Svetlana Simtchouk

Globular adiponectin induces a pro-inflammatory response in human astrocytic cells

Neuroinflammation, mediated in part by activated brain astrocytes, plays a critical role in the development of neurodegenerative disorders, including Alzheimers disease (AD). Adiponectin is the most abundant adipokine secreted from adipose tissue and has been reported to exert both anti- and pro-inflammatory effects in peripheral tissues; however, the effects of adiponectin on astrocytes remain unknown. Shifts in peripheral concentrations of adipokines, including adiponectin, could contribute to the observed link between midlife adiposity and increased AD risk. The aim of the present study was to characterize the effects of globular adiponectin (gAd) on pro-inflammatory cytokine mRNA expression and secretion in human U373 MG astrocytic cells and to explore the potential involvement of nuclear factor (NF)-κB, p38 mitogen-activated protein kinase (MAPK), extracellular signal-regulated kinase (ERK)1/2, c-Jun N-terminal kinase (JNK) and phosphatidylinositide 3-kinases (PI3K) signaling pathways in these processes. We demonstrated expression of adiponectin receptor 1 (adipoR1) and adipoR2 in U373 MG cells and primary human astrocytes. gAd induced secretion of interleukin (IL)-6 and monocyte chemoattractant protein (MCP)-1, and gene expression of IL-6, MCP-1, IL-1β and IL-8 in U373 MG cells. Using specific inhibitors, we found that NF-κB, p38MAPK and ERK1/2 pathways are involved in gAd-induced induction of cytokines with ERK1/2 contributing the most. These findings provide evidence that gAd may induce a pro-inflammatory phenotype in human astrocytes.

Mitochondrial transcription factor A (Tfam) is a pro-inflammatory extracellular signaling molecule recognized by brain microglia.

Microglia represent mononuclear phagocytes in the brain and perform immune surveillance, recognizing a number of signaling molecules released from surrounding cells in both healthy and pathological situations. The microglia interact with several damage-associated molecular pattern molecules (DAMPs) and recent data indicate that mitochondrial transcription factor A (Tfam) could act as a specific DAMP in peripheral tissues. This study tested the hypothesis that extracellular Tfam induces pro-inflammatory and cytotoxic responses of the microglia. Three different types of human mononuclear phagocytes were used to model human microglia: human peripheral blood monocytes from healthy donors, human THP-1 monocytic cells, and human primary microglia obtained from autopsy samples. When combined with interferon (IFN)-γ, recombinant human Tfam (rhTfam) induced secretions that were toxic to human SH-SY5Y neuroblastoma cells in all three models. Similar cytotoxic responses were observed when THP-1 cells and human microglia were exposed to human mitochondrial proteins in the presence of IFN-γ. rhTfam alone induced expression of pro-inflammatory cytokines interleukin (IL)-1β, IL-6 and IL-8 by THP-1 cells. This induction was further enhanced in the presence of IFN-γ. Upregulated secretion of IL-6 in response to rhTfam plus IFN-γ was confirmed in primary human microglia. Use of specific inhibitors showed that the rhTfam-induced cytotoxicity of human THP-1 cells depended partially on activation of c-Jun N-terminal kinase (JNK), but not p38 mitogen-activated protein kinase (MAPK). Overall, our data support the hypothesis that, in the human brain, Tfam could act as an intercellular signaling molecule that is recognized by the microglia to cause pro-inflammatory and cytotoxic responses.

Short-term high-intensity interval and moderate-intensity continuous training reduce leukocyte TLR4 in inactive adults at elevated risk of type 2 diabetes

