Palmitate Activates Insulin Signaling Pathway in Pancreatic Rat Islets

Objective: To investigate the action of palmitate on insulin receptor (IR) signaling pathway in rat pancreatic islets. The following proteins were studied: IR substrate-1 and -2 (IRS1 and IRS2), phosphatidylinositol 3-kinase, extracellular signal-regulated protein kinase-1 and -2 (ERK1/2), and signal transducer and activator of transcription 3 (STAT3). Methods: Immunoblotting and immunoprecipitation assays were used to evaluate the phosphorylation states of IRS1 and IRS2 (tyrosine [Tyr]), ERK1/2 (threonine 202 [Thr202]/Tyr204), and STAT3 (serine [Ser727]). Results: The exposure of rat pancreatic islets to 0.1-mmol/L palmitate for up to 30 minutes produced a significant increase of Tyr phosphorylation in IRS2 but not in IRS1. The association of phosphatidylinositol 3-kinase with IRS2 was also upregulated by palmitate. Exposure to 5.6-mmol/L glucose caused a gradual decrease in ERK1/2 (Thr202/Tyr204) and STAT3 (serine [Ser727]) phosphorylations after 30-minute incubation. The addition of palmitate (0.1 mmol/L), associated with 5.6-mmol/L glucose, abolished these latter effects of glucose after 15-minute incubation. Conclusions: Palmitate at physiological concentration associated with 5.6-mmol/L glucose activates IR signaling pathway in pancreatic A cells.

Prolonged exposure to palmitate impairs fatty acid oxidation despite activation of AMP-activated protein kinase in skeletal muscle cells

The aim of this study was to investigate the chronic effects of palmitate on fatty acid (FA) oxidation, AMPK/ACC phosphorylation/activation, intracellular lipid accumulation, and the molecular Mechanisms involved in these processes in skeletal muscle cells. Exposure of L6 myotubes for 8 h to 200, 400, 600, and 800 mu M of palmitate did rot affect cel viability but significantly reduced FA oxidation by similar to 26.5%, similar to 43.5%, similar to 50%, and similar to 47%, respectively. Interestingly, this occurred despite significant increases in AMPK (similar to 2.5-fold) and ACC (similar to 3-fold) phosphorylation and in malonyl-CoA decarboxylase activity (similar to 38-60%). Low concentrations of palmitate (50-100 mu M) caused an increase (similar to 30%) in CPT-I activity. However, as the concentration of palmitate increased, CPT-I activity decreased by similar to 32% after exposure for 8 h to 800 mu M of palmitate. Although FA uptake was reduced (similar to 35%) in cells exposed to increasing, palmitate concentrations, intracellular lipid accumulation increased in a dose-dependent manner, reaching values similar to 2.3-, similar to 3-, and 4-fold higher than control in muscle cells exposed to 400, 600, and 800 mu M palmitate, respectively. Interestingly, myotubes exposed to 400 mu M of palmitate for 1h increased basal glucose uptake and glycogen synthesis by similar to 40%. However, as time of incubation in the presence of palmitate progressed from 1 to 8h, these increases were abolished and a time-dependent inhibition of insulin-stimulated glucose uptake (similar to 65%) and glycogen synthesis (30%) was observed in myotubes. These findings may help explain the dysfunctional adaptations that occur in glucose and FA Metabolism in skeletal muscle under conditions of chronically elevated circulating levels of non-esterified FAs. Such as in obesity and Type 2 Diabetes.

GLP-1 Inhibits and Adrenaline Stimulates Glucagon Release by Differential Modulation of N- and L-Type Ca(2+) Channel-Dependent Exocytosis

Glucagon secretion is inhibited by glucagon-like peptide-1 (GLP-1) and stimulated by adrenaline. These opposing effects on glucagon secretion are mimicked by low (1-10 nM) and high (10 mu M) concentrations of forskolin, respectively. The expression of GLP-1 receptors in a cells is <0.2% of that in beta cells. The GLP-1-induced suppression of glucagon secretion is PKA dependent, is glucose independent, and does not involve paracrine effects mediated by insulin or somatostatin. GLP-1 is without much effect on a cell electrical activity but selectively inhibits N-type Ca(2+) channels and exocytosis. Adrenaline stimulates a cell electrical activity, increases [Ca(2+)] enhances L-type Ca(2+) channel activity, and accelerates exocytosis. The stimulatory effect is partially PKA independent and reduced in Epac2-deficient islets. We propose that GLP-1 inhibits glucagon secretion by PKA-dependent inhibition of the N-type Ca(2+) channels via a small increase in intracellular cAMP ([cAMP]). Adrenaline stimulates L-type Ca(2+) channel-dependent exocytosis by activation of the low-affinity cAMP sensor Epac2 via a large increase in [cAMP],.

