Samuel Gray

Protein kinase C activation: isozyme-specific effects on metabolism and cardiovascular complications in diabetes

Abstract Protein kinase C (PKC) is a family of multifunctional isoenzymes, activated by diacylglycerols (DAGs), which play a central role in signal transduction and intracellular crosstalk by phosphorylating at serine/threonine residues an array of substrates, including cell-surface receptors, enzymes, contractile proteins, transcription factors and other kinases. Individual isozymes vary in their pattern of tissue and subcellular distribution, function and Ca2+/phospholipid cofactor requirements, and in diabetes there is widespread activation of the DAG-PKC pathway in metabolic, cardiovascular and renal tissues. In liver, muscle and adipose tissue, PKC isozymes have been implicated both as mediators and inhibitors of insulin action. Activation of DAG-sensitive PKC isoforms, such as PKC-θ and PKC-ɛ, down-regulates insulin receptor signalling and could be an important biochemical mechanism linking dysregulated lipid metabolism and insulin resistance in muscle. On the other hand, atypical PKC isozymes, such as PKC-ζ and PKC-λ, have been identified as downstream targets of PI-3-kinase involved in insulin-stimulated glucose uptake, especially in adipocytes. Glucose-induced de novo synthesis of (palmitate-rich) DAG and sustained isozyme-selective PKC activation (especially but not exclusively PKC-β) has been strongly implicated in the pathogenesis of diabetic microangiopathy and macroangiopathy through a host of undesirable effects on endothelial function, VSM contractility and growth, angiogenesis, gene transcription (in part by MAP-kinase activation) and vascular permeability. Interventions that increase DAG metabolism (e. g. vitamin E) and/or inhibit PKC isozymes (e. g. the β-selective inhibitor LY333 531) ameliorate the biochemical and functional consequences of DAG-PKC activation in experimental diabetes, for example improving retinal blood flow and albuminuria in parallel with reductions in membrane-associated PKC isozyme activities. Thus, a greater understanding of the functional diversity and pathophysiological regulation of PKC isozymes is likely to have important clinical and therapeutic benefits. [Diabetologia (2001) 44: 659–673]

Exendin-4 increases insulin sensitivity via a PI-3-kinase-dependent mechanism: contrasting effects of GLP-1.

The insulinotropic agent, exendin-4, is a long-acting analogue of glucagon-like peptide-1 (GLP-1) which improves glucose tolerance in humans and animals with diabetes, but the underlying mechanisms and the effects of exendin-4 on peripheral (muscle/fat) insulin action are unclear. Previous in vivo and clinical studies have been difficult to interpret because of complex, simultaneous changes in insulin and glucagon levels and possible effects on hepatic metabolism. Thus, the comparative effects of exendin-4 and GLP-1 on insulin-stimulated 2-[3H]deoxyglucose (2-DOG) uptake were measured in fully differentiated L6 myotubes and 3T3-adipocytes, including co-incubation with inhibitors of the PI-3-kinase (wortmannin) and mitogen-activated protein (MAP) kinase (PD098059) pathways. In L6 myotubes, there was a concentration-dependent and PI-3-kinase-dependent increase in insulin-stimulated 2-DOG uptake with exendin-4 and GLP-1, e.g. for exendin-4 the C(I-200) value (concentration of insulin required to increase 2-DOG uptake 2-fold) decreased from 1.3 +/- 1.4 x 10(-7)M (insulin alone, n=16) to 5.9 +/- 1.3 x 10(-8)M (insulin+exendin-4 0.1nM, n=18, P<0.03). A similar insulin-sensitizing effect was observed with exendin-4 in 3T3-adipocytes, but GLP-1 had no effect on adipocyte insulin sensitivity. In conclusion, this is the first direct evidence showing that exendin-4 increases insulin-stimulated glucose uptake in muscle and fat derived cells via a pathway that involves PI-3-kinase activation. Furthermore, the contrasting responses of exendin and GLP-1 in 3T3-adipocytes suggest that the peripheral insulin-sensitizing effect of exendin-4 (in contrast to the insulinotropic effect) does not involve the GLP-1 receptor pathway.

