The Insulin Secretory Effect of the Human Duodenal Mucosa

Vol. 59, No.1 Printed in U.S.A.

G .-\~TH O E~TEH O L OGY

Cop;· ri~ht © 1970 by The Will iams & Wilkins Co.

THE INSULIN SECRETORY EFFECT OF THE HUMAN DUODENAL MUCOSA J. FASEL, M.D. H. HADJIKHANJ, AND J.-P. FELBER, M.D. Clinique Medicate Universitaire and Departement de Biochimie Clinique, Hopital Cantonal Universitaire, Lausanne, Switzerland

A study of the mechanism of action of hormonal factors of the intestinal mucosa on insulin release by the pancreas was undertaken by taking biopsies of the duodenal mucosa in 6 normal subjects during the course of an oral glucose tolerance test (100 g per os). Aqueous extracts of these biopsies were injected into the pancreaticoduodenal artery of rats. The injection of the extracts of biopsies removed 30 and 60 min after the glucose load was followed by an important insulin release, whereas the extracts of biopsies taken before and 180 min after glucose load showed only a slight effect on insulin release. The glucose concentration of these extracts was checked and cannot be at the origin of the insulin secretion. This study suggests the formation, in the duodenal mucosa, under the influence of an oral glucose load, of a factor of hormonal character which stimulates the release of insulin by the pancreas. The possibility of the activation of a prohormone or of the rapid synthesis of a hormone is discussed. It is known that the duodenal mucosa produces a series of hormones. Of them, secretin, pancreozymin, and an intestinal factor possessing glucagon-like immunoreactivity have been shown to stimulate insulin release by the pancreas. 1 - 5 Evidence has been reported that glucose given orally provokes a greater release of insulin than glucose given intravenously. 6 ' 7 It has been suggested that a humoral substance is released from the intestine during glucose absorption which stimulates the release of insulin from the pancreatic islets. Recent studies have demonstrated that increased plasma levels of Received June 26, 1969. Accepted January 23, 1970.

Address requests for reprints to: Dr. J.-P. Felber, Departement de Biochimie Clinique, Hopital Cantonal Universitaire, Lausanne, Switzerland. This work was supported in part by grants from the Nestle Company S.A., Vevey, Switzerland, and the Swiss National Fund for Scientific Research (request no. 5334.3) . The authors wish to thank Dr. N. Bilic for his collaboration and Mr. B. Ess for his technical assistance. 109

secretin and, in some cases, of pancreozymin and of glucagon-like immunoreactivity follow oral glucose administration. 8 ' 9 Similarly, the presence of material possessing ,6-cytotropic effect has been demonstrated in the intestinal mucosa. 10 ' 11 The experimental data reported here demonstrate the effect of the presence of glucose in the intestine on the production of insulinogenic material by the duodenal mucosa and the time relationship with carbohydrate load. 12

Material and Methods Six normal subjects were submitted simultaneously to a glucose tolerance test and to a series of biopsies of the duodenal mucosa. The subjects were kept fasting for 12 hr. After performing the Charles Debray method of duodenal intubation with the peroral hydraulic biopsy capsule for multiple sampling, the subjects took 100 g of glucose per os. Glycemia was measured 0, 30, 60, and 180 min after the glucose load. A first biopsy of the mucosa was taken before the test and the others 30, 60, and 180 min after the ingestion of glucose. Each fragment of the biopsy was weighed

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vein. This method allowed the injection of substances directly into the pancreatic arterv and the analysis of blood coming from th~ pancreas of the rat. Blood samples were first taken before injection of the mucosal extract and again 1, 5, 10, and 15 min afterward. Insu. linemia and glycemia were measured in each sample. fusulin was measured by radioimmu. noassay 13 and glucose by the glucose-oxidase method. 14 Glucose was also measured directlv in the extracts of the duodenal mucosa. To verify the sensitivity of the method, in. creasing concentrations of secretin (Boots Pure Drugs Company, Nottingham, England) were injected into the pancreaticoduodenal artery of rats (fig. 1). The injection of 0.2 U of secretin produced a rather weak insulin re. lease, whereas insulin secretion increased when greater amounts of secretin were injected.

