Protein kinase C-lambda knockout in embryonic stem cells and adipocytes impairs insulin-stimulated glucose transport

Atypical protein kinase C (aPKC) isoforms have been suggested to mediate insulin effects on glucose transport in adipocytes and other cells. To more rigorously test this hypothesis, we generated mouse embryonic stem (ES) cells and ES-derived adipocytes in which both aPKC-lambda alleles were knocked out by recombinant methods. Insulin activated PKC-lambda and stimulated glucose transport in wild-type (WT) PKC-lambda(+/+), but not in knockout PKC-lambda(-/-), ES cells. However, insulin-stimulated glucose transport was rescued by expression of WT PKC-lambda in PKC-lambda(-/-) ES cells. Surprisingly, insulin-induced increases in both PKC-lambda activity and glucose transport were dependent on activation of proline-rich tyrosine protein kinase 2, the ERK pathway, and phospholipase D (PLD) but were independent of phosphatidylinositol 3-kinase (PI3K) in PKC-lambda(+/+) ES cells. Interestingly, this dependency was completely reversed after differentiation of ES cells to adipocytes, i.e. insulin effects on PKC-lambda and glucose transport were dependent on PI3K, rather than proline-rich tyrosine protein kinase 2/ERK/PLD. As in ES cells, insulin effects on glucose transport were absent in PKC-lambda(-/-) adipocytes but were rescued by expression of WT PKC-lambda in these adipocytes. Our findings suggest that insulin activates aPKCs and glucose transport in ES cells by a newly recognized PI3K-independent ERK/PLD-dependent pathway and provide a compelling line of evidence suggesting that aPKCs are required for insulin-stimulated glucose transport, regardless of whether aPKCs are activated by PI3K-dependent or PI3K-independent mechanisms.     

Role of PKC isoforms in glucose transport in 3T3-L1 adipocytes: insignificance of atypical PKC

To elucidate the involvement of protein kinase C (PKC) isoforms in insulin-induced and phorbol ester-induced glucose transport, we expressed several PKC isoforms, conventional PKC-alpha, novel PKC-delta, and atypical PKC isoforms of PKC-lambda and PKC-zeta, and their mutants in 3T3-L1 adipocytes using an adenovirus-mediated gene transduction system. Endogenous expression and the activities of PKC-alpha and PKC-lambda/zeta, but not of PKC-delta, were detected in 3T3-L1 adipocytes. Overexpression of each wild-type PKC isoform induced a large amount of PKC activity in 3T3-L1 adipocytes. Phorbol 12-myristrate 13-acetate (PMA) activated PKC-alpha and exogenous PKC-delta but not atypical PKC-lambda/zeta. Insulin also activated the overexpressed PKC-delta but not PKC-alpha. Expression of the wild-type PKC-alpha or PKC-delta resulted in significant increases in glucose transport activity in the basal and PMA-stimulated states. Dominant-negative PKC-alpha expression, which inhibited the PMA activation of PKC-alpha, decreased in PMA-stimulated glucose transport. Glucose transport activity in the insulin-stimulated state was increased by the expression of PKC-delta but not of PKC-alpha. These findings demonstrate that both conventional and novel PKC isoforms are involved in PMA-stimulated glucose transport and that other novel PKC isoforms could participate in PMA-stimulated and insulin-stimulated glucose transport. Atypical PKC-lambda/zeta was not significantly activated by insulin, and expression of the wild-type, constitutively active, and dominant-negative mutants of atypical PKC did not affect either basal or insulin-stimulated glucose transport. Thus atypical PKC enzymes do not play a major role in insulin-stimulated glucose transport in 3T3-L1 adipocytes.     

Requirements for pYXXM motifs in Cbl for binding to the p85 subunit of phosphatidylinositol 3-kinase and Crk, and activation of atypical protein kinase C and glucose transport during insulin action in 3T3/L1 adipocytes

