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Influence of maternal diabetes on lipid metabolism in neonatal rat lung
132
Biochimica et Biophysics @ Elsevier/North-Holland
BBA
572 (1979)
Biomedical
132-138
Press
57294
INFLUENCE NEONATAL
R.A.
Acta,
RHOADES
OF MATERNAL RAT LUNG
*, D.A.
FILLER
DIABETES
IN
and B. VANNATA
Department of Physiology, Indiana University Street, Indianapolis, IN 46202 (U.S.A.) (Received
ON LIPID METABOLISM
School of Medicine,
1100 West Michigan
July 4th, 1978)
Key words: Diabetes;
Lipid metabolism;Pregnancy;
Phospholipid;
(Neonatal
rat lung)
Summary
The effect of maternal diabetes on tissue constituents, lipid metabolism and glucose utilization was examined in l-day old rat lungs. Maternal diabetes was induced by intravenous injection of streptozotocin (50 mg/kg body weight) into pregnant Long-Evans hooded rats on the 10th day of gestation resulting in a maternal serum glucose concentration which was 3-fold higher than that of controls. Neonates from diabetic mothers showed a significant decrease in body weight (14%), lung weight (32%) and lung protein concentration (30%). Glycogen, DNA, and lipid content of the lungs were significantly elevated in neonates from diabetic mothers. The percent of total phospholipid made up of phosphatidylcholine was not altered, but the percentage of disaturated phosphatidylcholine was decreased (25%). The activity of the CDPcholine pathway enzymes (choline kinase, cholinephosphate cytidylyltransferase and choline phosphotransferase) also showed a marked increase in lungs of neonates from diabetic mothers. Lung slices of neonates from diabetic mothers showed depressed in vitro incorporation of [U-14C]glucose into neutral lipids and decreased oxidation to CO?. The results of these investigations show that maternal diabetes interferes with the structural and metabolic processes by which the undifferentiated lung becomes functional at birth.
Introduction A major cause of mortality in the neonatal period is respiratory distress syndrome [ 11. This disease is characterized by biochemical alterations and a deficiency in lung surfactant, which is a surface-active material rich in phospholipids that coats the inner surface of the alveoli and imparts stability to the
* To whom
reprint requestsshould
be addressed.
133
lung [ 11. Recent evidence indicates that the respiratory distress syndrome in infants from diabetic mothers is 6 times higher than that from non-diabetic are not mothers [2]. Presently the mechanisms leading to this dysfunction known. One possible mechanism however, is that high maternal glucose crosses the placenta, stimulates insulin release by the fetal pancreas and in some way interferes with the fetal lung lipid metabolism. It is not yet clear whether it is the high endogenous insulin which causes the disturbances or the high levels of exogenous glucose. Several studies have shown the importance of substrate utilization for lipid metabolism in the lung [ 3-61. Glucose plays a central role in lipid metabolism by serving as an energy fuel, by supplying NADPH for reductive biosynthesis of lipids, and by supplying the glycerol backbone for lipid synthesis. In addition, glucose provides the C-2 units for the novo fatty acid synthesis [7]. Factors which control the utilization of glucose for lipid synthesis in the developing lung have not been well defined. The aim of this investigation was to specifically examine the influence of maternal diabetes on lung lipid metabolism in neonates and on glucose utilization for lipid synthesis. Materials and Methods Animals. Long-Evans hooded rats (Blue Spruce Farms, Altmont, N.Y.) were injected on the 10th day of pregnancy with streptozotocin (50 mg/kg) via the tail vein. Streptozotocin (Sigma, St. Louis, MO.) was prepared with sterile saline and buffered to pH 4.5 with citric acid. Controls were injected with sterile saline/citric acid (pH 4.5). Rats were given both food and water ad libitum. In addition, the streptozotocin-treated animals were given access to 0.9% NaCl. All animals were maintained on a 12 h light-dark cycle at 23°C. Both neonates and mothers were killed 1 day after birth. The mothers were killed by intraperitoneal injection of sodium pentobarbitol (60 mg/kg body weight) with rapid exsanguination via the carotid artery and thoracotomy. The l-day-old neonates were killed, by decapitation. Cellular constituents. Blood samples were centrifuged at 600 Xg for 5 min; the serum deproteinized with 2% HC104, and then stored at -80°C. Cellular constituents were taken from lung tissue which was freeze-clamped and stored at -80°C until measured. Maternal and neonatal serum glucose concentrations were determined by the method of Bergmeyer [ 81. Lung protein was measured by the method of Lowry et al. [9] using bovine serum albumin as a standard. Nucleic acid content was measured by the method of Schneider et al. [lo] using calf thymus nucleic acid as the standard. Lung glycogen was measured by the method of Lo et al. [ll]. Lipid extraction and analysis. Tissue lipids were extracted by the method of Folch et al. [ 121, and then concentrated to dryness under Nz and resuspended in CHC13/CH30H (2 : 1, v/v). Phospholipids were separated from neutral lipids by silicic acid chromatography (Bio-Sil A, 100-200 mesh; Bio-Rad Laboratories, Richmond, Calif.) [13]. Columns were 8 cm of silicic acid (activated for 12 h at 100°C) in Pasteur pipets and were previously washed with 4-5 ml CH,OH, CH3COCH3 and CHC13. An aliquot of the Folch lipid extract was applied to the columns; neutral lipids were eluted with 4 ml CHCIJ and phos-
134
pholipids were eluted with 4 ml CHJOH. Phosphatidylcholine and phosphatidylethanolamine were isolated by thin-layer chromatography on silica gel H PA) developed with CHC13/CH30H/ (Applied Science, State College, CH&OOH/HzO (25 : 15 : 4 ; 2, v/v). The spots were visualized with IZ vapor and aspirated into test tubes; the phosphorous content was determined for phospholipid quantitation [ 141. Disaturated phosphatidylcholine was separated from the lipid mixture by the method of Mason et al. [15]. Assay of enzyme actiuities. The enzymes of the CDPcholine pathway were examined in cell-free lung homogenates. Lung tissue was homogenized in 6 ~01s. 0.125 M KCl, 20 mM Tris-HCl (pH 7.4). The homogenate was centrifuged to produce a 100 000 X g supernatant fraction and the microsomal pellet, which was used for enzyme assay. Choline kinase was measured by the method of Weinhold et al. [16]. Choline was separated from the product, phosphorylcholine, by paper chromatography developed in ethanol/isopropanol/30% NH40H (6.5 : 2 : 3.5, v/v). For cholinephosphate cytidylyltransferase, the method of Feldman and Weinhold (Feldman, D.A. and Weinhold, P.A., personal communication) was used; the enzyme activity was measured in the reverse direction using CDPcholine as the substrate. Choline phosphotransferase was measured by the method of Oldenborg and van Golde [17], using 1,2diolein as the substrate. Tissue incubation. Lungs from neonates were trimmed of extraneous tissue, blotted and quickly weighed to the nearest 10 mg. Tissue slices (approx. 75-90 mg; 0.5 mm thickness) were prepared with a Stadie-Riggs hand microtome. Visceral pleura, major vessels and large bronchi were excluded from the slices. Slices were incubated for 2 h in 25 ml incubation flask (Kontes Glass Co., Vineland, N.J.) containing a rubber stopper fitted with a center well to collect CO*. The incubation medium (5 ml) contained 25 pmol glucose, 2 &i [U-‘“Clglucose (specific activity 15.6 Ci/mol; New England Nuclear, Boston, Mass.) in Krebs-Henseleit bicarbonate buffer [ 181. Detailed procedures for incubation and CO, trapping have been described elsewhere [ 71. All 14COZ measurements were made by cutting the center wells, placing these in scintillation vials and adding 15 ml Aquasol II (New England Nuclear, Boston, Mass.). Lipids were extracted and separated as described above, and the radioactivity was counted in 15 ml toluene-base solution with 4 g/l Omnifluor (New England Nuclear, Boston, Mass.). Results The streptozotocin-treated mothers showed the presence of glucose in the urine two days following the injection. Serum glucose of the treated mothers 1 day after delivery averaged 25.9 mM f 1.5 S.E. and the control animals averaged 8.1+ 0.4, which clearly demonstrates glucose intolerance during gestation. The l-day neonates from untreated mothers showed the classical hypoglycemic state (3.8 mM f 0.2 SE.) while the neonates from diabetic mothers were even more hypoglycemic (2.1 mM f 0.2 S.E.). Maternal diabetes also caused a significant decrease in body (14%) and lung weight (32%), and had a marked effect on lung constituents of the neonates (Table I). Lung protein was 30% lower in neonates from diabetic mothers, while liver protein was
135 TABLE EFFECT
I OF MATERNAL
DIABETES
ON l-DAY
Values are mean + S.E. (n = 10 unless otherwise Measurement
Control
Neonate body weight (9) Neonate lung weight (9) Protein (mg/mg tissue) Liver protein (mg/mg tissue) DNA (pg/mg tissue) Protein/DNA (Mg/@z) Glycogen (mg/lOO mg tissue)
6.49 0.19 0.13 0.17 6.85 5.20 1.58
* Significant
from controls
+ f + ? f f +
OLD RAT LUNGS
indicated
by figures in parentheses). Diabetic
0.1 0.12 0.003 0.01 0.4 1.5 0.1
(P < 0.05 by Student’s
(12)
5.57 0.13 0.09 0.18 10.12 1.50 1.76
+ 0.3 * (18) f 0.01 * k 0.01 * f 0.01 f 0.6 * -I 0.5 * + 0.09 *
t-test).
