, Metabolism, and Nutrition Section (A.R., M.C.G., F.Q.N.), Veterans Affairs Medical Center,Minneapolis, Minnesota 55417; and Departments of Medicine (M.C.G., F.Q.N.) and Food Science andNutrition (M.C.G.), University of Minnesota, Minneapolis, Minnesota 55455

Introduction: We are interested in the metabolic response to ingested macronutrients and theinteraction between macronutrients in meals. Recently, we have determined the insulin and glu-cose response to ingestion of lard, olive oil, or safflower oil, fat sources varying in fatty acidcomposition and carbohydrate (CHO), in the form of potato.

Objective: Our aim was to determine the effect of these dietary fats ingested alone or with potatoon glucagon, glucagon-like peptide-1 (GLP-1) (7-37 and 7-36 amide), and total and acyl-ghrelinconcentrations.

Methods: Healthy subjects ingested 25 g fat (lard, olive oil, or safflower oil), 50 g CHO (potato), 25 gfat with 50 g CHO, or water only. Glucagon, GLP-1 (7-37 and 7-36 amide), and total and acyl-ghrelinresponses were determined over 4 h.

Results: All fats when ingested alone increased glucagon. Glucagon increases were dramaticallyattenuated when fats were ingested with the potato. GLP-1 increased after all meals, but wasgreatest when fats were ingested alone. The fat-stimulated increase was completely negated whenfats were ingested with potato. Both acyl and total ghrelin decreased when only fats were ingested,as expected. When potato was ingested with any of the fats, the fat-induced decrease in acyl-ghrelin response also was essentially negated. Paradoxically, ghrelin increased when potato alonewas ingested.

Conclusions: The current data indicate that the glucagon, GLP-1 and ghrelin responses to ingestedfats, varying in fatty acid composition, are significantly affected by co-ingestion of CHO. Overall,the interaction between ingested foods in general is likely to be complex. (J Clin Endocrinol Metab95: 3385–3391, 2010)

Our laboratory is interested in the metabolic effect ofmacronutrients when ingested individually and in

combinations. Recently, we have determined that whenlard, olive oil, or safflower oil, fats considered to be highin saturated, monounsaturated, and polyunsaturatedfatty acids, respectively, are ingested with potato as asource of carbohydrate, the quantitative glucose and in-sulin area responses were the same as when potato is in-gested alone (1). However, the increase in both was de-

layed, reduced, and prolonged when potato was ingestedwith any of the fats. The ingestion of each of the three fatswith potato decreased the nonesterified fatty acids con-centration, and the decrease correlated with the change ininsulin concentration, as expected. All three fats when in-gested alone did modestly increase the insulin concentra-tion when compared with ingestion of water alone. TheC-peptide data confirmed that this represented a stimula-tion of insulin secretion. When lard, olive oil, or safflower

ISSN Print 0021-972X ISSN Online 1945-7197Printed in U.S.A.Copyright © 2010 by The Endocrine Societydoi: 10.1210/jc.2009-2559 Received December 1, 2009. Accepted April 9, 2010.First Published Online May 5, 2010

Abbreviation: GLP-1, Glucagon-like peptide-1.

O R I G I N A L A R T I C L E

E n d o c r i n e R e s e a r c h

J Clin Endocrinol Metab, July 2010, 95(7):3385–3391 jcem.endojournals.org 3385

The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 26 August 2014. at 00:30 For personal use only. No other uses without permission. . All rights reserved.

oil was ingested with the potato, there was an acceleratedrise in triacylglycerol concentration.

In addition to the determination of the above (1), wedetermined the effect of these dietary fats ingested alone orwith potato on the glucagon, glucagon-like peptide-1(GLP-1) (7-37 and 7-36 amide), and total and acyl-ghrelinconcentrations. These are hormones known to respond tofood intake. The results form the basis for the presentreport.

We consider such studies to be important because theyhelp to elucidate the physiological effects of various di-etary fats and how they interact with other macronutri-ents. Current data strongly emphasize that these nutrientsshould be considered not solely as a source of energy butalso as regulators of fuel homeostasis.

