Induction of control genes in intestinal gluconeogenesis is sequential during fasting and maximal in diabetes

Gilles Mithieux, Isabelle Bady, Amandine Gautier, Martine Croset, Fabienne Rajas, and Carine Zitoun

Institut National de la Santé et de la Recherche Médicale 449, Faculté Laennec, 69372 Lyon, France

Submitted 1 July 2003 ; accepted in final form 9 October 2003


    ABSTRACT
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
We studied in rats the expression of genes involved in gluconeogenesis from glutamine and glycerol in the small intestine (SI) during fasting and diabetes. From Northern blot and enzymatic studies, we report that only phosphoenolpyruvate carboxykinase (PEPCK) activity is induced at 24 h of fasting, whereas glucose-6-phosphatase (G-6-Pase) activity is induced only from 48 h. Both genes then plateau, whereas glutaminase and glycerokinase strikingly rebound between 48 and 72 h. The two latter genes are fully expressed in streptozotocin-diabetic rats. From arteriovenous balance and isotopic techniques, we show that the SI does not release glucose at 24 h of fasting and that SI gluconeogenesis contributes to 35% of total glucose production in 72-h-fasted rats. The new findings are that 1) the SI can quantitatively account for up to one-third of glucose production in prolonged fasting; 2) the induction of PEPCK is not sufficient by itself to trigger SI gluconeogenesis; 3) G-6-Pase likely plays a crucial role in this process; and 4) glutaminase and glycerokinase may play a key potentiating role in the latest times of fasting and in diabetes.

glucose-6-phosphatase; phosphoenolpyruvate carboxykinase; glutaminase; glycerokinase


AT VARIANCE WITH THE PREVIOUS VIEW that only the liver and kidney are gluconeogenic organs because both are the only organs to express glucose-6-phosphatase (G-6-Pase) (1, 29, 32), we recently demonstrated (35) that the small intestine (SI) also expresses the enzyme in humans and rats. In addition, the SI G-6-Pase gene is strongly induced in 48-h-fasted and streptozotocin-induced diabetic rats (35), just as it is in both the liver and the kidney (31). We then showed that this confers on the SI the capacity to contribute to ~20% of total glucose production in 48-h-fasted rats (11). We further demonstrated that glutamine is the main precursor of glucose synthesized in the SI (11), making glutaminase and phosphoenolpyruvate carboxykinase (PEPCK) two major control genes in SI gluconeogenesis (30, 36). On the other hand, the expression, without induction in insulinopenia, of the glycerokinase gene may account for the lesser role of glycerol as a possible glucose precursor in the SI (11). In contrast, alanine and lactate, i.e., the two major liver gluconeogenic substrates, are not glucose precursors in the rat SI (11).

In previous studies related to fasting, we reported that, after a period of induction lasting 48 h, G-6-Pase activity is surprisingly decreased at 72 h in the liver of rats, whereas in contrast it continuously increases in the kidney during the same time (28). This suggests that the liver might have a decreasing role and/or that other sources of glucose, e.g., the kidney (33), might have increasing roles in whole body glucose production during fasting (29). An additional intriguing observation is that, after near-total depletion during the first 2 days of fasting, a rebound of liver glycogen stores occurs at 72 h of fasting in rats (27). Noteworthy, a paradoxical storage of glycogen also takes place in the liver of alloxan- and streptozotocin-diabetic rats, which in addition is persistent during fasting (14, 15, 40). Because portal glucose plays a major role in liver glycogen deposition either as a substrate or as a regulator (3, 18, 21), the idea that the SI might produce more and more glucose as long as fasting lasts or in diabetes has constituted an attractive hypothesis. Because the metabolic changes accompanying fasting take place progressively compared with diabetes, we have taken advantage of the former to have a better understanding of the molecular mechanisms underlying SI gluconeogenesis in these dramatic insulinopenic states.

