Increased Protein Kinase C Activity Is Linked to Reduced Insulin Receptor Autophosphorylation in Liver of Starved Rats*

Phosphorylation of the insulin receptor B-subunit on serinelthreonine residues by protein kinase C reduces both receptor kinase activity and insulin action in cul-tured cells. Whether this mechanism regulates insulin action in intact animals was investigated in rats ren-dered insulin-resistant by 3 days of starvation. Insulin-stimulated autophosphorylation of the partially purified hepatic insulin receptor &subunit was decreased by 45% in starved animals compared to fed controls. This autophosphorylation defect was entirely reversed by removal of pre-existing phosphate from the receptor with alkaline phosphatase, suggesting that increased basal phosphorylation on serineithreonine residues may cause the decreased receptor tyrosine kinase activity. Tryptic removal of a C-terminal region of the receptor &subunit containing the Ser/Thr phosphorylation sites similarly normalized receptor autophosphorylation. To investigate which may be responsible for such increased Ser/Thr phosphorylation


Phosphorylation
of the insulin receptor B-subunit on serinelthreonine residues by protein kinase C reduces both receptor kinase activity and insulin action in cultured cells. Whether this mechanism regulates insulin action in intact animals was investigated in rats rendered insulin-resistant by 3 days of starvation. Insulinstimulated autophosphorylation of the partially purified hepatic insulin receptor &subunit was decreased by 45% in starved animals compared to fed controls. This autophosphorylation defect was entirely reversed by removal of pre-existing phosphate from the receptor with alkaline phosphatase, suggesting that increased basal phosphorylation on serineithreonine residues may cause the decreased receptor tyrosine kinase activity.
Tryptic removal of a C-terminal region of the receptor &subunit containing the Ser/Thr phosphorylation sites similarly normalized receptor autophosphorylation.
To investigate which kinase(s) may be responsible for such increased Ser/Thr phosphorylation in vivo, protein kinase C and CAMP-dependent protein kinase A in liver were studied. A a-fold increase in protein kinase C activity was found in both cytosol and membrane extracts from starved rats as compared to controls, while protein kinase A activity was diminished in the cytosol of starved rats. A parallel increase in protein kinase C was demonstrated by immunoblotting with a polyclonal antibody which recognizes several protein kinase C isoforms. These findings suggest that in starved, insulin-resistant animals, an increase in hepatic protein kinase C activity is associated with increased Ser/Thr phosphorylation which in turn decreases autophosphorylation and function of the insulin receptor kinase.
The insulin receptor possesses intrinsic tyrosine kinase activity which appears to be essential for signal transmission (1,2). In patients with non-insulin-dependent diabetes mel- litus (NIDDM),' receptor kinase activity is reduced by 40-80% (3)(4)(5). In obese patients with NIDDM, weight loss improves insulin resistance and the diabetic state and is associated with parallel improvement in the kinase activity (6), suggesting that the phosphorylation defect in these patients is caused by a regulatory rather than a genetic change in the receptor. We and others have shown that regulation of the intrinsic kinase activity at the receptor depends on the phosphorylation state of the p-subunit of the receptor (7,8).
Autophosphorylation of tyrosine residues increases kinase activity, whereas serine phosphorylation by other kinases leads to a decrease in the tyrosine kinase activity. Serine phosphorylation can be catalyzed by CAMP-dependent protein kinase (9) or by the Ca*+-and phospholipid-dependent protein kinase C (8-10). Starvation induces insulin resistance (11) and, in parallel, a reduction in insulin receptor kinase activity (12)(13)(14). In this study, we have examined starvation-induced changes of the insulin receptor and protein kinase C systems in rat liver and found evidence in this model of insulin resistance that the decrease in receptor tyrosine kinase may be mediated through Ser/Thr phosphorylation of the receptor by protein kinase C. NaF (100 mM), Na4P207 (10 mM), and EDTA (5 mM). Insulin binding capacity of the WGA-purified fractions was I30 -+ 12 pmol and 190 f 8 pmol of insulin/mg of protein for the fed (n = 4) and starved (n = 4) animals, respectively.