Cbl is phosphorylated by the insulin receptor and reportedly functions within the flotillin/CAP/Cbl/Crk/C3G/TC10 complex during insulin-stimulated glucose transport in 3T3/L1 adipocytes. Cbl, via pYXXM motifs at tyrosine-371 and tyrosine-731, also activates phosphatidylinositol (PI) 3-kinase, which is required to activate atypical protein kinase C (aPKC) and glucose transport during thiazolidinedione action in 3T3/L1 and human adipocytes [Miura et al. (2003) Biochemistry 42, 14335-14341]. Presently, we have examined the importance of Cbl in activating PI 3-kinase and aPKC during insulin action in 3T3/L1 adipocytes by expressing Y371F and Y731F Cbl mutants, which nullify pYXXM binding of Cbl to SH2 domains of downstream effectors. Interestingly, these mutants inhibited insulin-induced increases in (a) binding of Cbl to both Crk and the p85 subunit of PI 3-kinase, (b) activation of Cbl-dependent PI 3-kinase, (c) activation and translocation of aPKC to the plasma membrane, (d) translocation of Glut4 to the plasma membrane, (e) and glucose transport. Importantly, coexpression of wild-type Cbl reversed the inhibitory effects of Cbl mutants. In contrast to Cbl-dependent PI 3-kinase, Cbl mutants did not significantly inhibit the activation of PI 3-kinase by IRS-1, which is also required during insulin action. Our findings suggest that (a) Cbl uses pYXXM motifs to simultaneously activate PI 3-kinase and Crk/C3G/TC10 pathways and (b) Cbl, along with IRS-1, functions upstream of PI 3-kinase and aPKCs during insulin-stimulated glucose transport in 3T3/L1 adipocytes.     

Insulin substrates 1 and 2 are corequired for activation of atypical protein kinase C and Cbl-dependent phosphatidylinositol 3-kinase during insulin action in immortalized brown adipocytes

Phosphatidylinositol 3-kinase (PI3K)-dependent activation of atypical protein kinase C (aPKC) is required for insulin-stimulated glucose transport. Although insulin receptor substrate-1 (IRS-1) and IRS-2, among other factors, activate PI3K, there is little information on the relative roles of IRS-1and IRS-2 during aPKC activation by insulin action in specific cell types. Presently, we have used immortalized brown adipocytes in which either IRS-1 or IRS-2 has been knocked out by recombinant methods to examine IRS-1 and IRS-2 requirements for activation of aPKC. We have also used these adipocytes to see if IRS-1 and IRS-2 are required for activation of Cbl, which is required for insulin-stimulated glucose transport and has been found to function upstream of both PI3K/aPKC and Crk during thiazolidinedione action in 3T3/L1 adipocytes [Miura et al. (2003) Biochemistry 42, 14335]. In brown adipocytes in which either IRS-1 or IRS-2 was knocked out, insulin-induced increases in aPKC activity and glucose transport were markedly diminished. These effects of insulin on aPKC and glucose transport were fully restored by retroviral-mediated expression of IRS-1 or IRS-2 in their respective knockout cells. Knockout of IRS-1 or IRS-2 also inhibited insulin-induced increases in Cbl binding to the p85 subunit of PI3K, which, along with IRS-1/2, may be required for activation of PI3K, aPKC, and glucose transport during insulin action in 3T3/L1 adipocytes. These findings provide evidence that directly links both IRS-1 and IRS-2 to aPKC activation in immortalized brown adipocytes, and further suggest that IRS-1 and IRS-2 are required for the activation of Cbl/PI3K during insulin action in these cells.     

Okadaic acid activates atypical protein kinase C (zeta/lambda) in rat and 3T3/L1 adipocytes. An apparent requirement for activation of Glut4 translocation and glucose transport.

Okadaic acid, an inhibitor of protein phosphatases 1 and 2A, is known to provoke insulin-like effects on GLUT4 translocation and glucose transport, but the underlying mechanism is obscure. Presently, we found in both rat adipocytes and 3T3/L1 adipocytes that okadaic acid provoked partial insulin-like increases in glucose transport, which were inhibited by phosphatidylinositol (PI) 3-kinase inhibitors, wortmannin and LY294002, and inhibitors of atypical protein kinase C (PKC) isoforms, zeta and lambda. Moreover, in both cell types, okadaic acid provoked increases in the activity of immunoprecipitable PKC-zeta/lambda by a PI 3-kinase-dependent mechanism. In keeping with apparent PI 3-kinase dependence of stimulatory effects of okadaic acid on glucose transport and PKC-zeta/lambda activity, okadaic acid provoked insulin-like increases in membrane PI 3-kinase activity in rat adipocytes; the mechanism for PI 3-kinase activation was uncertain, however, because it was not apparent in phosphotyrosine immunoprecipitates. Of further note, okadaic acid provoked partial insulin-like increases in the translocation of hemagglutinin antigen-tagged GLUT4 to the plasma membrane in transiently transfected rat adipocytes, and these stimulatory effects on hemagglutinin antigen-tagged GLUT4 translocation were inhibited by co-expression of kinase-inactive forms of PKC-zeta and PKC-lambda but not by a double mutant (T308A, S473A), activation-resistant form of protein kinase B. Our findings suggest that, as with insulin, PI 3-kinase-dependent atypical PKCs, zeta and lambda, are required for okadaic acid-induced increases in GLUT4 translocation and glucose transport in rat adipocytes and 3T3/L1 adipocytes.     

