TIBS - February 1979
This work was supported by the Ministry of Education of Japan and the Yamada Science Foundation.
The role of insulin in fatty acid nietaholism
Boyer, P. D., Chance,
B., Ernster, L., Mitchell, P., Racker, E. and Slater, E. C. (1977) Annu. Rev. Biochem. 46, 955-1026 2 Hille, B. (1976) Annu. Rev. Physiol. 38, 139-159 3 Kagawa, Y. (1976) J. Cell. Physiol. 89, 569-573 4 Yoshida, M., Sone, N., Hirata, H. and Kagawa, Y. (1975) J. Biol. C/rem. 250, 7910-7916 5 Kagawa, Y., Sone, N., Yoshida, M., Hirata, H. and Okamoto, H. (1976) J. Biochem. 80, 141-151 6 Sone, N., Yoshida, M., Hirata, H. and Kagawa, Y. (197.5) J. Biol. Chem. 250, 7917-7923 7 Hirata, H., Sone, N., Yoshida, M. and. Kagawa, Y. (1976) Biochem. Biophys. Res Commun. 69, 665-671 8 [email protected]
M. F. and Raidt, H. (1975) Nature (London) 255, 256-259 9 Nelson, N. (1976) Biochim. Biophys. Acta 456, 314-338 10 Verschoor, G. J., Van der Sluis, P. R. and Slater, E. C. (1977) Biochim. Biophys. Acto 462, 438-449 11 Baird, B. A. and Hammes, G. G. (1976) J. Biol. Chem. 251, 6953-6962 12 Pedersen, P. L. (1975) J. Bioenergerics 6, 243-275 13 Bragg, P. D. and Hou, C. (1975) Arch. Biochem. Biophys. 167, 3 1l-321 14 Kagawa, Y. (1974) in Methods in Membrane Bmlogy (Korn, E. D., ed.), Vol. 1, pp. 201-269, Plenum Press, New York 15 Wakabayashi, T., Kubota, M., Yoshida, M. and Kagawa, Y. (1977) J. Mol. Biol. 117,511-519 16 Spitzberg, V. and Haworth, R. (1977) Biochim. Biophys. Acta 492, 237-240 17 Yoshida, M., Sone, N., Hirata, H. and Kagawa, Y. (1977) J. Biol Chem. 252, 3480-3485 18 Yoshida, M., Okamoto, H., Sone. N., Hirata, H. and Kagawa, Y. (1977) Proc. Nat. Acad. Sci. U.S.A. 74, 936940 19 Kagawa, Y. and Ariga, T. (1977) J. Biochem. 81, 1161-1165 20 Hirata, H., Sone, N., Yoshida, M. and Kagawa, Y. (1977) J. Supramolec. Struct. 6, 77-84 21 Okamoto, H., Sone, N., Hirata, H. Yoshida, M. and Kagawa, Y. (1977) J. Biol. Chem. 252, 6125-6131 22 Wachter, E., Sebald, W. and Tzagoloff, A. (1977) in Genetics and Biogenesis of Mitochondria (Bandlow, W., Scheweyen, R. J., Wolf, K. and Kaudewitz, F., eds) pp. 441-449, Walter de Gruyter & Co., Berlin, New York 23 Jagendorf, A. T. and Uribe, E. (1966) Proc. Nat. Acad. Sci. U.S.A. 55, 170-177 24 Racker, E, and Stoeckenius, W. (1974) J. Biol. Chem. 249, 662-663 25 Griffiths, D. E., Hyams, R. L. and Partis, M. D. (1977) FEBS Lett. 78, 155-160 26 Sone, N., Yoshida, M., Hirata, H. and Kagawa, Y. (1977) J. Biol. Chem. 252, 29562960 27 Kagawa, Y., Ohno, K., Yoshida, M., Takeuchi, Y, and Sone, N. (1977) Fed. Proc. 36, 1815-1818
Insulin is now recognized to have several sites of action in the regulation of adipose and liver fatty
ences both the b +-+ a interconversion and enzyme secretion from the adipocyte. Insulin increases the activity of lipoprotein lipase in vitro in tissues from starved rats, and lipolytic hormones oppose this effect . This suggests some form of acute control of the enzyme, but regulation by a protein kinase/phosphatase phosphorylation/dephosphorylation cycle appears unlikely . Provision of fatty acyl precursors for adipocyte triacylglycerol synthesis is thus geared to the secretion of insulin which maintains lipoprotein lipase activity. The provision of glycerol and dihydroxyacetone phosphate as precursors of the triacylglycerol glycerol moiety is also dependent upon insulin since, in rat at least, these are almost entirely derived from glucose. The uptake from glucose from the circulation by the rat adipocyte is promoted very strongly by insulin. This is The balance between triacylglycerol mainly attributable to stimulation of synthesis and mobilization in adipose tissue facilitated diffusion across the plasma Circulating fat destined for uptake and membrane on a specific carrier. Insulin storage by adipocytes is presented as thus primes the adipocyte for triacylchylomicra or very low density lipo- glycerol synthesis by provision of the proteins. Fat leaving adipose tissue is in necessary precursors. The question then the form of non-esterified fatty acids. arises, does insulin or other hormones