- Email: [email protected]
Mechanism of insulin action on control of fatty acid synthesis independent of glucose transport
Mechanism of Insulin Action on Control of Fatty Acid Synthesis Independent of Glucose Transport By M. L. HALPERIN AND B. H. ROBINSON
Iusulin has been found to cause a 30.fold increase in the rate of glucose conversion to fatty acid in white adipose tissue incubated in vitro. In the presence of maximally effective insulin concentrations, glucose conversion to fatty acid was limited by the accumulation of cytoplasmic NADH,, which effectively lowered intracegular pyruvate concentration. Insulin, in the absence of glucose, stimulated py-
ruvate conversion to fatty acid in adipose tissue from both normal fed and starved rats. Therefore, insulin augments the rate of fatty acid synthesis both by increasing the supply of substrate (pyruvate) and also by directly increasing pyruvate incorporation into fatty acid by a mechanism distinct from the known stimulation of glucose transport. (Metabolism 20: No. 1, January, 78-86, 1971)
I
T WAS FIRST DEMONSTRATED by Krahll that the uptake of glucose by adipose tissue is stimulated by the addition of insulin in vitro. Winegrad and Renold2 drew attention to the fact that insulin markedly augments fatty acid synthesis and oxidation of carbon 1 of glucose. It has been assumed until very recently that this effect of insulin requires the presence of glucose and can be explained in terms of the demonstrated action of insulin on glucose uptake. Jeanrenaud and Renold3 reported that the pattern of glucose metabolism in vitro is essentially the same whether glucose uptake is stimulated by insulin or by increasing the concentration of glucose in the incubation medium. Contrary to the preceding report, the results of Leonards and Landau4 revealed that a greater proportion of the carbon flow was directed to fatty acid when insulin rather than high glucose concentrations stimulated glucose metabolism. These results suggest that insulin might play a role in favoring fatty acid synthesis from intracellular glucose. Our results reported herein support this latter conclusion, since insulin stimulates glucose conversion to fatty acid both by increasing glucose transport (to supply intracellular pyruvate) and by augmenting pyruvate conversion to fatty acid.
Pathways
of Glucose Metabolism
The major pathways of glucose metabolism in the adipocyte of the rat are From the University of Toronto School of Medicine, St. Michael’s Hospital, and the Univdrsity of Toronto Department of Biochemistry, Toronto, Canada. Supported by Grant MRC MA3363 from the Medical Research Council of Canada and the St. Michael’s Hospital Research Sociaty. M. L. HALPERIN, M.D.C.M., F.R.C.P.(C): Assistant Professor of Medicine, University of Toronto School of Medicine; Attending Staff, St. Michael’s Hospital, Toronto, Canada; B. H. ROBINSON, PH.D.: MRC Postdoctoral Fellow, Department of Biochemistry, University of Toronto, Toronto, Canada. 78
METABOLISM,VOL. 20, No, 1 (JANUARY), 1971
CONTROL
OF FATTY
ACID SYNTHESIS
Fig. 1.-Pathways
79
of glucose metabolism in adipocyte.
reviewed in Fig. 1. The major control seems to be at the glucose transport step, regulated primarily by the presence of insulin. Once glucose has crossed the plasma membrane of the adipocyte it could be metabolized along several potential pathways. It could be converted to glycogen, and this pathway is particularly important in the fasted-refed rat as glycogen contents can increase by lOO-fold in this nutritional state. 5 Glucose could be oxidized in the hexose monophosphate shunt with the production of NADPHB; alternatively, glucose could be converted to alpha glycerol phosphate, the precursor of glyceride glycerol. Glucose could be metabolized in the glycolytic pathway to produce pyruvate plus cytoplasmic NADH2. The two major fates of pyruvate would be either lactate production or conversion to fatty acid. Pyruvate carboxylase and pyruvate dehydrogenase are specially important in this latter pathway. It is important to emphasize at this point that whenever pyruvate is formed from glucose in the cytoplasm so also is NADH2. There are four primary pathways for the removal of this reducing power from NADH*; these appear on the right side of Fig. 1: ( 1) lactate formation; (2) Alpha glycerol phosphate formation; (3) fatty acid synthesis, whereby reducing power from NADHz is transferred to NADP by means of both the NAD- and NADP-linked malate dehydrogenases; and (4) oxidation of reducing power of NADHB by mitochondria. In the intact tissue, there must be a balance between production and utilization rates of reducing equivalents. In white adipose tissue, the generation of reducing equivalents during the formation of acetyl CoA from glucose greatly exceeds their requirements for the reductive synthesis of fatty acids, lactate, and alpha glycerol phosphate .6-8 Accumulation of the reducing power during lipogenesis from glucose might regulate the flow in this pathway. Further since the lactate/pyruvate output ratio of adipose tissue incubated in vitro with insulin is increased,Q this suggests that the cytoplasmic component of the redox potential is elevated. The fact that the fatty acid synthesis rate from pyruvate is manyfold greater than that from lactatelO-I4 could be explained by the slow rate of oxidation of cytoplasmic NADH? in white adipose tissue. Since the studies of Flatt and Ba116v7and Rognstad and Katz* have quantitated the pathways mentioned above, we thought it necessary to obtain
80
HALPERIN
I NADH
DHAP
o
MALATE
OAA
I
i I I I
i
AND ROBINSON
FPH2
FP
I
NAD
NADH
Mitochondria
Fig. Z.-Possible chondria.
pathways of NADH transport into white adipose tissue mito-
information on the potential pathways for cytoplasmic-reducing port into mitochondria of white adipose tissue.
power trans-
Studies of Adipose Tissue Mitochondria
Mitochondria for these studies were prepared from adipocytes isolated by the technique of Rodbell. l5 These mitochondria were physiologically intact, as judged from the P:O ratios and the respiratory control ratiosI Borst17 has reviewed the mechanisms that have been proposed for the oxidation of cytoplasmic NADHz by mitochondria. In most tissues,l* including adipose tissue,16 there is a permeability barrier to NADHz unless mitochondria are subjected to treatment with hypoosmotic solutions. Pathways involving substrate cycles are required to translocate the cytoplasmic NADHz-reducing power. These cycles are illustrated in Figs. 2 and 3. Figure 2 is divided into three sections (A, B, and C) to facilitate the discussion. These mitochon-
Fig,
3.-Borst
cycle, A.A.T.: aspartate amino transferase. E.C. 2.6.1.1.
