Systemic Immuno-metabolic alterations in chronic obstructive pulmonary disease (COPD).

BACKGROUND: Metabolic adaptation in immune cells is necessary to modulate immune cell function as it is intricately coupled with intracellular metabolism. We aimed to characterize the metabolic state of human peripheral blood mononuclear cells (PBMCs) after long-term exposure to tobacco smoke in smokers with preserved lung function and COPD subjects. METHODS: PBMCs were isolated from healthy non-smokers (HNS), healthy smokers (HS) and COPD subjects, cultured and the mitochondrial respiration while utilizing glucose (glycolysis), fatty acids (ß-oxidation) or pyruvate (direct Krebs' cycle substrate) was measured using the XFp Extracellular Flux Analyzer. Plasma levels of inflammatory cytokines IFN-γ, IL-17, TNF-α, IL-5, IL-9 and IFN-α were measured using flow cytometry. RAW264.7 cells were exposed to cigarette smoke condensate (CSC) for 1 h and its effect on cell viability, cellular metabolism and phagocytosis ability were also studied. Patient's data was analyzed using the Mann Whitney U test, whereas Student's t test was performed to analyze the in-vitro data. RESULTS: PBMCs from COPD subjects showed a significant decrease in extracellular acidification rate (ECAR) while utilizing glucose as compared to HNS (151.9 Vs 215%). Mitochondrial oxygen consumption rate (OCR) on palmitate or pyruvate was also found to be significantly lower in COPD subjects as compared to HS and a strong positive correlation between palmitate OCR in PBMCs and FEV1 (r = 0.74, p < 0.05) and FVC (r = 0.79, p < 0.05) values in HS was observed. The metabolic shift towards fatty acid metabolism in healthy smokers promoted an inflammatory cytokine response with a greater increase in the levels of IL-5, IL-9 and IFN-α as compared to IFN-γ, IL-17 and TNF-α. In-vitro experiments with RAW 264.7 cells showed similar metabolic alterations and a reduced ability to phagocytose Streptococcus pneumonia and Haemophilus influenza after cigarette smoke exposure in the presence of glucose or palmitate. CONCLUSIONS: These findings indicate a metabolic basis for the inflammatory response in COPD and could suggest a new therapeutic target for controlling the immune response and delaying the onset of disease. TRIAL REGISTRATION: This observational study was retrospectively registered in the Clinical Trails Registry - India (ICMR - NIMS) on 19th January 2018 with the registration number CTRI/2018/01/011441 .

Short-term cigarette smoke exposure leads to metabolic alterations in lung alveolar cells.

Cigarette smoke (CS)-induced alveolar destruction and energy metabolism changes are known contributors to the pathophysiology of chronic obstructive pulmonary disease (COPD). This study examines the effect of CS exposure on metabolism in alveolar type II cells. Male A/J mice (8 wk old) were exposed to CS generated from a smoking machine for 4 or 8 weeks, and a recovery group was exposed to CS for 8 weeks and allowed to recover for 2 weeks. Alveolar type II cells were isolated from air- or CS- exposed mice. Acute CS exposure led to a reversible airspace enlargement in A/J mice as measured by the increase in mean linear intercept, indicative of alveolar destruction. The effect of CS exposure on cellular respiration was studied using the XF Extracellular Flux Analyzer. A decrease in respiration while metabolizing glucose was observed in the CS-exposed group, indicating altered glycolysis that was compensated by an increase in palmitate utilization; palmitate utilization was accompanied by an increase in the expression of CD36 and carnitine-palmitoyl transferase 1 in type II alveolar cells for the transport of palmitate into the cells and into mitochondria, respectively. The increase in palmitate use for energy production likely affects the surfactant biosynthesis pathway, as evidenced by the decrease in phosphatidylcholine levels and the increase in phospholipase A2 activity after CS exposure. These findings help our understanding of the mechanism underlying the surfactant deficiency observed in smokers and provide a target to delay the onset of COPD.

Metabolic shift in lung alveolar cell mitochondria following acrolein exposure.