unchanged, suggesting some selectivity in the effects of maternal diabetes. The marked decrease in protein/DNA ratio, coupled with increased DNA levels, suggest the presence of more undifferentiated lung cells in the neonates from diabetic mothers. Both glycogen (Table I) and lipid in lungs from neonates of diabetic mothers showed a significant increase (especially lung phospholipid, Table II). Although the percentages of phosphatidylcholine and phosphatidylethanolamine were not altered, the percentage of phosphatidylcholine made up of disaturated phosphatidylcholine showed a significant (25%) decrease in lungs from neonates of diabetic mothers. The activities of the CDPcholine pathway enzymes are presented in Table III. Both cholinephosphate cytidylyltransferase and choline phosphotransferase activities were stimulated by the presence of lipids in the assay mixture. For the former enzyme, phosphatidylglycerol served as an activator and for the latter enzyme 1,2diolein served as substrate. The specific activities of all three enzymes in the CDPcholine pathway were significantly higher in neonates from diabetic mothers. The utilization of [U-i4C]glucose by neonatal lung slices as assessed by its oxidation to CO? and incorporation into various lipid components is shown in Table IV. Glucose is readily oxidized to CO2 by fetal lungs, and most of the radioactivity incorporated into lipids from glucose appeared in lung phospholipids. Maternal diabetes significantly depressed glucose oxidation (21%) and
TABLE
II
INFLUENCE
OF MATERNAL
DIABETES
Values are mean ? S.E. Figures in parentheses
ON LUNG PHOSPHOLIPID represent
number
OF l-DAY
of determinations.
Lung phospholipid
Control
Total lipid (mg/me Protein) Phospholipid (mg/mg protein) Phosphatidylcholine (mg/mg phospholipid) Disaturated PhosphatidylchoIine (mg/mg phosphatidylcholine) Phosphatidylethanolamine (mg/mg phospholipid)
0.28 0.19 0.52 0.58 0.11
* Statistically
significant
from control
(P < 0.05 by Student’s
NEONATE
t-test).
f + f f f
Diabetic 0.05 0.01 0.02 0.04 0.005
(6) (6) (5) (5) (5)
0.60 0.48 0.57 0.43 0.12
f 2 f f t
0.02 0.01 0.03 0.06 0.02
* (6) * (6) (5) * (5) (5)
136 TABLE
III
ACTIVITIES
OF CDPCHOLKNE
Values are expressed
PATHWAY
as mean f S.E.
ENZYMES
(n = lO/group).
IN THE NEONATAL
Diabetic
mothers. Cytidylyltransferase lipid activator was 0.25 transferase lipid substrate was 1.0 mM diolein. Enzyme
refers to l-day
LUNG neonatal
mM phosphatidylgiycerol
nmolfmin
Diabetic
Choline khrase
0.74
+_0.04
1.09
t 0.06
*
Cho~nephosphate cytidylyi~~~erase + lipid activation - lipid activation
2.22
+ 0.14
2.94
+_0.09
*
1.19
r 0.07
1.52
+ 0.95
*
0.08 0.02
t 0.01 i 0.003
0.28 0.07
+ 0.03 + 0.01
* *
* P < 0.01
TABLE
by Student’s
phospho-
per mg
Control
Choline phosphotransferase + lipid substrate -lipid substrate
lungs from diabetic
and choline
t-test.
IV
UTILIZATION MOTHERS
OF
III-14CJGLUCOSE
BY
l-DAY
NEONATAL
Values are mean +- S.E., expressed as nmol glucose equivalent/100 100-150 mg) were incubated for 2 h at 37°C in 5 ml calcium-free 7.4) contahring 2 &Ci [U-‘4Clghrcose and 25 pmol glucose. Metaboiite
Control
co2
321.3 78 59.1 17.7
Total lipid Phosphoiipid Neutral lipid
LUNG
LSICES
FROM
DIABETIC
mg tissue per 2 h. Lung slices (approx. Krebs-Henseleit bicarbonate buffer (pH
Diabetic + 18.7 + 5.8 t 2.7 ? 0.8
* Mean values were significantly
different
251.6 69 55.9 12.9
+ 18.5 * t 4.2 + 1.9 + 0.8 *
from control
(P < 0.05
by Student’s
t-test).
