Effectsof Insulin Upon Fatty AcidsSynthesisFrom Pyruvate, Lactate, and Glucosein Rat Mammary Cells1 Y. T. YANG and R. L. BALDWIN Department of Animal Science University of California, Davis 95616
from fasted-high carbohydrate refed rats. Stimulation of fatty acid synthesis from pyruvate by adipose tissue in vitro has been closely studied by Halperin (6). The importance of insulin in maintaining lactation in rats has been documented (14). In vitro responses of glucose metabolism in mammary tissue to insulin are much smaller than in adipose tissue (16, 22). Martin and Baldwin (15) concluded from mammary metabolite patterns in normal and diabetic laetaring rats that the most limiting function in mammary ceils during insulin insufficiency is not glucose transport. There was a correlation between insulin status and intracellular reJox state. Our objectives were to extend observations of Martin and Baldwin by investigating the effects of insulin on glucose, pyruvate, and lactate metabolisms and their interactions under altered intracellular redox states in isolated mammary cells in vitro.
Abstract
In isolated rat mammary secretory cells, insulin stimulated fatty acid synthesis from pyruvate three times, stimulated glucose conversion to fatty acids 1.2 to 1.5 times, and decreased lactate conversion to fatty acids 20 to 30%. Incubation of glucose and pyruvate together depressed fatty acid synthesis from glucose not attributable to isotope dilution. Glucose stimulated conversion o~ pyruvate-2-14carbon to fatty acids without significantly affecting pyruvate1-14carbon conversion to 14-carbon dioxide. At differing concentrations, the electron accepters phenazine methosulfate and N,N,N',NP-tetramethyl-p-phenyl ene-diamine alleviated the depression by insulin of lactate conversion to fatty acids. The data support concepts that: (1) insulin acts at important sites other than or in addition to glucose transport in regulating mammary secretory cell metabolism and, particularly, fatty acid synthesis; (2) insulin actions upon fatty acid synthesis can vary dependent upon cellular redox state (insulin increases fatty acid synthesis in cells with a low redox state and decreases fatty acid synthesis in cells in a very reduced~ state); and (3) pyruvate depresses glucose carbon flux through the EmbdenMeyerhof pathway.
Materials and Methods Chemicals. All radioactive substrates were purchased from New England Chemical Company. Collagenase (lot Cis 207) was purchased from Worthington Chemical Company. Other biological chemicals were purchased from Sigma Chemical Company. Animals. First lactation Sprague-Dawley rats with 8 to 12 Dups were maintained on Purina laboratory chow. Animals were killed by decapitation between 16 and 28 days postparturn, and abdominal and inguinal mammary
Introduction
TABLE 1. Composition of media.
The most notable effects of insulin on adipose tissue metabolism are augmentation of hicose uptake and inhibition of fatty acid moilization (2, 9). Insulin may increase fatty acid synthesis in adipose tissue independent of glucose uptake. Jungas (10) and Halperin (4) demonstrated that insulin increases conversion of endogenous substrate, presumably glycogen, into fatty, acids in adipose tissue
Dispersion medium Krebs-Ringer bicarbonate buffer (pH 7.4) Bovine serum albumin (V) Acid casein hydrolysate Methionine Tryptophane Glucose Collagenase
Received August 7, 1974. a Supported in part by USPHS-NIH Grant AM07672.
100 ml 2 g 20 mg Img 1 mg 200 mg 100 mg
Washing medium same as dispersion medium but without glucose and collagenase. Medium for metabolic studies same as washing medium with desired concentration of substrat~ added.
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YANG A N D B A L D W I N
glands were removed for cell preparation. Preparation of isolated cells. Mammary cells were prepared by a procedure reported previously (16). Mammary tissues were trimmed to remove connective and adipose tissues, diced with scissors, and washed in KrebsRinger bicarbonate buffer. The tissues were then minced with scissors and washed again with buffer. Twelve grams of minced tissue were placed in a 250 ml Ehrlel~myer flash containing 40 ml of dispersion medium (Table 1). The flask was stoppered after flushing with an oxygen:carbon dioxide (95:5) mixture and placed in a metabolic shaker set at 100 strokes per min and 38 C. Every 15 min, 50 nag glucose in 2.5 ml buffer were added. Then the flask was flushed with mixed gas, stoppered, and incubated again. After the tissue was dispersed (60 to 75 min), contents of the flask were stirred with a plastic rod and gently strained through a nylon cloth into 50 ml plastic centrifuge tubes. Tissue remaining on the nylon cloth was placed in a flask with 40 ml of washing medium (Table 1), stirred, and then strained through nylon cloth into centrifuge tubes. This procedure increased cell yields significantly. After eentrifugation at half speed in an International clinical centrifuge at room tempera~ r e for 2 min, the supernatants were discarded. Fat adhering to the centrifuge tube walls was wiped away with tissue paper. The packed cells were resuspended in 45 ml of washing medium and centrifuged again. This washing was repeated five times. After the final wash, cells were resuspended in washing medium, and a portion of the cell suspension was centrifuged in a graduated centrifuge tube at full speed for 10 rain in the clinical centrifuge to determine packed cell volume. The remaining cell suspension was diluted to a final cell concentration of .03 nil packed cells/ml suspension. Recoveries of cells ranged from 15 to 20%. The cell preparations were free of nuclei and broken cell debris. More than 95% of the cells excluded trypan blue. Metabolic rates were linear for more than 2 h. Responses to iiasulin were maintained throughout the incubation period. Methods of incubation and analyses for labeled carbon dioxide and fatty acids have been reported (16). Variation among triplicate incubation flasks was usually within 5% of the mean. Results
Effect of insulin upon lactate and pyruvate utilization. Incorporation rates of [2-~4C] pyruvate and [2-14C] lactate into CO2 and fatty JOURNAL OF DAIRY SCIENCE, VOL. 58, NO. 3
•
6
20
0
0
o
5
10
15
10
20 0
24
16
18
12
12
8
6
4
20
S
10
15
20
PYRUVATE CONCENTRATION (raM)
40
-
o 0
30
0
10
20
-
i
J
30
40
L A C T A T E CONCENTRATION (raM)
Fla. 1. Effects of pyruvate and ~lactate c~)neentrations and insulin upo~ rates of fatty acid synthesis and pyruvate and lactate oxidation. Pyruvate-2-~'C and [2-1~C] lactate at varying concentrations incubated for 90 min at 38 C in ~.0 ml of incubation medium containing .02- .03 ml isolated mammary cells with ( e-,e ) and without ((D-C)) 2 milliunit crystalline insulin. Fatty acid synthesis expressed as #g atoms pyruvate or lactate carbon-2 incorporated into fatty acids per h by 1 ml packed mammary cells. CX:h production expressed as /~g atoms pyruvate or lactate carbon 2 incorporated into carbon dioxide per h by 1 ml packed mammary cells. acids in mammary cells are in Fig. 1. Fatty acid incorporation rates from [2-z'*C] pyruvate were increased with increasing medium pyrurate concentration up to 20 raM. Insulin at 1 milliunit per ml stimulated rates of fatty acid synthesis from pyruvate at all concentrations. However, relative response appeared to decline gradually as pyruvate concentration was T~mLE 2. Glucose and lactate metabolism and interactions in mammary cellsL ¢Lgatomlabeled carbon incorporated ( hr X ml cells) "~ CO2 Fatty acids --
Substrates [1-1~C] lactate [1-a'C] lactate -}- glucose [2-1'C] lactate [2-'~C] lactate + glucose [U-~'C] glucose [U-~'C] glucose + lactate
Insulin
+
--
+
Insulin Insulin Insulin
45.5
38.7
......
