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Effect of hypothyroidism on glucose transport and metabolism in rat small intestine
76
Biochirnica et Biophysica A cta, 1179 (1993) 76-80 © 1993 Elsevier Science Publishers B.V. All rights reserved 0167-4889/93/$06.00
BBAMCR 13445
Effect of hypothyroidism on glucose transport and metabolism in rat small intestine Samir M. Khoja
a
and George L. Kellett b
a Department of Biochemistry, King Abdulaziz Uniuersity, Jeddah (Saudi Arabia) and b Department of Biology, University of York, Heslington, York (UK) (Received 5 January 1993)
Key words: Glucose transport; Hypothyroidism; (Rat small intestine)
The effect of experimental hypothyroidism on the absorption, transmural transport and metabolism of glucose was studied by perfusion of isolated loops of rat jejunum in vitro. When expressed on a dry weight basis, the rate of absorption was enhanced by 32% (P < 0.01); when expressed on a length basis there was no significant change, since the enhancement per unit weight was almost exactly compensated by a diminution in mass per unit length in the hypothyroid state. When expressed in either units, there was a significant enhancement in transmural transport (+ 123% and + 77%, respectively, both P < 0.001), which reflected in part a diminution in the rate of glucose utilization ( - 2 9 % , P < 0.01 and -43%, P < 0.001, respectively). The changes in glucose utilization were matched by changes in lactate production. Three factors contributed to the diminution in glucose utilization in the hypothyroid state: a diminution in the concentration of 6-phosphofructo-l-kinase ( - 3 5 % , P < 0.05), and increase in the S0.5 of 6-phosphofructo-l-kinase for fructose 6-phosphate from 0.4 to 0.6 mM and a fall in the mucosal concentration of fructose 2,6-bisphosphate ( - 56%, P < 0.05). From the point of view of the whole animal, there is little if any change in the capacity of the intestine to absorb glucose from the lumen, but there is a large enhancement of transmural transport that is metabolically driven.
Introduction Glucose crosses the small intestine by two routes; after absorption across the brush border membrane, it is either transported across the basolateral m e m b r a n e unchanged or it is metabolized predominantly to lactate for subsequent hepatic gluconeogenesis, the latter route being marginally more important in quantitative terms in fed rats [1-6]. Changes in transport may therefore occur either through changes in the transport steps themselves or as a secondary consequence of changes in metabolism. The importance of the two routes is illustrated in the regulation of intestinal transport by insulin. Thus the injection of normal, fed rats with insulin [5] or the incubation of isolated jejunal loops in vitro with despentapeptide-insulin [6] caused 23% and 32% inhibition, respectively, of glucose absorption from the lumen, but no change in the rate of glucose utilization.
Correspondence to: S.M. Khoja, P.O. Box 6781, Jeddah 21452, Saudi Arabia. Abbreviations: FT3, free tri-iodothyronine; FT4, free thyroxine; TT3, total tri-iodothyronine; TT 4, total thyroxine.
In contrast, when rats were injected with anti-insulin serum, the rate of glucose utilization was inhibited by 79%, so that the proportion of glucose translocated unchanged across the intestine increased from 45 to 80%. There have been several studies of the effect of thyroid status on glucose transport by the small intestine. However, the results were conflicting, with different workers finding enhancement, no effect or inhibition of transport (see Ref. 7 for a review). The lack of consensus may, in part, reflect the fact that the methods used did not permit the relationship between metabolism and transport to be examined. Yet it is well known that thyroid status has a profound effect on sugar metabolism [7]. Recently, Ardawi and Jalalah [8] have reported that isolated enterocytes from the small intestine of hypothyroid rats also show diminished glucose utilization. Furthermore, plasma insulin levels are diminished in hypothyroid rats and, as noted, we have previously shown that glycolysis and glucose transport in rat small intestine are regulated by insulin and that the rate-limiting enzyme of glycolysis in this tissue is 6-phosphofructo-l-kinase [5,6]. In addition, fructose 2,6-bisphosphate plays an important role in the regulation of enterocyte glycolysis [9,10]. We have therefore
77 studied the underlying mechanisms responsible for the changes in intestinal metabolism that occur in hypothyroid rats and have explored the relationship between the changes in metabolism and the concomitant changes in transport. Materials and Methods
Animals Male Wistar rats (180-198 g) were fed ad libitum on a standard laboratory chow (Bantin and Kingman, Hull, UK) with free access to water. Animals were divided into two groups: a hypothyroid group (n = 15) and a pair-fed control group (n = 15). Experimental hypothyroidism was induced by administration of 6-n-propyl-2-thiouracil for 4-5 weeks. The drug was dissolved in warm ethanol and was added to the drinking water at a final concentration of 0.5 mg/ml. Ethanol was added to the drinking water of the pair-fed control rats at the same final concentration (1%). 6-n-propyl-2thiouracil is an anti-thyroid drug; it blocks hormone synthesis at the steps of iodine organification and iodotyrosine coupling and also inhibits the extrathyroidal conversion of thyroxine to tri-iodothyronine [11,121.
Chemicals and enzymes All biochemicals and enzymes were obtained from Sigma Chemicals, Poole, UK or from BoehringerMannheim (Mannheim, Germany) and were used without further purification. All other chemicals were of the analytical grade and obtained from BDH, Poole, UK.
Measurement of intestinal absorption and metabolism Glucose absorption, transmural transport and metabolism were determined in isolated jejunal loops in vitro as described in detail by Kellett and Barker [13]. Loops were perfused for 30 min with 5 mM glucose present on both sides of the intestine. Rates were expressed in both ~ m o l / h per g dry wt. and ~ m o l / h per cm.
