Rapid response of protein synthesis to insulin in 3T3 cells: Effects of protein kinase C depletion and differences from the response to serum repletion

Quiescent 3T3 cells grown in media containing 4% foetal calf serum showed different responses to insulin and to serum repletion (to 12%). Insulin stimulated protein synthesis within 1 h and this early response was insensitive to actinomycin D. The later insulin response showed progressive sensitivity to actinomycin D. The serum response was slower, not occurring until 1 h, and was inhibited by actinomycin D. Depletion of cell protein kinase C by pre-treatment with phorbol ester caused a total block of the immediate response to insulin but had little effect on the response to serum or the later response to insulin. Acute phorbol ester treatment stimulated protein synthesis.     

Insulin regulation of protein biosynthesis in differentiated 3T3 adipocytes. Regulation of glyceraldehyde-3-phosphate dehydrogenase

The effect of insulin on protein biosynthesis was examined in differentiated 3T3-L1 and 3T3-F442A adipocytes. Insulin altered the relative rate of synthesis of specific proteins independent of its ability to hasten conversion of the fibroblast (preadipocyte) phenotype to the adipocyte phenotype. Although more than one pattern of response to insulin was observed, we focused on the induction of a Mr 33,000 protein which was identified as the glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Exposure of 3T3 adipocytes to insulin throughout differentiation specifically increased GAPDH activity and protein content by 2- to 3-fold as compared to 3T3 adipocytes differentiated in the absence of insulin. These changes in enzyme activity and content could be accounted for by a 4-fold increase in the relative rate of synthesis of GAPDH and a 9-fold increase in hybridizable mRNA levels. Within 2 h of insulin addition to 3T3 adipocytes differentiated in the absence of hormone, hybridizable GAPDH mRNA levels increased 3-fold, and within 24 h GAPDH mRNA levels increased 8-fold, and [35S] methionine incorporation into GAPDH protein increased 5-fold. The increase in GAPDH mRNA and GAPDH biosynthesis could be demonstrated using physiologic concentrations of insulin (0.24 nM), indicating that these effects are mediated through a specific interaction with the insulin receptor. These studies demonstrate that insulin, as the sole hormonal perturbant, can increase the synthesis of certain 3T3 adipocyte proteins by altering the cellular content of a specific mRNA.     

Isoproterenol stimulates monocyte chemoattractant protein-1 expression and secretion in 3T3-L1 adipocytes

Recently, monocyte chemoattractant protein (MCP)-1 has been characterized as a novel adipocytokine upregulated in obesity and insulin resistance which impairs insulin signaling in muscle and fat in vitro. Growing evidence, on the other hand, suggests that increased activity of the sympathetic nervous system is an integral part in the development of insulin resistance. In the current study, the impact of the beta-adrenergic agonist isoproterenol on MCP-1 mRNA synthesis and secretion was determined in 3T3-L1 adipocytes. Interestingly, isoproterenol increased MCP-1 secretion 3-fold. Furthermore, 10 microM isoproterenol acutely induced MCP-1 mRNA by up to 5.3-fold in a time-dependent fashion with significant stimulation seen at concentrations as low as 0.3 microM effector. Studies using pharmacological inhibitors suggested that basal and isoproterenol-induced MCP-1 expressions are mediated via beta-adrenergic receptors and protein kinase A. Moreover, acute activation of adenylyl cyclase by forskolin was sufficient to mimic the effects of isoproterenol. Taken together, our results demonstrate that isoproterenol induces MCP-1 expression and secretion via a classical GS-protein-coupled pathway and support the notion that MCP-1 might be an interesting novel candidate linking obesity and insulin resistance.     

Effect of phenylarsine oxide on protein synthesis in 3T3-L1 adipocytes

In this report, we show that insulin stimulated the incorporation of tracer [3H]leucine into protein of 3T3-L1 adipocytes within 2 min of insulin addition. The concentration of insulin required to elicit 50% activation was 4nM. Phenylarsine oxide, an inhibitor of insulin-stimulated glucose transport, blocked not only insulin-stimulated protein synthesis but constitutive protein synthesis as well (Ki, 3 microM). Importantly, protein synthesis was not required for insulin-activated glucose transport since cycloheximide added either before or after insulin had no effect on the stimulated rates of glucose transport.     

