Masked and overt autoantibodies specific to the DPD epitope of 65-kDa glutamate decarboxylase (GAD65-DPD) are associated with preserved ß-cell functional reserve in ketosis-prone diabetes.

CONTEXT: Ketosis-prone diabetes (KPD), defined by presentation with diabetic ketoacidosis (DKA), comprises 4 subgroups based on the presence or absence of islet cell autoantibodies (A(-) or A(+)) and ß-cell functional reserve (ß(-) or ß(+)). Among A(+) KPD, autoantibody epitope reactivity to 65-kDa glutamate decarboxylase (GAD65), defined by monoclonal GAD65Ab(DPD), was associated with greater ß-cell functional reserve. In a majority of healthy individuals, GAD65Ab are present in the sera but are masked by anti-idiotypic antibodies; in contrast, overtly GAD65Ab-positive patients with autoimmune type 1 diabetes patients lack these anti-idiotypic antibodies. OBJECTIVE: Our objective was to determine the presence of masked and overt GAD65Ab(DPD) in relation to ß-cell function and genetic risk factors in KPD patients. DESIGN: We investigated the associations of masked and overt GAD65Ab(DPD) with ß-cell functional reserve, and their relationship with human leukocyte antigen (HLA) class II haplotypes linked to autoimmune diabetes susceptibility or resistance, in a large KPD cohort. PATIENTS: Adult KPD patients (n = 384) were followed longitudinally in a research clinic. MAIN OUTCOME MEASURES: ß-Cell function, autoantibody status, GAD65Ab epitopes, and HLA class II haplotypes were evaluated. RESULTS: Overall, KPD patients with ß-cell functional reserve (ß(+) subgroups) showed significantly higher frequency of masked GAD65Ab(DPD) than patients without ß-cell functional reserve (ß(-) subgroups): 112 of 144 (79%) compared with 59 of 100 (59%), respectively (P = .002). Masked or overt GAD65Ab(DPD) were also more frequent among autoantibody-positive patients with preserved ß-cell functional reserve (A(+)ß(+) KPD) than those lacking ß-cell function (A(+)ß(-) KPD): 77% compared with 55% (P = .01). The susceptibility HLA haplotypes DQA1*0301/DQB1*0302 and DQA1*0301/DQB1*0201 were associated with absence of overt or masked GAD65Ab(DPD) (odds Ratios 2.3 and 2.2, respectively). CONCLUSIONS: Masked GAD65Ab(DPD) are strongly associated with preserved ß-cell functional reserve among patients with KPD. Absence of GAD65Ab(DPD) reactivity is associated with 2 HLA class II susceptibility haplotypes for autoimmune type 1 diabetes.

ZnT8 autoantibody titers in type 1 diabetes patients decline rapidly after clinical onset.

Autoantibodies to the islet-specific zinc transporter isoform 8 (ZnT8) are detected in the majority of type 1 diabetes patients prior to and at clinical diagnosis. The presence of ZnT8Ab after diagnosis has not been investigated. This study analyzed the autoantibody response to ZnT8 in regard to age at onset and disease duration. Two new onset type 1 diabetes patient cohorts with different age distributions at onset (2-17 and 15-34 years of age at onset), a longitudinal subset of the younger type 1 diabetes patient cohort (n = 32), and a cohort of GAD65Ab-positive LADA patients (n = 47) was analyzed for the presence of autoantibodies directed to the two major isoforms, ZnT8-Arginine (ZnT8R) and ZnT8-Tryptophan (ZnT8W). The majority of type 1 diabetes patients tested positive for ZnT8Ab to both isoforms. ZnT8Ab titers were significantly higher in the younger type 1 diabetes patients as compared with the older cohort (ZnT8RAb at a median of 148 and 29 U/ml, respectively, p < 0.001) (ZnT8WAb at a median of 145 and 58 U/ml, respectively, p < 0.01). ZnT8RAb and ZnT8WAb titers were significantly lower in the LADA patients (ZnT8RAb at a median of 14 U/ml, ZnT8WAb at a median of 25 U/ml) as compared with either type 1 diabetes cohorts. In our longitudinal analysis of type 1 diabetes patients after clinical diagnosis, ZnT8Ab levels to both isoforms declined significantly during the initial year of disease (ZnT8RAb from a median of 320-162 U/ml, p = 0.0001; ZnT8WAb from a median of 128-46 U/ml, p = 0.0011). The antibody titers further declined during the following 4 years (p < 0.0001). We conclude that ZnT8Ab presents a useful marker for type 1 diabetes, especially in younger patients at disease diagnosis.

