Biochimicrr et Biophysics Acta, (1977) Zf,Y--224 @ Elsevier:North-Hollatld Biomedieaf Press
BBA 570%
EFFECT OF EXPERIMENTAL DIABETES AND INSULIN ON LIPID METABOLISM IK THE ISOLATED PERFUSED RAT LUNG
MICHAEL
A. MOXLEY
and ELLIOT
i)epartmcnt of Biochemistry, Ht*d., St. Louis. MO. 63103 (Received
February
St. Louis (U.S.A.)
J. LONG~~a~E Ckiuersity
SchooE of Medicine,
1402 S. Grund
8th, 19777)
Summary The isolated perfused rat lung was used as a model to study the possible hormonal re~lation of lipid metabolism in the mamrn~iall adult lung. Experimental diabetes, whether induced by alfoxan or streptozotocin, decreased the incorporation of [IJ-‘4Cfglucose into neutral lipids and phospholipids of both the surfactant fraction and the residual fraction of the lung by 60---80%. Glucose incorporation into phosphatidyl~holi~e and phosphatidylglycerol is decreased in experimental diabetes in both the surfactant and residual fractions to a comparable degree. Glucose incorporation is decreased in both the fatty acid and the glycerophosphocholine moieties of phosphatidylcholine isolated from the surfactant and residual fractions. Insulin treatment of normal animals 30 or 15 min prior to perfusion resulted in an approximate doubling of the in~o~oratio~ of glucose into the phosphatidyl~holine and phosphatidylglycerol isolated from the surfactant and residual fractions of the lung. The incorporation of glucose into palmitic acid isolated from phosphatidyl~holine was also shown to increase similarly. The results of these investigations indicate that insulin may play a role in regulating the synthesis of the important lipid components of the mamm~ian pulmon~ surfactant complex.
Introduction ft. has become increasingly apparent that lipid metabolism in the lung is of considerabfe importance in maintaining the normal function of that organ. It has been demonstra~d that the surface activity of the rnarnn~~i~ surfactant complex is attributable to the presence of the lipid components of the complex [l-4]. However, little information regarding those factors which may influence the metabolism of these lipid components is available. Because of the demonstrated importance of glucose as a substrate in the mammalian lung
219
[5-S] we are investigating the influence of hormones on glucose metabolism in the lung. The effects of chemically induced diabetes on the incorporation of glucose into the lipid fractions of the isolated perfused rat lung have been previously reported [9,10]. This report deals with the extension of those studies to include the determination of the effects of experimental diabetes and of insulin excess on the synthesis of the lipid components of the pulmonary surfactant complex. Materials and Methods The surgical procedures and perfusion technique utilized in these experiments have been described [7,10]. Male, Wistar strain rats (Hilltop Lab Animals, Inc., Scottsdale, Pa.) 150~-250 g were treated with alloxan (Sigma Chemical Co., St. Louis, MO.) or streptozotocin (Upjohn, Kalamazoo, Mich.) as previously described [lo] to induce diabetes. Lungs from diabetic animals were perfused for 1 h with a medium containing 5.6 mM glucose and 50 PCi of [U-14C]glucose (New England Nuclear, Boston, Mass.) Animals of the same strain, sex and age were used as controls. To determine the effects of insulin on lipid metabolism in the lungs of normal animals, rats received a single intravenous dose (4 I.U.) of regular insulin (Eli Lilly and Co., Indianapolis, Ind.) via the tail vein 15 or 30 min before the blood flow to the lungs was interrupted during the surgical procedure. Control animals were injected with physiological saline and the lungs were perfused as described. Following perfusion, lungs were removed from the perfusion apparatus, and frozen in liquid nitrogen. A portion of the tissue was taken for determination of tissue protein according to Lowry et al. [ll]. The remainder of the tissue was homogenized with a Ten Broeck tissue grinder in a medium consisting of 154 mM NaCl, 10 mM Tris * HCl and 1 mM EDTA, pH 7.4. The separation of the surfactant fraction from the residual fraction of the lung was accomplished using the discontinuous sucrose gradient centrifugation method of Frosolono et al. [ 121 as modified and described by