Exhaustive Exercise Increases Inflammatory Response via Toll Like Receptor-4 and NF-kappa Bp65 Pathway in Rat Adipose Tissue

Cytokines (IL-6, IL-10, and TNF-alpha) are increased after exhaustive exercise in the retroperitoneal adipose tissue (RPAT) and mesenteric adipose tissue (MEAT). An exhaustive acute exercise protocol induces inflammation in adipose tissue that lasts 6 h after the exercise has ended. It is well-established that this protocol increases circulating plasma levels of non-esterified fatty acids (NEFAs) and lipopolysaccharides (LPS), compounds that are important in stimulating signaling via toll like receptor-4 (TLR-4) in different type cells. In the present study, we investigated the regulation of TLR-4 and DNA-binding of nuclear factor-kappa Bp65 (NF-kappa Bp65) in different depots of adipose tissue in rats after exhaustive exercise. Rats were killed by decapitation immediately (E0 group, n = 6), 2 (E2 group, n = 6), and 6 h (E6 group, n = 6) after the exhaustive exercise, which consisted of running on a treadmill (approximately 70% V(O2max)) for 50 min and then running at an elevated rate that increased at 1 m/min, until exhaustion. The control group (C group, n = 6) was not subjected to exercise. In RPAT, TLR-4, MYD-88, and IkB alpha increased in the E2 group after exercise. MYD-88 and TRAF6 remained increased in the E6 group in comparison with the control group. DNA-binding of NF-kappa Bp65 was not altered. In MEAT, TLR-4, MYD-88, TRAF6, and DNA-binding of NF-kappa Bp65 were increased only in the E6 group. In conclusion, we have shown that increases in pro-inflammatory cytokines in adipose tissue pads after exhaustive exercise may be mediated via TLR-4 signaling, leading to increases in NF-kappa Bp65 binding to DNA in MEAT. J. Cell. Physiol. 226: 1604-1607, 2011. (C) 2010 Wiley-Liss, Inc.

Interaction with calmodulin is important for the secretion of thimet oligopeptidase following stimulation

Thimet oligopeptidase (EC 3.4.24.15; EP24.15) was originally described as a neuropeptide-metabolizing enzyme, highly expressed in the brain, kidneys and neuroendocrine tissue. EP24.15 lacks a typical signal peptide sequence for entry into the secretory pathway and is secreted by cells via an unconventional and unknown mechanism. In this study, we identified a novel calcium-dependent interaction between EP24.15 and calmodulin, which is important for the stimulated, but not constitutive, secretion of EP24.15. We demonstrated that, in vitro, EP24.15 and calmodulin physically interact only in the presence of Ca(2+), with an estimated K(d) value of 0.52 mu m. Confocal microscopy confirmed that EP24.15 colocalizes with calmodulin in the cytosol of resting HEK293 cells. This colocalization markedly increases when cells are treated with either the calcium ionophore A23187 or the protein kinase A activator forskolin. Overexpression of calmodulin in HEK293 cells is sufficient to greatly increase the A23187-stimulated secretion of EP24.15, which can be inhibited by the calmodulin inhibitor calmidazolium. The specific inhibition of protein kinase A with KT5720 reduces the A23187-stimulated secretion of EP24.15 and inhibits the synergistic effects of forskolin with A23187. Treatment with calmidazolium and KT5720 nearly abolishes the stimulatory effects of A23187 on EP24.15 secretion. Together, these data suggest that the interaction between EP24.15 and calmodulin is regulated within cells and is important for the stimulated secretion of EP24.15 from HEK293 cells.