Subjects and Methods

Nine healthy subjects (four females and five males) received lardas a test meal, 12 healthy subjects (six females and six males)received olive oil as a test meal, and 11 healthy subjects (sixfemales and five males) received safflower oil as a test meal. Allsubjects gave informed consent before participating in the study.The study was approved by the Minneapolis Department of Vet-erans Affairs Medical Center and the University of MinnesotaCommittee on Human Subjects. The mean age of the subjectswas 25 yr (range, 18–38); the mean body mass index was 23kg/m2 (range, 19–29), with mean lean body mass of 54 kg (range,37–67). Lean body mass was determined using a portable bodyimpedance analyzer (RJL Systems, Clinton Township, MI). Allparticipants had normal thyroid, liver, kidney function, lipidprofiles, and glycated hemoglobin. Subjects were studied in aSpecial Diagnostic and Treatment Unit, which is similar to aclinical research center. Subjects were instructed to eat a lightmeal the evening before the test and to fast after 2000 h. After anovernight fast of 12 h, an indwelling catheter was inserted intoan antecubital or forearm vein and kept patent with iv saline.Subjects ingested a test meal consisting of lard, olive oil, or saf-flower oil with or without mashed potatoes and mashed potatoesalone. On a separate occasion, they received water only. Eachsubject completed all four arms of the study. Generally, the 4-dstudy was conducted over a 2- to 3-wk period.

Boiled, peeled white potatoes (250 g raw weight) were givenin an amount to equal to 50 g starch (2). They were cooked10–15 min in a microwave oven before being served. Theamount of fat consumed either alone or mixed with potatoes was25 g. The oils were ingested at room temperature. Lard wasmelted to a liquid state, which then was ingested. The meals weregiven in a random order at 0815 h. Each fat, or water only, wasingested within 1 min. When the meal contained potato with orwithout fat, the meals were ingested within approximately 5 min.Patients were allowed to consume water and coffee ad libitum.Some of the subjects drank coffee after the test meal, others didnot, but this was randomly distributed. If they drank coffee, theydrank coffee during all four components of the study. Althoughthe amount of water was not quantified, when questioned, thesubjects indicated that similar amounts of water/coffee were con-sumed for all three test meals. Baseline blood samples were ob-

tained at 0745, 0800, and 0815 h. The test meal was ingested at0815 h. Blood was collected every 15 min after the beginning ofeach meal for the first 2 h, then every 30 min for the third andfourth hours.

Glucagon and active and total ghrelin were determined byRIA using kits purchased from Linco Research (purchased byMillipore, Billerica, MA). The biologically active forms of GLP-1[GLP-1 (7-36 amide) and GLP-1 (7-37)] were quantified using anELISA kit from Linco Research (purchased by Millipore). For thedetermination of GLP-1, the blood was collected in ice-coldVacutainerEDTA-plasmatubestowhichanappropriateamountofdipeptidyl-peptidase-4 inhibitor had already been added. Theblood was mixed with dipeptidyl-peptidase-4 inhibitor less than30 sec after collection. It then was immediately returned to an icebath and centrifuged.

For all data, the net integrated 240-min area responses, usingthe fasting values as baseline, were calculated using a computerprogram based on the trapezoid rule. Statistics were determinedusing repeated measures ANOVA with Prism 4 software for theMacintosh (GraphPad, La Jolla, CA). This was followed by Stu-dent’s t test for paired variates with the Microsoft Office Excel2007 program (Microsoft Corporation, Richmond, WA) for PC.A P value of �0.05 was the criterion for significance. Data arepresented as means � SEM.

Results

Glucagon response (Fig. 1)The plasma glucagon concentration decreased slowly

when only water was ingested. This decrease was less pro-nounced after potato alone and any potato � fat combi-nation meal. Lard, olive oil, and safflower oil ingestedindependently increased the glucagon concentration. Theincrease was greatest after olive oil ingestion.

The net integrated glucagon area response after inges-tion of water was negative. The glucagon area responseafter ingestion of potato was significantly less negative inall the three arms of the study when compared with thearea after water ingestion. The same was true for the glu-cagon area response after potato � lard ingestion. Theglucagon area after ingested potato was not different whencompared with potato � lard. The glucagon area re-sponses after potato � olive oil and potato � safflower oil,although slightly negative, were not different when com-pared with the potato alone or water. After ingestion oflard, olive oil, or safflower oil, the glucagon area responsewas positive and significantly higher when compared withthe glucagon area after water, potato, or potato � any fat.The area response after olive oil was considerably higherthan that after lard or safflower oil ingestion.

