Taming the sphinx: Mechanisms of cellular sphingolipid homeostasis.

Sphingolipids are important structural membrane components of eukaryotic cells, and potent signaling molecules. As such, their levels must be maintained to optimize cellular functions in different cellular membranes. Here, we review the current knowledge of homeostatic sphingolipid regulation. We describe recent studies in Saccharomyces cerevisiae that have provided insights into how cells sense changes in sphingolipid levels in the plasma membrane and acutely regulate sphingolipid biosynthesis by altering signaling pathways. We also discuss how cellular trafficking has emerged as an important determinant of sphingolipid homeostasis. Finally, we highlight areas where work is still needed to elucidate the mechanisms of sphingolipid regulation and the physiological functions of such regulatory networks, especially in mammalian cells. This article is part of a Special Issue entitled: The cellular lipid landscape edited by Tim P. Levine and Anant K. Menon.

Insulin signalling in hepatocytes of humans with type 2 diabetes: excessive production and activity of protein kinase C-ι (PKC-ι) and dependent processes and reversal by PKC-ι inhibitors.

AIMS/HYPOTHESIS: We examined the role of protein kinase C-ι (PKC-ι) in mediating alterations in the abundance of enzymes in hepatocytes of type 2 diabetic humans that contribute importantly to the development of lipid and carbohydrate abnormalities in type 2 diabetes. METHODS: We examined (1) insulin signalling in isolated hepatocytes of non-diabetic and type 2 diabetic humans and (2) the effects of two newly developed small molecule PKC-ι inhibitors on aberrant signalling and downstream processes. RESULTS: In contrast with PKC-ι deficiency in diabetic muscle, which diminishes glucose transport, PKC-ι in diabetic hepatocytes was overproduced and overactive, basally and after insulin treatment, and, moreover, was accompanied by increased abundance of PKC-ι-dependent lipogenic, proinflammatory and gluconeogenic enzymes. Heightened PKC-ι activity most likely reflected heightened activity of IRS-2-dependent phosphatidylinositol 3-kinase (PI3K), as IRS-1 levels and IRS-1/PI3K activity were markedly diminished. Importantly, insulin-stimulated PKC-ι abundance and its overabundance in diabetic hepatocytes was reversed in vitro by both insulin deprivation and PKC-ι inhibitors; this suggested operation of an insulin-driven, feed-forward/positive-feedback mechanism. In contrast with PKC-ι, protein kinase B (Akt2) activity and activation by insulin was diminished, apparently reflecting IRS-1 deficiency. Treatment of diabetic hepatocytes with PKC-ι/λ inhibitors diminished abundance of lipogenic, proinflammatory and gluconeogenic enzymes. CONCLUSIONS/INTERPRETATION: Our findings suggest that a vicious cycle of PKC-ι overactivity and overproduction exists in hepatocytes of humans with type 2 diabetes and contributes importantly to maintaining overactivity of lipogenic, proinflammatory and gluconeogenic pathways, which underlies the lipid and carbohydrate abnormalities in type 2 diabetes.

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.

Role of atypical protein kinase C in activation of sterol regulatory element binding protein-1c and nuclear factor kappa B (NFkappaB) in liver of rodents used as a model of diabetes, and relationships to hyperlipidaemia and insulin resistance.

AIMS/HYPOTHESIS: Previous findings in rodents used as a model of diabetes suggest that insulin activation of atypical protein kinase C (aPKC) is impaired in muscle, but, unexpectedly, conserved in liver, despite impaired hepatic protein kinase B (PKB/Akt) activation. Moreover, aPKC at least partly regulates two major transactivators: (1) hepatic sterol receptor binding protein-1c (SREBP-1c), which controls lipid synthesis; and (2) nuclear factor kappa B (NFkappaB), which promotes inflammation and systemic insulin resistance. METHODS: In Goto-Kakizaki rats used as a model of type 2 diabetes, we examined: (1) whether differences in hepatic aPKC and PKB activation reflect differences in activation of IRS-1- and IRS-2-dependent phosphatidylinositol 3-kinase (PI3K); (2) whether hepatic SREBP-1c and NFkappaB are excessively activated by aPKC; and (3) metabolic consequences of excessive activation of hepatic aPKC, SREBP-1c and NFkappaB. RESULTS: In liver, as well as in muscle, IRS-2/PI3K activation by insulin was intact, whereas IRS-1/PI3K activation by insulin was impaired. Moreover, hepatic IRS-2 is known to control hepatic aPKC during insulin activation. Against this background, selective inhibition of hepatic aPKC by adenoviral-mediated expression of mRNA encoding kinase-inactive aPKC or short hairpin RNA targeting Irs2 mRNA and partially depleting hepatic IRS-2 diminished hepatic SREBP-1c production and NFkappaB activities, concomitantly improving serum lipids and insulin signalling in muscle and liver. Similar improvements in SREBP-1c, NFkappaB and insulin signalling were seen in ob/ob mice following inhibition of hepatic aPKC. CONCLUSIONS/INTERPRETATION: In diabetic rodent liver, diminished PKB activation may largely reflect impaired IRS-1/PI3K activation, while conserved aPKC activation reflects retained IRS-2/PI3K activity. Hepatic aPKC may also contribute importantly to excessive SREPB-1c and NFkappaB activities. Excessive hepatic aPKC-dependent activation of SREBP-1c and NFkappaB may contribute importantly to hyperlipidaemia and systemic insulin resistance.

