Intracellular Mediators of Insulin Action

Intracellular Mediators of Insulin Action

W . Kirby Gottschalk and Leonard Jarett

University of Pennsylvania School of Medicine. Department of Pathology and Laboratory Medicine

I . Introduction ................................................................. I1 . The First Direct Evidence for Mediators of Insulin Action ................... 111 . Tissue Sources of Insulin Mediators .........................................

A . Plasma Membrane Sources .............................................. 8 . Whole Cells and Intact Tissue ...........................................

2 . Muscle ................................................................ 3 . Liver ..................................................................

IV . Enzymes Modulated by Insulin Mediators ................................... A . Glycogen Synthase ...................................................... B . Pyruvate Dehydrogenase ................................................ C . Glucose-6-Phosphatase .................................................. D . Phospholipid Methyitransferase ......................................... E . Acetyl-CoA Carboxylase ................................................. F . (Ca" + Mg2')-ATPase . ................................................ G . CAMP-Dependent Protein Kinase ........................................ H . Enzymes of cAMP Metabolism ..........................................

2 . Low-K, cAMP Phosphodiesterase .................................... V . Evidence for a Family of Mediators of Insulin Action ........................

VI . Physicochemical Properties of Insulin Mediators ............................ VII . Physiologic and Pathophysiologic Roles of Insulin Mediators ...............

VIII . Conclusion ..................................................................

1 . Adipocytes ...........................................................

1 . Adenylate Cyclase ....................................................

229 231 233 233 236 236 236 237 240 240 241 242 242 243 243 244 244 244 245 245 248 251 252

I . INTRODUCTION

Insulin. the major anabolic hormone of the body. has pleiotropic effects on cellular metabolism . Within seconds of interacting with its receptor. insulin promotes changes at the plasma membrane that result in increased transport of carbohydrates. amino acids. fatty acids. and ions; within minutes. it redirects metabolism by activating anabolic pathways and inhibiting catabolic events; and. over a period of hours. it exerts control over nuclear events resulting in

Diabetes/Metabolism Reviews. Vol . 1. No . 3. 229-259 (1985) 0 1985 by John Wiley & Sons. Inc . CCC 0742-4221/85/03229-31$04.00

230 INSULIN MEDIATORS

increased cell growth. Despite much intense effort spanning nearly the 60 years since insulin’s discovery, a detailed picture of the mechanisms of insulin action has yet.to emerge.

One early theory of insulin action proposed that insulin affected metabolism by interacting directly with intracellular enzymes.’ By the late 1940s, however, attention was directed at events occurring at the plasma membrane by the discoveries that (1) radioactively labeled insulin became attached to the surface of sensitive cells2z3 and (2) insulin promoted an increased rate of hexose transport into many sensitive cells.44 As a result of these discoveries, it was proposed that most of the actions of insulin on metabolism were consequences of the hormone’s effects on membrane transport systems. However, this unitarian model of insulin action became untenable after the discovery by Villar-Palasi and Larner that the effects of insulin on glycogen synthase could be dissociated from the hormone’s effects on glucose ~ p t a k e . ~

Conclusive proof that the binding of insulin to its receptor initiates the cellular response to insulin was adduced by Freychet et a1.,8,9 who developed techniques of labeling insulin to high specific activity and methodology to study the specific binding of ligands to their receptors. A variety of signals, including membrane hyperpolarization, cyclic nucleotides, ions, and hydrogen peroxide, have been proposed to mediate the effects of insulin. None of these, however, has been found to mimic insulin’s pleiotropic effects on both intact cells and in broken cell preparations, and, thus, none are likely to represent physiologicalIy relevant insulin second messengers.

Recently, attention has focused on a family of low-molecular-weight substances whose synthesis and/or release is enhanced by the interaction of insulin with its receptors on the surfaces of sensitive cells. Larner first discussed the theoretical implications of intracellular mediators of insulin action, lo and, with his co-workers, was the first to identify material with the biochemical properties of an insulin mediator in muscle extracts. At about the same time, but independently of Larner, Turakulov et al. l1 partially purified a low-molecular-weight material from livers of insulin-treated rats that regulated mitochondria1 Ca’+ transport. Also working independently of Larner, Jarett’s laboratory was the first to directly demonstrate the production by insulin of a mediator from the plasma membrane: they showed, in a subcellular system, that the interaction of insulin with its receptor on isolated plasma membranes brought about the generation and/or release of a low-molecular-weight material that could activate pyruvate dehydrogenase in isolated mitochondria. l2

The remainder of this review will focus on six topics. First, the initial experiments in which the mediators of insulin action were first detected will be discussed. Next the variety of tissue sources from which mediators have been obtained will be reviewed, followed by a description of the insulin-sensitive enzymes whose activities are modulated by the mediators. Fourth, evidence will be presented which clearly demonstrates that the second messengers of insulin action are a family of closely related molecules, not just a single entity. Fifth, the physicochemical properties of the mediators, as they are presently known, are summarized. Finally, the physiologic and pathologic importance of these mediators is discussed.

GOTTSCHALK AND JARETT 231

11. THE FIRST DIRECT EVIDENCE FOR MEDIATORS OF INSULIN ACTION

Larner’s proposal for an intracellular mediator of insulin action was based on several findings. First, Villar-Palasi and Larner showed that glycogen synthase could be activated by insulin in the absence of glucose in the media, that is, without concurrently stimulating the transport system and without, therefore, increasing intracellular glucose levels, which could ”push” the glycogen synthase r e a ~ t i o n . ~ , ~ ~ , ’ ~ These independent effects of insulin on two insulin- sensitive events could be explained by an insulin-dependent mediator.” In addition, Larner took note of the facts that (1) insulin inactivated CAMP-dependent protein kinase and (2) basal cAMP levels were not detectably affected by insulin, while it blocked the increase of cAMP elicited by glycogenolytic hormones. Larner suggested that these observations could be explained by the insulin- promoted generation of an intracellular messenger that was distinct from cAMP but interconvertible with it. lo As initially envisioned, this intermediate would block the activation of CAMP-dependent protein kinase by preventing increases in cAMP levels promoted by glycogenolytic hormones, as well as by acting as a sink for cAMP formed under basal conditions. In fact, it was later found that insulin inactivated the CAMP-dependent protein kinase both by promoting the formation of the holoenzyme (inactive) form and by desensitizing the holoenzyme to stimulation by CAMP.'^,'^

Larner’s laboratory provided the first direct experimental evidence for the putative intracellular messenger of insulin action. Larner et al. extracted from skeletal muscle of insulin-treated rabbits a low-molecular-weight, heat- and acid- stable material that inhibited CAMP-dependent protein kinase.17 This material was shown to be distinct from a cAMP antagonist18 by chromatography on Dowex-1, and, although it co-eluted from this column with cGMP, the protein kinase inhibitory material was much less stable than the cyclic nucleotide at pH 7, and was therefore distinct from it. In addition, unlike the protein kinase inhibitor that had previously been isolated in Kreb’s laboratory,” the inhibitor identified by Larner et al. was soluble in TCA and its amounts or activity were increased by insulin treatment of the rabbits before extraction. When the muscle extracts were subjected to paper chromatography to remove nucleotides and then sized on gel filtration columns, material in the same column fraction that possessed the protein kinase inhibitory material also activated glycogen synthase. 2o

Evidence was first provided by Jarett’s laboratory that insulin mediators are generated and/or released by the plasma membrane subsequent to the interaction of insulin with its receptor. Jarett and Smith developed the first plasma membrane system that responded directly to insulin in vitro.21 They showed that insulin enhanced the magnesium-dependent hydrolysis of ATP by isolated adipocyte plasma membranes. Since protein phosphorylation, especially of insulin-sensitive enzymes, plays an important role in intracellular regulation,22 the effects of insulin on the phosphorylation of adipocyte plasma membranes were examined. The addition of insulin to assay mixtures containing plasma membranes and [y-32P]ATP inhibited phosphorylation of membranes, in a con-


centration-dependent manner, compared with control mixtures.23 The effect was specific for insulin and was detected as early as 30 s after the addition of insulin.