GLP-1 response (Fig. 2)The plasma GLP-1 concentration remained stable after

ingestion of water only. After ingestion of lard, olive oil,or safflower oil alone, the GLP-1 concentration increased

3386 Radulescu et al. Effect of Diet on Glucagon and GLP-1 J Clin Endocrinol Metab, July 2010, 95(7):3385–3391


and remained elevated for the duration of the study. Whenlard, olive oil, or safflower oil was ingested with potato,there was only a small increase in GLP-1. That is, theincrease was blunted when potato was added to the fats.The plasma GLP-1 concentration increased slightly in abiphasic response after potato ingestion.

Potato ingestion resulted in an in-crease in GLP-1 area, but this was notstatistically significant when comparedwith water. The GLP-1 net area re-sponses after ingestion of lard, olive oil,and safflower oil were significantlyhigher when compared with the netarea response after ingestion of the re-spective water, potato, or potato � fatmeals. Interestingly, the area responseafter olive oil ingestion was greater thanafter lard or safflower oil ingestion. Thenet area responses after potato and po-tato � lard, olive oil, or safflower oilwere positive and not different. All werehigher when compared with the area re-sponse after water ingestion, but thiswas only statistically significant whenany of the fats were present.

Total ghrelin response (Fig. 3)The total ghrelin concentration did

not change significantly when onlywater was ingested. After the ingestionof potato alone, the total ghrelin de-creased to a nadir at 1–1.5 h, returnedto the fasting value at 2 h, and contin-ued to increase, remaining elevated atthe termination of the study. After lardor safflower oil ingestion, the total gh-relin decreased, reached a nadir at 3.5 h,and remained below baseline at the endof the study. After olive oil ingestion,the total ghrelin concentration dec-reased in a similar fashion, reaching anadir at 2.5 h and remaining below thefasting level at the termination of thestudy. When potato was ingested withlard, olive oil, and safflower oil, respec-tively, there was only a small and tran-sient initial decrease in total ghrelinconcentration.

The total ghrelin net area responsewas slightly positive after potato inges-tion, but this was not statistically sig-nificantly different from the net area re-sponse after water ingestion. The area

responses after lard and potato � lard were negative. Thiswas statistically significantly less than the area responseafter water or potato ingestion. A similar response wasobtained with olive oil. The area response after saffloweroil ingestion was negative and also statistically signifi-cantly different when compared with the area response

FIG. 1. Time course of serum glucagon concentrations. Time points are means � SEM. Insets,Net integrated areas under the curve after test meal ingestion. Bars with different lettersindicate that data are statistically significantly different (P � 0.05).



after water or potato ingestion. However, when potatowas ingested with safflower oil, the total ghrelin area re-sponse was not statistically significantly different whencompared with the net areas after water or potato inges-tion. Potato � lard, potato � olive oil, and potato � saf-flower oil ingestion resulted in a similar decrease in totalghrelin area when compared with only lard, olive oil, andsafflower oil ingestion, respectively.

Acyl-ghrelin response (Fig. 4)When only water was ingested, the acyl-ghrelin con-

centration remained stable. After lard, olive oil, or saf-flower oil ingestion, the acyl-ghrelin decreased and re-mained below the baseline value at the end of the study.After potato ingestion, the acyl-ghrelin decreased andreached a nadir at 1 h, which was followed by a reboundto above the fasting value. The response to potato � lard,potato � olive oil, or potato � safflower oil was similar tothat of potato alone, except the rebound was delayed andsmaller.

The net acyl-ghrelin area response was positive afterwater or potato, and the areas were essentially identical.The acyl-ghrelin area responses were negative after inges-tion of lard, olive oil, and safflower oil and were statisti-cally significantly lower when compared with the areaafter water and potato � lard, olive oil, or safflower oil.When potato was ingested with any of the dietary fats, thedecrease in acyl-ghrelin response induced by the ingestedfat alone was essentially negated. The net acyl-ghrelin ar-eas after potato � lard, potato � olive oil, or potato �safflower oil were not different when compared with po-tato alone or water.