exert acute control over the enzymatic Triacylglycerol synthesis system responsible for triacylglycerol The activity of lipoprotein lipase in the assembly? Being mindful of analogies capillary bed of adipose tissue appears to between the hormonal control of the determine the rate at which the tissue can glycogen and triacylglycerol mobilizing extract fatty acids from circulating lipo- systems, similar analogies between the proteins. The enzyme has a short half-’ control of glycogen synthesis and fatty life, requiring continuous synthesis by acid esterification might be predicted. adipocytes to maintain the levels of When rat adipocytes are incubated with activity observed in the fed state. After palmitate and [14C]fructose as precursors, food deprivation, lipoprotein lipase insulin appears to redirect adipocyte activity is considerably decreased. Re- metabolism of fructose to favour glyceride establishment of activity on refeeding synthesis . Furthermore, relatively brief correlates closely with plasma insulin [I]. exposure of rat adipocytes to catecholWhen refeeding is commenced after amine lipolytic hormones results in det starvation, two forms of the enzyme are creased glycerol phosphate acyltransferase seen in adipose tissue; an b form which and phosphatidate phosphohydrolase is extracellular and a b form which‘appears activities 16, 71. Insulin antagonizes these to be the intracellular precursor of a . actions (Sooranna, Cheng and Saggerson, It has been proposed that insulin influ- unpublished). Thus, it is possible that these enzymes might exist in forms that David Saggerson is at the Department of Bioare interconvertible by hormones. Much chemistry, University College London, Gower further work is necessary to clarify this. Street, London WCIE 6BT, U.K. @Elsevter/North-HollandBiomedicalPress 1979
That insulin plays a major role in the regulation of carbohydrate metabolism is long established. In this short review I have outlined some of our developing knowledge about the actions of this hormone in controlling the metabolism of lipids and the way in which insulin influences the interplay between carbohydrate and lipid metabolism. Some mention of glucagon is bound to enter the discussion since many investigators would support the concept of glucagon and insulin acting as mutual antagonists in bihormonal regulation of many processes in addition to blood glucose homeostasis. For lack of sufficient information from other species, virtually all the arguments advanced refer to the rat. Furthermore, to keep the article concise, I have restricted my attention to adipose tissue and the liver.
34 Triacylgl”ycerol mobilization Triacylglycerol mobilization is attributed to the intracellular ‘hormonesensitive lipase’ which may actually encompass triacylglycerol, diacylglycerol, monoacylglycerol and cholesterol ester hydrolase activities in the same enzyme . The properties of hormone-sensitive lipase have been extensively reviewed recently . In rat adipose tissue a number of hormones can activate the lipase. These include the catecholamines (acting through /3-receptors), glucagon and corticotropin. The most likely activation pathcyclase stimulation, way is adenylate intracellular cyclic AMP accumulation, of cyclic AMP-dependent activation protein kinase followed by phosphorylation and activation of the lipase. Insulin is able to antagonize the lipolytic action of these hormones extremely rapidly and it was established quite early that this antilipolytic action of insulin, to which the tissue is exquisitely sensitive, can be separated from the action of the hormone on glucose transport. Non-esterified fatty acid release from incubated adipose tissue is suppressed by insulin even in the absence of glucose. Although under certain conditions, insulin can suppress lipolytic hormone-induced increases in adipocyte cyclic AMP, it now seems untenable to propose this as the sole mechanism for the antilipolytic action ofinsulin [9, lo]. No convincing molecular mechanism to explain this action of insulin has yet emerged.