CONTROL
Table l.-Effect
Expt.
81
OF FATTY ACID SYNTHESIS
of TMPD on Rate of Glucose, Lactate, and Pyruvate Conversion to Fatty Acid*
Additions to Incubation
Rate of +I Incorporation (pg Atom *H/hr./Gm.
Medium
into Fatty Acid wet weight)
1
glucose glucose + TMPD
30.8 c 2.1 37.1 I? 2.07
2
lactate lactate + TMPD
4.6 + 1.4 9.8 2 3.2t
3
pyruvate pyruvate + TMPD
4
glucose + pyruvate glucose + pyruvate
35.1 r 5.4 26.7 k 4.2t 39.7 +- 4.0 35.9 k 4.2
+ TMPD
* Fat pads were preincubated for 30 min. in medium containing insulin (10 mU./ml.) and then transferred to fresh similar medium and where added TMPD (75 JNII), glucose (10 mM), lactate (10 mM), pyruvate (10 mM), and aH,O (0.2 mC/ml.) Results are given as mean k SEM for four paired observations. t p < .Ol for paired observations.
dria did not metabolize reducing power from NADH2, nor were they able to oxidize alpha glycerol phosphate to any significant degree, suggesting that pathways A and B were of very low activity in this tissue. The third pathway (Fig. 3, “Borst shuttle”)-that is, a malate/oxaloacetate cycle modified to include a double transamination-did not operate to any significant degree. This cycle was limited by the low activity of the intramitochondrial aspartate amino transferase in these mitochondria .I6 Further support for this concept was obtained from the fact that these mitochondria failed to metabolize malate in the presence of glutamate, probably because of the low activity of this intramitochondrial transaminase.lG Therefore, by virtue of the fact that this tissue can only oxidize cytoplasmic NADH2 at a very slow rate, this could be important as a potential control mechanism for fatty acid snythesis. Studies of Role Synthesis
of Cytoplasmic
Redox
Potential in Control
of Fatty Acid
All the evidence cited so far has been indirect concerning the role of the cytoplasmic NADH/NAD ratio on the control of fatty acid synthesis in white adipose tissue. In order to obtain direct evidence an agent was required that would permit the oxidation of cytoplasmic reducing power from NADHz but not from NADPH, by adipose tissue mitochondria. This substance should not be a substrate for fatty acid synthesis, should be nontoxic, and should have no other actions. NNN’N’-tetramethyl-p-phenylenediamine (TMPD) proved to be such an agent. Proof of its action can be obtained from Fig. 3 of Ref. 13 and Table 3 of Ref. 16. The addition of TMPD to fat pads incubated in the presence of glucose resulted in a lowering of the cytoplasmic redox potential. If this ratio plays a role in the control of fatty acid synthesis from substrates that produce cytoplasmic NADH2 then TMPD could be expected to increase fatty acid synthesis from glucose and lactate but not from pyruvate. This is seen in Table 1.
HALPERIN
82 Table Z.-Effect
AND ROBINSON
of Pyruvate, Lactate, and TMPD on Rates of Pyruvate Output and Fatty Acid Synthesis*
Additions to Incubation Medium
Rate of Pyruvate Output (!moles/hr./Gm. wet wt.)
1
insulin insulin + pyruvate
0.40 + .07 -
30.1 52.7
f 4.1 rf: 6.3t
2
insulin insulin + lactate
0.24 ?I .03 0.67 r .05t
24.8 32.6
k 1.3 z!z 1.6i
3
insulin insulin + TMPD
0.56 f. .05 1.41 * .1st
30.8 37.1
f 2.1 + 2.oi
4
insulin TMPD
0.30 t .02 0.46 Z!I.06
40.3 f 6.2 4.71 k .37t
Expt.
Rate of 8H Incorporation Acids (,,g. atoms/hr./Gm.
into Fatty wet wt.)
* Fat pads were preincubated for 30 min. in medium containing glucose (10 mM) and then incubated in fresh medium containing glucose (10 mM), aH,O (0.2 mC./ml.), and where added insulin (10 mU./ml.), TMPD (75 pM), pyruvate (5 mM) and lactate (5 mM). Results are given as mean k SEM for six groups of paired fat pads. t p < .Ol for paired observations.
We next asked the question, what is the mechanism whereby an increased cytoplasmic redox potential decreases the rate of fatty acid synthesis? The answer seems to be, by decreasing the intracellular pyruvate concentration. The evidence for this is presented in Table 2 and Ref. 19. Fatty acid synthesis by adipose tissue in the presence of glucose and optimal insulin levels was increased a further 20-75 per cent by the addition of lactate, pyruvate, or TMPD to the incubation medium. Cytoplasmic NADH,/NAD as estimated by the lactate to pyruvate output ratio was decreased by TMPD (to a third of the control level) and presumably also by pyruvate. However, lactate would be expected to increase it. Medium pyruvate concentrations, and by inference the intracellular pyruvate concentration, was increased in all these conditions. Therefore, fatty acid synthesis rates correlated with pyruvate concentrations rather than the NADHJNAD. In support of this, TMPD should not increase fatty acid synthesis when both glucose and pyruvate are present in the incubation medium, as the intracellular pyruvate concentration will already be markedly elevated. This is seen in Table 1 (experiment 4). Is the conclusion justified that the intracellular pyruvate concentration controls fatty acid synthesis from glucose, or are additional factors important? To test this an experiment was designed whereby pyruvate outputs from glucose-and therefore intracellular pyruvate concentrations-would be equal under two different experimental conditions, and the rate of glucose incorporation into fatty acid measured. The two conditions were glucose plus TMPD and glucose plus insulin. At equal pyruvate output rates, glucose conversion to fatty acid was eightfold greater in the presence of insulin as compared with TMPD in both fasted and normal fed rats; see Table 2 and Ref. 19. These results suggested that insulin not only increases glucose transport, but also augments pyruvate conversion to fatty acid. Therefore, although the control of the intra-
83
CONTROL OF FATTY ACID SYNTHESIS
Table 3.Effect
of Insolin on Pyruvate Conversion to Fatty Acid Rate of Pyruvate. 247 Incorporation into Fatty Acid (ml.rmoles/hr./Gm. wet wt.)