Discussion

In the first publication from this study, we have reportedfor the first time that lard, olive oil, and safflower oil in-gested alone all increase the insulin concentration whencompared with ingestion of water alone, although the in-crease is modest (1). The C-peptide data confirmed thatthis represented a stimulation of insulin secretion. Themechanism is totally unknown. The observation that theplasma glucose did not change is compatible with a fattyacid-induced insulin resistance, or the increase may nothave been sufficient to affect glucose metabolism.

In the same study, we now report that the ingestion oflard, olive oil, or safflower oil also greatly stimulated anincrease in GLP-1. Thus, the fat-stimulated insulin re-sponse could be mediated, at least in part, by a rise in theGLP-1 concentration. However, if a rise in GLP-1 is mech-anistically important, the fact that, quantitatively and dy-namically, the responses of GLP-1 and insulin were dif-ferent suggests that if GLP-1 is playing a role, it is a minorrole, and only a modest increase in GLP-1 concentrationis sufficient to stimulate insulin secretion.

It has been reported that ingestion of fats or glucoseindependently stimulates a rise in GLP-1 (3, 4). It also hasbeen reported that an increase in the plasma glucose con-centration amplifies the GLP-1 stimulatory effect on in-sulin secretion (5). Thus, we would have expected the in-gestion of potato with lard, olive oil, or safflower oil to

FIG. 2. Time course of serum GLP-1 concentrations. Time points aremeans � SEM. Insets, Net integrated areas under the curve after testmeal ingestion. Bars with different letters indicate that data arestatistically significantly different (P � 0.05).



have an additive effect in stimulating a rise in GLP-1 con-centration. This was not the case. The addition of potatoessentially extinguished the GLP-1 response to fat, i.e. in-stead of an additive effect the rise in GLP-1 was dimin-

ished. Interestingly, Vollmer et al. (6)have reported that the GLP-1 responseto a mixed meal was attenuated whenthe plasma glucose was clamped at ap-proximately 160 mg/dl using a glucoseinfusion protocol. The authors specu-lated that the attenuation could be ex-plained by a hyperglycemic inhibitionof gastric emptying, and this is the rea-son the GLP-1 response to meals is im-paired in people with type 2 diabetes.Our data suggest that an elevated glu-cose also could have impaired theGLP-1 response to the ingested fat. Thisis an issue that needs to be investigatedfurther.

A rise in glucagon concentration oc-curs with ingestion of proteins, fruc-tose, and galactose (7–10). All are glu-coneogenic substrates. The present dataindicate that orally administered di-etary fats also result in an increase inglucagon concentration. In a previousstudy, we also demonstrated progres-sively larger glucagon area responseswith increasing amounts of butter wheningested with potato in individuals withtype 2 diabetes (11). Thus, all ingestedmacronutrient constituents studied todate raise the plasma glucagon, with theexception of glucose. Ingested glucoselowers the glucagon concentration.

In the present study, the ingestion ofpotato alone also modestly increasedthe glucagon concentration when com-pared with the water control. Mostlikely, this was due to the small amountof protein present in potato (8). The ad-dition of potato to lard, olive oil, or saf-flower oil abrogated the glucagon re-sponse observed after the ingestion offat alone. This can be explained by apotato-induced increase in circulatingglucose and insulin concentrations be-cause both are reported inhibitors ofglucagon release (12).

Whether GLP-1 directly inhibits glu-cagon secretion is uncertain (13, 14).The current data strongly suggest that

GLP-1 does not inhibit glucagon secretion. When any ofthe fats was ingested, both GLP-1 and glucagon increased.It also is of interest that the insulin, GLP-1, and glucagon

FIG. 3. Time course of serum total ghrelin concentrations. Time points are means � SEM.Insets, Net integrated areas under the curve after test meal ingestion. Bars with differentletters indicate that data are statistically significantly different (P � 0.05).



concentrations were increased, butthe glucose concentration remainedentirely stable. As we have pointed outpreviously, the role of glucagon in reg-ulating the glucose concentration re-mains elusive, except in the presence ofhypoglycemia (15).