Animal insulin therapy induces a biased insulin antibody response that persists for years after introduction of human insulin.

Administration of exogenous insulin for the treatment of diabetes is often accompanied by the development of insulin antibodies (IA). These antibodies may affect the patient's requirement for insulin by acting as an insulin binding reservoir. The improvement of insulin purification in the 1970s and the development of human recombinant insulin both reduced the incidence of IA and their binding levels. This study investigates the parameters affecting IA frequency and binding levels in a cohort of type 1 diabetes (T1D) patients. All patients were treated with human recombinant insulin. About half of the patients had received animal insulin prior to the introduction of human recombinant insulin. We tested the IA frequency and binding level for all serum samples. IA were further analyzed for their epitope specificity comparing human and porcine insulin binding. We found that T1D patients who received animal insulin in the past show significantly higher IA binding levels as compared to patients treated exclusively with human recombinant insulin (IA binding level of 0.9 and 0.25 index, respectively, P = 0.005). T1D patients who received animal insulin in the past showed a relative bias towards porcine insulin, as compared to T1D patients who were treated with human recombinant insulin exclusively (P < 0.0001). We conclude that IA binding level and epitope specificity are biased by treatment with animal insulin. This bias remains for over 20 years after animal insulin treatment is terminated.

Comparison of three assays for the detection of GAD65Ab-specific anti-idiotypic antibodies.

Anti-idiotypic antibodies (anti-Id) to autoantibodies are present in several autoimmune diseases and are hypothesized to have regulatory function. Recently we reported the presence of anti-Id to a major type 1 diabetes-associated autoantibody (GAD65Ab) in sera of healthy individuals. Our current assay for the detection of GAD65Ab-specific anti-Id requires the initial removal of anti-Id from the sera using immobilized monoclonal GAD65Ab, followed by detection of the now exposed GAD65Ab. However, anti-Id in samples that are GAD65Ab-negative cannot be detected in this assay. Furthermore, we cannot distinguish between serum GAD65Ab and the monoclonal GAD65Ab used in the absorption of anti-Id. In this study we evaluated two novel detection assays for GAD65Ab-specific anti-Id. The biotin/streptavidin based absorption assay utilizes the strong interaction of biotin and streptavidin to prevent possible leakage of the immobilized antibody. Moreover, this assay format allows to identify the origin of the detected GAD65Ab. The ECL-based assay allows the direct detection of anti-Id independent of the presence of GAD65Ab. We analyzed new-onset type 1 diabetes patients (n=133) and matched healthy controls (n=178) for the presence of GAD65Ab-specific anti-Id using both new detection assays and the original absorption assay. We found that all three assays can distinguish between the type 1 diabetes cohort and the healthy control samples. The biotin/streptavidin assay allowed us to positively exclude the monoclonal GAD65Ab as the source of the detected GAD65Ab. While the original absorption assay showed the highest sensitivity and specificity, the ECL format showed the highest peak signal-to-noise ratio and excellent linear correlation, making this assay our first choice for quantification of anti-Id.

Peroxisome proliferator-activated receptor-gamma agonist improves skeletal muscle insulin signaling in the pregestational intrauterine growth-restricted rat offspring.