Combined thiazolidinedione-metformin treatment synergistically improves insulin signalling to insulin receptor substrate-1-dependent phosphatidylinositol 3-kinase, atypical protein kinase C and protein kinase B/Akt in human diabetic muscle.

AIMS/HYPOTHESES: Insulin-stimulated glucose transport in muscle is impaired in type 2 diabetes, presumably reflecting reduced activation of atypical protein kinase C (aPKC) and protein kinase B (PKB/Akt). As previously shown, reductions in aPKC activation are seen at sub-maximal and maximal insulin stimulation, reductions in PKB activation are best seen at sub-maximal insulin stimulation and aPKC reductions at maximal insulin are partly improved by thiazolidinedione or metformin treatment. However, effects of combined thiazolidinedione-metformin treatment on aPKC or PKB activation by sub-maximal and maximal insulin are unknown. METHODS: Type 2 diabetic patients were examined before and 5 to 6 weeks after combined thiazolidinedione-metformin therapy for activation of muscle aPKC and PKBbeta and their upstream activators, the insulin receptor (IR) and IRS-1-associated phosphatidylinositol 3-kinase (PI3K), during euglycaemic-hyperinsulinaemic clamp studies conducted with sub-maximal (400-500 pmol/l) and maximal (1400 pmol/l) insulin concentrations. RESULTS: Following combined thiazolidinedione-metformin therapy, increases in glucose disposal and increases in sub-maximal and maximal insulin-induced activities of all four muscle signalling factors, IR, IRS-1-dependent PI3K (IRS-1/PI3K), aPKC and PKBbeta, were observed. Increases in PKBbeta enzyme activity were accompanied by increases in phosphorylation of PKB and its substrate, AS160, which is needed for glucose transport. Despite improved aPKC activity, muscle aPKC levels, which are diminished in type 2 diabetes, were not altered. CONCLUSIONS/INTERPRETATION: Combined thiazolidinedione-metformin treatment markedly improves sub-maximal and maximal insulin signalling to IR, IRS-1/PI3K, aPKC and PKBbeta in type 2 diabetic muscle. These improvements exceed those previously reported after treatment with either agent alone.

Contrasting insulin dose-dependent defects in activation of atypical protein kinase C and protein kinase B/Akt in muscles of obese diabetic humans.

AIMS/HYPOTHESIS: Insulin-stimulated glucose transport in muscle is impaired in obesity and type 2 diabetes, but alterations in levels of relevant signalling factors, i.e. atypical protein kinase C (aPKC) and protein kinase B (PKB/Akt), are still uncertain. Clamp studies using maximal insulin concentrations have revealed defects in activation of aPKC, but not PKB, in both obese non-diabetic and obese diabetic subjects. In contrast, clamp studies using submaximal insulin concentrations revealed defects in PKB activation/phosphorylation in obese non-diabetic and diabetic subjects, but changes in aPKC were not reported. The aim of this study was to test the hypothesis that dose-related effects of insulin may account for the reported differences in insulin signalling to PKB in diabetic muscle. SUBJECTS AND METHODS: We compared enzymatic activation of aPKC and PKB, and PKB phosphorylation (threonine-308 and serine-473) during hyperinsulinaemic-euglycaemic clamp studies using both submaximal (400-500 pmol/l) and maximal (1400 pmol/l) insulin levels in non-diabetic control and obese diabetic subjects. RESULTS: In lean control subjects, the submaximal insulin concentration increased aPKC activity and glucose disposal to approximately 50% of the maximal level and PKBbeta activity to 25% of the maximal level, but PKBalpha activity was not increased. In these subjects, phosphorylation of PKBalpha and PKBbeta was increased to near-maximal levels at submaximal insulin concentrations. In obese diabetic subjects, whereas aPKC activation was defective at submaximal and maximal insulin concentrations, PKBbeta activation and the phosphorylation of PKBbeta and PKBalpha were defective at submaximal, but not maximal, insulin concentrations. CONCLUSIONS/INTERPRETATIONS: Defective PKBbeta activation/phosphorylation, seen on submaximal insulin stimulation in diabetic muscle, may largely reflect impaired activation of insulin signalling factors present in concentrations greater than those needed for full PKB activation/phosphorylation. Defective aPKC activation, seen at all insulin levels, appears to reflect, at least partly, an impaired action of distal factors needed for aPKC activation, or poor aPKC responsiveness.