Sanders and Longmore [13]. The surfactant fraction obtained by this method contains the total extracellular and intracellular surfactant of the lung. The remaining tissue components comprise the residual fraction. Following this procedure the lipids were extracted from the two fractions according to the method of Folch et al. [14]. Initial separation of the lipids was accomplished using column chromatography on silicic Pa.). Neutral lipids were acid (Unisil, Clarkson Chemical Co., Williamsport, eluted from a 2 g column with 50 ml of chloroform and phospholipids with 100 ml of methanol. When the phospholipids were to be further fractionated they were eluted with 50 ml of chloroform, 25 ml of tetrahydrofuran/dimethoxymethane/methanol/water (10 : 6 : 4 : 1, v/v) and 100 ml of methanol followed by separation by thin-layer chromatography on silica gel H as adapted by Godinez et al. [15]. Phospholipid phosphorus was quantitated using the method of Bartlett as modified by Dittmer and Wells [16] and specific radioactivities were obtained by counting suitable aliquots of the lipid fractions using liquid scintillation counting techniques. Efficiency of counting was determined by the channels ratio method. Fatty acids of the isolated phosphatidyl-
220
choline were isolated as methyl esters by transeste~fication [17]. Specific activity of the radiolabeled pafmitate was determined by using a specially designed effluent splitter and trapping device on the gas chromatograph. Quantitation of glucose incorporation into lung lipids is based on the specific activity of the media glucose and is expressed as glucose incorporated per g protein per h or as the specific activity of the isolated compounds. All phospholipid specific activities were corrected to a substrate specific activity of 2.5 * 10” dpm/pmol. Results are presented as the mean plus or minus the standard error of the mean of the number of perfusions performed. Results Previous reports from this laboratory indicated that experimental diabetes significantly lowered glucose incorporation into the total neutral and total phosphoiipid fraction of the isolated perfused rat lung. The results shown in Table I demonstrate that this effect is evident in both the surfactant and residual fractions of the lung. The rate of incorporation of glucose into the neutral lipids of both the surfactant fraction and the residual fraction of alloxan diabetic lungs is 19.3 and 13.2% of the control rate, respectively, while the rate of incorporation into the total phospholipids of the fractions is 12.1 and 18.4% that observed in normal lungs. Table I illustrates that similar results are obtained when diabetes is induced by streptozotoein treatment. Work from a number of laboratories indicates that certain disaturated species of ~~hosphatidylcholine [ 2,3] and phosphatidylgiycerol [ 18,191 are responsible for the surface activity of the mammalian surfactant complex. Table II illustrates that the specific activities of phosphatidylcholine and phosphatidylglycerol isolated from perfused streptozotocin diabetic rat lungs is considerably lower than found in normal lungs. The fact that the difference in residual phosphatidylglycerol is statistically insignificant is probably a reflection of errors in isolating a lipid which is only 2-3% of the lipid of the residual fraction [ 131. In similar experiments with alloxan diabetic animals the rate of incorporation of glucose into either phosphatidylcholine or phosphatidyl-
TABLE f U-14C1 BETIC The
I GLUCOSE AND
results
g protein
INCORPORATION
INTO
STREPTO%OTOCIN-DIABETIC from
three
alloxan-diabetic
LIPIDS
OF
LUNGS
FROM
NORMAL,
ALLOXAN-DIA-
RATS and
six streptozotocin-diabrtic
rats are expressed
in nmol
glucose/
per h. Neutral
lipids
Phospholipids
~._.. Surfactant
38.4 7.4
Residual
+_ 10.3 t
2.9
co.05
Normal Streptozotocin P value
diabetic
536.6 74.4
* 152.5 +_
23.4
co.05
Surfactant
Residual
335.3
+ 50.5
2360.0
40.7
+ 15.3
434.3
co.005
29.5
+_
4.6
472.7
i
77.3
302.0
11.6
t
1.7
134.3
F
33.5
64.4
co.025
co.02
co.02
+ 154.5 t
154.3
BBA 570%
EFFECT OF EXPERIMENTAL DIABETES AND INSULIN ON LIPID METABOLISM IK THE ISOLATED PERFUSED RAT LUNG
MICHAEL
A. MOXLEY
and ELLIOT
i)epartmcnt of Biochemistry, Ht*d., St. Louis. MO. 63103 (Received
February
St. Louis (U.S.A.)
