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Effect of capsaicin on intestinal glucose metabolism in vitro
Toxicology Letters, 3 (1979) 279~-283 o ~l~evier~No~~h-Holl~~d Biomedical Press
EFFECT OF CAPSAICIN IN VITRO
ON INTESTINAL
279
GLUCOSE
METAROLISM
SUMMARY
The role of capsaicin upon intestinal glucose utilization and lactic acid production was studied in everted sacs of rat and hamster jejunum, In bath the rat and the hamster, glucose transport was respectively inhibited by 45.8 and 38.7% when 14.0 mg% caps&+ was incubate at pH 7.4 for 60 min. However, when intestinal glucose metabolism was diminished by reducing the pH of the incubation medium from 7.4 to 5.0, the inhibitory effect of capsaicin was significantly reduced. Direct determination of the extent of the intestinal glucose metabolism as affected by capsaicin revealed a significantly (P < 0.005) greater glucose utilization (23.0% in the rat and 24.9% in the hamster) and lactic acid formation (13.8% in the rat and 88.4% in the hamster). These results indicate that the depression of intestinal glucose transport by capsaicin may in part be due to a greater metabolic breakdown of glucose into lactic acid.
INTRODUCTION
Capsaicin, the pungent principle in red pepper, has been shown to cause a dose-related inhibition of intestinal glucose transport in both the rat and the hamster [4]. However, Krebs-Henseleit-bicarbonate buffer (pH 7.4) was used as the incubation medium: with this, part of the absorbed glucose is metabolized by the intestine with accumulation of lactic acid in the serosal fluid of everted intestinal sac [6, 8, 10, 111. Accordingly, it is possible that the observed inhibition of glucose transport by capsaicin may be due partly to increased utilization of glucose by the gut wall, resulting in a reduction of glucose transported into the serosal medium. It has been shown that the pH optimum of hexokinase, the rate-limiting enzyme of intestinal glucose metabolism 171, ranges from 7.5-8.0 [3]. At the lower pH, intestinal glucose metabolism should be small. The objectives
280
of the present study, therefore, were: (1) To investigate whether, in the presence of capsaicin, there is increased glucose transport from the mucosal to the serosal side of everted rat and hamster small intestine, resulting in a greater concentration gradient when intestinal metabolism is reduced by lowering the pH of the incubation medium to pH 5.0; also to compare the degree of inhibition of glucose transport by capsaicin at pH 5.0 and at pH 7.4. (2) To assess directly the metabolism of intestine as affected by capsaicin by determining the amount of glucose utilized and lactic acid produced. MATERIALS
AND METHODS
The everted sacs [9] of the upper jejunum of both the rat and the hamster were employed for the transport studies [4, 91. The sacs were filled with 0.5 ml incubation medium (serosal fluid). The length of each filled sac was about 3 cm. Each preparation was incubated at 37°C for 60 min in lo-ml incubation medium (mucosal fluid) with or without 14.0 mg% capsaicin (Sigma). The intestinal sacs were continuously aerated with 5% CO2 + 95% OZ. At the beginning and end of incubation, the glucose and lactic acid concentration of pooled mucosa, serosa, and whole gut were determined. The difference between the initial and final amount of glucose and lactic acid in the whole system was calculated as the amount of glucose utilized and lactic acid produced by the gut tissue. As incubation medium, Krebs-Henseleit bicarbonate buffer at pH 7,4 or 5.0 was used. The pH of the buffer was brought to the desired level by addition of hydrochloric acid. D-Glucose (both mucosal and serosal solution) was employed as transport substrate. Glucose was determined by glucose oxidase method employing the test-kit of Worthington Chemical and lactic acid by the method of Barker and Summerson [I]. Parameters for active intestinal glucose transport were concentration differences of the substrates between the serosal and mucosal fluid of the everted sacs and serosal transfer of substrate against a concentration gradient (mg glucose and lactic acid per g wet weight tissue), RESULTS
AND DISCUSSION
Intestinal glucose transport of rat and hamster is shown in Fig. 1. It is evident that the everted sacs of both rat and hamster jejunum transported glucose against a steeper concentration gradient if the pH of the incubation medium were reduced from pH 7.4 to pH 5.0. The presence of 14.0 mg% capsaicin incubated for 60 min with normal intestinal metabolism (pH 7.4) caused a 458 and 38.7% reduction in glucose transport for the rat and the hamster, respectively. However, with depressed intestinal metabolism (pH LO), the inhibitory effect of capsaicin is demonstrated to be only 16.0% in the rat and 17.1% in the hamster. Because glucose is metabolized to a considerable extent by the small
281 HAMSTER RAl
pH5.0
Fig. 1. Transport of glucose by everted sacs of upper jejunum of rat and hamster as affected by pH and capsaicin. The black columns represent the amount of substrate being transported against a concentration gradient from the mucosal (M) to serosal (S) side of the intestinal wall. Initial glucose concentration = 135.0 mg%. The values are expressed as means r S.E.M. from at least 10 preparations.