With this aim, we studied the expression of key genes/enzyme activities involved in gluconeogenesis and in the metabolism of SI glucose precursors, i.e., glutamine and glycerol, in fasted and in diabetic rats. In parallel, we quantified SI glucose production at fasting times not studied up to now, i.e., at 24 and 72 h. Because our preceding studies had considered only three regions, i.e., duodenum, jejunum, and ileum, we have specified better the expression profile of the G-6-Pase gene along the SI by dividing it into six parts (1 for the duodenum, 3 for the jejunum, and 2 for the ileum). Briefly, we report that SI gluconeogenesis does not take place at 24 h of fasting but may represent up to one-third of total glucose production at 72 h of fasting in rats, that PEPCK is not sufficient by itself to trigger SI glucose production, and that G-6-Pase likely plays the key role in this process. Furthermore, glutaminase and glycerokinase may have a crucial potentiating effect on SI gluconeogenesis in prolonged fasting and in diabetes.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Animals. Male Sprague-Dawley rats (260-280 g) were housed for 3 days with water and standard laboratory chow (50% starch, 23.5% proteins, 5% lipids, 4% cellulose, 5.5% mineral salts, 12% water, based on weight; UAR, Epinay sur Orge, France). Rats were deprived of food for 6- [postabsorptive (PA) state], 24-, 48-, and 72 h with free access to water. Diabetic rats were used 3 days after a single injection of streptozotocin (70 mg/kg body wt; Sigma, La Verpillière, France). Rats were anesthetized with a single intraperitoneal injection of pentobarbital sodium (6 mg/100 g body wt) and were used either for the determination of glucose kinetics or for intestine sampling. All protocols described in this study were performed according to the rules of our local ethics committee for animal experimentation at Claude Bernard University-Lyon I.

Intestine sampling. The abdomen of anesthetized rats was immediately incised, and the total intestine was rapidly removed from the abdominal cavity. It was cut into eight fragments. The first 10 cm beyond the pylorus were considered as duodenum. After removal of the cecum and the colon (large intestine), the remaining SI was divided into five fragments (representing a mean of ~15 cm each): jejunum 1, 2, and 3 and ileum 1 and 2. For all fragments, only the inner part was retained and studied; i.e., 2 cm at each edge were cut off and discarded. Inner parts were rinsed using saline solution and frozen at liquid nitrogen temperature to be stored at -80°C until use.

G-6-Pase and PEPCK mRNA analysis. Total mRNA was extracted from intestine samples by following the protocol described by Sambrook et al. (39). RNA was quantified by ultraviolet absorbance at 260 nm (260:280 ratio was >1.8), and 2 µg were electrophoresed to check the quality of the preparation. The procedure to analyze and quantify G-6-Pase and PEPCK mRNA by Northern blot was described in our previous reports (31, 35).

Determination of enzyme activities. Frozen intestine samples were reduced to powder at liquid nitrogen temperature. The powder was homogenized in 10 mM HEPES and 0.25 M sucrose, pH 7.4 (9 vol/g tissue) by ultrasonication. G-6-Pase activity was directly assayed in homogenates for 10 min at 30°C at pH 7.3 in the presence of a saturating glucose 6-phosphate concentration (20 mM). Released Pi was determined by complexometry (4). Glutaminase isoenzymes, i.e., Pi-dependent and maleate-activated forms, were assayed in homogenates obtained as described above. The former activity was estimated from an incubation for 60 min at 37°C in a mixture composed of 40 mM Tris·HCl, 20 mM glutamine, 0.2 mM EDTA, 0.02% (wt/vol) BSA, and 140 mM phosphate, pH 8.3. The latter was determined from an incubation mixture composed of 50 mM maleate, 10 mM glutamine, 0.2 mM EDTA, and 0.02% (wt/vol) BSA, pH 6.6. Glutamate was then assayed in the presence of glutamate dehydrogenase via the generation of NADH + H+ (34). PEPCK activity was assayed in the supernatant of homogenates obtained by centrifugation for 1 h at 100,000 g using the decarboxylation assay previously described by Jomain-Baum and Schramm (23) and Rajas et al. (36). Glycerokinase maximal velocity (Vmax) was determined in 100,000-g supernatants of homogenates according to the procedure described by Bergmeyer et al. (5) and Croset et al. (11). Glutamate-pyruvate transaminase (GPT) was assayed under Vmax conditions in 100,000-g supernatants obtained from SI homogenates (6). The two isoforms of glycerol-3-phosphate dehydrogenase (G3PDH), i.e., the cytosolic form (NAD-linked) and the mitochondrial (FAD-linked) were assayed in the cytosol, and a mitochondrial-enriched fraction was prepared from the respective SI homogenates, respectively (17). The Vmax of cytosolic G3PDH was assayed according to Bergmeyer et al. (5). The mitochondrial G3PDH Vmax was determined as described by Gardner (17).

Glutaminase protein analysis. Whole protein extracts were prepared with frozen tissues as described above. Forty micrograms of the protein extracts were separated by electrophoresis (SDS-PAGE), and the glutaminase protein was detected by Western blotting using an anti-kidney-type glutaminase antibody at a 1:500 dilution (generous gift of N. P. Curthoys).