Function and dysfunction of aPKC isoforms for glucose transport in insulin-sensitive and insulin-resistant states

Considerable evidence suggests that atypical protein kinase C isoforms (aPKCs), serving downstream of insulin receptor substrates and phosphatidylinositol (PI) 3-kinase, are required for insulin-stimulated glucose transport in skeletal muscle and adipocytes. More recent findings further suggest that aPKCs are activated and required for glucose transport responses while serving downstream of 1) proline-rich tyrosine kinase-2, extracellular signal-regulated kinase, and phospholipase D, as during the actions of high concentrations of carbohydrates (glucose, sorbitol) and agents that activate 5'-AMP-activated protein kinase (exercise, 5-amino-imidazole-4-carboxamide-1-beta-D-riboside, dinitrophenol), and 2) Cbl-dependent PI 3-kinase, as during the action of insulin-sensitizing thiazolidinediones. It therefore seems reasonable to postulate that, regardless of the initial mechanism, aPKCs may serve as terminal molecular switches for activating glucose transport responses. This postulation is of critical importance, as it now appears that insulin-stimulated aPKC activation is compromised in various states of insulin resistance.     

Cbl PYXXM motifs activate the P85 subunit of phosphatidylinositol 3-kinase, Crk, atypical protein kinase C, and glucose transport during thiazolidinedione action in 3T3/L1 and human adipocytes

The thiazolidinedione (TZD), rosiglitazone, has previously been found to tyrosine-phosphorylate Cbl and activate Cbl-dependent phosphatidylinositol (PI) 3-kinase and atypical protein kinase Cs (aPKCs) while stimulating glucose transport in 3T3/L1 adipocytes. Presently, the role of Cbl in rosiglitazone action was further assessed in both 3T3/L1 and human adipocytes by expressing Y371F and/or Y731F mutant forms of Cbl that nullified the functionality of canonical pYXXM motifs in Cbl. These mutants diminished the interaction of Cbl with the p85 subunit of PI 3-kinase and inhibited subsequent increases in Cbl-dependent PI 3-kinase activity, aPKC activity, and glucose transport. These mutants also inhibited the interaction of Cbl with Crk, which has been implicated in the activation of other PI 3-kinase-independent signaling factors that have been found to be required during activation of glucose transport by insulin and other agonists. We conclude that pYXXM motifs in Cbl serve to activate PI 3-kinase-dependent and possibly PI 3-kinase-independent pathways that are required for TZD-dependent glucose transport in adipocytes.     

Dependence of insulin-stimulated glucose transporter 4 translocation on 3-phosphoinositide-dependent protein kinase-1 and its target threonine-410 in the activation loop of protein kinase C-zeta.

Previous studies have suggested that 1) atypical protein kinase C (PKC) isoforms are required for insulin stimulation of glucose transport, and 2) 3-phosphoinositide-dependent protein kinase-1 (PDK-1) is required for activation of atypical PKCs. Presently, we evaluated the role of PDK-1, both in the activation of PKC-zeta, and the translocation of epitope-tagged glucose transporter 4 (GLUT4) to the plasma membrane, during insulin action in transiently transfected rat adipocytes. Overexpression of wild-type PDK-1 provoked increases in the activity of cotransfected hemagglutinin (HA)-tagged PKC-zeta and concomitantly enhanced HA-tagged GLUT4 translocation. Expression of both kinase-inactive PDK-1 and an activation-resistant form of PKC-zeta that is mutated at Thr-410, the immediate target of PDK-1 in the activation loop of PKC-zeta, inhibited insulin-induced increases in both HA-PKC-zeta activity and HA-GLUT4 translocation to the same extent as kinase-inactive PKC-zeta. Moreover, the inhibitory effects of kinase-inactive PDK-1 were fully reversed by cotransfection of wild-type PDK-1 and partly reversed by wild-type PKC-zeta, but not by wild-type PKB. In contrast to the T410A PKC-zeta mutant, an analogous double mutant of PKB (T308A/S473A) that is resistant to PDK-1 activation had only a small effect on insulin-stimulated HA-GLUT4 translocation and did not inhibit HA-GLUT4 translocation induced by overexpression of wild-type PDK-1. Our findings suggest that both PDK-1 and its downstream target, Thr-410 in the activation loop of PKC-zeta, are required for insulin-stimulated glucose transport.     