Metformin improves atypical protein kinase C activation by insulin and phosphatidylinositol-3,4,5-(PO4)3 in muscle of diabetic subjects.

AIMS/HYPOTHESIS: Metformin is widely used for treating type 2 diabetes mellitus, but its actions are poorly understood. In addition to diminishing hepatic glucose output, metformin, in muscle, activates 5'-AMP-activated protein kinase (AMPK), which alone increases glucose uptake and glycolysis, diminishes lipid synthesis, and increases oxidation of fatty acids. Moreover, such lipid effects may improve insulin sensitivity and insulin-stimulated glucose uptake. Nevertheless, the effects of metformin on insulin-sensitive signalling factors in human muscle have only been partly characterised to date. Interestingly, other substances that activate AMPK, e.g., aminoimidazole-4-carboxamide-1-beta-D: -riboside (AICAR), simultaneously activate atypical protein kinase C (aPKC), which appears to be required for the glucose transport effects of AICAR and insulin. METHODS: Since aPKC activation is defective in type 2 diabetes, we evaluated effects of metformin therapy on aPKC activity in muscles of diabetic subjects during hyperinsulinaemic-euglycaemic clamp studies. RESULTS: After metformin therapy for 1 month, basal aPKC activity increased in muscle, with little or no change in insulin-stimulated aPKC activity. Metformin therapy for 8 to 12 months improved insulin-stimulated, as well as basal aPKC activity in muscle. In contrast, IRS-1-dependent phosphatidylinositol (PI) 3-kinase activity and Ser473 phosphorylation of protein kinase B were not altered by metformin therapy, whereas the responsiveness of muscle aPKC to PI-3,4,5-(PO(4))(3), the lipid product of PI 3-kinase, was improved. CONCLUSIONS/INTERPRETATION: These findings suggest that the activation of AMPK by metformin is accompanied by increases in aPKC activity and responsiveness in skeletal muscle, which may contribute to the therapeutic effects of metformin.

Insulin signalling downstream of protein kinase B is potentiated by 5'AMP-activated protein kinase in rat hearts in vivo.

AIMS/HYPOTHESIS: 5'AMP-activated protein kinase (AMPK) and insulin stimulate glucose transport in heart and muscle. AMPK acts in an additive manner with insulin to increase glucose uptake, thereby suggesting that AMPK activation may be a useful strategy for ameliorating glucose uptake, especially in cases of insulin resistance. In order to characterise interactions between the insulin- and AMPK-signalling pathways, we investigated the effects of AMPK activation on insulin signalling in the rat heart in vivo. METHODS: Male rats (350-400 g) were injected with 1 g/kg 5-aminoimidazole-4-carboxamide-1-beta-D-ribofuranoside (AICAR) or 250 mg/kg metformin in order to activate AMPK. Rats were administered insulin 30 min later and after another 30 min their hearts were removed. The activities and phosphorylation levels of components of the insulin-signalling pathway were subsequently analysed in individual rat hearts. RESULTS: AICAR and metformin administration activated AMPK and enhanced insulin signalling downstream of protein kinase B in rat hearts in vivo. Insulin-induced phosphorylation of glycogen synthase kinase 3 (GSK3) beta, p70 S6 kinase (p70S6K)(Thr389) and IRS1(Ser636/639) were significantly increased following AMPK activation. To the best of our knowledge, this is the first report of heightened insulin responses of GSK3beta and p70S6K following AMPK activation. In addition, we found that AMPK inhibits insulin stimulation of IRS1-associated phosphatidylinositol 3-kinase activity, and that AMPK activates atypical protein kinase C and extracellular signal-regulated kinase in the heart. CONCLUSIONS/INTERPRETATIONS: Our data are indicative of differential effects of AMPK on the activation of components in the cardiac insulin-signalling pathway. These intriguing observations are critical for characterisation of the crosstalk between AMPK and insulin signalling.

Atypical protein kinase C in insulin action and insulin resistance.