Determination of intestinal and total endogenous glucose production. All procedures to quantify SI glucose fluxes by use of [3-3H]glucose infusion in anesthetized rats were described in detail in our previous report (11). The body temperature was maintained at 37.5°C by means of a rectal probe-monitored heating blanket. At the end of a 90-min infusion, the blood was simultaneously sampled in the carotid artery and superior mesenteric vein and immediately centrifuged at 4°C to separate plasma. Glucose concentration and 3H radioactivity were determined from these plasma samples. Preliminary experiments involving blood sampling at different time points (not shown) indicated that a steady state of [3-3H]glucose specific activity (SA) and glycemia was obtained during the last 60 min of the 90-min infusion experiment. Intestinal blood flows (IBF) were determined in separate groups of animals under conditions similar to those in glucose kinetics experiments, using a radiolabeled microsphere technique as described previously (11). The fractional extraction (FX) of glucose across the SI was calculated as ([3-3H]glucose SAartery x glucose concentrationartery) - ([3-3H]glucose SAvein x glucose concentrationvein)/([3-3H]glucose SAartery x glucose concentrationartery). Intestinal glucose uptake (IGU) was calculated as IBF x glucose concentrationartery x FX, and intestinal glucose balance (IGB) was calculated as IBF x (glucose concentrationartery - glucose concentrationvein). Intestinal glucose release (IGR) was calculated as IGU - IGB. Total endogenous glucose production (EGP) was obtained from the [3-3H]glucose infusion rate and the arterial blood glucose SA (11).


    RESULTS
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Molecular and enzymatic studies. G-6-Pase and PEPCK mRNA levels were both markedly increased during fasting. The increase was significant from 24 h and peaked at 48 h (Fig. 1). The analysis shown was performed from jejunum 2 mRNA, but the results were comparable in other parts of the SI (not shown). In the four parts of the SI in which G-6-Pase is expressed in the fed state, i.e., duodenum, jejunum 1, jejunum 2, and jejunum 3, the G-6-Pase Vmax was not significantly increased at 24 h. In contrast, it was markedly increased by two to three times at 48 and 72 h of fasting (Fig. 2). In agreement with previous results (35), G-6-Pase activity was not expressed in ileum (1 and 2) in the fed postabsorptive (PA) state. Expression took place, albeit at a lower level than in duodenum and jejunum, in the fasting states (Fig. 2). PEPCK Vmax was assayed in the two SI parts of strongest expression in the fed state, i.e., jejunum 1 and 2 (36). In contrast with G-6-Pase activity, PEPCK was markedly increased by two to three times from 24 h of fasting (Fig. 3).



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Fig. 1. Effect of fasting on glucose-6-phosphatase (G-6-Pase; A) and phosphoenolpyruvate carboxykinase (PEPCK; B) mRNA abundances in the rat small intestine (SI). Analysis was performed from 20 µg of total mRNA extracted from jejunum 2. Top: representative Northern blot [postabsorptive (PA) to 72 h from left to right]. Middle: densitometric analysis carried out from 3 animals at each time. Densitometry was performed compared with a standard mRNA preparation analyzed in parallel in each blot. Data are expressed as arbitrary units (AU) relative to this standard. *Significantly different from PA value, P < 0.05. C: Northern blot experiment performed using a cDNA probe for glyceraldehyde-3-phosphate dehydrogenase (GAPDH).

 


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Fig. 2. Effect of fasting on the G-6-Pase enzyme along the SI. A: expression of enzyme activity as a function of fasting time in the 6 parts of SI sampled as described in MATERIALS AND METHODS. Duo, duodenum; Jej, jejunum; Il, ileum). B: gradient of expression of G-6-Pase along the SI under a noninduced condition (PA rat, light gray bar) and an induced condition (48-h-fasted, dark gray column). Results are means ± SE from 6 rats at each fasting time. *Significantly different from PA value, P < 0.05.

 


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Fig. 3. Time course of SI PEPCK activity during fasting in rat. Analysis was performed on jejunum 1 (left) and jejunum 2 (right). Results are means ± SE (n = 6). *Significance as in Fig. 2.