73.7 13.1
71.2 11.3
...... 1.0
5.1 160
4.7 218
42.0 165
256
167
194
140
176
.7 51.1
a 0.03 ml cells were incubated in 2 ml medium for 90 rain at 38 C; substrate cencentrations were 5 mM and insulin 1 m unit per nil. Values are the average of triplicates from a typical run.
339
I N S U L I N ACTIONS IN MAMMARY CELLS
TABLE 3. Glucose and pyruvatc metabolism and interactions in mammary eellsL gglatom labeled carbon incorporated (h × ml cells) -~ CO~ Fatty aeids --
Substrates [ 1-"C] pyruvate [ 1-~'C] pyruvate + glucose [2-a4C] pyruvate [2-~*C] pyruvate + glucose [U-a'C] glucose [U-~'C] glucose + pyruvate
+
--
+
Insulin Insulin Insulin Insulin 107
121
......
117 33.6
134 35
"" 3.4
"8.8
28.8 228
29.6 348
12.2 134
28.0 228
9
24
91
141
Experimental conditions as described in Table 2. increased. Rates of [2-14C] pyruvate oxidation reached a maximum at a pyruvate concentration of 10 raM. Insulin had no apparent effect on [2-14C] pyruvate oxidation to CO2. Rates of [2-14C] lactate incorporation into fatty acids were maximal at lactate concentrations of 10 mM and 20 mM. Labeled CO 2 production reached a maximum at 20 mM lactate. Addition of insulin significantly depressed [2-~C] lactate incorporation into fatty acids and CO~.
Interactions among glucose, lactate, pyrurate, and insulin. Addition of glucose increased [1-~4C] lactate conversion to ~4CO2 while decreasing [2-~C] lactate oxidation (,Table 2). Glucose greatly enhanced r a t e s of [2-~4C] lactate incorporation into fatty acids. Addition of insulin resulted in a further increase. Glucose addition depressed [2-~4C] lactate conversion to x4CO2. Addition of lactate to incubations containing [U-14C] glucose decreased incorporation of glucose carbon into fatty acids and decreased responses to insulin. Data in
Table 2 are from a typical run. This experiment was repeated four times, and responses were the same. Typical rather than mean ± SE data are presented because differences in metabolic rates and magnitudes of insulin responses of different cell preparations are large, and averaging obscures the pattern. Typical is representative in this context and not most dramatic. In the example selected, neither insulin responses nor metabolic rate were as large as in our most dramatic run. These same comments apply to .data in other tables. Addition of glucose slightly decreased oxidation of [2-14C] pyruvate (Table 3). Fatty acid synthesis from [2-1'4C] pyruvate increased several times in the presence of glucose while oxidation of [1-14C] pyruvate was not affected by addition of glucose. Conversion of [U-1'4C] glucose to COs was depressed by addition of pyruvate, and rates of fatty acid synthesis from [U-14C] glucose were depressed. Effects of insulin on [U-~'4C] glucose and [2-1~C] pyruvate incorporations into fatty acids were stimulatory when these substrates were incubated separately or in combination. Carbon dioxide and fatty acid formation from [1-14C] glucose and [6-1~C] glucose were measured in the presence or absence of pyruvate or lactate (Table 4). [1-~4C] glucose oxidation was increased by lactate and markedly reduced by pyruvate. [6-1'4C] glucose oxidation was decreased by lactate and pyruvate to the same extent. Fatty acid synthesis from [1-~4C] glucose and [6-1';C] glucose was decreased to 50 and 70%, respectively, by lactate and to 5% by pyruvate. The ratio of i4CO2 derived from [1-1~C] glucose and [6-14C] glucose and relative pentose cycle activity (PC%) calculated from fatty acid synthesis (11) were increased markedly by lactate. Pyruvate had only minor effects on these. Addition of insulin increased fatty acid synthesis and apparent pentose cycle
TABLE 4. Effects of pyruvate and lactate on the metabolism of glucose-l-~4C and glucose-6-~4C in mammary cells'.
Substrate added Glucose Glucose + pyruvate Glucose + lactate
Insulin -+ -+ -+
/zg/atom labeled carbon incorporated (h × ml cells) -a [1-1~C] glucose [6-1'C] glucose COs Fatty acids CO.~ Fatty acids
G-1-C - G-6-C
56.0 84.2 17.3 28.2 61.2 87.1
8.1 15.3 9.6 11.3 32.2 39'.6
25.8 37.1 1.22 3.31 12.8 17.2
6.9 5.5 1.8 2.5 1.9 2.2
72.0 116.3 3.0 8.9 ,50,8 80.2
--~
~'CO~.
PC~
34.3 41.6 32.4 36.1 49.8 56.6
"Experimental conditions as described in Tab]e 2. JOURNAL OF DAIRY SCIENCE, VOL. 58, NO. 3
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YANG AND B A L D W I N
TABLE 5. Effects of pyruvate and PMS on glucose carbon metabolism in mammary cells*. Substrate added Glucose
Insulin -+
~g/atom labeled carbon incorporated (h × ml cells) -~ [U-~C] glucose [1-1~C] glucose [6-1~C] glucose CO., Fatty acids CO.. Fatty acids CO2 Fatty acids 170 267
84.3 158.0
42.4 54.3
242 250
28.0 33.0
765 82.5
3.2 12.3 20.6 24.2
13.4 20.0
4.4 2.8
32.6 77.0
.74 .67
1.92 2.20
18.0 20.4
17.2 30.0
.46 1.58
1.49 2.26
82.6 86,5
.37 .44
1.59 1.53
Glucose
+ PMS
-
-
+ Glucose + pyruvate Glucose + pyruvate + PMS
-+
-
-
+
49.4 88.5 256 240
1.37 6.01 12.1 14.7
*Experimental conditions as described in Table 2. Phenazine methosulfate (PMS) added at concentration of .1 InM. percentage in all cases. Data in Table 5 indicate that phenazine methosuIfate (PMS) increased [U-14C] glucose oxidation to CO2. This was mainly due to increased pentose cycle flux as indicated by the greatly elevated ratio of l~COz derived from [1-14C] glucose and [6-1'C] glucose. Fatty acid synthesis from [U-~4C] glucose was depressed to less than half by PMS. Effects of pyruvate on glucose metabolism were similar to those in Table 3. Fatty acid synthesis from [U-14C] glucose was depressed much lower by pyruvate than by PMS. When PMS was presented in addition to pyruvate, rates of labeled glucose carbon oxidation were similar to that with PMS alone. The inhibitory effect of pyruvate upon fatty acid synthesis from glucose was partially relieved by PMS. Stimulatory effects of insulin on glucose metabolism were high in the presence of pyruvate but were diminished by PMS. When 2.5 mM lactate and pyruvate were presented simultaneously, rates of fatty acid synthesis from [2-14C] pyruvate were half of those when 5 mM pyruvate was presented
alone (Table 6). Incorporation of 2.5 m M [2-1~C] lactate into fatty acid was several times higher when incubated with 2.5 mM pyruvate than when 5 mM lactate alone served as substrate. Insulin stimulated fatty acid incorporation from [2-1~C] pyruvate and [2-~4C] lactate labeled carbons to the same extent when these substrates are incubated together. In contrast, insulin depressed fatty acid synthesis from lactate when this substrate was incubated alone. Incorporation of [2-~4C] pyruvate labeled carbon into fatty acids was depressed bv N,N,N',N'-tetramethyt- p- p h e n y l e n edia,m i n e (TMPD) at 20 ttM. PMS inhibited both fatty acid synthesis from and oxidation of pyruvate at 20 and 100 /~M. Stimulatory effects of insulin on [2-r4C] pyruvate conversion to fatty acids persisted in the presence of PMS and TMPD. Twenty micromolar PMS increased [U-X4C] glucose oxidation to CO2 without affecting rate of fatty acid synthesis. PMS (100 /~M) inhibited fatty acid synthesis from glucose and abolished the response to insulin. T M P D (20 /~M) had no effect on [U-14C] glucose incorpo-
