Promoted More Effectively by D-Allose Than by Glucose (Regulation/Malonate/Cyclohexlmide/Mannose) DONNA B
Total Page:16
File Type:pdf, Size:1020Kb
Proc. Natl. Acad. Sci. USA Vol. 83, pp. 5858-5860, August 1986 Biochemistry Hexose transport control in a fibroblast metabolic mutant can be promoted more effectively by D-allose than by glucose (regulation/malonate/cyclohexlmide/mannose) DONNA B. ULLREY AND HERMAN M. KALCKAR Unit of Biochemistry in the Department of Chemistry, Boston University, Boston, MA 02215 Contributed by Herman M. Kalckar, April 28, 1986 ABSTRACT By studying the energy-requiring control of transport system (4), was able to induce a well-expressed the hexose transport system (the transport "curb") in a lung transport curb in various types of fibroblasts (5, 6, *). In the fibroblast mutant called the phosphoglucose isomerase mutant PGI mutant that is unable to respond to mannose or D- (because it is devoid of the enzyme phosphoglucose isomerase) glucosamine, D-allose turns out to develop a curb that, much the following features were noted. The aldohexose D-allose, if like glucose, is abolished by malonate. In the parental line, added over 20 hr to a culture of the mutant, promotes the 023, in which several D-aldohexoses are able to develop a development of an intense curb ofthe hexose transport system, hexose transport curb (1), D-allose also brings about a greatly surpassing that brought about by incubation with pronounced transport curb, which also seems to be energy glucose. The allose-mediated curb can be circumvented by requiring. As will be discussed in this paper, D-allose, in various metabolic inhibitors as well as by the presence of other amounts as low as 1 mM, exerts a highly pronounced curb in aldohexoses such as mannose. the PGI mutant surpassing that brought about by glucose. A regulatory energy-requiring system that we have called the hexose transport "curb" has been examined in a cultured MATERIALS AND METHODS hamster fibroblast mutant, DS7, devoid of the enzyme phosphoglucose isomerase (D-glucose-6-phosphate ketol- Cells used were a Chinese hamster lung fibroblast line lacking isomerase, EC 5.3.1.9) (the PGI mutant). The parental line of PGI (DS7) and its parental line (023) (see ref. 6). Both were the mutant, 023, uses glucose readily for energy metabolism. tested by the 4',6-diamidino-2-phenylindole stain method and The transport curb in 023 is promoted by feeding with glucose were found free of mycoplasma (D. V. Young, Bioassay as well as with mannose or D-glucosamine. In DS7, the PGI Systems Research Corp.). The cells were grown in Dulbec- mutant, the two latter hexoses can be used in energy co's modified Eagle's medium (DMEM) with 10% fetal metabolism, yet no transport curb ensues. Conversely, bovine serum. Before the uptake test the cells were rinsed glucose, unable to serve in energfymetabolism ofthis mutant, twice with sugar-free DMEM; the cells were then given remains a promoter of the curb of its transport system. The modified DMEM without pyruvate and with various sugars all-cis aldohexose D-allose has turned out to be the most replacing glucose and supplemented with 10% dialyzed fetal effective promoter of the hexose transport curb. This curb bovine serum (Sigma) for 16-20 hr. Chx (Sigma) was used at can be released by various metabolic inhibitors, such as 35 gM (7). Other additions are indicated for the individual malonate or cycloheximide (Chx). Addition of mannose in experiments. Sugars and malonate were obtained from Sig- excess will also prevent the allose-induced transport curb in ma. the PGI mutant. 3-O-Methylglucose Transport. Cultures were rinsed three A comparison of the down-regulatory patterns of the times with sugar-free and serum-free medium. They were hexose transport system, which we call the "mediated curb," then preloaded with 50 mM 3-O-methylglucose in DMEM between a fibroblast mutant, defective in PGI (the PGI without glucose or serum for 30 min at 370C. The cells were mutant), and its parental line shows the following differences. next rinsed rapidly with 10 ml of phosphate-buffered saline In the parental line, like fibroblast cultures from other (PBS) and then incubated 20 sec at 220C with 3-0- hamster lines (tumorigenic or not), glucose and other ['4C]methylglucose containing L-[3H]glucose to check for aldohexoses, such as D-glucosamine or D-mannose, "in- completeness of washing. After the transport test the cells duce" a marked curb of their own transport system (1, 2). were rinsed rapidly with ice-cold PBS. In some experiments Cultures deprived of sugars or fed fructose instead of D- the cold PBS contained 0.1 mM phloretin. The cells were aldohexose consistently showed much higher rates ofhexose