intestine, subsequent experiments were conducted to determine whether the effect of pH on intestinal glucose transport (Fig. 1) is due to an influence of pH on intestinal glucose metabolism. The data summarized in Table I show that incubation of jejunal sacs with capsaicin at pH 7.4 resulted in an accumulation of a considerably greater amount of lactic acid (13.8% in the rat and 88.4% in the hamster) and increased utilization of glucose (23.0% in the rat and 24.9% in the hamster) by gut tissue. In contrast, a significantly smaller amount of
TABLE I EFFECT OF CAPSAICIN ON GLUCOSE AND HAMSTER INTESTINE
UTILIZATION
AND LACTIC ACID PRODUCTION
The everted jejunum was incubated in 135.0 mg% glucose in KrebsHenseleit-HCOJ with or without 14.0 mg% capsaicin at 37OC for 60 min. Animals
Capsaicin cont. (mg%)
IN RAT
buffer. PH 7.4
Amount of substance (me/g tissue wet wt) Initial glucose before incubation
Rat
0 14.0
33.20 36.10
Hamster
0 14.0
100.93 91.51
? f
3.26 3.10
f 13.21 k 10.60
Remaining glucose after incubation
Glucose utilized
Lactic acid produced (corrected)a
22.25 k 1.73 22.63 + 2.02
10.95 f 0.51b 13.47 ? 0.42
10.91 12.41
97.20 86.85
r 7.15 r 5.10
3.73 + 0.32b 4.66 f 0.22
iThe values were corrected for the lactic acid produced in the absence of glucose. Significantly different from control, P < 0.005.
* 0.91b k 0.96
1.72 + 0.06 3.24 k 0.05b
282
cl
glucose
utilization
RAT
lactic
acid
formation
HAMSTER
in
.
conrrol
I
:apsotcln
Fig. 2. Lactic acid production and glucose utilization in rat and hamster incubated at 37°C (pH 5.0) in 135.0 mg% glucose-Krebs-Henseleit-HCO; without 14.0 mg% capsaicin for 60 min.
everted jejunum, buffer with or
glucose was being utilized and little lactic acid was being produced by jejunum incubated at pH 5.0. Fig. 2 shows that, when the pH of the incubation medium was lowered to 5.0, the amount of glucose utilized was decreased by 68.0% for the rat and 25.4% for the hamster. Similarly, lactic acid produced in these conditions was diminished by 70.3% for the rat and 23.3% for the hamster. In the presence of capsaicin, the amounts of glucose utilized and lactic acid produced were not significantly different from those of controls. Therefore, lowering the pH of the incubation medium can significantly diminish the intestinal metabolism which is otherwise stimulated by capsaicin. Accordingly, the stimulating effect of a low pH of the incubation medium upon serosal glucose transfer appears to be attributable to a reduction of glucose metabolism. The reason for an increase in lactic acid production in capsaicin-treated gut tissue is not clearly understood. It may be related to deficiency in energy balance produced by perturbation of oxidative metabolism [2]. It is thus possible that the mucosal cells may attempt to compensate for the depletion in adenine nucleotide production [5] by enhancing aerobic glucose breakdown, with consequent increase in lactic acid formation.
