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The effect of thermal injury on the regulation of phosphofructokinase in the mucosa of rat small intestine
Biochimie, 69
71
(1987) 71 - 73 © Soci~t~ de Chimie biologique/Elsevier, Paris
Short communication
The effect of thermal injury on the regulation of phosphofructokinase in the mucosa of rat small intestine Samir M. KHOJA and M. Salleh M. ARDAWI !
Department o f Biochemistry, Faculty o f Science and l Department o f Clinical Biochemistry, Faculty o f Medicine, King Abdulaziz University, Jeddah, Saudi Arabia (Received 10-9-1986, accepted 30-10-1986)
Summary - The effects of thermal injury (72 h post-injury), 72 h-partial (20070 less food) or full starvation on the regulation of phosphofructokinase in the mucosa of rat small intestine were studied. Thermal injury and 72 h-partial or full starvation decreased the activity ratio Vo.5/V, but the ratios obtained for thermally injured or fully starved rats were significantly lower than those of controls or partially starved rats. The susceptibility of phosphofructokinase to ATP inhibition was increased after thermal injury and 72 h-partial or full starvation compared to that of normal controls. However, these changes that occurred in the enzyme activities of the rat small intestine were mainly specific to injury per se but do not exclude the contribution of partial starvation during the same period of time. phosphofructokinase I regulatory properties / small intestine I thermal injury R6sum6 - Effets des brfilures sur la r6gulation de la phosphofructokinase dans la muqueuse de l'intestin gr~le du rat. Les effets des br~lures (72 h post-br~lure), 72 h-t~artiei (20% moins de nourriture) ou bien aprbs un je~ne complet sur la r~gulation de la phosphofructokinase dans la muqueuse de I'intestin gr~le du rat ont ~t~ dtudids. Les br~lures et 72 h-partiel ou bien aprbs un je~ne complet, diminuent l'activitd du rapport v o s/V, mais la valeur obtenue aprbs br~lure chez les rats qui o~ ~ eu moins de nourriture est bien infe'rieuie que chez les rats qui dtaient nourris partiellement. La susceptibilit~ de ia phosphofructokinase I'inhibition par I'A TP a augmentd aprbs br~lures et 72 h-partiel ou de je~ne complet comparde ~ celle des tdmoins. Cependant, les changements qui surviennent ~l l'activit~ des enzymes dans l'intestin gr~le des rats sont sp~cifiques aux br~iures per se mais elles n'excluent pas la contribution du je~ne partiel durant la m~me pdriode. phosphofructokinase I propri6t~s rigulatrices I intestin gr~le I br~lure
Introduction The epithelial cells of the small intestine are characterized by high rates of aerobic glycolysis, and glucose oxidation provides an important source of energy in the fed-state [1]. In a recent study [2], the small intestinal mucosa obtained from thermallyinjured rats exhibited a decrease in the maximal activities of hexokinase and phosphofructokinase
over the first five days post-injury. However, thermally-injured rats exhibited a diminished food intake (by about 20070) during the first three days post-injury [2], and it was not clear whether the observed changes in enzyme activity were specific to the effect of thermal injury alone and/or to the partial decrease in food intake. There is accumulating evidence to support the view that phosphofructokinase is the principle rate-
72
S.M. Khoja and M.S.M. Ardawi
limiting enzyme o f glycolysis in the epithelial cells o f the rat small intestine and that its activity under suboptimal conditions at pH 7.0 was decreased in starved or s t r e p t o z o t o c i n - d i a b e t i c rats [ 3 - 5 ] . In the present communication, we present the effect o f thermal injury on the regulatory properties of phosphofructokinase from the mucosa of rat small intestine and c o m p a r e these properties with those of partial (20070 less food for 72 h) starvation, 72 h-full starvation and normal controls.