Preparation of extracts Rats were anaesthetized with Sagatal (0.1 ml/100 g body weight). The jejunum was excised and mucosa was collected and directly frozen in liquid N 2. For 6-phosphofructo-l-kinase (EC 2.7.1.11), the frozen mucosa was homogenized with 3 vols. (v/w) of ice-cold (4°C) extraction buffer consisting of 50 mM Tris-HC1 (pH 8.0), 100 mM (NH4)2504, 30 mM KF, 5 mM 2-mercaptoethanol, 5 mM EDTA, 1 mM phenylmethanesulphonyl fluoride, 1 mM 6-amino-n-hexanoic acid and 0.5 mg of soybean trypsin inhibitor/ml. Tissue homogenates were centrifuged for 30 min at 75 000 × g in a Sorvall RC5C HS20 centrifuge at 4°C. The pellets
were discarded and the particle-free supernatants were used for chromatography. Tissue extracts were directly chromatographed on a column (2.5 x 50 cm) of Sephadex G-100, equilibrated with extraction buffer (see above). Partially purified 6-phosphofructo-l-kinase was collected with fraction collector and the fraction containing highest enzyme activity was used for assays. The extraction medium for hexokinase (EC 2.7.1.1) consisted of 50 mM triethanolamine/HCl, 1 mM EDTA, 2 mM MgC12 and 30 mM 2-mercaptoethanol at pH 7.0. The same extraction medium was used for pyruvate kinase (EC 2.7.1.40), except that the pH was 7.6. Frozen mucosa was homogenized with 5 vols. of ice-cold extraction medium by using a Polytron homogenizer (PCU-2) for 10-20 s at 0°C. The homogenates were centrifuged at 13500 x g for 2 min. and the resultant supernatants were used for assays.
Assay of enzyme activities The activity of 6-phosphofructo-l-kinase under optimal conditions at pH 8.0 was measured at 27°C according to the method of Ling et al. [14]. The regulatory properties of the enzyme at pH 7.0 were determined under the conditions defined by Jamal and Kellett [15]. One unit of enzyme activity is defined as the formation of 1 /xmol of fructose 1,6-bisphosphate per min. The activity of hexokinase was measured as described by Crabtree and Newsholme [16], whereas the activity of pyruvate kinase was measured as described by Zammit et al., [17]. The final volume of assay mixtures in both cases was 1.0 ml. All spectrophotometric measurements were performed in a Gilford recording spectrophotometer (model 240) at 25°C. Maximal enzyme activities are expressed as /zmol of substrate utilized/min per g tissue. Protein was determined by the method of Lowry et al. [18] with bovine serum albumin as standard.
Assay of fructose 2,6-bisphosphate Fructose 2,6-bisphosphate was measured in rat jejunal mucosa with the method of Van Schaftingen et al. [19] with stimulation to PPi-dependent phosphofructokinase (EC 2.7.1.90) purified from potato tubers.
Determination of concentrations of metabolites and plasma hormones Concentrations of metabolites in neutralized extracts of plasma were determined spectrophotometrically (with a Beckman DU-6 recording spectrophotometer) by standard enzymic methods: glucose by the coupled hexokinase/glucose 6-phosphate dehydrogenase method as described by Bergmeyer et al., [20] and lactate by the method of Gawehn and Bergmeyer [21]. All plasma hormone concentrations were measured by RIA technique (Dragnostic Products, Los Angeles, USA). Radioactivity was determined in a Beckman Gamma Counter (model 5500).
78 TABLE I
General characteristics of control and hypothyroid rats Values are presented as means_+ S.E. for 10-12 rats. Rats were treated as described in Materials and Methods.
Initial body wt. (g) Final body wt. (g) Small intestine wt. (% of body wt.) Small intestine length ( c m / g ) Fructose 2,6-bisphosphate ( n m o l / g ) Plasma Plasma Plasma Plasma Plasma Plasma Plasma Plasma
glucose ( m m o l / I ) lactate ( m m o l / l ) TT 3 ( n m o l / I ) FT 3 ( p m o l / l ) TT 4 ( n m o l / l ) FT 4 ( p m o l / l ) thyrotropin ( ~ U / m l ) insulin ( / ~ U / m l )
Control rats
Hypothyroid rats
189 +3 280 ±6 2.24±0.06 20.1 +0.5 2.94+0.14 8.02_ 0.23 2.14 + 0.07 1.46 5:0.06 2.69 _+0.11 70.21 + 3.01 29.33+ 1.19 5.50_+0.56 55.42±3.98
192 +3 241 ±6 * 2.10+0.05 25.3+0.6 * 1.88+0.09 * 7.86 + 0.25 2.28 + 0.08 0.44 + 0.02 * * 0.40 + 0.03 * * 6.62+ 0.38 * * 1.405:0.08 ** 20.87+ 1.80 ** 24.28+2.18 **
Statistical significance: * P < 0.05, ** P < 0.001.