The effect of early intervention with a peroxisome proliferator-activated receptor-gamma (PPARgamma) agonist on skeletal muscle GLUT4 translocation and insulin signaling was examined in intrauterine (IUGR) and postnatal (PNGR) growth-restricted pregestational female rat offspring. Rosiglitazone [11 mumol/day provided from postnatal day (PN)21 to PN60] improved skeletal muscle insulin sensitivity and GLUT4 translocation in prenatal nutrient restriction [50% calories from embryonic day (e)11 to e21; IUGR] with (IUGR+PNGR) and without (IUGR) postnatal nutrient restriction (50% calories from PN1 to PN21; PNGR) similar to that of control (ad libitum feeds throughout; Con) (n = 6 each). This was accomplished by diminished basal and improved insulin-responsive GLUT4 association with the plasma membrane in IUGR, IUGR+PNGR, and PNGR mimicking that in Con (P < 0.005). While no change in p85-phosphatidylinositol 3-kinase (PI3-K) and phosphatase and tensin homolog deleted on chromosome 10 (PTEN) was observed, a decrease in protein tyrosine phosphatase 1B (PTP1B; P < 0.0002) and SH2-containing protein tyrosine phosphatase 2 (SHP2; P < 0.05) contributing to the rosiglitazone-induced insulin sensitivity was seen only in IUGR+PNGR. In contrast, an increase in phosphorylated 5'-adenosine monophosphate kinase (pAMPK; P < 0.04) and insulin responsiveness of phosphorylated phosphoinositide-dependent protein kinase-1 (pPDK1; P < 0.05), pAkt (P < 0.01), and particularly pPKCzeta (P < 0.0001) and its corresponding enzyme activity (P < 0.005) were observed in all four experimental groups. We conclude that early introduction of PPARgamma agonist improved skeletal muscle activation of AMPK and insulin signaling, resulting in insulin-independent AMPK and insulin-responsive GLUT4 association with plasma membranes in IUGR, IUGR+PNGR, and PNGR adult offspring, similar to that of Con. These findings support a role for insulin sensitizers in preventing the subsequent development of gestational or type 2 diabetes mellitus in intrauterine and postnatal growth-restricted offspring.

Early exercise regimen improves insulin sensitivity in the intrauterine growth-restricted adult female rat offspring.

We examined the effect of early exercise training (Ex) on glucose kinetics, basal, and insulin-stimulated skeletal muscle (SKM) plasma membrane (PM) GLUT4 in pre- and/or postnatal nutrient-restricted adult rat offspring compared with sedentary (Sed) state. Pregestational control female (Ex CON vs. Sed CON) and offspring exposed to prenatal (Ex IUGR vs. Sed IUGR), postnatal (Ex PNGR vs. Sed PNGR), or pre- and postnatal (Ex IUGR + PNGR vs. Sed IUGR + PNGR) nutrient restriction were studied. The combined effect of exercise and pre/postnatal nutrition in the Ex IUGR demonstrated positive effects on basal and glucose-stimulated plasma insulin response (GSIR) with suppression of endogenous hepatic glucose production (HGP) compared with sedentary state. Ex PNGR was hyperglycemic after glucose challenge with no change in glucose-stimulated insulin production or HGP compared with sedentary state. Ex IUGR + PNGR remained glucose tolerant with unchanged glucose-stimulated insulin production but increased endogenous HGP compared with sedentary state. Basal SKM PM-associated GLUT4 was unchanged by exercise in all four groups. Whereas Ex PNGR and Ex IUGR + PNGR insulin responsiveness was similar to that of Ex CON, Ex IUGR remained nonresponsive to insulin. Early introduction of regular Ex in the pregestational female offspring had a positive effect on hepatic adaptation to GSIR and HGP in IUGR and IUGR + PNGR, with no effect in PNGR. Change in insulin responsiveness of SKM GLUT4 translocation was observed in exercised IUGR + PNGR and PNGR but not in exercised IUGR.

GAD65 autoantibody responses in Japanese latent autoimmune diabetes in adult patients.