Protein kinase C-ε mediates bradykinin-induced cyclooxygenase-2 expression in human airway smooth muscle cells1

We previously reported that proinflammatory mediator bradykinin (BK) induces cyclooxygenase (COX)‐2 expression in human airway smooth muscle (HASM), but the mechanism is unknown in any biological system. Here, we studied the role of specific protein kinase C (PKC) isozyme(s) in COX‐2 expression. Among the eight PKC isozymes present in HASM cells, the Ca2+‐independent PKC‐δ and ‐ε and the Ca2+‐dependent PKC‐α and ‐βI were translocated to the nucleus upon BK stimulation. BK‐induced COX‐2 expression and prostaglandin E2 (PGE2) accumulation were mimicked by the direct PKC activator phorbol 12‐myristate 13‐acetate (PMA) and inhibited by the broad spectrum PKC inhibitor bisindolylmaleimide I. However, the selective Ca2+‐dependent PKC isozyme inhibitor Go 6976 had no effect. Furthermore, the membrane‐permeable calcium chelator BAPTA‐AM had no effect on BK‐induced COX‐2 expression and COX activity despite its inhibition of PGE2 accumulation, suggesting the involvement of Ca2+‐independent PKC isozymes. Rottlerin, a PKC‐δ inhibitor, also had no effect, likely implicating PKC‐ε. BK‐stimulated transcriptional activation of a COX‐2 promoter reporter construct was enhanced by overexpression of wild‐type PKC‐ε and abolished by a dominant negative PKC‐ε, but it was not affected by wild‐type or dominant negative PKC‐α or ‐δ. Collectively, our results demonstrate that PKC‐ε mediates BK‐induced COX‐2 expression in HASM cells.

Tissue- and time-dependent effects of endothelin-1 on insulin-stimulated glucose uptake

Hyperendothelinaemia is associated with various insulin-resistant states, e.g., diabetes, obesity and heart failure, but whether endothelin-1 (ET-1) has a direct effect on insulin-mediated glucose uptake is unclear because the interpretation of in vivo metabolic studies is complicated by ET-1 effects on muscle blood flow and insulin secretion. This study investigated the effects of ET-1 (1-10 nM) on basal and insulin-stimulated 2-deoxy-D-[3H]glucose (2-DOG) uptake in cultured L6 myoblasts and 3T3-adipocytes. RT-PCR analysis showed that both cell types express ET(A) but not ET(B) receptors. ET-1 had no effect on basal (non-insulin-mediated) glucose transport, but there was evidence of a tissue- and time-dependent inhibitory effect of ET-1 on insulin-stimulated glucose uptake. Specifically, ET-1 10 nM had a transient (0.5 h) inhibitory effect on glucose uptake in 3T3 cells (C(I-150) [dose of insulin required to increase glucose uptake by 50%, relative to control 100%] increased from 89 +/- 14 nM to 270 +/- 12 nM at 30 mins, P < 0.05) but no effect on insulin sensitivity in L6 myoblasts (C(I-150) was 56 +/- 14 nM [control], 43 +/- 14 [30 mins] and 26 +/- 16 [2 h]). In conclusion, the inhibitory effect of ET-1 on insulin-stimulated glucose uptake is transient and occurs in 3T3-L1 adipocytes but not skeletal muscle-derived cells, perhaps reflecting tissue differences in ET(A)-receptor signaling. It is therefore unlikely that chronic hyperendothelinaemia has a direct insulin-antagonist effect contributing to peripheral (ie muscle/fat) insulin resistance in vivo.

Insulin action in skeletal muscle: isozyme-specific effects of protein kinase C.

Abstract: Protein kinase C (PKC) is a family of multifunctional isozymes that plays an important role in the regulation of intracellular insulin signal transduction in various insulin‐sensitive tissues. This article highlights current understanding on the mechanism of PKC‐induced insulin resistance in skeletal muscle, a major target site for insulin‐mediated glucose disposal. Initial, apparently contradictory findings on the role of PKC on insulin action can be explained on the basis that certain PKC isoforms (e.g., ‐ζ and ‐λ) have been identified as downstream targets of PI3‐kinase activation, while DAG‐sensitive PKCs (e.g., ‐θ and ‐ε) have negative regulatory effects on insulin signaling. Hence, pharmacological therapies targeting specific PKC isoforms could enhance insulin action and improve glycemic control in patients with impaired glucose tolerance and overt diabetes.

Increased skeletal muscle expression of PKC‐θ but not PKC‐α mRNA in type 2 diabetes: inverse relationship with in‐vivo insulin sensitivity

Background  Increases in PKC‐θ (the major isoenzymic form of PKC in skeletal muscle) protein and isozyme activity have been reported in skeletal muscle from patients with type 2 diabetes mellitus (T2DM) and dietary‐induced rodent models of insulin resistance, but the underlying biochemical mechanism is unclear and muscle PKC‐θ mRNA expression has not been previously reported in patients with T2DM or in relation to in‐vivo measurements of insulin sensitivity.

Effects of rosiglitazone and oleic acid on UCP‐3 expression in L6 myotubes

Aims: The regulation of uncoupling protein‐3 (UCP‐3) expression in muscle remains unclear, specifically in relation to dietary and drug treatments. The present study evaluated the effects of oleic acid and rosiglitazone on UCP‐3 mRNA expression in differentiated L6 myotubes.