Acrolein, an α,ß unsaturated electrophile, is an environmental pollutant released in ambient air from diesel exhausts and cooking oils. This study examines the role of acrolein in altering mitochondrial function and metabolism in lung-specific cells. RLE-6TN, H441, and primary alveolar type II (pAT2) cells were exposed to acrolein for 4 h, and its effect on mitochondrial oxygen consumption rates was studied by XF Extracellular Flux analysis. Low-dose acrolein exposure decreased mitochondrial respiration in a dose-dependent manner because of alteration in the metabolism of glucose in all the three cell types. Acrolein inhibited glyceraldehyde-3-phosphate dehydrogenase (GAPDH) activity, leading to decreased substrate availability for mitochondrial respiration in RLE-6TN, H441, and pAT2 cells; the reduced GAPDH activity was compensated in pAT2 cells by an increase in the activity of glucose-6-phosphate dehydrogenase, the regulatory control of the pentose phosphate pathway. The decrease in pyruvate from glucose metabolism resulted in utilization of alternative sources to support mitochondrial energy production: palmitate-BSA complex increased mitochondrial respiration in RLE-6TN and pAT2 cells. The presence of palmitate in alveolar cells for surfactant biosynthesis may prove to be the alternative fuel source for mitochondrial respiration. Accordingly, a decrease in phosphatidylcholine levels and an increase in phospholipase A2 activity were found in the alveolar cells after acrolein exposure. These findings have implications for understanding the decrease in surfactant levels frequently observed in pathophysiological situations with altered lung function following exposure to environmental toxicants.

Short-term cigarette smoke exposure induces reversible changes in energy metabolism and cellular redox status independent of inflammatory responses in mouse lungs.

Cigarette smoking leads to alteration in cellular redox status, a hallmark in the pathogenesis of chronic obstructive pulmonary disease. This study examines the role of cigarette smoke (CS) exposure in the impairment of energy metabolism and, consequently, mitochondrial dysfunction. Male A/J mice were exposed to CS generated by a smoking machine for 4 or 8 wk. A recovery group was exposed to CS for 8 wk and allowed to recover for 2 wk. Acute CS exposure altered lung glucose metabolism, entailing a decrease in the rate of glycolysis and an increase in the pentose phosphate pathway, as evidenced by altered expression and activity of GAPDH and glucose-6-phosphate dehydrogenase, respectively. Impairment of GAPDH was found to be due to glutathionylation of its catalytic site cysteines. Metabolic changes were associated with changes in cellular and mitochondrial redox status, assessed in terms of pyridine nucleotides and glutathione. CS exposure elicited an upregulation of the expression of complexes II, III, IV, and V and of the activity of complexes II, IV, and V. Microarray analysis of gene expression in mouse lungs after exposure to CS for 8 wk revealed upregulation of a group of genes involved in metabolism, electron transfer chain, oxidative phosphorylation, mitochondrial transport and dynamics, and redox regulation. These changes occurred independently of inflammatory responses. These findings have implications for the early onset of alterations in energy and redox metabolism upon acute lung exposure to CS.

Tobacco Smoke Exposure Impairs Brain Insulin/IGF Signaling: Potential Co-Factor Role in Neurodegeneration.

BACKGROUND: Human studies suggest tobacco smoking is a risk factor for cognitive impairment and neurodegeneration, including Alzheimer's disease (AD). However, experimental data linking tobacco smoke exposures to underlying mediators of neurodegeneration, including impairments in brain insulin and insulin-like growth factor (IGF) signaling in AD are lacking. OBJECTIVE: This study tests the hypothesis that cigarette smoke (CS) exposures can impair brain insulin/IGF signaling and alter expression of AD-associated proteins. METHODS: Adult male A/J mice were exposed to air for 8 weeks (A8), CS for 4 or 8 weeks (CS4, CS8), or CS8 followed by 2 weeks recovery (CS8+R). Gene expression was measured by qRT-PCR analysis and proteins were measured by multiplex bead-based or direct binding duplex ELISAs. RESULTS: CS exposure effects on insulin/IGF and insulin receptor substrate (IRS) proteins and phosphorylated proteins were striking compared with the mRNA. The main consequences of CS4 or CS8 exposures were to significantly reduce insulin R, IGF-1R, IRS-1, and tyrosine phosphorylated insulin R and IGF-1R proteins. Paradoxically, these effects were even greater in the CS8+R group. In addition, relative levels of S312-IRS-1, which inhibits downstream signaling, were increased in the CS4, CS8, and CS8+R groups. Correspondingly, CS and CS8+R exposures inhibited expression of proteins and phosphoproteins required for signaling through Akt, PRAS40, and/or p70S6K, increased AßPP-Aß, and reduced ASPH protein, which is a target of insulin/IGF-1 signaling. CONCLUSION: Secondhand CS exposures caused molecular and biochemical abnormalities in brain that overlap with the findings in AD, and many of these effects were sustained or worsened despite short-term CS withdrawal.

Tobacco Smoke-Induced Brain White Matter Myelin Dysfunction: Potential Co-Factor Role of Smoking in Neurodegeneration.