Expt.
1
pyruvate (0.25 mM) pyruvate (0.25 mM) + insulin
normal normal
2
pyruvate (0.25 mM) pyruvate (0.25 mM) + insulin
36 hr. fasted 36 hr. fasted
3
pyruvate (25 mM) pyruvate (0.25 mM) + insulin
normal normal
4
pyruvate (25 mM) pyruvate (0.25 mM) + insulin
36 hr. fasted
390 630
r +
4.1 r 2.6 r 10,700 8,200
36 hr. fasted
2,720 5,210
80 1lOf 1.3 0.7
2 1,550 iz 4,860: c +-
590 7907
* Fat pads were preincubated for 30 min. in medium containing the noted pyruvate concentration and then transferred to fresh medium containing pyruvate (concentration in parenthesis), pyruvate 2-Cl4 (0.2 &ml.> and where added, insulin 10 mU./ml. Incubation volume was 2.5 ml. for experiments 3 and 4 and 20 mI. for experiments 1 and 2. Results are given as mean + SEM. The number of observations with paired tissues are 4 for experiments 2, 3, and 4, and 15 for experiment 1. ? p < .Ol for paired observations. $ p < .05 for paired observations. cellular pyruvate concentration offers a potential control for the rate of glucose conversion to fatty acid, this system is not the only regulatory site.
E#ect of Insulin on Pyruvate Conversion to Fatty Acid There have been numerous reports on the failure of insulin to increase pyruvate conversion to fatty acid, with a few exceptions.1g-21 However, pyruvate concentrations used in all these studies were considerably higher than the normal intracellular concentration, which varies from 60 PM in the normal fed to 250 PM in the fasted state (Halperin unpublished observations.) We decided, therefore, to test the effect of insulin on pyruvate conversion to fatty acid using both physiological and very high pyruvate concentrations. The results are reported in Table 3 and Ref. 19. In adipose tissue from starved rats, pyruvate was converted to fatty acids at a rate approaching that obtained in adipose tissue from fed rats, provided the concentration of pyruvate was increased to 25 mhL12Jg It is therefore apparent that cells from fasted animals possess the enzymatic capacity for high rates of fatty acid synthesis. The inhibition of fatty acid synthesis activity is manifested when tissues from fasted rats are incubated in the presence of physiological levels of substrates. In adipose tissue from fed rats, insulin caused a twofold increase in pyruvate (250 PM) conversion to fatty acid, even when glucose was absent from the medium. Insulin caused an increased fatty acid synthesis rate from pyruvate at both high and low pyruvate concentrations if unlabeled glucose was present.l” The mechanism by which insulin stimulated fatty acid synthesis from pyruvate is unknown. However, when the pyruvate concentration was increased to 25 mM, insulin no longer stimulated pyruvate conversion to fatty acid. This suggests that insulin in some way lowered the binding affinity for pyruvate or one of the other
84
HALPERIN AND ROBINSON Table 4.-Effect
Addition~~~i~cubation
None
Insulin
of Insulin on Pyruvate Output and Fatty Acid Synthesis in the Adipocyte of the Fasted-Refed Rat* Rate of Pyruvate Output (pmole/hr./Gm. wet wt.)
0.33 I .09 0.16 k .04t
Rate
of aH Incorporation (pg. atoms/hr./Gm.
into Fatty Acids wet wt.)
12.9 rt 0.9 19.4 k 2.5t
*Fat pads were preincubated for 30 min. in a medium containing albumin (25 mg./ml.) in the absence of glucose and then transferred to fresh similar medium containing in addition 3H,O (0.3 mc./mI.) and where added, insulin (10 mU./mI.). Results are given as mean k SEM for eight paired observations. i p < .Ol for paired observations.