glucose ~~orporation into lung neutral lipids (27%) with no effect on glucose incorporation into lung phospholipids. Discussion In the present studies, maternal rats treated with streptozotoc~ midway through gestation became extremely glucose intolerant and fetuses from such rats have been shown to be hyperglycemic and hyperinsulinemic at term [ 191. The results of this study show that maternal diabetes had a pronounced effect on the neonatal lung, and also, caused a decreased body weight of the neonates. In contrast, human neonates from diabetic mothers are usually larger than those from normal mothers. This difference is explained by the fact that in the animal model the animals were in an uncontrolled, severely hyperglycemic state, whereas diabetes in the human is controlled by insulin therapy. The demonstration that the percentage disaturated phosphatidyl~holine is decreased in lungs of neonates from diabetic mothers is particularly important
137
and assumes functional significance since this species of lung phospholipid is the principal agent responsible for surface tension reducing properties in the su~ac~nt complex [ 1] . With the observation that the enzyme activity in the CDPcholine pathway was elevated while the percent disaturated phosphatidylcholine is decreased in the streptozotocin-treated group suggests that pathways other than those responsible for phospholipid synthesis may be affected such as the acylation and reacylation enzymes, lysophosphatidylcholine acyltransferase and lysophosphatidylcholine: lysophosphatidylcholine acyltransferase. Additional studies will be required for firm support of this. Farrell et al. [20] assessed fetal lung lecithin biosynthesis at 85% term from pregnant rhesus monkeys injected with streptozotocin midway through gestation. The amniotic fluid lecithin/sphingomyelin ratio and the rate of [ 14C]choline incorporation into lecithin in fetal lung slices were both significantly greater than age matched controls. However, lecithin concentration was not changed. These findings support our data on lecithin and the increased enzyme activity in the CDPcholine pathway from lungs of neonates from diabetic mothers. The pattern of glucose utilization in the neonatal lung from the diabetic rat was also altered. Particularly significant was the observation that glucose oxidation to COZ and the incorporation of glucose into neutral lipids decreased while glucose ~corpo~tion into lung phospholipid was un~h~ged. These data suggest that glucose is being conserved for glycerol 3-phosphate which is then used in phospholipid synthesis. Recently, Moxely and Longmore [21] using adult lungs from rats treated with streptozotocin showed that less glucose was incorporated into phosphatidylcholine isolated from lung surfactant. While further studies will be required to verify these findings in neonate lungs, the fact that glucose incorporation into phosphatidylcholine is also depressed in the adult lung suggests that diabetes has a selective action on phosphatidylcholine metabolism. Although fetuses from diabetic rats have been shown to be hyperglycemic and hyperinsulinemic at term, after birth neonates from both normal and diabetic mothers become hypoglycemic. Since neonates from our diabetic rats were more hypoglycemic, 24 h after birth than controls, one could argue the effects seen in this study could be due, in part, to acute hypoglycemia. We are currently completing additional studies which show that this is not the case. First, lung weight, glycogen, DNA, lipid and protein concentration from lungs of 21day rat fetuses (term 22 days) of diabetic mothers showed the same effects as that of the lday-old neonates; however, the 21day ‘fetuses were hyperglycemic. Secondly, neonatal rats made hypoglycemic by 48 h starvation do not show altered lung weight or changes in lipid, protein or DNA content. Moreover, lung glycogen decreased rather than increased. The above observations suggest an insulin effect rather than an acute effect from hypoglycemia. Thus, the data seen in these studies with maternal diabetes indicate that insulin may play an important role in regulating the processes by which the undifferentiated lung becomes structurally and metabolically functional. Acknowledgements We greatly
appreciate
the expert
technical
assistance
of D. Ryder
and K.
138
Buechler. This investigation was supported by N.I.H. grant HD 10670. R.A. Rhoades is supported by Research Career Development Award K04 HL 00317. References 1 2 3 4 5 6 1 8 9 10 11 12 13 14 15 16 11 18
Farrell, P.M. and Avery, M.E. (1915) Am. Rev. Resp. Dis. 111. 651-688 Robert, M.F., Neff, R.K., Hubell. J.P. (1916) N. Engl. J. Med. 294. 351-360 Tierney. D.F. (1914) Ann. Rev. Pbysiol. 36, 209-231 Sslisbury-Murphey, S., Rubenstein. D. and Beck. J.C. (1966) Am. J. Physiol. 211, 988-992 Scholz, R.W.. Woodward, B.M. and Rhoades. R.A. (1912) Am. J. Physiol. 223,991-996 Bassett, D.J.P. and Fisher, A.B. (1976) Am. J. Physiol. 231. 1521-1532
Schols, R.W. and Rhoades, R.A. (1911) Biochem. J. 124.2451-264 Bergmeyer. H.U.. Bernt. E., Schmidt, F. and Stork, H. (1914) Methods Enzymat. Anal. 3.1196-1201 Lowry, O.H.. Rosebrough. N.J., Farr, A.L. and Randall, R.J. (1951) J. Biol. Chem. 193, 265-211 Schneider, W.C. (1951) Methods Enzymol. 3.680-684 Lo. S.J., Russell, J.C. and Taylor, A.W. (1970) J. APP~. Physiol. 28. 334-239 Folch, J.. Lees, M. and Sloane-Stanley, G.H. (1951) J. Biol. Chem. 226, 491-509 Hirsch, J. and Ahrens, E.H., Jr. (1958) J. Biol. Chem. 233,311-322 Bartlett. G.R. (1969) J. Biol. Chem. 234. 466468 Mason, R.J., Nellenbogen, J. and Clements. J.A. (1916) J. Lipid Res. 17, 281-284 Weinhold, P.A.. Skinner, R.S. and Sanders, R.D. (1913) Biochim. Biophys. Acta 326, 43-41 Oldenborg, V. and van Golde. L.M.G. (1916) Biochhn. Biophys. Acta 441,433-442 Umbreit. W.W., Burris. R.H. and Stauffer. J.F. (1912) Manometric and Biochemical Techniques, 5th edn., pp. 144-141. Burgess Publishing, Minneapolis, Minn. 19 Pitkin. R.M. and Van Arden. D.E. (1914) Endocrinology 94.1241-1251 20 Epstein, M.F.. Farrell, P.M. and Chez, R.A. (1916) Pediatrics 51.122-728 21 Moxely, M.A. and Longmore, W.J. (111) Biochim. Biophys. Acta 488, 218-224
Biochimica et Biophysics @ Elsevier/North-Holland
BBA
572 (1979)
Biomedical
132-138
Press
57294
INFLUENCE NEONATAL
R.A.