These results were examined further by using SDS-PAGE to analyze the phosphoproteins of the labeled membrane^.^^,^^ Five phosphorylated bands, three minor bands, and two major bands of 120,000 and 42,000 daltons, re- spectively, were identified. Insulin promoted the decreased phosphorylation of the two major bands, and this decrease entirely accounted for the decreases observed with the intact membranes. The identity of the 120,000-dalton band has not been established to date, but the 42,000-dalton band was shown to be the a-subunit of pyruvate dehydrogenase (PDH), which was contained in mitochondria that contaminated the plasma membrane preparations to the extent of 3-10%. Insulin had no effect on the phosphorylation of the 42,000-dalton band when added to mitochondria alone, but the hormone’s effect was restored when plasma membranes were added back to the system. These findings suggested that the effects of insulin on the 42,000-dalton band were secondary to its interaction with the insulin receptor on the plasma membrane, resulting in the generation and/or release of a mediator that affected the a-subunit in mitochondria. In a recent report, Akers and Routtenberg26 showed that the addition of insulin to homogenates of rat hippocampal tissue promoted the dephosphorylation of the a-subunit of pyruvate dehydrogenase.

The effect of insulin in these subcellular systems is similar to its effects in vivo. Insulin treatment of rat adipocytes decreased phosphorylation of the a- subunit of the PDH, and this dephosphorylation was correlated with increased PDH It had been shown previously that PDH is a phosphoprotein whose activity is inhibited by increased phosphorylation, and conversely is stimulated by depho~phorylat ion.~~,~~ These results, together with the in vitro results from Jarett’s laboratory, suggested that the material whose generation/ release from the membrane was promoted by insulin might mediate insulin’s effects on the PDH complex and stimulate PDH activity concomitantly with promoting its dephosphorylation.

Seals and Jarett” showed that the addition of insulin to mixtures of adipocyte plasma membrane and mitochondria activated the PDH, compared with control mixtures receiving BSA in place of insulin. The concentration-effect curve of insulin action was biphasic, increasing over the concentration range 50-100 p,U/ml, and decreasing from 100 to 250 p,U/ml. The range of insulin’s effectiveness was similar to the physiologic range of insulin action on fat cell metabolism. The effect of insulin was specific for the intact hormone, since neither boiled insulin nor the desoctapeptide of insulin had any detectable effect on the enzyme when they were tested at the concentration at which native insulin was maximally active. The insulin-mimetic agents Concanavalin-A and anti-insulin receptor antibody also stimulated the PDH in dose-dependent, biphasic manners. None of the active agents had any detectable effects on PDH activity when they were added directly to mitochondria in the absence of plasma membrane. In addition, Seals and Czech33 and Kiechle et a1.34 showed that material which stimulated PDH activity could be recovered in the supernatant fraction after treating adipocyte plasma membranes with insulin and subsequently removing the membranes by centrifugation. This ruled out the possi-


bility that direct contact between mitochondria and plasma membranes in the presence of insulin was necessary for the insulin effect.

In collaboration with Larner’s group, Jarett and Seals35 found that the material extracted from rabbit skeletal muscle activated PDH in isolated adipocyte mitochondria without added plasma membranes, and that extracts from insulin- treated animals activated the enzyme to a greater extent than did extracts from control animals. These results suggested that the extracts of insulin-treated muscle contained an active principle that was similar to, if not identical with, the mediator generated by interaction of insulin with its receptors on the adipocyte plasma membrane.

Thus, by the early 1980s, nearly 60 years after the first reported isolation of insulin in homogenous form, evidence adduced in two laboratories, working independently of each other and following different experimental approaches, showed that insulin elicits at least some of its biological effects by promoting the generationhelease of unique intracellular mediator(s).

111. TISSUE SOURCES OF INSULIN MEDIATORS

Insulin mediators have been detected from a variety of sources, including plasma membranes from insulin-sensitive cells, whole cells, and intact tissue from insulin-treated animals. A number of enzymes and metabolic systems have been employed to detect mediators in these studies, including glycogen-synthase D phosphatase, CAMP-dependent protein kinase, pyruvate dehydrogenase phosphatase, adenylate cyclase, phospholipid methyltransferase, acetyl-CoA carboxylase, low-K, CAMP phosphodiesterase, glucose-6-phosphatase, anti- lipolysis, and lipogenesis. The manner in which insulin mediators affect these enzymes is discussed in greater detail in section IV.

A. Plasma Membrane Sources

Plasma membrane preparations have been employed as convenient sources of small quantities of insulin mediators in a number of laboratories. Table I.A. summarizes the membrane systems that have been used to generate mediators of insulin action and the enzymes whose activities these mediators have been found to modulate.

Preliminary characterization of both the generation of mediator activity and certain of the mediator’s physicochemical properties were conducted in two laboratories, both using rat adipocyte membranes. Kiechle et al.34 found that mediator activity was spontaneously released from adipocyte membranes that had been prepared in Tris buffer, and more activity, as judged by the stimulation of PDH, was released by membranes prepared in Tris buffers containing EDTA or EGTA than in Tris buffers alone. The mediator-activity content of the Tris- treated membranes could be depleted by repeated incubation and centrifugation of the membranes. The material release from Tris-treated membranes resulted in a linear dose-response curve on PDH activity, and was stable at 4°C for at least 16 h at pH 7.0 but not pH 7.4. It eluted as a single, low-molecular-weight peak during chromatography on Sephadex G-15 or G-25.


Table I. Sources of Insulin Mediator Enzymes and Metabolic

Source System Affected Reference A. Membrane sources Rat adipocyte PDH

LOW-K, PDE' (Ca2 + + Mg2 + )-ATPase Lipolysis Lipogenesis Intracellular cAMP accumulation

Adenylate cyclase Acetyl-CoA carboxylase Lipogenesis

Glucose-6-phosphatase

Rat liver PDH

Human placenta PDH

B. Intact cell and tissue sources Rat adipocyte PDH

Rabbit skeletal CAMP-dependent protein kinase muscle Glycogen synthase phosphatase Rat skeletal muscle PDH

LOW-K, PDE*

Adenylate cyclase

Phospholipid methyltransferase Lipolysis

Adenylate cyclase

Adenylate cyclase Phospholipid methyltransferase Mitochondria1 Ca2+ transport

Adenylate cyclase

Adenylate cyclase

LOW-K, PDE'

Rat heart PDH

Rat liver PDH

Rat hepatocytes PDH

Hepatoma cells PDH

LOW-K, PDE* Human monocytes PDH

IM-9 lymphocytes PDH Glucose-6-phosphatase

Adenylate cyclase

*Low-& cAMP phosphodiesterase. tMacaulay, Kelly, and L. Jarett, unpublished observations. $Parker and L. Jarett, unpublished observations. $Both t and 1 after treatment with dexamethasone.68

T , 1 t T 1 t

.1 t1.l t

T l . 1 1

T, 1 T 1 t T, .1 1 t 1 1 t

t t 1

38 49 96 45 45 45 37 38 41 46 43,44 44

47,48 49 20 20 52, 58 58 58 t t 57 57 58 58 t 11 69 69 67 t 67 44 44 71 fi

Membranes prepared in phosphate buffers did not spontaneously release insulin-dependent mediator, but insulin treatment of such membranes did result in the release of mediator a ~ t i v i t y . ~ ~ , ~ ' These results were confirmed by Kiechle et al. ,34 who also showed that the insulin-elicited material from phosphate-treated membranes eluted in the same position as the material derived from Tris-treated membranes during chromatography on Sephadex G-25. Seals and Czech showed that the mediator generated by insulin from phosphate-


treated membranes was sensitive to treatment with pro tease^,^^ was stable for short times (12 min) at high temperatures (100°C) and under basic conditions (0.1 N NaOH), and bound reversibly to both anion-exchange and hydroxyapatite columns at pH 7.4.36 They also found that mediator generated by super high concentrations of insulin displayed biphasic dose-response effects on pyruvate dehydrogenase activity, consistent with results of Seals and Jarett.I2 This result suggested either that high concentrations of mediator inhibited the PDH, or that insulin elicited the generation of both PDH-inhibiting and PDH-activating activities. The multiplicity of mediator activities generated by the interaction of insulin with plasma membranes will be discussed in section V.