intermediates at the rate-limiting step between pyruvate and fatty acid as the effect is overcome by raising the pyruvate concentration. The apparent critical concentration of pyruvate for fatty acid synthesis in tissues from starved animals is considerably higher than prevailing in adipose tissue from fed rats. This interpretation is based on the fact that insulin exerted no influence on the very low rates of fatty acid synthesis from 250 PM in pyruvate, a concentration which is much lower than the apparent K, in adipose tissue from starved animals; on the other hand, insulin significantly increased pyruvate incorporation into fatty acid when the pyruvate concentration was 25 mM in adipose
tissue from the starved rat.19 Pyruvate outputs and the estimated intracellular pyruvate concentrations in adipose tissue from starved rats are considerably higher than corresponding values in adipose tissue from fed rats. These differences may reflect the high apparent K, for pyruvate in adipose tissue from starved rats. Another line of evidence to support the hypothesis that insulin lowers the binding affinity for pyruvate in its conversion to fatty acid is seen in Table 4 using fasted-refed rats. These adipocytes have enough endogenous substrate (glycogen) to supply more glucose than this tissue can consume in two hours of incubation at maximal rates of glucose utilization. 3Hz0 incorporation into fatty acid was used to measure the fatty acid synthesis rate. Since glucose could not be detected in the incubation medium in any of the experiments reported in Tables 3 and 4 and Ref. 19, these effects of insulin described are independent of the action of insulin on glucose transport across the plasma membrane. Insulin caused a marked reduction in the rate of pyruvate output from the adipocyte of the fasted-refed rat, suggesting that the intracellular pyruvate concentration was lower when insulin was present. The fact that insulin increased the rate of fatty acid synthesis despite a lower intracellular pyruvate concentration is consistent with the hypothesis recently proposedlg that insulin may lower the binding affinity for pyruvate in its conversion to fatty acids. who has also reported an enhancement of glycogen conversion Jungas, into fatty acid by insulin in the adipocyte of the fasted-refed rat, has suggested that insulin may do so by increasing the activity of pyruvate dehydrogenase. Alternatively, inhibition of fatty acid synthesis in conditions where there is increased cyclic AMP concentration caused either by the addition of lipolytic hormones or theophylline, or in adipocytes of fasted or fasted-refed rats,
CONTROL
OF FATTY
ACID SYNTHESIS
85
Fig. 4.-Role of insulin in control of fatty acid synthesis. Vertical arrow indicates stimulation by insulin.
indicates a possible role for cyclic AMP or one of the products of lipolysis in the regulation of fatty acid synthesis. In summary, insulin at optimal concentration causes a 30-fold increase in the rate of fatty acid synthesis when epididymal adipose tissue of the rat is incubated in the presence of glucose. Insulin was thought to promote fatty acid synthesis by increasing glucose transport. The rate of fatty acid synthesis is limited under these conditions by the accumulation of cytoplasmic reducing power. This increased cytoplasmic redox potential limits fatty acid synthesis by lowering the intracellular pyruvate concentration. Insulin now seems to have an additional role in augmenting glucose conversion to fatty acid apart from increasing glucose transport. Insulin appears to increase the binding afiinity (lower the apparent K,,,) for pyruvate or one of its products at the rate-limiting step in pyruvate conversion to fatty acids (see Fig. 4). ACKNOWLEDGMENT The authors are very grateful to Drs. A. Angel, F. S. Rolleston, and I. B. Fritz for helpful advice and to Miss G. McGill and Mrs. Linda Richardson for skilled technical assistance.
REFERENCES 1. Krahl, M. E.: The effect of insulin and pituitary hormones on glucose uptake in muscle. Ann. N.Y. Acad. Sci. 54:649, 1951. 2. Winegrad, A. I., and Renold, A. E.: Studies on rat adipose tissue in vitro. J. Biol. Chem. 233:267, 1958. 3. Jeanrenaud, B. and Renold, A. E.: Studies on rat adipose tissue in vitro. J. Biol. Chem. 234:3082, 1959. 4. Leonards, J. R., and Landau, B. R.: A study on the evidence of metabolic patterns in rat adipose tissue: insulin versus glucose concentration. Arch. Biochem. 91: 194, 1960. 5. Frerichs, H., and Ball, E. G.: Studies on the metabolism of adipose tissue. XI. Activation of phosphorylase by agents which stimulate lipolysis. Biochemistry 1: 501, 1962. 6. Flatt, J. P., and Ball, E. G.: Studies on the metabolism of adipose tissue. J. Biol. Chem. 239:675, 1964.
7. Flatt, J. P., and Ball, E. G.: Studies on the metabolism of adipose tissue. J. Biol. Chem. 241:2862, 1966. 8. Rognstad, R., and Katz, J.: The balance of pyridine nucleotides and ATP in adipose tissue. Proc. Nat. Acad. Sci. U.S.A. 55: 1148, 1966. 9. Halperin M. L., and Denton, R. M.: Regulation of glycolysis and L-glycerol 3phosphate concentration in rat epididymal adipose tissue in vitro. Biochem. J. 113:207, 1969. 10. Kneer, P., and Ball, E. G.: Studies on the metabolism of adipose tissue. J. Biol. Chem. 243:2863, 1968. 11. Schmidt, K., and Katz, J.: Metabolism of pyruvate and L-lactate by rat adipose tissue. J. Biol. Chem. 244:2125, 1969. 12. Reshef, L., Hanson, R. W., and Ballard, F. J.: Glyceride-glycerol synthesis from pyruvate. J. Biol. Chem. 244:1994, 1969.
86 13. Halperin, M. L., and Robinson, B. H.: The role of the cytoplasmic redox potential in the control of fatty acid synthesis from glucose, pyruvate and lactate in white adipose tissue. Biochem. J. 116:235, 1970. 14. DelBoca, J., and Flatt, J. P.: Fatty acid synthesis from glucose and acetate and the control of lipogenesis in adipose tissue. Europ. J. Biochem. 11:127, 1969. 15’. Rodbell, M.: Metabolism of isolated fat cells. J. Biol. Chem. 239:375, 1964. 16. Robinson, B. H., and Halperin, M. L.: Transport of reduced nicotinamide-adenine dinucleotide into mitochondria of rat white adipose tissue. Biochem. J. 116:229, 1970. 17. Borst, P.: Functionelle und Morphologische Organisation der Zelle. Wurzburg:
HALPERIN
AND ROBINSON
Springer-Verlag. Hydrogen transport and transport metabolites. 1963, p. 137. 18. Lehninger, A. L.: Phosphorylation coupled to oxidation of dihydrodiphosphopyridine nucleotide. J. Biol. Chem. 190:345, 1951. 19. Halperin, M. L.: An additional role for insulin in the control of fatty acid synthesis independent of glucose transport. Canad. J. Biochem. 48:1228, 1970. 20. Fain, J. N.: Effect of puromycin on incubated adipose tissue and its response to dexamethasone, insulin and epinephrine. Biochim. Biophys. Acta 84:636, 1964. 21. Jungas, R. L.: Effects of insulin and epinephrine on pyruvate dehydrogenase in adipose tissue. Fed. Proc. 29:981, 1970.
