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The transport and phosphorylation of 2-deoxyglucose by rat lenses
Exp. Eye Res. (1973) 15, 245-248
The Transport and Phosphorylation of 2-Deoxyglucose by Rat Lenses JOHN
F. R.
KUCK,
JR.
Laboratory for Ophthalmic Research,Department of Ophthalmology, Emory University School of Medicine, Atlanta, Georgia30322, U.S.A. As in other tissues, f-deoxyglucose is transported into the rat lens as the free sugar which is then converted to the sugar phosphate. The phosphate accumulates within the lens without escaping and over a period of hours its concentration approaches about one half the concentration of free 2-deoxyglucose in the lens. At this level there is apparently some inhibition of uptake, especially phosphorylation. At an incubation time of only 2 hr the use of this tracer may be useful in studying factors which affect transport and phosphorylation of hexoses.
1. Introduction The uptake and phosphorylation of 2-deoxyglucose has been studied in yeast (Van Steven&k, 1968), human blood platelets (Solomon and Gaut, 1970), erythrocytes (Reinwein, Kalman and Park, 1957), bacteria (Dietz and Heppel, 1971), and hamster kidney cortex and everted jejunal rings (Elsas and MacDonnell, 1972). The lens has been little studied. Kern (1965) noted that 2deoxyglucose was taken up by the calf lens and that it was a competitor in the uptake of glucose. The findings of Elsas and MacDonnell suggested that in the lens 2-deoxyglucose might accumulate as the phosphate. The interesting possibility exists that either the free sugar or its phosphate might adversely affect lens carbohydrate metabolism sincesuch interference has been documented for a number of tissues(Webb, 1966).
2. Materials
and Methods
Rat lenses were incubated at 37” in a balanced salt medium containing 7 mM glucose, 1 mM 2-deoxyglucose and 2-deoxyglucose-U-14C at a level of 5 to 20 &i/100 ml depending upon the type of experiment. When the medium was buffered chiefly with bicarbonate,
the pH was maintained by gassingwith 95% Oa-5% CO,. Sometimesa Tris buffered medium was employed. After incubating between 30 min to 1 hr (longer for the long-term experiments), the lenses were removed, cleaned, weighed and then homogenized in Somogyi zinc sulfate solution with the later addition of an equivalent amount of barium hydroxide. When total sugar (i.e., free sugar plus sugar phosphate) was to be determined, the protein precipitant was 10% trichloroacetic acid. When sugar phosphate alone was to be determined, the Somogyi precipitate was extracted 2-3 times with alcohol to remove labeled free sugar and the dry precipitate treated with alkaline phosphatase to release free 2-deoxyglucose from the adsorbed phosphate. The radioactivity of small aliquots of such solutions was measured in a scintillation counter.
3. Results Results of counting lens extracts were converted to CL de&red asthe counts/min/ml of lens water. This value was divided by CM, the counts/min/ml for the incubation medium. In general CL/CM is the distribution ratio between the lens water and the incubation medium. The values for lens sugar phosphate were treated likewise; although in this casethere was no sugar phosphate in the medium, the division of CL 245
246
J.
F.
KUCK
for sugar phosphate by CM for medium sugar served to normalize the results so that values were of the same order of magnitude. Thus, all values are given as CL/CM which is to be taken as a normalized concentration permitting direct comparison of 2-deoxyglucose with its phosphate as well as with 3-0-methylglucose. The uptake of 2-deoxyglucose by the rat lens is rapid during the first %hr of incubation, slows down between 2 and 8 hr, but is still significant at 19 hr. The sugar phosphate accumulation is less than that of free sugar and by 19 hr is approaching a maximum. The uptake of 3-0-methylglucose is much like that of 2-deoxyglucose in the range of incubation time studied but there is no conversion to sugar phosphate (Table I). TABLE
I
The long-term uptake of Z-deoxyglucose and 3-0-mdhylgllucose by rat lenses
Time
(hr)
f--2DGlc
CL/CM -h--p7 2DGlcP0,
0.49 0.67 0.83 1.06 1.20 1.38
0.16 0.25 0.35 0.53 0.68 0.76
30 MG
B 1 2 4 8 129 19
0.41 0.51 0.72 0.83 1.04
Increasing the usual medium phosphate concentration three-fold has no effect on the uptake of 2-deoxyglucose or its phosphorylation (Table II). The uptake of tracer 2-deoxyglucose by the rat lens is slightly depressed by increasing concentra.tion of carrier sugar. The inhibition of phosphorylation is more marked (Table III). TABLE
The e&t
II
of increased medium phosphate concentration on the uptake and phosphwylation of 2-deoxyglucose by rat Zewes
I’hosphate
(mx)
1 3
In no case does increasing
r--------Free sugar
CL/CM *------7 Augar phosphate
0.28 0.30
phosphate
concentration
Total
0.08 0.07
from
1 to 3 rn~ have
sugar
0.36 0.37
a significant
effect.
