Insulin regulation of multiple ribonucleic acid species in human skeletal muscle in insulin-sensitive and insulin-resistant subjects.

In vivo short term (2 h) insulin-regulated gene expression was examined in skeletal muscle of persons with differing insulin sensitivities. Nine genes were analyzed by a S1 nuclease protection assay with multiple probes (multiple S1 nuclease protection assay) to allow the simultaneous examination of RNA abundances from the multiple genes. In insulin-sensitive individuals, 5 of these 9 genes were insulin responsive. RNA from the proto-oncogenes c-Ha-ras, c-myc, and c-src transiently increased 2- to 4-fold within 30 min of insulin infusion. In addition, the RNA abundance of myf-5, a muscle specific differentiation factor, increased 3-fold with a time course similar to that of c-Ha-ras, c-myc, and c-src. In contrast, type 1 protein phosphatase alpha (PPP1A) RNA levels decreased by 50% within 30 min. In insulin-resistant individuals, the RNA levels of c-Ha-ras and myf-5 did not increase, whereas c-src RNA did increase within 30 min of insulin infusion. RNA encoding c-myc transiently increased in both groups; however, this response was lower in insulin-resistant individuals than in insulin-sensitive individuals in a pattern similar to c-Ha-ras and myf-5. PPP1A RNA levels slightly increased in insulin-resistant individuals. In both insulin-sensitive and insulin-resistant persons, RNA quantities of GLUT4, c-jun, c-fos, and the insulin receptor did not change over the period of insulin infusion. However, overall RNA levels of the insulin receptor and c-jun were lower in insulin-resistant individuals.

Cloning and expression of PTP-PEST. A novel, human, nontransmembrane protein tyrosine phosphatase.

The polymerase chain reaction was used to amplify protein tyrosine phosphatase (PTPase)-related cDNA from a template of total RNA isolated from human skeletal muscle. A novel PTPase, which we term PTP-PEST, was detected by this method. The polymerase chain reaction fragment was used to screen two different HeLa cell libraries to obtain full length cDNA clones. The cDNA predicts a protein of 510 amino acids, approximately 60 kDa, that does not contain an obvious signal sequence or transmembrane segment suggesting it is a nonreceptor type enzyme. The PTPase domain is located in the N-terminal portion of the molecule and displays approximately 35% identity to other members of this family of enzymes. The C-terminal segment is rich in Pro, Glu, Asp, Ser, and Thr residues, possessing features of PEST motifs which have previously been identified in proteins with very short intracellular half-lives. The protein was expressed in Escherichia coli as a fusion product with glutathione S-transferase. Intrinsic activity was demonstrated in vitro against a variety of phosphotyrosine-containing substrates including BIRK, the autophosphorylated cytoplasmic kinase domain of the insulin receptor beta subunit. It did not dephosphorylate phosphoseryl-phosphorylase a. PTP-PEST mRNA is broadly distributed in a variety of cell lines. Stimulation of human rhabdomyosarcoma A204 cells, a transformed muscle line, with insulin led to an approximately 4-fold induction of PTP-PEST mRNA within 36 h.

Abnormal regulation of ribosomal protein S6 kinase by insulin in skeletal muscle of insulin-resistant humans.

Insulin resistance in Pima Indians appears to result from a post-receptor impairment of insulin signal transduction that affects only some responses to insulin. To identify the primary lesion responsible for insulin resistance, we investigated the influence of insulin on ribosomal protein S6 kinase activities in skeletal muscle of insulin-sensitive and insulin-resistant nondiabetic Pima Indians during a 2-h hyperinsulinemic, euglycemic clamp. In sensitive subjects, S6 kinase activity was transiently activated fivefold over basal activity by 45 min of insulin infusion. Although basal activities in the two groups were similar, the response to insulin was delayed and restricted to about threefold over basal in subjects resistant to insulin. Two major S6 kinase activities in extracts of human muscle were resolved by chromatography on Mono Q. Peak 1, which accounted for basal activity owes to an enzyme antigenically related to the 90-kD S6 kinase II, a member of the rsk gene family. The major insulin-stimulated S6 kinase eluted as peak 2 and is antigenically related to a 70-kD S6 kinase. Our results show that insulin resistance impairs signaling to the 70-kD S6 kinase.