Iusulin has been found to cause a 30.fold increase in the rate of glucose conversion to fatty acid in white adipose tissue incubated in vitro. In the presence of maximally effective insulin concentrations, glucose conversion to fatty acid was limited by the accumulation of cytoplasmic NADH,, which effectively lowered intracegular pyruvate concentration. Insulin, in the absence of glucose, stimulated py-
ruvate conversion to fatty acid in adipose tissue from both normal fed and starved rats. Therefore, insulin augments the rate of fatty acid synthesis both by increasing the supply of substrate (pyruvate) and also by directly increasing pyruvate incorporation into fatty acid by a mechanism distinct from the known stimulation of glucose transport. (Metabolism 20: No. 1, January, 78-86, 1971)
I
T WAS FIRST DEMONSTRATED by Krahll that the uptake of glucose by adipose tissue is stimulated by the addition of insulin in vitro. Winegrad and Renold2 drew attention to the fact that insulin markedly augments fatty acid synthesis and oxidation of carbon 1 of glucose. It has been assumed until very recently that this effect of insulin requires the presence of glucose and can be explained in terms of the demonstrated action of insulin on glucose uptake. Jeanrenaud and Renold3 reported that the pattern of glucose metabolism in vitro is essentially the same whether glucose uptake is stimulated by insulin or by increasing the concentration of glucose in the incubation medium. Contrary to the preceding report, the results of Leonards and Landau4 revealed that a greater proportion of the carbon flow was directed to fatty acid when insulin rather than high glucose concentrations stimulated glucose metabolism. These results suggest that insulin might play a role in favoring fatty acid synthesis from intracellular glucose. Our results reported herein support this latter conclusion, since insulin stimulates glucose conversion to fatty acid both by increasing glucose transport (to supply intracellular pyruvate) and by augmenting pyruvate conversion to fatty acid.
Pathways
of Glucose Metabolism
The major pathways of glucose metabolism in the adipocyte of the rat are From the University of Toronto School of Medicine, St. Michael’s Hospital, and the Univdrsity of Toronto Department of Biochemistry, Toronto, Canada. Supported by Grant MRC MA3363 from the Medical Research Council of Canada and the St. Michael’s Hospital Research Sociaty. M. L. HALPERIN, M.D.C.M., F.R.C.P.(C): Assistant Professor of Medicine, University of Toronto School of Medicine; Attending Staff, St. Michael’s Hospital, Toronto, Canada; B. H. ROBINSON, PH.D.: MRC Postdoctoral Fellow, Department of Biochemistry, University of Toronto, Toronto, Canada. 78
METABOLISM,VOL. 20, No, 1 (JANUARY), 1971
CONTROL
OF FATTY
ACID SYNTHESIS
Fig. 1.-Pathways
79
of glucose metabolism in adipocyte.
reviewed in Fig. 1. The major control seems to be at the glucose transport step, regulated primarily by the presence of insulin. Once glucose has crossed the plasma membrane of the adipocyte it could be metabolized along several potential pathways. It could be converted to glycogen, and this pathway is particularly important in the fasted-refed rat as glycogen contents can increase by lOO-fold in this nutritional state. 5 Glucose could be oxidized in the hexose monophosphate shunt with the production of NADPHB; alternatively, glucose could be converted to alpha glycerol phosphate, the precursor of glyceride glycerol. Glucose could be metabolized in the glycolytic pathway to produce pyruvate plus cytoplasmic NADH2. The two major fates of pyruvate would be either lactate production or conversion to fatty acid. Pyruvate carboxylase and pyruvate dehydrogenase are specially important in this latter pathway. It is important to emphasize at this point that whenever pyruvate is formed from glucose in the cytoplasm so also is NADH2. There are four primary pathways for the removal of this reducing power from NADH*; these appear on the right side of Fig. 1: ( 1) lactate formation; (2) Alpha glycerol phosphate formation; (3) fatty acid synthesis, whereby reducing power from NADHz is transferred to NADP by means of both the NAD- and NADP-linked malate dehydrogenases; and (4) oxidation of reducing power of NADHB by mitochondria. In the intact tissue, there must be a balance between production and utilization rates of reducing equivalents. In white adipose tissue, the generation of reducing equivalents during the formation of acetyl CoA from glucose greatly exceeds their requirements for the reductive synthesis of fatty acids, lactate, and alpha glycerol phosphate .6-8 Accumulation of the reducing power during lipogenesis from glucose might regulate the flow in this pathway. Further since the lactate/pyruvate output ratio of adipose tissue incubated in vitro with insulin is increased,Q this suggests that the cytoplasmic component of the redox potential is elevated. The fact that the fatty acid synthesis rate from pyruvate is manyfold greater than that from lactatelO-I4 could be explained by the slow rate of oxidation of cytoplasmic NADH? in white adipose tissue. Since the studies of Flatt and Ba116v7and Rognstad and Katz* have quantitated the pathways mentioned above, we thought it necessary to obtain
80
HALPERIN
I NADH
DHAP
o
MALATE
OAA
I
i I I I
i
AND ROBINSON
FPH2
FP
I
NAD
NADH
Mitochondria
Fig. Z.-Possible chondria.
pathways of NADH transport into white adipose tissue mito-
information on the potential pathways for cytoplasmic-reducing port into mitochondria of white adipose tissue.
power trans-
Studies of Adipose Tissue Mitochondria
Mitochondria for these studies were prepared from adipocytes isolated by the technique of Rodbell. l5 These mitochondria were physiologically intact, as judged from the P:O ratios and the respiratory control ratiosI Borst17 has reviewed the mechanisms that have been proposed for the oxidation of cytoplasmic NADHz by mitochondria. In most tissues,l* including adipose tissue,16 there is a permeability barrier to NADHz unless mitochondria are subjected to treatment with hypoosmotic solutions. Pathways involving substrate cycles are required to translocate the cytoplasmic NADHz-reducing power. These cycles are illustrated in Figs. 2 and 3. Figure 2 is divided into three sections (A, B, and C) to facilitate the discussion. These mitochon-
Fig,
3.-Borst
cycle, A.A.T.: aspartate amino transferase. E.C. 2.6.1.1.