TABLE 6. Pyruvate-2-a4C and lactate-2-1'G metabolism and interaction in mammary cells. ~
Substrates [2-~'C] pyruvate, 5mM [2-a4C] lactate, 5raM [2-a*C] pyruvate, 2.5mM + lactate, 2.5mM [2-~4C] lactate, 2.5ram + pyruvate, 2.5ram
~g/atom labeled carbon incorporated ( h ) < ml eel/s) -~ CO~ Fatty acids -- Insulin + Insulin -- Insulin + Insulin 35 16.2
39 14.4
3.2 1.04
8.6 .68
18.4
19.2
1.8
4.4
I6.6
I5.0
1.4
3.4
*Incubation conditions same as described in Table 2 except substrate levels varied as indicated. JOURNAL OF DAIRY SCIENCE, VOL. 58, N o . 3
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T~L~. 7. Effects of PMS and TMPD on [U-a'C] glucose, [2-~4C] lactate, and [2-~'C] pyruvate metabolism and responses to insulin in mammary cells. ~
Experiment aao. 1
Substrate (5raM) [2-~'C] lactate
. PMS
2
2-[2-~'C] pyruvate PMS TMPD [U-~C] glucose PMS TMPD
3
#g/atom labeled carbon incorporation (h × ml packed cells) PMS, TMPD CO,_, Fatty acids (~M) -- Insulin + Insulin -- Insulin + Insulin
[2-1'C] pyruvate PMS TMPD [2-~*C] lactate PMS TMPD [U-~*C] glucose PMS TMPD
".1 .5 2 10 50 . 20" 20 . 20" 20 . 100 100 ... 100 I00 . 10b" 100
13.1 12.8 13.7 15.4 17.6 16.0 28.9 25.7 27.0 189 282 197 30.1 25.2 20.4 17.9 13.8 18.5 161 278 182
11.3 11.8 12.0 13.5 14.3 14.7 29.2 24.4 29.1 278 331 300 31.5 22.2 19.8 14.5 11.9 18.5 218 258 248
1.0 1.0 1.0 .9 .8 .2 3.0 .5 2.5 116 129 217 2.47 .18 .51 1.48 .30 1.42 126 47.1 123
.7 .7 .8 1.2 1.0 .2 6.5 1.2 4.6 236 196 242 8.41 .27 2.58 .62 .30 2.4 200 45.3 218
*Incubation conditions as described in Table 2. PMS is phenazine methosulfate; TMPD is N,N,N', N'-tetra-methyl-#-pherlylenedimnine. Different cell preparations used in Experiments 1, 2, and 3. ration into CO2 and fattv acids. At 100 t~M, TMPD slightly increased glucose oxidation. The response of glucose metabolism to insulin was maintained in the presence of TMPD. Basal rates of [2-1~4C] lactate oxidation and fatty acid synthesis were not affected by TMPD. Thus, the inhibitory effect of insulin ou [2-14C] lactate incorporation into fatty acid was reversed by T M P D (Table 7). Lactate2-14C oxidation was slightly increased by lower concentrations of PMS. At high concentration, PMS decreased [2-14C] lactate oxidation. Rates of [2-14C] lactate incorporation into fatty acid were decreased by PMS at concentrations above 50 raM. The inhibitory effect of insulin on fatty acid synthesis from [2-x*C] lactate was diminished by PMS. Discussion
In a recent report, Halperin (6) indicated that insulin can increase fatty acid synthesis from pyruvate in the absence of glucose in rat adipose tissue. Insulin did not influence maximum fatty acid synthesis rate but decreased the pynavate concentration at which half maximum rates are observed. From other experimental data (7), Halperin suggested that intracellular fatty aeyl CoA concentration, which can be decreased by insulin, may inhibit trans-
port of intramitoehondrial citrate to the cytosol, and thereby decrease fatty acid synthesis. Jungas (10) reported that insulin sh.'ghtly stimulated fatty acid synthesis in rat adipose tissue with pyruvate or lactate. Oxidation of [1-14C] lactate to COs was also slightly increased by insulin in adipose tissue from high carbohydrate fed rats. Pyruvate dehydrogenase activity in rat adipose tissues, assayed by measuring rates of [1-a~C] pyruvate or [1-14C] lactate conversion to CO2 in tissue homogenares, was stimulated by preincubation of tissue with insulin and decreased by preincubation with epinephrine (10, 21). Data from this experiment show a similar stimulatory effect of insulin on fatty acid synthesis from [2-~4C] pyruvate in isolated rat mammary cells over a wide range of pyruvate concentrations. This effect of insulin in mammary cells seems not to have resulted from increased pyruvate dehydrogenase flux as indicated by a relatively minor effect of insulin on [1-14C] pyruvate oxidation to CO2 (Table 3). The inhibitol T effect of insulin on fatty acid synthesis from lactate in mammary tissue (Table 2) is contrary to observations in rat adipose tissue (10). The cause of this difference is not known. There are several metabolic differences between mammary and adipose tissues which JOURNAL OF DAIRY SCIENCE, VOL. 58, N o . 3
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YANG AND B A L D W I N
could contribute to the difference. In mam- does not. The positive effect of insulin on fatty mary cells, reducing equivalents as reduced acid synthesis from pyruvate was maintained nicotinaruide adenine dinucleotide (NADH) in PMS and TMPD treated cells. Lactate-2can be efficiently transported between mito- 14C incorporation into fatty acids was not afehrondria and the cytosol by the Borst shuttle fected by 100 t~M TMPD, but the response to (1). In adipose tissue, lack of a functional insulin was changed from an inhibitory one to Borst shuttle (12, 17) could limit the transfer a stimulatory one. PMS, at low concentrations, of NADH reducing equivalents from the eyto- slightly increased [g-~4C] lactate oxidation. sol to the mitochondrion. Accumulation of The basal rate of fatty acid synthesis was not NADH or a change in NADH/nicotinamide affected, but the negative response to insulin adenine dinucleotide (NAD) ratio may cause was decreased. At high concentrations, PMS secondary metabolic effects in adipose such as decreased fatty acid synthesis from [2-~4C] lacchanging pyruvate dehydrogenase activity (3, tate and addition of insulin did not show any 23). Further, insulin has antilipolytic activity effect. The observations further indicate that in adipose tissue. Thus, changes of intracellu- insulin can affect fatty acid synthesis indelar free fatty acid concentrations occur. This pendent of glucose uptake and that this action could lead to metabolic changes, particularly is related to intracellular redox state. Katz et al. (11) reported that PMS, at conin fatty acid synthesis (7, 18). The different responses to insulin in fatty eentrations within the range of this study, inacid synthesis from [2-14C] pyruvate and creased fatty acid synthesis from lactate in rat [2-1"C] lactate were not due to differences in mammary and adipose tissue slices. This result intracellular pyruvate concentration and pyru- was not found in the present experiment with vate dehydrogenase flux since when concen- mammary cells. The reason for this discrepantrations of pyruvate and lactate were such that cy is not known. Inhibition of glucose utilization by pyruvate rates of carbon incorporation into fatty acids from these two substrates were comparable, has been observed by several investigators (8, insulin stimulated [2-x4C] pyruvate incorpora- 19, 24). The data in Table 3 indicate that total tion into fatty acids (Fig. 1). Furthermore, in- incorporation of two carbon units from glucose sulin had only minor effects upon rates of and pyruvate, when incubated together (12.2 [1-1'4C] pyruvate and [1-14C] lactate