9 except the crystallization step was omitted. The crude 2,5-AM1- ol, in 5 mm ammonium borate (ph 9), was purified on Dowex-

Transcription

1 Proc. Natl. Acad. Sci. USA Vol. 8, pp , July 1983 Biochemistry Regulation of carbohydrate metabolism by 2,5-anhydro-D-mannitol (gluconeogenesis/glycolysis/fructose-1,6-bisphosphatase/phosphofructokinase 1/pyruvate kinase) PATRICIO T. RIQUELME, MARY ELLEN WERNETTE-ANMIIOND, NANCY M. KNEER, AND ENRY A. LARDY Institute for Enyme Research and the Department of Biochemistry. University of Wisconsin. Madison, Wisconsin 5376 Contributed by enry A. Lardy, April 25, 1983 ABSTRACT In hepatocytes isolated from fasted rats, 2,5-anhydromannitol inhibits gluconeogenesis from lactate plus pyruvate and from substrates that enter the gluconeogenic pathay as triose phosphate. This fructose analog has no effect, hoever, on gluconeogenesis from xylitol, a substrate that enters the pathay primarily as fructose 6-phosphate. The sensitivity of gluconeogenesis to 2,5-anhydromannitol depends on the substrate metabolied; concentrations of 2,5-anhydromannitol required for 5% inhibition increase in the order lactate plus pyruvate < dihydroxyacetone < glycerol < sorbitol < fructose. The inhibition by 2,5-anhydromannitol of gluconeogenesis from dihydroxyacetone is accompanied by an increase in lactate formation and by to distinct crossovers in gluconeogenic-glycolytic metabolite patterns i.e., increases in pyruvate concentrations ith decreases in phosphoenolpyruvate and increases in fructose-1,6-bisphosphate concentrations ith little change in fructose 6-phosphate. In addition, 2,5-anhydromannitol blocks the ability of glucagon to stimulate gluconeogenesis and inhibit lactate production from dihydroxyacetone. 2,5-Anhydromannitol decreases cellular fructose 2,6-bisphosphate content in hepatocytes; therefore the effects of the fructose analog are not mediated by fructose 2,6-bisphosphate, a naturally occurring allosteric regulator. 2,5-Anhydromannitol also inhibits gluconeogenesis in hepatocytes isolated from fasted diabetic rats, but higher concentrations of the analog are required. 2,5-Anhydro-D-mannitol (2,5-AM-ol), an analog of P-D-fructose locked in the furan ring structure, is phosphorylated by fructokinase to form 2,5-AM-ol-l-P (1, 2). O2( 5 O 2,5 - ANYDRO-D- MANNITOL O R-D- FRUCTOSE /3-D-FRUCTOSE- I-P /3 -D- FRUCTOSE- 1,6-P. x PO3 PO3 Because 2,5-AM-ol is symmetrical, the monophosphate product can be considered an analog of both fructose-i-p and fructose-6-p (3). Because of the stability of its ring structure, 2,5- AMI-ol monophosphate cannot be cleaved bv aldolase in a manner similar to that of fructose-l-p, nor can it act as a substrate for phosphoglucoisomerase and be converted to glucose-6-p in a manner similar to that of fructose-6-p. 2,5-AM-ol monophosphate is a substrate for phosphofructokinase 1 (, 5). The resulting product, 2,5-AM-ol bisphosphate, is an analog of 8- fructose-1,6-p2 rather than a-fructose-1,6-p2 and, as such, is not hydrolyed readily by fructose-1,6-bisphosphatase, hich The publication costs of this article ere defrayed in part by page charge payment. This article must therefore be herebv marked "adcertiseinleut 'in accordance ith 18 U. S.C. 173 solely to indicate this fact. y PO= prefers the a anomer (6). The bisphosphate compound, thus, should accumulate ithin the cell. In vitro experiments have shon that 2,5-AM-ol bisphosphate is a competitive inhibitor of fructose-1,6-bisphosphatase (7, 8). In vie of the described findings, the potential of 2,5-AM-ol to act as a regulator of gluconeogenesis and glycolysis as examined in isolated rat hepatocytes. METODS AND MATERIALS Synthesis of 2,5-AM-ol. 2,5-AM-ol as prepared as in ref. 9 except the crystalliation step as omitted. The crude 2,5-AM1- ol, in 5 mm ammonium borate (p 9), as purified on Doex- I-X-8 (borate) (1). The unretained material, after deioniation and repeated concentration by evaporation of methanol, shoed only one spot on paper or thin-layer chromatography (5, 9). 