Low-level Laser Therapy Improves Skeletal Muscle Performance, Decreases Skeletal Muscle Damage and Modulates mRNA Expression of COX-1 and COX-2 in a Dose-dependent Manner

We tested if modulation in mRNA expression of cyclooxygenase isoforms (COX-1 and COX-2) can be related to protective effects of phototherapy in skeletal muscle. Thirty male Wistar rats were divided into five groups receiving either one of four laser doses (0.1, 0.3, 1.0 and 3.0 J) or a no-treatment control group. Laser irradiation (904 nm, 15 mW average power) was performed immediately before the first contraction for treated groups. Electrical stimulation was used to induce six tetanic tibial anterior muscle contractions. Immediately after sixth contraction, blood samples were collected to evaluate creatine kinase activity and muscles were dissected and frozen in liquid nitrogen to evaluate mRNA expression of COX-1 and COX-2. The 1.0 and 3.0 J groups showed significant enhancement (P < 0.01) in total work performed in six tetanic contractions compared with control group. All laser groups, except the 3.0 J group, presented significantly lower post-exercise CK activity than control group. Additionally, 1.0 J group showed increased COX-1 and decreased COX-2 mRNA expression compared with control group and 0.1, 0.3 and 3.0 J laser groups (P < 0.01). We conclude that pre-exercise infrared laser irradiation with dose of 1.0 J enhances skeletal muscle performance and decreases post-exercise skeletal muscle damage and inflammation.

Extracellular Signal-Regulated Kinase 1/2 Activation, via Downregulation of Mitogen-Activated Protein Kinase Phosphatase 1, Mediates Sex Differences in Desoxycorticosterone Acetate-Salt Hypertension Vascular Reactivity

Extracellular signal-regulated kinase (ERK) 1/2 has been reported to play a role in vascular dysfunction associated with mineralocorticoid hypertension. We hypothesized that, compared with female rats, an upregulation of ERK1/2 signaling in the vasculature of male rats contributes to augmented contractile responses in mineralocorticoid hypertension. Uninephrectomized male and female Sprague-Dawley rats received desoxycorticosterone acetate (DOCA) pellets (200 mg per animal) and saline to drink for 3 weeks. Control uninephrectomized rats received tap water to drink. Blood pressure, measured by telemetry, was significantly higher in male DOCA rats (191 +/- 3 mm Hg) compared with female DOCA rats (172 +/- 7 mm Hg; n=5). DOCA treatment resulted in augmented contractile responses to phenylephrine in aorta (22 +/- 3 mN; n=6) and small mesenteric arteries (13 +/- 2 mN; n=6) from male DOCA rats versus uninephrectomized male rats (16 +/- 3 and 10 +/- 2 mN, respectively; P<0.05) and female DOCA rats (15 +/- 1 and 11 +/- 1 mN, respectively). ERK1/2 inhibition with PD-98059 (10 mu mol/L) abrogated increased contraction to phenylephrine in aorta (14 +/- 2 mN) and small mesenteric arteries (10 +/- 2 mN) from male DOCA rats, without any effects in arteries from male uninephrectomized or female animals. Compared with the other groups, phosphorylated ERK1/2 levels were increased in the aorta from male DOCA rats, whereas mitogen-activated protein kinase phosphatase 1 expression was decreased. Interleukin-10 plasma levels, which positively regulate mitogen-activated protein kinase phosphatase 1 activity, were reduced in male DOCA-salt rats. We speculate that augmented vascular reactivity in male hypertensive rats is mediated via activation of the ERK1/2 pathway. In addition, mitogen-activated protein kinase phosphatase 1 and interleukin 10 play regulatory roles in this process. (Hypertension. 2010; 55: 172-179.)

Putative antinociceptive action of nitric oxide in the caudal part of the spinal trigeminal nucleus during chronic carrageenan-induced arthritis in the rat temporomandibular joint