conver- q- 9/2 ---- 16.7) is less than the stun (3.4 qsions to 14CO2 via the pyruvate dehydrogenase 134/2 ---- 70.4) of each incubated alone. This reaction while producing these contrasting re- observation plus consideration of the conversions of [1-14C] glucose and [6-14C] glucose sponses in fatty acid synthesis. It is much more likely that the effects of lac- into fatty acids (Table 4), indicates that the tate and pyruvate upon intraccllular redox effect of pyruvate upon glucose metabolism is state modify the action(s) of insulin on fatty not attributable to isotope dilution. Williamson (24) reported that pyruvate deacid synthesis in mammary cells. The implication is that insulin stimulates fatty acid synthe- pressed glucose oxidation in perfused rat heart, sis in cells in a less reduced state as is caused From a cross-over profile of tissue metabolites, by pyruvate, and reduces fatty acid synthesis he suggested that the site of inhibition was bein highly reduced cells as are produced by lac- tween a-glyeeraldehyde-P and phosphoglyceric tate. Phenazine methosulfate and TMPD have acid. The decrease in [2-a4C] pyruvate oxidation been used to modify intracellular redox states in vitro (5, 12, 22). PMS acts as a general to CO2 caused by glucose was not caused by electron carrier in accepting electrons from isotope dilution as indicated by increased both NADH and NADPH and transferring [1-x*C] pyruvate oxidation. In the presence of these to molecular oxygen. TMPD acts as an glucose, increased [2-a4C] pyruvate incorporaelectron carrier in transferring electrons be- tion into fatty acids could be attributable to tween NADH and cytochrome b5 reductase an increased supply of NADPH from the pen(20) and between cytochrome b and cyto- rose phosphate pathway. Glucose greatly inchromo c (13). Effects of PMS and TMPD on creased the incorporation of [2-a4C] lactate inmammary cell metabolism are concentration to fatty acid. This may also be due to increased dependent (Table 7). Pyruvate-2-~4C incorpo- NADPH supply as indicated by the elevated ration into fatty acids was depressed by both flux of glucose carbon through the pentose PMS and TMPD. PMS has more severe effects cycle in the presence of lactate (Table 4). than TMPD probably due to the fact that PMS Lactate-l-~4C oxidation was increased and depressed intracellular NADPH which is re- [2-~4C] lactate oxidation was decreased in the quired for fatty acid synthesis while TMPD presence of glucose, suggesting a shift in laeJOURNAL OF DAIItV SCIENCE, VOL. 58, NO. 3
I N S U L I N A C T I O N S IN MAMMARY CELLS
tate carbon metabolism from tricarboxylic acid cycle oxidation to fatty acid synthesis. Lactate increased [1-~4C] glucose and decreased [6-rdC] glucose oxidation to CO 2 without substantially affecting [U-~dC] glucose oxidation. This observation suggests isotope dilution and carbgn exchange. References
(1) Borst, P. 1963. Functionelle mad morphologische organisation der zelle, p. 137. W/irzburg: Springer-Verlag. (2) Crofford, O. D., and A. E. Reno,ld. 1965. Glucose uptake by incubated rat epididymat adipose tissue, characteristie of glucose transpose system and actions of insulin. J. Biol. Chem. 240:3237. (3) Garland, P. B., and P. J. Dandle. 1964. Control of pyruvate dehydrogenase in the perfused rat heart by the intracellular coneentration of acetyl-coenzyme A. Biochem. I. 91:6(3. (4) Halperin, M. L, 1970. An additional role for insulin in tile control of fatty acid synthesis independent of glucose transport. Can. J. Biochem. 48:1229. (5) Halperin, M. L., and B. H. Robinson. 1970. The role of the cytoplasmic redox i°°" tential in the control of fatty acid synthesis from glucose, pyruvate, and lactate in white adipose tissue. Bioehem. J. 116:235. (6) Halperin, M. 1971. Studies on the conversion of pyruvate into fatty acids in white adipose tissue. Biochem. J. 124:615. (7) Halperin, M. L., B. H. Robinson, and I. B. Fritz. 1972. Effects of paJmitoyl CoA on citrate and malate transport by rat liver mitochondrla. Prec. Nat. Aoad. Sei. 69:1003. (8) Hirsch, P. F., H. Baruch, and I. L. Chaikoff. 1954. The relation of glucose oxidation to lipogenesis in mammary tissue. J. Bi01. Chem. 210:785. (9) Jungas, B. L., and E, G. Ball. 1963. Studies on the metabolism of adipose tissue. XII. The effects of insulin and epinephrine on free fatty aeid and glycerol production in the presence and absence of glucose. Biochemistry 2:383. (10) Jungas, R. L. 1970. Effect of insulin oI~ fatty acid synthesis from pymvate, lactate, or endogenous sources in adipose tissue: Evidence for the hormonal regulation of pyruvato dehydrogenase. Endocrinology 86: 1368. (11) Katz, |., L. R. 'Bernard, and G. E. Bartsch. 1066. The pentose cycle, triosephosphate isomerization and lipogenesis in rat adipose
343
tissue. J. Biol. Chem. 241:727. (12) Katz, J., and P. A. Wals. 1970. Effect of phenazine methosuLfate on lipogenesis. J. Biol. Chem. 245:2546. (13) Lee, C. P., G. L. Scottocasa, and L. Ernster. 1967. Use of artificial electron accepters for abbreviated phosphorylating electron transport: Flavin-cytoehrome e. In Methods in enzymology, R. W. Estabrook and M. E. Ptdlman, ed. 10:33. New York Academic Press, Inc. (14) Martin, R. |., and B. L. Baldwin. 1971. Effects of alloxan diabetes on lactational perrefinance and mammary tissue metabolism in the rat. Endecrinoiogy 88:863. (15) Martin, R. 1-, and I~. L. Baldwin. 1971. Effects of insulin and anti-insulin serum treatments on levels of metabolites in rat mammary glands. Endocrinology 88:868. 16) Martin, R. |., and R. L. Baldwin. 1971. Effects of insulin on isolated rat: mammary cell metabolism: Glucose utilizatkm and metabolite patterns. Endocrinology 89:1263. 17) l~obinson, B. It., and M. L. IIalperin. 1970. Trm~sport of reduced nicotinamide-adenine dinueleotide into mitochondria of rat white adipose tissue. Biochem. J. 116:229. (18) Sehmidt, K., and ]. Katz. 1969. Metabolism of pyrnvate and L-lactate by rat adipose tissue. 1. Biol. Chem. 244:2125. (19) Smith, 2. W., and R. F. Glascock. 1969. The effects of acetate and of pyruvate on the pathways of glucose catabolh;m in lactating mammary tissue. J. Dairy Res. 36:455. (20) Strittmatter, P. C. 1963. Mi,~osomal eytochrome b., and cytochrome b~ reduetase. In The enzymes, M. A. Landy and K. Myrb~ck, ed. 8:113. New York Academic Press, Inc. (21) Taylor, S. I., and B. L. Jungas. 1972. Effects of antilipolytic agents on F,yruvate dehydrogenase activity of rat apidose tissue. Fed. Prec. 31:244. (22) Waiters, E., and P. McLean. 2t968. Effect of alloxan-diabetes and treatment with antiinsulin serum on pathways of glucose metabolism in lactating rat n~muuary gland. Bioehem. J. 109:407. (23) Wfeland, O., B. von laguw-Westermann, and B. StukowskL 1969. Kinetic and regulatory properties o~ heart musele pyruvato dehydrogenase. Hoppe-Seyter's Z. Physiol. Chem. 350: 329. (24) Wflliamson, J. R. 1965. Glycolytie control mechanism. I. Iulqibitiou of glycolysis by acetate and pyruvate m the isolated perfused rat heart. I. Biol. Chem. 240:2308.