Fatty acid synthesis in adipose tissue Adipose tissue from rat and some other species synthesizes long-chain fatty acids from carbohydrate precursors. There are various reasons why the body contribution of adipose tissue to this process compared with liver may have been overstated previously. Nevertheless, adipose tissue lipogenesis can be extensive and is also sensitive to insulin. After very short periods of exposure to physiological concentrations of insulin the rate of conversion of [‘“Clglucose into fatty acids by rat adipose tissue is greatly stimulated (lo- to 20-fold increases are quite common). With this precursor, activation of glucose transport into the cell plays an important part in the insulin stimulation. However, insulin effects distal to precursor entry can be seen. Insulin can stimulate [l*C]fructose incorporation into fatty acids without appreciably changing fructose uptake, can increase [14C]pyruvate incorporation into fatty acids, and when 3H,0 is used to
TIBS - February 1979 measure synthesis, insulin stimulation of fatty acid synthesis from endogenous precursors can also be seen. Pyruvate dehydrogenase and acetyl CoA carboxylase have been implicated as sites at which this insulin stimulation is exerted (see [l l] for a recent review). Both enzymes can exist in two interconvertible forms. Pyruvate dehydrogenase is subject to control by a phosphorylation/dephosphorylation covalent modification cycle with the dephosphoenzyme being ,the active form (PDH,). Acetyl CoA carboxylase is only active when its protomers (about 4 x IO5 daltons) combine into long filamentous polymers (about 8 x lo6 daltons). After exposure of adipose tissue pieces to insulin the proportion of both these enzymes in their respective active forms is increased. Again, the molecular mechanisms underlying these effects of insulin are not established.
Hepatic fatty acid synthesis This process is subject to acute control by blood-borne substrates and by direct actions of hormones on the liver. In addition, a number of lipogenic enzymes show considerable adaptation when the dietary or hormonal balance is changed. It is now known that blood glucose is not readily used as a direct precursor for hepatic fatty acid synthesis. Lactate and some amino acids may be much better in this respect [ 12,131. Furthermore, l*Clabel from exogenous substrates is extensively diluted by endogenous substrate carbon, probably from glycogen, which makes a substantial contribution to carbon. flow into fatty acids. For this reason it is essential to quantitate hepatic fatty acid synthesis by measurement of 3H,0 incorporation. Results obtained by any other method should be treated with caution. Regulation by blood-borne substrates Various laboratories have reported inhibition of fatty acid synthesis in hepatocytes or perfused liver when extracellular non-esterified fatty acid concentrations are raised. In viva this would normally be secondary to changes in adipose tissue metabolism discussed above. Although the inhibitory effects of different fatty acids correlate well with the effectiveness of their respective CoA esters as inhibitors of acetyl CoA carboxylase , measurements of the total tissue levels of fatty acyl CoA do not generally support this as being the mechanism of the inhibition. However, interpretation of these measurements is different because
of the likely intracellular compartmentation and binding of the fatty acyl CoA. The proportion of liver pyruvate dehydrogenase in the active form is inversely related to plasma non-esterified fatty acid concentration [15,16]. Glucose appears to exert a regulatory or ‘initiating’ role in hepatic fatty acid synthesis  over and above its limited role as a direct precursor. In addition the content of liver glycogen, which relates to the insulin status, also appears to exert a controlling influence on fatty acid synthesis . These effects are not understood. Acute eflects of hormones AS early as 1948 Bloch and Kramer reported that insulin increased the incorporation of [14C]acetate into fatty acids in liver slices. Many subsequent studies have confirmed this finding and have shown inhibition of the process by glucagon. Interpretation of these findings is problematical. It is difficult at present to assess whether insulin really has a direct stimulatory action on hepatic fatty acid synthesis when this is measured by 3H,0 incorporation. Topping and Mayes  found that insulin antagonized the inhibitory effects of non-esterified fatty acids on the process. Jeanrenaud et al.  have observed a rapid stimulatory effect of insulin but this could only be seen in livers taken from anti-insulin serumtreated animals. Inhibitory effects of glucagon  or dibutyryl cyclic AMP on 3H,0 incorporation appear to have been more widely observed. It remains to be fully established whether or not insulin and glucagon have rapid, mutually antagonistic, actions on this process. The proportion of pyruvate dehydrogenase in the active .form is increased by insulin in perfused liver [ 161. Heparic acetyl CoA carboxylase can be phosphorylated by an endogenous protein kinase  and incorporation of [32P]phosphate from [Y-~~P]ATP is accompanied by loss of activity. Acute inactivation of the enzyme by glucagon, adrenaline or angiotensin II has been reported, but it is unknown whether these effects are due to phosphorylation or depolymerization. Unlike adipose tissue, no activation of the liver enzyme by insulin has been reported. In short, our knowledge of acute regulation of hepatic fatty acid synthesis is surprisingly sparse. Adaptation of lipogenic enzymes of