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300

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0 1

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Fw. 1. Stimulation of insulin secretion measured in the portal vein of rats after injection of increasing concentrations of secretin into the pancreaticoduodena l artery.

and homogenized in one-tenth of its weight of isoton ic NaCl. After homogenization at 0 C, the extracts were centrifuged at 3000 g in a refri gerated centrifuge . The supernatant fluid was kept for measurement of insulin and glucose. In addition, 0.2 ml of it was injected into the pancreaticoduodenal artery of a rat according to the following technique. Wistar rats were an esthetized by intraperitoneal injection of Nembutal (pentobarbital sodium, Abbott Laboratories, North Chicago, Ill.). After laparotomy, the hepatic and left gastric arteries were ligated at their origin. The splenic artery was ligated at the hilus of the spleen in order to keep the arterial vascularization of the tail of the pancreas. The celiac axis was catheterized below the pancreaticoduodenal artery. The extracts were injected into the celiac axis and blood was removed from the portal

Results Insulin release by the rat pancreas after the injection of a total extract of human mucosa removed from the subjects during the oral glucose tolerance test is shown in table 1. The injection of extracts of the duodenal mucosa removed before glucose loading was followed by an insignificant rise in immunoreactive insulin (IRD. IRI remained low during the entire 15-min period of observation of the rat. In con· trast, the extracts of duodenal mucosa taken 30 and 60 min after glucose loading possessed a marked insulinogenic activity, as demonstrated by a great rise of IRI fol· lowing the injection of these extracts into the rat. Fragments removed 180 min after glucose administration showed only a small stimulatory effect on IRI release. The mean values for the 6 subjects are shown graphically in figure 2. In order to verify the specificity of the stimulation of insulin release by the duodenal extracts, aqueous extracts of human abdominal muscle were injected under the same conditions (table 2). No rise in insu· linemia was observed in the blood taken from the portal vein of the rats after the injection of these extracts. The glucose levels of the extracts of duodenal mucosa were checked and were found to be low, both before and after glu· cose administration. The values were be·

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INSULIN SECRETORY EFFECT OF MUCOSA

July 1970

tween 50 and 70 mg per 100 ml, measured by the glucose-oxidase method.

Discussion The results demonstrate that aqueous extracts of human duodenal mucosa stimulate insulin release when injected into the pancreaticoduodenal artery of the rat. TABLE

The insulinogenic effect of these extracts is strongly enhanced after glucose administration. The nature of the substance contained in the duodenal mucosa extracts is not known . Although the possibility of a glucose metabolite cannot be completely excluded, it seems unlikely. The insulin-

1. Insulinemia in the portal vein of the rat after injection of duodenal

mucosa extracts into the pancreaticoduodenal artery" Insulinemia in the portal vein of the rae

Subject no.

Time of blood removal from the portal vein of the rat

Biopsy removed before glucose load

Biopsy removed 30 min after glucose load

Biopsy removed 60 min after glucose load

Biopsy removed 180 min after glucose load

"U/ml

min

1

0 1 5 10 15

110 100 60 105 110

100 290 220 122 220

115 239 191 154 155

112 132 207 119

2

0 1 5 10 15

130 150 147 158 77

130 260 145 235 90

78 170 185 95 170

132 155 145 170 130

3

0 1 5 10 15

100 78 95 86 70

72 430 190 175 180

142 500 365 500 300

96 105 82 155 116

4

0 1 5 10 15

40 48 54 50 55

41 267 125 122

68 350 254 122 155

105 146 135 170 136

0 1 5 10 15

40 171 118 90 Ill

232 255 122 143

77 98 92 98 153

124 174 151 87 170

0 1 5 10 15

34 28 85 53 48

109 500 500 500 500

63 53 53 53 114

5

6

77

107 258 500 311

"Biopsies of the duodenal mucosa were taken before and at various times during oral glucose tolerance tests in 6 normal human subjects. 'Human duodenal biopsy removed at time intervals after glucose load (100 g per os).