 

The Pi-dependent form of glutaminase was not altered upon 24-h fasting and was slightly decreased in 48-h-fasted rats. Noteworthy, a further 40% increase was observed between 48 and 72 h of fasting (Fig. 4A). In parallel, quantification of glutaminase protein by Western blotting revealed that the amount of glutaminase was lower in 48-h-fasted rats than in other groups (Fig. 4A, inset). In diabetic rats, the Pi-dependent glutaminase activity was also 40% higher than that in 48-h-fasted rats. Furthermore, it was significantly higher (by 10%) than the glutaminase activity in normal PA rats (Fig. 4A). The maleate-dependent form was significantly decreased by ~20% in 48-h- and 72-h-fasted and diabetic rats compared with PA rats, whereas it was not affected in 24-h-fasted rats (Fig. 4A). GPT activity was slightly decreased throughout fasting (-20% at 48 h) but not in diabetes (Fig. 4B).



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Fig. 4. Time course of glutaminase (A), glutamate-pyruvate transaminase (GPT; B), glycerokinase (C), and G3PDH (D) during fasting and diabetes in rat SI. In A, Pi-dependent glutaminase activity is given by the gray bars, and the maleate-dependent glutaminase activity by open bars. Top inset: representative Western blot performed from 40 µg of total proteins extracted from jejunum 2. In D, open bars represent mitochondrial G3PDH activity and grey bars cytosolic G3PDH activity. In all panels, data found in diabetes are given by hatched bars on the right. Results are means ± SE of 4-6 animals per time. *Different from PA value (P < 0.05); °different from 48-h-fasted value (P < 0.05).

 

Glycerokinase activity was similar in fed PA and 24-h-fasted rats. Noteworthy, as noted for glutaminase, it was significantly decreased in 48-h-fasted rats, and it was markedly restored at 72 h of fasting. This represented an 80% increase between 48 and 72 h (Fig. 4C). Glycerokinase activity was comparable in 72-h-fasted and diabetic rats. G3PDH activities [cytosolic (NAD-dependent) and mitochondrial (FAD-dependent)] were not significantly altered by fasting or diabetes (Fig. 4D).

Increasing contribution of SI to glucose production during fasting. Because the IGF data in fed PA, 48-h-fasted, and diabetic rats have been previously published (11), we report here only on analogous studies performed in 24-h- and 72-h-fasted rats. In 24-h-fasted rats, [3-3H]glucose SA was not decreased in the mesenteric vein compared with the artery (Table 1). As previously argued (11), this constituted a first indication that no newly synthesized unlabeled glucose molecule had been released in the blood by the SI. The mean IGU, calculated from the individual FX and IBF, was in the same order range as the IGB. Consequently, the mean IGR was not different from zero: -4.4 ± 3.0 µmol·kg-1·min-1 (Table 1). These data strongly suggested that there was no detectable EGP by the SI in the 24-h-fasted state.


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Table 1. Intestinal glucose fluxes in fasted rats

 

In contrast, in 72-h-fasted rats, the [3-3H]glucose SA was markedly lower by 9% (P < 0.01) in the mesenteric vein compared with the artery (Table 1). This indicated that unlabeled glucose had been released by the SI. In agreement with the latter, there was no difference between glucose concentrations in the vein and those in the artery. Accordingly, the IGR was equal to the IGU, i.e., 16.9 ± 4.0 µmol·kg-1·min-1, representing 35% of total EGP (Table 1).


    DISCUSSION
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The results presented here point out the crucial quantitative role, previously unsuspected, of the SI in whole body glucose homeostasis during prolonged fasting. Particularly, in agreement with the lesser role of the liver in glucose production in long-term food deprivation (29) and further extending our previous work (11), we demonstrate that SI gluconeogenesis increasingly contributes to systemic glucose production after the first 24 h of fasting, accounting for ~20% of EGP at 48 h (11) and reaching 35% of total EGP in 72-h-fasted rats. It is of note that glycogen stores rebound in the liver at this time (27). In this former work, we had suggested that an inhibition of liver G-6-Pase flux, depending on both a decrease in gene expression and a metabolite inhibition of enzyme activity, might account, at least in part, for the liver glycogen rebound (27). It must be underlined that the release of glucose in the portal blood at this time may have a key role in the liver G-6-Pase activity suppression (18, 26, 38). It should be emphasized that the gradual replacement of liver gluconeogenesis by SI gluconeogenesis within the splanchnic bed during fasting, allowing the liver to spare glucose via a partial rebound of glycogen stores, could constitute a crucial mechanism of adaptation during lasting food deprivation.