Exercise can have anti-inflammatory effects in obesity, but the optimal type and intensity of exercise are not clear. This study compared short-term high-intensity interval training (HIIT) with moderate-intensity continuous training (MICT) in terms of improvement in cardiorespiratory fitness, markers of inflammation, and glucose control in previously inactive adults at elevated risk of developing type 2 diabetes. Thirty-nine inactive, overweight/obese adults (32 women) were randomly assigned to 10 sessions over 2 wk of progressive HIIT (n = 20, four to ten 1-min sessions at ∼90% peak heart rate, 1-min rest periods) or MICT (n = 19, 20-50 min at ∼65% peak heart rate). Before and 3 days after training, participants performed a peak O2 uptake test, and fasting blood samples were obtained. Both HIIT (1.8 ± 0.4 vs. 1.9 ± 0.4 l/min, pre vs. post) and MICT (1.8 ± 0.5 vs. 1.9 ± 0.5 l/min, pre vs. post) improved peak O2 uptake (P < 0.001) and lowered plasma fructosamine (P < 0.05). Toll-like receptor (TLR) 4 (TLR4) expression was reduced on lymphocytes and monocytes after both HIIT and MICT (P < 0.05) and on neutrophils after MICT (P < 0.01). TLR2 on lymphocytes was reduced after HIIT and MICT (P < 0.05). Plasma inflammatory cytokines were unchanged after training in both groups, but MICT led to a reduction in fasting plasma glucose (P < 0.05, 5.9 ± 1.0 vs. 5.6 ± 1.0 mmol/l, pre vs. post). Ten days of either HIIT or MICT can improve cardiorespiratory fitness and glucose control and lead to reductions in TLR2 and TLR4 expression. MICT, which involved a longer duration of exercise, may be superior for reducing fasting glucose.

Hyporesponsiveness to the anti-inflammatory action of interleukin-10 in type 2 diabetes.

Chronic low-grade inflammation contributes to the pathology and complications of type 2 diabetes (T2D). Interleukin-10 (IL10), an anti-inflammatory cytokine, is suggested to play a protective role in T2D. However, the impact of T2D on IL10 function has not been previously assessed. We examined the ability of IL10 to inhibit inflammation in human T2D immune cells and explored underlying mechanisms using macrophage models. IL10 was less effective at inhibiting tumour necrosis factor (TNF)-α secretion in T2D whole blood cultures, which was not explained by altered IL10 receptor surface expression. These findings were observed in macrophages exposed to high glucose, which demonstrated similar IL10 resistance or hyporesponsiveness. These findings were also not explained by changes in IL10 receptor protein or other downstream signaling proteins. High glucose was also shown to impair the ability of IL10 to activate STAT3, a downstream signaling protein of IL10. Treatment with the SHIP1 agonist, AQX-MN100, reversed IL10 hyporesponsiveness in macrophages cultured in high glucose and showed equal effectiveness at different glucose conditions. This data supports the idea that IL10 hyporesponsiveness may contribute to chronic inflammation in T2D. These novel findings suggest that strategies aimed to overcome IL10 hyporesponsiveness may hold therapeutic potential for reducing inflammation in T2D.

Human Adipose Tissue Conditioned Media from Lean Subjects Is Protective against H2O2 Induced Neurotoxicity in Human SH-SY5Y Neuronal Cells

Adipose tissue secretes numerous hormone-like factors, which are known as adipokines. Adipokine receptors have been identified in the central nervous system but the potential role of adipokine signaling in neuroprotection is unclear. The aim of this study is to determine (1) Whether adipokines secreted from cultured adipose tissue of lean humans is protective against oxidative stress-induced neurotoxicity in human SH-SY5Y neuronal cells; and (2) To explore potential signaling pathways involved in these processes. Adipose tissue conditioned media (ATCM) from healthy lean subjects completely prevented H2O2 induced neurotoxicity, while this effect is lost after heating ATCM. ATCM activated the phosphorylation of ERK1/2, JNK and Akt at serine 308 in SH-SY5Y cells. PD98059 (25 µM), SP600125 (5 µM) and LY29400 (20 µM) partially blocked the protective effects of ATCM against H2O2 induced neurotoxicity. Findings demonstrate that heat-sensitive factors secreted from human adipose tissue of lean subjects are protective against H2O2 induced neurotoxicity and ERK1/2, JNK, and PI3K signaling pathways are involved in these processes. In conclusion, this study demonstrates preliminary but encouraging data to further support that adipose tissue secreted factors from lean human subjects might possess neuroprotective properties and unravel the specific roles of ERK1/2, JNK and PI3K in these processes.

