The importance of triglyceride hydrolysis for the release of gastric inhibitory polypeptide

GASTROENTEROLOGY

1981;80:108-11

The Importance of Triglyceride Hydrolysis for the Release of Gastric Inhibitory Polypeptide STUART

A. ROSS

Division of Internal Alberta, Canada

and

Medicine,

ELDON

Faculty

A. SHAFFER

of Medicine,

Gastric inhibitory polypeptide is released from the small intestine after the ingestion of fat, but it is not known if triglyceride itself or one of its hydrolytic products is the stimulus to gastric inhibitory polypeptide secretion. Children with cystic fibrosis and defective fat Jipolysis were studied to help define the exact stimulus to gastric inhibitory polypeptide secretion. Pancreatic enzyme therapy was withheld from the children with cystic fibrosis during these tests. Ten normal children and 10 children with cystic fibrosis each ingested corn oil and serum immunoreactive gastric inhibitory polypeptide measured. The normal children had a lo-fold increase in serum gastric inhibitory polypeptide levels after the triglyceride, but no increase in immunoreactive gastric inhibitory polypeptide occurred in the children with cystic fibrosis. When three of the children with cystic fibrosis received their pancreatic enzymes and then ingested the triglyceride, gastric inhibitory polypeptide values increased lo-fold. To assess the relative importance of the products of triglyceride hydrolysis and the chain length of the component fatty acids, 6 normal adults consumed, on separate days, 40 mmol of corn oil, medium-chain triglycerides, long-chain fatty acids, or glycerol. Long-chain fatty acids caused a fourfold increase and triglyceride a 12-fold increase in gastric inhibitory polypeptide Jevels. There was no increase after medium-chain triReceived February 8, 1980. Accepted August 19, 1980. Address requests for reprints to: Dr. S. A. Ross, Division of Internal Medicine, Faculty of Medicine, University of Calgary, 2500 University Drive, N.W., Calgary, Alberta, Canada TZN lN4. This work was supported in part by the Canadian Diabetes Association and the Medical Research Council of Canada Grant MT-5080. The authors wish to thank Ms. Coral Bernstein for her excellent technical assistance, Dr. John Brown for supplies of his antiserum, the patients of the Alberta Children’s Hospital Cystic Fibrosis Clinic and the children of the W.J. Collett School who participated in the study, and Mrs. Sharon Sabey for preparation of the manuscript. 0 1981 by the American Gastroenterological Association 0018-5085/81/010108-04$2.50

University

of Calgary,

Calgary,

glyceride or glycerol. This indicates that long-chain fatty acids-the end product of triglyceride hydrolysis-are a stimulus to gastric inhibitory polypeptide secretion; that this release is apparently proportional to the quantity of long-chain fatty acid present; and that hydrolysis of triglyceride is required before gastric inhibitory polypeptide release can normally occur after fat ingestion. Gastric inhibitory polypeptide (GIP) is an enteric hormone primarily located in the duodenal mucosa (1). The main physiologic role described for GIP is that of augmentation of insulin release after the ingestion of food. Three main stimuli are known to release GIP: glucose, galactose, and triglyceride (2-4). However, it is not known whether the main stimulus to GIP secretion is triglyceride itself or a product of triglyceride hydrolysis: glycerol, /?-monoglyceride, or free fatty acids. To examine the precise signal for GIP secretion, we studied the effect of triglyceride ingestion in patients with known fat maldigestion, and then we determined the relative importance of its lipolytic products in normal human volunteers.

Methods Subjects Cystic fibrosis patients. Ten children (7-17 yr) with proven cystic fibrosis requiring pancreatic enzyme replacement, were compared to 15 normal children, matched for age, sex, and weight. The diagnosis of cystic fibrosis was based on three elevated sweat chloride concentrations (>i’O mEq/L) by using quantitative pilocarpine iontophoresis (5). All patients required pancreatic enzyme replacement for steatorrhea. Suppurative pulmonary complications of cystic fibrosis were not a major factor in these children, although all had radiologic changes compatible with the chronic lung disease associated with cystic fibrosis. On the day of the tests, all medication, including pancreatic enzyme therapy, was withheld from the children with cystic fibrosis.

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January 1981

Adult volunteers. Six normal male adults between the ages of 20 and 24 yr took part in the study; all were of ideal weight. Informed oral and written consent was obtained from the parents of the children and from the adult volunteers. The protocol was approved in December, 1978, by the Ethics Committee established by the University of Calgary.