It must be mentioned here that determination of glucose kinetics from tritiated glucose tracers is prone to possible pitfalls, related to the putative occurrence of glucose recycling through three futile cycles: the glucose/glucose 6-phosphate cycle (cycle 1), the fructose 6-phosphate/fructose 1,6-bisphosphate cycle (cycle 2), and the pyruvate/phosphoenolpyruvate cycle (cycle 3) (thoroughly reviewed in Ref. 44). Specifically, it cannot be formally excluded that the detritiation of [3-3H]glucose (utilized here to quantify SI glucose production) could be partially due to glucose recycling through cycle 2. However, the question of glucose cycling has received considerable attention in the past. It is now accepted that recycling through cycles 2 and 3 is virtually negligible in vivo in numerous species, including rat (see Ref. 24 for a review). On the other hand, cycling through cycle 1 may take place under certain situations (such as the fed state) and account for a partial detritiation of [2-3H]glucose, but in no way it can affect the detritiation of [3-3H]glucose. Moreover, we have previously shown that the greater part of glucose production by the SI in 48-h-fasted rats can be accounted for by the net incorporation of carbon-labeled glutamine and glycerol precursors (11). It is therefore likely that the 35% contribution of the SI to EGP found here represents true gluconeogenesis and not glucose cycling.

Noteworthy, the diabetic state closely resembles the 72-h-fasted situation, e.g., regarding the paradoxical liver glycogen storage (14, 15, 40) and net IGB [close to nil, even slightly negative, i.e., in the sense of a net glucose release (11)]. It is thus of interest to compare intestinal glucose fluxes in both situations. With regard to the diabetic state, the release of glucose by the SI is ascertained because of 1) the significant decrease in the [3-3H]glucose specific activity in the portal compared with the arterial blood; 2) the slight but significant increase in the plasma glucose concentration in the portal vs. the arterial blood; and 3) the net flux of glutamine carbons into glucose by passage through the SI (11). However, we previously failed to accurately quantify IGR because of the insufficient precision of tracer methodology in the determination of the fractional extraction of glucose and, consequently, of IGU (11). To obviate this problem, we have here calculated IGU in diabetic rats from lactate balance determination (not shown) on the basis of the previous works of Windmueller and Spaeth (43), who reported the molar ratio of glucose uptake to lactate release in a similar insulinopenic state. Strikingly, combined with the net glucose balance, this has allowed us to estimate an IGR accounting for 39% of total EGP, i.e., in the same order range as that determined in the 72-h-fasting state (35%; see Table 1). Even if such a calculation may appear speculative, it suggests that the contribution of the SI to EGP should be quantitatively comparable in these two insulinopenic states, representing at least one-third of EGP. However, it must be emphasized that, in contrast with the fasted state where the beneficial role of the SI in maintaining whole body glucose homeostasis seems obvious, the SI glucose production in the diabetic state might rather constitute a worsening factor in the dysregulations of glucose metabolism. Indeed, in addition to the increased expression of intestinal monosaccharide transporters, reported to occur in experimental diabetes in animals (8, 13) and in type 2 diabetes in humans (12), which likely enhances intestinal glucose absorption in the postprandial state, the occurrence of SI gluconeogenesis may further add to the complexity of the disease by causing more glucose to enter the system in the postabsorptive and/or postprandial situations.

It is of note that we could not detect glucose production by the SI at 24 h of fasting, whereas the SI PEPCK gene, which is generally considered the main regulatory gene in the commencement of gluconeogenesis in the liver, is already induced at both the mRNA and enzyme levels at this time (results of Figs. 1 and 3). However, several roles have been proposed for PEPCK in the SI, e.g., replenishment of the pyruvate pool (41, 42). The latter may allow the portal production of lactate and alanine (42) to be sustained, which may further be used by the liver gluconeogenesis (30). This pathway might be especially operative during short-term (24-h) fasting. A putative involvement of PEPCK in glyceroneogenesis, to maintain an active level of reesterification of free fatty acids during fasting, has also been suggested (36, 37). In contrast with PEPCK, the G-6-Pase activity is not increased before 48 h of fasting (Fig. 2), even if gene induction at the mRNA level occurs at 24 h (Fig. 1). This feature is true in the four major parts (duodenum and 3 jejunum) of the SI relating to gluconeogenesis, i.e., of strongest expression of G-6-Pase and/or PEPCK in the fed, 48-h-fasted, and streptozotocin-diabetic states (35, 36). The concomitant G-6-Pase activity induction and SI glucose production at 48 h of fasting (11), but not at 24 h, strongly suggests that the G-6-Pase gene plays a more crucial role than the PEPCK gene in the occurrence of gluconeogenesis in the SI. It must be specified that we have not studied the expression of fructose-1,6-bisphosphatase gene and activity here, because, as previously noted (11), the latter is expressed at very substantial, nonlimiting levels in the SI in various species (2, 7, 16).