extracted with ethanol and the extracts were assayed for transport (1). radioactivity in a scintillation counter. The results were The transport curb is energy-requiring and it also depends expressed as pmol/mg of cell protein per 20 sec (from on protein synthesis, since the curb is released by inhibitors duplicate samples). of oxidative phosphorylation as well as by inhibitors of Galactose Uptake Test. Cultures were rinsed three times protein synthesis (1-3). with PBS at 370C, incubated 5 or 10 min at 370C with 0.1 mM The D-aldohexoses that can induce a transport curb in the [14C]galactose, rinsed, and analyzed as described (2, 7). The PGI mutant are much more restricted. Neither D-glUCOS- results are expressed as nmol/mg of cell protein per 5 or 10 amine nor mannose was able to elicit a transport curb; only in the tables. Radiochemicals were glucose or galactose has retained this ability (1, 2). min, as stated respective Surprisingly enough, the all-cis hexose D-allose, suppos- from New England Nuclear. edly a nonmetabolizable hexose, albeit a ligand ofthe hexose Abbreviations: Chx, cycloheximide; PGI, phosphoglucose isomer- ase. The publication costs of this article were defrayed in part by page charge *Ullrey, D. B. & Kalckar, H. M. (1986) 86th Meeting of the payment. This article must therefore be hereby marked "advertisement" American Society of Microbiology, March 23-28, 1986, abstr. in accordance with 18 U.S.C. §1734 solely to indicate this fact. K195, p. 226. 5858 Downloaded by guest on September 23, 2021 Biochemistry: Ullrey and Kalckar Proc. Natl. Acad. Sci. USA 83 (1986) 5859 Table 1. Regulation of hexose uptake in 023 and DS7 cultures Table 3. Glucose and allose curb of hexose transport in the PGI mutant nmol/mg of protein per 10 min 3-O-[14C]Methylglucose transport, Sugar DS7 023 Sugar pmol/mg of protein per 20 sec Ratio Fructose (22 mM) 5.34 4.79 First incubation (18 hr) D-Glucosamine (5 mM) 4.36 2.12 None 35.94 Glucose (22 mM) 1.81 2.25 Fructose (22 mM) 47.62 Allose (22 mM) 1.06 1.30 Glucose (22 mM) 13.36 Allose (22 mM) 8.34 Near-confluent cultures were fed various hexoses with 10% Allose (5 mM) 8.18 dialyzed fetal calf serum over 20 hr. Uptake tests were performed Second incubation (7 hr)* with 0.1 mM [U-_4C]galactose at 370C for 10 min. Fructose (22 mM) 45.95 1.02 + Chx 47.06 RESULTS AND DISCUSSION Glucose (22 mM) 20.51 + Chx 55.00 2.68 Table 1 indicates that allose elicits an even stronger transport Allose (22 mM) 17.39 2.62 curb than glucose in the PGI mutant and the parental strain. + Chx 45.63 The fact that the allose-induced transport curb is released by Confluent DS7 cultures were maintained for 18 hr in sugar-free malonate (Table 2) indicates that this is a true curb and not medium containing L-glutamine and supplemented with 10% dialyzed simple toxicity. This is supported by the prevention of the fetal calf serum. Subsequently, 22 mM hexoses were added with or establishment of the allose-induced curb by Chx (Table 3). without 35 uM Chx; this refeeding period spanned only 7 hr. Confluent DS7 cultures were incubated for 18 hr in growth Transport tests were then carried out with 0.01 mM 3-0-[14C]- medium containing fructose, glucose, allose, or no sugar. The methylglucose for 20 sec at 23°C. sugar concentrations were 22 mM; however, an extra pair of *Sugar and 35 ,M Chx were added to original sugar-free samples for allose incubation mixtures with only 5 mM of this sugar was 7 hr. added (Table 3, top). A second set of hexose-starved DS7 cultures was exposed to Chx (35 gM) over 7 hr in the It should be emphasized here that the intense transport presence of 22 mM fructose, glucose, or allose and then curb that develops after 20 hr of exposure to D-allose is not analyzed. It can be seen in Table 3, bottom, that Chx is able a plain toxic action by this sugar but a regulatory effect. This to forestall the onset of the hexose transport curb, including appears from the fact that the additional presence of meta- that elicited by allose. bolic inhibitors over the same extended span of time still Table 4 illustrates the competition between allose and permitted the culture to manifest unbridled transport, as mannose. Since this mutant catabolizes mannose rapidly (1, determined in the transport test. 2), a large excess of this sugar was used. In the presence of In general, the biochemical literature on D-allose seems 22 mM mannose, allose fails to elicit a transport curb. The rather sparse. In an important study about 10 years ago (4) it lactic acid generated per mg of cell protein over 18 hr was found that D-allose (3H-labeled) acts as a transport ligand amounted to 3.7-4.3 ,umol from pyruvate and an additional in the hexose transport system ofadipose fat cells.