Short-term Exercise Training Alters Leukocyte Chemokine Receptors in Obese Adults.

Obesity is characterized by chronic low-grade inflammation driven by activation and tissue infiltration of circulating leukocytes. Although exercise has anti-inflammatory effects, the impact of exercise on mediators of leukocyte migration is unclear. Purpose To determine the impact of high-intensity interval training (HIIT) versus moderate-intensity continuous training (MICT), in the absence of weight/fat loss, on circulating chemokines and leukocyte chemokine receptors. Methods Thirty-seven inactive obese adults were randomized to 2 wk (10 sessions) of HIIT or MICT with fasting blood samples collected before and after training. Plasma concentration of C-C motif chemokine ligand 2 (CCL2; also known as monocyte chemoattractant protein-1), CCL3 (also known as macrophage inflammatory protein-1alpha), and C-X-C motif ligand 8 (CXCL8; also known as interleukin-8) were determined and the chemokine receptors CCR2, CCR5, and CXCR2 were measured on monocytes, neutrophils, and T cells. Results MICT reduced the percentage of monocytes positive for CCR2 and reduced surface protein expression of CXCR2 on monocytes (both P < 0.05), whereas HIIT increased CCR5 surface protein expression and percentage CCR5 positive monocytes and neutrophils (all P < 0.05) along with increasing the percentage of T cells that were positive for CCR5 (P < 0.05). There were no significant changes in circulating chemokines, percent body fat or visceral adipose tissue. Conclusions Exercise, in the absence of weight/fat loss and without changes in circulating chemokines, has direct effects on leukocytes in obese adults with HIIT and MICT resulting in different responses. MICT may reduce monocyte migration potential through downregulation of CCR2 and CXCR2, whereas HIIT may increase potential for CCR5-mediated monocyte, neutrophil, and T-cell infiltration. The impact of different exercise protocols on leukocyte trafficking to tissues in obesity warrants further research.

One-week high-fat diet leads to reduced toll-like receptor 2 expression and function in young healthy men

Toll-like receptor 2 (TLR2) is implicated in inflammatory responses to high-fat diet (HFD)-induced obesity in rodents, but human HFD studies examining TLR2-mediated immune responses are lacking. Our aim was to determine whether HFD affected TLR2 function in humans. We hypothesized that a short-term HFD in humans would impair TLR2-mediated immune function. Fasting blood samples were obtained from healthy young men (N = 9) before and after a 7-day HFD. Toll-like receptor 2 function was assessed in ex vivo whole blood cultures stimulated with the TLR2 agonist N-palmitoyl-S-[2,3-bis[palmitoyloxy]-[2RS]-propyl]-[R]-cysteinyl-[S]-seryl-[S]-lysyl-[S]-lysyl-[S]-lysyl-[S]-lysine (Pam3-Cys-SK4). Peripheral blood mononuclear cells (PBMCs) were isolated to examine TLR2, TLR4, and p47 subunit of nicotinamide adenine dinucleotide phosphate oxidase (p47(phox)) protein expression via Western blotting. Pam3-Cys-SK4-stimulated secretion of interleukin-1β (-35%, P = .005), interleukin-6 (-32%, P = .01), and tumor necrosis factor-α (-33%, P = .06) was reduced following the HFD. High-fat diet resulted in decreased TLR2 (P = .049) and p47(phox) (P = .037) protein expression from PBMCs. To mimic lipid overload ex vivo, follow-up experiments were performed in whole blood cultures exposed to a mixture of free fatty acids for 24 hours; and surface protein expression of TLR2 and TLR4 on CD14+ monocytes was measured by flow cytometry. Free fatty acid exposure for 24 hours ex vivo reduced monocyte TLR2 levels by about 20% (P = .028). A 7-day HFD in young healthy men resulted in impaired TLR2 function. Decreased TLR2 and p47(phox) protein expression in PBMCs, possibly due to excess free fatty acids, may mediate this response. Our current findings indicate that impaired TLR2 response after HFD might be partially responsible for increased risk of infection in diet-induced obesity.