OBJECTIVE: To determine whether development of insulin requirement in patients with latent autoimmune diabetes in adults (LADA) is accompanied with the emergence of a type 1 diabetes-like autoimmune response. RESEARCH DESIGN AND METHODS: We correlated beta-cell-specific autoimmunity reflected in autoantibodies to the 65-kDa isoform of GAD (GAD65) with insulin requirement. We determined GAD65Ab epitope specificities in type 1 diabetic patients, LADA patients without insulin requirement (nonprogressed), and LADA patients that had developed insulin requirement (progressed). RESULTS: Recognition of a type 1 diabetes-specific GAD65Ab epitope was more pronounced in type 1 diabetic patients than in nonprogressed (P < 0.001) or progressed (P < 0.01) LADA patients, with no significant differences between the two LADA cohorts. These differences were particularly pronounced in samples with GAD65Ab titers <1,000 units/ml, with no differences in epitope specificities in samples with higher GAD65Ab titers. Disease duration (initial diabetes diagnosis until sample collection or development of insulin requirement) in nonprogressed and progressed LADA patients, respectively, was not correlated with epitope specificity, suggesting lack of epitope maturation. This was supported by epitope analyses of longitudinal samples from LADA patients during progression to insulin requirement. CONCLUSIONS: First, the GAD65Ab-specific autoimmune reaction in type 1 diabetic patients with low and moderate GAD65Ab titers differs from that in LADA patients, irrespective of insulin requirement. Second, the GAD65Ab-specific autoimmune response in LADA patients does not change after their initial diabetes diagnosis. Finally, LADA patients with high GAD65Ab titers resemble type 1 diabetic patients in their GAD65Ab epitope specificity.

The lack of anti-idiotypic antibodies, not the presence of the corresponding autoantibodies to glutamate decarboxylase, defines type 1 diabetes.

Autoantibodies to glutamate decarboxylase 65 (GAD65Ab) are commonly believed to be a major characteristic for type 1 diabetes (T1D). We investigated the presence of GAD65Ab in healthy individuals (n = 238) and first-degree relatives (FDRs) of T1D patients (n = 27) who tested negative for GAD65Ab in conventional RIAs. Sera were applied to affinity columns coated with GAD65-specific mAbs to absorb anti-idiotypic antibodies (anti-Ids). The absorbed sera were analyzed for binding to GAD65 by RIAs. Both healthy individuals and FDRs present GAD65Ab that are inhibited by anti-Id, masking them in conventional detection methods. The presence of GAD65Ab-specific anti-Ids was confirmed by competitive ELISA. Remarkably, T1D patients (n = 54) and Stiff Person Syndrome patients (n = 8) show a specific lack of anti-Ids to disease-associated GAD65Ab epitopes. Purified anti-Ids from healthy individuals and FDRs inhibited the binding of GAD65Ab from T1D patients to GAD65. We conclude that masked GAD65Ab are present in the healthy population and that a lack of particular anti-Ids, rather than GAD65Ab per se, is a characteristic of T1D. The lack of these inhibitory antibodies may contribute to T cell activation by GAD65Ab.

Modulation of diabetes in NOD mice by GAD65-specific monoclonal antibodies is epitope specific and accompanied by anti-idiotypic antibodies.

Type 1 diabetes is caused by the autoimmune destruction of pancreatic beta cells. Here we show that administration of a human monoclonal antibody (b96.11) specific to the 65-kDa isoform of glutamate decarboxylase (GAD65) to prediabetic non-obese diabetic (NOD) mice significantly delays the onset of autoimmune diabetes. We found this effect to be epitope-specific, as only b96.11 showed this therapeutic property, while a GAD65-specific human monoclonal control antibody (b78) derived from the same patient, but specific to a different determinant of GAD65, had no significant effect on the progression of disease. Administration of b96.11 or b78 to NOD mice was accompanied by the generation of anti-idiotypic antibodies. Importantly, the induced anti-idiotypic antibodies were specific for the immunizing antibody and blocked the binding of GAD65 by the respective antibody. These findings suggest a potential role for the internal image of the GAD65 determinant recognized by b96.11 in the anti-idiotypic antibody, supporting an immunomodulatory role for GAD65-specific autoantibodies, as originally postulated by Jerne.

Effect of ethanol on inflammatory responses. Implications for pancreatitis.