Materials and methods All biochemicals were obtained from either Sigma or Boehringer-Mannheim and used without further purification. All chemicals were analytical reagent grade from BDH. Animals were male Wistar rats (200-230 g) either maintained on a standard laboratory diet (Oxoid, modified 41 B) fed ad libitum, allowed 20070 less food (72 hpartial starvation) or starved for 72 h with free access to water. Thermal injury (about 33070 of total surface area) was produced on the dorsal area of the rat as described by Herndon et al. [6], and rats were sacrificed 3 days post-injury. Extracts of intestinal mucosa were prepared as follows: rats were anesthetized with Sagatal (0.1 ml/100 g of body wt). The jejunal mucosa was collected as described previously [7] and homogenized directly in a Potter-Elvehjem homogenizer with 5 vol. (v/w) of extraction buffer consisting of 50 mM Tris-HC1 buffer, pH 8.0, containing 100 mM (NH4)2804, 30 mM KF, l mM 2-mercaptoethanol, 1 mM EDTA, and, as proteinase inhibitors" I mM phenylmethylsulfonyl fluoride; 1 raM 6-amino-n-hexanoic acid and 0.5 mg/mi of soybean trypsin inhibitor. The homogenate was centrifuged at 75000 x g for 30 rain at 4°C. The pellet was discarded, and the particle-free supernatant was stored on ice until use. Phosphofructokinase activity under optimal conditions at pH 8.0 was measured at 27°C as described in [8]. Regulatory properties of the enzyme at pH 7.0 were deter-
mined as described in [9]. One unit of activity is defined as the production of 1 ~mol of fructose-l,6-bis-phosphate/min.
Results and Discussion It has been established that in rat intestinal mucosa, 48 h-starvation affected the regulatory properties of phosphofructokinase [3]. However, such properties were not studied in thermally-injured or partially-starved rats. The study of the latter will clarify whether the observed changes in the activity of phosphofructokinase were mainly as a result of thermal injury alone a n d / o r due to the partial decrease in food intake that was observed during the first 3 days post-injury [2]. For the purpose of studying this effect, the activity observed at pH 7.0, v, is expressed as a ratio to that observed under optimal conditions at pH 8.0, V, by the value (v0.s/V) thus separating the regulatory properties from specific enzyme activities. Table I shows that thermal injury and 72 h-partial or full-starvation decreased the ratio Vo.5/V, but those ratios from both thermally-injured and fully-starved rats were significantly lower than those o f 72 h-partiallystarved rats. Fig. I shows that in the jejunal mucosa obtained from injured rats, the fructose-6-phosphate saturation curve, in the presence of 2.5 mM A T P , is shifted to higher concentrations; from an apparent K m of 0.58 m M in the control to 0.83 m M in the injured rats (P < 0.001). Similarly, 72 h-partial or full-starvation increased the apparent K m by about 15% (not significant) and 38°70 (P < 0.001), respectively. This increase in the apparent K m values is further indicated by the increased susceptibility to inhibition of jejunal phosphofructokinase by A T P after thermal injury or 72-partialor full-starvation (Fig. 2). The latter observation is consistent with another report [3].
Table I. Maximal activity and activity ratios of phosphofructokinase of intestinal mucosa under different conditions. Animal status
Enzyme activity (nmol/min/mg of protein)
Vo.s/V
Fed (control) Injured (72 h-post thermal injury) Partially-starved (20070 less food for 72 h) Starved (72 h-full starvation)
132.0 + 7.6 90.2_+3.8 118.0-+5.2 87.1 +4.4
0.39 + 0.02 0.19+0.03' ** 0.28-+0.01" 0.19+_0.02' **
(4) (5)* (5) (4)*
For experimental details see Materials and Methods. Activities were measured at 27°C. Results are presented as means + S.D., with the numbers of animals used given in parentheses. The statistical significance (Student's t test) of differences between control and experimental values is indicated by *: (P< 0.05), ** : (P< 0.001) and between partial-starvation (20% less food for 72 h) and injured or 72 h-starvation is indicated by "1" : (P<0.005).