283 ACKNOWLEDGEMENT
The author for secretarial
would like to extend his appreciation assistance.
to Miss Wilai Limpasuk
REFERENCES 1 S.B. Barker and W.H. Summerson, The calorimetric determination of lactic acid in biological material, J. Biol Chem., 138 (1941) 535-554. 2 P. Chudapongse and W. Janthasoot, Studies on the effect of capsaicin on metabolic reactions of isolated rat liver mitochondria, Toxicol. Appl. Pharmacol., 37 (1976) 263-270. 3 C. Long, Studies involving enzymic phosphorylation, I. The hexokinase activity of rat tissue, Biochem. J., 52 (1952) 407-415. 4 Y. Monsereenusorn and T. Glinsukon. Inhibitory effect of capsaicin on intestinal glucose absorption in vitro, Food Cosmet. Toxicol., 16 (1978) 469-473. 5 Y. Monsereenusorn and T. Glinsukon, Possible mode of inhibitory action of capsaicin on intestinal glucose absorption in vitro, Presented at 7th Physiological Society Meeting, Songkla, Thailand, 1978, Abstr. No. 7, p. 12. 6 H. Newey, D.H. Smyth and B.C. Whaler, The absorption of glucose by the in vitro intestinal preparation, J. Physiol., 129 (1955) l-11. 7 K.W. Wahle, D.G. Armstrong and H.S.A. Sherratt, The metabolism of the small intestine: glycolysis in the mucosa of the small intestine of the sheep, Comp. Biochem. Physiol., 38 B (1971) 5-20. 8 T.H. Wilson, The role of lactic acid production in glucose absorption from the intestine, J. Biol. Chem., 222 (1956) 751-763. 9 T.H. Wilson and G. Wiseman, The use of sacs of everted small intestine for the study of the transference of substances from the mucosal to the serosal surface, J. Physiol. 123 (1954) 116-125. 10 T.H. Wilson, Concentration gradients of lactate, hydrogen, and some other ions across the intestine in vitro, Biochem. J., 56 (1954) 521-527. 11 T.H. Wilson and G. Wiseman, Metabolic activity of the small intestine of the rat and golden hamster (Mesocricetus auratus). J. Physiol., 123 (1954) 126-130.
EFFECT OF CAPSAICIN IN VITRO
ON INTESTINAL
279
GLUCOSE
METAROLISM
SUMMARY
The role of capsaicin upon intestinal glucose utilization and lactic acid production was studied in everted sacs of rat and hamster jejunum, In bath the rat and the hamster, glucose transport was respectively inhibited by 45.8 and 38.7% when 14.0 mg% caps&+ was incubate at pH 7.4 for 60 min. However, when intestinal glucose metabolism was diminished by reducing the pH of the incubation medium from 7.4 to 5.0, the inhibitory effect of capsaicin was significantly reduced. Direct determination of the extent of the intestinal glucose metabolism as affected by capsaicin revealed a significantly (P < 0.005) greater glucose utilization (23.0% in the rat and 24.9% in the hamster) and lactic acid formation (13.8% in the rat and 88.4% in the hamster). These results indicate that the depression of intestinal glucose transport by capsaicin may in part be due to a greater metabolic breakdown of glucose into lactic acid.