J. LONG~~a~E Ckiuersity
SchooE of Medicine,
1402 S. Grund
8th, 19777)
Summary The isolated perfused rat lung was used as a model to study the possible hormonal re~lation of lipid metabolism in the mamrn~iall adult lung. Experimental diabetes, whether induced by alfoxan or streptozotocin, decreased the incorporation of [IJ-‘4Cfglucose into neutral lipids and phospholipids of both the surfactant fraction and the residual fraction of the lung by 60---80%. Glucose incorporation into phosphatidyl~holi~e and phosphatidylglycerol is decreased in experimental diabetes in both the surfactant and residual fractions to a comparable degree. Glucose incorporation is decreased in both the fatty acid and the glycerophosphocholine moieties of phosphatidylcholine isolated from the surfactant and residual fractions. Insulin treatment of normal animals 30 or 15 min prior to perfusion resulted in an approximate doubling of the in~o~oratio~ of glucose into the phosphatidyl~holine and phosphatidylglycerol isolated from the surfactant and residual fractions of the lung. The incorporation of glucose into palmitic acid isolated from phosphatidyl~holine was also shown to increase similarly. The results of these investigations indicate that insulin may play a role in regulating the synthesis of the important lipid components of the mamm~ian pulmon~ surfactant complex.
Introduction ft. has become increasingly apparent that lipid metabolism in the lung is of considerabfe importance in maintaining the normal function of that organ. It has been demonstra~d that the surface activity of the rnarnn~~i~ surfactant complex is attributable to the presence of the lipid components of the complex [l-4]. However, little information regarding those factors which may influence the metabolism of these lipid components is available. Because of the demonstrated importance of glucose as a substrate in the mammalian lung
219
[5-S] we are investigating the influence of hormones on glucose metabolism in the lung. The effects of chemically induced diabetes on the incorporation of glucose into the lipid fractions of the isolated perfused rat lung have been previously reported [9,10]. This report deals with the extension of those studies to include the determination of the effects of experimental diabetes and of insulin excess on the synthesis of the lipid components of the pulmonary surfactant complex. Materials and Methods The surgical procedures and perfusion technique utilized in these experiments have been described [7,10]. Male, Wistar strain rats (Hilltop Lab Animals, Inc., Scottsdale, Pa.) 150~-250 g were treated with alloxan (Sigma Chemical Co., St. Louis, MO.) or streptozotocin (Upjohn, Kalamazoo, Mich.) as previously described [lo] to induce diabetes. Lungs from diabetic animals were perfused for 1 h with a medium containing 5.6 mM glucose and 50 PCi of [U-14C]glucose (New England Nuclear, Boston, Mass.) Animals of the same strain, sex and age were used as controls. To determine the effects of insulin on lipid metabolism in the lungs of normal animals, rats received a single intravenous dose (4 I.U.) of regular insulin (Eli Lilly and Co., Indianapolis, Ind.) via the tail vein 15 or 30 min before the blood flow to the lungs was interrupted during the surgical procedure. Control animals were injected with physiological saline and the lungs were perfused as described. Following perfusion, lungs were removed from the perfusion apparatus, and frozen in liquid nitrogen. A portion of the tissue was taken for determination of tissue protein according to Lowry et al. [ll]. The remainder of the tissue was homogenized with a Ten Broeck tissue grinder in a medium consisting of 154 mM NaCl, 10 mM Tris * HCl and 1 mM EDTA, pH 7.4. The separation of the surfactant fraction from the residual fraction of the lung was accomplished using the discontinuous sucrose gradient centrifugation method of Frosolono et al. [ 121 as modified and described by Sanders and Longmore [13]. The surfactant fraction