Enhanced intestinal fat absorption in diabetic chinese hamsters

Diabetologia, Dec 1973

Summary Intestinal absorption of glyoeryl tri-[1-14C]-oleate and [1-14C]-oleic acid, measured by serial determinations of blood radioactivity after oral administration of the compounds in peanut oil, was significantly greater in non-ketotic diabetic Chinese hamsters than in nondiabetic controls. Incorporation of [1-14C]-oleic acid into tissue lipids by jejunal slices in vitro was equal in both groups on a unit tissue weight basis. The data suggest that intraluminal hydrolysis does not differ between diabetic and non-diabetic animals. Overall uptake of free fatty acids may be enhanced in diabetics as a consequence of increased small intestinal mass.

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Enhanced intestinal fat absorption in diabetic chinese hamsters

Enhanced Intestinal Fat Absorption in Diabetic Chinese Hamsters T.M. Parkinson Diabetes Atherosclerosis Research The Upjohn Company Kalamazoo Michigan U.S.A. Summary. Intestinal absorption of glyceryl tri-[1-14C]cleat| and [1-1aC]-oleic acid, measured by serial determinations of blood radioactivity after oral administration of the compounds in peanut oil, was significantly greater in non-ketotie diabetic Chinese hamsters than in nondiabetic controls. Incorporation of [1-~dC]-oleic acid into tissue lipids by jejunal slices in vitro was equal in both groups on a unit tissue weight basis. The data suggest that intraluminal hydrolysis does not differ between diabetic and non-diabetic animals. Overall uptake oI free fatty acids may be enhanced in diabetics as a consequence of increased small intestinal mass. Chinese hamster; Cricetulus griseus; diabetes; intestinal absorption; triglyeeride; fatty acid; blood radioactivity; intestinal lipid synthesis - M e t h o d s Animals Animals of both sexes were selected for s t u d y from the Upjohn colony of Chinese hamsters described previously b y Gerritsen and Dulin [ 9 ]. Non-ketotic diabetic animals were characterized b y a consistent Tes-Tape| value of + 4 for urine glucose. Glucosuria correlates well with blood sugar in these animals and is an acceptable measurement of diabetes [10]. Diabetic animals were m a t c h e d with non-diabetic animals of the same sex and a p p r o x i m a t e age. All animals were maintained on Purina Mouse Breeder Chow and fasted for 24 h before use. B o d y weights of fasted diabetic and non-diabetic animals did not differ significantly. Searle Corp.) dissolved in 0.2 ml p e a n u t oil. Blood samples were t a k e n from the orbital sinus [ 11 ] at intervals up to 8 h. 25 ~l of each sample was added immediately to a glass scintillation vial containing 0.2 ml saline using an Eppendorf pipet. One drop of n-octanol was added to prevent foaming and the diluted blood was bleached with 0.2 m130 ~/ohydrogen peroxide. Bleached samples were dissolved in 2 ml Solucne-100| (Packard I n s t r u m e n t Company, Inc.) and counted in 15 ml of Diotol scintillation fluid [ 12 ] in a TriCarb| Model 3375 liquid scintillation spectrometer (Packard I n s t r u m e n t Company, Inc.). Counting efficiency was determined using a [14C]-toluene standard and appropriate blanks and averaged 71%. The small intestine was excised under ether anesthesia, rinsed in ice cold saline and everted over a glass rod. Approximately 5 cm of the proximal jejunum was cut into rings 1 - - 2 m m wide, excluding segments with visible l y m p h nodes [ 13 ]. Rings from individual diabetic animals and non-diabetic controls were pooled and k e p t in Krebs-Ringer bicarbonate buffer p H 7.4, gassed with 95% 02--5 % CO2, at room t e m p e r a t u r e until used (approximately 10 rain). Incubation medium was Krebs-Ringcr bicarbonate buffer, p H 7.4, containing 0.7--0.8 ~Ci [1-14C]-oleic acid (sp act 59.7 mCi) (Amersham/Searle Corp.) per 3 ml. The olcic acid was dissolved