Effects of cannabinoid receptors on skeletal muscle oxidative pathways

The endocannabinoids, a recently discovered endogenous, lipid derived, signaling system regulating energy metabolism, have effects on central and peripheral energy metabolism predominantly via the cannabinoid receptor type 1 (CB1). CB1 is expressed centrally in the hypothalamus and nucleus accumbens and peripherally in adipocytes and skeletal muscle. This study determined the effect of endocannabinoids on the expression of genes regulating energy metabolism in human skeletal muscle. Primary cultures of myotubes (lean and obese; n = 3/group) were treated with the cannabinoid receptor agonist, anandamide (AEA) (0.2 and 5 μM) and the CB1 specific antagonist AM251 (0.2 and 5 μM) separately and in combination for 24 h. The expression of mRNA for AMP-activated protein kinase (AMPK) alpha 1 (α1) and alpha 2 (α2), pyruvate dehydrogenase kinase 4 (PDK4) and peroxisome proliferator-activated receptor-γ co-activator-1α (PGC-1α) were determined using ‘Real Time’ RT-PCR. AMPKα1 mRNA increased in lean and obese myotubes in response to AM251 (P < 0.05). AEA inhibited the effect of AM251 on AMPKα1 mRNA levels in myotubes from lean and obese subjects (P < 0.05); the dose–response curve was shifted to the left in the obese. In response to AM251, irrespective of the presence of AEA, PDK4 expression was decreased in lean and obese myotubes (P < 0.05). Taken together these data suggest that endocannabinoids regulate pathways affecting skeletal muscle oxidation, effects particularly evident in myotubes from obese individuals.

Ecological niche modelling and DNA sequencing support a new, morphologically cryptic beetle species unveiled by DNA barcoding

Background: DNA sequencing techniques used to estimate biodiversity, such as DNA barcoding, may reveal cryptic species. However, disagreements between barcoding and morphological data have already led to controversy. Species delimitation should therefore not be based on mtDNA alone. Here, we explore the use of nDNA and bioclimatic modelling in a new species of aquatic beetle revealed by mtDNA sequence data.

Methodology/Principal Findings: The aquatic beetle fauna of Australia is characterised by high degrees of endemism, including local radiations such as the genus Antiporus. Antiporus femoralis was previously considered to exist in two disjunct, but morphologically indistinguishable populations in south-western and south-eastern Australia. We constructed a phylogeny of Antiporus and detected a deep split between these populations. Diagnostic characters from the highly variable nuclear protein encoding arginine kinase gene confirmed the presence of two isolated populations. We then used ecological niche modelling to examine the climatic niche characteristics of the two populations. All results support the status of the two populations as distinct species. We describe the south-western species as Antiporus occidentalis sp.n.

Conclusion/Significance: In addition to nDNA sequence data and extended use of mitochondrial sequences, ecological niche modelling has great potential for delineating morphologically cryptic species. © 2011 Hawlitschek et al.

Insulin-stimulated insulin recetor substrate-2-associated phosphatidylinositol 3-kinase activity is enhanced in human skeletal muscle after exercise.

Exercise increases skeletal muscle insulin action but the underlying mechanisms mediating this are equivocal. In mouse skeletal muscle, prior exercise enhances insulin-stimulated insulin receptor substrate-2 (IRS-2) signaling (Diabetes 2002;51:479-83), but it is unknown if this also occurs in humans. Hyperinsulinemic-euglycemic clamps were performed on 7 untrained males at rest and immediately after 60 minutes of cycling exercise at ~75% Vo_2peak. Muscle biopsies were obtained at basal, immediately after exercise, and at 30 and 120 minutes of hyperinsulinemia. Insulin infusion increased (P < .05) insulin receptor tyrosine phosphorylation similarly in both the rest and exercise trials. Under resting conditions, insulin infusion resulted in a small, but non–statistically significant increase in IRS-2–associated phosphatidylinositol 3 (PI 3)–kinase activity over basal levels. Exercise per se decreased (P < .05) IRS-2–associated PI 3–kinase activity. After exercise, insulin-stimulated IRS-2–associated PI 3–kinase activity tended to increase at 30 minutes and further increased (P < .05) at 120 minutes when compared with the resting trial. Insulin increased (P < .05) Akt Ser⁴⁷³ and GSK-3α/β Ser²¹/Ser⁹ phosphorylation in both trials, with the response tending to be higher in the exercise trial. In conclusion, in the immediate period after an acute bout of exercise, insulin-stimulated IRS-2 signaling is enhanced in human skeletal muscle.