Thiazolidinedione treatment enhances insulin effects on protein kinase C-zeta /lambda activation and glucose transport in adipocytes of nondiabetic and Goto-Kakizaki type II diabetic rats

We evaluated effects of the thiazolidinedione, rosiglitazone, on insulin-induced activation of protein kinase C (PKC)-zeta/lambda and glucose transport in adipocytes of Goto-Kakizaki (GK)-diabetic and nondiabetic rats. Insulin effects on PKC-zeta/lambda and 2-deoxyglucose uptake were diminished by approximately 50% in GK adipocytes, as compared with control adipocytes. This defect in insulin-induced PKC-zeta/lambda activation was associated with diminished activation of IRS-1-dependent phosphatidylinositol (PI) 3-kinase, and was accompanied by diminished phosphorylation of threonine 410 in the activation loop of PKC-zeta; in contrast, protein kinase B (PKB) activation and phosphorylation were not significantly altered. Rosiglitazone completely reversed defects in insulin-stimulated 2-deoxyglucose uptake, PKCzeta/lambda enzyme activity and PKC-zeta threonine 410 phosphorylation, but had no effect on PI 3-kinase activation or PKB activation/phosphorylation in GK adipocytes. Similarly, in adipocytes of nondiabetic rats, rosiglitazone provoked increases in insulin-stimulated 2-deoxyglucose uptake, PKC-zeta/lambda enzyme activity and phosphorylation of both threonine 410 activation loop and threonine 560 autophosphorylation sites in PKC-zeta, but had no effect on PI 3-kinase activation or PKB activation/phosphorylation. Our findings suggest that (a) decreased effects of insulin on glucose transport in adipocytes of GK-diabetic rats are due at least in part to diminished phosphorylation/activation of PKC-zeta/lambda, and (b) thiazolidinediones enhance glucose transport responses to insulin in adipocytes of both diabetic and nondiabetic rats through increases in phosphorylation/activation of PKC-zeta/lambda.     

An inhibitor of p38 mitogen-activated protein kinase prevents insulin-stimulated glucose transport but not glucose transporter translocation in 3T3-L1 adipocytes and L6 myotubes

The precise mechanisms underlying insulin-stimulated glucose transport still require investigation. Here we assessed the effect of SB203580, an inhibitor of the p38 MAP kinase family, on insulin-stimulated glucose transport in 3T3-L1 adipocytes and L6 myotubes. We found that SB203580, but not its inactive analogue (SB202474), prevented insulin-stimulated glucose transport in both cell types with an IC50 similar to that for inhibition of p38 MAP kinase (0.6 microM). Basal glucose uptake was not affected. Moreover, SB203580 added only during the transport assay did not inhibit basal or insulin-stimulated transport. SB203580 did not inhibit insulin-stimulated translocation of the glucose transporters GLUT1 or GLUT4 in 3T3-L1 adipocytes as assessed by immunoblotting of subcellular fractions or by immunofluorescence of membrane lawns. L6 muscle cells expressing GLUT4 tagged on an extracellular domain with a Myc epitope (GLUT4myc) were used to assess the functional insertion of GLUT4 into the plasma membrane. SB203580 did not affect the insulin-induced gain in GLUT4myc exposure at the cell surface but largely reduced the stimulation of glucose uptake. SB203580 had no effect on insulin-dependent insulin receptor substrate-1 phosphorylation, association of the p85 subunit of phosphatidylinositol 3-kinase with insulin receptor substrate-1, nor on phosphatidylinositol 3-kinase, Akt1, Akt2, or Akt3 activities in 3T3-L1 adipocytes. In conclusion, in the presence of SB203580, insulin caused normal translocation and cell surface membrane insertion of glucose transporters without stimulating glucose transport. We propose that insulin stimulates two independent signals contributing to stimulation of glucose transport: phosphatidylinositol 3-kinase leads to glucose transporter translocation and a pathway involving p38 MAP kinase leads to activation of the recruited glucose transporter at the membrane.     