The uptake of 2-deoxyglucose increases linearly with temperature between 3 and 45” with a slight fall off at higher temperatures. The phosphorylation of 2-deoxyglucose is increased markedly by rising temperature up to 37” but is significantly below normal (37”) at 45” (Table IV).
RAT
LENS
TRANSPORT
OF
247
2.DEOXYGLUCOSE
The effect of aging (expressed as lens weight) on the uptake and phosphorylation of 2-deoxyglucose is shown in Table V. The very young and rapidly growing lens takes up and phosphorylates the sugar much more actively than the older lens. After the lens weight reaches 20 mg (75 g body weight, 5 weeks old) the decrease in phosphorylation is small. In the last column the sugar phosphate is expressed as percent of total sugar in the lens. For lenses of intermediate age the accumulated sugar phosphate is about one-third of the total sugar. Young lenses are more efbcient in phosphorylation and older lenses are less efficient. TABLE
III
The e$ect of carrier concentration on the uptuke ad phmphorylation
of 2deoxyglwose
CL/CM Carrier
(mM)
Free sugar
Sugar phosphate
0.45 0.39 0.39 0.42 0.39 0.40
0.17 0.15
04m3* 0.63 1.25 2.50 5.0 10.0
Total
0.62 0.54 0.57 0.56 0.50 0.47
0.18 0.13
0.11 0.07
* No added TABLE
sugar
carrier. IV
The eff&t of incubation temperature on the uptake and phosphorylatim
of 2-deoxyglucose
CL/CM Temperature
----h----.----, r--Free sugar Sugar
(“C)
0.19
3 24 37 45
Total
phosphate
0.03 0.08 0.21 0.15
0.43 0.49 0.54
TABLE
The uptake and phosphorylation
sugar
0.22 0.51 0.70 0.69
V
of 2-deoxyglucose by aging rat lenses CL/CM
Lens weight
14
19 41 60
(mg)
(--------A.-Free sugar
Sugar
phosphate
0.42 0.35 0.22
0.41 0.17 0.12
0.19
0.05
Total
043 0.52 0.34 0.24
---sugar
o/o efficiency
50 33 36 20
248
J . F . KUCK
4. Discussion The handling of 2-deoxyglucose by the lens has an unusual feature in that the free sugar is transported into the lens where it is phosphorylated to the sugar phosphate which is not further metabolized. The accumulated sugar phosphate is unable to escapefrom the lens and yet appears to have little effect on the continuing transport and phosphorylation, at least during a short incubation. Thus the use of this tracer may serve asan index for monitoring in one experiment the effects of conditions which may modify transport and/or phosphorylation. For instance the temperature survey (Table IV) showsthat the uptake is affected by the temperature in a linear fashion while phosphorylation showsa sharp break above 37”, probably due to inactivation of the hexokinase. This is further evidence that the transport of 2-deoxyglucose, in agreement with the mechanismfound for other tissues,is independent of phosphorylation. ACKNOWLEDGMENT
This work wassupported by PHS grant No. EY 00260from the National Eye Institute. REFERENCES Dietz, G. W. and Heppel, L. A. (1971). J. Bid. Chem. 246,288l. Elsas, L. J. and MacDonnell, R. C. (1972). B&him. Biuphys. Acta 255,948. Kern, H. L. (1965). Invest. O~~~~Z. 4,680. Reinwein, D., Kalman, C. F. and Park, C. R. (1957). Fed. Proc. Fed. Amer. Sot. Eq. Biol. 16, 237. Solomon, H. M. and Gaut, Z. N. (1970). B&&m. Pharmacol. 19,263l. Van Steveninok, J. (1968). B&him. Bkvphys. Acta 163,386. Webb, J. L. (1966). In Enzyme and Meiabolic Inhibitors, Vol. II, p. 386. Academic Press, London.
