Let us unite against COVID-19 - a New Zealand perspective.

Novel coronavirus 2019 (COVID-19) has shaken the existence of mankind worldwide, including that of New Zealand. In comparison to other countries, New Zealand has had a very low number of confirmed and probable cases as well as COVID-19-related deaths. New Zealand closed its borders and rapidly declared a stringent lockdown to eliminate COVID-19. The country's 'go hard, go early' policy serves as an exemplar for the rest of the world to date. The mysterious nature of COVID-19 has caused tremendous stress and uncertainty leading to universal conflict between public health and state economy. Mental health services and non-government organisations have been proactive in the fight against COVID-19. Though there has been no significant rise in referrals to secondary mental health services to date (4 May 2020), a rapid surge in mental health presentations is widely anticipated. Telehealth may prove to be an efficient and cost-effective tool for the provision of future health services.

Disseminated rhinosporidiosis.

INTRODUCTION: Rhinosporidiosis primarily affects the mucous membranes of the nose and nasopharynx. The disseminated form of this chronic fungal disease is extremely rare. CASE REPORT: The authors present a case of disseminated rhinosporidiosis in an immunocompetent patient with involvement of the skin, subcutaneous tissue, muscle, bone, penis and urethra, and with a long-standing primary lesion in the nose. DISCUSSION: A late or atypical presentation of rhinosporidiosis may cause diagnostic dilemma. Fine needle aspiration cytology of the tumoural lesions may establish the diagnosis. Histopathology is confirmatory. The subcutaneous masses may be solid or cystic. Ulceroproliferative lesions need to be differentiated from malignancies. CONCLUSION: This is the first reported case of truly disseminated rhinosporidiosis with simultaneous involvement of multiple anatomically unrelated sites in a single patient. This is also the first reported case of cystic rhinosporidiosis. The possibility and sequelae of spontaneous regression of rhinosporidiosis are also discussed.

Leprosy mimicry of lupus vulgaris and misdiagnosis of leprosy--a case report.

Leprosy and tuberculosis (TB) both are still rampant in India. Leprosy predominantly presents through skin manifestations whereas cutaneous manifestations of TB though not so frequent but are not rare. Lupus vulgaris (LV), the commonest of all cutaneous manifestations of TB, mimics leprosy very closely and may prompt the examiner to misdiagnose leprosy, especially, by health workers (HW), in a field situation, where leprosy is diagnosed and treated on clinical basis alone as per NLEP guidelines. Because of existing stigmata, such wrong diagnosis can put the patient and the party under psychological stress and creates unnecessary complications.

Looked after children in Dublin and their mental health needs.

Children in care in Ireland have increased by 27% in the last decade. This population is recognized to be among the most vulnerable. This study aims to describe their placement histories, service use and mental health needs. Data was obtained on 174 children (56.5% of eligible sample) with a mean age of 10.83 (SD = 5.04). 114 (65.5%) were in care for three years or more. 29 (16.7%) did not have a SW and 49 (37.7%) had no GP 50 (28.7%) were attending CAMHS. Long term care, frequent placement changes and residential setting were significantly related with poorer outcomes and increased MH contact. Given the increase in numbers in care and the overall decrease in resource allocation to health and social care, individual care planning and prioritizing of resources are essential.

AICAR and metformin, but not exercise, increase muscle glucose transport through AMPK-, ERK-, and PDK1-dependent activation of atypical PKC.