2,5- AM-ol as stored froen as an aqueous solution (2 M) containing.1mm EDTA. Isolation and Incubation of epatocytes. Unless otherise noted, cells ere isolated from livers of normal rats fasted 2 hr and ere incubated as previously described (11). Ca>2 (1.3 mm) as included in all incubation mixtures. Diabetes as induced in rats fasted overnight by intravenous injection of alloxan at mg/kg of body eight and as verified by blood glucose concentrations greater than 3 mg/1 ml after 1 eek. epatocytes isolated from livers of diabetic rats fasted 26-3 hr ere incubated in Krebs-enseleit medium containing 1.3% bovine serum albumin ith oxygenation at 37C for 5 min to deplete residual glycogen stores, then ashed and incubated as usual. Basal glucose production as )umol per min per g et eight. Similar preliminary incubation of hepatocytes isolated from fasted normal rats did not alter their behavior. Measurement of Glucose and Lactate Production. Glucose and lactate ere determined enymatically as described (11). Determination of Gluconeogenic-Glycolytic Intermediates. epatocytes (.5-.5 g et eight in 12 ml total volume) ere incubated under 95% 2/5% CO2 in sealed 125-ml plastic bottles. After 15 min, to 5-ml aliquots ere removed from each flask and the hepatocytes ere concentrated through a bromodecane/dodecane mixture into 1. ml of 6% perehloric acid/1 mm isocitrate as in ref. 12. An additional 1.2 ml of each suspension as added to perchloric acid directly for determinations of total glucose and pyruvate concentrations. Duplicate samples ere pooled and neutralied ith KO. Metabolites in the neutralied extracts, except for fructose- 1,6-P2, ere assayed spectrophotometrically bv established envmatic methods (13). Fructose-1,6-P, as assaved ith pig kidney fructose-1,6-bisphosphatase (.5 unit/ml final concentration) and coupled to NADP production via phosphoglucose isomerase and glucose-6-p dehydrogenase. Pig kidney fructose- Abbreviations: 2,5-ANi-ol, 2,5-anhydromannitol; dibutvrvl-canip, N6,2'- dil)utv\rvrladenosinie 3',5'-cNvclic m*onophosphlate. I? 31

2 32 Biochemistry: Riquelme et al. Proc. Natl. Acad. Sci. USA 8 (1983) minated by the addition of.15 ml of.55 M NaO. Fructose- 2,6-P2 concentrations ere assayed in neutralied extracts by the activation of pyrophosphate:fructose-6-p phosphotransferase as in ref. 16. Extracts from hepatocytes incubated ith 2,5-12 AM-ol did not interfere ith the assay of fructose-2,6-p2 standards. Fructose-2,6-P2 as synthesied by dicyclohexylcarbodiimide treatment of fructose-1,6-p2, by slight modifications of Z the procedure for the synthesis of fructose-2-p (17), and purified essentially as described in ref. 18 except that triethyl- ammonium bicarbonate as used in the rechromatography step. 8 Materials. Sources of materials, in addition to those in ref. I-~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ 11, are glucosamine, Pfanstiehl Laboratories (Waukegan, IL); 2F alloxan monohydrate and N6,2'-dibutyryladenosine 3',5'-cyclic monophosphate (dibutyryl-camp), Sigma. Ui '5 2. 2,5- ANYDROMANNITOL, mm FIG. 1. Effects of increasing concentrations of 2,5-AM-ol on gluconeogenesis and hormonal stimulation of gluconeogenesis from 1 mm lactate plus 1 mm pyruvate. epatocytes isolated from normal 2-hrfasted rats ere incubated ith substrate and effectors for 3 min. The points, ith the exception of the 1% control point, are the means + SEM of averaged duplicates from three different cell preparations. The rate of glucose production from 1 mm lactate plus 1 mm pyruvate is.31 ±.2,umol per min per g et eight of cells. *, No hormone; o,.5,um glucagon; A, 5 um norepinephrine. RESULTS