The Transport and Phosphorylation of 2-Deoxyglucose by Rat Lenses JOHN
F. R.
KUCK,
JR.
Laboratory for Ophthalmic Research,Department of Ophthalmology, Emory University School of Medicine, Atlanta, Georgia30322, U.S.A. As in other tissues, f-deoxyglucose is transported into the rat lens as the free sugar which is then converted to the sugar phosphate. The phosphate accumulates within the lens without escaping and over a period of hours its concentration approaches about one half the concentration of free 2-deoxyglucose in the lens. At this level there is apparently some inhibition of uptake, especially phosphorylation. At an incubation time of only 2 hr the use of this tracer may be useful in studying factors which affect transport and phosphorylation of hexoses.
1. Introduction The uptake and phosphorylation of 2-deoxyglucose has been studied in yeast (Van Steven&k, 1968), human blood platelets (Solomon and Gaut, 1970), erythrocytes (Reinwein, Kalman and Park, 1957), bacteria (Dietz and Heppel, 1971), and hamster kidney cortex and everted jejunal rings (Elsas and MacDonnell, 1972). The lens has been little studied. Kern (1965) noted that 2deoxyglucose was taken up by the calf lens and that it was a competitor in the uptake of glucose. The findings of Elsas and MacDonnell suggested that in the lens 2-deoxyglucose might accumulate as the phosphate. The interesting possibility exists that either the free sugar or its phosphate might adversely affect lens carbohydrate metabolism sincesuch interference has been documented for a number of tissues(Webb, 1966).
2. Materials
and Methods
Rat lenses were incubated at 37” in a balanced salt medium containing 7 mM glucose, 1 mM 2-deoxyglucose and 2-deoxyglucose-U-14C at a level of 5 to 20 &i/100 ml depending upon the type of experiment. When the medium was buffered chiefly with bicarbonate,
the pH was maintained by gassingwith 95% Oa-5% CO,. Sometimesa Tris buffered medium was employed. After incubating between 30 min to 1 hr (longer for the long-term experiments), the lenses were removed, cleaned, weighed and then homogenized in Somogyi zinc sulfate solution with the later addition of an equivalent amount of barium hydroxide. When total sugar (i.e., free sugar plus sugar phosphate) was to be determined, the protein precipitant was 10% trichloroacetic acid. When sugar phosphate alone was to be determined, the Somogyi precipitate was extracted 2-3 times with alcohol to remove labeled free sugar and the dry precipitate treated with alkaline phosphatase to release free 2-deoxyglucose from the adsorbed phosphate. The radioactivity of small aliquots of such solutions was measured in a scintillation counter.
3. Results Results of counting lens extracts were converted to CL de&red asthe counts/min/ml of lens water. This value was divided by CM, the counts/min/ml for the incubation medium. In general CL/CM is the distribution ratio between the lens water and the incubation medium. The values for lens sugar phosphate were treated likewise; although in this casethere was no sugar phosphate in the medium, the division of CL 245
246
J.
F.
KUCK
for sugar phosphate by CM for medium sugar served to normalize the results so that values were of the same order of magnitude. Thus, all values are given as CL/CM which is to be taken as a normalized concentration permitting direct comparison of 2-deoxyglucose with its phosphate as well as with 3-0-methylglucose. The uptake of 2-deoxyglucose by the rat lens is rapid during the first %hr of incubation, slows down between 2 and 8 hr, but is still significant at 19 hr. The sugar phosphate accumulation is less than that of free sugar and by 19 hr is approaching a maximum. The uptake of 3-0-methylglucose is much like that of 2-deoxyglucose in the range of incubation time studied but there is no conversion to sugar phosphate (Table I). TABLE
I
The long-term uptake of Z-deoxyglucose and 3-0-mdhylgllucose by rat lenses
Time
(hr)
f--2DGlc
CL/CM -h--p7 2DGlcP0,
0.49 0.67 0.83 1.06 1.20 1.38
0.16 0.25 0.35 0.53 0.68 0.76
30 MG
B 1 2 4 8 129 19
0.41 0.51 0.72 0.83 1.04
Increasing the usual medium phosphate concentration three-fold has no effect on the uptake of 2-deoxyglucose or its phosphorylation (Table II). The uptake of tracer 2-deoxyglucose by the rat lens is slightly depressed by increasing concentra.tion of carrier sugar. The inhibition of phosphorylation is more marked (Table III). TABLE
The e&t
II
of increased medium phosphate concentration on the uptake and phosphwylation of 2-deoxyglucose by rat Zewes
I’hosphate
(mx)
1 3
In no case does increasing
r--------Free sugar
CL/CM *------7 Augar phosphate
0.28 0.30
phosphate
concentration
Total
0.08 0.07
from
1 to 3 rn~ have
sugar
0.36 0.37
a significant
effect.