EXPERIMENTAL PROCEDURES
To compare receptor autophosphorylation, equal amounts of receptor (500 pmol of insulin binding capacity) from fed and starved animals were incubated with [r-"*P]ATP and Mn+ in the absence and presence of 10e9 or 10e7 M insulin. In both preparations, the insulin-stimulated autophosphorylated receptor p-subunit was selectively immunoprecipitated with anti-phosphotyrosine antibody and appeared as a 95-kDa band on the autoradiograms of SDS gels (Fig. 1 Equal amounts (500 pmol of insulin binding capacity of WGA-purified insulin receptor from liver of rats starved for 72 h and fed ad &turn, were incubated with 10d9 or lo-' insulin. The phosphorylation reaction was started by adding [-y-"'P]ATP and Mn*+ and terminated after 2 min. The insulin receptor was immunoprecipitated with anti-phosphotyrosine antibody, analyzed on SDS-PAGE gels, and visualized by autoradiography. The autoradiographs are shown in the upper part, and results of scanning of four pairs of animals are summarized in the graph (in relative units).
the receptor prior to the autophosphorylation reaction. First, the WGA-purified receptors were incubated with immobilized alkaline phosphatase under conditions previously shown to remove from the receptor phosphate added by protein kinase C-catalyzed serine/threonine phosphorylation (8). WGA preparations treated with active or inactive alkaline phosphatase from fed and starved animals were subjected to insulin stimulation under the same conditions described above. Dephosphorylation of the insulin receptor caused an increase in insulin-stimulated autophosphorylation of the P-subunit of the insulin receptor from livers of both fed and starved rats. The increase was almost 4-fold (355%) in receptors from starved animals and only 2-fold (182%) in receptors from fed animals. Thus, the treatment with alkaline phosphatase reversed the defect in phosphorylation found on receptors of starved animals, such that insulin-stimulated autophosphorylation was essentially equalized between receptors of starved and fed rats (Fig. 2).
Second, the WGA preparations were incubated with trypsin under conditions previously shown to remove a lo-kDa fragment from the C terminus of the P-subunit of the insulin receptor (15). This lo-kDa peptide contains threonine-1336 (16) which has recently been identified as a phosphorylation site by protein kinase C, and also contains at least one of the serine sites of phosphorylation (18).' The trypsin-treated WGA-purified receptor preparation from liver of fed and starved rats were stimulated with 100 nM insulin under conditions identical with those described above and compared to each other as well as to non-trypsin-treated preparations. Trypsin-treated P-subunit migrated at M, = 85, about 10,000 lower than the nondigested P-subunit. As in the dephosphorylation with alkaline phosphatase, removal of the M, = 10,000 peptide normalized the decrease in autophosphorylation of the P-subunit seen in starved animals equalizing it to that of fed controls (Fig. 3). Autophosphorylation of the WGA-purified insulin receptor from livers of starved and fed rats before and after incubation of the receptor with alkaline phosphatase-agarose for 1 h at 4 "C to remove all phosphate added by protein kinase C activation. The receptors were phosphorylated as described in the legend to Fig.  1. The two first groups of bars on the left are experiments performed with the intact receptor; the two groups of bars on the right are with receptor that was dephosphorylated by alkaline phosphatase. In each case, the solid bars represent the stimulation with 1 nM insulin; the hatched bar the stimulation with 100 nM insulin. In all cases, no phosphorylation was detected in the absence of insulin. These autoradiographs were scanned, and the bar graph summarizes the results of four such experiments (mean + SD.).

FIG. 3. Autophosphorylation
of the insulin receptor purified from starved and fed rats before and after mild trypsinization.

Autophosphorylation
of the WGA-purified insulin receptor from liver of fed and starved rats was performed as in Fig. 1 (first two lanes). This experiment was repeated after incubation of the receptor with TPCK-treated trypsin for 1 min at 22 "C and stopping the trypsinization with aprotinin before autophosphorylation (last two lanes). All lanes are of receptors stimulated with insulin. The experiment was repeated twice in two pairs of animals. The difference in autophosphorylation between fed and starved was 26 + 6% before trypsinization and -5 f 4% after removal of the lo-kDa piece.
from Fed and Starved Rats-The results of the autophosphorylation experiments shown above suggest that increased serinelthreonine phosphorylation of the P-subunit may be the cause of the decrease in autophosphorylation of the insulin receptor. Two serine kinases have been implicated in phosphorylation of the insulin receptor on serinelthreonine residues: CAMP-dependent protein kinase (protein kinase A), and calcium-and phospholipid-dependent protein kinase (protein kinase C). Therefore, we measured the activity of these two enzymes following starvation. Livers of both starved and fed rats were homogenized in the presence of multiple protease inhibitors and the kinases were partially purified on a DEAE-Sephacel (see "Experimental Procedures").