Statistical analysis of results
creased (P < 0.001). The plasma concentrations of insulin were significantly decreased ( - 128%, P < 0.001). When expressed on a dry weight basis, experimental hypothyroidism caused significant increases in the rates of glucose absorption ( + 32%, P < 0.01) and transmural transport ( + 123%, P < 0.001), whereas it caused a significant decrease in the rate of glucose utilization ( - 29%, P < 0.01; Table II). The rate of lactate release into the serosal medium was significantly decreased ( - 7 8 % , P < 0.01) so as the total rate of lactate production ( - 74%, P < 0.01). The percentage of glucose utilized converted into lactate was also significantly reduced (31.3 + 6.5 to 12.0 + 1.7%, P < 0.05) in hypothyroid rats (Table II). In contrast, when the data were expressed on a length basis, there was no significant change in the rate of glucose absorption. However, there was a significant change in all other rates, especially that of glucose utilization, which was diminished by 43% (P < 0.001). Table III shows the maximal activities of hexoki-
Results are given as mean + S.E. for the indicated number of observations. Statistical significance was assessed using Student's t-test. Results Table I details the general characteristics of the rats made hypothyroid by administration of 6-n-propyl-2thiouracil. Thus the gain of the body weight of the hypothyroid rats was significantly less than that of their pair-fed control (P < 0.05). The mucosal concentration of fructose 2,6-bisphosphate was significantly decreased ( - 5 6 % , P < 0.05) in hypothyroid rats. No significant differences were seen in the plasma concentrations of glucose and lactate. The plasma concentrations of total tri-iodothyronine (TT3), free tri-iodothyronine (FT3), total thyroxine (TT4) and free thyroxine (FT 4) were significantly decreased (P < 0.001) in hypothyroid rats, whereas that of thyrotropin was significantly inT A B L E II
The rates of glucose absorption, transmural transport and metabolism measured in isolated jejunal loops from control and hypothyroid rats Experimental details are given in Materials and Methods. Rates are given as means + S.E. for 7 rats. Rate:
Glucose Absorption Transmural transport Utilization Lactate Release into the luminal peffusate Release into the serosal medium Total rate of lactate production Glucose utilized converted into lactate (%)
~ m o l / h per g dry wt.
~ m o l / h per cm
Control rats
Hypothyroid rats
Control rats
Hypothyroid rats
413 167 246
547 372 175
20.6+0.8 8.3+0.7 12.2+0.8
21.6+1.1 14.7+ 1.0 * * * 6.9+0.4 * * *
2.0:t:0.6 5.9 ± 1.4 8.0 ± 1.9
0.6+0.2 * 1.0 ± 0.04 * * 1.6 + 0.2 * *
+17 + 14 +15
41 + 119 + 160 + 31.3+
12 29 38 6.5
+ 2 9 ** +25 *** + 1 0 **
15 + 26 ± 41 ± 12.0±
4 * 1** 5 ** 1.7 *
P values are given for the comparison of experimental and normal control perfusions: * P < 0.05; * * P < 0.01, * * * P < 0.001.
79 TABLE III
Maximal activities of hexokinase, 6-phosphofructo-l-kinase and pyrurate kinase in intestinal mucosal scrapings of control and hypothyroid rats Values are presented as means+S.E, with the number of rats given in parentheses. For experimental details, see Materials and Methods. Enzyme
Activity ( U / g tissue wt.)
Hexokinase
6-Phosphofructo-l-kinase Pyruvate kinase
Control rats
Hypothyroid rats
2.165:0.08 (6) 11.62:t: 0.30 (8) 38.21 5:1.49 (6)
1.485:0.06 * (6) 7.55 5:0.29 * * (8) 20.115:1.78 * * (6)
Values significantly different from controls are shown: * P < 0.01, ** P < 0.001.
nase, 6-phosphofructo-l-kinase and pyruvate kinase in intestinal mucosa of hypothyroid and pair-fed control rats. The activities of these enzymes measured were significantly decreased ( - 4 6 % , P < 0.01 for hexokinase; - 5 4 % , P < 0.001 for 6-phosphofructo-l-kinase; - 90%, P < 0.001 for pyruvate kinase) in the intestinal mucosa of the hypothyroid rats (Table III). The regulatory properties of 6-phosphofructo-1kinase from intestinal mucosa partially purified by chromatography on Sephadex G-100 are presented in Fig. 1. Fig. 1A shows the fructose 6-phosphate saturation curve of mucosal 6-phosphofructo-l-kinase measured under suboptimal conditions in the presence of 2.5 mM ATP at pH 7.0. In hypothyroid rats; the curve is displaced to higher fructose 6-phosphate concentrations and as a result, S0.5, the concentration of fructose 6-phosphate required to achieve half-maximal saturation, was shifted from 0.4 mM in the controls to 0.6 mM in the hypothyroid rats. This increase in S0.5 is a
reflection of an increased sensitivity to inhibition by ATP in hypothyroid rats (Fig 1B). Discussion
The present study shows that the rates of glucose transport and metabolism were changed in the whole intestine of hypothyroid compared with control rats. The rates of glucose utilization and lactate production were strongly inhibited, whether expressed on a dry weight or length basis. Three factors contributing to this inhibition were established. First, the concentrations of three key regulatory glycolytic enzymes, namely hexokinase, 6-phosphofructo-l-kinase and pyruvate kinase, were significantly diminished (Table III). Secondly, the regulatory properties of 6-phosphofructo-1kinase, which has been shown to be the principal rate-limiting enzyme of glycolysis in rat small intestine [5], were altered. In particular, the enzyme in hypothyroid rats showed a modest increase in susceptibility to inhibition by ATP (Fig. 1). It should be noted that this appeared to be a permanent change in the properties of the enzyme, independent of changes in the concentrations of effectors, since the assays were performed using chromatographed enzyme. Such a permanent change in properties might arise through phosphorylation as happens for the liver isoenzyme [22], antiserum to which strongly cross-reacts with the intestinal isoenzyme [23]. Thirdly, the concentration of fructose 2,6bisphosphate, a powerful activator of 6-phosphofructo-l-kinase, was diminished in hypothyroid rats. This finding is therefore consistent with the idea that fructose 2,6-bisphosphate plays an important role in the regulation of 6-phosphofructo-l-kinase and glycolysis in rat small intestine [24]. Since glycolysis in rat
1.0
®
~°I ® ~l> 0.5,
>1 > o.s
o
0.5 [Fructose-6-phosphatal (mM)
1.o 20
I
1
I
I
2 3 4 [ATP] (raM) Fig. 1. Regulatory properties of mucosal 6-phosphofructo-l-kinase in hypothyroid and control rats. The e n z y m e w a s p a r t i a l l y purified on Sephadex G-100; assays were performed at pH 7.0 as described in Materials and Methods and the activity at pH 7.0, v, is e x p r e s s e d as a ratio to that at pH 8.0, V. (A) Fructose 6-phosphate saturation curve determined at 2.5 raM ATP; (B) ATP inhibition curve determined at 0.5 mM fructose 6-phosphate. (e) Control rats; (o) hypothyroid rats.
