Method for depletion of mitochondria DNA in human bronchial epithelial cells

Mitochondria are increasingly recognized to play a role in the airway inflammation of asthma. Model systems to study the role of mitochondrial gene expression in bronchial epithelium are lacking. Here, we create custom bronchial epithelial cell lines that are depleted of mitochondrial DNA. One week of ethidium bromide (EtBr) treatment led to ∼95 % reduction of mtDNA copy number (mtDNA-CN) in cells, which was further reduced by addition of 25 µM 2′,3′-dideoxycytidin (ddC). Treatment for up to three weeks with EtBr and ddC led to near complete loss of mtDNA. The basal oxygen consumption rate (OCR) of mtDNA-depleted BET-1A and BEAS-2B cells dropped to near zero. Glycolysis measured by extracellular acidification rate (ECAR) increased ∼two-fold in cells when mtDNA was eliminated. BET-1A ρ0 and BEAS-2B ρ0 cells were cultured for two months, frozen and thawed, cultured for two more months, and maintained near zero mtDNA-CN. Mitochondrial DNA–depleted BET-1A ρ0 and BEAS-2B ρ0 cell lines are viable, lack the capacity for aerobic respiration, and increase glycolysis. • BET-1A and BEAS-2B cells were treated with ethidium bromide (EtBr) with or without 2′,3′-dideoxycytidine (ddC) to create cells lacking mitochondrial DNA (mtDNA). • Cells’ mtDNA copy number relative to nuclear DNA (nDNA) were verified by quantitative polymerase chain reaction (qPCR). • Cells were also assessed for oxidative phosphorylation by measures of oxygen consumption using the Seahorse analyzer

Microparticles release by adipocytes act as "find-me" signals to promote macrophage migration.

Macrophage infiltration of adipose tissue during weight gain is a central event leading to the metabolic complications of obesity. However, what are the mechanisms attracting professional phagocytes to obese adipose tissue remains poorly understood. Here, we demonstrate that adipocyte-derived microparticles (MPs) are critical "find-me" signals for recruitment of monocytes and macrophages. Supernatants from stressed adipocytes stimulated the attraction of monocyte cells and primary macrophages. The activation of caspase 3 was required for release of these signals. Adipocytes exposed to saturated fatty acids showed marked release of MPs into the supernatant while common genetic mouse models of obesity demonstrate high levels of circulating adipocyte-derived MPs. The release of MPs was highly regulated and dependent on caspase 3 and Rho-associated kinase. Further analysis identified these MPs as a central chemoattractant in vitro and in vivo. In addition, intravenously transplanting circulating MPs from the ob/ob mice lead to activation of monocytes in circulation and adipose tissue of the wild type mice. These data identify adipocyte-derived MPs as novel "find me" signals that contributes to macrophage infiltration associated with obesity

Effects of gastric bypass surgery on expression of glucose transporters and fibrotic biomarkers in kidney of diabetic fatty rats

BACKGROUND: Diabetic nephropathy is the leading cause of chronic kidney disease. Observational studies suggest Roux-en-Y gastric bypass (RYGB) reduces progression of diabetic nephropathy. OBJECTIVES: To unravel the mechanisms by which RYGB is beneficial and protective for diabetic nephropathy. SETTING: Academic laboratories. METHODS: Forty-eight Zucker diabetic fatty rats were randomized to RYGB, sham surgery (SHAM), or pair-fed (PF) groups. An oral glucose tolerance test was performed at 25 days post intervention and kidneys were harvested at 30 days. Primary outcome measures included expression of key genes and proteins in the glucose transport, oxidative stress, inflammation, and fibrosis pathways. RESULTS: Thirty days post intervention, RYGB rats weighed 349 ± 8 g, which was lower than SHAM (436 ± 14 g, P < .001), but not PF (374 ± 18 g) rats. RYGB rats had lower fasting glucose than PF animals and improved homeostatic model assessment of insulin resistance compared with PF and SHAM groups. These enhanced metabolic outcomes were accompanied by reduced sodium-glucose co-transporter 1 (Sglt1) gene expression (-23% versus PF, P = .01) in the kidney of RYGB rats. Expression of Sglt2, Glut1, or Glut2 mRNA, or oxidative stress and inflammation markers did not differ significantly. However, RYGB surgery induced a 19% lower expression of transforming growth factor (Tgfβ) mRNA (P = .004) compared with SHAM treated animals. Notably, adenosine monophosphate-activated protein kinase phosphorylation was increased (P = .04) in kidneys of the RYGB surgery animals. CONCLUSIONS: Improvement of hyperglycemia after RYGB may reduce the glucose load on the kidney leading to a downregulation of specific glucose transporters. RYGB surgery may also attenuate kidney fibrosis through the adenosine monophosphate-activated protein kinase/TGFβ pathway.status: publishe