Rat liver membranes have also been used in a number of investigations as sources of insulin mediator activity. Jarett and K i e ~ h l e ~ ~ showed that liver membranes spontaneously released material that activated either liver or adipocyte mitochondria1 pyruvate dehydrogenase. Insulin treatment of a liver particulate fraction in phosphate buffer enhanced the generation of activities that modulate PDH,3840 adenylate c y ~ l a s e , ~ ' , ~ ~ acetyl-CoA c a r b o ~ y l a s e , ~ ~ , ~ ~ and glucose-6- p h ~ s p h a t a s e . ~ ~ Ethanol extraction of the lyophilized supernatant of insulin-treated membranes yielded an ethanol-insoluble fraction that stimulated PDH and acetyl-CoA carboxylase and an ethanol-soluble fraction that inhibited PDH and adenylate c y c l a ~ e ~ ~ and acetyl-CoA carboxyla~e.~~ The PDH-stimulating material was active when either adipocyte or liver mitochondria were used in the assay.41

Placenta is an insulin-responsive tissue, rich in insulin receptors, and thus a potentially useful source of insulin mediators. Two groups have shown that physiologically relevant concentrations of insulin promote the generation of PDH-modulating activity from human placental membranes. Sakamoto et al.43 reported that activity was detectable only after gel filtration of the supernatant fractions of insulin-treated membranes, and was not observed in the crude supernatants before fractionation. No stimulating activity was detected, either before or after chromatography, in supernatant fractions from the control membranes, and only PDH-inhibiting activity was detected after chromatography. This inhibitory material was not characterized further.

Suzuki et al.44 also found PDH-stimulating material from placental membranes. However, in contrast to the results of Sakamoto et al., activity was detected both in the crude supernatant fractions and after gel filtration; no significant amounts of inhibiting material was observed after gel filtration of either control or insulin-treated supernatants. The reason for this discrepancy was not clear. Suzuki et al. also showed that, similarly to PDH mediator from adipocyte membranes,36 the PDH-stimulating material from placental membranes bound reversibly to both anion-exchange and hydroxyapatite chromatographic supports.

Two groups have used assays that are based on intact cells, rather than cell homogenates or partially purified enzymes, to examine the effects of insulin mediators extracted from plasma membranes on metabolic systems, such as lipolysis, which are not completely amenable to in vitro analysis. Zhang et al.45 tested extracts of insulin-treated adipocyte plasma membranes for their ability to mimic insulin action on intact adipocytes. Before testing, insulin was removed from the extracts by gel filtration chromatography. The extracts inhibited both basal and hormone (glucagon + forsko1in)-stimulated CAMP accumulation. The


basis of this action is not understood, however, since the extract had no effects on either adenylate cyclase or low-K, CAMP phosphodiesterase in broken cell preparations. The extracts also inhibited both basal and hormone-stimulated lipolysis and stimulated lipogenesis in a dose-dependent manner. Whether or not all three biological effects are due to the same substances awaits further purification.

An insulin-dependent lipogenic activity was also recovered from a liver particulate fraction by Car0 et a1.& These workers tested for lipogenesis using intact cultured hepatocytes, and they found that, in addition to lipogenesis, activity in the supernatant fraction also promoted insulin-receptor downregulation. These biological effects were not due to contaminating insulin in the supernatant fraction, since no insulin was detected in the supernatants by RIA, or when 1251-insulin was used to generate the mediator substance. The active principles have not been characterized further, and its (their) relationship to other mediator activities, especially the lipogenic activity reported by Zhang et al.,45 awaits purification and chemical characterization of the respective active materials.

Plasma membranes from both liver and adipocytes have been used extensively as sources of insulin mediators in studies examining the physiologic roles played by the mediators. These results are reviewed in section VIII. Most of the physicochemical properties of the insulin mediators have been determined using material generated from plasma membranes, and these results are summarized in section VI.

B. Whole Cells and Intact Tissue

The presence of mediators in tissue extracts, in quantities that are dependent on the physiologic condition of the tissue, is one proof that these substances subserve biologically important roles. In addition, intact tissues are probably the only sources rich enough to provide the amounts of material that are needed for the full chemical characterization and identification of insulin mediators. Table I.B. summarizes the tissues from which insulin mediators have been extracted and the enzymes whose activities they modulate.

2. Adipocytes Kiechle et al.47 showed that PDH-stimulating mediator activity could be

extracted from intact adipocytes, and that more activity was extractable from insulin-treated cells than from control cells that had been treated with BSA. These results were confirmed by Seals and Czech.48 Kiechle and Jarett49 also demonstrated that an insulin-dependent mediator of low-Y, CAMP phosphodiesterase was extractable from intact adipocytes. This material had no measurable effects on a high-Km CAMP phosphodiesterase from adipocytes, consistent with the reported lack of effect of insulin treatment of intact cells on this enzyme.50

2. Muscle

Lamer et al.17 were the first to demonstrate the presence of insulin mediator activity in insulin-treated tissue. They observed that extracts of skeletal muscle from insulin-treated rabbits inhibited cAMP-dependent protein kinase to a greater extent than muscle extracts from control animals. The crude extract was frac-


tionated by heat denaturation in acid to remove the bulk of the protein, by paper chromatography to remove nucleotides, and then gel filtered on Sephadex G-25.“ Mediator activity eluted from the G-25 column as a single peak, in the molecular-weight range of 1500 daltons. It specifically inhibited CAMP-dependent protein kinase and had no detectable effects on CAMP-independent kinases. Material in this column fraction also stimulated glycogen synthase D phosphatase and pyruvate dehydrogenase.

Recently, by employing combinations of gel filtration and HPLC ion-exchange chromatography, Thompson et al.52 and Cheng et a1.53J03 have reported the purification to near homogeneity of both the CAMP-dependent protein kinase and the PDH-stimulating mediator activities.

Jarett’s laboratory has identified four mediator activities in extracts of rat m ~ s c l e . ~ ” ~ Both skeletal and heart muscle extracts possess PDH-stimulating and adenylate cyclase-inhibiting mediator activities, and the levels of both were enhanced by insulin treatment. Insulin treatment also enhanced the levels in skeletal muscle extracts of both a phospholipid methyltransferase-inhibiting mediator (K. L. Kelly, S. L. Macaulay, and L. Jarett, unpublished observations) and a Iow-K, CAMP phosphodiesterase-stimulating mediator,58 but the effect of heart extracts on these enzymes has not yet been studied. On the basis of dilution assays, heart appears to be the richest source of the PDH and the adenylate cyclase mediators yet examined. Thompson et al.52 and Cheng et al.’03 have also identified a PDH-stimulating mediator in rat skeletal muscle.

The effects of the skeletal muscle extract on adipocyte metabolism have been examined using a whole (intact) cell assay system. Partially purified mediator stimulated PDH activity in a dose-dependent f a ~ h i o n ~ ~ , ~ ~ and, in addition, stimulated glycogen synthase but had no effect on either glucose oxidation or glucose t r a n ~ p o r t . ~ ~ Mediator from skeletal muscle also inhibited isoproterenol- stimulated phospholipid methyltransferase and lipolysis (K. L. Kelly, S. L. Macaulay, and L. Jarett, unpublished observations). Effects on either adenylate cyclase or lipid metabolism have not been examined to date.

3. Liver In a series of studies on the action of insulin on liver mitochondria, Gain-

utdinov et a1.11,6042 reported the presence in liver extracts of a low-molecular- weight regulator of mitochondrial Ca2+ transport. The activity, which stimulated mitochondrial Ca2+ uptake in vitro, was enhanced in liver extracts of insulin-treated rats. In addition to, or perhaps as a result of, its effects on mitochondrial Ca2+ transport, the insulin-dependent activity inhibited epinephrine-stimulated oxidation of pyruvate and succinate in rat liver mitochondria in vitro. It also exhibited a number of in vivo biological effects that are relevant to insulin action, including antagonizing both gluconeogenesis and epinephrine- stimulated glucose release from rat liver slices, and lowering blood glucose levels in rats made hyperglycemic by alloxan treatment. It was partially purified by anion-exchange and gel filtration chromatography and shown to possess the properties of a heat-stable glycopeptide; 43 amino acids were identified after acid hydrolysis of the active material.60 However, no evidence was provided that these amino acids were part of the mediator molecule. Further studies are


required to establish the relationship of this mediator to the other mediators discussed in this review.

A number of researchers have shown that insulin affects nuclear functions. Purrello et al.63 demonstrated that insulin activated a nucleoside triphosphatase located on the nuclear envelope, and further showed that treatment with insulin or insulin-mimetic agents decreased 32P-incorporation into nuclear envelope protein^.^,^^ This may be one way in which insulin regulates mRNA metabolism. In this context, it is of interest that Horvat66 reported that insulin promoted the generation of a low-molecular-weight moiety in liver that stimulated nuclear RNA polymerase activity.