in 2 - - 3 drops ethanol, converted to the sodium salt with 1 drop of 0.1 h r N a O H and dissolved in buffer to give the desired final concentration. Five rings of intestine were incubated in 3 ml of medium for 60 min at 37~ under 95% 0 2 - - 5 % COs. Incubation was stopped b y placing the flasks in an ice water bath. Tissue was rinsed in buffer, blotted d r y o n filter paper and weighed. Rings from each flask were homogenized in glass screw cap tissue grinders (Kontes Glass Co.) in 5 ml chloroform-methanol (2:1, v/v) and stood for 1.5--2 h. The tubes were centrifuged and the supernatant was removed and washed with Diabetologia Oleic Acid Incorporation in vitro Triglycerides are resynthesized from absorbed free f a t t y acids and 2-monoglycerides prior to being re2 ml of 0.073% MgCl~. 6 H~O. Washed lower organic phase was evaporated under N~ and the residue was redissolved in chloroform-methanol. One ml was pipetted into scintillation vials, evaporated and dissolved in 15 ml I)iotol. The remainder was evaporated, redissolved in a few drops of chloroform-methanol and chromatographed on pre-coated silica gel TLC plates (Brinkmann Instruments, Inc.) in hexane-ethyl etheracetic acid (90:10:1, v/v]v). Spots were detected by iodine vapour and those corresponding to phospholipids and monoglycerides (origin), diglycerides, cholesterol, f a t t y acids, triglycerides and cholesterol esters were identified by chromatographing known standards on the same plates. Spots were scraped into scintillation vials, shaken with 15 ml Diotol containing 3% water for 1 h to elute lipids from the silica gel and counted. Counting efficiency for both the Diotol and Diotol + 3 % water systems was determined using a [zdC]-toluene standard and averaged 87 and 85%, respectively. Statistical Methods Because of unequal variances in the diabetic and non-diabetic groups, intestinal absorption of glyceryl trioleate and oMe acid was evaluated by nonparametric statistics using the Wilcoxon rank sum test [ 14 ]. Oleic acid incorporation data were evaluated by a oneway analysis of variance using Dunnett's t test [ 15 ]. Results Intestinal Absorption in vivo Radioactivity from administered glyceryl tri-[1ZdC]-oleate appeared in the blood of diabetic hamsters significantly f a s t e r t h a n in non-diabetic controls through the 3 h sampling period (Fig. 1). Peak blood levels occurred between 3 and 4 h in both groups. Total area under the radioactivity-time curve from 0--8 h was approximately 3 times greater in diabetics. Inspection of the total curve areas for each animal showed no correlation between triglyceride absorption and body weight over the weight range of 17.8--31.7 g. Intraluminal hydrolysis of long-chain f a t t y acid triglycerides by pancreatic lipase precedes f a t t y acid absorption [ 16 ]. In order to distinguish at least indirectly between possible enhanced hydrolysis of glyceryl tri-[1AdC]-oleate and enhanced absorption of released [1-14C]-oleie acid, absorption of administered free f a t t y acid was determined. Radioactivity was significantly higher in diabetic animals t h a n in nondiabetics over the entire 8 h sampling period and was maximal at 3 h in both groups (Fig. 2). Total radioactivity was approximately 2.5 times greater in diabetics t h a n in non-diabetics. There was no correlation between absorption and body weight over the weight range of 21.0--38.5 g. 1-J-t = m >Ira I-,. 