BACKGROUND/AIMS: Alcohol use alters inflammatory cell responses. While alcohol has direct effects on pancreatic acinar cells, activation of inflammatory cells is a major component of the pathology of alcoholic pancreatitis. METHODS: The effects of acute or chronic alcohol exposure were evaluated in human monocytes on the production of TNFalpha or IL-10 production, pro-inflammatory gene and nuclear factor-kappaB (NF-kappaB) activation. RESULTS: Moderate, acute alcohol consumption or equivalent doses of alcohol in vitro had anti-inflammatory effects on monocyte activation via inhibition of pro-inflammatory genes and NF-kappaB activation, inhibition of TNFalpha production and augmentation of the anti-inflammatory cytokine, IL-10. In contrast, acute alcohol treatment augmented NF-kappaB activation and TNFalpha production and inhibited IL-10 levels in the presence of complex stimulation with combined TLR2 and TLR4 ligands. Prolonged alcohol exposure also resulted in an increase in NF-kappaB and TNFalpha production in response to TLR4 stimulation with LPS. CONCLUSION: These results suggest that alcohol can either attenuate or promote inflammatory responses that are critical in pancreatitis. Our results support the hypothesis that both acute alcohol intake in the presence of complex stimuli (such as necrotic cells) and chronic alcohol exposure result in hyper-responsiveness of monocytes to inflammatory signals and may contribute to increased inflammation in pancreatitis.

Transgenerational inheritance of the insulin-resistant phenotype in embryo-transferred intrauterine growth-restricted adult female rat offspring.

To determine mechanisms underlying the transgenerational presence of metabolic perturbations in the intrauterine growth-restricted second-generation adult females (F2 IUGR) despite normalizing the in utero metabolic environment, we examined in vivo glucose kinetics and in vitro skeletal muscle postinsulin receptor signaling after embryo transfer of first generation (F1 IUGR) to control maternal environment. Female F2 rats, procreated by F1 pre- and postnatally nutrient- and growth-restricted (IUGR) mothers but embryo transferred to gestate in control mothers, were compared with similarly gestating age- and sex-matched control (CON) F2 progeny. Although there were no differences in birth weight or postnatal growth patterns, the F2 IUGR had increased hepatic weight, fasting hyperglycemia, hyperinsulinemia, and unsuppressed hepatic glucose production, with no change in glucose futile cycling or clearance, compared with F2 CON. These hormonal and metabolic aberrations were associated with increased skeletal muscle total GLUT4 and pAkt concentrations but decreased plasma membrane-associated GLUT4, total pPKCzeta, and PKCzeta enzyme activity, with no change in total SHP2 and PTP1B concentrations in IUGR F2 compared with F2 CON. We conclude that transgenerational presence of aberrant glucose/insulin metabolism and skeletal muscle insulin signaling of the adult F2 IUGR female offspring is independent of the immediate intrauterine environment, supporting nutritionally induced heritable mechanisms contributing to the epidemic of type 2 diabetes mellitus.

TLR2- and TLR4-mediated signals determine attenuation or augmentation of inflammation by acute alcohol in monocytes.

Most pathogens express ligands for multiple TLRs that share common downstream signaling. In this study, we investigated the effects of acute alcohol on inflammatory pathways induced by TLR2 or TLR4 ligands and their combination. In human monocytes, alcohol attenuated TLR4- but not TLR2-induced TNF-alpha protein and mRNA levels and NF-kappaB activation. In contrast, acute alcohol augmented TNF-alpha production when both TLR2 and TLR4 ligands were present. IL-1R-associated kinase (IRAK)-1 activity was reduced by alcohol in TLR4, but it was augmented in TLR2- plus TLR4-stimulated cells. IRAK-monocyte, an inhibitor of IRAK-1, was induced in TLR4, but it was reduced in TLR2- plus TLR4-stimulated monocytes by alcohol. This was supported by decreased IRAK-1:TRAF6 association in TLR4 induced but sustained presence of IRAK-1:TRAF6 complexes in TLR2- plus TLR4-stimulated monocytes after alcohol treatment. Phosphorylation of MAPKs such as ERK1/2 was selectively inhibited by acute alcohol in TLR4-stimulated cells. In contrast, JNK phosphorylation as well as AP-1 nuclear binding were augmented by acute alcohol in the presence of combined TLR4 and TLR2 stimulation. Consistent with this result, the JNK inhibitor prevented alcohol-induced augmentation of TNF-alpha production. These results suggest that acute alcohol attenuates TLR4-induced inflammation via inhibition of IRAK-1 and ERK1/2 kinases and increases in IRAK-monocyte levels in monocytes. Conversely, in the presence of TLR2 and TLR4 ligands, acute alcohol augments inflammatory responses via IRAK-1 activation and JNK phosphorylation. Thus, the complexity of TLR-mediated signals may determine attenuation or augmentation of inflammatory responses by acute alcohol.