JOURNAL OF DAIRY SCIENCE, V o L 58, N o . 3
from fasted-high carbohydrate refed rats. Stimulation of fatty acid synthesis from pyruvate by adipose tissue in vitro has been closely studied by Halperin (6). The importance of insulin in maintaining lactation in rats has been documented (14). In vitro responses of glucose metabolism in mammary tissue to insulin are much smaller than in adipose tissue (16, 22). Martin and Baldwin (15) concluded from mammary metabolite patterns in normal and diabetic laetaring rats that the most limiting function in mammary ceils during insulin insufficiency is not glucose transport. There was a correlation between insulin status and intracellular reJox state. Our objectives were to extend observations of Martin and Baldwin by investigating the effects of insulin on glucose, pyruvate, and lactate metabolisms and their interactions under altered intracellular redox states in isolated mammary cells in vitro.
Abstract
In isolated rat mammary secretory cells, insulin stimulated fatty acid synthesis from pyruvate three times, stimulated glucose conversion to fatty acids 1.2 to 1.5 times, and decreased lactate conversion to fatty acids 20 to 30%. Incubation of glucose and pyruvate together depressed fatty acid synthesis from glucose not attributable to isotope dilution. Glucose stimulated conversion o~ pyruvate-2-14carbon to fatty acids without significantly affecting pyruvate1-14carbon conversion to 14-carbon dioxide. At differing concentrations, the electron accepters phenazine methosulfate and N,N,N',NP-tetramethyl-p-phenyl ene-diamine alleviated the depression by insulin of lactate conversion to fatty acids. The data support concepts that: (1) insulin acts at important sites other than or in addition to glucose transport in regulating mammary secretory cell metabolism and, particularly, fatty acid synthesis; (2) insulin actions upon fatty acid synthesis can vary dependent upon cellular redox state (insulin increases fatty acid synthesis in cells with a low redox state and decreases fatty acid synthesis in cells in a very reduced~ state); and (3) pyruvate depresses glucose carbon flux through the EmbdenMeyerhof pathway.
Materials and Methods Chemicals. All radioactive substrates were purchased from New England Chemical Company. Collagenase (lot Cis 207) was purchased from Worthington Chemical Company. Other biological chemicals were purchased from Sigma Chemical Company. Animals. First lactation Sprague-Dawley rats with 8 to 12 Dups were maintained on Purina laboratory chow. Animals were killed by decapitation between 16 and 28 days postparturn, and abdominal and inguinal mammary
Introduction
TABLE 1. Composition of media.
The most notable effects of insulin on adipose tissue metabolism are augmentation of hicose uptake and inhibition of fatty acid moilization (2, 9). Insulin may increase fatty acid synthesis in adipose tissue independent of glucose uptake. Jungas (10) and Halperin (4) demonstrated that insulin increases conversion of endogenous substrate, presumably glycogen, into fatty, acids in adipose tissue
Dispersion medium Krebs-Ringer bicarbonate buffer (pH 7.4) Bovine serum albumin (V) Acid casein hydrolysate Methionine Tryptophane Glucose Collagenase
Received August 7, 1974. a Supported in part by USPHS-NIH Grant AM07672.
100 ml 2 g 20 mg Img 1 mg 200 mg 100 mg
Washing medium same as dispersion medium but without glucose and collagenase. Medium for metabolic studies same as washing medium with desired concentration of substrat~ added.
337
YANG A N D B A L D W I N
glands were removed for cell preparation. Preparation of isolated cells. Mammary cells were prepared by a procedure reported previously (16). Mammary tissues were trimmed to remove connective and adipose tissues, diced with scissors, and washed in KrebsRinger bicarbonate buffer. The tissues were then minced with scissors and washed again with buffer. Twelve grams of minced tissue were placed in a 250 ml Ehrlel~myer flash containing 40 ml of dispersion medium (Table 1). The flask was stoppered after flushing with an oxygen:carbon dioxide (95:5) mixture and placed in a metabolic shaker set at 100 strokes per min and 38 C. Every 15 min, 50 nag glucose in 2.5 ml buffer were added. Then the flask was flushed with mixed gas, stoppered, and incubated again. After the tissue was dispersed (60 to 75 min), contents of the flask were stirred with a plastic rod and gently strained through a nylon cloth into 50 ml plastic centrifuge tubes. Tissue remaining on the nylon cloth was placed in a flask with 40 ml of washing medium (Table 1), stirred, and then strained through nylon cloth into centrifuge tubes. This procedure increased cell yields significantly. After eentrifugation at half speed in an International clinical centrifuge at room tempera~ r e for 2 min, the supernatants were discarded. Fat adhering to the centrifuge tube walls was wiped away with tissue paper. The packed cells were resuspended in 45 ml of washing medium and centrifuged again. This washing was repeated five times. After the final wash, cells were resuspended in washing medium, and a portion of the cell suspension was centrifuged in a graduated centrifuge tube at full speed for 10 rain in the clinical centrifuge to determine packed cell volume. The remaining cell suspension was diluted to a final cell concentration of .03 nil packed cells/ml suspension. Recoveries of cells ranged from 15 to 20%. The cell preparations were free of nuclei and broken cell debris. More than 95% of the cells excluded trypan blue. Metabolic rates were linear for more than 2 h. Responses to iiasulin were maintained throughout the incubation period. Methods of incubation and analyses for labeled carbon dioxide and fatty acids have been reported (16). Variation among triplicate incubation flasks was usually within 5% of the mean. Results
Effect of insulin upon lactate and pyruvate utilization. Incorporation rates of [2-~4C] pyruvate and [2-14C] lactate into CO2 and fatty JOURNAL OF DAIRY SCIENCE, VOL. 58, NO. 3
•
6
20
0
0
o
5
10
15
10
20 0
24
16
18
12
12
8
6
4
20
S
10
15
20
PYRUVATE CONCENTRATION (raM)
40
-
o 0
30
0
10
20
-
i
J
30
40
L A C T A T E CONCENTRATION (raM)
Fla. 1. Effects of pyruvate and ~lactate c~)neentrations and insulin upo~ rates of fatty acid synthesis and pyruvate and lactate oxidation. Pyruvate-2-~'C and [2-1~C] lactate at varying concentrations incubated for 90 min at 38 C in ~.0 ml of incubation medium containing .02- .03 ml isolated mammary cells with ( e-,e ) and without ((D-C)) 2 milliunit crystalline insulin. Fatty acid synthesis expressed as #g atoms pyruvate or lactate carbon-2 incorporated into fatty acids per h by 1 ml packed mammary cells. CX:h production expressed as /~g atoms pyruvate or lactate carbon 2 incorporated into carbon dioxide per h by 1 ml packed mammary cells. acids in mammary cells are in Fig. 1. Fatty acid incorporation rates from [2-z'*C] pyruvate were increased with increasing medium pyrurate concentration up to 20 raM. Insulin at 1 milliunit per ml stimulated rates of fatty acid synthesis from pyruvate at all concentrations. However, relative response appeared to decline gradually as pyruvate concentration was T~mLE 2. Glucose and lactate metabolism and interactions in mammary cellsL ¢Lgatomlabeled carbon incorporated ( hr X ml cells) "~ CO2 Fatty acids --
Substrates [1-1~C] lactate [1-a'C] lactate -}- glucose [2-1'C] lactate [2-'~C] lactate + glucose [U-~'C] glucose [U-~'C] glucose + lactate
Insulin
+
--
+
Insulin Insulin Insulin
45.5
38.7
......
73.7 13.1
71.2 11.3
...... 1.0
5.1 160
4.7 218
42.0 165
256
167
194
140
176
.7 51.1
a 0.03 ml cells were incubated in 2 ml medium for 90 rain at 38 C; substrate cencentrations were 5 mM and insulin 1 m unit per nil. Values are the average of triplicates from a typical run.