obtained by this method contains the total extracellular and intracellular surfactant of the lung. The remaining tissue components comprise the residual fraction. Following this procedure the lipids were extracted from the two fractions according to the method of Folch et al. [14]. Initial separation of the lipids was accomplished using column chromatography on silicic Pa.). Neutral lipids were acid (Unisil, Clarkson Chemical Co., Williamsport, eluted from a 2 g column with 50 ml of chloroform and phospholipids with 100 ml of methanol. When the phospholipids were to be further fractionated they were eluted with 50 ml of chloroform, 25 ml of tetrahydrofuran/dimethoxymethane/methanol/water (10 : 6 : 4 : 1, v/v) and 100 ml of methanol followed by separation by thin-layer chromatography on silica gel H as adapted by Godinez et al. [15]. Phospholipid phosphorus was quantitated using the method of Bartlett as modified by Dittmer and Wells [16] and specific radioactivities were obtained by counting suitable aliquots of the lipid fractions using liquid scintillation counting techniques. Efficiency of counting was determined by the channels ratio method. Fatty acids of the isolated phosphatidyl-
220
choline were isolated as methyl esters by transeste~fication [17]. Specific activity of the radiolabeled pafmitate was determined by using a specially designed effluent splitter and trapping device on the gas chromatograph. Quantitation of glucose incorporation into lung lipids is based on the specific activity of the media glucose and is expressed as glucose incorporated per g protein per h or as the specific activity of the isolated compounds. All phospholipid specific activities were corrected to a substrate specific activity of 2.5 * 10” dpm/pmol. Results are presented as the mean plus or minus the standard error of the mean of the number of perfusions performed. Results Previous reports from this laboratory indicated that experimental diabetes significantly lowered glucose incorporation into the total neutral and total phosphoiipid fraction of the isolated perfused rat lung. The results shown in Table I demonstrate that this effect is evident in both the surfactant and residual fractions of the lung. The rate of incorporation of glucose into the neutral lipids of both the surfactant fraction and the residual fraction of alloxan diabetic lungs is 19.3 and 13.2% of the control rate, respectively, while the rate of incorporation into the total phospholipids of the fractions is 12.1 and 18.4% that observed in normal lungs. Table I illustrates that similar results are obtained when diabetes is induced by streptozotoein treatment. Work from a number of laboratories indicates that certain disaturated species of ~~hosphatidylcholine [ 2,3] and phosphatidylgiycerol [ 18,191 are responsible for the surface activity of the mammalian surfactant complex. Table II illustrates that the specific activities of phosphatidylcholine and phosphatidylglycerol isolated from perfused streptozotocin diabetic rat lungs is considerably lower than found in normal lungs. The fact that the difference in residual phosphatidylglycerol is statistically insignificant is probably a reflection of errors in isolating a lipid which is only 2-3% of the lipid of the residual fraction [ 131. In similar experiments with alloxan diabetic animals the rate of incorporation of glucose into either phosphatidylcholine or phosphatidyl-
TABLE f U-14C1 BETIC The
I GLUCOSE AND
results
g protein
INCORPORATION
INTO
STREPTO%OTOCIN-DIABETIC from
three
alloxan-diabetic
LIPIDS
OF
LUNGS
FROM
NORMAL,
ALLOXAN-DIA-
RATS and
six streptozotocin-diabrtic
rats are expressed
in nmol
glucose/
per h. Neutral
lipids
Phospholipids
~._.. Surfactant
38.4 7.4
Residual
+_ 10.3 t
2.9
co.05
Normal Streptozotocin P value
diabetic
536.6 74.4
* 152.5 +_
23.4
co.05
Surfactant
Residual
335.3
+ 50.5
2360.0
40.7
+ 15.3
434.3
co.005
29.5
+_
4.6
472.7
i
77.3
302.0
11.6
t
1.7
134.3
F
33.5
64.4
co.025
co.02
co.02
+ 154.5 t
154.3