In starvation or diabetes the activities a ‘lipogenic set’ of enzymes are
TIBS - February 1979
strikingly decreased and this is corrected by refeeding or insulin therapy respectively. Immunotitration of rat-liver extracts with specific antibodies has established that these changes in fatty acid synthetase, acetyl CoA carboxylase NADP-malate dehydrogenase, ATP-citrate lyase and the pentose phosphate pathway dehydrogenases are due to changes in the amount of enzyme protein. In several cases [‘“Clleucine labelling experiments in vivo have further characterized these changes as being due to altered rates of enzyme synthesis. It is of course uncertain whether these changes themselves influence the capacity of the liver to synthesize fatty acids or are secondary to preceding acute controls. It’has been suggested that the amounts of these enzymes in rat liver are controlled by the relative concentrations of glucagon and insulin [21,22] since insulin is necessary to permit re-establishment of lipogenic enzyme levels on refeeding and glucagon antagonizes restoration. Other workers though, have provided evidence that other factors, perhaps the levels of hepatic carbohydrate metabolites, may also contribute to the restoration of these enzymes after starvation or diabetes. Studies with hepatic cells in tissue culture are presently under way in several laboratories to establish more clearly the hormonal and metabolite requirements for induction and repression of these e-nzymes. /
carnitine acyltransferase reactions. These are implicated since treatments which bring about a ketogenic profile in the liver increase ketone body formation from oleate or (-)octanoylcarnitine but not from octanoate. Also carnitine acyltransferase inhibitors reverse the pattern of increased fatty acid oxidation and decreased esterification observed in a ketotic liver. The carnitine content of ketotic liver is increased for reasons that are unclear at present. Presumably this facilitates the carnitine acyltransferase reaction. Recently  physiological concentrations .of malonyl CoA were found to inhibit carnitine acyltransferase in rat liver. This may represent an important metabolic signal since in turn, glucagon decreases malonly CoA content of hepatocytes as well as decreasing fatty acid synthesis [ 191. Two factors link hepatic ketogenesis and fatty acid synthesis in an inverse relationship. These are malonyl CoA and glycogen. The rate of fatty acid synthesis is essentially linearly correlated to both, whereas ketogenesis increases as glycogen decreases . In some cases direct effects of insulin to decrease, and glucagon to increase, ketogenesis in the perfused rat liver have been observed. Whether these effects relate to the changes caused by hormonal manipulation in vivo remains to be established.
Hepatic‘ triacylglycerol secretion
A fuller review of this area may be found in . All ketotic states are characterized by some deficiency of insulin. Any sustained increase in ketone body formation by the liver requires increased delivery of non-esterified fatty acids from adipose tissue (see section on adipose tissue lipolysis). However, it is now recognized that there must be a’ simultaneous alteration in liver metabolism to allow this extra fatty acid to be converted into ketone bodies. This is seen with perfused livers isolated from starved or diabetic rats in which ketogenesis supported by a given fatty acid load is greatly increased. Furthermore, this change can be rapidly produced by treatment of animals with anti-insulin serum or glucagon . Earlier suggestions that ketogenesis was controlled in a manner secondary to fatty acid esterification, perhaps by modulation of the glycerol phosphate concentration, have given way to the proposal that major control over ketogenesis is exerted in the oxidative sequence itself, probably at the
An understanding of insulin control of these processes in vivo is complicated by the fact that some degree of supression in insulin-deficient states may be accompanied by decreased peripheral utilization of plasma triacylglycerol and increased provision to the liver of non-esterified fatty acids, the esterification precursor. This frequently results in triacylglycerol accumulation in the liver in starvation and diabetes. Perfused livers taken from starved, diabetic or anti-insulin serumtreated rats show a diminished secretion of lipoprotein triacylglycerol. This is a complex multistage process and at present there is no clear view as to which steps are altered in these conditions. A direct effect of insulin to enhance very low density lipoprotein triacylglycerol synthesis and secretion by the perfused liver has been reported . Conversely glucagon and dibutyryl cyclic AMP inhibit triacylglycerol output : Again at present this is a poorly understood area where much work is expected in the future.