In the present study, both acyl andtotal ghrelin were suppressed for theduration of the study when lard, oliveoil, or safflower oil was ingested. Inges-tion of potato or potato plus lard, oliveoil, or safflower oil resulted in only asmall transient decrease, followed by arebound increase in both. In contrast, ina study by Foster-Schubert et al. (16),lipid ingestion suppressed total andacyl-ghrelin less effectively than did car-bohydrate ingestion. However, the testmeals consisted of isocaloric beveragesof mixed macronutrient compositionwith 80% of the calories derived fromcarbohydrate or lipids. Similar to re-sults of our study, the total and acyl-ghrelin rebound was most pronouncedafter carbohydrate ingestion.

It has been reported that the ghrelinconcentration is suppressed by insulin(17–19). In the present study, the insu-lin concentration cannot explain the bi-phasic acyl ghrelin response to potatoor potato � any fat. In addition, theghrelin concentration remained sup-pressed after ingestion of each fat, al-though the insulin concentration wasalways much lower after any fat inges-tion than after potato or potato � fatingestion. Thus, it is difficult to explainthe current data based on a quantitativeregulation of ghrelin by insulin. A cer-tain amount of insulin may be necessaryto maintain the mechanism by whichingested nutrients regulate ghrelin se-cretion. However, a rise in insulin dur-ing the meal is not necessary. Our dataare compatible with such a concept.The rebound increase in ghrelin whenpotato or potato plus fat was ingestedstill remains to be explained.

Considerable evidence implicatesghrelin suppression by ingested nutri-ents in postprandial satiation (20). Inour study, the satiety data (1) did not

FIG. 4. Time course of serum acyl-ghrelin concentrations. Time points are means � SEM.Insets, Net integrated areas under the curve after test meal ingestion. Bars with differentletters indicate that data are statistically significantly different (P � 0.05).



correlate with the ghrelin data. The ghrelin suppressionafter ingestion of fat or potato � fat meals vs. ingestion ofpotato alone was not associated with a decrease of thedesire to eat, degree of hunger or proposed food intake, orwith an increase of fullness sensation.

SummaryThere is a complex interaction between ingested fats

and carbohydrates in regard to the regulation of glucagon,GLP-1, and ghrelin as well as for insulin. We have deter-mined that dietary fats ingested alone stimulate a rise inglucagon and GLP-1 concentration. However, the gluca-gon and GLP-1 increases were dramatically attenuatedwhen the fats were ingested with the potato. The ghrelinconcentration was decreased in response to fat ingestedalone. Ingested potato only modestly and transiently de-creased the ghrelin concentrations. The interaction of fatswith potato did not change significantly the ghrelin re-sponse to ingested fat alone.

Acknowledgments

The authors thank the subjects for participating in the study; thestaff of the Special Diagnostic and Treatment Unit, the staff of theClinical Chemistry Laboratory, and the staff of the NuclearMedicine Department for their assistance; Heidi Hoover, R.D.,M.S., for help with food preparation; and Linda Hartich, M.T.,for technical advice and laboratory assistance. We thank Dr.Michael Kuzkowski for advice on statistical analysis of the data.

Address all correspondence and requests for reprints to:Frank Q. Nuttall, M.D., Ph.D., Endocrine, Metabolism, and Nu-trition Section (111G), VA Medical Center, One Veterans Drive,Minneapolis, Minnesota 55417. E-mail: [email protected].

This work was supported by Merit Review funds from theDepartment of Veterans Affairs and a grant from the NationalPork Board.

F.Q.N. and M.C.G. designed the study. A.R. collected thedata. All three authors analyzed the data and contributed to thewriting of the manuscript.

Disclosure Summary: There is no conflict of interest.