Protocol Subjects were fasted overnight for a minimum of 12 h before each study. The patients were allowed to sit or recline throughout the test. Venous blood samples were drawn through an indwelling needle placed in the anticubital vein, kept patent by a slow infusion of 155 mM NaCl. A basal blood sample was drawn immediately before the administration of the test substance. Blood samples were then drawn at 30,60,90, and 120 min for estimation of immunoreactive gastric inhibitory polypeptide (IRGIP). Aprotinin, 0.1 ml 1000 KIU (Novo Industries, Copenhagen, Denmark) was added to each 1 ml of whole blood at the time of collection. The samples were centrifuged at 4’C, and the serum was stored at -20°C. Cystic fibrosis study. Both the normal and CF children drank 40 mmol of triglyceride in the form of Lipomul (Upjohn Company, Kalamazoo, Mich.) containing predominantly the glycerides of linoleic acid C18:2 and oleic acid C18:l. On a separate occasion, 3 of the children with cystic fibrosis received their pancreatic enzyme therapy at the same time as ingesting the triglyceride. Adult volunteers. The adult volunteers were given triglycerides and on a separate occasion, one of the hydrolytic products of triglyceride to assess the response of GIP to these substances (Table 1). In addition, the importance of chain length was investigated by the administration of medium-chain triglycerides. Each subject received on a separate day 40 mmol of: Lipomul, medium-chain triglycerides as MCT oil-containing 75% tricaprylic acid C:8 and 15% tricapric acid C:lO-long-chain fatty acids (as oleic acid), glycerol, or monoglyceride (Atmul. 124-Atlas Chemical Industries Canada, Ltd.). The substances were instilled via a nasogastric tube except for the monoglyceride, which was eaten as a solid from a spoon. A minimum of 0.5 h was allowed before the initial blood samples were taken after the insertion of the tube.

Gastric

Inhibitory

Polypeptide

Immunoassay

High purified GIP (UBC II), kindly provided by Dr. John Brown of the University of British Columbia, was used for iodination and as the radioimmunoassay standard. Gastric inhibitory polypeptide was iodinated using chloramine T and immediately purified on microfine silicate (QUSO-G32 Philadelphia Quartz Company, Philadelphia, Pa.). Before an immunoassay, the radioactive GIP was further purified on a QAE 25 Sephadex column. To elute the tracer from the column, a gradient mixer was used to provide a gradient of between 0.15 and 0.05 M NaCl. Two thousand counts per minute of radioactive GIP were added to each assay tube, and after the separation of the immunoassay, each tube was counted for 10 min.

109

The GIP antiserum (GP 24) used in the studies was kindly provided by Dr. John Brown. This antiserum did not cross-react with glucagon, secretin insulin, cholecystokinin, vasoactive intestinal polypeptide, or gastrin at concentrations >lO pg/ml. The detection limit for four replicates was 25 pg/ml of serum. The assay buffer was 0.05 M PO,, pH 8.5, with 0.25% HSA and 1000 KIU/ml of aprotinin. The l-ml incubation volume was made up of 100 4 plasma, 500 ~1 radioactive GIP, 100 4 antibody, and incubation buffer. One hundred microliters of charcoal-dehormonized human plasma was added to the standard curve. The assay was incubated for 72 h at 4’C and separated by using a slurry of 0.5 g% dextran-coated charcoal. The initial binding without added antigen was 35%, and nonspecific binding in the absence of antigen was less than 5.2%. The interassay coefficient of variation was 4.8%. All samples were measured within the same assay. The standards were prepared in assay buffer and stored at -70’C for a period of no longer than 8 m. The inhibitory capacity of the standards remained unchanged in the immunoassay standard curves over this period. Dilutions of acid extracts of human and canine duodenum revealed parallel dilution curves in the immunoassay compared with the antigen standards. This parallelism was present when hormone-free plasma or buffer alone was present in the standard curves. Addition of up to 1 ng of antigen to serum resulted in recovery of >98% of the added antigen. Statistical significance of the data was evaluated by the Student’s t-test for paired or unpaired data, where applicable. The total hormone response to the test substance was assessed by measuring the area beneath the hormone response curve and the statistical significance of any rise assessed by the Student’s t-test.