Activators of 5'-AMP-activated protein kinase (AMPK) 5-aminoimidazole-4-carboxamide-1-beta-d-ribofuranoside (AICAR), metformin, and exercise activate atypical protein kinase C (aPKC) and ERK and stimulate glucose transport in muscle by uncertain mechanisms. Here, in cultured L6 myotubes: AICAR- and metformin-induced activation of AMPK was required for activation of aPKC and ERK; aPKC activation involved and required phosphoinositide-dependent kinase 1 (PDK1) phosphorylation of Thr410-PKC-zeta; aPKC Thr410 phosphorylation and activation also required MEK1-dependent ERK; and glucose transport effects of AICAR and metformin were inhibited by expression of dominant-negative AMPK, kinase-inactive PDK1, MEK1 inhibitors, kinase-inactive PKC-zeta, and RNA interference (RNAi)-mediated knockdown of PKC-zeta. In mice, muscle-specific aPKC (PKC-lambda) depletion by conditional gene targeting impaired AICAR-stimulated glucose disposal and stimulatory effects of both AICAR and metformin on 2-deoxyglucose/glucose uptake in muscle in vivo and AICAR stimulation of 2-[(3)H]deoxyglucose uptake in isolated extensor digitorum longus muscle; however, AMPK activation was unimpaired. In marked contrast to AICAR and metformin, treadmill exercise-induced stimulation of 2-deoxyglucose/glucose uptake was not inhibited in aPKC-knockout mice. Finally, in intact rodents, AICAR and metformin activated aPKC in muscle, but not in liver, despite activating AMPK in both tissues. The findings demonstrate that in muscle AICAR and metformin activate aPKC via sequential activation of AMPK, ERK, and PDK1 and the AMPK/ERK/PDK1/aPKC pathway is required for metformin- and AICAR-stimulated increases in glucose transport. On the other hand, although aPKC is activated by treadmill exercise, this activation is not required for exercise-induced increases in glucose transport, and therefore may be a redundant mechanism.

Prevalence of anti HCV, HBsAg and HIV antibodies in high risk recipients of blood and blood products.

Along with hepatitis B virus (HBV) and human immunodeficiency virus (HIV), Hepatitis C virus (HCV) is emerging as a major transfusion hazard. 22 cases of haemophilia (A 19, B 3) and 20 cases of thalassaemia (2 16, E(2) 4) constituted the study group. Patients tested for anti HCV (using third generation ELISA), HBsAg and antibodies to HIV I and II. Prevalence of anti HCV was 54.5% in haemophilics and 5% in thalassaemics. HBsAg was detected in 9.09% haemophilics and 5% thalassaemics. No anti HIV was detected in this cohort. Anti HCV seropositivity in haemophilics has increased compare to previous studies.

Impaired activation of protein kinase C-zeta by insulin and phosphatidylinositol-3,4,5-(PO4)3 in cultured preadipocyte-derived adipocytes and myotubes of obese subjects.

Insulin resistance in obesity is partly due to diminished glucose transport in myocytes and adipocytes, but underlying mechanisms are uncertain. Insulin-stimulated glucose transport requires activation of phosphatidylinositol (PI) 3-kinase (3K), operating downstream of insulin receptor substrate-1. PI3K stimulates glucose transport through increases in PI-3,4,5-(PO(4))(3) (PIP(3)), which activates atypical protein kinase C (aPKC) and protein kinase B (PKB/Akt). However, previous studies suggest that activation of aPKC, but not PKB, is impaired in intact muscles and cultured myocytes of obese subjects. Presently, we examined insulin activation of glucose transport and signaling factors in cultured adipocytes derived from preadipocytes harvested during elective liposuction in lean and obese women. Relative to adipocytes of lean women, insulin-stimulated [(3)H]2-deoxyglucose uptake and activation of insulin receptor substrate-1/PI3K and aPKCs, but not PKB, were diminished in adipocytes of obese women. Additionally, the direct activation of aPKCs by PIP(3) in vitro was diminished in aPKCs isolated from adipocytes of obese women. Similar impairment in aPKC activation by PIP(3) was observed in cultured myocytes of obese glucose-intolerant subjects. These findings suggest the presence of defects in PI3K and aPKC activation that persist in cultured cells and limit insulin-stimulated glucose transport in adipocytes and myocytes of obese subjects.

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.

Rosiglitazone, insulin treatment, and fasting correct defective activation of protein kinase C-zeta/lambda by insulin in vastus lateralis muscles and adipocytes of diabetic rats.