Effects of 2,5-AM-ol on Gluconeogenesis and Glycolysis in epatocytes. Increasing concentrations of 2,5-AM-ol progressively decrease the rate of glucose production from 1 mm lactate plus 1 mm pyruvate; the concentration required for 5% inhibition of the rate is mm (Fig. 1). At concentrations greater than 2 mm, glucose synthesis from the combination substrate is almost negligible (data not shon). Although rates of gluconeogenesis are substantially loered in the presence of 2,5-AM-ol, the ability of norepinephrine or glucagon to stimulate those rates is retained at concentrations of the analog as high as 1 mm. igher concentrations of 2,5-AM-ol can prevent the response to the catecholamine. The effects of 2,5-AM-ol on gluconeogenesis from substrates that enter the Embden-Meyerhof pathay as triose phosphate or fructose-6-p are compared in Fig. 2. 2,5-AM-ol inhibits gluconeogenesis from 2.5 mm dihydroxyacetone, glycerol, sorbitol, or fructose, but at concentrations as high as 2 mm (Fig. 2) or 1 mm (data not shon) it does not affect gluconeogenesis from 2.5 mm xylitol. Because xylitol is metabolied via the pentose pathay and enters the gluconeogenic pathay primarily as fructose-6-p, 2,5-AM-ol does not appear to inhibit gluconeogenesis at a site subsequent to fructose-1,6-bisphosphatase. Although gluconeogenesis from each of the substrates entering as triose phosphate is affected by the analog, higher concen- 1,6-bisphosphatase as purified as in ref. 1 ith an additional heating step at 62C for 5 min (15). Standard additions of fructose-1,6-p2 gave the expected absorbance changes and demonstrated that AMP in the cell extracts is not sufficient to inhibit the fructose-1,6-bisphosphatase. Determination of Fructose-2,6-P2 Content of epatocytes. epatocyte incubations ere performed as usual but ere ter -i 3 -I 2,5-ANYDROMANNITOL, mm FIG. 2. Effects of 2,5-AM-ol on gluconeogenesis from substrates that enter the gluconeogenic pathay at triose phosphate or fructose-6-p. Incubation conditions ere the same as those for Fig. 1. The points are the means ± SEM of averaged duplicates from three different cell preparations ith the exception of those in the xylitol series; they represent the means from to different cell preparations. Rates of glucose production, expressed as jmol per min per g et eight of cells, from the substrates in the absence of 2,5-AM-ol are glycerol,.6 ±.5; dihydroxyacetone (DA),.81 ±.1; sorbitol,.87 ±.2; fructose, 1.23 ±.8; xylitol,.7.

3 Biochemistry: Riquelme et al. Proc. Natl. Acad. Sci. USA 8 (1983) 33 - U LL I- U. -j L) cn IL CD 2,5- ANYDROMANNITOL, mm FIG. 3. Effects of 2,5-AM-ol on glucose and lactate formation from 2.5 mm dihydroxyacetone in the absence and presence of glucagon. Incubation conditions ere the same as those for Fig. 1. The points are the means ± SEM of averaged duplicates from four different cell preparations. Control rates of glucose and lactate production from 2.5 mm dihydroxyacetone, respectively, ere.77 ±. and.61 ±.,tmol per min per g et eight of cells. o and *, glucose formation; A and A, lactate formation; e and *, no hormone; o and A, plus.5,tm glucagon. trations of 2,5-AM-ol are required for comparable inhibition from these substrates than from lactate plus pyruvate (Figs. 1 and 2). The concentration of 2,5-AM-ol required for 5% inhibition of gluconeogenesis from dihydroxyacetone (.5-1. mm) is severalfold higher than that observed for lactate plus pyruvate (Fig. 1). Gluconeogenesis from glycerol or sorbitol, substrates that require oxidation prior to conversion to triose phosphate, is even less sensitive to inhibition by 2,5-AM-ol. While gluconeogenesis from fructose, like that from dihydroxyacetone, does not require oxidation of the substrate prior to conversion to triose phosphate, it is least sensitive to the analog. * Concomitant ith the decrease in glucose formation from dihydroxyacetone (Fig. 2), an increase in lactate formation is observed (Fig. 3). The ability of glucagon to increase glucose production and simultaneously to decrease lactate production from dihydroxyacetone is ell documented (11, 19-23). In the presence of mm 2,5-AM-ol, the metabolic responses to glucagon are abolished (Fig. 3). In hepatocytes incubated ith dihydroxyacetone in the absence of extracellular Ca2", responses to norepinephrine or glucagon are similar (11). 