0 m dM: 0=. en. I 8 I 8 leased into the lymph in chylomicrons [ 16 ]. This intracellular metabolic phase of fat absorption was compared in diabetic and non-diabetic hamsters by determining the incorporation of [1-xaC]-oleic acid into triglycerides and other lipid fractions b y slices of intestine i n vitro. Overall results of three separate experiments indicate no difference in total incorporation per mg wet weight of intestine (Table 1). Incubated and non-incubated intestinal rings were dried in a v a c u u m desiceator over PeOn. W a t e r content of both diabetic and non-diabetic tissue was 85%; hence total incorporation also did not differ significantly on a d r y tissue weight basis. Distribution of radioactivity in the various lipid fractions was equivalent. Discussion Blood specific radioactivity is not greater in the diabetic animals because of haemoconeentration. Sirek and Sirek [ 17 ] reported t h a t haematocrit and total serum protein are identical in non-diabetic and non-ketotic diabetic Chinese hamsters. I also found no significant difference in haematocrits of diabetic and non-diabetic animals used in these studies (diabetics 49  2.8, non-diabetics 46  3.2, m e a n =LS. D.). free oleic acid in diabetic animals indicates t h a t enhanced triolein absorption p r o b a b l y is not solely the result of increased intral~minal hydrolysis, although this m a y contribute to the overM1 process. Biochemical [ 20 ] and morphological [ 21, 22 ] studies have shown t h a t u p t a k e of micellar f a t t y acids from the intestinal lumen is an energy-independent diffusion process. The kinetic characteristics of the entry step have been described as typical of a saturation phenomenon [23], and hence uptake should depend on total absorptive surface area. I n addition Cardell et al. [ 24 ] have proposed t h a t intracellular synthesis and sequest r a t i o n of triglycerides on smooth endoplasmie reticulum maintains the f a t t y acid diffusion gradient and permits continued absorption. This process also would depend on cell mass. Therefore, it is possible t h a t enhanced intestinal f a t absorption in the diabetic Chinese h a m s t e r is a consequence of increased small intestinal mass. Preliminary data indicate diabetic hamster small intestine is larger, expressed as wet weight or percent b o d y weight, t h a n non-diabetic h a m s t e r small intestine. I f Change in blood radioactivity following a test dose of labelled f a t or f a t t y acid is often used b o t h clinically and in experimental animals as an estimate of f a t absorption [ 18 ]. Studies of plasma triglyceride hydrolysis a n d u p t a k e b y liver a n d peripheral tissue have not been done in Chinese hamsters and I assume in interpreting the blood radioactivity curves in the present s t u d y t h a t rate of removal of triglycerides, which should be the p r e d o m i n a n t 14C-labelled chemical species, is similar in diabetic and non-diabetic animals. T h a t this is so is suggested b y similar shapes of the curves for b o t h groups, b u t this would have to be confirmed b y direct m e a s u r e m e n t such as o M c acid clearance after intravenous administration. Hence, differences in p e a k and total radioactivity are a t t r i b u t e d to differences in intestinal f a t hydrolysis or absorption. Since absorption of triolein was enhanced rather t h a n decreased, pancreatic lipase secretion does not appear to be impaired in diabetic hamsters. Exoerine pancreatic function has not been evaluated in Chinese hamsters, although decreased plasma insulin response to feeding [ 9 ] and decreased beta cell mass and b e t a cell degranulation [19] suggest endocrine pancreas dysfunction. ~larkedly increased absorption of administered this is so, although intracellular incorporation of [1-14C]-oleie acid into triglyeerides did not differ per unit of intestinal weight, incorporation would be expected to be greater overall, leading to enhanced chylomicron formation and release. Such an effect of increased gut size on hexose t r a n s p o r t in alloxan- and streptozotoein-induced diabetic rats has been reported b y Schedl and Wilson [ 4 ]. Acknowledgement. I thank Mr. T. I-Ionohan for skillful technical assistance. 