PDH- and low-K, cAMP phosphodiesterase-stimulating and adenylate cyclase- and phospholipid methyltransferase-inhibiting mediator activities have been extracted from rat liver by Macaulay et a1.54,58 (S. L. Macaulay, K. L. Kelly, and L. Jarett, unpublished observations). More activity was recovered from livers of rats treated with insulin than from control rats. On the basis of their behavior during purification, the PDH- and adenylate cyclase-modulating activities appear identical to those extracted from rat skeletal muscle. Parker et al.67 treated H4-11-EC3’ hepatoma cells with insulin and found increased amounts of PDH- and low-K, cAMP phosphodiesterase-stimulating activity. The activities were heat- and acid-stable, and possessed apparent molecular weights of 1000-2000. Similarly to the material extracted from adipocyte membranes,36 the PDH-stimulating mediator extracted from H4-11-EC3’ cells bound reversibly to an anion-exchange resin (Dowex-1). This is in contrast to the behavior of the PDH- and adenylate cyclase-modulating mediators extracted from muscle and intact liver by Macaulay et al., which bound to cation but not anion exchangers,% and may reflect tissue differences in the detailed structures of these mediators.

A strikingly unique feature of the H4-11-EC3‘ cell mediator is that the dose- response curve of insulin-promoted production of mediator activity increased linearly to a plateau level and did not exhibit biphasic behavior. This result has been confirmed by Esau and Koontz,68 who used Reuber H-35 hepatoma cells. In addition, these workers reported that cultures which were treated with dexamethasone before incubation with insulin produced, in an insulin-dependent manner, both PDH-activating and PDH-inhibiting activities. Dexamethasone treatment alone did not result in generation of the inhibiting activity. The implications of these results on the mechanism of generation of mediator activity, and on the multiplicity of mediator activities elicited by insulin, will be discussed in section V.

Saltie140 has also reported the extraction of insulin-dependent PDH- and adenylate cyclase-modulating mediator activities from rat hepatocytes, but not enough information is available to allow a comparison between these materials and those reported by other workers.

A cAMP antagonist, which inhibits both CAMP-dependent protein kinase and adenylate cyclase and activates phosphoprotein phosphatase and PDH, has been identified in rat hepatocyte extracts by W a ~ n e r . ~ ~ The activities were elicited in response to treatment with either epinephrine or insulin, but were not present in extracts of control cells. Pretreatment of rats with indomethacin


resulted in diminished recoveries of the adenylate cyclase inhibitor after epinephrine stimulation of the isolated hepatocytes, but recoveries could be restored to normal levels by the addition of prostaglandin E l (PGE-1) during epinephrine treatment. PGE-1 itself was without effect on adenylate cyclase in these studies, and could be separated from the mediator activity by anion- exchange chromatography. It is possible that PGE-1 either forms an integral part of the mediator or is essential for the generation of the mediators. Was- ner has reported that partially purified mediator contains both prostaglandin and inositol phosphate.70 The active component(s) in Wasner’s preparation differs biologically from the CAMP-lowering activity described by Zhang et al.,45 since the activity described by Zhang et al. was apparent only when intact cells were employed for assay, while Wasner’s cAMP antagonist was report- edly active when either membranes or intact hepatocytes were used. The activity prepared by Wasner also differed chemically from the adenylate cyclase mediator reported by Jarett’s laboratory in that it was extractable into organic solvents whereas the activity prepared by the Jarett group was soluble only in aqueous buffers (unpublished results). Other properties of the cAMP antagonist studied by Wasner differed significantly from those reported by other laboratories in this field, and it appears that this prostaglandinlike material is, in fact, biochemically distinct from the other adenylate cyclase-inhibiting activities discussed in this review.

Human peripheral blood cells are richly endowed with insulin receptors. Since the generation of insulin mediators depends on ligand-receptor interactions, it was expected that these cells might provide an abundant source of insulin-dependent mediator. In addition, knowledge of the mediator generation system from human sources might have application in the study of abnormal conditions in man.

Insulin treatment of IM-9 lymphocytes resulted in decreased PDH-stimulating activity in the cell extract compared with the extracts of control cells.71 Although these results were unexpected, they are consistent with the fact that insulin has no detectable postbinding biological effects on these cells. The further implications of these results will be discussed in section V. In contrast, insulin treatment enhanced the amounts of PDH-stimulating activity that was extracted from human m o n ~ c y t e s . ~ ~ These cells also yielded increased amounts of glucose-6-phosphatase-inhibiting activity after insulin treatment. Both activities are low-molecular-weight moieties (1000-2000 daltons), are heat- and acid- stable, and both bind reversibly to the anion-exchange resin Dowex-1 and to hydroxyapatite in ways reminiscent of the PDH-stimulating activity from adipocyte membrane^.^^

In summary, mediator activity has been extracted from a variety of sources, including plasma membranes, whole cells, and intact tissues, and in each case (with the exception of IM-9 cells) treatment with physiologic concentrations of insulin enhanced the amount or activity of the putative mediators that were extracted. These results substantiated the proposal that insulin promotes the generation and/or release of intracellular messengers, which appear to mediate many of insulin’s effects on metabolism. The physiologic and pathophysiologic importance of these activities are discussed in section VIII.


IV. ENZYMES MODULATED BY INSULIN MEDIATORS

The insulin-sensitive enzymes whose activities are known to be modulated by insulin mediator activities, in ways that mimic the effects of insulin treatment of intact cells or tissues, are listed in TablCII. For some time it had been thought that insulin exerted its effects on metabolism solely by promoting the phosphorylation or dephosphorylation of its target enzymes. In fact, the activities of many insulin-sensitive enzymes are regulated by such means, including glycogen synthase and pyruvate dehydrogenase. However, there are exceptions to this model of insulin action, such as adenylate cyclase, and in such cases, insulin mediators are thought to interact directly with the enzymes and regulate their activities allosterically.

Glycogen Synthase

Insulin treatment of intact cells results in dephosphorylation and, concomitantly, activation of glycogen synthase. This could occur by inhibition of a protein kinase, by activation of the glycogen synthase D phosphatase, or both. Larner et aLZ0 have demonstrated effects of partially purified mediator preparations from skeletal muscle on both CAMP-dependent protein kinase and phosphoprotein phosphatase activities when glycogen synthase was used as the substrate. Mediator effects on CAMP-dependent protein kinase are discussed below. Mediator effects on the glycogen synthase D phosphatase have been directly observed by monitoring the release of 32Pi from labeled phosphorylase-a using purified phosphatase from muscle.53 Mediator activity that stimulated this

Table 11. Insulin

Insulin-Sensitive Enzymes Affected by Mediators in Same Manner as

Effects of Enzyme Source Phosphorylation Dephosphorylation

PDH Rat adipocytes 1 t .1 t Rat liver

Adenylate cyclase Rat adipocytes ? ? Rat liver ? ?

LOW-Y, PDE Rat adipocytes ? ? CAMP-dependent Rabbit muscle ? ? protein hnase

Glycogen synthase Rat adipocytes 1 Glycogen synthase Rat adipocytes ?

Acetyl CoA carboxylase Rat liver t , 1" Glucose-6-phosphatase Rat liver t Phospholipid Rat adipocytes t

phosphatase

methyltransferase

t ?

1 .1

*Glucagon and epinephrine promote increased phosphorylation and concomitant activation; insulin inhibits acetyl-CoA carboxylase and promotes increased phosphorylation at a site that is distinct from those phosphorylated under the influence of glucagon and epinephrine. The role of the insulin-promoted phosphorylation in inactivating the enzyme has not been conclusively established.


phosphatase has been partially p~rified,’~ and by the criteria of gel filtration chromatography it appeared larger than, and therefore distinct from, either the PDH-stimulating or the protein kinase-inhibiting activities.