339
I N S U L I N ACTIONS IN MAMMARY CELLS
TABLE 3. Glucose and pyruvatc metabolism and interactions in mammary eellsL gglatom labeled carbon incorporated (h × ml cells) -~ CO~ Fatty aeids --
Substrates [ 1-"C] pyruvate [ 1-~'C] pyruvate + glucose [2-a4C] pyruvate [2-~*C] pyruvate + glucose [U-a'C] glucose [U-~'C] glucose + pyruvate
+
--
+
Insulin Insulin Insulin Insulin 107
121
......
117 33.6
134 35
"" 3.4
"8.8
28.8 228
29.6 348
12.2 134
28.0 228
9
24
91
141
Experimental conditions as described in Table 2. increased. Rates of [2-14C] pyruvate oxidation reached a maximum at a pyruvate concentration of 10 raM. Insulin had no apparent effect on [2-14C] pyruvate oxidation to CO2. Rates of [2-14C] lactate incorporation into fatty acids were maximal at lactate concentrations of 10 mM and 20 mM. Labeled CO 2 production reached a maximum at 20 mM lactate. Addition of insulin significantly depressed [2-~C] lactate incorporation into fatty acids and CO~.
Interactions among glucose, lactate, pyrurate, and insulin. Addition of glucose increased [1-~4C] lactate conversion to ~4CO2 while decreasing [2-~C] lactate oxidation (,Table 2). Glucose greatly enhanced r a t e s of [2-~4C] lactate incorporation into fatty acids. Addition of insulin resulted in a further increase. Glucose addition depressed [2-~4C] lactate conversion to x4CO2. Addition of lactate to incubations containing [U-14C] glucose decreased incorporation of glucose carbon into fatty acids and decreased responses to insulin. Data in
Table 2 are from a typical run. This experiment was repeated four times, and responses were the same. Typical rather than mean ± SE data are presented because differences in metabolic rates and magnitudes of insulin responses of different cell preparations are large, and averaging obscures the pattern. Typical is representative in this context and not most dramatic. In the example selected, neither insulin responses nor metabolic rate were as large as in our most dramatic run. These same comments apply to .data in other tables. Addition of glucose slightly decreased oxidation of [2-14C] pyruvate (Table 3). Fatty acid synthesis from [2-1'4C] pyruvate increased several times in the presence of glucose while oxidation of [1-14C] pyruvate was not affected by addition of glucose. Conversion of [U-1'4C] glucose to COs was depressed by addition of pyruvate, and rates of fatty acid synthesis from [U-14C] glucose were depressed. Effects of insulin on [U-~'4C] glucose and [2-1~C] pyruvate incorporations into fatty acids were stimulatory when these substrates were incubated separately or in combination. Carbon dioxide and fatty acid formation from [1-14C] glucose and [6-1~C] glucose were measured in the presence or absence of pyruvate or lactate (Table 4). [1-~4C] glucose oxidation was increased by lactate and markedly reduced by pyruvate. [6-1'4C] glucose oxidation was decreased by lactate and pyruvate to the same extent. Fatty acid synthesis from [1-~4C] glucose and [6-1';C] glucose was decreased to 50 and 70%, respectively, by lactate and to 5% by pyruvate. The ratio of i4CO2 derived from [1-1~C] glucose and [6-14C] glucose and relative pentose cycle activity (PC%) calculated from fatty acid synthesis (11) were increased markedly by lactate. Pyruvate had only minor effects on these. Addition of insulin increased fatty acid synthesis and apparent pentose cycle
TABLE 4. Effects of pyruvate and lactate on the metabolism of glucose-l-~4C and glucose-6-~4C in mammary cells'.
Substrate added Glucose Glucose + pyruvate Glucose + lactate
Insulin -+ -+ -+
/zg/atom labeled carbon incorporated (h × ml cells) -a [1-1~C] glucose [6-1'C] glucose COs Fatty acids CO.~ Fatty acids
G-1-C - G-6-C
56.0 84.2 17.3 28.2 61.2 87.1
8.1 15.3 9.6 11.3 32.2 39'.6
25.8 37.1 1.22 3.31 12.8 17.2
6.9 5.5 1.8 2.5 1.9 2.2
72.0 116.3 3.0 8.9 ,50,8 80.2
--~
~'CO~.
PC~
34.3 41.6 32.4 36.1 49.8 56.6
"Experimental conditions as described in Tab]e 2. JOURNAL OF DAIRY SCIENCE, VOL. 58, NO. 3
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YANG AND B A L D W I N
TABLE 5. Effects of pyruvate and PMS on glucose carbon metabolism in mammary cells*. Substrate added Glucose
Insulin -+
~g/atom labeled carbon incorporated (h × ml cells) -~ [U-~C] glucose [1-1~C] glucose [6-1~C] glucose CO., Fatty acids CO.. Fatty acids CO2 Fatty acids 170 267
84.3 158.0
42.4 54.3
242 250
28.0 33.0
765 82.5
3.2 12.3 20.6 24.2
13.4 20.0
4.4 2.8
32.6 77.0
.74 .67
1.92 2.20
18.0 20.4
17.2 30.0
.46 1.58
1.49 2.26
82.6 86,5
.37 .44
1.59 1.53
Glucose
+ PMS
-
-
+ Glucose + pyruvate Glucose + pyruvate + PMS
-+
-
-
+
49.4 88.5 256 240
1.37 6.01 12.1 14.7
*Experimental conditions as described in Table 2. Phenazine methosulfate (PMS) added at concentration of .1 InM. percentage in all cases. Data in Table 5 indicate that phenazine methosuIfate (PMS) increased [U-14C] glucose oxidation to CO2. This was mainly due to increased pentose cycle flux as indicated by the greatly elevated ratio of l~COz derived from [1-14C] glucose and [6-1'C] glucose. Fatty acid synthesis from [U-~4C] glucose was depressed to less than half by PMS. Effects of pyruvate on glucose metabolism were similar to those in Table 3. Fatty acid synthesis from [U-14C] glucose was depressed much lower by pyruvate than by PMS. When PMS was presented in addition to pyruvate, rates of labeled glucose carbon oxidation were similar to that with PMS alone. The inhibitory effect of pyruvate upon fatty acid synthesis from glucose was partially relieved by PMS. Stimulatory effects of insulin on glucose metabolism were high in the presence of pyruvate but were diminished by PMS. When 2.5 mM lactate and pyruvate were presented simultaneously, rates of fatty acid synthesis from [2-14C] pyruvate were half of those when 5 mM pyruvate was presented
alone (Table 6). Incorporation of 2.5 m M [2-1~C] lactate into fatty acid was several times higher when incubated with 2.5 mM pyruvate than when 5 mM lactate alone served as substrate. Insulin stimulated fatty acid incorporation from [2-1~C] pyruvate and [2-~4C] lactate labeled carbons to the same extent when these substrates are incubated together. In contrast, insulin depressed fatty acid synthesis from lactate when this substrate was incubated alone. Incorporation of [2-~4C] pyruvate labeled carbon into fatty acids was depressed bv N,N,N',N'-tetramethyt- p- p h e n y l e n edia,m i n e (TMPD) at 20 ttM. PMS inhibited both fatty acid synthesis from and oxidation of pyruvate at 20 and 100 /~M. Stimulatory effects of insulin on [2-r4C] pyruvate conversion to fatty acids persisted in the presence of PMS and TMPD. Twenty micromolar PMS increased [U-X4C] glucose oxidation to CO2 without affecting rate of fatty acid synthesis. PMS (100 /~M) inhibited fatty acid synthesis from glucose and abolished the response to insulin. T M P D (20 /~M) had no effect on [U-14C] glucose incorpo-
TABLE 6. Pyruvate-2-a4C and lactate-2-1'G metabolism and interaction in mammary cells. ~