Use of a multiple S1 nuclease protection assay to monitor changes in RNA levels for type 1 phosphatase and several proto-oncogenes in response to insulin.

Changes in insulin-regulated gene expression occur in a time- and tissue-dependent fashion. To monitor these changes we have adapted the S1 nuclease protection assay to allow simultaneous estimation of multiple RNA species in a single sample by using synthetic oligonucleotides of various lengths as probes for specific RNA species, which can then be resolved by electrophoresis. The multiple S1 nuclease protection assay was used to assess the influence of insulin on the RNA concentrations of 12 different genes in human skeletal muscle. Estimates obtained by this assay were comparable with those obtained by Northern analysis. RNA levels for proto-oncogene c-src displayed a transient 4-fold increase, whereas RNA levels for type 1 protein phosphatase were suppressed by 50% during the same time period. RNAs corresponding to known insulin-responsive genes such as c-fos, c-myc, c-Ha-ras, and c-src displayed rapid and transient 2-4-fold increases between 30 and 60 min as detected by either Northern analysis or the multiple S1 nuclease protection assay. In addition, RNA levels for the insulin receptor, Glut-4, Glut-3, and c-jun were apparently unaffected by exposure of the cells to insulin.

Defective insulin response of phosphorylase phosphatase in insulin-resistant humans.

Insulin-stimulated glycogen synthase activity in human muscle is reduced in insulin-resistant subjects. Insulin regulation of human muscle glycogen synthase may require activation of a type-1 protein phosphatase (PP-1). We investigated the change of phosphorylase phosphatase and glycogen synthase activities in muscle biopsies obtained during a 2-h hyperinsulinemic euglycemic clamp in 12 insulin-sensitive (group S) and 8 insulin-resistant (group R) subjects. Fasting phosphorylase phosphatase activity was lower in group R than in group S, and did not increase significantly with insulin infusion in group R until 20 min. In group S, phosphorylase phosphatase was significantly stimulated by 10 min, remaining significantly higher than in group R at all time points. The insulin-mediated changes in phosphatase activities were not decreased by 3 nM okadaic acid but were completely inhibited by 1 microM okadaic acid, thereby verifying that insulin-stimulated phosphorylase phosphatase is accounted for by a PP-1. Subcellular fractionation demonstrated reduced fasting PP-1 activities in both the glycogen and cytosolic fractions of muscle obtained from subjects in group R compared to those in group S. These results suggest that insulin activation of PP-1 could contribute to the stimulation of glycogen synthase by this hormone in human muscle. Lower fasting PP-1 activity in cytosol and glycogen fractions plus lower insulin-stimulated PP-1 activity could explain, in part, reduced insulin-stimulated glycogen synthase in skeletal muscle of insulin-resistant subjects.

Abnormal regulation of protein tyrosine phosphatase activities in skeletal muscle of insulin-resistant humans.

Insulin resistance in skeletal muscle may be an expression of the genetic basis of a common form of non-insulin-dependent diabetes mellitus (NIDDM) in humans. Impaired insulin action results from an apparent postreceptor defect in insulin signal transduction that limits the influence of the hormone on various protein serine/threonine kinases and phosphatases that are thought to contribute to the mechanism by which insulin affects intracellular events. The fact that numerous responses to insulin are affected suggests that the cause of insulin resistance involves an early step in insulin action. Therefore, we examined the influence of insulin on protein tyrosine phosphatase (PTPase) activities, which may counteract the protein tyrosine kinase activity of the insulin receptor in skeletal muscle of insulin-sensitive and insulin-resistant humans. Insulin infusion in vivo produced a rapid 25% suppression of soluble-PTPase activity in muscle of insulin-sensitive subjects, but this response was severely impaired in subjects who were insulin resistant. Insulin did not affect PTPase activity in the particulate fraction of muscle from either group, but basal particulate activity was 33% higher in resistant subjects than in sensitive subjects. Either or both of these abnormal characteristics of PTPase activities could be central to the causes of insulin resistance and NIDDM.

Activation of skeletal muscle casein kinase II by insulin is not diminished in subjects with insulin resistance.