Cytokines and alcohol.

Cytokines are multifunctional proteins that play a critical role in cellular communication and activation. Cytokines have been classified as being proinflammatory (T helper 1, Th1) or anti-inflammatory (T helper 2, Th2) depending on their effects on the immune system. However, cytokines impact a variety of tissues in a complex manner that regulates inflammation, cell death, and cell proliferation and migration as well as healing mechanisms. Ethanol (alcohol) is known to alter cytokine levels in a variety of tissues including plasma, lung, liver, and brain. Studies on human monocyte responses to pathogens reveal ethanol disruption of cytokine production depending upon the pathogen and duration of alcohol consumption, with multiple pathogens and chronic ethanol promoting inflammatory cytokine production. In lung, cytokine production is disrupted by ethanol exacerbating respiratory distress syndrome with greatly increased expression of transforming growth factor beta (TGFbeta). Alcoholic liver disease involves an inflammatory hepatitis and an exaggerated Th1 response with increases in tumor necrosis factor alpha (TNFalpha). Recent studies suggest that the transition from Th1 to Th2 cytokines contribute to hepatic fibrosis and cirrhosis. Cytokines affect the brain and likely contribute to changes in the central nervous system that contribute to long-term changes in behavior and neurodegeneration. Together these studies suggest that ethanol disruption of cytokines and inflammation contribute in multiple ways to a diversity of alcoholic pathologies.

Perturbed skeletal muscle insulin signaling in the adult female intrauterine growth-restricted rat.

To determine the molecular mechanism(s) linking fetal adaptations in intrauterine growth restriction (IUGR) to adult maladaptations of type 2 diabetes mellitus, we investigated the effect of prenatal seminutrient restriction, modified by early postnatal ad libitum access to nutrients (CM/SP) or seminutrient restriction (SM/SP), vs. early postnatal seminutrient restriction alone (SM/CP) or control nutrition (CM/CP) on the skeletal muscle postreceptor insulin-signaling pathway in the adult offspring. The altered in utero hormonal/metabolic milieu was associated with no change in basal total IRS-1, p85, and p110beta subunits of PI 3-kinase, PKCtheta, and PKCzeta concentrations but an increase in basal IRS-2 (P < 0.05) only in the CM/SP group and an increase in basal phospho (p)-PDK-1 (P < 0.05), p-Akt (P < 0.05), and p-PKCzeta (P < 0.05) concentrations in the CM/SP and SM/SP groups. Insulin-stimulated increases in p-PDK-1 (P < 0.05) and p-Akt (P < 0.0007), with no increase in p-PKCzeta, were seen in both CM/SP and SM/SP groups. SHP2 (P < 0.03) and PTP1B (P < 0.03) increased only in SM/SP with no change in PTEN in CM/SP and SM/SP groups. Aberrations in kinase and phosphatase moieties in the adult IUGR offspring were initiated in utero but further sculpted by the early postnatal nutritional state. Although the CM/SP group demonstrated enhanced kinase activation, the SM/SP group revealed an added increase in phosphatase concentrations with the net result of heightened basal insulin sensitivity in both groups. The inability to further respond to exogenous insulin was due to the key molecular distal roadblock consisting of resistance to phosphorylate and activate PKCzeta necessary for GLUT4 translocation. This protective adaptation may become maladaptive and serve as a forerunner for gestational and type 2 diabetes mellitus.