Atypical protein kinases C (PKCs), zeta and lambda, and protein kinase B (PKB) are thought to function downstream of phosphatidylinositol 3-kinase (PI 3-kinase) and regulate glucose transport during insulin action in skeletal muscle and adipocytes. Insulin-stimulated glucose transport is defective in type II diabetes mellitus, and this defect is ameliorated by thiazolidinediones and lowering of blood glucose by chronic insulin therapy or short-term fasting. Presently, we evaluated the effects of these insulin-sensitizing modalities on the activation of insulin receptor substrate-1 (IRS-1)-dependent PI 3-kinase, PKC-zeta/lambda, and PKB in vastus lateralis skeletal muscles and adipocytes of nondiabetic and Goto-Kakizaki (GK) diabetic rats. Insulin provoked rapid increases in the activity of PI 3-kinase, PKC-zeta/lambda, and PKB in muscles and adipocytes of nondiabetic rats, but increases in IRS-1-dependent PI 3-kinase and PKC-zeta/lambda, but not PKB, activity were substantially diminished in GK muscles and adipocytes. Rosiglitazone treatment for 10-14 days, 10-day insulin treatment, and 60-h fasting reversed defects in PKC-zeta/lambda activation in GK muscles and adipocytes and increased glucose transport in GK adipocytes, without necessarily increasing IRS-1-dependent PI 3-kinase or PKB activation. Our findings suggest that insulin-sensitizing modalities, viz. thiazolidinediones, chronic insulin treatment, and short-term fasting, similarly improve defects in insulin-stimulated glucose transport at least partly by correcting defects in insulin-induced activation of PKC-zeta/lambda.

Insulin and PIP3 activate PKC-zeta by mechanisms that are both dependent and independent of phosphorylation of activation loop (T410) and autophosphorylation (T560) sites.

Activation of protein kinase C-zeta (PKC-zeta) by insulin requires phosphatidylinositol (PI) 3-kinase-dependent increases in phosphatidylinositol-3,4,5-(PO(4))(3) (PIP(3)) and phosphorylation of activation loop and autophosphorylation sites, but actual mechanisms are uncertain. Presently, we examined: (a) acute effects of insulin on threonine (T)-410 loop phosphorylation and (b) effects of (i) alanine (A) and glutamate (E) mutations at T410 loop and T560 autophosphorylation sites and (ii) N-terminal truncation on insulin-induced activation of PKC-zeta. Insulin acutely increased T410 loop phosphorylation, suggesting enhanced action of 3-phosphoinositide-dependent protein kinase-1 (PDK-1). Despite increasing in vitro autophosphorylation of wild-type PKC-zeta and T410E-PKC-zeta, insulin and PIP(3) did not stimulate autophosphorylation of T560A, T560E, T410A/T560E, T410E/T560A, or T410E/T560E mutant forms of PKC-zeta; thus, T560 appeared to be the sole autophosphorylation site. Activating effects of insulin and/or PIP(3) on enzyme activity were completely abolished in T410A-PKC-zeta, partially compromised in T560A-PKC-zeta, T410E/T560A-PKC-zeta, and T410A/T560E-PKC-zeta, and largely intact in T410E-PKC-zeta, T560E-PKC-zeta, and T410E/T560E-PKC-zeta. Activation of the T410E/T560E mutant suggested a phosphorylation-independent mechanism. As functional correlates, insulin effects on epitope-tagged GLUT4 translocation were compromised by expression of T410A-PKC-zeta, T560A-PKC-zeta, T410E/T560A, and T410A/T560E-PKC-zeta but not T410E-PKC-zeta, T560E-PKC-zeta, or T410E/T560E-PKC-zeta. Insulin, but not PIP(3), activated truncated, pseudosubstrate-lacking forms of PKC-zeta and PKC-lambda by a wortmannin-sensitive mechanism, apparently involving PI 3-kinase/PDK-1-dependent phosphorylations but independent of PIP(3)-dependent conformational activation. Our findings suggest that insulin, via PIP(3), provokes increases in PKC-zeta enzyme activity through (a) PDK-1-dependent T410 loop phosphorylation, (b) T560 autophosphorylation, and (c) phosphorylation-independent/conformational-dependent relief of pseudosubstrate autoinhibition.