In order to investigate a putative role for nitric oxide (NO) in the central nociceptive processing following carrageenan-induced arthritis in the rat temporomandibular joint (TMJ), we analyzed the immunoreactivity, gene expression and activity of nitric oxide synthases (NOS) in the caudal part of the spinal trigeminal nucleus (Sp5C) during the acute (24 h), chronic (15 days) and chronic-active (14 days-24 h) arthritis. In addition, evaluation of head-withdrawal threshold was carried out in all phases of arthritis under chronic inhibition of nNOS with the selective inhibitor 7-nitroindazole (7-NI). Neurons with nNOS-like immunoreactivity (nNOS-LI) were concentrated mainly in the lamina II of the Sp5C, showing no significant statistical difference during arthritis. Only a discrete percentage of nNOS-LI neurons expressed Fos immunoreactivity. The mRNA expression for both nNOS and endothelial nitric oxide synthases (eNOS) presented no noticeable differences among the groups. No expression of inducible nitric oxide synthase (iNOS) was detected in the Sp5C by either immunohistochemistry or reverse-transcription polymerase chain reaction (RTPCR). Ca(2+)-dependent NOS activity in the ipsilateral Sp5C was significantly higher (108.3 +/- 49.2%; P<0.01) in animals during the chronic arthritis. Interestingly, this increased activity was completely abolished 24 h later, in the chronic-active arthritis. Finally, head-withdrawal threshold decreased significantly in the chronic arthritis in animals under 7-NI chronic inhibition. In conclusion, nNOS immunoreactivity and mRNA expression are stable in the Sp5C during TMJ arthritis evolution, but its activity significantly increases in the chronic-phases supporting an antinociceptive role of the nNOS as evidenced by pain threshold experiment. (C) 2009 Elsevier B.V. All rights reserved.

Age-related changes in cerebellar phosphatase-1 reduce Na,K-ATPase activity

We evaluated whether changes in protein content and activity of PP-1 and PP-2A were the mechanism underneath the basal age-related reduction in alpha(2/3)-Na,K-ATPase activity in rats cerebella and whether this occurred through the cyclic GMP-PKG pathway. PP1 activity, but not its expression, increased with age, whereas PP-2 was not changed. The activity Of ot2/3-Na,K-ATPase varied with age. and there was a negative association between the PP-1 and alpha(2/3)-Na,K-ATPase activities. In young rats, the inhibition of PP-1 and PP-2A by okadaic acid (OA) increased in a dose-dependent manner alpha(1)- and alpha(2/3)-Na,K-ATPase, but had no effect on Mg-ATPase activity. A direct stimulation of PKG with 8-Br-cyclic GMP did not surmount the effect of OA. This analogue of cyclic GMP inhibited PP-1 activity only, indicating that at least part of the increase in alpha(1)- and alpha(2/3)-Na,K-ATPase activity induced by OA was mediated by the cyclic GMP-PKG-PP-1 cascade. Taking into account that PP1 inhibition increased alpha(2/3)-Na,K-ATPase activity, we propose that an age-related increase in PP-1 activity due to a decrease in cyclic GMP-PKG modulation plays a role for the age-related reduction of alpha(2/3)-Na,K-ATPase activity in rat cerebellum. (C) 2007 Elsevier Inc. All rights reserved.

Pivotal Role for Platelet-Activating Factor Receptor in CD36 Expression and oxLDL Uptake by Human Monocytes/Macrophages

The uptake of oxLDL by CD36 is not regulated by intracellular levels of cholesterol, leading to macrophage differentiation into foam cells which play a major role in atherosclerosis. Furthermore, oxLDL competes with PAF in macrophages for binding to PAF receptors (PAFR). Here we investigated the involvement of PAFR in CD36 expression and uptake of oxLDL by human monocytes/macrophages. Adherent peripheral blood mononuclear cells were treated with PAFR-antagonists (WEB2170, CV3988); inhibitors of ERK1/2 (PD98059), p38 (SB203580), JNK (SP600125) or diluents, before stimulation with oxLDL or PAF. After 24 h, uptake of FITC oxLDL and expression of CD36 was determined by flow cytometry and phosphorylation of MAP-kinases by Western blot. It was shown that the uptake of oxLDL was reduced by PAFR antagonists. CD36 expression was up-regulated by oxLDL, an effect reversed by PAFR antagonists. The up-regulation of CD36 and oxLDL uptake both required MAP-kinases activation. The oxLDL induced ERK1/2 and JNK but not p38 phosphorylation was reversed by PAFR-antagonists suggesting that oxLDL signalling involves PAFR dependent and independent pathways. In macrophages from PAFR(-/-) mice, oxLDL was unable to up-regulate CD36 expression and the oxLDL uptake was reduced compared to wild type. These results suggest that oxLDL interacts with PAFR in macrophages to increase CD36 expression and oxLDL uptake. Whereas pharmacological intervention at the level of PAFR would be beneficial in atherosclerosis remains to be determined. Copyright (C) 2011 S. Karger AG, Basel