Activation of the ERK pathway and atypical protein kinase C isoforms in exercise- and aminoimidazole-4-carboxamide-1-beta-D-riboside (AICAR)-stimulated glucose transport

Exercise increases glucose transport in muscle by activating 5'-AMP-activated protein kinase (AMPK), but subsequent events are unclear. Presently, we examined the possibility that AMPK increases glucose transport through atypical protein kinase Cs (aPKCs) by activating proline-rich tyrosine kinase-2 (PYK2), ERK pathway components, and phospholipase D (PLD). In mice, treadmill exercise rapidly activated ERK and aPKCs in mouse vastus lateralis muscles. In rat extensor digitorum longus (EDL) muscles, (a) AMPK activator, 5-aminoimidazole-4-carboxamide-1-beta-d-riboside (AICAR), activated PYK2, ERK and aPKCs; (b) effects of AICAR on ERK and aPKCs were blocked by tyrosine kinase inhibitor, genistein, and MEK1 inhibitor, PD98059; and (c) effects of AICAR on aPKCs and 2-deoxyglucose (2-DOG) uptake were inhibited by genistein, PD98059, and PLD-inhibitor, 1-butanol. Similarly, in L6 myotubes, (a) AICAR activated PYK2, ERK, PLD, and aPKCs; (b) effects of AICAR on ERK were inhibited by genistein, PD98059, and expression of dominant-negative PYK2; (c) effects of AICAR on PLD were inhibited by MEK1 inhibitor UO126; (d) effects of AICAR on aPKCs were inhibited by genistein, PD98059, 1-butanol, and expression of dominant-negative forms of PYK2, GRB2, SOS, RAS, RAF, and ERK; and (e) effects of AICAR on 2DOG uptake/GLUT4 translocation were inhibited by genistein, PD98059, UO126, 1-butanol, cell-permeable myristoylated PKC-zeta pseudosubstrate, and expression of kinase-inactive RAF, ERK, and PKC-zeta. AMPK activator dinitrophenol had effects on ERK, aPKCs, and 2-DOG uptake similar to those of AICAR. Our findings suggest that effects of exercise on glucose transport that are dependent on AMPK are mediated via PYK2, the ERK pathway, PLD, and aPKCs.     

Identification of a Role for CLASP2 in Insulin Action

Insulin stimulates the mobilization of glucose transporter 4 (GLUT4) storage vesicles to the plasma membrane, resulting in an influx of glucose into target tissues such as muscle and fat. We present evidence that CLIP-associating protein 2 (CLASP2), a protein previously unassociated with insulin action, is responsive to insulin stimulation. Using mass spectrometry-based protein identification combined with phosphoantibody immunoprecipitation in L6 myotubes, we detected a 4.8-fold increase of CLASP2 in the anti-phosphoserine immunoprecipitates upon insulin stimulation. Western blotting of CLASP2 immunoprecipitates with the phosphoantibody confirmed the finding that CLASP2 undergoes insulin-stimulated phosphorylation, and a number of novel phosphorylation sites were identified. Confocal imaging of L6 myotubes revealed that CLASP2 colocalizes with GLUT4 at the plasma membrane within areas of insulin-mediated cortical actin remodeling. CLASP2 is responsible for directing the distal end of microtubules to the cell cortex, and it has been shown that GLUT4 travels along microtubule tracks. In support of the concept that CLASP2 plays a role in the trafficking of GLUT4 at the cell periphery, CLASP2 knockdown by siRNA in L6 myotubes interfered with insulin-stimulated GLUT4 localization to the plasma membrane. Furthermore, siRNA mediated knockdown of CLASP2 in 3T3-L1 adipocytes inhibited insulin-stimulated glucose transport. We therefore propose a new model for CLASP2 in insulin action, where CLASP2 directs the delivery of GLUT4 to cell cortex landing zones important for insulin action.     

Glucose activates protein kinase C-zeta /lambda through proline-rich tyrosine kinase-2, extracellular signal-regulated kinase, and phospholipase D: a novel mechanism for activating glucose transporter translocation.