Effects of adenoviral gene transfer of wild-type, constitutively active, and kinase-defective protein kinase C-lambda on insulin-stimulated glucose transport in L6 myotubes.

We used adenoviral gene transfer methods to evaluate the role of atypical protein kinase Cs (PKCs) during insulin stimulation of glucose transport in L6 myotubes. Expression of wild-type PKC-lambda potentiated maximal and half-maximal effects of insulin on 2-deoxyglucose uptake, but did not alter basal uptake. Expression of constitutively active PKC-lambda enhanced basal 2-deoxyglucose uptake to virtually the same extent as that observed during insulin treatment. In contrast, expression of kinase-defective PKC-lambda completely blocked insulin-stimulated, but not basal, 2-deoxyglucose uptake. Similar to alterations in glucose transport, constitutively active PKC-lambda mimicked, and kinase-defective PKC-lambda completely inhibited, insulin effects on GLUT4 glucose transporter translocation to the plasma membrane. Expression of kinase-defective PKC-lambda, in addition to inhibition of atypical PKC enzyme activity, was attended by paradoxical increases in GLUT4 and GLUT1 glucose transporter levels and insulin-stimulated protein kinase B enzyme activity. Our findings suggest that in L6 myotubes, 1) atypical PKCs are required and sufficient for insulin-stimulated GLUT4 translocation and glucose transport; and 2) activation of protein kinase B in the absence of activation of atypical PKCs is insufficient for insulin-induced activation of glucose transport.

Glucose activates mitogen-activated protein kinase (extracellular signal-regulated kinase) through proline-rich tyrosine kinase-2 and the Glut1 glucose transporter.

Glucose serves as both a nutrient and regulator of physiological and pathological processes. Presently, we found that glucose and certain sugars rapidly activated extracellular signal-regulated kinase (ERK) by a mechanism that was: (a) independent of glucose uptake/metabolism and protein kinase C but nevertheless cytochalasin B-inhibitable; (b) dependent upon proline-rich tyrosine kinase-2 (PYK2), GRB2, SOS, RAS, RAF, and MEK1; and (c) amplified by overexpression of the Glut1, but not Glut2, Glut3, or Glut4, glucose transporter. This amplifying effect was independent of glucose uptake but dependent on residues 463-468, IASGFR, in the Glut1 C terminus. Accordingly, glucose effects on ERK were amplified by expression of Glut4/Glut1 or Glut2/Glut1 chimeras containing IASGFR but not by Glut1/Glut4 or Glut1/Glut2 chimeras lacking these residues. Also, deletion of Glut1 residues 469-492 was without effect, but mutations involving serine 465 or arginine 468 yielded dominant-negative forms that inhibited glucose-dependent ERK activation. Glucose stimulated the phosphorylation of tyrosine residues 402 and 881 in PYK2 and binding of PYK2 to Myc-Glut1. Our findings suggest that: (a) glucose activates the GRB2/SOS/RAS/RAF/MEK1/ERK pathway by a mechanism that requires PYK2 and residues 463-468, IASGFR, in the Glut1 C terminus and (b) Glut1 serves as a sensor, transducer, and amplifier for glucose signaling to PYK2 and ERK.

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.

Protein kinase C-zeta and phosphoinositide-dependent protein kinase-1 are required for insulin-induced activation of ERK in rat adipocytes.