BACKGROUND: Meta-analysis studies showed that smokers have increased risk for developing Alzheimer's disease (AD) compared with non-smokers, and neuroimaging studies revealed that smoking damages white matter structural integrity. OBJECTIVE: The present study characterizes the effects of side-stream (second hand) cigarette smoke (CS) exposures on the expression of genes that regulate oligodendrocyte myelin-synthesis, maturation, and maintenance and neuroglial functions. METHODS: Adult male A/J mice were exposed to air (8 weeks; A8), CS (4 or 8 weeks; CS4, CS8), or CS8 followed by 2 weeks recovery (CS8 + R). The frontal lobes were used for histology and qRT-PCR analysis. RESULTS: Luxol fast blue, Hematoxylin and Eosin stained histological sections revealed CS-associated reductions in myelin staining intensity and narrowing of the corpus callosum. CS exposures broadly decreased mRNA levels of immature and mature oligodendrocyte myelin-associated, neuroglial, and oligodendrocyte-related transcription factors. These effects were more prominent in the CS8 compared with CS4 group, suggesting that molecular abnormalities linked to white matter atrophy and myelin loss worsen with duration of CS exposure. Recovery normalized or upregulated less than 25% of the suppressed genes; in most cases, inhibition of gene expression was either sustained or exacerbated. CONCLUSION: CS exposures broadly inhibit expression of genes needed for myelin synthesis and maintenance. These adverse effects often were not reversed by short-term CS withdrawal. The results support the hypothesis that smoking contributes to white matter degeneration, and therefore could be a key risk factor for a number of neurodegenerative diseases, including AD.

Cigarette Smoke-Induced Alterations in Frontal White Matter Lipid Profiles Demonstrated by MALDI-Imaging Mass Spectrometry: Relevance to Alzheimer's Disease.

BACKGROUND: Meta-analysis has shown that smokers have significantly increased risks for Alzheimer's disease (AD), and neuroimaging studies showed that smoking alters white matter (WM) structural integrity. OBJECTIVE: Herein, we characterize the effects of cigarette smoke (CS) exposures and withdrawal on WM myelin lipid composition using matrix assisted laser desorption and ionization-imaging mass spectrometry (MALDI-IMS). METHODS: Young adult male A/J mice were exposed to air (8 weeks; A8), CS (4 or 8 weeks; CS4, CS8), or CS8 followed by 2 weeks recovery (CS8 + R). Frontal lobe WM was examined for indices of lipid and protein oxidation and lipid profile alterations by MALDI-IMS. Lipid ions were identified by MS/MS with the LIPID MAPS prediction tools database. Inter-group comparisons were made using principal component analysis and R-generated heatmap. RESULTS: CS increased lipid and protein adducts such that higher levels were present in CS8 compared with CS4 samples. CS8 + R reversed CS8 effects and normalized the levels of oxidative stress. MALDI-IMS demonstrated striking CS-associated alterations in WM lipid profiles characterized by either reductions or increases in phospholipids (phosphatidylinositol, phosphatidylserine, phosphatidylcholine, or phosphatidylethanolamine) and sphingolipids (sulfatides), and partial reversal of CS's inhibitory effects with recovery. The heatmap hierarchical dendrogram and PCA distinguished CS exposure, duration, and withdrawal effects on WM lipid profiles. CONCLUSION: CS-mediated WM degeneration is associated with lipid peroxidation, protein oxidative injury, and alterations in myelin lipid composition, including shifts in phospholipids and sphingolipids needed for membrane integrity, plasticity, and intracellular signaling. Future goals are to delineate WM abnormalities in AD using MALDI-IMS, and couple the findings with MRI-mass spectroscopy to improve in vivo diagnostics and early detection of brain biochemical responses to treatment.

PI3K/AKT signaling regulates bioenergetics in immortalized hepatocytes.

Regulation of cellular bioenergetics by PI3K/AKT signaling was examined in isogenic hepatocyte cell lines lacking the major inhibitor of PI3K/AKT signaling, PTEN (phosphatase and tensin homolog deleted on chromosome 10). PI3K/AKT signaling was manipulated using the activator (IGF-1) and the inhibitor (LY 294002) of the PI3K/AKT pathway. Activation of PI3K/AKT signaling resulted in an enhanced anaerobic glycolysis and mitochondrial respiration. AKT, when phosphorylated and activated, translocated to mitochondria and localized within the membrane structure of mitochondria, where it phosphorylated a number of mitochondrial-resident proteins including the subunits α and ß of ATP synthase. Inhibition of GSK3ß by either phosphorylation by AKT or lithium chloride resulted in activation of pyruvate dehydrogenase, i.e., a decrease in its phosphorylated form. AKT-dependent phosphorylation of ATP synthase subunits α and ß resulted in an increased complex activity. AKT translocation to mitochondria was associated with an increased expression and activity of complex I. These data suggest that the mitochondrial signaling pathway AKT/GSK3ß/PDH, AKT-dependent phosphorylation of ATP synthase, and upregulation of mitochondrial complex I expression and activity are involved in the control of mitochondrial bioenergetics by increasing substrate availability and regulating the mitochondrial catalytic/energy-transducing capacity.