Impaired activation of AMP-Kinase and fatty acid oxidation by globular adiponectin in cultured human skeletal muscle of obese type 2 diabetics

Adiponectin is an adipocyte-derived hormone associated with antidiabetic actions. In rodent skeletal muscle, globular adiponectin (gAD) activates AMP-kinase (AMPK) and stimulates fatty acid oxidation effects mediated through the adiponectin receptors, AdipoR1 and AdipoR2. In the present study, we examined the mRNA expression of adiponectin receptors and the effects of gAD on AMPK activity and fatty acid oxidation in skeletal muscle myotubes from lean, obese, and obese type 2 diabetic subjects. Myotubes from all groups expressed approximately 4.5-fold more AdipoR1 mRNA than AdipoR2, and obese subjects tended to have higher AdipoR1 expression (P = 0.052). In lean myotubes, gAD activates AMPK[alpha]1 and -[alpha]2 by increasing Thr172 phosphorylation, an effect associated with increased acetyl-coenzyme A carboxylase (ACC[beta]) Ser221 phosphorylation and enhanced rates of fatty acid oxidation, effects similar to those observed after pharmacological AMPK activation by 5-aminoimidazole-4-carboxamide riboside. In obese myotubes, the activation of AMPK signaling by gAD at low concentrations (0.1 [mu]g/ml) was blunted, but higher concentrations (0.5 [mu]g/ml) stimulated AMPK[alpha]1 and -[alpha]2 activities, AMPK and ACC[beta] phosphorylation, and fatty acid oxidation. In obese type 2 diabetic myotubes, high concentrations of gAD stimulated AMPK[alpha]1 activity and AMPK phosphorylation; however, ACC[beta] phosphorylation and fatty acid oxidation were unaffected. Reduced activation of AMPK signaling and fatty acid oxidation in obese and obese diabetic myotubes was not associated with reduced protein expression of AMPK[alpha] and ACC[beta] or the expression and activity of the upstream AMPK kinase, LKB1. These data suggest that reduced activation of AMPK by gAD in obese and obese type 2 diabetic subjects is not caused by reduced adiponectin receptor expression but that aspects downstream of the receptor may inhibit AMPK signaling.

Identification of an integral plasma membrane pool of protein kinase C in mammalian tissues and cells

Protein kinase C (PKC) is a family of serine/threonine protein kinases that are pivotal in cellular regulation. Since its discovery in 1977, PKCs have been known as cytosolic and peripheral membrane proteins. However, there are reports that PKC can insert into phospholipids vesicles in vitro. Given the intimate relationship between the plasma membrane and the activation of PKC, it is important to determine whether such “membrane-inserted” form of PKC exists in mammalian cells or tissues. Here, we report the identification of an integral plasma membrane pool for all the 10 PKC isozymes in vivo by their ability to partition into the detergent-rich phase in Triton X-114 phase partitioning, and by their resistance to extractions with 0.2 M sodium carbonate (pH 11.5), 2 M urea and 2 M sodium chloride. The endogenous integral membrane pool of PKC in mouse fibroblasts is found to be acutely regulated by phorbol ester or diacylglycerol, suggesting that this pool of PKC may participate in cellular processes known to be regulated by PKC. At least for PKCα, the C2–V3 region at the regulatory domain of the kinase is responsible for membrane integration. Further exploration of the function of this novel integral plasma membrane pool of PKC will not only shed new light on molecular mechanisms underlying its cellular functions but also provide new strategies for pharmaceutical modulation of this important group of kinases.

Src homology 3-domain growth factor receptor-bound 2-like (endophilin) interacting protein 1, a novel neuronal protein that regulates energy balance

To identify genes involved in the central regulation of energy balance, we compared hypothalamic mRNA from lean and obese Psammomys obesus, a polygenic model of obesity, using differential display PCR. One mRNA transcript was observed to be elevated in obese, and obese diabetic, P. obesus compared with lean animals and was subsequently found to be increased 4-fold in the hypothalamus of lethal yellow agouti (Ay/a) mice, a murine model of obesity and diabetes. Intracerebroventricular infusion of antisense oligonucleotide targeted to this transcript selectively suppressed its hypothalamic mRNA levels and resulted in loss of body weight in both P. obesus and Sprague Dawley rats. Reductions in body weight were mediated by profoundly reduced food intake without a concomitant reduction in metabolic rate. Yeast two-hybrid screening, and confirmation in mammalian cells by bioluminescence resonance energy transfer analysis, demonstrated that the protein it encodes interacts with endophilins, mediators of synaptic vesicle recycling and receptor endocytosis in the brain. We therefore named this transcript Src homology 3-domain growth factor receptor-bound 2-like (endophilin) interacting protein 1 (SGIP1). SGIP1 encodes a large proline-rich protein that is expressed predominantly in the brain and is highly conserved between species. Together these data suggest that SGIP1 is an important and novel member of the group of neuronal molecules required for the regulation of energy homeostasis.

A pseudosymmetric cell adhesion regulatory domain in the β7 tail of the integrin α4β7 that interacts with focal adhesion kinase and src

The β7 integrins α4β7 and Eβ7 play key roles in forming the gut-associated lymphoid tissue, and contribute to chronic inflammation. The α4β7 integrin-mediated adhesion of activated lymphocytes is largely due to a transient increase in avidity from ligand-induced clustering of α4β7 at the cell-surface. Here, we report that L and D enantiomers of a cell-permeable peptide YDRREY encompassing residues 735-740 of the cytoplasmic tail of the β7 subunit inhibit the adhesion of T cells to β7 integrin ligands. The YDRREY peptide abrogated mucosal addressin cell adhesion molecule-1-induced clustering of α4β7 on the surface of activated T cells. A mutated form of the YDRREY peptide carrying either single or double conservative mutations at Tyr⁷³⁵Phe and Tyr⁷⁴⁰Phe was unable to inhibit T cell adhesion, suggesting that both tandem tyrosines are critical for activity. The YDRREY peptide was bound and phosphorylated by focal adhesion kinase and src, which may serve to sequester cytoskeletal proteins to the cytoplasmic domain of 4β7. The quasi-palindromic sequence YDRREY within the β7 cytoplasmic tail constitutes a cell adhesion regulatory domain that modulates the interaction of β7-expressing leukocytes with their endothelial and epithelial ligands. Cell-permeable peptidomimetics based on this motif have utility as anti-inflammatory reagents for the treatment of chronic inflammatory disease.

