AMP-Activated Protein Kinase (AMPK) Mediates Nutrient Regulation of Thioredoxin-Interacting Protein (TXNIP) in Pancreatic Beta-Cells

Thioredoxin-interacting protein (TXNIP) regulates critical biological processes including inflammation, stress and apoptosis. TXNIP is upregulated by glucose and is a critical mediator of hyperglycemia-induced beta-cell apoptosis in diabetes. In contrast, the saturated long-chain fatty acid palmitate, although toxic to the beta-cell, inhibits TXNIP expression. The mechanisms involved in the opposing effects of glucose and fatty acids on TXNIP expression are unknown. We found that both palmitate and oleate inhibited TXNIP in a rat beta-cell line and islets. Palmitate inhibition of TXNIP was independent of fatty acid beta-oxidation or esterification. AMP-activated protein kinase (AMPK) has an important role in cellular energy sensing and control of metabolic homeostasis; therefore we investigated its involvement in nutrient regulation of TXNIP. As expected, glucose inhibited whereas palmitate stimulated AMPK. Pharmacologic activators of AMPK mimicked fatty acids by inhibiting TXNIP. AMPK knockdown increased TXNIP expression in presence of high glucose with and without palmitate, indicating that nutrient (glucose and fatty acids) effects on TXNIP are mediated in part via modulation of AMPK activity. TXNIP is transcriptionally regulated by carbohydrate response element-binding protein (ChREBP). Palmitate inhibited glucose-stimulated ChREBP nuclear entry and recruitment to the Txnip promoter, thereby inhibiting Txnip transcription. We conclude that AMPK is an important regulator of Txnip transcription via modulation of ChREBP activity. The divergent effects of glucose and fatty acids on TXNIP expression result in part from their opposing effects on AMPK activity. In light of the important role of TXNIP in beta-cell apoptosis, its inhibition by fatty acids can be regarded as an adaptive/protective response to glucolipotoxicity. The finding that AMPK mediates nutrient regulation of TXNIP may have important implications for the pathophysiology and treatment of diabetes

Glucose Amplifies Fatty Acid-Induced Endoplasmic Reticulum Stress in Pancreatic β-Cells via Activation of mTORC1

BACKGROUND: Palmitate is a potent inducer of endoplasmic reticulum (ER) stress in beta-cells. In type 2 diabetes, glucose amplifies fatty-acid toxicity for pancreatic beta-cells, leading to beta-cell dysfunction and death. Why glucose exacerbates beta-cell lipotoxicity is largely unknown. Glucose stimulates mTORC1, an important nutrient sensor involved in the regulation of cellular stress. Our study tested the hypothesis that glucose augments lipotoxicity by stimulating mTORC1 leading to increased beta-cell ER stress. PRINCIPAL FINDINGS: We found that glucose amplifies palmitate-induced ER stress by increasing IRE1alpha protein levels and activating the JNK pathway, leading to increased beta-cell apoptosis. Moreover, glucose increased mTORC1 activity and its inhibition by rapamycin decreased beta-cell apoptosis under conditions of glucolipotoxicity. Inhibition of mTORC1 by rapamycin did not affect proinsulin and total protein synthesis in beta-cells incubated at high glucose with palmitate. However, it decreased IRE1alpha expression and signaling and inhibited JNK pathway activation. In TSC2-deficient mouse embryonic fibroblasts, in which mTORC1 is constitutively active, mTORC1 regulated the stimulation of JNK by ER stressors, but not in response to anisomycin, which activates JNK independent of ER stress. Finally, we found that JNK inhibition decreased beta-cell apoptosis under conditions of glucolipotoxicity. CONCLUSIONS/SIGNIFICANCE: Collectively, our findings suggest that mTORC1 mediates glucose amplification of lipotoxicity, acting through activation of ER stress and JNK. Thus, mTORC1 is an important transducer of ER stress in beta-cell glucolipotoxicity. Moreover, in stressed beta-cells mTORC1 inhibition decreases IRE1alpha protein expression and JNK activity without affecting ER protein load, suggesting that mTORC1 regulates the beta-cell stress response to glucose and fatty acids by modulating the synthesis and activity of specific proteins involved in the execution of the ER stress response. This novel paradigm may have important implications for understanding beta-cell failure in type 2 diabetes

Pulmonary surfactant phosphatidylglycerol inhibits respiratory syncytial virus–induced inflammation and infection

Respiratory syncytial virus (RSV) is the most common cause of hospitalization for respiratory tract infection in young children. It is also a significant cause of morbidity and mortality in elderly individuals and in persons with asthma and chronic obstructive pulmonary disease. Currently, no reliable vaccine or simple RSV antiviral therapy is available. Recently, we determined that the minor pulmonary surfactant phospholipid, palmitoyl-oleoyl-phosphatidylglycerol (POPG), could markedly attenuate inflammatory responses induced by lipopolysaccharide through direct interactions with the Toll-like receptor 4 (TLR4) interacting proteins CD14 and MD-2. CD14 and TLR4 have been implicated in the host response to RSV. Treatment of bronchial epithelial cells with POPG significantly inhibited interleukin-6 and -8 production, as well as the cytopathic effects induced by RSV. The phospholipid bound RSV with high affinity and inhibited viral attachment to HEp2 cells. POPG blocked viral plaque formation in vitro by 4 log units, and markedly suppressed the expansion of plaques from cells preinfected with the virus. Administration of POPG to mice, concomitant with viral infection, almost completely eliminated the recovery of virus from the lungs at 3 and 5 days after infection, and abrogated IFN-γ (IFN-γ) production and the enhanced expression of surfactant protein D (SP-D). These findings demonstrate an important approach to prevention and treatment of RSV infections using exogenous administration of a specific surfactant phospholipid