CONTROL
Table l.-Effect
Expt.
81
OF FATTY ACID SYNTHESIS
of TMPD on Rate of Glucose, Lactate, and Pyruvate Conversion to Fatty Acid*
Additions to Incubation
Rate of +I Incorporation (pg Atom *H/hr./Gm.
Medium
into Fatty Acid wet weight)
1
glucose glucose + TMPD
30.8 c 2.1 37.1 I? 2.07
2
lactate lactate + TMPD
4.6 + 1.4 9.8 2 3.2t
3
pyruvate pyruvate + TMPD
4
glucose + pyruvate glucose + pyruvate
35.1 r 5.4 26.7 k 4.2t 39.7 +- 4.0 35.9 k 4.2
+ TMPD
* Fat pads were preincubated for 30 min. in medium containing insulin (10 mU./ml.) and then transferred to fresh similar medium and where added TMPD (75 JNII), glucose (10 mM), lactate (10 mM), pyruvate (10 mM), and aH,O (0.2 mC/ml.) Results are given as mean k SEM for four paired observations. t p < .Ol for paired observations.
dria did not metabolize reducing power from NADH2, nor were they able to oxidize alpha glycerol phosphate to any significant degree, suggesting that pathways A and B were of very low activity in this tissue. The third pathway (Fig. 3, “Borst shuttle”)-that is, a malate/oxaloacetate cycle modified to include a double transamination-did not operate to any significant degree. This cycle was limited by the low activity of the intramitochondrial aspartate amino transferase in these mitochondria .I6 Further support for this concept was obtained from the fact that these mitochondria failed to metabolize malate in the presence of glutamate, probably because of the low activity of this intramitochondrial transaminase.lG Therefore, by virtue of the fact that this tissue can only oxidize cytoplasmic NADH2 at a very slow rate, this could be important as a potential control mechanism for fatty acid snythesis. Studies of Role Synthesis
of Cytoplasmic
Redox
Potential in Control
of Fatty Acid
All the evidence cited so far has been indirect concerning the role of the cytoplasmic NADH/NAD ratio on the control of fatty acid synthesis in white adipose tissue. In order to obtain direct evidence an agent was required that would permit the oxidation of cytoplasmic reducing power from NADHz but not from NADPH, by adipose tissue mitochondria. This substance should not be a substrate for fatty acid synthesis, should be nontoxic, and should have no other actions. NNN’N’-tetramethyl-p-phenylenediamine (TMPD) proved to be such an agent. Proof of its action can be obtained from Fig. 3 of Ref. 13 and Table 3 of Ref. 16. The addition of TMPD to fat pads incubated in the presence of glucose resulted in a lowering of the cytoplasmic redox potential. If this ratio plays a role in the control of fatty acid synthesis from substrates that produce cytoplasmic NADH2 then TMPD could be expected to increase fatty acid synthesis from glucose and lactate but not from pyruvate. This is seen in Table 1.
HALPERIN
82 Table Z.-Effect
AND ROBINSON
of Pyruvate, Lactate, and TMPD on Rates of Pyruvate Output and Fatty Acid Synthesis*
Additions to Incubation Medium
Rate of Pyruvate Output (!moles/hr./Gm. wet wt.)
1
insulin insulin + pyruvate
0.40 + .07 -
30.1 52.7
f 4.1 rf: 6.3t
2
insulin insulin + lactate
0.24 ?I .03 0.67 r .05t
24.8 32.6
k 1.3 z!z 1.6i
3
insulin insulin + TMPD
0.56 f. .05 1.41 * .1st
30.8 37.1
f 2.1 + 2.oi
4
insulin TMPD
0.30 t .02 0.46 Z!I.06
40.3 f 6.2 4.71 k .37t
Expt.
Rate of 8H Incorporation Acids (,,g. atoms/hr./Gm.
into Fatty wet wt.)
* Fat pads were preincubated for 30 min. in medium containing glucose (10 mM) and then incubated in fresh medium containing glucose (10 mM), aH,O (0.2 mC./ml.), and where added insulin (10 mU./ml.), TMPD (75 pM), pyruvate (5 mM) and lactate (5 mM). Results are given as mean k SEM for six groups of paired fat pads. t p < .Ol for paired observations.
We next asked the question, what is the mechanism whereby an increased cytoplasmic redox potential decreases the rate of fatty acid synthesis? The answer seems to be, by decreasing the intracellular pyruvate concentration. The evidence for this is presented in Table 2 and Ref. 19. Fatty acid synthesis by adipose tissue in the presence of glucose and optimal insulin levels was increased a further 20-75 per cent by the addition of lactate, pyruvate, or TMPD to the incubation medium. Cytoplasmic NADH,/NAD as estimated by the lactate to pyruvate output ratio was decreased by TMPD (to a third of the control level) and presumably also by pyruvate. However, lactate would be expected to increase it. Medium pyruvate concentrations, and by inference the intracellular pyruvate concentration, was increased in all these conditions. Therefore, fatty acid synthesis rates correlated with pyruvate concentrations rather than the NADHJNAD. In support of this, TMPD should not increase fatty acid synthesis when both glucose and pyruvate are present in the incubation medium, as the intracellular pyruvate concentration will already be markedly elevated. This is seen in Table 1 (experiment 4). Is the conclusion justified that the intracellular pyruvate concentration controls fatty acid synthesis from glucose, or are additional factors important? To test this an experiment was designed whereby pyruvate outputs from glucose-and therefore intracellular pyruvate concentrations-would be equal under two different experimental conditions, and the rate of glucose incorporation into fatty acid measured. The two conditions were glucose plus TMPD and glucose plus insulin. At equal pyruvate output rates, glucose conversion to fatty acid was eightfold greater in the presence of insulin as compared with TMPD in both fasted and normal fed rats; see Table 2 and Ref. 19. These results suggested that insulin not only increases glucose transport, but also augments pyruvate conversion to fatty acid. Therefore, although the control of the intra-
83
CONTROL OF FATTY ACID SYNTHESIS
Table 3.Effect
of Insolin on Pyruvate Conversion to Fatty Acid Rate of Pyruvate. 247 Incorporation into Fatty Acid (ml.rmoles/hr./Gm. wet wt.)