On performance of transform domain adaptive filters with Markov-2 inputs

In this paper, the analysis for the performance of the discrete Fourier transform LMS adaptive filter (DFT-LMS) and the discrete cosine transform LMS adaptive filter (DCT-LMS) for the Markov-2 inputs is presented. To improve the convergence property of the least mean squares (LMS) adaptive filter, the DFT-LMS and DCT-LMS preprocess the inputs with the fixed orthogonal transforms and power normalization. We derive the asymptotic results for the eigenvalues and eigenvalue distributions of the preprocessed input autocorrelation matrices with DFT-LMS and DCT-LMS for Markov-2 inputs. These results explicitly show the superior decorrelation property of DCT-LMS over that of DFT-LMS, and also provide the upper bounds for the eigenvalue spreads of the finite-length DFT-LMS and DCT-LMS adaptive filters. Simulation results are demonstrated to support the analytic results.

Effect of training on activation of extracellular signal-regulated kinase 1/2 and p38 mitogen-activated protein kinase pathways in rat soleus muscle

1. The effect of a chronic programme of either low- or moderate-to-high-intensity treadmill running on the activation of the extracellular-signal regulated protein kinase (ERK1/2) and the p38 mitogen-activated protein kinase (MAPK) pathways was determined in rat muscle. 2. Sprague-Dawley rats were assigned to one of three groups: (i) sedentary (NT; n = 8); (ii) low-intensity training (8 m/min; LIT; n = 16); and (iii) moderate-to-high-intensity training (28 m/min; HIT;n = 16). The training regimens were planned so that animals covered the same distance and had similar glycogen utilization for both LIT and HIT exercise sessions. 3. A single bout of LIT or HIT following 8 weeks of training led to a twofold increase in the phosphorylation of ERK1/2 (P = 0.048) and a two- to threefold increase in p38 MAPK (P = 0.005). Extracellular signal-regulated kinase 1/2 phosphorylation in muscle sampled 48 h after the last exercise bout was similar to sedentary values, while p38 MAPK phosphorylation was 70-80% lower than sedentary. One bout of LIT or HIT increased total ERK1/2 and p38 MAPK expression, with the magnitude of this increase being independent of prior exercise intensity or duration. Extracellular signal-regulated kinase 1/2 expression was increased three- to fourfold in muscle sampled 48 h after the last exercise bout irrespective of the prior training programme (P = 0.027), but p38 MAPK expression was approximately 90% lower than sedentary values. 4. In conclusion, exercise-training of different intensities/durations results in selective postexercise activation of intracellular signalling pathways, which may be one mechanism regulating specific adaptations induced by diverse training programmes.

Granulocyte colony-stimulating factor receptor signalling via Janus kinase 2/signal transducer and activator of transcription 3 in ovarian cancer

Ovarian cancer remains a major cause of cancer mortality in women, with only limited understanding of disease aetiology at the molecular level. Granulocyte colony-stimulating factor (G-CSF) is a key regulator of both normal and emergency haematopoiesis, and is used clinically to aid haematopoietic recovery following ablative therapies for a variety of solid tumours including ovarian cancer.

Targeting CD44, ABCG2 and CD133 markers using aptamers: in silico analysis of CD133 extracellular domain 2 and its aptamer

The application of nucleic acid aptamers for the diagnosis and therapy of cancer stem cells (CSCs) is expanding. The current study truncated and probed various existing aptamers against CSC markers CD44, ABCG2 and CD133 in retinoblastoma (RB) primary cells, cell lines, a breast cancer cell line and MCF7-sphere. Truncated CD44 aptamer retained its specific binding to cancer cells, ABCG2+ve MCF7-spheres and CD133+ve RB cells. Similarly, ABCG2 and CD133 aptamers showed higher affinity to ABCG2+ve, CD133+ve cells than the negative population and cell lines. All aptamers appreciably reduced viability of up to 50% and 32% of the primary RB tumor cells and cell lines, respectively. Colony formation of MCF7, RB cell lines and MCF7-sphere growth were inhibited significantly. Structure prediction, simulation of CD133 extracellular domain 2 (ExD2) and A15 followed by docking to comprehend the potential interaction revealed hydrogen bonds and non bonded interactions between them. This information could be used to improve the A15 aptamer to gain more interactions with CD133. Thus approaches undertaken here can be applied universally for cell-specific targeting, and the aptamers studied against CSC markers deserve further in vivo studies.