Regulation o f phosphofructokinase
73
1.0 1.0
=.1> 0.5 >1> 0.5
•
05
14)
!
20
i
I
I
I
1
2
3
4
ATP (raM)
F6 P (raM)
Fig. 1. Effect of thermal injury, 72 h-partial- or full-starvation on the dependence of phosphofructokinase activity on the fructose-6-phosphateconcentration. Enzymeactivitywas assayed at pH 7.0 in the presence of ATP (2.5 mM) as described in the Materials and Methods section, with crude (particle-free)extracts derived from the jejunal mucosa of fed ( o ); thermally-injured (e); 72 h-partially- (E2) or fully-starved (A) animals. Each point represents the mean of 4-5 separate animals.
Fig. 2. Effect of thermal injury, 72 h-partial- or full-starvation on the dependence of phosphofructokinaseactivityon the ATP concentration. Enzyme activity was assayed at pH 7.0 in the presence of fructose-6-phosphate (0.5 raM) as described in the Materials and Methodssection, with crude (particle-free)extracts derived from the jejunal mucosa of fed ( o ); thermally-injured (o); 72 h-partially- ([]) or fully-starved (A) animals. Each point is the mean of 4-5 separate animals.
Therefore, it is concluded that the changes observed in the maximal activities of phosphofructokinase in the intestinal mucosa of thermally-injured rats are mainly specific to the injury per se but do not exclude the contribution of partial starvation that occurred over the first 72 h post-injury.
References
X~.~,~,ltALllff , IL I l G ~
Ukkll
l l ~ ' l . , I U l Ll~t.g L I I G L
III~.eUUCU,~
llU-
lated enterocytes obtained from thermally injured rats exhibited a diminished rate of glucose metabolism [2]. Since there is accumulating evidence to support the view that phosphofructokinase is the principle rate-limiting enzyme of glycolysis in the small intestinal mucosa [3, 4, 10], the above decrease in glucose utilization can be explained by a combination of changes in the specific activity and in the regulatory properties of the enzyme. The latter is indicated by the increased susceptibility of mucosal phosphofructokinase to inhibition by A T P (Fig. 2). Inhibition of glucose utilization by the small intestinal mucosa in thermally-injured rats (during the first 72 h post-injury) may permit more glucose to enter the body as glucose rather than as lactate, which requires hepatic gluconeogenesis [ 11]. Therefore, it is possible that this allows more glucose to be directed towards tissue repair and the immune system [l 2].
1 Windmueller H.G. (1984) in: GlutamineMetabolism in Mammalian Tissues (Haussinger E. & Sies H., eds.), Springer Verlag, Heidelberg, pp. 61-77 2 Ardawi M.S.M. & Newsholme E.A. (1986) AXetabolism, (in press) 3 Jamal A. & Kellett G.L. (1983) Biochem. J. 210, 129-135 4 Jamal A. & Kellett G.L. (1983) Diabetologia 25, 355-359 5 Jamal A., Kellett G.L. & Robertson J.P. (1984) Biochem. J. 218, 459-464 6 Herndon D.N., Wilmore D.W. & Mason D. (1978) J. Surg. Res. 25, 394-403 7 Khoja S.M., Beach N.L. & Kellett G.L. (1983) Biochem. J. 211,373-379 8 Ling K.H., Marcus F. & Lardy H.A. (1965) J. Biol. Chem. 240, 1893-1899 9 Hussey C.R., Liddle P.F., Ardron D. & Kellett G.L. (1977) Eur. J. Biochem. 80, 497-506 l0 Hanson P.J. & Parsons D.S. (1978) J. Physiol. (London) 278, 55-67 11 Newsholme E.A. & Leech A.R. (1983) in: Biochemistry for the Medical Sciences, John Wiley & Sons, New York, 444-454 12 Ardawi M.S.M. & Newsholme E.A. (1985) Essays Biochem. 21, Academic Press, New York, 1-44
71
(1987) 71 - 73 © Soci~t~ de Chimie biologique/Elsevier, Paris
Short communication
The effect of thermal injury on the regulation of phosphofructokinase in the mucosa of rat small intestine Samir M. KHOJA and M. Salleh M. ARDAWI !