Interestingly, SI glucose production further markedly increases between 48 and 72 h of fasting, whereas both G-6-Pase and PEPCK activities plateau within this period. We have thus studied the expression of the specific enzymes involved in the metabolism of the two substrates of SI gluconeogenesis, i.e., glutamine and glycerol, respectively. Regarding glutamine utilization, a slight decrease in the major rate-limiting Pi-dependent form of glutaminase occurs between 24 and 48 h of fasting, in agreement with previously reported data (25). Interestingly, a marked reincrease occurs at 72 h. Our results from Western blotting strongly suggest that this biphasic evolution pattern is, at least in part, dependent on a decrease and reinduction of glutaminase protein expression. These data are in strong agreement with the previous observation that the capacity of glutamine utilization of the SI is potentiated in long-term-compared with short-term-fasted dog (9). On the other hand, we report that GPT, which is responsible for glutamate transamination to {alpha}-ketoglutarate and incorporation into the tricarboxylic acid cycle, is slightly decreased when fasting is prolonged. This is also in keeping with previous results (22). However, this should not alter glutamine utilization, because the huge GPT activity expressed cannot be rate limiting in regard to glutaminase, irrespective of the fasting time. In the same manner, the rate-limiting enzyme activity responsible for glycerol utilization (i.e., glycerokinase) decreases up to 48 h of fasting to further reincrease to a level similar to the fed level at 72 h. On the other hand, neither the cytosolic nor the mitochondrial G3PDH, the enzymes responsible for the conversion of glycerol 3-phosphate in dihydroxy-acetone phosphate and integration in the gluconeogenesis pathway, is significantly affected upon fasting. The reinduction of both rate-limiting enzymes responsible for the utilization of the two SI glucose precursors after 48 h, in addition to the sustained overexpression of both G-6-Pase and PEPCK, is in keeping with the further augmentation of SI glucose production at 72 h of fasting. It is of note that all enzymes involved in the metabolism of SI glucose precursors, particularly the two rate-limiting glutaminase and glycerokinase (Fig. 4), and in gluconeogenesis, i.e., G-6-Pase and PEPCK (35, 36), are fully expressed in the diabetic situation. This is in line with the estimation (see above) that IGR might be quantitatively comparable in both the 72-h-fasted and the diabetic states.

In conclusion, we report here that the SI increasingly contributes to whole body glucose production when fasting is prolonged in rat, up to a participation representing 35% of total EGP at 72 h. Together with the likely concomitant, increasing role of the kidney (1, 31, 33), this strongly suggests that the role of the liver in systemic glucose production during fasting may be, at the very least in rat, far less important than previously thought. Our results also strongly suggest that the SI may contribute to about one-third of total EGP in the diabetic state, which may further add to the complexity of the disease. We show that the induction of PEPCK activity is not sufficient by itself to trigger SI glucose production and that the G-6-Pase enzyme likely plays the major role in this phenomenon. Glutaminase and glycerokinase, accounting for the utilization of the two SI glucose precursors, may play additional potentiating roles at the latest times of fasting and in diabetes. These data further point out the essential role of the SI in whole body glucose production in insulinopenic states.


    ACKNOWLEDGMENTS
 
We greatly appreciated helpful discussions with Dr. M. Beylot about glucose tracer analysis.


    FOOTNOTES
 

Address for reprint requests and other correspondence: G. Mithieux, INSERM 449, Faculté Laennec, 69372 Lyon, cedex 08, France (e-mail: mithieux{at}laennec.univ-lyon1.fr).