In both fed and starved animals, protein kinase C activity in liver was higher in membrane as compared to cytosol (Fig.  4). Protein kinase C activity was higher in solubilized liver membranes of starved rats compared to fed controls (81 + 17 All liver extracts were prepared as in Fig. 4. 200 pg of protein from liver extracts pooled from four starved and four fed animals were analyzed by electrophoresis as well as a similar amount from fed rat brain. The proteins were blotted on nitrocellulose paper and probed with anti-protein kinase C antibody raised in rabbits against a peptide corresponding to sequence 280-292 of brain protein kinase C. Visualization was achieved by '"'I-protein A and autoradiography. uersus 43 f 11 pmol of "P incorporated 1 mg of protein/min (n = 8, p < 0.01)). Activity was also higher in cytosol of livers of starved rats compared to ad libitum fed controls (38 + 3 versus 23 + 4 pmol of "'P incorporated/mg of protein/min, n = 8, p < 0.01). The ratio of activity of protein kinase C in membrane/cytosol was 1.86 in fed rats and 2.13 in starved rats, perhaps suggesting a slightly larger portion of protein kinase C to be membrane-bound in starved rats.
Unlike protein kinase C, protein kinase A activity was decreased by starvation in rat liver. Cytosolic protein kinase A activity decreased from 42 + 10 pmol of "P/mg of protein/ min to 21 f 2.4 pmol of "P/mg of liver during starvation rats.
Immunoblotting of Protein Kinase C-To determine whether the increased protein kinase C activity in liver of starved rats was due to increased amounts of the enzyme or increased specific activity, we measured the amount of immunodetectable protein kinase C in liver extracts from both fed and starved rats. Cytosolic and membrane extracts from four fed and starved rats were pooled, and samples containing equal amounts of protein were analyzed by SDS-PAGE and electroblotted to nitrocellulose. Probing was done using polyclonal antibody against peptide 0442 of bovine brain protein kinase C (19), and visualization was with ""I-protein A and autoradiography. In contrast to previous reports which suggested that protein kinase C is of lower M, in liver (20), we identified an undegraded form with M, = 82, equal to that found in brain (Fig. 5). In addition, liver contained multiple lower M, bands which are presumably proteolytic degradation products. As shown in Fig. 5, there was a 180% increase (by densitometry) in the nondegraded form of protein kinase C membrane extracts of starved animals. However, the degraded form of M, = 55,000 was more abundant in fed rat liver membrane extracts. Similar results were seen in cytosol where protein kinase C migrated at the same M, as in the membrane fractions and the M, = 82,000 band was more abundant (200%) in starved animals compared to fed animals. DISCUSSION Regulation of transmembrane signalling by receptor phosphorylation is a mechanism common to several different receptor systems including the family of receptors which are tyrosine kinases. The tyrosine kinase activity of both the EGF and insulin receptors are under positive and negative control by the state of receptor phosphorylation. Autophosphorylation of the insulin receptor on tyrosine residues enhances tyrosine kinase activity (7). In contrast, activation of both protein kinase A and protein kinase C in intact cells leads to Ser/Thr phosphorylation of the insulin receptor which is associated with reduced tyrosine kinase activity and a decrease in insulin action (8)(9)(10). The insulin receptor is phosphorylated in vitro by protein kinase C, and this phosphorylation leads to a 65% decrease in its protein tyrosine kinase activity (10). A similar reduction is seen in insulin receptor purified from hepatoma cells which have been pretreated with a protein kinase C activator, and, in this case, treatment of the receptor tyrosine kinase with alkaline phosphatase reverses protein kinase C-mediated receptor phosphorylation and increases receptor kinase activity (8). In the case of the EGF receptor, the regulatory role of tyrosine phosphorylation remains debated, but serine/threonine phosphorylation, catalyzed at least in part by protein kinase C, reduces kinase activity and alters receptor binding (23). Threonine-654, located in the EGF receptor cytoplasmic domain, appears to be a major site of protein kinase C phosphorylation. In this study, the role of serine/threonine phosphorylation in the starvation-induced decrease in the tyrosine kinase activity of rat liver insulin receptor was examined. In agreement with other reports (12)(13)(14), we found that insulinstimulated autophosphorylation of insulin receptor from liver of rats starved for 72 h was decreased by -50% compared to that of animals fed ad libitum. To probe the possibility that the reduced kinase activity seen in starved animals was a result of phosphorylation on serine/threonine residues, the in vitro autophosphorylation was repeated with receptors where pre-existing phosphate was removed, either by preincubation of the receptor with alkaline phosphatase or by removal of a lo-kDa piece of the C terminus of the P-subunit containing threonine-1336.
