The effect of volatile fatty acids on plasma glucose concentration

Comp. Biochera. Physiol., 1966, Vol. 18, pp. 527 to 536. Pergamon Press Ltd. Printed in Great Britain

THE EFFECT OF VOLATILE FATTY ACIDS ON PLASMA GLUCOSE CONCENTRATION* R. W. P H I L L I P S t and A. L. BLACK Department of Physiological Sciences, School of Veterinary Medicine, University of California, Davis, California (Received 7 ffanuary 1966) A b s t r a c t - - 1 . Butyrate causes a marked increase in plasma glucose in lambs and

a remission of hypoglycemic convulsions. 2. Propionate causes a similar remission but a considerably smaller plasma glucose increase. 3. Neither of these compounds materially affects plasma glucose levels in other species. 4. Acetate and/g-hydroxybutyrate had no effect on plasma glucose concentration in any species. 5. Lamb liver perfusion results were variable as regards a direct action of butyrate on liver glycogen. INTRODUCTION VERY little ingested carbohydrate is absorbed as such in ruminants (Schambye, 1951) since the rumen microflora readily attack these materials and metabolize them to smaller compounds. The volatile fatty acids, acetic, propionic, and butyric acids, are considered to be major products of this rumen fermentation and to be a major energy source in ruminant metabolism (Hungate et al., 1961). In spite of the lack of alimentary carbohydrate, the ruminants have a plasma glucose concentration (60-80 mg per cent) only slightly below other mammals and a relatively high rate of glucose utilization (Annison & White, 1961 ; Jarrett, Jones & Potter, 1964). This is accomplished by an active process of gluconeogenesis from absorbed nutrients, of which only propionate, among the volatile fatty acids, has been shown to provide a significant net source of glucose (Pennington, 1952, 1954; Annison et al., 1963 ; Black et al., 1961 ; Leng & Annison, 1963). Several workers have shown that intravenous infusion of proprionate and butyrate may increase the plasma glucose concentration (Reid, 1951; Potter, 1952; Kronfeld, 1957; Ash et al., 1964). The exact mechanism by which butyrate causes a glucose increase remains * This study was supported in part by a grant from the National Institutes of Health RGO8183. The senior author (R. W. Phillips) was a trainee on PHS training grant 2G-633-PATC-3. t Current address : Department of Physiology, Colorado State University, Fort Collins, Colorado. 527

528

R.W. PHILLIPSANDA. L. BLACK

obscure, but it is due, at least in part, to an activation of liver phosphorylase and subsequent breakdown of glycogen (Phillips et al., 1965). MATERIALS AND METHODS Experimental animals were mixed whiteface lambs and Holstein calves from 5 to 90 days old. The dogs and the horse were mature animals of mixed breeding. All animals had sterile polyethylene catheters inserted into the jugular vein to allow frequent blood sampling with minimum disturbance for the animals. Catheter ends were fitted with a Tuohy-Borst adapter, a two-way stopcock, and the entire assemblage was anchored with a simple subcutaneous suture. The patency of the catheter was maintained by injection of heparinized (12 units/cm ~) sterile saline into the catheter after removing each blood sample. T h e volatile fatty acids and fl-hydroxybutyrate, a ketone body, were administered intravenously as a 2-5 N solution, neutralized to pH 7.2 with NaOH; 2.5 mmoles/kg body wt. were given at each injection. Blood samples were taken at frequent intervals after the injection of the volatile fatty acids, immediately placed in heparinized, fluorinized tubes, chilled and centrifuged. T h e plasma was removed and frozen for subsequent analysis. Plasma glucose concentration was measured by a glucose oxidase method (Campbell & Kronfeld, 1961) after precipitation of the proteins with stable tungstic acid (Hycel Corporation). Ketone body concentration was measured in the distillate from the plasma protein-free filtrate using a method described by Reid (1960). Liver phosphorylase and glycogen were assayed by the methods of Illingworth & Cori (1953) and Hansen et al. (1952), respectively. Insulin (Regular Illetin, Ely Lilly Co.) was given at the rate of 4 units/kg/hr until convulsions appeared at which time one of the volatile fatty acids or fl-hydroxybutyric was administered through the jugular catheter. The liver perfusion apparatus used to study in vitro responses to metabolites has been described in detail elsewhere (Phillips, 1965). RESULTS In the initial experiments the animals were made hypoglycemic with insulin before administration of the volatile fatty acids. This was done in part to confirm earlier work (Potter, 1952) and in part to test the hypothesis that butyrate is hyperglycemic only in conditions of hypoglycemia (Kronfeld, 1956). The results with lamb 73, shown in Fig. 1, essentially duplicate Potter's experiment in which butyrate was injected intravenously followed by propionate and a second butyrate injection. It can be seen that the injection of butyrate caused an increase in plasma glucose concentration, and that this effect was greater with butyrate than it was with an equimolar amount of propionate. An even greater response was obtained in a second lamb (No. 27), and again the effect of butyrate exceeded that of propionate. In the third lamb (No. 70), the sequence was reversed, injecting first propionate followed by butyrate and, finally, acetate. The response to the first propionate injection was relatively great, about a twofold increase in plasma glucose, but the subsequent response to butyrate was at least as great, if not greater.