2,5-AMol is equally potent in inhibiting gluconeogenesis from dihydroxyacetone in the absence of added Ca2>, and the effects of * The possibility that 2,5-AM-ol could be exerting its effects by decreasing cellular ATP content as examined in hepatocytes metaboliing 1 mm lactate plus 1 mm pyruvate, 2.5 mm dihydroxyacetone, or 2.5 mm fructose. While addition of.5 mm 2,5-AM-ol causes an 8% inhibition of gluconeogenesis from lactate plus pyruvate or a 5-6% inhibition of gluconeogenesis from dihvdroxvacetone, it causes less than a 1% decrease in cellular ATP concentrations hen each of the above substrates is being metabolied. OR r U. I-i PYR PEP 2PGA 3PGA Tnose P FBP F6P G6P Gic A/LXg FIG.. Effect of 2,5-AM-ol on metabolite concentrations in hepatocytes incubated ith dihydroxyacetone. epatocytes ere incubated for 15 min ith 5 mm dihydroxyacetone in the absence or presence of.5 mm 2,5-AM-ol. Glucose and pyruvate ere determined as total concentrations and phosphorylated intermediates ere determined in concentrated cell extracts. Each value represents the mean ± SEM of four different hepatocyte preparations. PYR, pyruvate; PEP, phosphoenolpyruvate; 2PGA, 2-phosphoglycerate; 3PGA, 3-phosphoglycerate; triose P, triose phosphates; FBP, fructose-1,6-p2; F6P, fructose-6-p; G6P, glucose-6-p; Glc, glucose. either hormone on the metabolism of dihydroxyacetone are abolished by mm 2,5-AM-ol (data not shon). Effects of 2,5-AM-ol on Gluconeogenic-Glycolytic Intermediates. In Fig., metabolite profiles in hepatocytes incubated ith dihvdroxvacetone and.5 mm 2,5-AM-ol sho to distinct crossovers that could account for the observed decreases in gluconeogenesis and increases in glycolysis. The increase of fructose-1,6-p2 ith little change of fructose-6-p and a decrease of glucose-6-p is consistent ith an inhibition of fructose-1,6-bisphosphatase, activation of phosphofructokinase, or both. Although 2,5-AM-ol bisphosphate has been reported to inhibit aldolase (2), the accumulation of fructose-1,6- P2 from dihydroxyacetone indicates that such an inhibition is not significant in these experiments. The second crossover, observed as an increase in pyruvate production and a decrease in phosphoenolpyruvate accumulation, corresponds to an activation of pyruvate kinase. Effects of 2,5-AM-ol on epatocyte Fructose-2,6-P2 Content. Fructose-2,6-P2, an inhibitor of fructose-1,6-bisphosphatase and a stimulator of phosphofructokinase, is a potent physiological regulator of gluconeogenesis and glycolysis (for revie, see ref. 25). Glucose, fructose, dihydroxyacetone, or xylitol elicits an increase in hepatocyte concentrations of fructose-2,6-p2 (cf. refs and Table 1). Dibutyryl-cAMP abolishes the ability of these substrates to increase fructose-2,6-p2 content above basal levels (data not shon), similar to the observed effects of glucagon in the presence of glucose (25). Because an elevation of hepatic fructose-2,6-p2 by 2,5-AMol could explain the observed inhibition of gluconeogenesis and stimulation of glycolysis (3-32), direct measurements of fructose-2,6-p2 ere performed. At concentrations as lo as.1 mm, 2,5-AM-ol blocks most of the dihydroxvacetone-induced increase of fructose-2,6-p2 concentrations and significantly reduces the increase caused by glucose or by fructose (Table 1). 2,5-AM-ol also eliminates the small increase of fructose-2,6-p2 content caused by xylitol, but it does not inhibit gluconeogenesis from this substrate (Fig. 2 and Table 1). Alterations in fruc-