The latter experiment was based on the recent work by Lewis et al.' suggesting that protein kinase C phosphorylates the insulin receptor at threonine-1336 adjacent to the C terminus of the cytoplasmic portion of the psubunit of the receptor, as well as on previous reports that found an increased phosphorylation on serine residues after phosphorylation of the insulin receptor by the purified kinase C in a cell free system (10) or after treatment of cells by phorbol esters (8). Both enzymatic manipulations led to a normalization or equalization of insulin-stimulated autophosphorylation of the receptor, suggesting that phosphate on Ser/Thr residues of the receptor could explain the decrease in tyrosine kinase level and that the model of regulation by multisite phosphorylation may be valid in the intact animal. Starvation-induced insulin resistance is not the only reversible state of insulin resistance accompanied by a reversible decrease in insulin receptor kinase activity. In obese subjects with NIDDM, there is a 50-80s reduction in kinase activity of the insulin receptor from adipocytes, and this can almost be completely reversed upon weight loss. This correlates with correction of the glucose disposal rate as measured during a euglycemic clamp (6). This reversible decrease was suggested to result from an increased proportion of receptors that bind insulin but lack tyrosine kinase activity, rather than a diminished kinase activity of all the receptors (21). Although not tested, the increase in proportion of tyrosine kinase-deficient receptors in that study could well be a result of increased serine phosphorylation by protein kinase C. Increased protein kinase C activity alone or combined with a genetic propensity to an increase in Ser/Thr phosphorylation of the receptor, could lead to the altered receptor function observed in patients with NIDDM.
The defect in kinase activity of receptor purified from livers of streptozotocin diabetic rats is another example of a reversible alteration in kinase activity, which in this case is partially restored by treatment of the rats with insulin (22). In data not shown, we performed a similar set of experiments to those used to characterize the starved animals and were unable to document similar changes. While we found a reduction in the receptor autophosphorylation, this defect was not reversed by alkaline phosphatase treatment, nor was protein kinase C activity in liver increased. Thus, the proposed model of regulation of the insulin receptor kinase may not be applicable to all forms of acquired or genetic models of insulin resistance in animals.
The finding of an increase in activity of protein kinase C in liver extracts of starved rats was not due to an increase in the activity of a nonspecific serine phosphatase, as starvation induced an opposite effect on protein kinase A. The higher protein kinase C activity was paralleled by an increase in the amount of the intact enzyme as demonstrated by immunoblotting.
An opposite effect was seen in the amount of the major degradation products of protein kinase C as well as a change in the pattern of degradation.
These results could be explained by a difference in proteolytic activity toward protein kinase C in livers of starved rats, or alternatively, they can be explained by a differential induction of protein kinase C isoenzymes by starvation. Protein kinase C activity is present in at least seven different isozymes (24)(25)(26)(27)(28). Each of these has a molecular weight -80,000 and is composed of a M, = 50,000 catalytic domain and a M, = 30,000 regulatory domain. Recently, it has been shown that the cleavage of these different isozymes by trypsin and calpain differs with Type III being the most resistant (25,26). The proteolytic fragments generated include a 50-kDa fragment which is fully active, but is no longer regulated by diacylglycerol, Ca*+, and phospholipids, as well as 67-to 74-kDa fragments which retain partial dependence on these co-factors (29). Although the antibody we used recognizes at least three isoenzymes, if starvation induces production of one isoenzyme which is differentially proteolyzed, this could account for the findings regarding protein kinase C in liver.
Rapid and extensive degradation of protein kinase C in liver extracts has led to several erroneous results. Previous work found only low activity in liver compared to other organs on a weight basis (30). Azhar et al. (20) used multiple protease inhibitors and a mild detergent to unmask a much higher activity of protein kinase C in rat liver. The same group purified protein kinase C and determined it to be composed of subunits with molecular weight of 64,000 and described three isoenzymes with nearly identical enzymatic properties. In this work, we were able to demonstrate by immunoblotting that the intact protein kinase C in liver is of identical molecular weight as that in brain and that the previously described 64-kDa subunits of the enzymes are probably degradation products.
Protein kinase C is thought to play a major role in control of a wide variety of processes (28). Increased activity in starvation would certainly have a broader influence beyond the effects described here. For example, the EGF receptor in hepatocytes of starved rats is reported to have decreased binding and tyrosine kinase activity (31) that could result from starvation-induced protein kinase C activation. Phosphorylation of glycogen synthase by protein kinase C has been shown to lead to its inactivation (32) and may be involved in the decrease in glycogen synthase activity in starvation.
Although several experimental lines of evidence reported in this study support the hypothesis that the model of regulation of insulin receptor tyrosine kinase in vitro is correct for the intact animal, direct evidence is still lacking. This task awaits direct measurements of phosphoserine and phosphothreonine levels in hepatic insulin receptors.