Short hairpin RNA targeting 2B gene of coxsackievirus B3 exhibits potential antiviral effects both <it>in vitro</it> and <it>in vivo</it>

<p>Abstract</p> <p>Background</p> <p>Coxsackievirus B3 is an important infectious agent of viral myocarditis, pancreatitis and aseptic meningitis, but there are no specific antiviral therapeutic reagents in clinical use. RNA interference-based technology has been developed to prevent the viral infection.</p> <p>Methods</p> <p>To evaluate the impact of RNA interference on viral replication, cytopathogenicity and animal survival, short hairpin RNAs targeting the viral 2B region (shRNA-2B) expressed by a recombinant vector (pGCL-2B) or a recombinant lentivirus (Lenti-2B) were tansfected in HeLa cells or transduced in mice infected with CVB3.</p> <p>Results</p> <p>ShRNA-2B exhibited a significant effect on inhibition of viral production in HeLa cells. Furthermore, shRNA-2B improved mouse survival rate, reduced the viral tissues titers and attenuated tissue damage compared with those of the shRNA-NC treated control group. Lenti-2B displayed more effective role in inhibition of viral replication than pGCL-2B <it>in vivo</it>.</p> <p>Conclusions</p> <p>Coxsackievirus B3 2B is an effective target of gene silencing against coxsackievirus B3 infection, suggesting that shRNA-2B is a potential agent for further development into a treatment for enterviral diseases.</p

Vertical transmission of hepatitis C virus in a hospital in southern Brazil Transmissão vertical do vírus da hepatite C em um hospital do sul do Brasil

BACKGROUND: There still are controversies concerning the vertical transmission of hepatitis C virus. AIM: To evaluate the prevalence of antibodies against hepatitis C virus in pregnant women, as well as the rate of vertical transmission of this virus. PATIENTS AND METHODS: Between August 1998 and November 1999, 1,090 consecutive pregnant women were screened for anti-hepatitis C virus; positive results were confirmed by the polymerase chain reaction assay. Patient's viral load was evaluated by the branched deoxyribonucleic acid assay. Hepatitis C virus genotype was identified by direct sequencing of the polymerase chain reaction amplification products. The same tests were performed in the children born from infected mothers at the 1st and 6th month of life. RESULTS: Of the 1,090 mothers surveyed, 29 were positive for anti-hepatitis C virus (prevalence of 2.66%). Twenty-five patients presented with hepatitis C virus RNA, with a median hepatitis C virus viral load of 3.132 ± 5.891 MEq/mL. Twenty-two patients (six human immunodeficiency virus-coinfected) were followed and gave birth to 23 children; 18 of them had blood samples tested at the 1st month of life, and 22, at the 6th month. Vertical transmission rate was 5.56%; it affected a girl who had hepatitis C virus RNA detectable only in the 1st month sample (41.570 MEq/mL). The mother who transmitted hepatitis C virus was coinfected with human immunodeficiency virus and presented with an hepatitis C virus viral load of 3.765 MEq/mL, with 100% homology with her daughter's hepatitis C virus genotype. CONCLUSION: These results suggest that the prevalence of hepatitis C virus infection in pregnant women should not be neglected, and early diagnosis of vertical transmission and the follow up of infected children should be emphasized.<br>RACIONAL: Ainda existem controvérsias em relação à transmissão vertical do vírus da hepatite C (VHC). OBJETIVO: Avaliar a prevalência dos anticorpos contra o VHC (anti-VHC) em mulheres grávidas, bem como a percentagem de transmissão vertical observada. PACIENTES E MÉTODOS: Entre agosto de 1998 e novembro de 1999, 1.090 mulheres grávidas consecutivas realizaram a determinação do anti-VHC. A confirmação do teste foi feita pela reação em cadeia da polimerase. A carga viral foi determinada pelo b-DNA e o genótipo por seqüenciamento. Os mesmos testes foram realizados no 1º e 6º mês de vida, nas crianças nascidas de mães infectadas. RESULTADOS: Das 1.090 mães estudadas, 29 apresentaram positividade para o anti-VHC (prevalência de 2,66%). Em 25 pacientes foi demonstrado o RNA do VHC, sendo que a carga viral média foi de 3,132 ± 5,891 MEq/mL. Vinte e duas pacientes (6 co-infectadas com o vírus da imunodeficiência humana) foram seguidas e deram à luz a 23 crianças, das quais 18 tiveram seu sangue testado no 1º mês e 22 no 6º mês. Foi observada transmissão vertical em 5,56% dos casos. Assim, em uma criança do sexo feminino foi detectado o RNA do VHC (41,570 MEq/mL). A mãe desta criança estava co-infectada pelo vírus da imunodeficiência humana e apresentava carga viral de 3,765 MEq/mL, com 100% de homologia no genótipo viral. CONCLUSÃO: Estes resultados sugerem que a prevalência da infecção pelo VHC em gestantes não deve ser negligenciada e que um diagnóstico precoce e o seguimento das crianças infectadas deve ser preconizado