Kinetic analysis of cytochrome P450 reductase fromArtemisia annua reveals accelerated rates of NADPH‐dependent flavin reduction

Cytochrome P450 reductase from Artemisia annua (aaCPR) is a diflavin enzyme that has been employed for the microbial synthesis of artemisinic acid (a semi‐synthetic precursor of the anti‐malarial drug, artemisinin) based on its ability to transfer electrons to the cytochrome P450 monooxygenase, CYP71AV1. We have isolated recombinant aaCPR (with the N‐terminal transmembrane motif removed) from Escherichia coli and compared its kinetic and thermodynamic properties with other CPR orthologues, most notably human CPR. The FAD and FMN redox potentials and the macroscopic kinetic constants associated with cytochrome c3+ reduction for aaCPR are comparable to that of other CPR orthologues, with the exception that the apparent binding affinity for the oxidized coenzyme is ~ 30‐fold weaker compared to human CPR. CPR from A. annua shows a 3.5‐fold increase in uncoupled NADPH oxidation compared to human CPR and a strong preference (85 100‐fold) for NADPH over NADH. Strikingly, reduction of the enzyme by the first and second equivalent of NADPH is much faster in aaCPR, with rates of > 500 and 17 s−1 at 6 °C. We also optically detect a charge‐transfer species that rapidly forms in < 3 ms and then persists during the reductive half reaction. Additional stopped‐flow kinetic studies with NADH and (R)‐[4‐2H]NADPH suggest that the accelerated rate of flavin reduction is attributed to the relatively weak binding affinity of aaCPR for NADP+.

Aromatic substitution of the FAD‐shielding tryptophan reveals its differential role in regulating electron flux in methionine synthase reductase and cytochrome P450 reductase

Methionine synthase reductase (MSR) and cytochrome P450 reductase (CPR) transfer reducing equivalents from NADPH via an FAD and FMN cofactor to a redox partner protein. In both enzymes, hydride transfer from NADPH to FAD requires displacement of a conserved tryptophan that lies coplanar to the FAD isoalloxazine ring. Swapping the tryptophan for a smaller aromatic side chain revealed a distinct role for the residue in regulating MSR and CPR catalysis. MSR W697F and W697Y showed enhanced catalysis, noted by increases in kcat and kcat/Km(NADPH) for steady‐state cytochrome c3+ reduction and a 10‐fold increase in the rate constant (kobs1) associated with hydride transfer. Elevated primary kinetic isotope effects on kobs1 for W697F and W697Y suggest that preceding isotopically insensitive steps like displacement of W697 are less rate determining. MSR W697Y, but not MSR W697F, showed detectable formation of the disemiquinone intermediate, indicating that the polarity of the aromatic side chain influences the rate of interflavin electron transfer. By contrast, the CPR variants (W676F and W676Y) displayed modest decreases in cytochrome c3+ reduction, a 30‐ and 3.5‐fold decrease in the rate of FAD reduction, accumulation of a FADH2–NADP+ charge‐transfer complex and dramatically suppressed rates of interflavin electron transfer. We conclude for MSR that hydride transfer is ‘gated’ by the free energy required to disrupt dispersion forces between the FAD isoalloxazine ring and W697. By contrast, the bulky indole ring of W676 accelerates catalysis in CPR by lowering the energy barrier for displacement of the oxidized nicotinamide ring coplanar with the FAD.

Role of amyloid β in the induction of lipolysis and secretion of adipokines from human adipose tissue

Amyloid-β (Aβ) peptide, which is generated from proteolytic cleavage of amyloid precursor protein (APP), is a key molecule involved in the pathology of Alzheimer disease. Both APP and Aβ peptides are expressed in adipose tissue, however it is currently unclear whether Aβ can affect the key functions of adipose tissue. We aimed to explore whether Aβ affected lipolysis and adipokine secretion in cultured human adipose tissue. We found that Aβ25–35, which contains the main functional domain of the Aβ, stimulated lipolysis via PKA and ERK1/2-dependent pathways and that Aβ25–35 induced leptin and IL-6 secretion. It is concluded that Aβ peptide exerts functional effects on adipose tissue that may lead to increased release of free fatty acids and pro-inflammatory adipokines.