Insulin controls glucose uptake by translocating GLUT4 and other glucose transporters to the plasma membrane in muscle and adipose tissues by a mechanism that appears to require protein kinase C (PKC)-zeta/lambda operating downstream of phosphatidylinositol 3-kinase. In diabetes mellitus, insulin-stimulated glucose uptake is diminished, but with hyperglycemia, uptake is maintained but by uncertain mechanisms. Presently, we found that glucose acutely activated PKC-zeta/lambda in rat adipocytes and rat skeletal muscle preparations by a mechanism that was independent of phosphatidylinositol 3-kinase but, interestingly, dependent on the apparently sequential activation of the dantrolene-sensitive, nonreceptor proline-rich tyrosine kinase-2; components of the extracellular signal-regulated kinase (ERK) pathway, including, GRB2, SOS, RAS, RAF, MEK1 and ERK1/2; and, most interestingly, phospholipase D, thus yielding increases in phosphatidic acid, a known activator of PKC-zeta/lambda. This activation of PKC-zeta/lambda, moreover, appeared to be required for glucose-induced increases in GLUT4 translocation and glucose transport in adipocytes and muscle cells. Our findings suggest the operation of a novel pathway for activating PKC-zeta/lambda and glucose transport.     

cDNA cloning and functional characterization of a novel splice variant of c-Cbl-associated protein from mouse skeletal muscle

c-Cbl-associated protein (CAP) is an SH3-containing adapter protein that binds to the proto-oncogene c-Cbl. Recent work suggests that signaling through these molecules is involved in the regulation of insulin-stimulated glucose uptake in 3T3-L1 adipocytes. Skeletal muscle is the major site of insulin-stimulated glucose disposal but there have been no reports of CAP function in this tissue. Using RT-PCR of mouse skeletal muscle RNA, we discovered a novel splice variant of CAP (CAPSM; GenBank Accession No. AF521593) that is different from the adipocyte form by inclusion of a novel 168 bp fragment. This fragment encodes a peptide sequence that shows very high similarity with exon 25 of the human homologue of CAP (SORBS1). To understand the function of CAPSM in glucose uptake regulation, L6 myotubes were transfected with either CAPSM or a truncated CAPSM devoid of all three SH3-binding domains (CAPDeltaSH3), which prevents CAP association with c-Cbl. Transfection with CAPDeltaSH3 decreased insulin-stimulated 2-deoxyglucose (2-DG) uptake and reduced c-Cbl phosphorylation. In contrast, transfection of L6 myotubes with CAPDeltaSH3 had no effect on dinitrophenol (DNP)- or hypoxia-stimulated glucose uptake, stimuli that work through insulin-independent mechanisms for the regulation of glucose uptake. These data demonstrate the existence of a novel CAP isoform expressed in skeletal muscle, and suggest the involvement of the CAP/Cbl pathway in the regulation of insulin-stimulated glucose uptake in L6 myotubes.     

A dominant-negative p38 MAPK mutant and novel selective inhibitors of p38 MAPK reduce insulin-stimulated glucose uptake in 3T3-L1 adipocytes without affecting GLUT4 translocation

Participation of p38 mitogen-activated protein kinase (p38) in insulin-induced glucose uptake was suggested using pyridinylimidazole p38 inhibitors (e.g. SB203580). However, the role of p38 in insulin action remains controversial. We further test p38 participation in glucose uptake using a dominant-negative p38 mutant and two novel pharmacological p38 inhibitors related to but different from SB203580. We present the structures and activities of the azaazulene pharmacophores A291077 and A304000. p38 kinase activity was inhibited in vitro by A291077 and A304000 (IC(50) = 0.6 and 4.7 microm). At higher concentrations A291077 but not A304000 inhibited JNK2alpha (IC(50) = 3.5 microm). Pretreatment of 3T3-L1 adipocytes and L6 myotubes expressing GLUT4myc (L6-GLUT4myc myotubes) with A291077, A304000, SB202190, or SB203580 reduced insulin-stimulated glucose uptake by 50-60%, whereas chemical analogues inert toward p38 were ineffective. Expression of an inducible, dominant-negative p38 mutant in 3T3-L1 adipocytes reduced insulin-stimulated glucose uptake. GLUT4 translocation to the cell surface, immunodetected on plasma membrane lawns of 3T3-L1 adipocytes or on intact L6-GLUT4myc myotubes, was not altered by chemical or molecular inhibition of p38. We propose that p38 contributes to enhancing GLUT4 activity, thereby increasing glucose uptake. In addition, the azaazulene class of inhibitors described will be useful to decipher cellular actions of p38 and JNK.     