The mechanisms used by insulin to activate the multifunctional intracellular effectors, extracellular signal-regulated kinases 1 and 2 (ERK1/2), are only partly understood and appear to vary in different cell types. Presently, in rat adipocytes, we found that insulin-induced activation of ERK was blocked (a) by chemical inhibitors of both phosphatidylinositol 3-kinase (PI3K) and protein kinase C (PKC)-zeta, and, moreover, (b) by transient expression of both dominant-negative Deltap85 PI3K subunit and kinase-inactive PKC-zeta. Further, insulin effects on ERK were inhibited by kinase-inactive 3-phosphoinositide-dependent protein kinase-1 (PDK-1), and by mutation of Thr-410 in the activation loop of PKC-zeta, which is the target of PDK-1 and is essential for PI3K/PDK-1-dependent activation of PKC-zeta. In addition to requirements for PI3K, PDK-1, and PKC-zeta, we found that a tyrosine kinase (presumably the insulin receptor), the SH2 domain of GRB2, SOS, RAS, RAF, and MEK1 were required for insulin effects on ERK in the rat adipocyte. Our findings therefore suggested that PDK-1 and PKC-zeta serve as a downstream effectors of PI3K, and act in conjunction with GRB2, SOS, RAS, and RAF, to activate MEK and ERK during insulin action in rat 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.

Effects of knockout of the protein kinase C beta gene on glucose transport and glucose homeostasis.

The beta-isoform of protein kinase C (PKC) has paradoxically been suggested to be important for both insulin action and insulin resistance as well as for contributing to the pathogenesis of diabetic complications. Presently, we evaluated the effects of knockout of the PKCbeta gene on overall glucose homeostasis and insulin regulation of glucose transport. To evaluate subtle differences in glucose homeostasis in vivo, knockout mice were extensively backcrossed in C57BL/6 mice to diminish genetic differences other than the absence of the PKCbeta gene. PKCbeta-/- knockout offspring obtained through this backcrossing had 10% lower blood glucose levels than those observed in PKCbeta+/+ wild-type offspring in both the fasting state and 30 min after i.p. injection of glucose despite having similar or slightly lower serum insulin levels. Also, compared with commercially obtained C57BL/6-129/SV hybrid control mice, serum glucose levels were similar, and serum insulin levels were similar or slightly lower, in C57BL/6-129/SV hybrid PKCbeta knockout mice in fasting and fed states and after i.p. glucose administration. In keeping with a tendency for slightly lower serum glucose and/or insulin levels in PKCbeta knockout mice, insulin-stimulated 2-deoxyglucose (2-DOG) uptake was enhanced by 50-100% in isolated adipocytes; basal and insulin-stimulated epitope-tagged GLUT4 translocations in adipocytes were increased by 41% and 27%, respectively; and basal 2-DOG uptake was mildly increased by 20-25% in soleus muscles incubated in vitro. The reason for increased 2-DOG uptake and/or GLUT4 translocation in these tissues was uncertain, as there were no significant alterations in phosphatidylinositol 3-kinase activity or activation or in levels of GLUT1 or GLUT4 glucose transporters or other PKC isoforms. On the other hand, increases in 2-DOG uptake may have been partly caused by the loss of PKCbeta1, rather than PKCbeta2, as transient expression of PKCbeta1 selectively inhibited insulin-stimulated translocation of epitope-tagged GLUT4 in adipocytes prepared from PKCbeta knockout mice. Our findings suggest that 1) PKCbeta is not required for insulin-stimulated glucose transport; 2) overall glucose homeostasis in vivo is mildly enhanced by knockout of the PKCbeta gene; 3) glucose transport is increased in some tissues in PKCbeta knockout mice; and 4) increased glucose transport may be partly due to loss of PKCbeta1, which negatively modulates insulin-stimulated GLUT4 translocation.

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.

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.

RO 31-8220 activates c-Jun N-terminal kinase and glycogen synthase in rat adipocytes and L6 myotubes. Comparison to actions of insulin.