1. Laszt , L. , Vogel , H. : Resorption of glucose from the small intestine of alloxan-diabetie rats . Nature (Lend.) 157 , 551 -- 552 ( 1946 ) 9.. Crane, 1 %.K.: An effect of alloxan-diabetes on the active transport of sugars by rat small intestine, in vitro . Biochem biophys. Res. Commun . 4 , 436 -- 440 ( 1961 ) 3. Olsen , W.A. , Rosenberg , I . g . : Intestinal transport of sugars and amino acids in diabetic rats . J. clin. Invest . 49 , 96 -- 105 ( 1970 ) 4. Schedl , H.P. , Wilson, H . D . : Effects of diabetes on intestinal growth and hexose transport in the rat . Amer. J. Physiol . 220 , 1739 -- 1745 ( 1971 ) 5. Lal , D. , Schedl , H . P . : Regulation of intestinal amino acid transport . Gastroenterol . 64 , 758 ( 1973 ) 6. Vinnik , I.E. , Kern , F. , Jr. , Sussman , K . E . : The effect of diabetes mellitus and insulin on glucose absorption by the small intestine in man . J. Lab. clin. Med . 66 , 131 -- 136 ( 1965 ) 7. Schneider , L.E. , Schedl , H. P. : Diabetes and intestinal calcium absorption in the rat . Amer. J. Physiol . 223 , 1319 -- 1323 ( 1972 ) 8. Corsini , G. , Gandolfi , E. , Bonechi , I. , Cerri , B. : F a t absorption in diabetes mellitus . Diabetes 16 , 455 -- 461 ( 1967 ) 9. Gerritsen , G.C. , Dulin , W . E . : Characterization of diabetes in the Chinese hamster . Diabetologia 3 , 74 -- 84 ( 1967 ) 10. Dulin , W.E. , Gerritsen , G.C. : Interaction of genetics and environment on diabetes in the Chinese hamster as compared with h u m a n and other diabetic animal species. I n : Nutrition and Diabetes Mellitus . VI. Capri Conference, Capri, 1972 . (Eds. Froesch, E.R. , Yudkin , J. ) Acta Diabetologia Latina 9 , Suppl . 1 , 48 -- 84 ( 1972 ) 11. Riley , V. : Adaptation of the orbital bleeding technic to rapid serial blood studies . Prec. Soc. exp. Biol . (N.Y.) 104 , 751 -- 754 ( 1960 ) 12. Herberg , R . J . : Determination of carbon-14 and tritium in blood and other whole tissues . Anal. Chem . 32 , 42 -- 46 ( 1960 ) 13. Crane , R . K . , Martdelstam , P. : The active transport of sugars b y various preparations of hamster intestine . Biochim. biophys. Acta (Amst .) 45 , 460 -- 476 ( 1960 ) 14. Steel , R.G.D. , Torrie , J . H . : Principles and procedures of statistics, pp. 402 -- 403 . :New York: McGrawHill Book Co., Inc . 1960 15. Ibid ., pp. 111 -- 112 16. Senior , J . R . : I n t e s t i n a l absorption of fats . J. Lipid Res . 5 , 495 -- 521 ( 1964 ) 17. Sirek , O.V. , Sirek , A. : The colony of Chinese hamsters of the C.H . Best Institute . A review of experimental work . Diabetologia 3 , 65 -- 73 ( 1967 ) 18. Wiseman , G. : Absorption from the intestine , pp. 85 -- 147 . New York: Academic Press, Inc. 1964 19. Carpenter , A.-M. , Gerritsen , G.C. , Dulin , W.E. , Lazarow , A. : Islet and beta cell volumes in diabetic Chinese hamsters and their non-diabetic siblings . Diabetologia 3 , 92 -- 96 ( 1967 ) 20. Johnston , J.M. , BorgstrSm , B. : The intestinal absorption and metabolism of micellar solutions of lipids . Biochim. biophys. Acta (Amst .) 84 , 412 -- 423 ( 1964 ) 21. Strauss , E. , Ire , S. : Autoradiographic and biochemical study of linolenie acid-C1. absorption b y hamster intestine from mixed micelles in vitro . J. Cell Biol . 27 , 101A ( 1965 ) 22. Strauss , E . W . : Electron microscopic study of intestinal fat absorption in vitro, from mixed micelles containing linolenie acid, monoolein, and bile salt . J. Lipid l~es. 7 , 307 -- 323 ( 1966 ) 23. Lyon , I. : Studies on transmural potentials in vitro in relation to intestinal absorption V. Kinetic characteristics of lipid interactions with rat gut . Biochim. biophys. Acta (Amst .) 163 , 75 -- 84 ( 1968 ) 24. Cardell , R . R . , Jr., Badenhausen , S. , Porter , K . R . : Intestinal triglyceride absorption in the rat . An electron microscopical study . J. Cell Biol . 34 , 123 -- 155 ( 1967 )

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T. M. Parkinson. Enhanced intestinal fat absorption in diabetic chinese hamsters, Diabetologia, 1973, 505-508, DOI: 10.1007/BF00461697