80 small intestine is known to be regulated by insulin [5,6] and as the plasma insulin concentration in hypothyroid rats is about 44% of that in control rats, it seems likely that changes in insulin levels, in addition to the changes in thyroid hormone levels, were also involved in the regulation of the metabolic changes observed. The absolute rate of glucose oxidation, given by the rate of glucose utilization - 0.5 x total rate of lactate production, was unchanged in the hypothyroid state, in agreement with the report of Bronk and Parsons [25]. Table II shows that both the rates of glucose absorption and transmural transport were significantly enhanced when expressed on dry weight basis. However, when the rate of absorption was expressed on per cm of intestine basis, no significant change was observed. Thus the enhancement of absorption per unit weight was almost exactly compensated by a diminution in the mass of intestine per unit length in the hypothyroid state. Since the overall length of intestine was unaltered, then, from the physiological point of view, it is unlikely that there is any change in the capacity of the whole intestine in vivo to absorb glucose from the lumen in hypothyroid state. Nevertheless, the enhancement of transmural transport across the vascular side was still as much as 77% (P < 0.001) when expressed on a length basis, since 68% of the glucose absorbed was transported unchanged compared with about 40% in control rats. This reflects the strong inhibition of glucose utilization and shows clearly the interrelationship of metabolism and transport in this tissue. References 1 Remesy, C., Demigne, C. and Autrene, J. (1978) Biochem. J. 170, 321-329.
2 Shapiro, A. and Shapiro, B. (1979) Biochim. Biophys. Acta 586, 123-127. 3 Pritchard, P.J. and Porteous, J.W. (1977) Biochem. J. 164, 1-14. 4 Nicholls, T.J., Leese, H.J. and Bronk, J.R. (1983) Biochem. J. 212, 183-187. 5 Kellett, G.L., Jamal, A., Robertson, J.P. and Wollen, N. (1984) Biochem. J. 219, 1027-1035. 6 Wo|len, N. and Kellett, G.L. (1988) Gut 29, 1064-1069. 7 Levin, R.J. (1969) J. Endocrinol. 45, 315-348. 8 Ardawi, M.S.M. and Jalahah, S.M. (1991) Clin. Sci. 81,347-355. 9 Hue, L. and Rider, M.H. (1987) Biochem. J. 245, 313-324. 10 Lowry, M. and Kellett, G.L. (1989) Biochem. J. 259, 624-626. 11 Ingbar, S.H. and Woeber, K.A. (1981) in Williams, R.H., (ed.), The thyroid gland, Textbook of endocrinology, pp. 117-247, W.B. Saunders, Philadelphia. 12 Hedge, G.A., Colby, H.D. and Goodman, R.L. (1987) Clinical endocrine physiology, W.B. Saunders, Philadelphia. 13 Kellett, G.L. and Barker, E.D. (1989) Biochim. Biophys. Acta 979, 311-315. 14 Ling, K.H., Marcus, F. and Lardy, H.A. (1965) J. Biol. Chem. 240, 1893-1899. 15 Jamal, A. and Kellett, G.L. (1983) Biochem. J. 210, 129-135. 16 Crabtree, B. and Newsholme, E.A. (1972) Biochem. J. 126, 49-58. 17 Zammit, V.A., Beis, I. and Newsholme, E.A. (1978) Biochem. J. 174, 989-998. 18 Lowry, O.H., Rosebrough, N.J., Farr, A.L. and Randall, R.J. (1951) J. Biol. Chem. 193, 265-275. 19 Van Schaftingen, E., Lederer, B., Bartrons, R. and Hers, H.G. (1982) Eur. J. Biochem. 129, 191-195. 20 Bergmeyer, H.U., Bernt, E., Schmidt, F. and Stork, H. (1974) in Methods of Enzymatic Analysis (Bergmeyer, H.U., ed.), pp. 1196-1202, Academic Press, London. 21 Gawehn, K. and Bergmeyer, H.U. (1974) in Methods of Enzymatic Analysis (Bergmeyer, H.U., ed.), pp. 1492-1495, Academic Press, London. 22 Kagimoto, T. and Uyeda, K. (1979) J. Biol. Chem. 254, 5584-5587. 23 Khoja, S.M. and Kellett, G.L. (1983) Biochem. J. 215, 335-341. 24 Khoja, S.M. (1986) Comp. Biochem. Physiol. 85B, 337-341. 25 Bronk, J.R. and Parsons, D.S. (1965) J. Physiol. 179, 323-332.