Expt.
1
pyruvate (0.25 mM) pyruvate (0.25 mM) + insulin
normal normal
2
pyruvate (0.25 mM) pyruvate (0.25 mM) + insulin
36 hr. fasted 36 hr. fasted
3
pyruvate (25 mM) pyruvate (0.25 mM) + insulin
normal normal
4
pyruvate (25 mM) pyruvate (0.25 mM) + insulin
36 hr. fasted
390 630
r +
4.1 r 2.6 r 10,700 8,200
36 hr. fasted
2,720 5,210
80 1lOf 1.3 0.7
2 1,550 iz 4,860: c +-
590 7907
* Fat pads were preincubated for 30 min. in medium containing the noted pyruvate concentration and then transferred to fresh medium containing pyruvate (concentration in parenthesis), pyruvate 2-Cl4 (0.2 &ml.> and where added, insulin 10 mU./ml. Incubation volume was 2.5 ml. for experiments 3 and 4 and 20 mI. for experiments 1 and 2. Results are given as mean + SEM. The number of observations with paired tissues are 4 for experiments 2, 3, and 4, and 15 for experiment 1. ? p < .Ol for paired observations. $ p < .05 for paired observations. cellular pyruvate concentration offers a potential control for the rate of glucose conversion to fatty acid, this system is not the only regulatory site.
E#ect of Insulin on Pyruvate Conversion to Fatty Acid There have been numerous reports on the failure of insulin to increase pyruvate conversion to fatty acid, with a few exceptions.1g-21 However, pyruvate concentrations used in all these studies were considerably higher than the normal intracellular concentration, which varies from 60 PM in the normal fed to 250 PM in the fasted state (Halperin unpublished observations.) We decided, therefore, to test the effect of insulin on pyruvate conversion to fatty acid using both physiological and very high pyruvate concentrations. The results are reported in Table 3 and Ref. 19. In adipose tissue from starved rats, pyruvate was converted to fatty acids at a rate approaching that obtained in adipose tissue from fed rats, provided the concentration of pyruvate was increased to 25 mhL12Jg It is therefore apparent that cells from fasted animals possess the enzymatic capacity for high rates of fatty acid synthesis. The inhibition of fatty acid synthesis activity is manifested when tissues from fasted rats are incubated in the presence of physiological levels of substrates. In adipose tissue from fed rats, insulin caused a twofold increase in pyruvate (250 PM) conversion to fatty acid, even when glucose was absent from the medium. Insulin caused an increased fatty acid synthesis rate from pyruvate at both high and low pyruvate concentrations if unlabeled glucose was present.l” The mechanism by which insulin stimulated fatty acid synthesis from pyruvate is unknown. However, when the pyruvate concentration was increased to 25 mM, insulin no longer stimulated pyruvate conversion to fatty acid. This suggests that insulin in some way lowered the binding affinity for pyruvate or one of the other
84
HALPERIN AND ROBINSON Table 4.-Effect
Addition~~~i~cubation
None
Insulin
of Insulin on Pyruvate Output and Fatty Acid Synthesis in the Adipocyte of the Fasted-Refed Rat* Rate of Pyruvate Output (pmole/hr./Gm. wet wt.)
0.33 I .09 0.16 k .04t
Rate
of aH Incorporation (pg. atoms/hr./Gm.
into Fatty Acids wet wt.)
12.9 rt 0.9 19.4 k 2.5t
*Fat pads were preincubated for 30 min. in a medium containing albumin (25 mg./ml.) in the absence of glucose and then transferred to fresh similar medium containing in addition 3H,O (0.3 mc./mI.) and where added, insulin (10 mU./mI.). Results are given as mean k SEM for eight paired observations. i p < .Ol for paired observations.
intermediates at the rate-limiting step between pyruvate and fatty acid as the effect is overcome by raising the pyruvate concentration. The apparent critical concentration of pyruvate for fatty acid synthesis in tissues from starved animals is considerably higher than prevailing in adipose tissue from fed rats. This interpretation is based on the fact that insulin exerted no influence on the very low rates of fatty acid synthesis from 250 PM in pyruvate, a concentration which is much lower than the apparent K, in adipose tissue from starved animals; on the other hand, insulin significantly increased pyruvate incorporation into fatty acid when the pyruvate concentration was 25 mM in adipose
tissue from the starved rat.19 Pyruvate outputs and the estimated intracellular pyruvate concentrations in adipose tissue from starved rats are considerably higher than corresponding values in adipose tissue from fed rats. These differences may reflect the high apparent K, for pyruvate in adipose tissue from starved rats. Another line of evidence to support the hypothesis that insulin lowers the binding affinity for pyruvate in its conversion to fatty acid is seen in Table 4 using fasted-refed rats. These adipocytes have enough endogenous substrate (glycogen) to supply more glucose than this tissue can consume in two hours of incubation at maximal rates of glucose utilization. 3Hz0 incorporation into fatty acid was used to measure the fatty acid synthesis rate. Since glucose could not be detected in the incubation medium in any of the experiments reported in Tables 3 and 4 and Ref. 19, these effects of insulin described are independent of the action of insulin on glucose transport across the plasma membrane. Insulin caused a marked reduction in the rate of pyruvate output from the adipocyte of the fasted-refed rat, suggesting that the intracellular pyruvate concentration was lower when insulin was present. The fact that insulin increased the rate of fatty acid synthesis despite a lower intracellular pyruvate concentration is consistent with the hypothesis recently proposedlg that insulin may lower the binding affinity for pyruvate in its conversion to fatty acids. who has also reported an enhancement of glycogen conversion Jungas, into fatty acid by insulin in the adipocyte of the fasted-refed rat, has suggested that insulin may do so by increasing the activity of pyruvate dehydrogenase. Alternatively, inhibition of fatty acid synthesis in conditions where there is increased cyclic AMP concentration caused either by the addition of lipolytic hormones or theophylline, or in adipocytes of fasted or fasted-refed rats,
CONTROL
OF FATTY
ACID SYNTHESIS
85
Fig. 4.-Role of insulin in control of fatty acid synthesis. Vertical arrow indicates stimulation by insulin.