4 3 Biochemistry: Riquelme et al. Table 1. Fructose-2,6-P2 concentrations in hepatocytes from fasted rats after incubation ith,.1,.25, and.5 mm 2,5-AM-ol Fructose-2,6-P2, nmol/g et t Substrate mm.1 mm.25 mm.5 mm None.3 ±. Glucose, 2 mm 1.6 ± ±.7* 2.3 ±.2t ;6 ± Olt Fructose, 2.5 mm 1.3 ± ±.2t 1. ± O.1t.6 ± O.lt DA, 2.5 mm 9.7 ± ± O.t. ±.lt. ± O.lt Xylitol, 2.5 mm. 2. ±.6.7 ±.1* ;5 ±.1*. ±.1* Incubation conditions ere the same as those for Fig. 1. Each value represents the mean ± SEM from three different hepatocyte preparations, except for the incubations ith dihydroxyacetone (DA), hich represent four preparations. *P <.1 in comparison ith no added 2,5-AM-ol. tp <.5 in comparison ith no added 2,5-AM-ol. tose-2,6-p2 concentrations are therefore not responsible for the inhibition of gluconeogenesis and the enhancement of glycolysis observed in the presence of 2,5-AM-ol; Effects of 2,5-AM-ol on Gluconeogenesis in Diabetic Rat epatocytes. In hepatocytes isolated from diabetic rats, 2,5-AMol inhibits gluconeogenesis from lactate plus pyruvate or from dihydroxyacetone, but not from xylitol (Fig. 5). Gluconeogenesis from lactate plus pyruvate is much less sensitive to inhibition by 2,5-AM-ol in hepatocytes from diabetic rats than in hepatocytes from normal rats, but gluconeogenesis from dihydroxyacetone is only slightly less sensitive. Concentrations of 2,5-AM-ol > 1. mm are required for 5% inhibition from either substrate. The inhibition of gluconeogenesis from dihydroxyacetone is accompanied by an increase in lactate production: the control rate of lactate production (.36 ±.5,umol per min per g et eight, n = 5) as increased by 67% in the presence of 2 mm 2,5-AM-ol. In contrast to the ability of 2,5-AMol to abolish the effects of glucagon on the metabolism of dihydroxyacetone in normal hepatocytes, the fructose analog reduces, but does not eliminate, the effects of dibutyryl-camp in cells from diabetic rats. 2,5-ANYDROMANNITOL, FIG. 5. Effect of 2,5-AM-ol on gluconeogenesis in hepatocytes from diabetic rats. epatocyte preparation and incubations ere performed as described in the text and each point represents the mean ± SEM of six hepatocyte preparations. (A) Gluconeogenesis from 5 mm dihydroxyacetone is shon in the absence (o) and presence (o) of 5,.tM dibutyryl-camp. (B) Gluconeogenesis from 2.5 mm xylitol (A) and 1 mm lactate plus 1 mm pyruvate-(). Control rates of gluconeogenesis, expressed as I&mol per min per g et eight of cells, in the absence of 2,5- AM-ol are dihydroxyacetone, 1.13 ±.11; xylitol,.98 ±.7; lactate plus pyruvate,.91 ±.1. mm Proc. Natl. Acad. Sci. USA 8- (1983) DISCUSSION 2,5-AM-ol inhibits hepatocyte gluconeogenesis from substrates that enter the pathay prior to fructose-1,6-bisphosphatase, but it does not inhibit glucose production from xylitol, a substrate that enters predominantly as fructose-6-p. This indicates that phosphoglucoisomerase and glucose-6-phosphatase are not involved in the inhibition of gluconeogenesis by 2,5-AM-ol and is consistent ith a report that 2,5-AM-ol fails to inhibit glucose production, from galactose (33), a substrate that is converted to glucose via glucose-l-p and glucose-6-p. Gluconeogenesis from substrates that enter the Embden- Meyerhof pathay as triose phosphate is less sensitive to inhibition by 2,5-AM-ol- than that from lactate plus pyruvate. The folloing order of sensitivity has been observed: dihydroxyacetone > glycerol > sorbitol > fructose (Fig. 2). Because glycerol and sorbitol require oxidation prior to conversion to glucose, utiliation of these substrates is already limited by the necessity for reoxidation of excessive cytosolic NAD (3), and this may