Results Cystic Fibrosis Study The changes in serum GIP concentrations in the normal and cystic fibrosis children are shown in Figure 1. The fasting GIP values were not significantly different in the normal subjects compared to the children with cystic fibrosis: normals 110+15 pg/ ml, with cystic fibrosis 102f8 pg/ml. When the normal subjects ingested the triglyceride, GIP values rose significantly (p < O.OOl), reaching a mean peak value of 1080+80 pg/ml at 90 min. The children with cystic fibrosis demonstrated no increase in serum GIP concentrations from basal levels after the ingestion of triglyceride. When 3 of the children with cystic fibrosis ingested pancreatic enzymes and then triglyceride, IRGIP levels promptly increased lo-fold, achieving a response similar to that seen in the normal children. Adult Volunteers The changes in GIP secretion are shown in Figure 2. The basal GIP values in all the studies were

110

GASTROENTEROLOGY

ROSS AND SHAFFER

Table 1.

1200-

Six Normal Adults Quantity (ml)

Substrates triglyceride

(mmol)

40 40 40 40 40

50 18 13 3 5O

Triglyceride (corn oil) Medium-chain

Vol. 80, No. 1

(MCT oil)

Long-chain fatty acids (oleic acid) Glycerol Monoglyceride

1000TRIGLYCERIDE

800 IRGIP w/ml 600 -

a Solid at 23%

not significantly different between the subjects. Triglyceride resulted in a l&fold increase in serum GIP by 90 min. The long-chain fatty acids caused a fourfold increase in GIP levels at 90 min. In contrast, GIP levels did not change after the administration of medium-chain triglyceride, monoglyceride, or glycerol. The integrated total hormone response of the test substances in both groups, as assessed by the area beneath the hormone response curve, are shown in Table 2.

The key nutrients stimulating the release of GIP from the intestine are glucose, galactose, and triglyceride (3,4,6). Although triglyceride is considered to be one of the most potent stimuli to GIP secretion, it is not known whether the GIP secreted is in re-

1200

t TRIGLYCERIDE

,I I

p
P n-N)

IRGIP w/ml

LONGCHAIN FATTY ACID

MEDIUM-CHAIN TRIGLYCERIDE

30

60

90

Figure 2. Immunoreactive GIP (IRGIP) response to triglyceride and the hydrolytic products of triglyceride in 6 normal subjects. Serum IRGIP concentrations, mean f SEM in response to ingesting 40 mmoles of each of the following test substances: triglycerides (circles), long-chain fatty acids (diamonds), medium-chain triglyceride (inverted triangles), monoglyceride (squares), glycerol (upright triangles).

to triglyceride itself or to a hydrolytic product of triglyceride. This work has demonstrated that

sponse

hydrolysis of dietary fat is necessary for GIP release. When children with cystic fibrosis ingest triglyceride, the associated pancreatic exocrine failure causes fat maldigestion, and no GIP is released. When hydrolysis of triglyceride is achieved by the addition of pancreatic enzymes, the GIP response in the children with cystic fibrosis more than equals the response observed in normal children. The previously reported observation that hypersecretion of GIP occurs in children with cystic fibrosis after the ingestion of glucose would indicate that there is no

Table

2.

Integrated

GIP Response to Test Substances

Subjects

MINUTES Figure 1. Immunoreactive GIP (IRGIP) secretion following triglyceride ingestion in normal and cystic fibrosis children. Serum IRGIP concentrations mean &SJXM for: 10 normal children (solid circles), 10 children with cystic fibrosis (open triangles), 3 children with cystic fibrosis who had taken pancreatic enzymes immediately before the study (open squares).

120

MINUTES

Cystic fibrosis study

Adult volunteer study

Normal children Children with cystic fibrosis Children with cystic fibrosis + Pancreatic enzymes Triglyceride FFA MCT, glycerol, /?-monoglyceride

Area beneath hormone response curve (mg/mJ.min) 46.3f 11.0 No significant rise 49.2 ZIZ 9.0 70.7 f 5.4 28.7 f 6.7