Department o f Biochemistry, Faculty o f Science and l Department o f Clinical Biochemistry, Faculty o f Medicine, King Abdulaziz University, Jeddah, Saudi Arabia (Received 10-9-1986, accepted 30-10-1986)
Summary - The effects of thermal injury (72 h post-injury), 72 h-partial (20070 less food) or full starvation on the regulation of phosphofructokinase in the mucosa of rat small intestine were studied. Thermal injury and 72 h-partial or full starvation decreased the activity ratio Vo.5/V, but the ratios obtained for thermally injured or fully starved rats were significantly lower than those of controls or partially starved rats. The susceptibility of phosphofructokinase to ATP inhibition was increased after thermal injury and 72 h-partial or full starvation compared to that of normal controls. However, these changes that occurred in the enzyme activities of the rat small intestine were mainly specific to injury per se but do not exclude the contribution of partial starvation during the same period of time. phosphofructokinase I regulatory properties / small intestine I thermal injury R6sum6 - Effets des brfilures sur la r6gulation de la phosphofructokinase dans la muqueuse de l'intestin gr~le du rat. Les effets des br~lures (72 h post-br~lure), 72 h-t~artiei (20% moins de nourriture) ou bien aprbs un je~ne complet sur la r~gulation de la phosphofructokinase dans la muqueuse de I'intestin gr~le du rat ont ~t~ dtudids. Les br~lures et 72 h-partiel ou bien aprbs un je~ne complet, diminuent l'activitd du rapport v o s/V, mais la valeur obtenue aprbs br~lure chez les rats qui o~ ~ eu moins de nourriture est bien infe'rieuie que chez les rats qui dtaient nourris partiellement. La susceptibilit~ de ia phosphofructokinase I'inhibition par I'A TP a augmentd aprbs br~lures et 72 h-partiel ou de je~ne complet comparde ~ celle des tdmoins. Cependant, les changements qui surviennent ~l l'activit~ des enzymes dans l'intestin gr~le des rats sont sp~cifiques aux br~iures per se mais elles n'excluent pas la contribution du je~ne partiel durant la m~me pdriode. phosphofructokinase I propri6t~s rigulatrices I intestin gr~le I br~lure
Introduction The epithelial cells of the small intestine are characterized by high rates of aerobic glycolysis, and glucose oxidation provides an important source of energy in the fed-state [1]. In a recent study [2], the small intestinal mucosa obtained from thermallyinjured rats exhibited a decrease in the maximal activities of hexokinase and phosphofructokinase
over the first five days post-injury. However, thermally-injured rats exhibited a diminished food intake (by about 20070) during the first three days post-injury [2], and it was not clear whether the observed changes in enzyme activity were specific to the effect of thermal injury alone and/or to the partial decrease in food intake. There is accumulating evidence to support the view that phosphofructokinase is the principle rate-
72
S.M. Khoja and M.S.M. Ardawi
limiting enzyme o f glycolysis in the epithelial cells o f the rat small intestine and that its activity under suboptimal conditions at pH 7.0 was decreased in starved or s t r e p t o z o t o c i n - d i a b e t i c rats [ 3 - 5 ] . In the present communication, we present the effect o f thermal injury on the regulatory properties of phosphofructokinase from the mucosa of rat small intestine and c o m p a r e these properties with those of partial (20070 less food for 72 h) starvation, 72 h-full starvation and normal controls.
