Carbohydrate ingestion attenuates the reduction in complex cognitive function and cerebral blood flow during prolonged passive heat stress in humans.

PURPOSE: To determine whether carbohydrate ingestion would reduce cognitive dysfunction in humans following long duration passive heat stress (PHS) versus consuming electrolytes alone. METHODS: Fifteen young (27 ± 4 y) healthy adults were exposed to 120 min of PHS through the use of a liquid perfused suit (50 °C) on two randomized visits. Subjects consumed fluids supplemented with electrolytes (E) or electrolytes + carbohydrates (E + C). Pre- and post-heat stress, body mass (BM) and plasma osmolality (pOsm) were measured. Heart rate (HR), blood pressure (BP), Physiological Strain Index (PSI), core temperature (Tc), plasma glucose, respiration rate (RR), end-tidal CO2 (PetCO2) and internal carotid artery (ICA) blood flow were recorded at baseline and every 15 min of heat stress. Cognitive function was assessed via the Automated Neuropsychological Assessment Metric at baseline and at 30- and 120 min during heat stress. RESULTS: There were no significant differences between fluid conditions for BM, pOsm, PSI, Tc, RR or PetCO2. Plasma glucose was ∼75% greater in the E + C condition compared to the E condition after 90 min of PHS (P < 0.05). Cognitive function (120 min) was impaired following PHS only in E condition (P < 0.05) and performance on complex cognitive tasks were better by ∼22-340% in the E + C vs. E (P < 0.05). Compared to the E condition, HR and BP were lower and ICA blood flow, vascular conductance, and glucose delivery was ∼90% greater in the E + C after 90 min of PHS (P < 0.05). CONCLUSIONS: These data are the first to demonstrate that carbohydrate ingestion may have a protective effect on cognitive function during long duration PHS. Furthermore, this protection was associated with preserved ICA blood flow and glucose delivery to the brain.

Mouse glucose transporter 9 splice variants are expressed in adult liver and kidney and are up-regulated in diabetes.

A novel glucose transporter (GLUT), mouse GLUT9 (mGLUT9), was recently cloned from mouse 7-d embryonic cDNA. Several splice variants of mGLUT9 were described, two of which were cloned (mGLUT9a and mGLUT9a Delta 209-316). This study describes the cloning and characterization of another splice variant, mGLUT9b. Cloned from adult liver, mGLUT9b is identical to mGLUT9a except at the amino terminus. Based on analysis of the genomic structure, the different amino termini result from alternative transcriptional/translational start sites. Expression and localization of these two mGLUT9 splice variants were examined in control and diabetic adult mouse tissues and in cell lines. RT-PCR analysis demonstrated expression of mGLUT9a in several tissues whereas mGLUT9b was observed primarily in liver and kidney. Using a mGLUT9-specific antibody, Western blot analysis of total membrane fractions from liver and kidney detected a single, wide band, migrating at approximately 55 kDa. This band shifted to a lower molecular mass when deglycosylated with peptide-N-glycosidase F. Both forms were present in liver and kidney. Immunohistochemical localization demonstrated basolateral distribution of mGLUT9 in liver hepatocytes and the expression of mGLUT9 in specific tubules in the outer cortex of the kidney. To investigate the alternative amino termini, mGLUT9a and mGLUT9b were overexpressed in kidney epithelium cell lines. Subcellular fractions localized both forms to the plasma membrane. Immunofluorescent staining of polarized Madin Darby canine kidney cells overexpressing mGLUT9 depicted a basolateral distribution for both splice variants. Finally, mGLUT9 protein expression was significantly increased in the kidney and liver from streptozotocin-induced diabetic mice compared with nondiabetic animals.

Interleukin-1 stimulates beta-cell necrosis and release of the immunological adjuvant HMGB1.

BACKGROUND: There are at least two phases of beta-cell death during the development of autoimmune diabetes: an initiation event that results in the release of beta-cell-specific antigens, and a second, antigen-driven event in which beta-cell death is mediated by the actions of T lymphocytes. In this report, the mechanisms by which the macrophage-derived cytokine interleukin (IL)-1 induces beta-cell death are examined. IL-1, known to inhibit glucose-induced insulin secretion by stimulating inducible nitric oxide synthase expression and increased production of nitric oxide by beta-cells, also induces beta-cell death. METHODS AND FINDINGS: To ascertain the mechanisms of cell death, the effects of IL-1 and known activators of apoptosis on beta-cell viability were examined. While IL-1 stimulates beta-cell DNA damage, this cytokine fails to activate caspase-3 or to induce phosphatidylserine (PS) externalization; however, apoptosis inducers activate caspase-3 and the externalization of PS on beta-cells. In contrast, IL-1 stimulates the release of the immunological adjuvant high mobility group box 1 protein (HMGB1; a biochemical maker of necrosis) in a nitric oxide-dependent manner, while apoptosis inducers fail to stimulate HMGB1 release. The release of HMGB1 by beta-cells treated with IL-1 is not sensitive to caspase-3 inhibition, while inhibition of this caspase attenuates beta-cell death in response to known inducers of apoptosis. CONCLUSIONS: These findings indicate that IL-1 induces beta-cell necrosis and support the hypothesis that macrophage-derived cytokines may participate in the initial stages of diabetes development by inducing beta-cell death by a mechanism that promotes antigen release (necrosis) and islet inflammation (HMGB1 release).

The role and regulation of COX-2 during viral infection.