Biochirnica et Biophysica A cta, 1179 (1993) 76-80 © 1993 Elsevier Science Publishers B.V. All rights reserved 0167-4889/93/$06.00
BBAMCR 13445
Effect of hypothyroidism on glucose transport and metabolism in rat small intestine Samir M. Khoja
a
and George L. Kellett b
a Department of Biochemistry, King Abdulaziz Uniuersity, Jeddah (Saudi Arabia) and b Department of Biology, University of York, Heslington, York (UK) (Received 5 January 1993)
Key words: Glucose transport; Hypothyroidism; (Rat small intestine)
The effect of experimental hypothyroidism on the absorption, transmural transport and metabolism of glucose was studied by perfusion of isolated loops of rat jejunum in vitro. When expressed on a dry weight basis, the rate of absorption was enhanced by 32% (P < 0.01); when expressed on a length basis there was no significant change, since the enhancement per unit weight was almost exactly compensated by a diminution in mass per unit length in the hypothyroid state. When expressed in either units, there was a significant enhancement in transmural transport (+ 123% and + 77%, respectively, both P < 0.001), which reflected in part a diminution in the rate of glucose utilization ( - 2 9 % , P < 0.01 and -43%, P < 0.001, respectively). The changes in glucose utilization were matched by changes in lactate production. Three factors contributed to the diminution in glucose utilization in the hypothyroid state: a diminution in the concentration of 6-phosphofructo-l-kinase ( - 3 5 % , P < 0.05), and increase in the S0.5 of 6-phosphofructo-l-kinase for fructose 6-phosphate from 0.4 to 0.6 mM and a fall in the mucosal concentration of fructose 2,6-bisphosphate ( - 56%, P < 0.05). From the point of view of the whole animal, there is little if any change in the capacity of the intestine to absorb glucose from the lumen, but there is a large enhancement of transmural transport that is metabolically driven.
Introduction Glucose crosses the small intestine by two routes; after absorption across the brush border membrane, it is either transported across the basolateral m e m b r a n e unchanged or it is metabolized predominantly to lactate for subsequent hepatic gluconeogenesis, the latter route being marginally more important in quantitative terms in fed rats [1-6]. Changes in transport may therefore occur either through changes in the transport steps themselves or as a secondary consequence of changes in metabolism. The importance of the two routes is illustrated in the regulation of intestinal transport by insulin. Thus the injection of normal, fed rats with insulin [5] or the incubation of isolated jejunal loops in vitro with despentapeptide-insulin [6] caused 23% and 32% inhibition, respectively, of glucose absorption from the lumen, but no change in the rate of glucose utilization.
Correspondence to: S.M. Khoja, P.O. Box 6781, Jeddah 21452, Saudi Arabia. Abbreviations: FT3, free tri-iodothyronine; FT4, free thyroxine; TT3, total tri-iodothyronine; TT 4, total thyroxine.
In contrast, when rats were injected with anti-insulin serum, the rate of glucose utilization was inhibited by 79%, so that the proportion of glucose translocated unchanged across the intestine increased from 45 to 80%. There have been several studies of the effect of thyroid status on glucose transport by the small intestine. However, the results were conflicting, with different workers finding enhancement, no effect or inhibition of transport (see Ref. 7 for a review). The lack of consensus may, in part, reflect the fact that the methods used did not permit the relationship between metabolism and transport to be examined. Yet it is well known that thyroid status has a profound effect on sugar metabolism [7]. Recently, Ardawi and Jalalah [8] have reported that isolated enterocytes from the small intestine of hypothyroid rats also show diminished glucose utilization. Furthermore, plasma insulin levels are diminished in hypothyroid rats and, as noted, we have previously shown that glycolysis and glucose transport in rat small intestine are regulated by insulin and that the rate-limiting enzyme of glycolysis in this tissue is 6-phosphofructo-l-kinase [5,6]. In addition, fructose 2,6-bisphosphate plays an important role in the regulation of enterocyte glycolysis [9,10]. We have therefore
77 studied the underlying mechanisms responsible for the changes in intestinal metabolism that occur in hypothyroid rats and have explored the relationship between the changes in metabolism and the concomitant changes in transport. Materials and Methods
Animals Male Wistar rats (180-198 g) were fed ad libitum on a standard laboratory chow (Bantin and Kingman, Hull, UK) with free access to water. Animals were divided into two groups: a hypothyroid group (n = 15) and a pair-fed control group (n = 15). Experimental hypothyroidism was induced by administration of 6-n-propyl-2-thiouracil for 4-5 weeks. The drug was dissolved in warm ethanol and was added to the drinking water at a final concentration of 0.5 mg/ml. Ethanol was added to the drinking water of the pair-fed control rats at the same final concentration (1%). 6-n-propyl-2thiouracil is an anti-thyroid drug; it blocks hormone synthesis at the steps of iodine organification and iodotyrosine coupling and also inhibits the extrathyroidal conversion of thyroxine to tri-iodothyronine [11,121.
Chemicals and enzymes All biochemicals and enzymes were obtained from Sigma Chemicals, Poole, UK or from BoehringerMannheim (Mannheim, Germany) and were used without further purification. All other chemicals were of the analytical grade and obtained from BDH, Poole, UK.
Measurement of intestinal absorption and metabolism Glucose absorption, transmural transport and metabolism were determined in isolated jejunal loops in vitro as described in detail by Kellett and Barker [13]. Loops were perfused for 30 min with 5 mM glucose present on both sides of the intestine. Rates were expressed in both ~ m o l / h per g dry wt. and ~ m o l / h per cm.