Materials and methods All biochemicals were obtained from either Sigma or Boehringer-Mannheim and used without further purification. All chemicals were analytical reagent grade from BDH. Animals were male Wistar rats (200-230 g) either maintained on a standard laboratory diet (Oxoid, modified 41 B) fed ad libitum, allowed 20070 less food (72 hpartial starvation) or starved for 72 h with free access to water. Thermal injury (about 33070 of total surface area) was produced on the dorsal area of the rat as described by Herndon et al. [6], and rats were sacrificed 3 days post-injury. Extracts of intestinal mucosa were prepared as follows: rats were anesthetized with Sagatal (0.1 ml/100 g of body wt). The jejunal mucosa was collected as described previously [7] and homogenized directly in a Potter-Elvehjem homogenizer with 5 vol. (v/w) of extraction buffer consisting of 50 mM Tris-HC1 buffer, pH 8.0, containing 100 mM (NH4)2804, 30 mM KF, l mM 2-mercaptoethanol, 1 mM EDTA, and, as proteinase inhibitors" I mM phenylmethylsulfonyl fluoride; 1 raM 6-amino-n-hexanoic acid and 0.5 mg/mi of soybean trypsin inhibitor. The homogenate was centrifuged at 75000 x g for 30 rain at 4°C. The pellet was discarded, and the particle-free supernatant was stored on ice until use. Phosphofructokinase activity under optimal conditions at pH 8.0 was measured at 27°C as described in [8]. Regulatory properties of the enzyme at pH 7.0 were deter-
mined as described in [9]. One unit of activity is defined as the production of 1 ~mol of fructose-l,6-bis-phosphate/min.
Results and Discussion It has been established that in rat intestinal mucosa, 48 h-starvation affected the regulatory properties of phosphofructokinase [3]. However, such properties were not studied in thermally-injured or partially-starved rats. The study of the latter will clarify whether the observed changes in the activity of phosphofructokinase were mainly as a result of thermal injury alone a n d / o r due to the partial decrease in food intake that was observed during the first 3 days post-injury [2]. For the purpose of studying this effect, the activity observed at pH 7.0, v, is expressed as a ratio to that observed under optimal conditions at pH 8.0, V, by the value (v0.s/V) thus separating the regulatory properties from specific enzyme activities. Table I shows that thermal injury and 72 h-partial or full-starvation decreased the ratio Vo.5/V, but those ratios from both thermally-injured and fully-starved rats were significantly lower than those o f 72 h-partiallystarved rats. Fig. I shows that in the jejunal mucosa obtained from injured rats, the fructose-6-phosphate saturation curve, in the presence of 2.5 mM A T P , is shifted to higher concentrations; from an apparent K m of 0.58 m M in the control to 0.83 m M in the injured rats (P < 0.001). Similarly, 72 h-partial or full-starvation increased the apparent K m by about 15% (not significant) and 38°70 (P < 0.001), respectively. This increase in the apparent K m values is further indicated by the increased susceptibility to inhibition of jejunal phosphofructokinase by A T P after thermal injury or 72-partialor full-starvation (Fig. 2). The latter observation is consistent with another report [3].
Table I. Maximal activity and activity ratios of phosphofructokinase of intestinal mucosa under different conditions. Animal status
Enzyme activity (nmol/min/mg of protein)
Vo.s/V
Fed (control) Injured (72 h-post thermal injury) Partially-starved (20070 less food for 72 h) Starved (72 h-full starvation)
132.0 + 7.6 90.2_+3.8 118.0-+5.2 87.1 +4.4
0.39 + 0.02 0.19+0.03' ** 0.28-+0.01" 0.19+_0.02' **
(4) (5)* (5) (4)*
For experimental details see Materials and Methods. Activities were measured at 27°C. Results are presented as means + S.D., with the numbers of animals used given in parentheses. The statistical significance (Student's t test) of differences between control and experimental values is indicated by *: (P< 0.05), ** : (P< 0.001) and between partial-starvation (20% less food for 72 h) and injured or 72 h-starvation is indicated by "1" : (P<0.005).