INTRODUCTION
Capsaicin, the pungent principle in red pepper, has been shown to cause a dose-related inhibition of intestinal glucose transport in both the rat and the hamster [4]. However, Krebs-Henseleit-bicarbonate buffer (pH 7.4) was used as the incubation medium: with this, part of the absorbed glucose is metabolized by the intestine with accumulation of lactic acid in the serosal fluid of everted intestinal sac [6, 8, 10, 111. Accordingly, it is possible that the observed inhibition of glucose transport by capsaicin may be due partly to increased utilization of glucose by the gut wall, resulting in a reduction of glucose transported into the serosal medium. It has been shown that the pH optimum of hexokinase, the rate-limiting enzyme of intestinal glucose metabolism 171, ranges from 7.5-8.0 [3]. At the lower pH, intestinal glucose metabolism should be small. The objectives
280
of the present study, therefore, were: (1) To investigate whether, in the presence of capsaicin, there is increased glucose transport from the mucosal to the serosal side of everted rat and hamster small intestine, resulting in a greater concentration gradient when intestinal metabolism is reduced by lowering the pH of the incubation medium to pH 5.0; also to compare the degree of inhibition of glucose transport by capsaicin at pH 5.0 and at pH 7.4. (2) To assess directly the metabolism of intestine as affected by capsaicin by determining the amount of glucose utilized and lactic acid produced. MATERIALS
AND METHODS
The everted sacs [9] of the upper jejunum of both the rat and the hamster were employed for the transport studies [4, 91. The sacs were filled with 0.5 ml incubation medium (serosal fluid). The length of each filled sac was about 3 cm. Each preparation was incubated at 37°C for 60 min in lo-ml incubation medium (mucosal fluid) with or without 14.0 mg% capsaicin (Sigma). The intestinal sacs were continuously aerated with 5% CO2 + 95% OZ. At the beginning and end of incubation, the glucose and lactic acid concentration of pooled mucosa, serosa, and whole gut were determined. The difference between the initial and final amount of glucose and lactic acid in the whole system was calculated as the amount of glucose utilized and lactic acid produced by the gut tissue. As incubation medium, Krebs-Henseleit bicarbonate buffer at pH 7,4 or 5.0 was used. The pH of the buffer was brought to the desired level by addition of hydrochloric acid. D-Glucose (both mucosal and serosal solution) was employed as transport substrate. Glucose was determined by glucose oxidase method employing the test-kit of Worthington Chemical and lactic acid by the method of Barker and Summerson [I]. Parameters for active intestinal glucose transport were concentration differences of the substrates between the serosal and mucosal fluid of the everted sacs and serosal transfer of substrate against a concentration gradient (mg glucose and lactic acid per g wet weight tissue), RESULTS
AND DISCUSSION
Intestinal glucose transport of rat and hamster is shown in Fig. 1. It is evident that the everted sacs of both rat and hamster jejunum transported glucose against a steeper concentration gradient if the pH of the incubation medium were reduced from pH 7.4 to pH 5.0. The presence of 14.0 mg% capsaicin incubated for 60 min with normal intestinal metabolism (pH 7.4) caused a 458 and 38.7% reduction in glucose transport for the rat and the hamster, respectively. However, with depressed intestinal metabolism (pH LO), the inhibitory effect of capsaicin is demonstrated to be only 16.0% in the rat and 17.1% in the hamster. Because glucose is metabolized to a considerable extent by the small
281 HAMSTER RAl
pH5.0
Fig. 1. Transport of glucose by everted sacs of upper jejunum of rat and hamster as affected by pH and capsaicin. The black columns represent the amount of substrate being transported against a concentration gradient from the mucosal (M) to serosal (S) side of the intestinal wall. Initial glucose concentration = 135.0 mg%. The values are expressed as means r S.E.M. from at least 10 preparations.
intestine, subsequent experiments were conducted to determine whether the effect of pH on intestinal glucose transport (Fig. 1) is due to an influence of pH on intestinal glucose metabolism. The data summarized in Table I show that incubation of jejunal sacs with capsaicin at pH 7.4 resulted in an accumulation of a considerably greater amount of lactic acid (13.8% in the rat and 88.4% in the hamster) and increased utilization of glucose (23.0% in the rat and 24.9% in the hamster) by gut tissue. In contrast, a significantly smaller amount of
TABLE I EFFECT OF CAPSAICIN ON GLUCOSE AND HAMSTER INTESTINE
UTILIZATION
AND LACTIC ACID PRODUCTION
The everted jejunum was incubated in 135.0 mg% glucose in KrebsHenseleit-HCOJ with or without 14.0 mg% capsaicin at 37OC for 60 min. Animals
Capsaicin cont. (mg%)
IN RAT
buffer. PH 7.4
Amount of substance (me/g tissue wet wt) Initial glucose before incubation
Rat
0 14.0
33.20 36.10
Hamster
0 14.0
100.93 91.51
? f
3.26 3.10
f 13.21 k 10.60
Remaining glucose after incubation
Glucose utilized
Lactic acid produced (corrected)a
22.25 k 1.73 22.63 + 2.02
10.95 f 0.51b 13.47 ? 0.42
10.91 12.41
97.20 86.85
r 7.15 r 5.10
3.73 + 0.32b 4.66 f 0.22
iThe values were corrected for the lactic acid produced in the absence of glucose. Significantly different from control, P < 0.005.