Acta,
RHOADES
OF MATERNAL RAT LUNG
*, D.A.
FILLER
DIABETES
IN
and B. VANNATA
Department of Physiology, Indiana University Street, Indianapolis, IN 46202 (U.S.A.) (Received
ON LIPID METABOLISM
School of Medicine,
1100 West Michigan
July 4th, 1978)
Key words: Diabetes;
Lipid metabolism;Pregnancy;
Phospholipid;
(Neonatal
rat lung)
Summary
The effect of maternal diabetes on tissue constituents, lipid metabolism and glucose utilization was examined in l-day old rat lungs. Maternal diabetes was induced by intravenous injection of streptozotocin (50 mg/kg body weight) into pregnant Long-Evans hooded rats on the 10th day of gestation resulting in a maternal serum glucose concentration which was 3-fold higher than that of controls. Neonates from diabetic mothers showed a significant decrease in body weight (14%), lung weight (32%) and lung protein concentration (30%). Glycogen, DNA, and lipid content of the lungs were significantly elevated in neonates from diabetic mothers. The percent of total phospholipid made up of phosphatidylcholine was not altered, but the percentage of disaturated phosphatidylcholine was decreased (25%). The activity of the CDPcholine pathway enzymes (choline kinase, cholinephosphate cytidylyltransferase and choline phosphotransferase) also showed a marked increase in lungs of neonates from diabetic mothers. Lung slices of neonates from diabetic mothers showed depressed in vitro incorporation of [U-14C]glucose into neutral lipids and decreased oxidation to CO?. The results of these investigations show that maternal diabetes interferes with the structural and metabolic processes by which the undifferentiated lung becomes functional at birth.
Introduction A major cause of mortality in the neonatal period is respiratory distress syndrome [ 11. This disease is characterized by biochemical alterations and a deficiency in lung surfactant, which is a surface-active material rich in phospholipids that coats the inner surface of the alveoli and imparts stability to the
* To whom
reprint requestsshould
be addressed.
133
lung [ 11. Recent evidence indicates that the respiratory distress syndrome in infants from diabetic mothers is 6 times higher than that from non-diabetic are not mothers [2]. Presently the mechanisms leading to this dysfunction known. One possible mechanism however, is that high maternal glucose crosses the placenta, stimulates insulin release by the fetal pancreas and in some way interferes with the fetal lung lipid metabolism. It is not yet clear whether it is the high endogenous insulin which causes the disturbances or the high levels of exogenous glucose. Several studies have shown the importance of substrate utilization for lipid metabolism in the lung [ 3-61. Glucose plays a central role in lipid metabolism by serving as an energy fuel, by supplying NADPH for reductive biosynthesis of lipids, and by supplying the glycerol backbone for lipid synthesis. In addition, glucose provides the C-2 units for the novo fatty acid synthesis [7]. Factors which control the utilization of glucose for lipid synthesis in the developing lung have not been well defined. The aim of this investigation was to specifically examine the influence of maternal diabetes on lung lipid metabolism in neonates and on glucose utilization for lipid synthesis. Materials and Methods Animals. Long-Evans hooded rats (Blue Spruce Farms, Altmont, N.Y.) were injected on the 10th day of pregnancy with streptozotocin (50 mg/kg) via the tail vein. Streptozotocin (Sigma, St. Louis, MO.) was prepared with sterile saline and buffered to pH 4.5 with citric acid. Controls were injected with sterile saline/citric acid (pH 4.5). Rats were given both food and water ad libitum. In addition, the streptozotocin-treated animals were given access to 0.9% NaCl. All animals were maintained on a 12 h light-dark cycle at 23°C. Both neonates and mothers were killed 1 day after birth. The mothers were killed by intraperitoneal injection of sodium pentobarbitol (60 mg/kg body weight) with rapid exsanguination via the carotid artery and thoracotomy. The l-day-old neonates were killed, by decapitation. Cellular constituents. Blood samples were centrifuged at 600 Xg for 5 min; the serum deproteinized with 2% HC104, and then stored at -80°C. Cellular constituents were taken from lung tissue which was freeze-clamped and stored at -80°C until measured. Maternal and neonatal serum glucose concentrations were determined by the method of Bergmeyer [ 81. Lung protein was measured by the method of Lowry et al. [9] using bovine serum albumin as a standard. Nucleic acid