Preparation of extracts Rats were anaesthetized with Sagatal (0.1 ml/100 g body weight). The jejunum was excised and mucosa was collected and directly frozen in liquid N 2. For 6-phosphofructo-l-kinase (EC 2.7.1.11), the frozen mucosa was homogenized with 3 vols. (v/w) of ice-cold (4°C) extraction buffer consisting of 50 mM Tris-HC1 (pH 8.0), 100 mM (NH4)2504, 30 mM KF, 5 mM 2-mercaptoethanol, 5 mM EDTA, 1 mM phenylmethanesulphonyl fluoride, 1 mM 6-amino-n-hexanoic acid and 0.5 mg of soybean trypsin inhibitor/ml. Tissue homogenates were centrifuged for 30 min at 75 000 × g in a Sorvall RC5C HS20 centrifuge at 4°C. The pellets
were discarded and the particle-free supernatants were used for chromatography. Tissue extracts were directly chromatographed on a column (2.5 x 50 cm) of Sephadex G-100, equilibrated with extraction buffer (see above). Partially purified 6-phosphofructo-l-kinase was collected with fraction collector and the fraction containing highest enzyme activity was used for assays. The extraction medium for hexokinase (EC 2.7.1.1) consisted of 50 mM triethanolamine/HCl, 1 mM EDTA, 2 mM MgC12 and 30 mM 2-mercaptoethanol at pH 7.0. The same extraction medium was used for pyruvate kinase (EC 2.7.1.40), except that the pH was 7.6. Frozen mucosa was homogenized with 5 vols. of ice-cold extraction medium by using a Polytron homogenizer (PCU-2) for 10-20 s at 0°C. The homogenates were centrifuged at 13500 x g for 2 min. and the resultant supernatants were used for assays.
Assay of enzyme activities The activity of 6-phosphofructo-l-kinase under optimal conditions at pH 8.0 was measured at 27°C according to the method of Ling et al. [14]. The regulatory properties of the enzyme at pH 7.0 were determined under the conditions defined by Jamal and Kellett [15]. One unit of enzyme activity is defined as the formation of 1 /xmol of fructose 1,6-bisphosphate per min. The activity of hexokinase was measured as described by Crabtree and Newsholme [16], whereas the activity of pyruvate kinase was measured as described by Zammit et al., [17]. The final volume of assay mixtures in both cases was 1.0 ml. All spectrophotometric measurements were performed in a Gilford recording spectrophotometer (model 240) at 25°C. Maximal enzyme activities are expressed as /zmol of substrate utilized/min per g tissue. Protein was determined by the method of Lowry et al. [18] with bovine serum albumin as standard.
Assay of fructose 2,6-bisphosphate Fructose 2,6-bisphosphate was measured in rat jejunal mucosa with the method of Van Schaftingen et al. [19] with stimulation to PPi-dependent phosphofructokinase (EC 2.7.1.90) purified from potato tubers.
Determination of concentrations of metabolites and plasma hormones Concentrations of metabolites in neutralized extracts of plasma were determined spectrophotometrically (with a Beckman DU-6 recording spectrophotometer) by standard enzymic methods: glucose by the coupled hexokinase/glucose 6-phosphate dehydrogenase method as described by Bergmeyer et al., [20] and lactate by the method of Gawehn and Bergmeyer [21]. All plasma hormone concentrations were measured by RIA technique (Dragnostic Products, Los Angeles, USA). Radioactivity was determined in a Beckman Gamma Counter (model 5500).
78 TABLE I
General characteristics of control and hypothyroid rats Values are presented as means_+ S.E. for 10-12 rats. Rats were treated as described in Materials and Methods.
Initial body wt. (g) Final body wt. (g) Small intestine wt. (% of body wt.) Small intestine length ( c m / g ) Fructose 2,6-bisphosphate ( n m o l / g ) Plasma Plasma Plasma Plasma Plasma Plasma Plasma Plasma
glucose ( m m o l / I ) lactate ( m m o l / l ) TT 3 ( n m o l / I ) FT 3 ( p m o l / l ) TT 4 ( n m o l / l ) FT 4 ( p m o l / l ) thyrotropin ( ~ U / m l ) insulin ( / ~ U / m l )
Control rats
Hypothyroid rats
189 +3 280 ±6 2.24±0.06 20.1 +0.5 2.94+0.14 8.02_ 0.23 2.14 + 0.07 1.46 5:0.06 2.69 _+0.11 70.21 + 3.01 29.33+ 1.19 5.50_+0.56 55.42±3.98
192 +3 241 ±6 * 2.10+0.05 25.3+0.6 * 1.88+0.09 * 7.86 + 0.25 2.28 + 0.08 0.44 + 0.02 * * 0.40 + 0.03 * * 6.62+ 0.38 * * 1.405:0.08 ** 20.87+ 1.80 ** 24.28+2.18 **
Statistical significance: * P < 0.05, ** P < 0.001.