Laminin-alpha1 globular domains 3 and 4 induce heterotrimeric G protein binding to alpha-syntrophin's PDZ domain and alter intracellular Ca2+ in muscle.

Alpha-syntrophin is a component of the dystrophin glycoprotein complex (DGC). It is firmly attached to the dystrophin cytoskeleton via a unique COOH-terminal domain and is associated indirectly with alpha-dystroglycan, which binds to extracellular matrix laminin. Syntrophin contains two pleckstrin homology (PH) domains and one PDZ domain. Because PH domains of other proteins are known to bind the betagamma-subunits of the heterotrimeric G proteins, whether this is also a property of syntrophin was investigated. Isolated syntrophin from rabbit skeletal muscle binds bovine brain Gbetagamma-subunits in gel blot overlay experiments. Laminin-1-Sepharose or specific antibodies against syntrophin, alpha- and beta-dystroglycan, or dystrophin precipitate a complex with Gbetagamma from crude skeletal muscle microsomes. Bacterially expressed syntrophin fusion proteins and truncation mutants allowed mapping of Gbetagamma binding to syntrophin's PDZ domain; this is a novel function for PDZ domains. When laminin-1 is bound, maximal binding of Gsalpha and Gbetagamma occurs and active Gsalpha, measured as GTP-gamma35S bound, decreases. Because intracellular Ca2+ is elevated in Duchenne muscular dystrophy and Gsalpha is known to activate the dihydropyridine receptor Ca2+ channel, whether laminin also altered intracellular Ca2+ was investigated. Laminin-1 decreases active (GTP-gammaS-bound) Gsalpha, and the Ca2+ channel is inhibited by laminin-1. The laminin alpha1-chain globular domains 4 and 5 region, the region bound by DGC alpha-dystroglycan, is sufficient to cause an effect, and an antibody that specifically blocks laminin binding to alpha-dystroglycan inhibits Gbeta binding by syntrophin in C2C12 myotubes. These observations suggest that DGC is a matrix laminin, G protein-coupled receptor.

Hepatitis C core and nonstructural 3 proteins trigger toll-like receptor 2-mediated pathways and inflammatory activation.

BACKGROUND AND AIMS: Recent evidence suggests that toll-like receptors (TLRs) recognize certain viruses. We reported that hepatitis C virus (HCV) core and nonstructural 3 (NS3) proteins activate inflammatory pathways in monocytes. The aim of this study was to investigate the role of TLRs in innate immune cell activation by core and NS3 proteins. METHODS: Human monocytes, human embryonic kidney cells transfected with TLR2, and peritoneal macrophages from TLR2, MyD88 knockout, and wild-type mice were studied to determine intracellular signaling and proinflammatory cytokine induction by HCV proteins. RESULTS: HCV core and NS3 proteins triggered inflammatory cell activation via the pattern recognition receptor TLR2 and failed to activate macrophages from TLR2 or MyD88-deficient mice. HCV core and NS3 induced interleukin (IL)-1 receptor-associated kinase (IRAK) activity, phosphorylation of p38, extracellular regulated (ERK), and c-jun N-terminal (JNK) kinases and induced AP-1 activation. Activation of nuclear factor-kappaB by core and NS3 was associated with increased IkappaBalpha phosphorylation. TLR2-mediated cell activation was dependent on the conformation of core and NS3 proteins and required sequences in the regions of aa 2-122 in core and aa 1450-1643 in NS3. Although cellular uptake of core and NS3 proteins was independent of TLR2 expression, cell activation required TLR2. HCV core protein and TLR2 showed intracellular colocalization. The hyper-elevated TNF-alpha induction by TLR2 ligands in monocytes of HCV-infected patients was not due to increased TLR2 expression. CONCLUSIONS: HCV core and NS3 proteins trigger inflammatory pathways via TLR2 that may affect viral recognition and contribute to activation of the innate immune system.

Selective priming to Toll-like receptor 4 (TLR4), not TLR2, ligands by P. acnes involves up-regulation of MD-2 in mice.