The Effects of Propionate and Valerate on Insulin Responsiveness for Glucose Uptake in 3T3-L1 Adipocytes and C2C12 Myotubes via G Protein-Coupled Receptor 41

Since insulin resistance can lead to hyperglycemia, improving glucose uptake into target tissues is critical for regulating blood glucose levels. Among the free fatty acid receptor (FFAR) family of G protein-coupled receptors, GPR41 is known to be the Gα_i/o-coupled receptor for short-chain fatty acids (SCFAs) such as propionic acid (C3) and valeric acid (C5). This study aimed to investigate the role of GPR41 in modulating basal and insulin-stimulated glucose uptake in insulin-sensitive cells including adipocytes and skeletal muscle cells. Expression of GPR41 mRNA and protein was increased with maximal expression at differentiation day 8 for 3T3-L1 adipocytes and day 6 for C2C12 myotubes. GPR41 protein was also expressed in adipose tissues and skeletal muscle. After analyzing dose-response relationship, 300 µM propionic acid or 500 µM valeric acid for 30 min incubation was used for the measurement of glucose uptake. Both propionic acid and valeric acid increased insulin-stimulated glucose uptake in 3T3-L1 adipocyte, which did not occur in cells transfected with siRNA for GPR41 (siGPR41). In C2C12 myotubes, these SCFAs increased basal glucose uptake, but did not potentiate insulin-stimulated glucose uptake, and siGPR41 treatment reduced valerate-stimulated basal glucose uptake. Therefore, these findings indicate that GPR41 plays a role in insulin responsiveness enhanced by both propionic and valeric acids on glucose uptake in 3T3-L1 adipocytes and C2C12 myotubes, and in valerate-induced increase in basal glucose uptake in C2C12 myotubes.     

Insulin activates protein kinases C-zeta and C-lambda by an autophosphorylation-dependent mechanism and stimulates their translocation to GLUT4 vesicles and other membrane fractions in rat adipocytes.

In rat adipocytes, insulin provoked rapid increases in (a) endogenous immunoprecipitable combined protein kinase C (PKC)-zeta/lambda activity in plasma membranes and microsomes and (b) immunoreactive PKC-zeta and PKC-lambda in GLUT4 vesicles. Activity and autophosphorylation of immunoprecipitable epitope-tagged PKC-zeta and PKC-lambda were also increased by insulin in situ and phosphatidylinositol 3,4,5-(PO(4))(3) (PIP(3)) in vitro. Because phosphoinositide-dependent kinase-1 (PDK-1) is required for phosphorylation of activation loops of PKC-zeta and protein kinase B, we compared their activation. Both RO 31-8220 and myristoylated PKC-zeta pseudosubstrate blocked insulin-induced activation and autophosphorylation of PKC-zeta/lambda but did not inhibit PDK-1-dependent (a) protein kinase B phosphorylation/activation or (b) threonine 410 phosphorylation in the activation loop of PKC-zeta. Also, insulin in situ and PIP(3) in vitro activated and stimulated autophosphorylation of a PKC-zeta mutant, in which threonine 410 is replaced by glutamate (but not by an inactivating alanine) and cannot be activated by PDK-1. Surprisingly, insulin activated a truncated PKC-zeta that lacks the regulatory (presumably PIP(3)-binding) domain; this may reflect PIP(3) effects on PDK-1 or transphosphorylation by endogenous full-length PKC-zeta. Our findings suggest that insulin activates both PKC-zeta and PKC-lambda in plasma membranes, microsomes, and GLUT4 vesicles by a mechanism requiring increases in PIP(3), PDK-1-dependent phosphorylation of activation loop sites in PKC-zeta and lambda, and subsequent autophosphorylation and/or transphosphorylation.     