Statistical analysis of results
creased (P < 0.001). The plasma concentrations of insulin were significantly decreased ( - 128%, P < 0.001). When expressed on a dry weight basis, experimental hypothyroidism caused significant increases in the rates of glucose absorption ( + 32%, P < 0.01) and transmural transport ( + 123%, P < 0.001), whereas it caused a significant decrease in the rate of glucose utilization ( - 29%, P < 0.01; Table II). The rate of lactate release into the serosal medium was significantly decreased ( - 7 8 % , P < 0.01) so as the total rate of lactate production ( - 74%, P < 0.01). The percentage of glucose utilized converted into lactate was also significantly reduced (31.3 + 6.5 to 12.0 + 1.7%, P < 0.05) in hypothyroid rats (Table II). In contrast, when the data were expressed on a length basis, there was no significant change in the rate of glucose absorption. However, there was a significant change in all other rates, especially that of glucose utilization, which was diminished by 43% (P < 0.001). Table III shows the maximal activities of hexoki-
Results are given as mean + S.E. for the indicated number of observations. Statistical significance was assessed using Student's t-test. Results Table I details the general characteristics of the rats made hypothyroid by administration of 6-n-propyl-2thiouracil. Thus the gain of the body weight of the hypothyroid rats was significantly less than that of their pair-fed control (P < 0.05). The mucosal concentration of fructose 2,6-bisphosphate was significantly decreased ( - 5 6 % , P < 0.05) in hypothyroid rats. No significant differences were seen in the plasma concentrations of glucose and lactate. The plasma concentrations of total tri-iodothyronine (TT3), free tri-iodothyronine (FT3), total thyroxine (TT4) and free thyroxine (FT 4) were significantly decreased (P < 0.001) in hypothyroid rats, whereas that of thyrotropin was significantly inT A B L E II
The rates of glucose absorption, transmural transport and metabolism measured in isolated jejunal loops from control and hypothyroid rats Experimental details are given in Materials and Methods. Rates are given as means + S.E. for 7 rats. Rate:
Glucose Absorption Transmural transport Utilization Lactate Release into the luminal peffusate Release into the serosal medium Total rate of lactate production Glucose utilized converted into lactate (%)
~ m o l / h per g dry wt.
~ m o l / h per cm
Control rats
Hypothyroid rats
Control rats
Hypothyroid rats
413 167 246
547 372 175
20.6+0.8 8.3+0.7 12.2+0.8
21.6+1.1 14.7+ 1.0 * * * 6.9+0.4 * * *
2.0:t:0.6 5.9 ± 1.4 8.0 ± 1.9
0.6+0.2 * 1.0 ± 0.04 * * 1.6 + 0.2 * *
+17 + 14 +15
41 + 119 + 160 + 31.3+
12 29 38 6.5
+ 2 9 ** +25 *** + 1 0 **
15 + 26 ± 41 ± 12.0±
4 * 1** 5 ** 1.7 *
P values are given for the comparison of experimental and normal control perfusions: * P < 0.05; * * P < 0.01, * * * P < 0.001.
79 TABLE III
Maximal activities of hexokinase, 6-phosphofructo-l-kinase and pyrurate kinase in intestinal mucosal scrapings of control and hypothyroid rats Values are presented as means+S.E, with the number of rats given in parentheses. For experimental details, see Materials and Methods. Enzyme
Activity ( U / g tissue wt.)
Hexokinase
6-Phosphofructo-l-kinase Pyruvate kinase
Control rats
Hypothyroid rats
2.165:0.08 (6) 11.62:t: 0.30 (8) 38.21 5:1.49 (6)
1.485:0.06 * (6) 7.55 5:0.29 * * (8) 20.115:1.78 * * (6)
Values significantly different from controls are shown: * P < 0.01, ** P < 0.001.
nase, 6-phosphofructo-l-kinase and pyruvate kinase in intestinal mucosa of hypothyroid and pair-fed control rats. The activities of these enzymes measured were significantly decreased ( - 4 6 % , P < 0.01 for hexokinase; - 5 4 % , P < 0.001 for 6-phosphofructo-l-kinase; - 90%, P < 0.001 for pyruvate kinase) in the intestinal mucosa of the hypothyroid rats (Table III). The regulatory properties of 6-phosphofructo-1kinase from intestinal mucosa partially purified by chromatography on Sephadex G-100 are presented in Fig. 1. Fig. 1A shows the fructose 6-phosphate saturation curve of mucosal 6-phosphofructo-l-kinase measured under suboptimal conditions in the presence of 2.5 mM ATP at pH 7.0. In hypothyroid rats; the curve is displaced to higher fructose 6-phosphate concentrations and as a result, S0.5, the concentration of fructose 6-phosphate required to achieve half-maximal saturation, was shifted from 0.4 mM in the controls to 0.6 mM in the hypothyroid rats. This increase in S0.5 is a
reflection of an increased sensitivity to inhibition by ATP in hypothyroid rats (Fig 1B). Discussion
The present study shows that the rates of glucose transport and metabolism were changed in the whole intestine of hypothyroid compared with control rats. The rates of glucose utilization and lactate production were strongly inhibited, whether expressed on a dry weight or length basis. Three factors contributing to this inhibition were established. First, the concentrations of three key regulatory glycolytic enzymes, namely hexokinase, 6-phosphofructo-l-kinase and pyruvate kinase, were significantly diminished (Table III). Secondly, the regulatory properties of 6-phosphofructo-1kinase, which has been shown to be the principal rate-limiting enzyme of glycolysis in rat small intestine [5], were altered. In particular, the enzyme in hypothyroid rats showed a modest increase in susceptibility to inhibition by ATP (Fig. 1). It should be noted that this appeared to be a permanent change in the properties of the enzyme, independent of changes in the concentrations of effectors, since the assays were performed using chromatographed enzyme. Such a permanent change in properties might arise through phosphorylation as happens for the liver isoenzyme [22], antiserum to which strongly cross-reacts with the intestinal isoenzyme [23]. Thirdly, the concentration of fructose 2,6bisphosphate, a powerful activator of 6-phosphofructo-l-kinase, was diminished in hypothyroid rats. This finding is therefore consistent with the idea that fructose 2,6-bisphosphate plays an important role in the regulation of 6-phosphofructo-l-kinase and glycolysis in rat small intestine [24]. Since glycolysis in rat
1.0
®
~°I ® ~l> 0.5,
>1 > o.s
o
0.5 [Fructose-6-phosphatal (mM)
1.o 20
I
1
I
I
2 3 4 [ATP] (raM) Fig. 1. Regulatory properties of mucosal 6-phosphofructo-l-kinase in hypothyroid and control rats. The e n z y m e w a s p a r t i a l l y purified on Sephadex G-100; assays were performed at pH 7.0 as described in Materials and Methods and the activity at pH 7.0, v, is e x p r e s s e d as a ratio to that at pH 8.0, V. (A) Fructose 6-phosphate saturation curve determined at 2.5 raM ATP; (B) ATP inhibition curve determined at 0.5 mM fructose 6-phosphate. (e) Control rats; (o) hypothyroid rats.