Differential expression of apoptosis-related genes from death receptor pathway in chronic myeloproliferative diseases

Background Chronic myeloproliferative disorders (MPDs) are clonal haematopoietic stem cell malignancies characterised by an accumulation of mature myeloid cells in bone marrow and peripheral blood. Deregulation of the apoptotic machinery may be associated with MPD physiopathology. Aims To evaluate expression of death receptors` family members, mononuclear cell apoptosis resistance, and JAK2 allele burden. Subjects and Methods Bone marrow haematopoietic progenitor CD34 cells were separated using the Ficoll-hypaque protocol followed by the Miltenyi CD34 isolation kit, and peripheral blood leukocytes were separated by the Haes-Steril method. Total RNA was extracted by the Trizol method, the High Capacity Kit was used to synthesise cDNA, and real-time PCR was performed using SybrGreen in ABIPrism 7500 equipment. The results of gene expression quantification are given as 2(-Delta Delta Ct). The JAK2 V617F mutation was detected by real-time allelic discrimination PCR assay. Peripheral blood mononuclear cells (PBMCs) were isolated by the Ficoll-hypaque protocol and cultured in the presence of apoptosis inducers. Results In CD34 cells, there was mRNA overexpression for fas, faim and c-flip in polycythaemia vera (PV), essential thrombocythaemia (ET) and primary myelofibrosis (PMF), as well as fasl in PMF, and dr4 levels were increased in ET. In leukocytes, fas, c-flip and trail levels were increased in PV, and dr5 expression was decreased in ET. There was an association between dr5 and fasl expression and JAK2V617F mutation. PBMCs from patients with PV, ET or PMF showed resistance to apoptosis inducers. Conclusions The results indicate deregulation of apoptosis gene expression, which may be associated with MPD pathogenesis leading to accumulation of myeloid cells in MPDs.

Differential kinase requirement for enhancement of Fc gamma R-mediated phagocytosis in alveolar macrophages by leukotriene B(4) vs. D(4)

In alveolar macrophages, leukotriene (IT) B(4) and cysteinyl LTs (LTC(4), LTD(4) and LTE(4)) both enhance Fc gamma receptor (Fc gamma R)-mediated phagocytosis. In the present study we investigated the role of specific PKC isoforms (PKC-alpha and -delta), the MAP kinases p38 and ERK 1/2, and PI3K in mediating the potentiation of Fc gamma R-mediated phagocytosis induced by addition of leukotrienes to the AMs. It was found that exogenously added LTB(4) and LTD(4) both enhanced PKC-delta and -alpha phosphorylation during Fc gamma R engagement. Studies with isoform-selective inhibitors indicated that exogenous LTB(4) effects were dependent on both PKC-alpha and -delta, while LTD(4) effects were exclusively due to PKC-delta activation. Although both exogenous LTB(4) and LTD(4) enhanced p38 and ERK 1/2 activation, LTB(4) required only ERK 1/2, while LTD(4) required only p38 activation. Activation by both LTs was dependent on PI3K activation. Effects of endogenous LTs on kinase activation were also investigated using selective LT receptor antagonists. Endogenous LTB(4) contributed to Fc gamma R-mediated activation of PKC-alpha, ERK 1/2 and PI3K, while endogenous cysLTs contributes to activation of PKC-delta, p38 and PI3K. Taken together, our data show that the capacities of LTB(4) and LTD(4) to enhance Fc gamma R-mediated phagocytosis reflect their differential activation of specific kinase programs. (C) 2008 Elsevier Ltd. All rights reserved.