Insulin fails to stimulate muscle protein synthesis in sepsis despite unimpaired signaling to 4E-BP1 and S6K1

Induction of sepsis in rats causes an inhibition of protein synthesis in skeletal muscle that is resistant to the stimulatory actions of insulin. To gain a better understanding of the underlying reason for this lack of response, the present study was undertaken to investigate sepsis-induced alterations in insulin signaling to regulatory components of mRNA translation. Experiments were performed in perfused hindlimb preparations from rats 5 days after induction of a septic abscess. Sepsis resulted in a 50% reduction in protein synthesis in the gastrocnemius. Protein synthesis in muscles from septic rats, but not controls, was unresponsive to stimulation by insulin. The insulin-induced hyperphosphorylation response of the translation repressor protein 4E-binding protein 1 (4E-BP1) and of the 70-kDa S6 kinase (S6K1) (1), two targets of insulin action on mRNA translation, was unimpaired in gastrocnemius of septic rats. Hyperphosphorylation of 4E-BP1 in response to insulin resulted in its dissociation from the inactive eukaryotic initiation factor (eIF)4E. 4E-BP1 complex in both control and septic rats. However, assembly of the active eIF4F complex as assessed by the association of eIF4E with eIF4G did not follow the pattern predicted by the increased availability of eIF4E resulting from changes in the phosphorylation of 4E-BP1. Indeed, sepsis caused a dramatic reduction in the amount of eIF4G associated with eIF4E in the presence or absence of insulin. Thus the inability of insulin to stimulate protein synthesis during sepsis may be related to a defect in signaling to a step in translation initiation involved in assembly of an active eIF4F complex.     

Temporally graded requirement for protein synthesis following memory reactivation

Learning of new information is transformed into long-lasting memory through a process known as consolidation, which requires protein synthesis. Classical theory held that once consolidated, memory was insensitive to disruption. However, old memories that are insensitive to protein synthesis inhibitors can become vulnerable if they are recalled (reactivated). These findings led to a new hypothesis that when an old memory is reactivated, it again becomes labile and, similar to a newly formed memory, requires a process of reconsolidation in order to be maintained. Here, we show that the requirement for protein synthesis of a reactivated memory is evident only when the memory is recent. In fact, memory vulnerability decreases as the time between the original training and the recall increases.     

Glycogen synthase kinase 3beta is a novel regulator of high glucose- and high insulin-induced extracellular matrix protein synthesis in renal proximal tubular epithelial cells

High glucose (30 mM) and high insulin (1 nM), pathogenic factors of type 2 diabetes, increased mRNA expression and synthesis of lamininbeta1 and fibronectin after 24 h of incubation in kidney proximal tubular epithelial (MCT) cells. We tested the hypothesis that inactivation of glycogen synthase kinase 3beta (GSK3beta) by high glucose and high insulin induces increase in synthesis of laminin beta1 via activation of eIF2Bepsilon. Both high glucose and high insulin induced Ser-9 phosphorylation and inactivation of GSK3beta at 2 h that lasted for up to 48 h. This was associated with dephosphorylation of eIF2Bepsilon and eEF2, and increase in phosphorylation of 4E-BP1 and eIF4E. Expression of the kinase-dead mutant of GSK3beta or constitutively active kinase led to increased and diminished laminin beta1 synthesis, respectively. Incubation with selective kinase inhibitors showed that high glucose- and high insulin-induced laminin beta1 synthesis and phosphorylation of GSK3beta were dependent on PI 3-kinase, Erk, and mTOR. High glucose and high insulin augmented activation of Akt, Erk, and p70S6 kinase. Dominant negative Akt, but not dominant negative p70S6 kinase, inhibited GSK3beta phosphorylation induced by high glucose and high insulin, suggesting Akt but not p70S6 kinase was upstream of GSK3beta. Status of GSK3beta was examined in vivo in renal cortex of db/db mice with type 2 diabetes at 2 weeks and 2 months of diabetes. Diabetic mice showed increased phosphorylation of renal cortical GSK3beta and decreased phosphorylation of eIF2Bepsilon, which correlated with renal hypertrophy at 2 weeks, and increased laminin beta1 and fibronectin protein content at 2 months. GSK3beta and eIF2Bepsilon play a role in augmented protein synthesis associated with high glucose- and high insulin-stimulated hypertrophy and matrix accumulation in renal disease in type 2 diabetes.     

Glucose transport by cultured human fibroblasts: regulation by phorbol esters and insulin.