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


    REFERENCES
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Adrogue H. Glucose homeostasis and the kidney. Kidney Int 42: 1266-1282, 1992.[ISI][Medline]
  2. Anderson J. Glucose metabolism in jejunal mucosa of fed, fasted, and streptozotocin-diabetic rats. Am J Physiol 226: 226-229, 1974.[Free Full Text]
  3. Assimacopoulos-Jeannet F and Jeanrenault B. Insulin activates 6-phosphofructo-2-kinase and pyruvate kinase in the liver. J Biol Chem 265: 7202-7206, 1990.[Abstract/Free Full Text]
  4. Baginsky E, Foa P, and Zak B. Glucose-6-phosphatase. In: Methods of Enzymatic Analysis, edited by Bergmeyer HU. New York: Academic, 1974, p. 876-880.
  5. Bergmeyer H, Gawehn K, and Grassl M. Enzymes as biochemical reagents. In: Methods of Enzymatic Analysis, edited by Bergmeyer HU. New York: Academic, 1974, p. 425-522.
  6. Bergmeyer HU and Bernt E. Glutamate-pyruvate transaminase. In: Methods of Enzymatic Analysis, edited by Bergmeyer HU. New York: Academic, 1974, p. 732-750.
  7. Bismut H, Hers H, and Van Schaftingen E. Conversion of fructose to glucose in rabbit small intestine. Eur J Biochem 213: 721-726, 1993.[Abstract]
  8. Burant C, Flink S, De Paoli A, Chen J, Lee W, Heoliger M, Buse J, and Chang E. Small intestine hexose transport in experimental diabetes. Increased transporter mRNA and protein expression in enterocytes. J Clin Invest 93: 578-585, 1994.[ISI][Medline]
  9. Cersosimo E, Williams P, and Radosewith P. Role of glutamine in adaptations in nitrogen metabolism during fasting. Am J Physiol Endocrinol Metab 250: E622-E628, 1986.[Abstract/Free Full Text]
  10. Chatelain F, Pegorier J, Minassian C, Bruni N, Tarpin S, Girard J, and Mithieux G. Development and regulation of glucose-6-phosphatase gene expression in rat liver, intestine and kidney. Diabetes 47: 882-889, 1998.[Abstract]
  11. Croset M, Rajas F, Zitoun C, Hurot J, Montano S, and Mithieux G. Rat small intestine is an insulin-sensitive gluconeogenic organ. Diabetes 50: 740-746, 2001.[Abstract/Free Full Text]
  12. Dyer J, Wood IS, Palejwala A, Ellis A, and Shirazi-Beechey SP. Expression of monosaccharide transporters in intestine of diabetic humans. Am J Physiol Gastrointest Liver Physiol 282: G241-G248, 2002.[Abstract/Free Full Text]
  13. Fedorak R, Cheeseman CI, Thomson AB, and Porter VM. Altered glucose carrier expression: mechanism of intestinal adaptation during streptozotocin-induced diabetes in rats. Am J Physiol Gastrointest Liver Physiol 261: G585-G591, 1991.[Abstract/Free Full Text]
  14. Friedmann B, Goodman EH Jr, and Weinhouse S. Liver glycogen synthesis in intact alloxan diabetic rats. J Biol Chem 238: 2899-2905, 1963.[Free Full Text]
  15. Friedmann B, Goodman EH Jr, and Weinhouse S. Dietary and hormonal effects on gluconeogenesis in the rat. J Biol Chem 240: 3729-3735, 1965.[Free Full Text]
  16. Froesh R. Essential fructosuria and hereditary fructose intolerance. In: The Metabolic Basis of Inherited Disease, edited by Stanbury J, Wyngaarden J, and Fredrickson D. New York: McGraw-Hill, 1972, p. 131-138.
  17. Gardner R. A sensitive colorimetric assay for mitochondrial {alpha}-glycero-phosphate dehydrogenase. Anal Biochem 59: 272-276, 1974.[ISI][Medline]
  18. Guignot L and Mithieux G. Mechanisms by which insulin, associated or not with glucose, may inhibit hepatic glucose production in the rat. Am J Physiol Endocrinol Metab 277: E984-E989, 1999.[Abstract/Free Full Text]
  19. Hahn P and Smale FA. Phosphoenolpyruvate carboxykinase in the small intestine of developing rodents. J Nutr 115: 986-989, 1982.
  20. Hahn P and Wei-Ning H. Gluconeogenesis from lactate in the small intestinal mucosa of suckling rats. Pediatr Res 20: 1321-1323, 1986.[Abstract]
  21. Halimi S, Assimacopoulos-Jeannet F, Terretaz J, and Jeanrenault B. Differential effect of steady-state hyperinsulinemia and hyperglycemia on hepatic glycogenolysis and glycolysis in rats. Diabetologia 30: 268-272, 1987.[ISI][Medline]
  22. Hsu L and Tarver H. Distribution of the activity of various hydrolases, transaminases, and glutamic dehydrogenases in the digestive tracts of fed and fasted rats. Gastroenterology 53: 78-87, 1967.[ISI][Medline]