Insulin resistance, which may precede the development of non-insulin-dependent diabetes mellitus in Pima Indians, appears to result from a postreceptor defect in signal transduction in skeletal muscle. To identify the putative postreceptor lesion responsible for insulin resistance in Pima Indians, we investigated the influence of insulin on the activity of casein kinase II (CKII) in skeletal muscle of seven insulin-sensitive, four insulin-resistant, nondiabetic, and five insulin-resistant diabetic Pima Indians during a 2 h hyperinsulinemic, euglycemic clamp. In sensitive subjects, CKII was transiently activated reaching a maximum over basal activity (42%) at 45 min before declining. CKII was also stimulated in resistant (19%) and diabetic (34%) subjects. Basal CKII activity in resistant subjects was 40% higher than in either sensitive or diabetic subjects, although the concentration of CKII protein, as determined by Western blotting, was equal among the three groups. Basal CKII activity was correlated with fasting plasma insulin concentrations, suggesting that the higher activity in resistant subjects resulted from insulin action. Extracts of muscle obtained from all three groups either before or after insulin administration were treated with immobilized alkaline phosphatase, which reduced and equalized CKII activity. These results suggest that insulin stimulates CKII activity in human skeletal muscle by a mechanism involving phosphorylation of either CKII or of an effector molecule, and support the idea that elevated basal activity in resistant subjects results from insulin action. It appears that the ability of insulin to activate CKII in skeletal muscle is not impaired in insulin-resistant Pima Indians, and that the biochemical lesion responsible for insulin resistance occurs either downstream from CKII or in a different pathway of insulin action.

Subunit structure of casein kinase II from bovine testis. Demonstration that the alpha and alpha' subunits are distinct polypeptides.

The relationship between the alpha and alpha' subunits of casein kinase II was studied. For this study, a rapid scheme for the purification of the enzyme from bovine testis was developed. Using a combination of chromatography on DEAE-cellulose, phosphocellulose, hydroxylapatite, gel filtration on Sephacryl S-300 and heparin-agarose, the enzyme was purified approximately 7,000-fold. The purification scheme was completed within 48 h and resulted in the purification of milligram quantities of casein kinase II from 1 kg of fresh bovine testis. The purified enzyme had high specific activity (3,000-5,000 nmol of phosphate transferred per min/mg protein) when assayed at 30 degrees C with ATP and the synthetic peptide RRRDDDSDDD as substrates. The isolated enzyme was a phosphoprotein with an alkali-labile phosphate content exceeding 2 mol/mol protein. By sodium dodecyl sulfate-polyacrylamide gel electrophoresis three polypeptides were apparent: alpha (Mr 45,000), alpha' (Mr 40,000), and beta (Mr 26,000). Several lines of evidence conclusively demonstrated that the alpha and alpha' subunits are distinct polypeptides. Two-dimensional maps of 125I-tryptic peptides derived from the two proteins were related, but distinct. An antipeptide antibody was raised in rabbits which reacted only with the alpha subunit on immunoblots and failed to react with either the alpha' or beta subunits. Direct comparison of peptide sequences obtained from the alpha and alpha' subunits revealed differences between the two polypeptides. The results of this study clearly demonstrate that the alpha and alpha' subunits of casein kinase II are not related by post-translational modification and are probably encoded by different genes.

Activation of casein kinase II in response to insulin and to epidermal growth factor.

Insulin treatment enhances casein kinase II (CKII) activity in 3T3-L1 mouse adipocytes and H4-IIE rat hepatoma cells, the magnitude of the activation varying from 30% to 150%. Activation of CKII was apparent after 5 min of exposure of 3T3-L1 cells to insulin, was maximal by 10 min, and persisted through 90 min. The insulin-stimulated activity was inhibited by low concentrations of heparin and was stimulated by spermine. Activation of CKII was effected by physiological concentrations of insulin (EC50 = 0.15 nM), suggesting that the effect is a true insulin response and not one mediated through insulin-like growth factor receptors. Epidermal growth factor (100 ng/ml for 10 min) also activated CKII in A431 human carcinoma cells, which is consistent with other observations that insulin and epidermal growth factor may have some common effects. Insulin stimulation of CKII activity was due to an increase in the maximal velocity of the kinase; the apparent Km for peptide substrate was not altered. Enhanced activity did not appear to result from increased synthesis of CKII protein, because cycloheximide did not block the effect and because an immunoblot developed with antiserum to CKII showed no effect of insulin on the cytosolic concentration of CKII. Because insulin-stimulated CKII activity was maintained after chromatography of cell extracts on Sephadex G-25, it is unlikely that the effect is mediated by a low-molecular-weight activator of the kinase. Rather, the results are consistent with the possibility that insulin activates CKII by promoting a covalent modification of the kinase.