account for the decreased sensitivity of gluconeogenesis to inhibition by 2,5-AM-ol. It is also possible that the increased concentrations of a-glycerophosphate in cells metaboliing glycerol or sorbitol (11, 3) are responsible for the decreased sensitivity. Because a-glycerophosphate is an allosteric inhibitor of phosphofructokinase 1 (28), its accumulation in cells could counteract the effects of lo concentrations of 2,5-AM-ol by decreasing 2,5-AM-ol-bisphosphate formation. That gluconeogenesis from fructose is least sensitive to inhibition by 2,5-AM-ol is best explained by competition beteen fructose and 2,5-AM-ol for phosphorylation by fructokinase and is consistent ith 2,5-AM-ol metabolism occurring via fructokinase in the liver (1, 2). The metabolite patterns (Fig. ) indicate that the major effects of 2,5-AM-ol are a decrease in the net conversion of fructose-1,6-p2 to fructose-6-p and an increase in the net conversion of phosphoenolpyruvate to pyruvate. Because 2,5-AM-ol elicits these effects ithout significant depletion of cellular ATP and because cellular fructose-2,6-p2 concentrations are decreased by the compound, the monophosphate and bisphosphate derivatives of the fructose analog are probably responsible. As ill be reported elsehere, these sugar phosphates are the primary products of 2,5-AM-ol metabolism in isolated hepatocytes. At the fructose-6-p/fructose-1,6-p2 site, 2,5-AM-ol bisphosphate may cause an inhibition of fructose-1,6-bisphosphatase in vivo analogous to that observed in vitro (7, 8) or it may mimic fructose-1,6-p2 as an allosteric activator of phosphofructokinase 1. Metabolite crossover analysis cannot distinguish beteen these effects, and either action could lead to the increased concentrations of fructose-i,6-p2. Under some conditions both enymes may be inhibited, because 2,5-AM-ol monophosphate may compete ith and thus inhibit the phosphorylation of fructose-6-p. The crossover beteen pyruvate and phosphoenolpyruvate is consistent ith a stimulation of pyruvate kinase activity. While the observed increase of fructose-1,6-p2 in cells metaboliing 2,5-AM-ol could account for this activation (35-37), direct effects of the 2,5-AM-ol bis- and monophosphates may also be important at this site. 2,5-AM-ol bisphosphate may act as an allosteric activator of pyruvate kinase, as does fructose-1,6-p2. 2,5-AM-ol monophosphate may also stimulate pyruvate kinase, since fructose-l-p is a knon activator of this enyme (38). The decrease of fructose-2,6-p2 concentrations in the presence of 2,5-AM-ol is most likely due to the presence of 2,5-AMol monophosphate. Because 2,5-AM-ol lacks the 2 O it cannot be phosphorylated in the 2 position but might act as an inhibitor

5 Biochemistry: Riquelme et al. of phosphofructokinase 2 and account for the ability of 2,5-AMol to decrease fructose-2,6-p2 concentrations to the basal values even hen substrates are present. Inhibition by 2,5-AM-ol of gluconeogenesis from lactate plus pyruvate is more pronounced in hepatocytes isolated from normal rats rather than in those from diabetic rats (Figs. 1 and 5). Decreases in phosphofructokinase and pyruvate kinase activities have been observed in diabetic livers (39-2); either of these changes could contribute to a diminished sensitivity to 2,5-AMol. The observation that gluconeogenesis from dihydroxyacetone is only slightly less sensitive to 2,5-AM-ol inhibition in the diabetic cells than in the normal controls indicates that the metabolism of the fructose analog is not seriously hindered in the diabetic hepatocytes despite the loer activity of phosphofructokinase. Therefore, the relative insensitivity of gluconeogenesis from lactate plus pyruvate to inhibition by 2,5-AM-ol probably reflects the decreased amounts of pyruvate kinase, because less reversal of gluconeogenesis could be