Substrates [2-~'C] pyruvate, 5mM [2-a4C] lactate, 5raM [2-a*C] pyruvate, 2.5mM + lactate, 2.5mM [2-~4C] lactate, 2.5ram + pyruvate, 2.5ram
~g/atom labeled carbon incorporated ( h ) < ml eel/s) -~ CO~ Fatty acids -- Insulin + Insulin -- Insulin + Insulin 35 16.2
39 14.4
3.2 1.04
8.6 .68
18.4
19.2
1.8
4.4
I6.6
I5.0
1.4
3.4
*Incubation conditions same as described in Table 2 except substrate levels varied as indicated. JOURNAL OF DAIRY SCIENCE, VOL. 58, N o . 3
INSULIN ACTIONS IN MAMMARY CELLS
341
T~L~. 7. Effects of PMS and TMPD on [U-a'C] glucose, [2-~4C] lactate, and [2-~'C] pyruvate metabolism and responses to insulin in mammary cells. ~
Experiment aao. 1
Substrate (5raM) [2-~'C] lactate
. PMS
2
2-[2-~'C] pyruvate PMS TMPD [U-~C] glucose PMS TMPD
3
#g/atom labeled carbon incorporation (h × ml packed cells) PMS, TMPD CO,_, Fatty acids (~M) -- Insulin + Insulin -- Insulin + Insulin
[2-1'C] pyruvate PMS TMPD [2-~*C] lactate PMS TMPD [U-~*C] glucose PMS TMPD
".1 .5 2 10 50 . 20" 20 . 20" 20 . 100 100 ... 100 I00 . 10b" 100
13.1 12.8 13.7 15.4 17.6 16.0 28.9 25.7 27.0 189 282 197 30.1 25.2 20.4 17.9 13.8 18.5 161 278 182
11.3 11.8 12.0 13.5 14.3 14.7 29.2 24.4 29.1 278 331 300 31.5 22.2 19.8 14.5 11.9 18.5 218 258 248
1.0 1.0 1.0 .9 .8 .2 3.0 .5 2.5 116 129 217 2.47 .18 .51 1.48 .30 1.42 126 47.1 123
.7 .7 .8 1.2 1.0 .2 6.5 1.2 4.6 236 196 242 8.41 .27 2.58 .62 .30 2.4 200 45.3 218
*Incubation conditions as described in Table 2. PMS is phenazine methosulfate; TMPD is N,N,N', N'-tetra-methyl-#-pherlylenedimnine. Different cell preparations used in Experiments 1, 2, and 3. ration into CO2 and fattv acids. At 100 t~M, TMPD slightly increased glucose oxidation. The response of glucose metabolism to insulin was maintained in the presence of TMPD. Basal rates of [2-1~4C] lactate oxidation and fatty acid synthesis were not affected by TMPD. Thus, the inhibitory effect of insulin ou [2-14C] lactate incorporation into fatty acid was reversed by T M P D (Table 7). Lactate2-14C oxidation was slightly increased by lower concentrations of PMS. At high concentration, PMS decreased [2-14C] lactate oxidation. Rates of [2-14C] lactate incorporation into fatty acid were decreased by PMS at concentrations above 50 raM. The inhibitory effect of insulin on fatty acid synthesis from [2-x*C] lactate was diminished by PMS. Discussion
In a recent report, Halperin (6) indicated that insulin can increase fatty acid synthesis from pyruvate in the absence of glucose in rat adipose tissue. Insulin did not influence maximum fatty acid synthesis rate but decreased the pynavate concentration at which half maximum rates are observed. From other experimental data (7), Halperin suggested that intracellular fatty aeyl CoA concentration, which can be decreased by insulin, may inhibit trans-
port of intramitoehondrial citrate to the cytosol, and thereby decrease fatty acid synthesis. Jungas (10) reported that insulin sh.'ghtly stimulated fatty acid synthesis in rat adipose tissue with pyruvate or lactate. Oxidation of [1-14C] lactate to COs was also slightly increased by insulin in adipose tissue from high carbohydrate fed rats. Pyruvate dehydrogenase activity in rat adipose tissues, assayed by measuring rates of [1-a~C] pyruvate or [1-14C] lactate conversion to CO2 in tissue homogenares, was stimulated by preincubation of tissue with insulin and decreased by preincubation with epinephrine (10, 21). Data from this experiment show a similar stimulatory effect of insulin on fatty acid synthesis from [2-~4C] pyruvate in isolated rat mammary cells over a wide range of pyruvate concentrations. This effect of insulin in mammary cells seems not to have resulted from increased pyruvate dehydrogenase flux as indicated by a relatively minor effect of insulin on [1-14C] pyruvate oxidation to CO2 (Table 3). The inhibitol T effect of insulin on fatty acid synthesis from lactate in mammary tissue (Table 2) is contrary to observations in rat adipose tissue (10). The cause of this difference is not known. There are several metabolic differences between mammary and adipose tissues which JOURNAL OF DAIRY SCIENCE, VOL. 58, N o . 3
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YANG AND B A L D W I N
could contribute to the difference. In mam- does not. The positive effect of insulin on fatty mary cells, reducing equivalents as reduced acid synthesis from pyruvate was maintained nicotinaruide adenine dinucleotide (NADH) in PMS and TMPD treated cells. Lactate-2can be efficiently transported between mito- 14C incorporation into fatty acids was not afehrondria and the cytosol by the Borst shuttle fected by 100 t~M TMPD, but the response to (1). In adipose tissue, lack of a functional insulin was changed from an inhibitory one to Borst shuttle (12, 17) could limit the transfer a stimulatory one. PMS, at low concentrations, of NADH reducing equivalents from the eyto- slightly increased [g-~4C] lactate oxidation. sol to the mitochondrion. Accumulation of The basal rate of fatty acid synthesis was not NADH or a change in NADH/nicotinamide affected, but the negative response to insulin adenine dinucleotide (NAD) ratio may cause was decreased. At high concentrations, PMS secondary metabolic effects in adipose such as decreased fatty acid synthesis from [2-~4C] lacchanging pyruvate dehydrogenase activity (3, tate and addition of insulin did not show any 23). Further, insulin has antilipolytic activity effect. The observations further indicate that in adipose tissue. Thus, changes of intracellu- insulin can affect fatty acid synthesis indelar free fatty acid concentrations occur. This pendent of glucose uptake and that this action could lead to metabolic changes, particularly is related to intracellular redox state. Katz et al. (11) reported that PMS, at conin fatty acid synthesis (7, 18). The different responses to insulin in fatty eentrations within the range of this study, inacid synthesis from [2-14C] pyruvate and creased fatty acid synthesis from lactate in rat [2-1"C] lactate were not due to differences in mammary and adipose tissue slices. This result intracellular pyruvate concentration and pyru- was not found in the present experiment with vate dehydrogenase flux since when concen- mammary cells. The reason for this discrepantrations of pyruvate and lactate were such that cy is not known. Inhibition of glucose utilization by pyruvate rates of carbon incorporation into fatty acids from these two substrates were comparable, has been observed by several investigators (8, insulin stimulated [2-x4C] pyruvate incorpora- 19, 24). The data in Table 3 indicate that total tion into fatty acids (Fig. 1). Furthermore, in- incorporation of two carbon units from glucose sulin had only minor effects upon rates of and pyruvate, when incubated together (12.2 [1-1'4C] pyruvate and [1-14C] lactate conver- q- 9/2 ---- 16.7) is less than the stun (3.4 qsions to 14CO2 via the pyruvate dehydrogenase 134/2 ---- 70.4) of each