Prostaglandins are lipid mediators, generated by cyclooxygenase (COX), that have been shown to participate in the regulation of virus replication and the modulation of inflammatory responses following infection. A number of studies support a role for PGE2 in the modulation of virus replication and virulence in a cell type and virus selective manner. Virus infection also stimulates the expression of a number of proinflammatory gene products, including COX-2, inducible nitric oxide synthase (iNOS) as well as proinflammatory cytokines. This review will focus on the mechanisms by which proinflammatory prostaglandin production regulates virus replication and virulence. In addition, the signaling pathways that are activated during a virus infection, and that regulate proinflammatory gene expression in macrophages will be reviewed. Specific attention will be placed on the ability of virus infection to activate multiple signaling cascades (such as PKR, MAPK, iPLA2, NF-kappaB) and how these pathways are integrated in the regulation of individual target gene expression.

Role of MAPK in the regulation of double-stranded RNA- and encephalomyocarditis virus-induced cyclooxygenase-2 expression by macrophages.

In response to virus infection or treatment with dsRNA, macrophages express the inducible form of cyclooxygenase-2 (COX-2) and produce proinflammatory prostaglandins. Recently, we have shown that NF-kappaB is required for encephalomyocarditis virus (EMCV)- and dsRNA-stimulated COX-2 expression in mouse macrophages. The dsRNA-dependent protein kinase R is not required for EMCV-stimulated COX-2 expression, suggesting the presence of protein kinase R-independent pathways in the regulation of this antiviral gene. In this study, the role of MAPK in the regulation of macrophage expression of cyclooxygenase-2 (COX)-2 in response to EMCV infection was examined. Treatment of mouse macrophages or RAW-264.7 cells with dsRNA or infection with EMCV stimulates the rapid activation of the MAPKs p38, JNK, and ERK. Inhibition of p38 and JNK activity results in attenuation while ERK inhibition does not modulate dsRNA- and EMCV-induced COX-2 expression and PGE2 production by macrophages. JNK and p38 appear to selectively regulate COX-2 expression, as inhibition of either kinase fails to prevent dsRNA- or EMCV-stimulated inducible NO synthase expression by macrophages. Using macrophages isolated from TLR3-deficient mice, we show that p38 and JNK activation and COX-2 expression in response to EMCV or poly(IC) does not require the presence the dsRNA receptor TLR3. These findings support a role for p38 and JNK in the selective regulation of COX-2 expression by macrophages in response to virus infection.

Regulation of membrane-associated iPLA2 activity by a novel PKC isoform in ventricular myocytes.

Thrombin stimulation of rabbit ventricular myocytes increases membrane-associated, Ca2+-independent phospholipase A2 (iPLA2) activity, resulting in accelerated hydrolysis of membrane plasmalogen phospholipids and increased production of arachidonic acid and lysoplasmenylcholine. This study was designed to investigate the signal transduction pathways involved in activation of membrane-associated iPLA2. Incubation of isolated membrane fractions suspended in Ca2+-free buffer with thrombin or phorbol 12-myristate 13-acetate resulted in a two- to threefold increase in iPLA2 activity. Prior treatment with the PKC inhibitor GF-109203X blocked iPLA2 activation by thrombin. These data suggest that a novel PKC isoform present in the membrane fraction modulates iPLA2 activity. Immunoblot analysis revealed a significant portion of PKC-epsilon present in the membrane fraction, but no other membrane-associated novel PKC isoform was detected by this method. These data indicate that activation of membrane-associated iPLA2 is mediated by a membrane-associated novel PKC isoform in thrombin-stimulated rabbit ventricular myocytes.

Regulation of cyclooxygenase-2 expression by macrophages in response to double-stranded RNA and viral infection.

In this study the regulation of macrophage expression of cyclooxygenase-2 (COX-2) in response to dsRNA and virus infection was examined. Treatment of RAW 264.7 macrophages with dsRNA results in COX-2 mRNA accumulation and protein expression and the production of PGE(2). Similar to dsRNA, encephalomyocarditis virus (EMCV) infection of RAW 264.7 cells stimulates COX-2 expression and PGE(2) accumulation. The dsRNA-dependent protein kinase (PKR), which has been shown to participate in the regulation of gene expression in response to dsRNA and virus infection, does not appear to participate in the regulation of COX-2 expression by macrophages. Expression of dominant negative mutants of PKR in RAW 264.7 cells fails to attenuate dsRNA- and EMCV-induced COX-2 expression or PGE(2) production. Furthermore, dsRNA and EMCV stimulate COX-2 expression and PGE(2) accumulation to similar levels in macrophages isolated from wild-type and PKR-deficient mice. Recently, a novel PKR-independent role for the calcium-independent phospholipase A(2) (iPLA(2)) in the regulation of inducible NO synthase expression by macrophages in response to virus infection has been identified. The selective iPLA(2) suicide substrate inhibitor bromoenol lactone prevents dsRNA- and EMCV-stimulated inducible NO synthase expression; however, bromoenol lactone does not attenuate dsRNA- or EMCV-induced COX-2 expression by macrophages. In contrast, inhibition of NF-kappaB activation prevents dsRNA-stimulated COX-2 expression and PGE(2) accumulation by macrophages. These findings indicate that virus infection and treatment with dsRNA stimulate COX-2 expression by a mechanism that requires the activation of NF-kappaB and that is independent of PKR or iPLA(2) activation.