Regulation o f phosphofructokinase
73
1.0 1.0
=.1> 0.5 >1> 0.5
•
05
14)
!
20
i
I
I
I
1
2
3
4
ATP (raM)
F6 P (raM)
Fig. 1. Effect of thermal injury, 72 h-partial- or full-starvation on the dependence of phosphofructokinase activity on the fructose-6-phosphateconcentration. Enzymeactivitywas assayed at pH 7.0 in the presence of ATP (2.5 mM) as described in the Materials and Methods section, with crude (particle-free)extracts derived from the jejunal mucosa of fed ( o ); thermally-injured (e); 72 h-partially- (E2) or fully-starved (A) animals. Each point represents the mean of 4-5 separate animals.
Fig. 2. Effect of thermal injury, 72 h-partial- or full-starvation on the dependence of phosphofructokinaseactivityon the ATP concentration. Enzyme activity was assayed at pH 7.0 in the presence of fructose-6-phosphate (0.5 raM) as described in the Materials and Methodssection, with crude (particle-free)extracts derived from the jejunal mucosa of fed ( o ); thermally-injured (o); 72 h-partially- ([]) or fully-starved (A) animals. Each point is the mean of 4-5 separate animals.
Therefore, it is concluded that the changes observed in the maximal activities of phosphofructokinase in the intestinal mucosa of thermally-injured rats are mainly specific to the injury per se but do not exclude the contribution of partial starvation that occurred over the first 72 h post-injury.
References
X~.~,~,ltALllff , IL I l G ~
Ukkll
l l ~ ' l . , I U l Ll~t.g L I I G L
III~.eUUCU,~
llU-
lated enterocytes obtained from thermally injured rats exhibited a diminished rate of glucose metabolism [2]. Since there is accumulating evidence to support the view that phosphofructokinase is the principle rate-limiting enzyme of glycolysis in the small intestinal mucosa [3, 4, 10], the above decrease in glucose utilization can be explained by a combination of changes in the specific activity and in the regulatory properties of the enzyme. The latter is indicated by the increased susceptibility of mucosal phosphofructokinase to inhibition by A T P (Fig. 2). Inhibition of glucose utilization by the small intestinal mucosa in thermally-injured rats (during the first 72 h post-injury) may permit more glucose to enter the body as glucose rather than as lactate, which requires hepatic gluconeogenesis [ 11]. Therefore, it is possible that this allows more glucose to be directed towards tissue repair and the immune system [l 2].
1 Windmueller H.G. (1984) in: GlutamineMetabolism in Mammalian Tissues (Haussinger E. & Sies H., eds.), Springer Verlag, Heidelberg, pp. 61-77 2 Ardawi M.S.M. & Newsholme E.A. (1986) AXetabolism, (in press) 3 Jamal A. & Kellett G.L. (1983) Biochem. J. 210, 129-135 4 Jamal A. & Kellett G.L. (1983) Diabetologia 25, 355-359 5 Jamal A., Kellett G.L. & Robertson J.P. (1984) Biochem. J. 218, 459-464 6 Herndon D.N., Wilmore D.W. & Mason D. (1978) J. Surg. Res. 25, 394-403 7 Khoja S.M., Beach N.L. & Kellett G.L. (1983) Biochem. J. 211,373-379 8 Ling K.H., Marcus F. & Lardy H.A. (1965) J. Biol. Chem. 240, 1893-1899 9 Hussey C.R., Liddle P.F., Ardron D. & Kellett G.L. (1977) Eur. J. Biochem. 80, 497-506 l0 Hanson P.J. & Parsons D.S. (1978) J. Physiol. (London) 278, 55-67 11 Newsholme E.A. & Leech A.R. (1983) in: Biochemistry for the Medical Sciences, John Wiley & Sons, New York, 444-454 12 Ardawi M.S.M. & Newsholme E.A. (1985) Essays Biochem. 21, Academic Press, New York, 1-44