B. Pyruvate Dehydrogenase

PDH is a hormonally regulated enzyme which catalyzes one of the rate- limiting steps in fatty acid biosynthesis. Numerous laboratories have made ex- tensive use of pyruvate dehydrogenase as a routine assay for the identification and characterization of insulin mediator activity. PDH is a multi-enzyme complex composed of five enzymes: pyruvate decarboxylase, dihydrolipoyl acetyl- transferase, dihydrolipoyl dehydrogenase, a CAMP-independent protein kinase, and a phosphoprotein phosphatase. The activity of the complex is regulated by the extent of phosphorylation of the a-subunit of the decarboxylase, which is determined by the competing effects of the PDH-kinase and the PDH-phosphatase. The dephosphorylated enzyme is the active form both in vitro and in vivo. The decarboxylase may be phosphorylated at any of three serine residues. Although only phosphorylation at the first residue inactivates the enzyme, more heavily phosphorylated forms are reactivated by the phosphatase more slowly than are more lightly phosphorylated forms. This shows that the phosphatase works on all three sites, and the first one phosphorylated is the one that controls the activity of the decarboxylase. The regulatory properties of the PDH complex have been reviewed recently by Reed” and Wieland.73

The insulin mediator could activate the a-subunit by either inhibiting the kinase or by activating the phosphatase. To decide between these possibilities, Popp et al.74 incubated plasma membrane/mitochondria mixtures with insulin or Con-A in the presence of sodium fluoride, a PDH-phosphatase inhibitor,75 or dichloroacetic acid, a PDH-kinase i n h i b i t ~ r . ~ ~ Sodium fluoride completely blocked the ability of either insulin or Con-A to activate PDH activity, whether or not ATP, a substrate for the kinase, was also present in the incubation mixture. Dichloroacetic acid (DCA) alone had no effect on enzyme activity in the absence of ATP, but stimulated activity, compared with controls without the kinase inhibitor, in the presence of ATP. In the absence of ATP, the addition of DCA had no effect on the stimulation of PDH activity by either insulin or Con-A. However, in the presence of added ATP the effects of DCA were addi- tive with those of either ligand. These data showed that stimulation of the a- subunit of pyruvate dehydrogenase by insulin mediator occurred via activation of the phosphoprotein phosphatase, and not by inhibiting the PDH-kinase. Similar studies, leading to the same conclusions, were performed with partially purified mediator preparations from a d i p ~ c y t e s , ~ ~ adipocyte plasma mem- b r a n e ~ , ~ ~ hepatoma cells,67 and human monocytes.44

Macaulay and Jarett77 have demonstrated this effect more directly by using mitochondria that had been prelabeled with [y-32P]ATP. As revealed by SDS- PAGE analysis, the a-subunit of the PDH complex was the only mitochondria1 protein detectably labeled. Insulin mediator, in the absence of added ATP, promoted the dephosphorylation of labeled mitochondria with a time course par- alleling the activation of pyruvate dehydrogenase measured on the same mitochondria. Both dephosphorylation and activation were abolished by so-


dium fluoride. In addition, Newman et al.7R have reported that supernatant fluids from insulin-treated adipocyte membranes stimulated the activity of purified PDH-phosphatase, but had no detectable effect on purified PDH-kinase.

Most routine assays of PDH activity in this and other laboratories have involved the use of broken mitochondria. One criticism of this practice was that such mitochondria did not accurately reflect physiologic conditions, and that the PDH complex was exposed to a number of agents that may artifactually modulate activity. Parker and Jarett54 addressed this problem by treating fully intact, respiring mitochondria with insulin mediators from liver membranes, hepatoma cells, IM-9 lymphocytes, and liver and muscle. In all cases except the IM-9 cells, mediators from insulin-treated sources stimulated PDH to greater extents than mediators from control sources. In addition, Jarett et a1.55,56 have shown that insulin activity from rat skeletal muscle stimulates pyruvate dehydrogenase in intact adipocytes. These results showed that mediators can elicit physiologically relevant responses of PDH in intact systems.

C. Glucose-6-Phosphatase

Glucose-6-phosphatase catalyzes a key reaction in carbohydrate metabolism. Activation of the enzyme increases glucose levels while inhibition leads to increased glucose-6-phosphate levels, which may feed into lipogenesis, among other pathways. Insulin has been shown to inhibit the activity of glucose-6- phosphatase from rat liver micro~omes ,~~ perhaps by promoting its dephosphorylation.80,s1 Suzuki et a1.42,44 have partially purified from human placenta and monocytes and from rat liver an activity that inhibits glucose-6-phosphatase. This mediator activity co-eluted from gel filtration, ion-exchange (Dowex-1), and hydroxyapatite columns with PDH-stimulating mediator activity. These results suggested that the same active principle may be responsible for both inhibition of glucose-6-phosphatase and stimulation of PDH, but this suggestion cannot be ascertained until both mediator substances have been purified and identified.

D. Phospholipid Methyltransferase

Phospholipid methyltransferse (PLMT) is a membrane-bound enzyme, which is ubiquitously distributed in mammalian tissues. It catalyzes the stepwise methylation of phosphatidylethanolamine to phosphatidylcholine in response to receptor-ligand interactions. PMLT appears to be important in the transduction of receptor-mediated signals and/or in mediating cellular responses, although the exact nature of its role in these processes is not understood (for review, see refs. 82 and 83). Glucagon, beta-adrenergic agonists, forskolin, human chorionic gonadotropin (hCG), and adrenocorticotropin (ACTH) stimulated PLMT activity, apparently via a CAMP-dependent process.8446 Partially purified PLMT from rat liver has been phosphorylated and activated in parallel fashion by exoge- nously added CAMP-dependent protein k i n a ~ e . ~ ~ These data are consistent with the proposal that the activity of PLMT may be regulated by phosphorylation- dephosphorylation.


Hormones such as ACTH and epinephrine, whose actions on adipocytes oppose those of insulin, cause a stimulation of adipocyte PLMT in a manner that can be mimicked by added dibutryl CAMP or forskolin. Physiologic concentrations of insulin antagonize the stimulatory effects of ACTH, epinephrine, and forskolin on PLMT, and insulin alone causes a concentration-dependent inhibition of PLMT (K. L. Kelly, E. H. A. Wong, and L. Jarett, submitted for publication). Partially purified preparations of insulin mediator from rat skeletal muscle and liver mimic this insulin effect on intact adipocytes, and more of this inhibitory activity is present in extracts from insulin-treated tissue than in extracts from control tissue. Mediator extracted from skeletal muscle also inhibited isoproterenol-stimulated PLMT in rat adipocyte and liver plasma membranes (K. L. Kelly, S. L. Macaulay, and L. Jarett, unpublished results). These results suggest that the inhibitory effects of insulin on adipocyte PLMT may be via dephosphorylation, mediated by an as yet unidentified insulin mediator.

E. Acetyl-CoA Carboxylase

The activity of acetyl-CoA carboxylase, which catalyzes a rate-limiting step in fatty acid biosynthesis, is hormonally regulated. Glucagon and epinephrine promoted CAMP-dependent phosphorylations of the enzyme, which resulted in its inactivation.88 Insulin activated acetyl-CoA carboxylase and, paradoxically, also promoted the enzyme's phosphorylation, but at a site distinct from those sensitive to glucose or epinephrine and via a CAMP-independent protein ki- n a ~ e . * ~ - ~ l It has not yet been conclusively demonstrated that the phosphorylation of acetyl-CoA carboxylase promoted by i n s u h is causally related to insulin's activation of this enzyme; the relationship between these events is currently under investigation in a number of laboratories.

Saltiel et al.40,41 recovered activities that modulated acetyl-CoA carboxylase in the supernatant fractions from insulin-treated liver particulate fractions. An acetyl-CoA carboxylase-stimulating activity was present in the insoluble residue after extraction of lyophilized supernatant fractions with ethanol, and an inhibiting mediator was detected in the ethanol-soluble portion.

F. (Ca2+ + Mg2+)-ATPase

Treatment of intact adipocytes or isolated adipocyte plasma membranes with physiologic concentrations of insulin resulted in the dephosphorylation of a phosphoprotein of 110,000 MW. This protein has been identified as a high- affinity (Ca2+ + Mg2+)-ATPase,y2 and may be the enzymatic basis for a high- affinity, calmodulin-sensitive plasma membrane Ca2 + -transport ~ y s t e m . ~ ~ , ~ ~ Physiologic concentrations of insulin inhibited both the (Ca2+ + Mg*+)-ATPase and Ca2+-transport in intact adipocytes as well as in isolated adipocyte mem- b r a n e ~ . ~ ~ In contrast, incubation of adipocyte membranes with a crude insulin mediator preparation derived from adipocyte membranes stimulated (Ca2 + + Mg2+)-ATPase fourfold and stimulated Ca2+ transport more than twofold.y6 These opposing effects of insulin and of insulin mediator remain unexplained. They may have been due to contaminants present in the membrane extract, and the effect of insulin mediators on this ATPase needs reinvestigation using insulin mediator preparations of greater purity.