The uptake of 2-deoxyglucose increases linearly with temperature between 3 and 45” with a slight fall off at higher temperatures. The phosphorylation of 2-deoxyglucose is increased markedly by rising temperature up to 37” but is significantly below normal (37”) at 45” (Table IV).
RAT
LENS
TRANSPORT
OF
247
2.DEOXYGLUCOSE
The effect of aging (expressed as lens weight) on the uptake and phosphorylation of 2-deoxyglucose is shown in Table V. The very young and rapidly growing lens takes up and phosphorylates the sugar much more actively than the older lens. After the lens weight reaches 20 mg (75 g body weight, 5 weeks old) the decrease in phosphorylation is small. In the last column the sugar phosphate is expressed as percent of total sugar in the lens. For lenses of intermediate age the accumulated sugar phosphate is about one-third of the total sugar. Young lenses are more efbcient in phosphorylation and older lenses are less efficient. TABLE
III
The e$ect of carrier concentration on the uptuke ad phmphorylation
of 2deoxyglwose
CL/CM Carrier
(mM)
Free sugar
Sugar phosphate
0.45 0.39 0.39 0.42 0.39 0.40
0.17 0.15
04m3* 0.63 1.25 2.50 5.0 10.0
Total
0.62 0.54 0.57 0.56 0.50 0.47
0.18 0.13
0.11 0.07
* No added TABLE
sugar
carrier. IV
The eff&t of incubation temperature on the uptake and phosphorylatim
of 2-deoxyglucose
CL/CM Temperature
----h----.----, r--Free sugar Sugar
(“C)
0.19
3 24 37 45
Total
phosphate
0.03 0.08 0.21 0.15
0.43 0.49 0.54
TABLE
The uptake and phosphorylation
sugar
0.22 0.51 0.70 0.69
V
of 2-deoxyglucose by aging rat lenses CL/CM
Lens weight
14
19 41 60
(mg)
(--------A.-Free sugar
Sugar
phosphate
0.42 0.35 0.22
0.41 0.17 0.12
0.19
0.05
Total
043 0.52 0.34 0.24
---sugar
o/o efficiency
50 33 36 20
248
J . F . KUCK
4. Discussion The handling of 2-deoxyglucose by the lens has an unusual feature in that the free sugar is transported into the lens where it is phosphorylated to the sugar phosphate which is not further metabolized. The accumulated sugar phosphate is unable to escapefrom the lens and yet appears to have little effect on the continuing transport and phosphorylation, at least during a short incubation. Thus the use of this tracer may serve asan index for monitoring in one experiment the effects of conditions which may modify transport and/or phosphorylation. For instance the temperature survey (Table IV) showsthat the uptake is affected by the temperature in a linear fashion while phosphorylation showsa sharp break above 37”, probably due to inactivation of the hexokinase. This is further evidence that the transport of 2-deoxyglucose, in agreement with the mechanismfound for other tissues,is independent of phosphorylation. ACKNOWLEDGMENT
This work wassupported by PHS grant No. EY 00260from the National Eye Institute. REFERENCES Dietz, G. W. and Heppel, L. A. (1971). J. Bid. Chem. 246,288l. Elsas, L. J. and MacDonnell, R. C. (1972). B&him. Biuphys. Acta 255,948. Kern, H. L. (1965). Invest. O~~~~Z. 4,680. Reinwein, D., Kalman, C. F. and Park, C. R. (1957). Fed. Proc. Fed. Amer. Sot. Eq. Biol. 16, 237. Solomon, H. M. and Gaut, Z. N. (1970). B&&m. Pharmacol. 19,263l. Van Steveninok, J. (1968). B&him. Bkvphys. Acta 163,386. Webb, J. L. (1966). In Enzyme and Meiabolic Inhibitors, Vol. II, p. 386. Academic Press, London.