References

1. Radulescu A, Hassan Y, Gannon MC, Nuttall FQ 2009 The degree ofsaturation of fatty acids in dietary fats does not affect the metabolicresponse to ingested carbohydrate. J Am Coll Nutr 28:286–295

2. U.S. Department of Agriculture 1984 Composition of foods raw,processed, prepared. Agriculture Handbook No. 8-11. Vegetables

and vegetable products. Washington, DC: U.S. Government PrintingOffice

3. Herrmann C, Goke R, Richter G, Fehmann HC, Arnold R, Goke B1995 Glucagon-like peptide-1 and glucose-dependent insulin-re-leasing polypeptide plasma levels in response to nutrients. Digestion56:117–126

4. Beysen C, Karpe F, Fielding BA, Clark A, Levy JC, Frayn KN 2002Interaction between specific fatty acids, GLP-1 and insulin secretionin humans. Diabetologia 45:1533–1541

5. Nathan DM, Schreiber E, Fogel H, Mojsov S, Habener JF 1992Insulinotropic action of glucagon like peptide-I-(7–37) in diabeticand nondiabetic subjects. Diabetes Care 15:270–276

6. Vollmer K, Gardiwal H, Menge BA, Goetze O, Deacon CF, SchmidtWE, Holst JJ, Meier JJ 2009 Hyperglycemia acutely lowers the post-prandial excursions of glucagon-like peptide-1 and gastric inhibi-tory polypeptide in humans. J Clin Endocrinol Metab 94:1379–1385

7. Krezowski PA, Nuttall FQ, Gannon MC, Bartosh NH 1986 Theeffect of protein ingestion on the metabolic response to oral glucosein normal individuals. Am J Clin Nutr 44:847–856

8. Westphal SA, Gannon MC, Nuttall FQ 1990 The metabolic re-sponse to glucose ingested with various amounts of protein. Am JClin Nutr 52:267–272

9. Nuttall FQ, Khan MA, Gannon MC 2000 Peripheral glucose ap-pearance rate following fructose ingestion in normal subjects. Me-tabolism 49:1565–1571

10. Gannon MC, Khan MA, Nuttall FQ 2001 Glucose appearance rateafter the ingestion of galactose. Metabolism 50:93–98

11. Gannon MC, Ercan N, Westphal SA, Nuttall FQ 1993 Effect ofadded fat on the plasma glucose and insulin response to ingestedpotato in individuals with NIDDM. Diabetes Care 16:874–880

12. Franklin I, Gromada J, Gjinovci A, Theander S, Wollheim CB 2005�-Cell secretary products activate �-cell ATP-dependent potassiumchannels to inhibit glucagon release. Diabetes 54:1808–1815

13. Meier JJ, Deacon CF, Schmidt WE, Holst JJ, Nauck MA 2007 Sup-pression of glucagon secretion is lower after oral glucose adminis-tration than during intravenous glucose administration in humansubjects. Diabetologia 50:806–813

14. Vollmer K, Holst JJ, Baller B, Ellrichmann M, Nauck MA, SchmidtWE, Meier JJ 2008 Predictors of incretin concentrations in subjectswith normal, impaired, and diabetic glucose tolerance. Diabetes 57:678–687

15. Nuttall FQ, Ngo A, Gannon MC 2008 Regulation of hepatic glucoseproduction and the role of gluconeogenesis in humans: is the rate ofgluconeogenesis constant? Diabetes Metab Res Rev 24:438–458

16. Foster-Schubert KE, Overduin J, Prudom CE, Liu J, Callahan HS,Gaylinn BD, Thorner MO, Cummings DE 2008 Acyl and total gh-relin are suppressed strongly by ingested proteins, weakly by lipids,and biphasically by carbohydrates. J Clin Endocrinol Metab 93:1971–1979

17. Flanagan DE, Evans ML, Monsod TP, Rife F, Heptulla RA,Tamborlane WV, Sherwin RS 2003 The influence of insulin on cir-culating ghrelin. Am J Physiol Endocrinol Metab 284:E313–E316

18. Mohlig M, Spranger J, Otto B, Ristow M, Tschop M, Pfeiffer AF2002 Euglycemic hyperinsulinemia, but not lipid infusion, decreasescirculating ghrelin levels in humans. J Endocrinol Invest 25:RC36–RC38

19. 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

20. Cummings DE 2006 Ghrelin and the short- and long-term regula-tion of appetite and body weight. Physiol Behav 89:71–84



the effect on glucagon, glucagon-like peptide-1, total and acyl-ghrelin of dietary fats ingested...

Documents