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TIME AFTER GLUCOSE LOAD (100 g p.o.l : MUCOSA TAKEN AT

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FASEL ET AL.

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linogenic effect of the duodenal mucosa extracts was enhanced after glucose load, whereas it was very weak before. This excludes the possibility that the substance(s) (possibly hormone) possessing the ~-cytotropic activity might exist as such during the fasting state and be released after glucose ingestion. Otherwise its concentration in the duodenal mucosa would be high before glucose absorption and low afterward. The observation that it appears after glucose ingestion may be compatible with the existence of either a prohormone, which may be activated during the passage of the food in the gut, or a rapid synthesis of the hormone(s). Further studies are needed to better understand this problem. REFERENCES

( 6 normal subjQcts)

FIG. 2. Fragments of duodenal mucosa were removed in 6 normal subjects before and 30, 60, and 180 min after oral glucose administration. The four curves represent the mean values of six plasma insulin levels of blood collected from the portal vein of rats injected with each of these six mucosal extracts. Blood was taken 0, 1, 5, 10, and 15 min after injection into the rat. TABLE 2. lnsulinemia in the portal vein of the rat after injection into the pancreaticoduodenal artery of extracts of human abdominal muscle

1. Unger RH , Ketterer H, Eisentraut AM, et a!:

2.

3.

4.

5.

6.

7.

sensitive mechanism of the islets of Langerhans is rather specific to glucose itsele 5 and a very high concentration of such metabolites would be necessary to stimulate the high insulin response which was observed. The possibility of a hormone factor such as secretin or some other gut hormone seems more likely. It is interesting to note that the insu-

8.

9.

10.

Effect of secretin on insulin secretion. Lancet 2: 24-26, 1966 Dupre J, Rojas L, White JG, et al: Effects of secretin on insulin and glucagon in portal and peripheral blood in man. Lancet 2:26-27, 1966 Pfeiffer EF, Telib M, Ammon J, et al : Direkte Stimulierung der Insulinsekretion in vitro durcl. Sekretin. Deutsch Med Wschr 90:1663- 1665, 1965 Unger RH , Ketterer H , Dupre J, et a! : The effects of secretin, pancreozymin, and gastrin on insulin and glucagon secretion in anesthetized dogs. J Clin Invest 46:630- 645, 1967 Unger RH , Ohneda A, Valverde I, et al: Characterization of the responses of circulating glucagon-like immunoreactivity to intraduodenal and intravenous administration of glucose. J Clin Invest 47:48-65, 1968 Eirich H, Stimmler L, Hlad CJ Jr, eta!: Plasma insulin response to oral and intravenous glucose administration . J Clin End ocr 24 :1076- 1082, 1962 Mcintyre N, Holdsworth CD, Turner DS : Intestinal factors in the control of insulin secretion . J Clin Endocr 25:1317-1324, 1965 Young JD, Lazarus L, Chisholm DJ: Secretin and pancreozymin-cholecystokinin after glucose. Lancet 2:914, 1968 Chisholm DJ, Young JD, Lazarus L : The gastrointestinal stimulus to insulin release. I. Secretin. J Clin Invest 48:1453-1460, 1969 Dupre J, Beck JC: Stimulation of release of insulin by an extract of intestinal mucosa . Diabetes 15:555-559, 1966

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INSULIN SECRETORY EFFECT OF MUCOSA

11. Haddad JG, Owen JA Jr : In vitro insulinogenic effect of material harvested from incubated gut loops of rats. Metabolism 18:71-72, 1969 12. Vannotti A, Hadjikhani H, Fasel J, et a!: The endocrine function of the intestinal mucosa . Amer J Proctol 20:68-71, 1969 13. Hales CN, Randle PJ: Immunoassay of insulin

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with insulin-antibody precipitate. Biochem J 88: 137-146, 1963 14. Hugget ASG, Nixon DA: Enzymatic determination of blood glucose. Biochem J 66:12P, 1956 15. Coore HG, Randle PJ: Regulation of insulin secretion studied with pieces of rabbit pancreas incubated in vitro. Biochem J 93:66-78, 1964