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There was very little effect from the second injection of propionate and no detectable response to acetate injection. To test the durability of the response to butyrate, two lambs were injected repeatedly for a period of more than 3 hr. The results, summarized in Fig. 2, show that the response diminished with time, the plasma glucose increase being only one-fifth as great after the fourth successive butyrate injection as it was after the first injection (lamb 46). In lamb 47 (Fig. 2) the plasma ketone body concentration was also measured and found to increase three- to fivefold immediately after each injection of butyrate. LAMB 73 B =' eUTYRATE P = PROPIONATE A = ACETATE -.~ • I N I T I A L PLASMA GLUCOSE CONC.

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Since it had been reported in earlier work (Kronfeld, 1957) that injection of 3-hydroxybutyrate was followed by increased blood glucose levels in sheep, two lambs were injected with this ketone body and the response compared to that evoked by an equimolar amount of butyrate. As shown in Fig. 3, injection of 3-hydroxybutyrate had no effect in either lamb, although injection of butyrate before or after the fl-hydroxybutyrate caused a marked increase in plasma glucose

530

R. W. PHILLIPS AND A. L. BLACK

concentration. These results indicate that the effect of butyrate on plasma glucose concentration is not mediated by the fl-hydroxybutyrate formed in butyrate metabolism.

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The discrepancy between the present results and those reported earlier (Kronfeld, 1957) may have been due to differences in experimental procedure. The lambs used in the present study had venous catheters implanted at least 24 hr prior to the experimental period. This technique allows frequent and rapid blood sampling or injection of metabolites with minimum disturbance to the animal. In contrast, sampling of blood by hypodermic puncture of the vessels would tend to excite the animals and it would be impossible to distinguish a hyperglycemic response to an injected metabolite from that caused indirectly via the sympathetic nervous system, due to animal excitement. In all of the preceding experiments the lambs were held until they were in severe insulin-induced hypoglycemia, either stage 3 or 4 of Reid (1951), before the volatile fatty acidswere injected. Invariably, the injection of either propionate or butyrate led to rapid recovery and within 10-15 min the lambs appeared

531

VOLATILE FATTY ACIDS AND PLASMA GLUCOSE

completely normal (see Figs. 4-7); however, they would lapse back into the hypoglycemic convulsions 45-60 min post-injection. The return to stage 3-4 hypoglycemia was quite rapid, occurring in less than 10 min from the first indication of depression. The relapses were caused by the continuing effect of the insulin originally injected to initiate the hypoglycemia. When the experiment was terminated, the lambs were injected with glucose (2.5 mM/kg) and given food. They were then able to maintain plasma glucose levels without recurrence of convulsions. LAMB 120

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Three young canes were used in parallel experiments to measure the response to butyrate in another ruminant species. The resuks, summarized in Fig. 8, show that in only one of the calves was there a definite increase in plasma glucose concentration and this was modest, about 25 mg per cent compared to the response seen in lambs, 76 mg per cent increase, on the average, following the first butyrate injection. Even though the injected volatile fatty acids in calves did not result in dramatic changes in plasma glucose concentration as seen in lambs, butyrate and propionate did cause the same marked remission of hypoglycemic convulsions. In all three calves, only 10-15 rain elapsed following butyrate or propionate

532

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injections until the animals were able to stand and walk about and appeared completely normal; as with the lambs, the calves relapsed into stage 3 or 4 of hypoglycemia (Reid, 1951) in 45-60 min and it was necessary to inject intravenously with glucose and offer feed to interrupt the recurrent convulsions. Intravenous injection of acetate had no effect on the hypoglycemic convulsions nor the plasma glucose (Fig. 8) in the calves.