No significant rise

January 1981

failure of the GIP-secreting cells in the child with cystic fibrosis (7). The only product of triglyceride hydrolysis capable of triggering GIP release was oleic acid. In the normal adult subjects studied, this long-chain fatty acid increased GIP levels in a fashion similar to triglyceride. Indeed, the value of GIP reached with the long-chain fatty acids was 2.5 times less that of the response noted with equimolar’administration of triglyceride, the hydrolysis of which would normally produce two or three long-chain fatty acids. Medium-chain triglyceride did not produce a rise in GIP, indicating that chain length is an important determinant of GIP secretion. However, weak stimulation of GIP has also been demonstrated, at least in dogs, after the administration of amino acids and medium chain triglycerides through a duodenal fistula (8,9). The failure of /3-monoglyceride to elicit GIP secretion in our studies would seem to indicate that this lipolytic product of triglyceride is not a stimulus to GIP secretion. Unlike the other test lipids, the /?monoglyceride was administered as a solid, and this could result in delayed delivery of this test substance from the stomach to the duodenum (10). Thus, during the 2 h of test procedure, the monoglyceride may not have reached the proximal small bowel in sufficient quantities to have any effect on GIP secretion. It is now apparent that GIP plays an essential role in the disposal of ingested nutrients, and it is the key intestinal hormonal stimulus for insulin release, causing an augmented insulin secretion in response to hyperglycemia and resulting in an increased disposal of carbohydrates (2,4,6). Gastric inhibitory polypeptide also appears to have anabolic effects on adipocytes by increasing the levels of lipoprotein lipase and by blocking the lipolytic properties of glucagon (11,12). Gastric inhibitory polypeptide may also influence the rate of absorption of nutrients from the small bowel (13). Failure of GIP release would therefore have far-reaching effects on carbohydrate metabolism and nutrition, particularly in the presence of endocrine and exocrine pancreatic

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111

insufficiency as occurs in cystic fibrosis. Thus, this demonstration that hydrolysis of fat is necessary for GIP secretion becomes considerably important when designing a diet for patients with maldigestion of fat and associated malnutrition. References 1. Brown

JC, Pederson RA, Jorpes E, Mutt V. Preparation of highly active enterogastrone. Can J Physiol Pharmacol 1969;47:113. 2. Dupre J, Ross SA, Watson D, Brown JC. Stimulation of insulin secretion of gastric inhibitory polypeptide in man. J Clin Endocrinol Metab 1973:37:826. 3. Ross SA, Brown JC, Dupre J. Hypersecretion of gastric inhibitory polypeptide following oral glucose in diabetes mellitus. Diabetes 1977;26:525. 4. Ross SA, Dupre J. Effects of ingestion of triglycerides or galactose on secretion of gastric inhibitory polypeptide and on responses to intravenous glucose in normal and diabetic subjects. Diabetes 1978;27:327. 5. Gibson LE. Cooke RE. A test for concentration of electrolytes in sweat in cystic fibrosis of the pancreas utilising pylocarpine by iontophoresis. Pediatrics 1959;23:545. 6. Brown JC, Dryburgh J, Ross SA, Duprd J. Identification and actions of gastric inhibitory polypeptide. Recent Prog Horm Res 1975;31:487. 7. Ross SA. McArthur RG, Morrison D. Hypersecretion of gastric inhibitory polypeptide in non-diabetic children with cystic fibrosis. Clin Res 1978;26:851A. 8. Thomas FB, Sinar D, Mazzaferri EL, et al. Selective release of gastric inhibitory polypeptide by intraduodenal amino acid perfusion in man. Gastroenterology 1978;74:1281. 9. O’Dorisio TM, Cataland S, Stevenson M, Mazzaferri EL. Gastric inhibitory polypeptide (GIP) intestinal distribution and stimulation by amino acids and medium-chain triglycerides. Dig Dis 1976;21:761. 10.Heading RC, Tothill P. McLaughlin GP, Shearman GP. Gastric emptying rate measurements in man. A double isotope scanning technique for similtaueous study of liquid and sohd components of a meal. Gastroenterology 1976;71:45. 11.Eckel RH, Fujimoto WY, Brunzell J. Gastric inhibitory polypeptide enhanced lipoprotein lipase activity in cultured preadipocytes. Diabetes 1979;28:1141. 12.Dupre J, Greenidge N, MacDonald TJ, et al. Inhibition of glucagon adipocytes by gastric inhibitory polypeptide. Metabolism 1976;25:1197. 13.Schusdziarra V, Ipp E, Harris V, et al. Studies of the physiology and pathophysiology of the pancreatic D cell. Metabolism 1978:27:1227.