Substrate specificity determinants for casein kinase II as deduced from studies with synthetic peptides.

The specificity of casein kinase II has been further defined by analyzing the kinetics of phosphorylation reactions using a number of different synthetic peptides as substrates. The best peptide substrates are those in which multiple acidic amino acids are present on both sides of the phosphorylatable serine or threonine. Acidic residues on the NH2-terminal side of the serine (threonine) greatly enhance the kinetic constants but are not absolutely required. Acidic residues on the COOH-terminal side of the serine (threonine) are absolutely required. One position for which the occupation of an acidic residue is especially critical is the position located 3 residues to the COOH terminus of the phosphate acceptor site, although the presence of an acidic amino acid in the positions that are 4 or 5 residues removed may also provide an appropriate structure that will serve as a substrate for the kinase. Aspartate serves as a better amino acid determinant than glutamate. A relatively short sequence of amino acids surrounding the phosphate acceptor site appears to serve as the basis for the specificity of casein kinase II. The peptides in this study were also assayed with casein kinase I and the casein kinase from the mammary gland so that the specificities of these kinases could be compared to that of casein kinase II.

Induction of casein kinase II during differentiation of 3T3-L1 cells.

The peptide Arg-Arg-Arg-Glu-Glu-Glu-Thr-Glu-Glu-Glu was shown to be a specific substrate for casein kinase II (CK II) in extracts of 3T3-L1 cells. Fractionation of a cell extract on DEAE-cellulose revealed only one peptide kinase and it eluted at the same salt concentration required to elute CK II. Consistent with the properties of CK II, the peptide kinase activity was inhibited by very low concentrations of heparin (Ki less than 6 nM) and it used GTP efficiently as a substrate. A Western blot, developed with antiserum to bovine thymus CK II, demonstrated the presence of CK II protein in 3T3-L1 extracts and that peptide kinase activity was directly related to the amount of CK II protein. The peptide was used to assay CK II activity in extracts of 3T3-L1 cells stimulated to differentiate into adipocytes. Differentiation produced a transient increase in CK II activity that reached a maximum (4-fold) on day 4. The increased activity was accounted for by increased CK II protein. Induction of CK II preceded the increase in total protein and was not the result of cell proliferation. CK II induction was coincident with induction of the insulin receptor, but, whereas insulin binding remained elevated, CK II activity declined after day 4. Agents that stimulate differentiation of 3T3-L1 cells did not cause induction of CK II in 3T3-C2 cells that do not differentiate. The transient nature of the induction of CK II suggests that the kinase may contribute to the process of differentiation rather than being a phenotypic change like that of the insulin receptor.

Activation of phosphofructokinase from rat tissues by 6-phosphogluconate and fructose 2,6-bisphosphate.

6-Phosphogluconate activates phosphofructokinase from liver, adipose tissue, kidney, and skeletal muscle by decreasing the apparent S0.5 for fructose 6-phosphate without affecting the maximum velocity. The response of phosphofructokinase to 6-phosphogluconate is hyperbolic, with apparent activation constants similar to concentrations of 6-phosphogluconate in tissues. Phosphofructokinase from these tissues is also activated by fructose 2,6-bisphosphate, but the apparent activation constants are much less than the concentrations of fructose 2,6-bisphosphate in tissues. Under most conditions, the effects of 6-phosphogluconate and fructose 2,6-bisphosphate are additive. However, with low concentrations of fructose 6-phosphate there is synergism between the effectors. Whereas fructose 2,6-bisphosphate overcomes the inhibition of phosphofructokinase by high concentrations of ATP, 6-phosphogluconate does not. Thus, the effectors probably act at different sites on the enzyme. The relative effect of 6-phosphogluconate is much greater on phosphofructokinase from the lipogenic tissues, adipose, and liver, than it is on the enzyme from kidney or skeletal muscle. Thus, the influence of 6-phosphogluconate on phosphofructokinase, which could coordinate the disposition of glucose 6-phosphate between the oxidative branch of the hexosemonophosphate pathway and glycolysis, may be important for lipogenesis.