Exercise training-induced improvement in skeletal muscle PGC-1α-mediated fat metabolism is independent of dietary glycemic index

OBJECTIVE: We hypothesized that a low glycemic diet combined with exercise would increase expression of nuclear regulators of fat transport and oxidation in insulin-resistant skeletal muscle. METHOD: Nineteen subjects (64±1 yrs; 34±1 kg/m(2)) were randomized to receive isocaloric high- (HiGIX; 80±0.6 units, N=10) or low-glycemic index (LoGIX; 40±0.3 units, N=9) diets combined with supervised exercise (1 h/d, 5 d/wk at ~85% HR(max)) for 12 weeks. Insulin sensitivity was determined by hyperinsulinemic-euglycemic clamp. Skeletal muscle biopsies were obtained before and after the intervention to assess fasting gene and protein expression. RESULTS: Weight loss was similar for both groups (9.5±1.3 kg). Likewise, improvements in insulin sensitivity (P<0.002), and PPARγ (P<0.002), PGC-1α (P=0.003), CD36 (P=0.003), FABP3 [mRNA, P=0.01 and protein, P=0.02], and CPT1B [mRNA, P=0.03 and protein, P=0.008] expression were similar for both interventions. Increased insulin sensitivity correlated with increased PGC-1α expression (P=0.04), and increased fasting fat oxidation correlated with increased FABP3 (P=0.04) and CPT1B (P=0.05) expression. CONCLUSIONS: An exercise/diet program resulting in an 8–10% weight loss improved insulin sensitivity and key molecular mechanisms in skeletal muscle that are controlled by PGC-1α. These effects were independent of the glycemic index of the diets

Effect of Roux-en-Y Gastric Bypass on the NLRP3 Inflammasome in Adipose Tissue from Obese Rats.

Obesity is associated with low-grade chronic inflammation. We hypothesized that Roux-en-Y gastric bypass (RYGB) surgery would reduce activation of the NLRP3 inflammasome in metabolically active adipose tissue (AT) of obese rats, and this change would be related to decreases in body weight and improved glycemic control.Omental, mesenteric and subcutaneous fat depots were collected from Sprague-Dawley rats: Sham control and RYGB; 90-days after surgery. NLRP3, caspase-1, apoptosis-associated speck-like protein (ASC), IL-1β, IL-18, IL-6 and MCP-1 gene and protein expression were quantified. Glucose metabolism was assessed by oral glucose tolerance test (OGTT).Compared to Sham surgery controls, RYGB surgery decreased IL-6, MCP-1, NLRP3, IL-18, caspase-1 and ASC in omental fat, and decreased IL-6, MCP1, IL-1β, IL-18, caspase-1 and ASC gene expression in mesenteric fat. We observed differential gene expression between visceral and subcutaneous fat for IL-6 and IL-1β, both being downregulated by RYGB in visceral, and upregulated in subcutaneous depots. These changes in gene expression were accompanied by a decrease in NLRP3, ASC, IL-18, caspase-1 and IL-1β protein expression in omental tissue. We found a positive correlation between caspase-1, ASC, MCP-1, IL-18 and IL-6 gene expression following surgery and glucose AUC response in omental fat, while the change in glucose AUC response correlated with caspase-1 gene expression in subcutaneous fat.This study demonstrates that bariatric surgery reverses inflammation in visceral adipose tissue by suppressing NLRP3 inflammasome activation. These are the first data to implicate the NLRP3 inflammasome in diabetes remission after RYGB surgery

Microparticles Release by Adipocytes Act as “Find-Me” Signals to Promote Macrophage Migration

<div><p>Macrophage infiltration of adipose tissue during weight gain is a central event leading to the metabolic complications of obesity. However, what are the mechanisms attracting professional phagocytes to obese adipose tissue remains poorly understood. Here, we demonstrate that adipocyte-derived microparticles (MPs) are critical “find-me” signals for recruitment of monocytes and macrophages. Supernatants from stressed adipocytes stimulated the attraction of monocyte cells and primary macrophages. The activation of caspase 3 was required for release of these signals. Adipocytes exposed to saturated fatty acids showed marked release of MPs into the supernatant while common genetic mouse models of obesity demonstrate high levels of circulating adipocyte-derived MPs. The release of MPs was highly regulated and dependent on caspase 3 and Rho-associated kinase. Further analysis identified these MPs as a central chemoattractant in vitro and in vivo. In addition, intravenously transplanting circulating MPs from the ob/ob mice lead to activation of monocytes in circulation and adipose tissue of the wild type mice. These data identify adipocyte-derived MPs as novel “find me” signals that contributes to macrophage infiltration associated with obesity.</p></div