This is by no means an exhaustive accound of lipid metabolic processes that are influenced by insulin. The list of effects of the hormone is continually growing. As for all other actions of insulin, there is at present no mechanism that is described in molecular terms. References 1 Cryer, A., Riley, S. E., Williams, E. R. and Robinson, D. S. (1974) Biochem. J. 140, 561-563. 2 Nilsson-Ehle, P., Garfinkel, A. S. and Schotz, M. S. (1976) Biochim. Biophys. Actu 431, 147-156. 3 Davies, P., Cryer, A. and Robinson, D. S. (1974) FEBS Lett. 45, 271-275 4 Khoo, J. C., Steinberg, D., Huang, J. J. and Vagelos, P. R. (1976) J. Biol. Chem. 251, 2882-2890 5 Sooranna, S. R. and Saggerson, E. D. (1975) Biochem. J. 150, 441-451 6 Sooranna, S. R. and Saggerson, E. D. (1976) FEBS Lett. 64, 36-39 7 Cheng, C. H. K. and Saggerson, E. D. (1978) FEBS Lett. 87, 65-68 8 Steinberg, D. (1976) Adv. Cyclic Nucleotide Res. 7, 157-198 9 Fain, J. N. and Rosenberg, L. (1972) Diabetes 21, Suppl. 2, 414427 10 Siddle, K. and Hales, C. N. (1974) Biochem. J. 142, 97-103 11 Denton, R. M., Bridges, B., Brownsey, R., Evans, G., Hughes, W. and Stansbie, D. (1977) Biochem. Sot. Trans. 5, 894-900 12 Salmon, D. M. W., Bowen, N. L. and Hems, D. A. (1974) Biochem. J. 142, 611-618 13 Clark, D. G., Rognstad, R. and Katz, J. (1974) J. Biol. Chem. 249, 2018-2036 14 Nilsson, A., Sundler, R. and Akesson, B. (1974) FEBS Lett. 45, 282-285 15 Wieland, 0. H., Patzelt, C. and Loffler, G. (1972) Eur. J. Biochem. 26, 426-433 16 Topping, D. L., Goheer, M. A., Coore, H. G. and Mayes, P. A. (1977) Biochem. Sot. Trans. 5, 1000-1001 17 Topping, D. L. and Mayes, P. A. (1976) Biochem. Sot. Trans. 4, 717 18 Assimacopoulos-Jeannet, F., Karakash, C., Lemarchand, Y. and Jeanrenaud, B. (1977) Biochim. Biophys. Acta 498, 91-101 19 Cook, G. A., Nielsen, R. C., Hawkins, R. A., Mehlman, M. A., Lakshmanan, M. R. and Veech, R. L. (1977) J. Biol. Chem. 252, 4421-4424 20 Lee, K. H. and Kim, K. H. (1977) J. Biol. Chem. 252, 1748-1751 21 Lakshmanan, M. R., Nepokroeff, C. M. and Porter, J. W. (1972) Proc. Nat. Acad. Sci. U.S.A.. 69, 35163519 22 Nepokroeff, C. M., Lakshmanan, M. R., Ness, G. C., Muesing, A., Kleinsek, A. and Porter, J. W. (1974) Arch. Biochem. Biophys. 162, 340-344 23 Mc.Garry, J. D. and Foster, D. W. (1977) Arch. Intern. Med. 137, 495-501 24 Mc.Garry, J. D., Wright, P. H. and Foster, D. W. (1975) J. Clin. Invest. 55, 1202-1209 25 Mc.Garry, J. D., Mannaerts, G. P. and Foster, D. W. (1977) J. Clin. Invest. 60, 265-270 26 Topping, D. L. and Mayes, P. A. (1972) Biochem. J. 126, 295-311 27 Heimberg, M., Weinstein, I. and Kohout, M. (1969) J. Biol. Chem. 244, 5131-5139