* 0.91b k 0.96
1.72 + 0.06 3.24 k 0.05b
282
cl
glucose
utilization
RAT
lactic
acid
formation
HAMSTER
in
.
conrrol
I
:apsotcln
Fig. 2. Lactic acid production and glucose utilization in rat and hamster incubated at 37°C (pH 5.0) in 135.0 mg% glucose-Krebs-Henseleit-HCO; without 14.0 mg% capsaicin for 60 min.
everted jejunum, buffer with or
glucose was being utilized and little lactic acid was being produced by jejunum incubated at pH 5.0. Fig. 2 shows that, when the pH of the incubation medium was lowered to 5.0, the amount of glucose utilized was decreased by 68.0% for the rat and 25.4% for the hamster. Similarly, lactic acid produced in these conditions was diminished by 70.3% for the rat and 23.3% for the hamster. In the presence of capsaicin, the amounts of glucose utilized and lactic acid produced were not significantly different from those of controls. Therefore, lowering the pH of the incubation medium can significantly diminish the intestinal metabolism which is otherwise stimulated by capsaicin. Accordingly, the stimulating effect of a low pH of the incubation medium upon serosal glucose transfer appears to be attributable to a reduction of glucose metabolism. The reason for an increase in lactic acid production in capsaicin-treated gut tissue is not clearly understood. It may be related to deficiency in energy balance produced by perturbation of oxidative metabolism [2]. It is thus possible that the mucosal cells may attempt to compensate for the depletion in adenine nucleotide production [5] by enhancing aerobic glucose breakdown, with consequent increase in lactic acid formation.
283 ACKNOWLEDGEMENT
The author for secretarial
would like to extend his appreciation assistance.
to Miss Wilai Limpasuk
REFERENCES 1 S.B. Barker and W.H. Summerson, The calorimetric determination of lactic acid in biological material, J. Biol Chem., 138 (1941) 535-554. 2 P. Chudapongse and W. Janthasoot, Studies on the effect of capsaicin on metabolic reactions of isolated rat liver mitochondria, Toxicol. Appl. Pharmacol., 37 (1976) 263-270. 3 C. Long, Studies involving enzymic phosphorylation, I. The hexokinase activity of rat tissue, Biochem. J., 52 (1952) 407-415. 4 Y. Monsereenusorn and T. Glinsukon. Inhibitory effect of capsaicin on intestinal glucose absorption in vitro, Food Cosmet. Toxicol., 16 (1978) 469-473. 5 Y. Monsereenusorn and T. Glinsukon, Possible mode of inhibitory action of capsaicin on intestinal glucose absorption in vitro, Presented at 7th Physiological Society Meeting, Songkla, Thailand, 1978, Abstr. No. 7, p. 12. 6 H. Newey, D.H. Smyth and B.C. Whaler, The absorption of glucose by the in vitro intestinal preparation, J. Physiol., 129 (1955) l-11. 7 K.W. Wahle, D.G. Armstrong and H.S.A. Sherratt, The metabolism of the small intestine: glycolysis in the mucosa of the small intestine of the sheep, Comp. Biochem. Physiol., 38 B (1971) 5-20. 8 T.H. Wilson, The role of lactic acid production in glucose absorption from the intestine, J. Biol. Chem., 222 (1956) 751-763. 9 T.H. Wilson and G. Wiseman, The use of sacs of everted small intestine for the study of the transference of substances from the mucosal to the serosal surface, J. Physiol. 123 (1954) 116-125. 10 T.H. Wilson, Concentration gradients of lactate, hydrogen, and some other ions across the intestine in vitro, Biochem. J., 56 (1954) 521-527. 11 T.H. Wilson and G. Wiseman, Metabolic activity of the small intestine of the rat and golden hamster (Mesocricetus auratus). J. Physiol., 123 (1954) 126-130.