G . CAMP-Dependent Protein Kinase

Larner’s laboratory demonstrated that insulin treatment of intact tissue inhibited CAMP-dependent protein kinase by maintaining the enzyme in its inactive, holoenzyme form and desensitizing it to the effects of CAMP.'^,^^ Larner et al. l7 were also the first to show that extracts of insulin-treated tissue contained an active principle that inhibited this protein kinase, and decreased 13H]cAMP binding to it. They have used this enzyme extensively as the basis of an assay for insulin mediators, and, as discussed above, have substantially purified a mediator activity that appears to inhibit CAMP-dependent protein kinase specifically. CAMP-dependent protein kinase has not been proven to be a phosphoprotein.

H. Enzymes of cAMP Metabolism

It has been extensively documented that insulin treatment lowers hormonally elevated cAMP levels in intact a d i p ~ c y t e s ~ ~ , ~ ~ and other tissues.98 Insulin may bring about this effect by either inhibiting hormone-stimulated cAMP biosynthesis or by enhancing the rate of cAMP degradation. Both adenylate cyclase and low-& cAMP phosphodiesterase activities are modulated by insulin mediators in subcellular systems. In this regard, however, it is important to recall that the CAMP-lowering mediator reported by Zhang et al.45 had no detectable direct effects on either enzyme in cell homogenates.

1. Adenylate Cyclase

Insulin inhibits both basal and hormonally stimulated adenylate cyclase activities in adipocyte and hepatocyte plasma membranes ,99,100 No firm evidence has been provided that adenylate cyclase is regulated by phosphorylation or dephosphorylation.

Saltiel et al.38 identified an inhibitor of adenylate cyclase in lyophilized supernatant fractions from insulin-treated liver particles. The inhibitor was ethanol soluble, and was distinguishable on this basis from an ethanol-insoluble, PDH- stimulating activity that was also present in the crude extract.

Adenylate cyclase inhibitors have also been identified in extracts of insulin- treated rat skeletal m ~ s c l e , ~ ~ , ~ ~ rat liver,58 and rat heart.57 However, significant differences in the gel filtration characteristics of this mediator were reported by Macaulay et a1.57,58 and Cheng et al.53 Macaulay et al. reported that the inhibitor eluted after PDH-stimulating activity during chromatography on Sephadex G- 15, with an apparent molecular weight of 1500, whereas Cheng et al. claim this mediator activity elutes before PDH-stimulating activity, with an apparent molecular weight of 3000-5000, during chromatography on Sephadex G-25. These discrepancies may reflect differences in the column buffers used67 and/or differences in the assay system. The cyclase-inhibiting activity isolated by Jarett’s laboratory also differed from that reported by Saltiel et al.3s in that the former was insoluble in 100% ethanol while the latter was soluble in ethanol.

As summarized above, both W a ~ n e r ~ ~ and Zhang et al.45 have detected activities that lowered hormone-elevated cAMP levels in whole cells. However,


the relationship of these activities with each other and with the adenylate cyclase-inhibiting activities reported by other investigators will not be clear until the respective active principles have been purified and identified.

2. Low-K, C A M P Phosphodiesterase

Insulin treatment of hepa t~cy te s~~ and a d i p o c y t e ~ ~ ~ , ' ~ ~ increased the low- K, cAMP phosphodiesterase activity in isolated microsome fractions and in adipocyte plasma membranes."' A mediator activity that stimulated low-K, cAMP phosphodiesterase has been identified in extracts of rat ad ip~cy tes ,~~ H4-11-EC3' hepatoma cells,67 and rat liver and skeletal muscle,58 as well as the supernatant fractions from rat adipocyte plasma membranes.49 In each case, recoveries of the mediator activity were enhanced in samples from insulin- treated cells or tissue. Low-K, cAMP phosphodiesterase has not been proven to be regulated by phosphorylation or dephosphorylation.

V. EVIDENCE FOR A FAMILY OF MEDIATORS OF INSULIN ACTION

This section summarizes the evidence, often alluded to in the previous discussion, that insulin generates a family of related compounds that mediate many of its actions.

Indirect evidence that insulin elicited the generation of both stimulating and inhibiting mediators was provided by two kinds of experiments. First, the dose-response curves of insulin or of insulin-mimetic agents with regard to mediator production were biphasic when plasma membrans were used to generate the mediators. 12,36*38,39 Second, dilution curves of the membrane supernatant fractions were also biphasic in the assays of PDH.36,39,44 acetyl-CoA carb~xylase,~~ and glucose-6-phosphatase.44 Larner et aL20 also showed that the insulin-dependent mediator from skeletal muscle produced a biphasic dose- response curve in the CAMP-dependent protein kinase assay. The mediator dose-response experiments suggested that the stimulating mediator was more active at low concentrations than was inhibiting activity, or that more stimulating mediator was generatedheleased at lower concentrations, whereas at higher concentrations more of the inhibiting mediator was produced.

The insulin-promoted generation of multiple mediators has been demonstrated more directly by both biological and biophysical means. Cheng et al. were the first to report the separation of stimulating from inhibiting mediator activities.lo2 They fractioned a partially purified mediator preparation into two zones by high-voltage paper electrophoresis. Activity in one zone inhibited CAMP-dependent protein kinase and stimulated glycogen synthase phosphatase, while activity in the other zone had the opposite effects. Larner's laboratory has recently reported the preparation of both the CAMP-dependent protein kinase-inhibiting mediator and the PDH-stimulating mediator.53 In addition, they have reported that the adenylate cyclase-inhibiting and glycogen synthase phosphoprotein phosphatase-stimulating mediator activities can be separated from the PDH and the protein kinase mediators by gel filtration on Sephadex G-25 columns.


Saltiel et al.3s were the first to resolve enzyme-stimulating from enzyme- inhibiting mediator activities by fractional extraction with ethanol. The ethanol- insoluble fraction from insulin-treated rat liver particles monotonically stimulated PDH and acetyl-CoA carboxylase activities, whereas the ethanol-soluble portion monotonically inhibited PDH, adenylate cyclase, and acetyl-CoA carboxylase. When the mediator activities were resolved from each other in this manner, insulin was observed to elicit the generation of both the PDH-stimulating and -inhibiting activities in a dose-dependent manner.

Macaulay et a1.57,58 have separated PDH-stimulating activity from adenylate cyclase-inhibiting activity by gel filtration. However, the adenylate cyclase mediator, whether extracted from rat liver or rat skeletal or heart muscle, behaved differently from those described by either Cheng et al. or Saltiel et al. First, both the PDH-stimulating and the adenylate cyclase-inhibiting activities were ethanol insoluble, in contradistinction to the ethanol-soluble adenylate cyclase-inhibiting activity reported from liver particles by Saltiel et al.3s Second, in contrast to the material described by Cheng et al.,53 the PDH and adenylate cyclase mediators were separated from each other by gel filtration in 50 mM formic acid on Sephadex G-15 columns, with the adenylate cyclase-modulating activity eluting after the PDH m e d i a t ~ r , ~ ~ , ~ ’ but they were not separated by chromatography on Sephadex G-25 columns. The nature of these differences in solubility and chromatographic properties is not understood at the present time. The differences probably do not reflect species differences in the structures of these cyclase mediators, however, since all three groups used rat t i s s ~ e ~ ~ , ~ ~ , ~ ~ , ~ ~ for their work.

Hepatoma cells produce at least four biologically distinguishable insulin mediator activities: low-K, cAMP phosphodiesterase-stimulating, adenylate cyclase-inhibiting, and PDH-stimulating and -inhibiting mediators. The production of the latter two mediators can be dissociated from one another by the conditions of cell culture. The promotion by insulin of the production of PDH- stimulating mediator activity from H4-11-EC3’ cells grown in standard media was not biphasic, but increased monotonically to a plateau at high insulin con- c e n t r a t i o n ~ . ~ ~ However, when the cells were first exposed to the glucocorticoid dexamethasone, they were able to produce a PDH-inhibiting mediator, in addition to the PDH-stimulating activity, in response to insulin.68 H4-11-EC3’ cells did not require exposure to dexamethasone to produce the 1ow-Q cAMP phosphodiesterase- or the adenylate cyclase-modulating activities in response to insulin. It is evident from these studies that the PDH-stimulating, PDH-inhibiting, and adenylate cyclase-inhibiting activities produced by hepatoma cells all represent physically different substances. Since both the PDH-stimulating and the low-K, cAMP phosphodiesterase-stimulating mediator activities elute at the same position from Sephadex G-25 columns in acid buffer, more work needs to be done to determine whether they are due to the same or different substances.