80 small intestine is known to be regulated by insulin [5,6] and as the plasma insulin concentration in hypothyroid rats is about 44% of that in control rats, it seems likely that changes in insulin levels, in addition to the changes in thyroid hormone levels, were also involved in the regulation of the metabolic changes observed. The absolute rate of glucose oxidation, given by the rate of glucose utilization - 0.5 x total rate of lactate production, was unchanged in the hypothyroid state, in agreement with the report of Bronk and Parsons [25]. Table II shows that both the rates of glucose absorption and transmural transport were significantly enhanced when expressed on dry weight basis. However, when the rate of absorption was expressed on per cm of intestine basis, no significant change was observed. Thus the enhancement of absorption per unit weight was almost exactly compensated by a diminution in the mass of intestine per unit length in the hypothyroid state. Since the overall length of intestine was unaltered, then, from the physiological point of view, it is unlikely that there is any change in the capacity of the whole intestine in vivo to absorb glucose from the lumen in hypothyroid state. Nevertheless, the enhancement of transmural transport across the vascular side was still as much as 77% (P < 0.001) when expressed on a length basis, since 68% of the glucose absorbed was transported unchanged compared with about 40% in control rats. This reflects the strong inhibition of glucose utilization and shows clearly the interrelationship of metabolism and transport in this tissue. References 1 Remesy, C., Demigne, C. and Autrene, J. (1978) Biochem. J. 170, 321-329.
2 Shapiro, A. and Shapiro, B. (1979) Biochim. Biophys. Acta 586, 123-127. 3 Pritchard, P.J. and Porteous, J.W. (1977) Biochem. J. 164, 1-14. 4 Nicholls, T.J., Leese, H.J. and Bronk, J.R. (1983) Biochem. J. 212, 183-187. 5 Kellett, G.L., Jamal, A., Robertson, J.P. and Wollen, N. (1984) Biochem. J. 219, 1027-1035. 6 Wo|len, N. and Kellett, G.L. (1988) Gut 29, 1064-1069. 7 Levin, R.J. (1969) J. Endocrinol. 45, 315-348. 8 Ardawi, M.S.M. and Jalahah, S.M. (1991) Clin. Sci. 81,347-355. 9 Hue, L. and Rider, M.H. (1987) Biochem. J. 245, 313-324. 10 Lowry, M. and Kellett, G.L. (1989) Biochem. J. 259, 624-626. 11 Ingbar, S.H. and Woeber, K.A. (1981) in Williams, R.H., (ed.), The thyroid gland, Textbook of endocrinology, pp. 117-247, W.B. Saunders, Philadelphia. 12 Hedge, G.A., Colby, H.D. and Goodman, R.L. (1987) Clinical endocrine physiology, W.B. Saunders, Philadelphia. 13 Kellett, G.L. and Barker, E.D. (1989) Biochim. Biophys. Acta 979, 311-315. 14 Ling, K.H., Marcus, F. and Lardy, H.A. (1965) J. Biol. Chem. 240, 1893-1899. 15 Jamal, A. and Kellett, G.L. (1983) Biochem. J. 210, 129-135. 16 Crabtree, B. and Newsholme, E.A. (1972) Biochem. J. 126, 49-58. 17 Zammit, V.A., Beis, I. and Newsholme, E.A. (1978) Biochem. J. 174, 989-998. 18 Lowry, O.H., Rosebrough, N.J., Farr, A.L. and Randall, R.J. (1951) J. Biol. Chem. 193, 265-275. 19 Van Schaftingen, E., Lederer, B., Bartrons, R. and Hers, H.G. (1982) Eur. J. Biochem. 129, 191-195. 20 Bergmeyer, H.U., Bernt, E., Schmidt, F. and Stork, H. (1974) in Methods of Enzymatic Analysis (Bergmeyer, H.U., ed.), pp. 1196-1202, Academic Press, London. 21 Gawehn, K. and Bergmeyer, H.U. (1974) in Methods of Enzymatic Analysis (Bergmeyer, H.U., ed.), pp. 1492-1495, Academic Press, London. 22 Kagimoto, T. and Uyeda, K. (1979) J. Biol. Chem. 254, 5584-5587. 23 Khoja, S.M. and Kellett, G.L. (1983) Biochem. J. 215, 335-341. 24 Khoja, S.M. (1986) Comp. Biochem. Physiol. 85B, 337-341. 25 Bronk, J.R. and Parsons, D.S. (1965) J. Physiol. 179, 323-332.