It now seems clear that aPKC (atypical protein kinase C) isoforms are required for insulin-stimulated glucose transport in muscle and adipocytes. Moreover, there are marked defects in the activation of aPKCs under a variety of insulin-resistant conditions in humans, monkeys and rodents. In humans, defects in aPKC in muscle are seen in Type II diabetes and its precursors, obesity, the obesity-associated polycystic ovary syndrome and impaired glucose tolerance. These defects in muscle aPKC activation are due to both impaired activation of insulin receptor substrate-1-dependent PI3K (phosphoinositide 3-kinase) and the direct activation of aPKCs by the lipid product of PI3K, PI-3,4,5-(PO4)3. Although it is still uncertain which underlying defect comes first, the resultant defect in aPKC activation in muscle most certainly contributes significantly to the development of skeletal muscle insulin resistance. Of further note, unlike the seemingly ubiquitous presence of defective aPKC activation in skeletal muscle in insulin-resistant states, the activation of aPKC is normal or increased in livers of Type II diabetic and obese rodents. The maintenance of aPKC activation in the liver may explain how insulin-dependent lipid synthesis is maintained in these states, as aPKCs function mainly in the activation of enzymes important for lipid synthesis. Thus increased activation of liver aPKC in hyperinsulinaemic states may contribute significantly to the development of hyperlipidaemia in insulin-resistant states.

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.

DGAT1 promoter polymorphism associated with alterations in body mass index, high density lipoprotein levels and blood pressure in Turkish women.

Triglyceride synthesis is catalyzed by acyl CoA:diacylglycerol acyltransferases (DGAT), microsomal enzymes that use diacylglycerol and fatty acyl CoAs as substrates. Because DGAT1 expression is up-regulated during adipocyte differentiation and DGAT1 deficiency is associated with leanness in mice, we hypothesized that alterations in DGAT1 expression may affect human body weight. We identified five polymorphisms in the human DGAT1 promoter and 5' non-coding sequence in a random Turkish population. Functional analysis of one common variant, C79T, revealed reduced promoter activity for the 79T allele in cultured cell lines. In 476 Turkish women, the 79T allele was associated with lower body mass index (BMI) (p = 0.004), conferring an odds ratio of 2.0 (95% CI = 1.30-3.07, p = 0.0001) for BMI

Introduction of human apolipoprotein E4 "domain interaction" into mouse apolipoprotein E.

Human apolipoprotein E4 (apoE4) binds preferentially to lower density lipoproteins, including very low density lipoproteins, and is associated with increased risk of atherosclerosis and neurodegenerative disorders, including Alzheimer's disease. This binding preference is the result of the presence of Arg-112, which causes Arg-61 in the amino-terminal domain to interact with Glu-255 in the carboxyl-terminal domain. ApoE2 and apoE3, which have Cys-112, bind preferentially to high density lipoproteins (HDL) and do not display apoE4 domain interaction. Mouse apoE, like apoE4, contains the equivalent of Arg-112 and Glu-255, but lacks the critical Arg-61 equivalent (it contains Thr-61). Thus, mouse apoE does not display apoE4 domain interaction and, as a result, behaves like human apoE3, including preferential binding to HDL. To assess the potential role of apoE4 domain interaction in atherosclerosis and neurodegeneration, we sought to introduce apoE4 domain interaction into mouse apoE. Replacing Thr-61 in mouse apoE with arginine converted the binding preference from HDL to very low density lipoproteins in vitro, suggesting that apoE4 domain interaction could be introduced into mouse apoE in vivo. Using gene targeting in embryonic stem cells, we created mice expressing Arg-61 apoE. Heterozygous Arg-61/wild-type apoE mice displayed two phenotypes found in human apoE4/E3 heterozygotes: preferential binding to lower density lipoproteins and reduced abundance of Arg-61 apoE in the plasma, reflecting its more rapid catabolism. These findings demonstrate the successful introduction of apoE4 domain interaction into mouse apoE in vivo. The Arg-61 apoE mouse model will allow the effects of apoE4 domain interaction in lipoprotein metabolism, atherosclerosis, and neurodegeneration to be determined.

Cloning of DGAT2, a second mammalian diacylglycerol acyltransferase, and related family members.