There are tissue differences in the structure of mediators that modulate the same enzyme in the same directions. This fact was shown by the contrasting behaviors of the H4-11-EC3’ and the rat muscle PDH-stimulating mediators in ethanol. The H4-11-EC3’ cell PDH-stimulating activity was completely soluble in 100% ethanol (Table III), in contrast to the ethanol insolubility of the PDH


Table 111. from H4-11-EC3’ Heuatoma Cells

Ethanol Fractionation of PDH-Stimulating Mediator

PDH Activity (nmollmgimin) Fraction Basal Control Insulin

Supernatant 8 f 0.14 11 -+ 0.39 20 ? 0.65 (EtOH soluble) Residue 8 ? 0.42 10 f 0.29 8 f 0.71 (EtOH insoluble) Starting material 13 * 0.32 24 +- 1

~ ~~ ~

Mediator was prepared as d e ~ c r i b e d . ~ ~ Extracts were lyophilized and portions were fractionated with ethanol. The ethanol- soluble and -insoluble fractions were separated by centrifugation and the ethanol portion was evaporated under a stream of N,. Both residues were resuspended in 1 mM formic acid to volumes equivalent to the volume of the original extract and were assayed for their effects on PDH according to Macaulay and Jarett.77 The data are means f SEM of triplicate determinations from a representative experiment. (J. C. Parker and L. Jarett, unpublished observations.)

mediators from liver particulate fractions38 and from rat liver and skeletal mus- c1e.57,58 In addition, the adenylate cyclase-inhibiting mediator from H4-11-EC3’ cells was also ethanol soluble, thus fractionating with the PDH-stimulating activity from this system.

The fact that mediator preparations from insulin-treated IM-9 lymphocytes stimulated PDH activity less than did similar preparations from untreated cells71 suggested that these cells produce only the PDH-inhibiting activity in response to insulin. This possibility was examined by fractionating IM-9 extracts with ethanol. Little PDH-stimulating activity was present in the ethanol-insoluble residue from insulin-treated cells, whereas significant amounts of stimulatory activity was present in the residues of control cells. Table IV shows that PDH- inhibiting activity was present in both ethanol supernatant fractions, however, and insulin treatment enhanced production of the inhibitor. Table V shows that

Table IV. Lymphocytes

Ethanol Fractionation of PDH Mediator from IM-9

PDH Activity (nmol/mg/min) Fraction Basal Control Insulin

Supernatant 6 f 0.41 5 f 0.40 3 -+ 0.34 (EtOH soluble) Residue 5 f 0.35 13 -+ 1 5 f 0.16 (EtOH insoluble) Starting material 23 f 1 9 ? 13

IM-9 lymphocytes were treated with insulin and extracted as described for H4-11-EC3‘ cells,67 and the lyophilized extract was fractionated with ethanol and prepared for assay as described in the legend to Table 111. The data are means ? SEM of triplicate determinations from a representative experiment. (J. C. Parker and L. Jarett, unpublished observations.)


Table V. Mediator from IM-9 Lymphocytes

Ethanol Fractionation of Adenylate Cyclase-Inhibiting

Adenylate Cyclase Activity Starting (pmol/mg/min)

Sample Material Residue Supernatant Buffer blank 747 2 3 825 -+ 46 742 ? 18

Insulin extract 484 & 2 462 f 21 688 * 7 IM-9 lymphocytes were treated and extracted as described in

the legend to Table IV. A buffer blank was carried through the fractionation process for control purposes. The fractions were assayed for their effects on adenylate cyclase as described in Macau- lay and Jarett.77 The data are means ? SEM of triplicate determinations from a representative experiment. (J. C. Parker and L. Jarett, unpublished observations.)

Control extract 336 5 32 322 * 30 727 * 20

1M-9 cells also produced less of an adenylate cyclase inhibitor in response to insulin and that, in contrast to the PDH-inhibitor produced by these cells, this material was insoluble in ethanol.

These data provide evidence that, within a given tissue, a number of mediator activities, which differ with respect to both their biological effects and, apparently, their chemical structures, were produced in response to the interaction of insulin with its receptor. In addition, tissue differences are revealed in the structure of at least some mediator activities that regulate the same enzyme, such as the PDH-modulating mediators from hepatoma cells and from intact rat tissue. The natures of these differences will be revealed only after the respective mediators have been purified to homogeneity and completely characterized. It is anticipated that these future studies will also help decide whether the mediator activities modulating acetyl-CoA carboxylase, glucose-6-phosphatase, or phospholipid methyltransferase are represented by distinctive entities or by previously identified mediator substances.

VI. PHY SICOCHEMICAL PROPERTIES OF INSULIN MEDIATORS

The insulin mediators that have been reported to date share a number of common properties. All are water soluble and are low-molecular-weight moieties, as judged by gel filtration in acid b ~ f f e r s , ~ ~ , ~ , ~ ~ , ~ ~ ~ ~ r ~ ~ , ~ ~ , ~ ~ , ~ ~ resistant to boiling, and are stable at acid pH but only for short times at neutral or basic pH. They appear not to be aromatic or to contain substantial aromatic portions since they are not detectably adsorbed onto charcoal. The PDH mediator has an isoelectric point of 4-553 (J. C . Parker and L. Jarett, unpublished observations); it adsorbs onto anion-exchange resins at pH 2 636,42t44,67*103 and onto cation exchangers at 5 pH 3.52,57,58

Information from several studies suggested that peptide bonds were essential for the biological activity of insulin mediators. The evidence for this was twofold. First, a proteolytic process has been implicated in the generation and/ or release of mediators from the plasma The insulin-promoted generation of PDH-modulating activity from adipocyte membranes was blocked


by serine protease inhibitors and arginine ester protease substrate analogues (PMSF, TAME, DFB, NPGB), while trypsin, in the absence of either insulin or protease inhibitors, promoted the release of a PDH-mediator activity.33 Material with gycogen synthase-stimulating activity was also generated by trypsin treatment of intact adipocytes;lo4 however, in this latter study, no evidence for the generation of glycogen synthase-inhibiting activity was found, suggesting that trypsin did not faithfully reproduce all of insulin's actions at the cell surface with respect to mediator production. These results must be interpreted cau- tiously. Although the results showed that material with insulin mediatorlike activity can be produced by perturbing the structure of the cell membranes with proteolytic agents, including insulin, they do not speak to the chemical nature of the material that is released by such perturbations, specifically whether such material possesses amino acids or peptide bonds. For example, treatment of intact adipocytes with phospholipase-C mimicked insulin's actions,'05 as did trypsinlo6 treatment. Thus, a direct relationship between proteolytic events at the plasma membrane and the chemical nature of insulin mediators has not been established.

Second, various proteases have been reported to reduce the biochemical effects of several mediators. However, since none of these studies proved that the detected inactivation of mediator activity was specifically due to protease action by inclusion of all of the appropriate controls, such as treatment with inactivated protease, treatment with protease in the presence of excess alternate substrate, and so on, care must be taken to avoid overinterpretation of these experiments. The activity of the PDH-stimulating mediator was destroyed upon exposure to trypsin33,42,43,107 or ~ h y m o t r y p s i n . ~ ~ , ~ ~ Trypsin also abolished the activities of the glucose-6-phosphatase-inhibiting mediator42 and of the CAMP- lowering mediator prepared by Zhang et al.45 However, neither trypsin nor chymotrypsin had any measurable effect on PDH-inhibiting activity extracted from liver membranes.lo7

The activities of PDH-stimulating mediators from intact rat skeletal muscle, heart, or liver were reduced only about 40% by specific pro tease^.^^ Trypsin was without effect on any of these mediator activities. Chymotrypsin and sub- tilisin were the most effective proteases tested. Heat inactivation and incubation at 4°C prevented protease-mediated inactivation of the mediators. Although a number of known protease inhibitors failed to prevent inactivation, TLCK was effective. Since only partially purified preparations of the mediators were used in these studies, the failure to detect more complete inactivation could have been due to the presence of contaminating materials in the extracts, which were resistant to protease digestion, and therefore still stimulated PDH activity.