content was measured by the method of Schneider et al. [lo] using calf thymus nucleic acid as the standard. Lung glycogen was measured by the method of Lo et al. [ll]. Lipid extraction and analysis. Tissue lipids were extracted by the method of Folch et al. [ 121, and then concentrated to dryness under Nz and resuspended in CHC13/CH30H (2 : 1, v/v). Phospholipids were separated from neutral lipids by silicic acid chromatography (Bio-Sil A, 100-200 mesh; Bio-Rad Laboratories, Richmond, Calif.) [13]. Columns were 8 cm of silicic acid (activated for 12 h at 100°C) in Pasteur pipets and were previously washed with 4-5 ml CH,OH, CH3COCH3 and CHC13. An aliquot of the Folch lipid extract was applied to the columns; neutral lipids were eluted with 4 ml CHCIJ and phos-
134
pholipids were eluted with 4 ml CHJOH. Phosphatidylcholine and phosphatidylethanolamine were isolated by thin-layer chromatography on silica gel H PA) developed with CHC13/CH30H/ (Applied Science, State College, CH&OOH/HzO (25 : 15 : 4 ; 2, v/v). The spots were visualized with IZ vapor and aspirated into test tubes; the phosphorous content was determined for phospholipid quantitation [ 141. Disaturated phosphatidylcholine was separated from the lipid mixture by the method of Mason et al. [15]. Assay of enzyme actiuities. The enzymes of the CDPcholine pathway were examined in cell-free lung homogenates. Lung tissue was homogenized in 6 ~01s. 0.125 M KCl, 20 mM Tris-HCl (pH 7.4). The homogenate was centrifuged to produce a 100 000 X g supernatant fraction and the microsomal pellet, which was used for enzyme assay. Choline kinase was measured by the method of Weinhold et al. [16]. Choline was separated from the product, phosphorylcholine, by paper chromatography developed in ethanol/isopropanol/30% NH40H (6.5 : 2 : 3.5, v/v). For cholinephosphate cytidylyltransferase, the method of Feldman and Weinhold (Feldman, D.A. and Weinhold, P.A., personal communication) was used; the enzyme activity was measured in the reverse direction using CDPcholine as the substrate. Choline phosphotransferase was measured by the method of Oldenborg and van Golde [17], using 1,2diolein as the substrate. Tissue incubation. Lungs from neonates were trimmed of extraneous tissue, blotted and quickly weighed to the nearest 10 mg. Tissue slices (approx. 75-90 mg; 0.5 mm thickness) were prepared with a Stadie-Riggs hand microtome. Visceral pleura, major vessels and large bronchi were excluded from the slices. Slices were incubated for 2 h in 25 ml incubation flask (Kontes Glass Co., Vineland, N.J.) containing a rubber stopper fitted with a center well to collect CO*. The incubation medium (5 ml) contained 25 pmol glucose, 2 &i [U-‘“Clglucose (specific activity 15.6 Ci/mol; New England Nuclear, Boston, Mass.) in Krebs-Henseleit bicarbonate buffer [ 181. Detailed procedures for incubation and CO, trapping have been described elsewhere [ 71. All 14COZ measurements were made by cutting the center wells, placing these in scintillation vials and adding 15 ml Aquasol II (New England Nuclear, Boston, Mass.). Lipids were extracted and separated as described above, and the radioactivity was counted in 15 ml toluene-base solution with 4 g/l Omnifluor (New England Nuclear, Boston, Mass.). Results The streptozotocin-treated mothers showed the presence of glucose in the urine two days following the injection. Serum glucose of the treated mothers 1 day after delivery averaged 25.9 mM f 1.5 S.E. and the control animals averaged 8.1+ 0.4, which clearly demonstrates glucose intolerance during gestation. The l-day neonates from untreated mothers showed the classical hypoglycemic state (3.8 mM f 0.2 SE.) while the neonates from diabetic mothers were even more hypoglycemic (2.1 mM f 0.2 S.E.). Maternal diabetes also caused a significant decrease in body (14%) and lung weight (32%), and had a marked effect on lung constituents of the neonates (Table I). Lung protein was 30% lower in neonates from diabetic mothers, while liver protein was
135 TABLE EFFECT
I OF MATERNAL
DIABETES
ON l-DAY
Values are mean + S.E. (n = 10 unless otherwise Measurement
Control
Neonate body weight (9) Neonate lung weight (9) Protein (mg/mg tissue) Liver protein (mg/mg tissue) DNA (pg/mg tissue) Protein/DNA (Mg/@z) Glycogen (mg/lOO mg tissue)
6.49 0.19 0.13 0.17 6.85 5.20 1.58
* Significant
from controls
+ f + ? f f +
OLD RAT LUNGS
indicated
by figures in parentheses). Diabetic
0.1 0.12 0.003 0.01 0.4 1.5 0.1
(P < 0.05 by Student’s
(12)
5.57 0.13 0.09 0.18 10.12 1.50 1.76
+ 0.3 * (18) f 0.01 * k 0.01 * f 0.01 f 0.6 * -I 0.5 * + 0.09 *
t-test).