Lipopolysaccharide (LPS) triggers cytokine production through Toll-like receptor 4 (TLR4), which shares downstream signaling pathways with TLR2. We investigated the roles of TLR2 and TLR4 in Propionibacterium acnes (P. acnes)-primed, LPS-induced liver damage using selective TLR ligands. Stock LPS induced interleukin 8 in both TLR4- and TLR2-expressing human embryonic kidney (HEK) 293 cells. Purified LPS (TLR4 ligand) activated HEK/TLR4 cells, while peptidoglycan and lipoteichoic acid (TLR2 ligands) activated HEK/TLR2 cells, respectively. In mice, P. acnes priming resulted in increased liver messenger RNA (mRNA) and serum levels of tumor necrosis factor alpha, interleukin 12, and interferon gamma (IFN-gamma) by both stock LPS and purified LPS challenges compared with nonprimed controls. In contrast, P. acnes failed to sensitize to TLR2 ligands (peptidoglycan + lipoteichoic acid). In the liver, P. acnes-priming was associated with up-regulation of TLR4 and MD-2 proteins, and subsequent LPS challenge further increased MD-2 and CD14 mRNA levels. The lack of sensitization to TLR2 ligands by P. acnes correlated with no increase in hepatic TLR1 or TLR6 mRNA. In vitro, P. acnes pretreatment desensitized RAW macrophages to a secondary stimulation via both TLR2 and TLR4. However, IFN-gamma could selectively prevent desensitization to TLR4 but not to TLR2 ligands. Furthermore, P. acnes induced production of IFN-gamma in vivo as well as in isolated splenocytes. In vitro, P. acnes-primed Hepa 1-6 hepatocytes but not RAW macrophages produced increased MD-2 and CD14 mRNA levels after an LPS challenge. In conclusion, P. acnes priming to selective TLR4-mediated liver injury is associated with up-regulation of TLR4 and MD-2 and is likely to involve IFN-gamma and prevent TLR4 desensitization by P. acnes.

Skeletal muscle signaling pathway through the dystrophin glycoprotein complex and Rac1.

The dystrophin glycoprotein complex has been proposed to be involved in signal transduction. Here we have shown that laminin binding causes syntrophin to recruit Rac1 from the rabbit skeletal muscle. Laminin-Sepharose and syntrophin-Sepharose bind a protein complex containing Rac1 from the muscle membranes. The presence of heparin, which inhibits laminin interactions, prevents recruitment of Rac1. The dystrophin glycoprotein complex recruits Rac1 via syntrophin through a Grb2.Sos1 complex. A syntrophin antibody also prevents recruitment of Rac1, suggesting that the signaling complex requires syntrophin. PAK1 is in turn bound by Rac1. c-Jun NH2-terminal kinase-p46 is phosphorylated and activated only when laminin is present, and the p54 isoform is activated when laminin is depleted or binding is inhibited with heparin. In the presence of laminin, c-Jun is activated in both skeletal muscle microsomes and in C2C12 myoblasts, and proliferation increases in C2C12 myoblasts. We postulate that this pathway signals muscle homeostasis and hypertrophy.

Dystrophin-glycoprotein complex and Ras and Rho GTPase signaling are altered in muscle atrophy.

The dystrophin-glycoprotein complex (DGC) is a sarcolemmal complex whose defects cause muscular dystrophies. The normal function of this complex is not clear. We have proposed that this is a signal transduction complex, signaling normal interactions with matrix laminin, and that the response is normal growth and homeostasis. If so, the complex and its signaling should be altered in other physiological states such as atrophy. The amount of some of the DGC proteins, including dystrophin, beta-dystroglycan, and alpha-sarcoglycan, is reduced significantly in rat skeletal muscle atrophy induced by tenotomy. Furthermore, H-Ras, RhoA, and Cdc42 decrease in expression levels and activities in muscle atrophy. When the small GTPases were assayed after laminin or beta-dystroglycan depletion, H-Ras, Rac1, and Cdc42 activities were reduced, suggesting a physical linkage between the DGC and the GTPases. Dominant-negative Cdc42, introduced with a retroviral vector, resulted in fibers that appeared atrophic. These data support a putative role for the DGC in transduction of mechanical signals in muscle.