Amino acids have been detected in the acid hydrolyzates of partially purified preparations of three mediators. Turakulov and Gainutdinov6' reported 43 amino acids after hydrolysis of an insulin-dependent mitochondria1 Ca2+ transport regulator. Twelve amino acids were detected after hydrolysis of a partially purified CAMP-dependent protein kinase-inhibiting mediator, and 14 amino acids were identified in the hydrolyzates of a partially purified preparation of the PDH-stimulating mediator.53 However, the biological activities of the preparations after hydrolysis were not reported in these studies, and it is not known if the mediators were, in fact, destroyed by these procedures. Also, although


both groups claim to have purified the mediators to near homogeneity, no evidence supporting this claim was presented. For these reasons, the signifi- cance of the reported amino acids to the biological activities of these mediators awaits positive identification of the mediator substances.

Larner has suggested that, if proteases generate mediators from the external face of the cell surface, the mediators would most likely be g1y~opeptides.l~~ Evidence has been adduced in support of this proposal. The activities of both the PDH-stimulating and -inhibiting mediators from rat liver membranes were destroyed by exposure to either neuraminidase or P-galactosidase. lo8,109 Alter- nate substrates protected the mediators from inactivation in these experiments. 108,109 These results suggested that the PDH-modulating mediators possess carbohydrate residues that are essential for their biological activities. However, Jarett's laboratory was unable to confirm these findings using PDH-stimulating mediator from intact rat liver or skeletal muscle (Macaulay and L. Jarett, unpublished results). This lack of inactivation could have been due to contaminants in the mediator preparation that interfered with the digestion.

Phospholipids and their derivatives have also been suggested to either be part of or to constitute insulin mediators. Wasner70 claims that the cAMP antagonist substance he has extracted from hepatocytes contained prostaglandin El , phosphate, and inositol. It is important to recall (c.f. section III.B.3) that this substance is probably chemically distinct from the other adenylate cyclase-inhibiting mediators discussed in this review. Begum et aI.lo9 have suggested that the PDH-modulating activity may be a prostaglandin derivative, since treatment of rats with indomethacin blocked the production of the PDH- stimulating mediator, and prostaglandin E2 overcame this block. In addition, they found that treatment of intact adipocytes with dexamethasone prevented PDH-stimulating mediator release from subsequently isolated plasma mem- b r a n e ~ . * ~ ~ Incubation of the adipocytes with arachidonic acid as well as dexamethasone partially prevented the dexamethasone effect. lo9 Prostaglandin E2, however, did not overcome the ability of dexamethasone to prevent the release of PDH-stimulating mediators. The multiplicity of effects of indomethacin, dexamethasone, prostaglandins, and arachidonic acids make it difficult to conclude that these insulin mediators are, in fact, prostaglandins or derivatives thereof.

Jarett's laboratory has shown that various phospholipids have effects on PDH and low-K, cAMP phosphodiesterase similar to those of several insulin mediators.'01,1*0,120 Farese et al. 111-114 have shown that insulin treatment of adipose tissue changed the concentrations of these same phospholipids. The differences in water solubility of the phospholipids and insulin mediators make it unlikely that the mediators are indeed phospholipid or phospholipid-like. How- ever, this does not rule out the possibility that phospholipids may be involved in regulating events related to insulin action.

In summary, circumstantial evidence suggests that mediators of insulin action may possess a variety of chemical entities, including peptides and carbohydrates, which are essential for their biological activities. There is, however, no definitive proof that mediators are, for instance, peptides or glycopeptides, and no such proof will be possible until mediator activities have been purified to homogeneity and their complete chemical characterization accomplished.

GOTTSCHALK AND JARETT 25 1

VII. PHYSIOLOGIC AND PATHOPHYSIOLOGIC ROLES OF INSULIN MEDIATORS

The results summarized above clearly demonstrate that insulin mediators subserve physiologically relevant roles in regulating metabolism. Thus, the mediators are generated from a variety of sources, including plasma membranes, cells, and intact tissues; their generationirelease is enhanced by insulin; they modulate the activities of a number of insulin-sensitive enzymes and metabolic systems in the same direction as insulin treatment; and they can affect both individual enzymes and metabolic systems in intact cells. Insulin mediators cannot mimic all of insulin's short-term effects on intact cells, however, since they have no detectable effects on glucose transport or oxidation, and their ability to mimic insulin's long-term effects has not yet been examined. The work summarized in this section provides additional evidence for the physiologic importance of these substances.

Diet clearly affects the generation or action of insulin mediators. Mem- branes from either adipocytes or liver taken from fat-fed rats released less PDH- stimulating activity, either spontaneously in Tris bufft!rs or in response to insulin, Con A, or trypsin, than membranes from carbohydrate-fed or control (chow-fed) rats. 108,115,116 Liver membranes from fat-fed rats also released less PDH-inhibiting activity.lo7 It is not known whether these results reflect reduced insulin binding to the membranes, which is known to occur as a result of high- fat diets, altered properties of the mediator or of its generationhelease mechanism, or all three.

Fasting, treatment with pharmacologic concentrations of glucocorticoids, and certain forms of diabetes all elicit insulin resistance. All have also been associated with the reduced generation of insulin mediators. Amatruda and Chang117 and Trowbridge et al."' showed that less PDH-stimulating mediator activity was released from the particulate fractions of livers from fasted rats than from control livers. Refeeding restored mediator release to control level^."^ Macaulay et al.57 also found that the recoveries of PDH- and adenylate cyclase-modulating activities from heart muscle of insulin-treated rats that had been starved for 72 h were significantly reduced compared with the yields from control (24-h starved) rats.

The release of both PDH-stimulating and -inhibiting activities from membranes was attenuated by prior treatment of the donor animals with dexameth- a ~ o n e . ' ~ ' , ~ ~ ~ This result is consistent with this hormone's ability to induce insulin resistance in vivo. However, it is in sharp contrast with dexamethasone's effects on cultured hematoma cells. In that system, exposure to the hormone enables the cells to generate PDH-inhibiting mediator in response to insulin, without diminishing their abilities to generate the PDH-stimulating mediator. The reason(s) for these discrepant results may not be resolved until the mechanisms of mediator generationiaction have been elucidated.

Amatruda and Chang117 have shown that streptozocin-induced diabetes in rats reduced the amounts of PDH-stimulating mediator activity that was released from liver membranes. Treatment of the diabetic rats with therapeutic concentrations of insulin restored mediator production to control levels.


The observation that the production or action of insulin mediators is affected by the physiologic or the pathologic state of the source of mediators, in directions that are consistent with the responsiveness of the sources to insulin, argues forcibly that these substances play important roles in mediating insulin’s effects and in regulating metabolism.

VIII. CONCLUSION

The results summarized in this review support the conclusion that the interaction of insulin with its receptor on the plasma membrane results in the generation of a family of low-molecular-weight moieties that mediate many, but not all, of the short-term actions of insulin. These substances are produced in appropriate, insulin-sensitive subcellular systems, as well as in intact cells and tissue. They affect the activities of a number of insulin-sensitive enzymes and metabolic systems, both in cell homogenates and in intact cells. The quantities of the mediators produced are dependent on the physiologic and pathologic state of the organism, and are reduced under conditions in which the sensitivity of other metabolic parameters to insulin are also reduced. At present, the chemical natures of these substances are not known, but, in light of the currently available information, are likely to be novel, although they may contain amino acids.

The studies relating insulin resistance to the reduced production of insulin mediators suggests that the mediators have potential clinical importance, both for increased understanding of the nature of the postreceptor defects involved in cases of insulin resistance, particularly type I1 diabetes and obesity, and for their possible therapeutic value in such cases.

Future directions are clear. The active principles responsible for mediator action must be purified to homogeneity and identified; the mechanisms of their generation must be established; their spectrum of action, short-term effects, long-term effects, etc., must be precisely defined; and the molecular basis for their effects must be determined.

Acknowledgment The authors express their appreciation to S. L. Macaulay, J . 0. Macaulay, J. A.

Smith, E. H. A. Wong, J. C. Parker, and K. L. Kelly for their contributions to the work reported in this review and for their criticism of the review.

This work has been supported by NIH grants AM28144 and AM07409 and a grant from the Juvenile Diabetes Foundation.

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Intracellular Mediators of Insulin Action