indicates a possible role for cyclic AMP or one of the products of lipolysis in the regulation of fatty acid synthesis. In summary, insulin at optimal concentration causes a 30-fold increase in the rate of fatty acid synthesis when epididymal adipose tissue of the rat is incubated in the presence of glucose. Insulin was thought to promote fatty acid synthesis by increasing glucose transport. The rate of fatty acid synthesis is limited under these conditions by the accumulation of cytoplasmic reducing power. This increased cytoplasmic redox potential limits fatty acid synthesis by lowering the intracellular pyruvate concentration. Insulin now seems to have an additional role in augmenting glucose conversion to fatty acid apart from increasing glucose transport. Insulin appears to increase the binding afiinity (lower the apparent K,,,) for pyruvate or one of its products at the rate-limiting step in pyruvate conversion to fatty acids (see Fig. 4). ACKNOWLEDGMENT The authors are very grateful to Drs. A. Angel, F. S. Rolleston, and I. B. Fritz for helpful advice and to Miss G. McGill and Mrs. Linda Richardson for skilled technical assistance.
REFERENCES 1. Krahl, M. E.: The effect of insulin and pituitary hormones on glucose uptake in muscle. Ann. N.Y. Acad. Sci. 54:649, 1951. 2. Winegrad, A. I., and Renold, A. E.: Studies on rat adipose tissue in vitro. J. Biol. Chem. 233:267, 1958. 3. Jeanrenaud, B. and Renold, A. E.: Studies on rat adipose tissue in vitro. J. Biol. Chem. 234:3082, 1959. 4. Leonards, J. R., and Landau, B. R.: A study on the evidence of metabolic patterns in rat adipose tissue: insulin versus glucose concentration. Arch. Biochem. 91: 194, 1960. 5. Frerichs, H., and Ball, E. G.: Studies on the metabolism of adipose tissue. XI. Activation of phosphorylase by agents which stimulate lipolysis. Biochemistry 1: 501, 1962. 6. Flatt, J. P., and Ball, E. G.: Studies on the metabolism of adipose tissue. J. Biol. Chem. 239:675, 1964.
7. Flatt, J. P., and Ball, E. G.: Studies on the metabolism of adipose tissue. J. Biol. Chem. 241:2862, 1966. 8. Rognstad, R., and Katz, J.: The balance of pyridine nucleotides and ATP in adipose tissue. Proc. Nat. Acad. Sci. U.S.A. 55: 1148, 1966. 9. Halperin M. L., and Denton, R. M.: Regulation of glycolysis and L-glycerol 3phosphate concentration in rat epididymal adipose tissue in vitro. Biochem. J. 113:207, 1969. 10. Kneer, P., and Ball, E. G.: Studies on the metabolism of adipose tissue. J. Biol. Chem. 243:2863, 1968. 11. Schmidt, K., and Katz, J.: Metabolism of pyruvate and L-lactate by rat adipose tissue. J. Biol. Chem. 244:2125, 1969. 12. Reshef, L., Hanson, R. W., and Ballard, F. J.: Glyceride-glycerol synthesis from pyruvate. J. Biol. Chem. 244:1994, 1969.
86 13. Halperin, M. L., and Robinson, B. H.: The role of the cytoplasmic redox potential in the control of fatty acid synthesis from glucose, pyruvate and lactate in white adipose tissue. Biochem. J. 116:235, 1970. 14. DelBoca, J., and Flatt, J. P.: Fatty acid synthesis from glucose and acetate and the control of lipogenesis in adipose tissue. Europ. J. Biochem. 11:127, 1969. 15’. Rodbell, M.: Metabolism of isolated fat cells. J. Biol. Chem. 239:375, 1964. 16. Robinson, B. H., and Halperin, M. L.: Transport of reduced nicotinamide-adenine dinucleotide into mitochondria of rat white adipose tissue. Biochem. J. 116:229, 1970. 17. Borst, P.: Functionelle und Morphologische Organisation der Zelle. Wurzburg:
HALPERIN
AND ROBINSON
Springer-Verlag. Hydrogen transport and transport metabolites. 1963, p. 137. 18. Lehninger, A. L.: Phosphorylation coupled to oxidation of dihydrodiphosphopyridine nucleotide. J. Biol. Chem. 190:345, 1951. 19. Halperin, M. L.: An additional role for insulin in the control of fatty acid synthesis independent of glucose transport. Canad. J. Biochem. 48:1228, 1970. 20. Fain, J. N.: Effect of puromycin on incubated adipose tissue and its response to dexamethasone, insulin and epinephrine. Biochim. Biophys. Acta 84:636, 1964. 21. Jungas, R. L.: Effects of insulin and epinephrine on pyruvate dehydrogenase in adipose tissue. Fed. Proc. 29:981, 1970.