Defining the contribution of AMP-activated protein kinase (AMPK) and protein kinase C (PKC) in regulation of glucose uptake by metformin in skeletal muscle cells

The importance of AMP-activated protein kinase (AMPK) and protein kinase C (PKC) as effectors of metformin (Met) action on glucose uptake (GU) in skeletal muscle cells was investigated. GU in L6 myotubes was stimulated 2-fold following 16 h of Met treatment and acutely enhanced by insulin in an additive fashion. Insulin-stimulated GU was sensitive to PI3K inhibition, whereas that induced by Met was not. Met and its related biguanide, phenformin, stimulated AMPK activation/phosphorylation to a level comparable with that induced by the AMPK activator, 5-amino-1-β-d-ribofuranosyl-imidazole-4-carboxamide (AICAR). However, the increase in GU elicited by AICAR was significantly lower than that induced by either biguanide. Expression of a constitutively active AMPK mimicked the effects of AICAR on GU, whereas a dominant interfering AMPK or shRNA silencing of AMPK prevented AICAR-stimulated GU and Met-induced AMPK signaling but only repressed biguanide-stimulated GU by ～20%. Consistent with this, analysis of GU in muscle cells from α1(-/-)/α2(-/-) AMPK-deficient mice revealed a significant retention of Met-stimulated GU, being reduced by ～35% compared with that of wild type cells. Atypical PKCs (aPKCs) have been implicated in Met-stimulated GU, and in line with this, Met and phenformin induced activation/phosphorylation of aPKC in L6 myotubes. However, although cellular depletion of aPKC (>90%) led to loss in biguanide-induced aPKC phosphorylation, it had no effect on Met-stimulated GU, whereas inhibitors targeting novel/conventional PKCs caused a significant reduction in biguanide-induced GU. Our findings indicate that although Met activates AMPK, a significant component of Met-stimulated GU in muscle cells is mediated via an AMPK-independent mechanism that involves novel/conventional PKCs.     

Knockdown of NYGGF4 increases glucose transport in C2C12 mice skeletal myocytes by activation IRS-1/PI3K/AKT insulin pathway

NYGGF4, an obesity-related gene, is proposed to be involved in the development of insulin resistance. Skeletal muscle is a primary target organ for insulin and NYGGF4 showed a relatively high expression level in skeletal muscle. Therefore, this study aimed to explore the effect of NYGGF4 on insulin sensitivity of skeletal muscle cells. RNA interference (RNAi) was adopted to silence NYGGF4 expression in mice C2C12 skeletal myocytes. A remarkably increased insulin-stimulated glucose uptake and GLUT4 translocation was observed in NYGGF4 silencing C2C12 cells. Importantly, the enhanced glucose uptake induced by NYGGF4 silencing could be abrogated by the PI3K inhibitor LY294002. In addition, the crucial molecules involved in PI3K insulin signaling pathway were detected by western blotting. The results showed that NYGGF4 knockdown dramatically activate the insulin-stimulated phosphorylation of IRS-1 and AKT. Taken together, these data demonstrate that NYGGF4 knockdown increases glucose transport in myocytes by activation of the IRS-1/PI3K/AKT insulin pathway.     

Neuregulin signaling on glucose transport in muscle cells

Neuregulin-1, a growth factor that potentiates myogenesis induces glucose transport through translocation of glucose transporters, in an additive manner to insulin, in muscle cells. In this study, we examined the signaling pathway required for a recombinant active neuregulin-1 isoform (rhHeregulin-beta(1), 177-244, HRG) to stimulate glucose uptake in L6E9 myotubes. The stimulatory effect of HRG required binding to ErbB3 in L6E9 myotubes. PI3K activity is required for HRG action in both muscle cells and tissue. In L6E9 myotubes, HRG stimulated PKBalpha, PKBgamma, and PKCzeta activities. TPCK, an inhibitor of PDK1, abolished both HRG- and insulin-induced glucose transport. To assess whether PKB was necessary for the effects of HRG on glucose uptake, cells were infected with adenoviruses encoding dominant negative mutants of PKBalpha. Dominant negative PKB reduced PKB activity and insulin-stimulated glucose transport but not HRG-induced glucose transport. In contrast, transduction of L6E9 myotubes with adenoviruses encoding a dominant negative kinase-inactive PKCzeta abolished both HRG- and insulin-stimulated glucose uptake. In soleus muscle, HRG induced PKCzeta, but not PKB phosphorylation. HRG also stimulated the activity of p70S6K, p38MAPK, and p42/p44MAPK and inhibition of p42/p44MAPK partially repressed HRG action on glucose uptake. HRG did not affect AMPKalpha(1) or AMPKalpha(2) activities. In all, HRG stimulated glucose transport in muscle cells by activation of a pathway that requires PI3K, PDK1, and PKCzeta, but not PKB, and that shows cross-talk with the MAPK pathway. The PI3K, PDK1, and PKCzeta pathway can be considered as an alternative mechanism, independent of insulin, to induce glucose uptake.