Studies involving the cloning and disruption of the gene for acyl-CoA:diacylglycerol acyltransferase (DGAT) have shown that alternative mechanisms exist for triglyceride synthesis. In this study, we cloned and characterized a second mammalian DGAT, DGAT2, which was identified by its homology to a DGAT in the fungus Mortierella rammaniana. DGAT2 is a member of a gene family that has no homology with DGAT1 and includes several mouse and human homologues that are candidates for additional DGAT genes. The expression of DGAT2 in insect cells stimulated triglyceride synthesis 6-fold in assays with cellular membranes, and DGAT2 activity was dependent on the presence of fatty acyl-CoA and diacylglycerol, indicating that this protein is a DGAT. Activity was not observed for acyl acceptors other than diacylglycerol. DGAT2 activity was inhibited by a high concentration (100 mm) of MgCl(2) in an in vitro assay, a characteristic that distinguishes DGAT2 from DGAT1. DGAT2 is expressed in many tissues with high expression levels in the liver and white adipose tissue, suggesting that it may play a significant role in mammalian triglyceride metabolism.

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.

Cholesterol esterification by host and parasite is essential for optimal proliferation of Toxoplasma gondii.

Upon host cell invasion the apicomplexan parasite Toxoplasma gondii resides in a specialized compartment termed the parasitophorous vacuole that is derived from the host cell membrane but modified by the parasite. Despite the segregation of the parasitophorous vacuole from the host endocytic network, the intravacuolar parasite has been shown to acquire cholesterol from the host cell. In order to characterize further the role of sterol metabolism in T. gondii biology, we focused our studies on the activity of acyl-CoA:cholesterol acyltransferase (ACAT), a key enzyme for maintaining the intracellular homeostasis of cholesterol through the formation of cholesterol esters. In this study, we demonstrate that ACAT and cholesterol esters play a crucial role in the optimal replication of T. gondii. Moreover, we identified ACAT activity in T. gondii that can be modulated by pharmacological ACAT inhibitors with a consequent detrimental effect on parasite replication.

Insulin-sensitive phospholipid signaling systems and glucose transport. Update II.

Insulin provokes rapid changes in phospholipid metabolism and thereby generates biologically active lipids that serve as intracellular signaling factors that regulate glucose transport and glycogen synthesis. These changes include: (i) activation of phosphatidylinositol 3-kinase (PI3K) and production of PIP3; (ii) PIP3-dependent activation of atypical protein kinase Cs (PKCs); (iii) PIP3-dependent activation of PKB; (iv) PI3K-dependent activation of phospholipase D and hydrolysis of phosphatidylcholine with subsequent increases in phosphatidic acid (PA) and diacylglycerol (DAG); (v) PI3K-independent activation of glycerol-3-phosphate acylytansferase and increases in de novo synthesis of PA and DAG; and (vi) activation of DAG-sensitive PKCs. Recent findings suggest that atypical PKCs and PKB serve as important positive regulators of insulin-stimulated glucose metabolism, whereas mechanisms that result in the activation of DAG-sensitive PKCs serve mainly as negative regulators of insulin signaling through PI3K. Atypical PKCs and PKB are rapidly activated by insulin in adipocytes, liver, skeletal muscles, and other cell types by a mechanism requiring PI3K and its downstream effector, 3-phosphoinositide-dependent protein kinase-1 (PDK-1), which, in conjunction with PIP3, phosphorylates critical threonine residues in the activation loops of atypical PKCs and PKB. PIP3 also promotes increases in autophosphorylation and allosteric activation of atypical PKCs. Atypical PKCs and perhaps PKB appear to be required for insulin-induced translocation of the GLUT 4 glucose transporter to the plasma membrane and subsequent glucose transport. PKB also appears to be the major regulator of glycogen synthase. Together, atypical PKCs and PKB serve as a potent, integrated PI3K/PDK-1-directed signaling system that is used by insulin to regulate glucose metabolism.

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.

Increased atherosclerosis in LDL receptor-null mice lacking ACAT1 in macrophages.

During atherogenesis, circulating macrophages migrate into the subendothelial space, internalize cholesterol-rich lipoproteins, and become foam cells by progressively accumulating cholesterol esters. The inhibition of macrophage acyl coenzyme A:cholesterol acyltransferase (ACAT), which catalyzes the formation of cholesterol esters, has been proposed as a strategy to reduce foam cell formation and to treat atherosclerosis. We show here, however, that hypercholesterolemic LDL receptor-deficient (LDLR(-/-)) mice reconstituted with ACAT1-deficient macrophages unexpectedly develop larger atherosclerotic lesions than control LDLR(-/-) mice. The ACAT1-deficient lesions have reduced macrophage immunostaining and more free cholesterol than control lesions. Our findings suggest that selective inhibition of ACAT1 in lesion macrophages in the setting of hyperlipidemia can lead to the accumulation of free cholesterol in the artery wall, and that this promotes, rather than inhibits, lesion development.