Effects of diets on concentrations of 6-phosphogluconate and fructose 2,6-bisphosphate in rat livers and an assay of fructose 2,6-bisphosphate with an improved method.

We determined the effects of diets that have different lipogenic potentials on hepatic concentrations of 6-phosphogluconate and fructose 2,6-bisphosphate, both of which activate hepatic phosphofructokinase. Diets high in carbohydrate increased concentrations of both effectors compared to a high protein (gluconeogenic) diet. The concentration of 6-phosphogluconate was associated with the lipogenic nature of the diet, and the range of its concentration matched that over which phosphofructokinase responds to 6-phosphogluconate in vitro. In contrast, the concentration of fructose of 2,6-bisphosphate was not associated with the lipogenic potential of the diets. Fructose 2,6-bisphosphate was either absent from liver or its concentration was 10- to 30-fold higher than the concentration that gives the maximal activation of phosphofructokinase in vitro. The results indicate that fructose 2,6-bisphosphate and 6-phosphogluconate have different roles in the regulation of phosphofructokinase. Fructose 2,6-bisphosphate may be involved in switching hepatic carbohydrate metabolism between gluconeogenesis and glycolysis, whereas changes in the concentration of 6-phosphogluconate may coordinate the disposition of glucose 6-phosphate between the oxidative branch of the hexosemonophosphate pathway and glycolysis. In the course of our studies, we improved an enzymatic assay for fructose 2,6-bisphosphate.

Regulation of hepatic phosphofructokinase by 6-phosphogluconate.

6-Phosphogluconate activates phosphofructokinase in extracts of rat liver by decreasing both the apparent S0.5 for fructose 6-phosphate and the degree of cooperative binding of fructose 6-phosphate. There is no effect on the maximum velocity. Enzyme activity is a hyperbolic function of the concentration of 6-phosphogluconate, and the apparent Km is 60 microM. Thus, a substantial activation of the enzyme is achieved with a physiological concentration of 6-phosphogluconate (40 microM). Activation of phosphofructokinase by 6-phosphogluconate is influenced by a low molecular weight factor(s) in liver extracts. Part of the influence may be attributed to fructose 2,6-bisphosphate which, in some conditions, acts synergistically with 6-phosphogluconate to activate phosphofructokinase. In the presence of a mixture of ATP, ADP, and AMP at physiological concentrations, the effects of 6-phosphogluconate (40 and 200 microM) and a saturating concentration of fructose 2,6-bisphosphate (400 nM) are more nearly additive. This result suggests that 6-phosphogluconate and fructose 2,6-bisphosphate act at different sites on the enzyme and that 6-phosphogluconate may contribute to the physiological regulation of hepatic phosphofructokinase. Regulation of phosphofructokinase by 6-phosphogluconate may provide a means by which the disposition of glucose 6-phosphate between the oxidative branch of the hexosemonophosphate pathway and glycolysis can be coordinated, an effect which may be important during hepatic lipogenesis.

Protein degradation in primary monolayer cultures of adult rat hepatocytes. Further evidence for the regulation of protein degradation by amino acids.

Intracellular protein degradation was measured in primary monolayer cultures of adult rat hepatocytes by the loss of radioactivity from proteins pulse-labeled in culture with L-[U-14C]valine. The fractional rate of protein degradation of total protein measured over 18 h was similar to the rate in vivo. The fraction rate of protein breakdown was altered significantly by varying the composition of the chase medium from a balanced salts medium (high rate of protein degradation) to a more complete medium (lower rate of protein degradation). Whereas the vitamin component of the complete medium had some effect, the major portion of the inhibition of protein degradation was due to the essential amino acids in the medium. This inhibition was found to depend on the presence of methonine, phenylalanine, and tryptophan, the latter two of which appear to be primarily responsible. The observed rate of protein breakdown was a function of the level of amino acids in the medium when they were varied in the physiological range for rat plasma. Our results suggest that the regulation of protein degradation by essential amino acids may be physiologically important in maintaining intracellular amino acid pools when the exogenous supply of amino acids is diminished.