The activation of c-Jun N-terminal kinase (JNK) by insulin and anisomycin has been reported to result in increases in glycogen synthase (GS) activity in rat skeletal muscle (Moxham et al., J Biol Chem, 1996, 271:30765-30773). In addition, the protein kinase C (PKC) inhibitor, RO 31-8220, has been reported to activate JNK in rat-1 fibroblasts (Beltman et al., J Biol Chem, 1996, 271:27018-27024). Presently, we found that the RO 31-8220, as well as insulin, activated JNK and GS and stimulated glucose incorporation into glycogen in rat adipocytes and L6 myotubes. In contrast to activation of JNK, RO 31-8220 inhibited extracellular response kinases 1 and 2 (ERK1/2) and had no significant effects on protein kinase B (PKB). Stimulatory effects of RO 31-8220 on JNK and glycogen metabolism were not explained by PKC inhibition, as other PKC inhibitors were without effect on glucose incorporation into glycogen and have no effect on JNK (Beltman et al., J Biol Chem, 1996, 271:27018). Insulin, on the other hand, activated JNK, as well as PKB and ERK1/2. However, stimulatory effects of insulin on GS and glucose incorporation into glycogen appeared to be fully intact and additive to those of RO 31-8220, despite the fact that insulin did not provoke additive increases in JNK activity above those observed with RO 31-8220 alone. Our findings suggest that JNK serves to activate GS during the action of RO 31-8220 in rat adipocytes and L6 myotubes; insulin, on the other hand, appears to activate GS largely independently of JNK.

Effects of transiently expressed atypical (zeta, lambda), conventional (alpha, beta) and novel (delta, epsilon) protein kinase C isoforms on insulin-stimulated translocation of epitope-tagged GLUT4 glucose transporters in rat adipocytes: specific interchangeable effects of protein kinases C-zeta and C-lambda.

Atypical protein kinase (PK)C isoforms, zeta and lambda, have been reported to be activated by insulin via phosphoinositide 3-kinase, and have been suggested to be required for insulin-stimulated glucose transport. Here, we have examined the effects of transiently expressed wild-type (WT), constitutively active (Constit) and kinase-inactive (KI) forms of atypical PKCs, zeta and lambda, on haemagglutinin antigen (HAA)-tagged glucose transporter 4 (GLUT4) translocation in rat adipocytes, and compared these effects with each other and with those of comparable forms of conventional (alpha, beta) and novel (delta, epsilon) PKCs, which have also been proposed to be required for insulin-stimulated glucose transport. KI-PKC-zeta evoked consistent, sizeable (overall mean of 65%) inhibitory effects on insulin-stimulated, but not basal or guanosine-5'-[gamma-thio]triphosphate-stimulated, HAA-GLUT4 translocation; moreover, inhibitory effects of KI-PKC-zeta were largely reversed by co-transfection of WT-PKC-zeta. Like KI-PKC-zeta, KI-PKC-lambda inhibited insulin-stimulated HAA-GLUT4 translocation by approx. 40-60%, and the combination of KI-PKC-zeta and KI-PKC-lambda caused nearly complete (85%) inhibition. Of particular interest is the fact that inhibitory effects of KI forms of PKC-zeta and PKC-lambda were largely reversed by the opposite WT forms, i.e. PKC-lambda and PKC-zeta respectively. In contrast with KI forms of atypical PKCs, KI forms of PKC-alpha, PKC-beta2, PKC-delta and PKC-epsilon had little or no effect on insulin-stimulated HAA-GLUT4 translocation. Concerning the question of sufficiency, overexpression of WT-PKC-zeta enhanced insulin effects on HAA-GLUT4 translocation, whereas WT forms of PKC-alpha, PKC-beta2, PKC-delta and PKC-epsilon did not affect GLUT4 translocation; furthermore, Constit PKC-zeta evoked increases in HAA-GLUT4 translocation approaching those of insulin, but Constit forms of PKC-alpha and PKC-beta2 were without effect. Our findings suggest that, among PKCs, the atypical PKCs, zeta and lambda, appear to be specifically, but interchangeably, required for insulin effects on HAA-GLUT4 translocation.