incubated alone. This reaction while producing these contrasting re- observation plus consideration of the conversions of [1-14C] glucose and [6-14C] glucose sponses in fatty acid synthesis. It is much more likely that the effects of lac- into fatty acids (Table 4), indicates that the tate and pyruvate upon intraccllular redox effect of pyruvate upon glucose metabolism is state modify the action(s) of insulin on fatty not attributable to isotope dilution. Williamson (24) reported that pyruvate deacid synthesis in mammary cells. The implication is that insulin stimulates fatty acid synthe- pressed glucose oxidation in perfused rat heart, sis in cells in a less reduced state as is caused From a cross-over profile of tissue metabolites, by pyruvate, and reduces fatty acid synthesis he suggested that the site of inhibition was bein highly reduced cells as are produced by lac- tween a-glyeeraldehyde-P and phosphoglyceric tate. Phenazine methosulfate and TMPD have acid. The decrease in [2-a4C] pyruvate oxidation been used to modify intracellular redox states in vitro (5, 12, 22). PMS acts as a general to CO2 caused by glucose was not caused by electron carrier in accepting electrons from isotope dilution as indicated by increased both NADH and NADPH and transferring [1-x*C] pyruvate oxidation. In the presence of these to molecular oxygen. TMPD acts as an glucose, increased [2-a4C] pyruvate incorporaelectron carrier in transferring electrons be- tion into fatty acids could be attributable to tween NADH and cytochrome b5 reductase an increased supply of NADPH from the pen(20) and between cytochrome b and cyto- rose phosphate pathway. Glucose greatly inchromo c (13). Effects of PMS and TMPD on creased the incorporation of [2-a4C] lactate inmammary cell metabolism are concentration to fatty acid. This may also be due to increased dependent (Table 7). Pyruvate-2-~4C incorpo- NADPH supply as indicated by the elevated ration into fatty acids was depressed by both flux of glucose carbon through the pentose PMS and TMPD. PMS has more severe effects cycle in the presence of lactate (Table 4). than TMPD probably due to the fact that PMS Lactate-l-~4C oxidation was increased and depressed intracellular NADPH which is re- [2-~4C] lactate oxidation was decreased in the quired for fatty acid synthesis while TMPD presence of glucose, suggesting a shift in laeJOURNAL OF DAIItV SCIENCE, VOL. 58, NO. 3
I N S U L I N A C T I O N S IN MAMMARY CELLS
tate carbon metabolism from tricarboxylic acid cycle oxidation to fatty acid synthesis. Lactate increased [1-~4C] glucose and decreased [6-rdC] glucose oxidation to CO 2 without substantially affecting [U-~dC] glucose oxidation. This observation suggests isotope dilution and carbgn exchange. References
(1) Borst, P. 1963. Functionelle mad morphologische organisation der zelle, p. 137. W/irzburg: Springer-Verlag. (2) Crofford, O. D., and A. E. Reno,ld. 1965. Glucose uptake by incubated rat epididymat adipose tissue, characteristie of glucose transpose system and actions of insulin. J. Biol. Chem. 240:3237. (3) Garland, P. B., and P. J. Dandle. 1964. Control of pyruvate dehydrogenase in the perfused rat heart by the intracellular coneentration of acetyl-coenzyme A. Biochem. I. 91:6(3. (4) Halperin, M. L, 1970. An additional role for insulin in tile control of fatty acid synthesis independent of glucose transport. Can. J. Biochem. 48:1229. (5) Halperin, M. L., and B. H. Robinson. 1970. The role of the cytoplasmic redox i°°" tential in the control of fatty acid synthesis from glucose, pyruvate, and lactate in white adipose tissue. Bioehem. J. 116:235. (6) Halperin, M. 1971. Studies on the conversion of pyruvate into fatty acids in white adipose tissue. Biochem. J. 124:615. (7) Halperin, M. L., B. H. Robinson, and I. B. Fritz. 1972. Effects of paJmitoyl CoA on citrate and malate transport by rat liver mitochondrla. Prec. Nat. Aoad. Sei. 69:1003. (8) Hirsch, P. F., H. Baruch, and I. L. Chaikoff. 1954. The relation of glucose oxidation to lipogenesis in mammary tissue. J. Bi01. Chem. 210:785. (9) Jungas, B. L., and E, G. Ball. 1963. Studies on the metabolism of adipose tissue. XII. The effects of insulin and epinephrine on free fatty aeid and glycerol production in the presence and absence of glucose. Biochemistry 2:383. (10) Jungas, R. L. 1970. Effect of insulin oI~ fatty acid synthesis from pymvate, lactate, or endogenous sources in adipose tissue: Evidence for the hormonal regulation of pyruvato dehydrogenase. Endocrinology 86: 1368. (11) Katz, |., L. R. 'Bernard, and G. E. Bartsch. 1066. The pentose cycle, triosephosphate isomerization and lipogenesis in rat adipose
343
tissue. J. Biol. Chem. 241:727. (12) Katz, J., and P. A. Wals. 1970. Effect of phenazine methosuLfate on lipogenesis. J. Biol. Chem. 245:2546. (13) Lee, C. P., G. L. Scottocasa, and L. Ernster. 1967. Use of artificial electron accepters for abbreviated phosphorylating electron transport: Flavin-cytoehrome e. In Methods in enzymology, R. W. Estabrook and M. E. Ptdlman, ed. 10:33. New York Academic Press, Inc. (14) Martin, R. |., and B. L. Baldwin. 1971. Effects of alloxan diabetes on lactational perrefinance and mammary tissue metabolism in the rat. Endecrinoiogy 88:863. (15) Martin, R. 1-, and I~. L. Baldwin. 1971. Effects of insulin and anti-insulin serum treatments on levels of metabolites in rat mammary glands. Endocrinology 88:868. 16) Martin, R. |., and R. L. Baldwin. 1971. Effects of insulin on isolated rat: mammary cell metabolism: Glucose utilizatkm and metabolite patterns. Endocrinology 89:1263. 17) l~obinson, B. It., and M. L. IIalperin. 1970. Trm~sport of reduced nicotinamide-adenine dinueleotide into mitochondria of rat white adipose tissue. Biochem. J. 116:229. (18) Sehmidt, K., and ]. Katz. 1969. Metabolism of pyrnvate and L-lactate by rat adipose tissue. 1. Biol. Chem. 244:2125. (19) Smith, 2. W., and R. F. Glascock. 1969. The effects of acetate and of pyruvate on the pathways of glucose catabolh;m in lactating mammary tissue. J. Dairy Res. 36:455. (20) Strittmatter, P. C. 1963. Mi,~osomal eytochrome b., and cytochrome b~ reduetase. In The enzymes, M. A. Landy and K. Myrb~ck, ed. 8:113. New York Academic Press, Inc. (21) Taylor, S. I., and B. L. Jungas. 1972. Effects of antilipolytic agents on F,yruvate dehydrogenase activity of rat apidose tissue. Fed. Prec. 31:244. (22) Waiters, E., and P. McLean. 2t968. Effect of alloxan-diabetes and treatment with antiinsulin serum on pathways of glucose metabolism in lactating rat n~muuary gland. Bioehem. J. 109:407. (23) Wfeland, O., B. von laguw-Westermann, and B. StukowskL 1969. Kinetic and regulatory properties o~ heart musele pyruvato dehydrogenase. Hoppe-Seyter's Z. Physiol. Chem. 350: 329. (24) Wflliamson, J. R. 1965. Glycolytie control mechanism. I. Iulqibitiou of glycolysis by acetate and pyruvate m the isolated perfused rat heart. I. Biol. Chem. 240:2308.
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