unchanged, suggesting some selectivity in the effects of maternal diabetes. The marked decrease in protein/DNA ratio, coupled with increased DNA levels, suggest the presence of more undifferentiated lung cells in the neonates from diabetic mothers. Both glycogen (Table I) and lipid in lungs from neonates of diabetic mothers showed a significant increase (especially lung phospholipid, Table II). Although the percentages of phosphatidylcholine and phosphatidylethanolamine were not altered, the percentage of phosphatidylcholine made up of disaturated phosphatidylcholine showed a significant (25%) decrease in lungs from neonates of diabetic mothers. The activities of the CDPcholine pathway enzymes are presented in Table III. Both cholinephosphate cytidylyltransferase and choline phosphotransferase activities were stimulated by the presence of lipids in the assay mixture. For the former enzyme, phosphatidylglycerol served as an activator and for the latter enzyme 1,2diolein served as substrate. The specific activities of all three enzymes in the CDPcholine pathway were significantly higher in neonates from diabetic mothers. The utilization of [U-i4C]glucose by neonatal lung slices as assessed by its oxidation to CO? and incorporation into various lipid components is shown in Table IV. Glucose is readily oxidized to CO2 by fetal lungs, and most of the radioactivity incorporated into lipids from glucose appeared in lung phospholipids. Maternal diabetes significantly depressed glucose oxidation (21%) and
TABLE
II
INFLUENCE
OF MATERNAL
DIABETES
Values are mean ? S.E. Figures in parentheses
ON LUNG PHOSPHOLIPID represent
number
OF l-DAY
of determinations.
Lung phospholipid
Control
Total lipid (mg/me Protein) Phospholipid (mg/mg protein) Phosphatidylcholine (mg/mg phospholipid) Disaturated PhosphatidylchoIine (mg/mg phosphatidylcholine) Phosphatidylethanolamine (mg/mg phospholipid)
0.28 0.19 0.52 0.58 0.11
* Statistically
significant
from control
(P < 0.05 by Student’s
NEONATE
t-test).
f + f f f
Diabetic 0.05 0.01 0.02 0.04 0.005
(6) (6) (5) (5) (5)
0.60 0.48 0.57 0.43 0.12
f 2 f f t
0.02 0.01 0.03 0.06 0.02
* (6) * (6) (5) * (5) (5)
136 TABLE
III
ACTIVITIES
OF CDPCHOLKNE
Values are expressed
PATHWAY
as mean f S.E.
ENZYMES
(n = lO/group).
IN THE NEONATAL
Diabetic
mothers. Cytidylyltransferase lipid activator was 0.25 transferase lipid substrate was 1.0 mM diolein. Enzyme
refers to l-day
LUNG neonatal
mM phosphatidylgiycerol
nmolfmin
Diabetic
Choline khrase
0.74
+_0.04
1.09
t 0.06
*
Cho~nephosphate cytidylyi~~~erase + lipid activation - lipid activation
2.22
+ 0.14
2.94
+_0.09
*
1.19
r 0.07
1.52
+ 0.95
*
0.08 0.02
t 0.01 i 0.003
0.28 0.07
+ 0.03 + 0.01
* *
* P < 0.01
TABLE
by Student’s
phospho-
per mg
Control
Choline phosphotransferase + lipid substrate -lipid substrate
lungs from diabetic
and choline
t-test.
IV
UTILIZATION MOTHERS
OF
III-14CJGLUCOSE
BY
l-DAY
NEONATAL
Values are mean +- S.E., expressed as nmol glucose equivalent/100 100-150 mg) were incubated for 2 h at 37°C in 5 ml calcium-free 7.4) contahring 2 &Ci [U-‘4Clghrcose and 25 pmol glucose. Metaboiite
Control
co2
321.3 78 59.1 17.7
Total lipid Phosphoiipid Neutral lipid
LUNG
LSICES
FROM
DIABETIC
mg tissue per 2 h. Lung slices (approx. Krebs-Henseleit bicarbonate buffer (pH
Diabetic + 18.7 + 5.8 t 2.7 ? 0.8
* Mean values were significantly
different
251.6 69 55.9 12.9
+ 18.5 * t 4.2 + 1.9 + 0.8 *
from control
(P < 0.05
by Student’s
t-test).