  23. Jomain-Baum M and Schramm V. Kinetic mechanism of phosphoenolpyruvate carboxykinase (GTP) from rat liver cytosol. J Biol Chem 253: 3648-3659, 1978.[Abstract]
  24. Katz J. Use of isotopes for the study of glucose metabolism in vivo. In: Techniques in Metabolic Research, edited by Kornberg HL. Amsterdam: Elsevier/North Holland, 1979, B207: p, 1-22.
  25. Kong S, Hall J, Cooper D, and McCauley R. Starvation alters the activity and mRNA level of glutaminase and glutamine synthetase in the rat intestine. J Nutr Biochem 11: 393-400, 2000.[CrossRef][ISI][Medline]
  26. Mevorach M, Giacca A, Aharon Y, Hawkins M, Shamoon H, and Rossetti L. Regulation of endogenous glucose production by glucose per se is impaired in type 2 diabetes mellitus. J Clin Invest 102: 744-753, 1998.[Abstract/Free Full Text]
  27. Minassian C, Ajzannay A, Riou J, and Mithieux G. Investigation of the mechanism of glycogen rebound in the liver of 72-hour fasted rats. J Biol Chem 269: 16585-16588, 1994.[Abstract/Free Full Text]
  28. Minassian C, Zitoun C, and Mithieux G. Differential time course of liver and kidney glucose-6 phosphatase activity during long-term fasting in rat correlates with differential time course of messenger RNA level. Mol Cell Biochem 155: 37-41, 1996.[ISI][Medline]
  29. Mithieux G. New knowledge regarding glucose-6 phosphatase gene and protein and their roles in the regulation of glucose metabolism. Eur J Endocrinol 136: 137-145, 1997.[ISI][Medline]
  30. Mithieux G. New data and concepts on glutamine and glucose metabolism in the gut. Curr Opin Clin Nutr Metab Care 4: 267-271, 2001.[CrossRef][ISI][Medline]
  31. Mithieux G, Vidal H, Zitoun C, Bruni N, Daniele N, and Minassian C. Glucose-6-phosphatase mRNA and activity are increased to the same extent in kidney and liver of diabetic rats. Diabetes 45: 891-896, 1996.[Abstract]
  32. Mittelman S and Bergman R. Liver glucose production in health and diabetes. Curr Opin Endocrinol Diabetes 5: 126-135, 1999.
  33. Owen OE, Felig P, Morgan AP, Wahren J, and Cahill GF. Liver and kidney metabolism during prolonged starvation. J Clin Invest 48: 574-583, 1969.[ISI][Medline]
  34. Passonneau J and Lowry OH. Specific methods and procedures. In: Enzymatic Analysis. A Pratical Guide, edited by Passonneau J. Totowa, NJ: Humana, 1993, p. 260-261.
  35. Rajas F, Bruni N, Montano S, Zitoun C, and Mithieux G. The glucose-6 phosphatase gene is expressed in human and rat small intestine: regulation of expression in fasted and diabetic rats. Gastroenterology 117: 132-139, 1999.[ISI][Medline]
  36. Rajas F, Croset M, Zitoun C, Montano S, and Mithieux G. Induction of PEPCK gene expression in insulinopenia in rat small intestine. Diabetes 49: 1165-1168, 2000.[Abstract]
  37. Reshef L, Hanson R, and Ballard F. A possible physiological role for glyceroneogenesis in rat adipose tissue. J Biol Chem 245: 5979-5984, 1970.[Abstract/Free Full Text]
  38. Rossetti L, Giaccari A, Barzilai N, Howard K, Sebel G, and Hu M. Mechanism by which hyperglycemia inhibits hepatic glucose production in conscious rats. J Clin Invest 92: 1126-1134, 1993.[ISI][Medline]
  39. Sambrook J, Frittsch E, and Maniatis T. Molecular Cloning: a Laboratory Manual. Cold Spring Harbor, NY: Cold Spring Harbor, 1989.
  40. Van de Werve G, Sestoft L, Folke M, and Kristensen LO. The onset of liver glycogen synthesis in fasted-refed rats. Diabetes 33: 944-949, 1984.[Abstract]
  41. Watford M. Glutamine metabolism in rat small intestine: synthesis of three-carbon products in isolated enterocytes. Biochim Biophys Acta 1200: 73-78, 1994.[ISI][Medline]
  42. Windmueller H. Metabolism of vascular and luminal glutamine by intestinal mucosa in vivo. In: Glutamine Metabolism in Mammalian Tissues, edited by Haüssinger D and Sies H. Berlin: Springer-Verlag, 1984, p. 61-67.
  43. Windmueller HG and Spaeth AE. Identification of ketone bodies and glutamine as the major respiratory fuels in vivo for postabsorptive rat small intestine. J Biol Chem 253: 69-76, 1978.[ISI][Medline]
  44. Wolfe RR. Specific applications: glucose metabolism. In: Tracers in Metabolic Research, edited by Wolfe RR. New York: Liss, 1984, chapt. 9, p. 113-125.