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FIG. 8. Plasma glucose concentration in fasted, insulin-induced hypoglycemic calves after the intravenous infusion of 2"5 mM/kg of the various volatile fatty acids. The symbols on the graphs, B (butyrate), P (propionate) and A (acetate), indicate the time of fatty acid injection. In two dogs and a horse no remission of hypoglycemic depression and very little alteration of the plasma glucose concentration was seen with any of the volatile fatty acids (Table 1). In an effort to determine what effect the plasma glucose concentration had on the glucogenicity of butyric acid, two 2-week-old nursing lambs were injected with butyrate or acetate ½ hr after the morning nursing when the initial plasma glucose was over 100 mg per cent. The response (see Fig. 9) was essentially the same as seen in the fasted insulin hypoglycemic lambs, butyrate causing an increase in plasma glucose of 60-80 mg per cent while acetate was without effect. These results do not support Kronfeld's hypothesis (1956) that butyrate causes an increase

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FIGS. 4-7. Lamb in hypoglycemic convulsions ; butyrate 2.5 mM/kg administered intravenously at time 0. Lamb had responded and was able to stand in 13 min (Fig. 5) and was completely free of hypoglycemic symptoms in 23 min (Fig. 6). Convulsions reoccurred 54 min post butyrate injection (Fig. 7).

533

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of blood glucose when the concentration of the latter is low and a decrease when the concentration is high. In the present study the effect of butyrate was to increase plasma glucose when it was initially either low (Figs. 1-3) or high (Fig. 9). T A B L E 1 - - E F F E C T OF I N T R A V ~ O U S INJECTION OF VOLATILE FATTY ACIDS ON PLASMA GLUCOSE CONCENTRATION IN NON*RUMINANT ANIMALS

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It has been shown that the injection of butyric acid can cause an activation of liver phosphorylase in the intact and adrenalectomized animal (Phillips et al., 1965). This response was not observed in studies with liver slices (Ash et al., 1964).

534

R. W. PHILLIPSANDA. L. BLACK

In an effort to clarify the relationship between plasma butyrate concentration and liver response three in situ liver perfusions were performed on lambs. The plasma glucose concentration, liver glycogen concentration and liver phosphorylase activity were measured at various times during the perfusion. The results, shown in Fig. 10, were variable presumably due to experimental conditions, sampling PERFUSION~1 •*qp

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FIG. 10. Plasma glucose concentration, liver glycogen concentration and liver phosphorylase activity during in situ liver perfusions. problems and bleeding from sample sites. In addition the glucose concentration was increasing rather rapidly in the perfusing medium so that it was difficult to establish an effect of butyrate, under these in vitro conditions, on the liver. It appears that butyrate may have caused an increase of phosphrylase in the first infusion but there is no definite evidence of a similar effect in the next two perfusions. In the third perfusion the liver appeared to respond to epinephrine but showed little or no response to butyrate injection. DISCUSSION

The volatile fatty acids, propionate and butyrate, markedly increased plasma glucose concentration in lambs when injected intravenously in hyperphysiological amounts and, simultaneously, the compounds relieved hypoglycemic convulsion.

VOLATILE FATTY ACIDS AND PLASMA GLUCOSE

535

It was also possible to relieve such convulsions in calves by volatile fatty acid injection but, in these ruminants, there was no concomitant increase in plasma glucose. This lack of effect of plasma glucose concentration in calves may indicate that the central nervous tissue can utilize butyrate as an energy source, a possibility supported by the fact that cerebral respiratory quotients of less than one have been measured in some ruminants (Davis et al., 1964). On the other hand, butyrate may be utilized by muscle and thereby have a sparing effect on glucose or glucose precursors which provide substrate for the brain. Whatever the response to butyrate it seems restricted to ruminants as it was not evident in limited trials using either the horse, another herbivore, or the dog, a carnivore. The fact that neither of these species is normally metabolically dependent on volatile fatty acids may account for the lack of response. It is also apparent that the increase in plasma glucose concentration following injection of butyrate does not depend on existing glucose concentration, as had been suggested (Kronfeld, 1956), but can occur in sheep with normal blood sugar levels (Ash et al., 1964) or in lambs under conditions of relative hyperglycemia, as shown in Fig. 9. The hyperglycemic response to butyrate in the intact lamb involves, at least in part, release of glucose from the liver. In the normal or adrenalectomized lambs, injection of butyrate was followed by increased levels of active phosphorylase, decreased levels of liver glycogen and hyperglycemia (Phillips et al., 1965). The response in adrenalectomized lambs eliminated epinephrine as a factor in the increased plasma glucose concentration but the possibility remained that some other factor, such as glucagon, may mediate the changes seen after butyrate injection. An effort was made to eliminate glucagon as a factor by perfusing lamb livers in situ. The results from these perfusions were too variable to permit valid conclusions so the possibility remains that butyrate may have an indirect effect on liver glycogenolysis mediated by glucagon (Lukens, 1959). It is also possible that glycogenolysis is so sensitive to environmental conditions that anoxia (Morgan & Parmeggiani, 1964), which is difficult to avoid in perfused organs, or some other condition may have effectively masked the response to butyrate during the perfusion. The possibility of a direct conversion of butyrate to glucose (Kronfeld, 1957) seems unlikely in view of the decreasing magnitude of the response with repeated injections. Studies in ruminants with C14-butyrate have also indicated that pathways for direct conversion of butyrate to glucose do not occur in the intact cow or sheep (Black et al., 1961 ; Annison et al., 1963). REFERENCES ANNISON E. F., LENG R. A., LINDSAYD. B. & WHITE R. R. (1963) The metabolism of acetic acid, propionic acid and butyric acid in sheep. Biochem. ft. 88, 248-252. ANNISONE. F. & WHITER. R. (1961) Glucose utilization in sheep. Biochem.ff. 80, 162-169. ASH R. W., PENNINGTONR. J. & REID R. S. (1964) The effect of short-chain fatty acids on blood glucose concentration in sheep. Biochem. ft. 90, 353-360. BLACKA. L., KLEIBERM. & BROWNALICE M. (1961) Butyrate metabolism in the lactating cow. ft. biol. Chem. 236, 2399-2403.

