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Biomedical subjects

R V Farese

Publications and source records attributed to R V Farese.

At least 19 recordsLinked to original sources
PubMed2006

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.

Biopsy↗
PubMed2006

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.

AMP-Activated Protein Kinases↗
PubMed2005

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.

AMP-Activated Protein Kinases↗
PubMed2005

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.

Animals↗
PubMed2004

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.

Adipocytes↗
PubMed2002

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 </= 20. Interestingly, after controlling for the influence of BMI, the 79T allele was also associated with higher plasma HDL cholesterol levels (p = 0.0006) and lower diastolic blood pressure (p = 0.019) in these women. No association was found in Turkish men (n = 846). Our findings suggest that genetic variation at the DGAT1 locus may influence BMI and other metabolic parameters associated with cardiovascular risk in selected human populations.

Acyltransferases↗
PubMed2001

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.

Animals↗
PubMed2001

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.

3T3 Cells↗
PubMed2001

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.

Adipocytes↗
PubMed2001

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.

Anilides↗
PubMed2001

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.

3-Phosphoinositide-Dependent Protein Kinases↗
PubMed2001

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.

Animals↗
PubMed2001

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.

Animals↗
PubMed2001

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.

Adipocytes↗
PubMed2000

Mammalian acyl-CoA:cholesterol acyltransferases.

Cholesterol, the chief sterol found in vertebrates, exists both as a free sterol and as a component of cholesterol esters, which are synthesized by acyl-CoA:cholesterol acyltransferase (ACAT) enzymes. Considerable knowledge concerning cholesterol ester metabolism has accumulated during the past century. However, rapid advances have occurred in the past 7 years since the cloning of an ACAT gene, including the discovery that two ACATs function in mammalian biology. A clearer picture of the functions of ACAT enzymes in cellular cholesterol metabolism and physiologic processes is now emerging. These insights may have relevance for the development of ACAT inhibitors for treating hypercholesterolemia or atherosclerosis in humans.

Amino Acid Sequence↗
PubMed2000

Increased atherosclerosis in hyperlipidemic mice deficient in alpha -tocopherol transfer protein and vitamin E.

Although lipid peroxidation in the subendothelial space has been hypothesized to play a central role in atherogenesis, the role of vitamin E in preventing lipid peroxidation and lesion development remains uncertain. Here we show that in atherosclerosis-susceptible apolipoprotein E knockout mice, vitamin E deficiency caused by disruption of the alpha-tocopherol transfer protein gene (Ttpa) increased the severity of atherosclerotic lesions in the proximal aorta. The increase was associated with increased levels of isoprostanes, a marker of lipid peroxidation, in aortic tissue. These results show that vitamin E deficiency promotes atherosclerosis in a susceptible setting and support the hypothesis that lipid peroxidation contributes to lesion development. Ttpa(-/-) mice are a genetic model of vitamin E deficiency and should be valuable for studying other diseases in which oxidative stress is thought to play a role.

Animals↗