The regulation of 3-O-methyl-D-glucose (OMG) uptake by insulin and phorbol esters was studied in cultured human skin fibroblasts. Insulin rapidly stimulated OMG uptake through a mechanism independent of new protein synthesis. Maximal insulin effect was reached in 30 min and remained constant up to 12 h. The protein kinase C activators 12-O-tetradecanoyl phorbol 13-acetate (TPA) and phorbol 12,13-dibutyrate (PdBU) promoted an initial rapid stimulation followed by a secondary long-term rise of OMG influx. This latter effect of phorbol esters on OMG influx began after 1 h, reached a maximum in 12-15 h, and was prevented by the simultaneous addition of protein synthesis inhibitors, suggesting that phorbol esters increased the synthesis of new glucose transporters. In accord with this interpretation, phorbol esters, but not insulin, increased mRNA levels for two distinct glucose transporters (GLUT1 and GLUT3) in human fibroblasts. Both the rapid and the long-term effects of phorbol esters on OMG influx were dose-dependent and half-maximal stimulations occurred at 15 nM for both PdBU and TPA. Kinetic analysis of OMG uptake indicated that both effects of phorbol esters were associated with an increase in the Vmax of the transport process, with no significant changes of the Km (4-6 mM). These results suggest that, in human fibroblasts, phorbol esters, unlike insulin, produce a long-term stimulation of OMG uptake, which is dependent upon protein synthesis and is associated with increased levels of GLUT1 and GLUT3 mRNA.     

Increased messenger RNA from protein synthesis inhibited human fibroblasts

Numerous reports have demonstrated that specific protein synthesis in response to specific inducers is markedly stimulated by a simultaneous brief exposure to protein synthesis inhibitors such as cycloheximide. This phenomenon is known as “superinduction” and is most often attributed to the accumulation of cytoplasmic messenger RNA during the inhibition period. Messenger RNA, as defined by rapid labeling, oligo (dt)-cellulose binding, and cell free protein synthesis stimulation was measured in cycloheximide treated human fibroblasts. In spite of a consistent 40% decrease in total polysomal ³H-uridine labeled RNA, a 1.5- to 2-fold increase in extractable mRNA was observed. These data provide direct evidence that protein synthesis inhibition stimulates the appearance of cytoplasmic mRNA and/or completely blocks its degradation and, are consistent with the hypothesis that mRNA accumulation partly underlies the superinduction phenomena.     

Effects of insulin,pertussis toxin and cholera toxin on protein synthesis and diacylglycerol production in 3T3 fibroblasts: Evidence for a G-protein mediated activation of phospholipase C in the insulin signal mechanism

The rapid increase in protein synthesis that occurs on addition of insulin (1 mU/ml) to stepped-down 3T3 cells was blocked by pre-incubation of the cells with pertussis toxin. Cholera toxin on the other hand stimulated protein synthesis and this effect was insensitive to actinomycin D and inhibited by pro-treatment of the cells with phorbol dibutyrate to deplete cell protein kinase C. Insulin was found to cause a rapid and transient increase in diacylglycerol (DAG) synthesis. The insulin-induced increase in diacylglycerol was blocked by pertussis toxin. Exogenous DAG (10 M) stimulated protein synthesis within 1 hour. The results suggest that insuIin stimulates ribosomal activity through a signal mechanism that involves a G-protein mediated activation of phospholipase C to increase DAG levels.     

GTP gamma S inhibits early c-myc protein accumulation but not DNA synthesis in Swiss 3T3 fibroblasts

Quiescent Swiss 3T3 fibroblasts stimulated with epidermal growth factor and insulin showed large transient increases in c-myc mRNA and c-myc protein accumulation which were maximal at about 2 h after addition of the co-mitogens. When the cells were loaded with 0.1 mM of guanosine 5'-O-(3-thiotriphosphate) (GTP gamma S) by transient permeabilisation immediately before mitogenic stimulation, the increase in c-myc mRNA was similar to that observed in unloaded cells but the corresponding c-myc protein peak was reduced by at least 95%. The GTP gamma S completely blocked incorporation of [35S]methionine into cell proteins for 3-4 h after addition of the mitogens, but not thereafter, and caused a delay in the subsequent onset of DNA synthesis by the same period. The data show that less than 5% of the early increase in c-myc protein normally observed after mitogenic stimulation is required for its obligatory role in the progression of cells to S phase implied by other evidence.     

Involvement of PI 3-kinase and activated ERK in facilitating insulin-stimulated triacylglycerol synthesis in hepatocytes

Triacylglycerol synthesis was studied in hepatocytes isolated from fasted/refed rats by EDTA perfusion. Insulin induced a 1.5-fold increase in glucose incorporation into triacylglycerol. Insulin-stimulated triacylglycerol synthesis and insulin-stimulated protein kinase B/Akt activity were inhibited by the phosphatidylinositol 3-kinase inhibitors wortmannin and LY 294002, and the mitogen-activated protein kinase kinase inhibitor PD 98059. Inhibition of p70 ribosomal protein-S6 kinase with rapamycin was without effect. Insulin-stimulated pyruvate dehydrogenase activity was abolished by phosphatidylinositol 3-kinase inhibitors. No effect of insulin on acetyl CoA carboxylase activity was observed.