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R. W. PHILLIPS AND A, L. BLACK

CAMPBELL L. A. ~ KRONFELD D. S. (1961) Estimation of low concentrations of plasma glucose using oxidase. Am, J. Vet. Res. 22, 587-589. DAVIS L. E., WESTFALLB. A. ~ DALE H. E. (1964) Central blood flow in the anesthetized goat: Method of measurement and normal values. Am. J. Vet. Res. 25, 1159-1165. HANSEN R. G., RUTTERW. J. ~ CRAINEE. M. (1952) A nephelometric method for the determination of glycogen. J. biol. Chem. 195, 127-132. HUNGATE R. E., MAH R. A. & SIMESENM. (1961) Rates of production of individual volatile fatty acids in the rumen of lactating cows. Appl. Microbiol. 9, 554-561. ILLINGWORTHn. ~; CORI G. T. (1953) Crystalline muscle phosphorylase. In Biochemical Preparations (Edited by SNELL E. E.), Vol. 3, pp. 4-7. Wiley, New York. JARRETT I. G., JONES G. B. & POTTER B. J. (1964) Changes in glucose utilization during development of the lamb. Biochem. J. 90, 189-194. KRONFELDD. S. (1956) Effect of butyrate administration on btood glucose in sheep. Nature, Lond. 178, 1290-1291. KRONFELD D. S. (1957) The effects on blood sugar and ketone bodies of butyrate, acetate and fl-OH butyrate infused into sheep. Aust. J. exp. Biol. reed. Sci. 35, 257-266. LENG R. A. & ANNISON E. F. (1936) Metabolism of acetate, propionate and butyrate by sheep-liver slices. Biochem. J. 86, 319-327. LUKENS F. D. W. (1959) The pancreas; insulin and glucagon. A. Rev. Physiol. 21, 445-474. MORGAN H. E. & PARMEGGIANIA. (1964) Control of Glycogen Metabolism. Ciba Foundation Symposium, pp. 256-258. PENNINGTON R. J. (1952) The metabolism of short-chain fatty acids in the sheep--I. Fatty acid utilization and ketone body production by tureen epithelium and other tissues. Biochem. J. 51, 251-258. PENNINGTON R. J. (1954) The metabolism of short-chain fatty acids in sheep. Biochem. J. 56, 410-416. PHILLIPS R. W. (1965) The nature of the glucogenic effect of butyrate. Ph.D. Thesis, University of California. PHILLIPS R. W., BLACKA. L. & MOLLER F. (1965) Butyrate induced glycogenolysis in hypoglycemic lambs. Life Sci. 4, 521-525. POTTER B. J. (1952) Relief of hypoglycemic convulsions with butyric acid. Nature, Lend. 170, 541. REID R. L. (1951) Studies on the carbohydrate metabolism of sheep--IV. Hypoglycaemic signs and their relationship to blood glucose. Aust. J. agric. Res. 2, 146-157. REID R. L. (1960) The determination of ketone bodies in blood. Analyst, Lond. 85, 265-271. SCHAMBYEP. (1951) Volatile acids and glucose in portal blood of sheep--III. The influence of orally administered glucose. Nord. VetMed. 3, 1003-1014.