glucose ~~orporation into lung neutral lipids (27%) with no effect on glucose incorporation into lung phospholipids. Discussion In the present studies, maternal rats treated with streptozotoc~ midway through gestation became extremely glucose intolerant and fetuses from such rats have been shown to be hyperglycemic and hyperinsulinemic at term [ 191. The results of this study show that maternal diabetes had a pronounced effect on the neonatal lung, and also, caused a decreased body weight of the neonates. In contrast, human neonates from diabetic mothers are usually larger than those from normal mothers. This difference is explained by the fact that in the animal model the animals were in an uncontrolled, severely hyperglycemic state, whereas diabetes in the human is controlled by insulin therapy. The demonstration that the percentage disaturated phosphatidyl~holine is decreased in lungs of neonates from diabetic mothers is particularly important
137
and assumes functional significance since this species of lung phospholipid is the principal agent responsible for surface tension reducing properties in the su~ac~nt complex [ 1] . With the observation that the enzyme activity in the CDPcholine pathway was elevated while the percent disaturated phosphatidylcholine is decreased in the streptozotocin-treated group suggests that pathways other than those responsible for phospholipid synthesis may be affected such as the acylation and reacylation enzymes, lysophosphatidylcholine acyltransferase and lysophosphatidylcholine: lysophosphatidylcholine acyltransferase. Additional studies will be required for firm support of this. Farrell et al. [20] assessed fetal lung lecithin biosynthesis at 85% term from pregnant rhesus monkeys injected with streptozotocin midway through gestation. The amniotic fluid lecithin/sphingomyelin ratio and the rate of [ 14C]choline incorporation into lecithin in fetal lung slices were both significantly greater than age matched controls. However, lecithin concentration was not changed. These findings support our data on lecithin and the increased enzyme activity in the CDPcholine pathway from lungs of neonates from diabetic mothers. The pattern of glucose utilization in the neonatal lung from the diabetic rat was also altered. Particularly significant was the observation that glucose oxidation to COZ and the incorporation of glucose into neutral lipids decreased while glucose ~corpo~tion into lung phospholipid was un~h~ged. These data suggest that glucose is being conserved for glycerol 3-phosphate which is then used in phospholipid synthesis. Recently, Moxely and Longmore [21] using adult lungs from rats treated with streptozotocin showed that less glucose was incorporated into phosphatidylcholine isolated from lung surfactant. While further studies will be required to verify these findings in neonate lungs, the fact that glucose incorporation into phosphatidylcholine is also depressed in the adult lung suggests that diabetes has a selective action on phosphatidylcholine metabolism. Although fetuses from diabetic rats have been shown to be hyperglycemic and hyperinsulinemic at term, after birth neonates from both normal and diabetic mothers become hypoglycemic. Since neonates from our diabetic rats were more hypoglycemic, 24 h after birth than controls, one could argue the effects seen in this study could be due, in part, to acute hypoglycemia. We are currently completing additional studies which show that this is not the case. First, lung weight, glycogen, DNA, lipid and protein concentration from lungs of 21day rat fetuses (term 22 days) of diabetic mothers showed the same effects as that of the lday-old neonates; however, the 21day ‘fetuses were hyperglycemic. Secondly, neonatal rats made hypoglycemic by 48 h starvation do not show altered lung weight or changes in lipid, protein or DNA content. Moreover, lung glycogen decreased rather than increased. The above observations suggest an insulin effect rather than an acute effect from hypoglycemia. Thus, the data seen in these studies with maternal diabetes indicate that insulin may play an important role in regulating the processes by which the undifferentiated lung becomes structurally and metabolically functional. Acknowledgements We greatly
appreciate
the expert
technical
assistance
of D. Ryder
and K.
138
Buechler. This investigation was supported by N.I.H. grant HD 10670. R.A. Rhoades is supported by Research Career Development Award K04 HL 00317. References 1 2 3 4 5 6 1 8 9 10 11 12 13 14 15 16 11 18
Farrell, P.M. and Avery, M.E. (1915) Am. Rev. Resp. Dis. 111. 651-688 Robert, M.F., Neff, R.K., Hubell. J.P. (1916) N. Engl. J. Med. 294. 351-360 Tierney. D.F. (1914) Ann. Rev. Pbysiol. 36, 209-231 Sslisbury-Murphey, S., Rubenstein. D. and Beck. J.C. (1966) Am. J. Physiol. 211, 988-992 Scholz, R.W.. Woodward, B.M. and Rhoades. R.A. (1912) Am. J. Physiol. 223,991-996 Bassett, D.J.P. and Fisher, A.B. (1976) Am. J. Physiol. 231. 1521-1532
Schols, R.W. and Rhoades, R.A. (1911) Biochem. J. 124.2451-264 Bergmeyer. H.U.. Bernt. E., Schmidt, F. and Stork, H. (1914) Methods Enzymat. Anal. 3.1196-1201 Lowry, O.H.. Rosebrough. N.J., Farr, A.L. and Randall, R.J. (1951) J. Biol. Chem. 193, 265-211 Schneider, W.C. (1951) Methods Enzymol. 3.680-684 Lo. S.J., Russell, J.C. and Taylor, A.W. (1970) J. APP~. Physiol. 28. 334-239 Folch, J.. Lees, M. and Sloane-Stanley, G.H. (1951) J. Biol. Chem. 226, 491-509 Hirsch, J. and Ahrens, E.H., Jr. (1958) J. Biol. Chem. 233,311-322 Bartlett. G.R. (1969) J. Biol. Chem. 234. 466468 Mason, R.J., Nellenbogen, J. and Clements. J.A. (1916) J. Lipid Res. 17, 281-284 Weinhold, P.A.. Skinner, R.S. and Sanders, R.D. (1913) Biochim. Biophys. Acta 326, 43-41 Oldenborg, V. and van Golde. L.M.G. (1916) Biochhn. Biophys. Acta 441,433-442 Umbreit. W.W., Burris. R.H. and Stauffer. J.F. (1912) Manometric and Biochemical Techniques, 5th edn., pp. 144-141. Burgess Publishing, Minneapolis, Minn. 19 Pitkin. R.M. and Van Arden. D.E. (1914) Endocrinology 94.1241-1251 20 Epstein, M.F.. Farrell, P.M. and Chez, R.A. (1916) Pediatrics 51.122-728 21 Moxely, M.A. and Longmore, W.J. (111) Biochim. Biophys. Acta 488, 218-224