ADCK3, an ancestral kinase, is mutated in a form of recessive ataxia associated with coenzyme Q(10) deficiency

Muscle coenzyme Q(10) (CoQ(10) or ubiquinone) deficiency has been identified in more than 20 patients with presumed autosomal-recessive ataxia. However, mutations in genes required for CoQ(10) biosynthetic pathway have been identified only in patients with infantile-onset multisystemic diseases or isolated nephropathy. Our SNP-based genome-wide scan in a large consanguineous family revealed a locus for autosomal-recessive ataxia at chromosome 1q41. The causative mutation is a homozygous splice-site mutation in the aarF-domain-containing kinase 3 gene (ADCK3). Five additional mutations in ADCK3 were found in three patients with sporadic ataxia, including one known to have CoQ(10) deficiency in muscle. All of the patients have childhood-onset cerebellar ataxia with slow progression, and three of six have mildly elevated lactate levels. ADCK3 is a mitochondrial protein homologous to the yeast COQ8 and the bacterial UbiB proteins, which are required for CoQ biosynthesis. Three out of four patients tested showed a low endogenous pool of CoQ(10) in their fibroblasts or lymphoblasts, and two out of three patients showed impaired ubiquinone synthesis, strongly suggesting that ADCK3 is also involved in CoQ(10) biosynthesis. The deleterious nature of the three identified missense changes was confirmed by the introduction of them at the corresponding positions of the yeast COQ8 gene. Finally, a phylogenetic analysis shows that ADCK3 belongs to the family of atypical kinases, which includes phosphomositide and choline kinases, suggesting that ADCK3 plays an indirect regulatory role in ubiquinone biosynthesis possibly as part of a feedback loop that regulates ATP production.

Exogenous insulin stimulates glycogen accumulation in Rhipicephalus (Boophilus) microplus embryo cell line BME26 via PI3K/AKT pathway

Ticks are obligatory blood-feeding arthropods and important vectors of both human and animal disease agents. Besides its metabolic role, insulin signaling pathway (ISP) is widely described as crucial for vertebrate and invertebrate embryogenesis, development and cell survival. In such cascade, Phosphatidylinositol 3-OH Kinase (PI3K) is hierarchically located upstream Protein Kinase B (PKB). To study the insulin-triggered pathway and its possible roles during embryogenesis we used a culture of embryonic Rhipicephalus microplus cells (BME26). Exogenous insulin elevated cell glycogen content in the absence of fetal calf serum (FCS) when compared to cells without treatment. Moreover, in the presence of PI3K inhibitors (Wortmannin or LY294002) these effects were blocked. We observed an increase in the relative expression level of PI3K`s regulatory subunit (p85), as determined by qRT-PCR. In the presence of PI3K inhibitors these effects on transcription were also reversed. Additionally, treatment with Wortmannin increased the expression level of the insulin-regulated downstream target glycogen synthase kinase 3 beta (GSK3 beta). The p85 subunit showed elevated transcription levels in ovaries from fully engorged females, but was differentially expressed during tick embryogenesis. These results strongly suggest the presence of an insulin responsive machinery in BME26 cells, and its correlation with carbohydrate/glycogen metabolism also during embryogenesis. (C) 2009 Published by Elsevier Inc.

Endurance training activates AMP-activated protein kinase, increases expression of uncoupling protein 2 and reduces insulin secretion from rat pancreatic islets

Endurance exercise is known to enhance peripheral insulin sensitivity and reduce insulin secretion. However, it is unknown whether the latter effect is due to the reduction in plasma substrate availability or alterations in beta-cell secretory machinery. Here, we tested the hypothesis that endurance exercise reduces insulin secretion by altering the intracellular energy-sensitive AMP-activated kinase (AMPK) signaling pathway. Male Wistar rats were submitted to endurance protocol training one, three, or five times per week, over 8 weeks. After that, pancreatic islets were isolated, and glucose-induced insulin secretion (GIIS), glucose transporter 2 (GLUT2) protein content, total and phosphorylated calmodulin kinase kinase (CaMKII), and AMPK levels as well as peroxisome proliferator-activated receptor-gamma coactivator-1-alpha (PGC-1 alpha) and uncoupling protein 2 (UCP2) content were measured. After 8 weeks, chronic endurance exercise reduced GIIS in a dose-response manner proportionally to weekly exercise frequency. Contrariwise, increases in GLUT2 protein content, CaMKII and AMPK phosphorylation levels were observed. These alterations were accompanied by an increase in UCP2 content, probably mediated by an enhancement in PGC-1 alpha protein expression. In conclusion, chronic endurance exercise induces adaptations in beta-cells leading to a reduction in GIIS, probably by activating the AMPK signaling pathway. Journal of Endocrinology (2011) 208, 257-264