accomplished by complete activation of this enyme. This paper demonstrates that 2,5-AM-ol can inhibit gluconeogenesis in isolated rat hepatocytes and implicates the key gluconeogenic and glycolytic enymes-fructose-1,6-bisphosphatase, phosphofructokinase, and pyruvate kinase-as possible sites here the mono- and bisphosphate forms of 2,5-AMol may be exerting an effect. More detailed studies of the metabolism of 2,5-AM-ol in hepatocytes and the effects of the phosphorylated derivatives of 2,5-AM-ol on isolated preparations of each of these key enymes ill be reported elsehere. This ork as supported by Grants AM 2,678 and AM 1,33 from the National Institutes of ealth. P.T.R. is a recipient of an Advanced Opportunity Felloshipfrom the Graduate School of the University of Wisconsin. 1. Raushel, F. M. & Cleland, W. W. (1973)J. Biol. Chem. 28, Raushel, F. M. & Cleland, W. W. (1977) Biochemistry 16, Gray, G. R. (1971) Biochemistry 1, Bar-Tana, J. & Cleland, W. W. (197)J. BioL Chem. 29, Koerner, T. A. W., Jr., Younathan, E. S., Ashour, A.-L. E. & Voll, R. J. (197) J. Biol Chem. 29, Frey, W. A., Fishbein, R., demaine, M. M. & Benkovic, S. J. (1977) Biochemistry 16, demaine, M. M. & Benkovic, S. J. (1972) Arch. Biochem. Biophys. 152, Marcus, C. J. (1976)J. BioL Chem. 251, orton, D. & Philips, K. D. (1976) in Methods in Carbohydrate Chemistry, eds. Whistler, R. L. & BeMiller, J. N. (Academic, Ne York), Vol. 7, pp Khym, J. K. & Zill, L. P. (1952)J. Am. Chem. Soc. 7, Kneer, N. M., Wagner, M. J. & Lardy,. A. (1979)J. BioL Chem. 25, Proc. Natl. Acad. Sci. USA 8 (1983) Cornell, N. W. (198) Anal. Biochem. 12, Bergmeyer,. U., ed. (197) Methods of Enymatic Analysis (Academic, Ne York), 3rd Ed., Vols Colombo, G. & Marcus, F. (1973)J. Biol. Chem. 28, Marcus, F. & aley, B. E. (1979) J. Biol. Chem. 25, Van Schaftingen, E., Lederer, B., Bartrons, R. & ers,.-g. (1982) Eur. J. Biochem. 129, Pontis,. G. & Fischer, C. L. (1963) Biochem. J. 89, Van Schaftingen, E. & ers,.-g. (1981) Eur. J. Biochem. 117, Blair, J. B., Cook, D. E. & Lardy,. A. (1973)J. Biol. Chem. 28, Clark, M. G., Kneer, N. M., Bosch, A. L. & Lardy,. A. (197) J. Biol. Chem. 29, Rognstad, R. (1976) Int. J. Biochem. 7, Pilkis, S. J., Riou, J. P. & Claus, T.. (1976)1. Biol. Chem. 251, Foster, J. L. & Blair, J. B. (1978) Arch. Biochem. Biophys. 189, Midelfort, C. F., Gupta, R. K. & Rose, I. A. (1976) Biochemistry 15, ers,.-g. & Van Schaftingen (1982) Biochem. J. 26, Van Schaftingen, E., ue, L. & ers,.-g. (198) Biochem. J. 192, Richards, C. S. & Uyeda, K. (1982) Biochem. Biophys. Res. Commun. 19, Claus, T.., Schlumpf, J. R., El-Maghrabi, M. R. & Pilkis, S. J. (1982) J. Biol. Chem. 257, ue, L., Blackmore, P. F., Shikama,., Robinson-Steiner, A. & Exton, J.. (1982)J. Biol. Chem. 257, Pilkis, S. J., El-Maghrabi, M. R., Pilkis, J. & Claus, T. (1981) J. Biol Chem. 256, Van Schaftingen, E. & ers,. -G. (1981) Proc. NatL. Acad. Sci. USA 78, Morikofer-Zeg, S., Stoecklin, F. B. & Walter, P. (1981) Biochem. Biophys. Res. Commun. 11, Stevens,. C. & Dills, W. L. (1981) Fed. Proc. Fed. Am. Soc. Exp. Biol., 82 (abstr. 379). 3. Berry, M. N., Kun, E. & Werner,. V. (1973) Eur. J. Biochem. 33, ess, B., aeckel, R. & Brand, K. (1966) Biochem. Biophys. Res. Commun. 2, Taylor, C. B. & Bailey, E. (1967) Biochem. J. 12, 32c-33c. 37. Weber, G., Lea, M. A., Convery,. J.. & Stamm, N. B. (1967) in Advances in Enyme -Regulation, ed. Weber, G. (Pergamon, London), Vol. 5, pp Eggleston, L. V. & Woods,. F. (197) FEBS Lett. 6, Tanaka, T., arano, Y., Morimura,. & Mori, R. (1965) Biochem. Biophys. Res. Commun. 21, Weber, G., Singhal, R. L., Stamm, N. B., Lea, M. A. & Fisher, E. A. (1966) in Advances in Enyme Regulation, ed. Weber, G. (Pergamon, London), Vol., pp Willms, B., Ben-Ami, P. & Soling,.-D. (197) orm. Metab. Res. 2, Feliu, J. E., ue, L. & ers,.-g. (1977) Eur. J. Biochem. 81,