Carbenoxolone Treatment Ameliorated Metabolic Syndrome in WNN/Ob Obese Rats but induced Severe Fat Loss and Glucose Intolerance in Learn Rats

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Carbenoxolone treatment ameliorated metabolic syndrome in WNIN/Ob obese rats, but induced severe fat loss and glucose intolerance in lean rats.

BACKGROUND: 11beta-hydroxysteroid dehydrogenase type 1 (11β-HSD1) regulates local glucocorticoid action in tissues by catalysing conversion of inactive glucocorticoids to active glucocorticoids. 11β-HSD1 inhibition ameliorates obesity and associated co-morbidities. Here, we tested the effect of 11β-HSD inhibitor, carbenoxolone (CBX) on obesity and associated comorbidities in obese rats of WNIN/Ob strain, a new animal model for genetic obesity. METHODOLOGY/PRINCIPAL FINDINGS: Subcutaneous injection of CBX (50 mg/kg body weight) or volume-matched vehicle was given once daily for four weeks to three month-old WNIN/Ob lean and obese rats (n = 6 for each phenotype and for each treatment). Body composition, plasma lipids and hormones were assayed. Hepatic steatosis, adipose tissue morphology, inflammation and fibrosis were also studied. Insulin resistance and glucose intolerance were determined along with tissue glycogen content. Gene expressions were determined in liver and adipose tissue. CBX significantly inhibited 11β-HSD1 activity in liver and adipose tissue of WNIN/Ob lean and obese rats. CBX significantly decreased body fat percentage, hypertriglyceridemia, hypercholesterolemia, insulin resistance in obese rats. CBX ameliorated hepatic steatosis, adipocyte hypertrophy, adipose tissue inflammation and fibrosis in obese rats. Tissue glycogen content was significantly decreased by CBX in liver and adipose tissue of obese rats. Severe fat loss and glucose- intolerance were observed in lean rats after CBX treatment. CONCLUSIONS/SIGNIFICANCE: We conclude that 11β-HSD1 inhibition by CBX decreases obesity and associated co-morbidities in WNIN/Ob obese rats. Our study supports the hypothesis that inhibition of 11β-HSD1 is a key strategy to treat metabolic syndrome. Severe fat loss and glucose -intolerance by CBX treatment in lean rats suggest that chronic 11β-HSD1 inhibition may lead to insulin resistance in normal conditions

Effect of carbenoxolone on adipose tissue gene expression quantified by semi quantitative reverse transcription PCR.

<p>(A). Stearoyl CoA desaturase 1 (SCD1). (B). Malic enzyme 1 (ME1). (C). Macrophage expressed gene (MPEG). (D). Lysosomal acid lipase (LIPA). (E). Beta3-Adrenergic receptor (β3-AR). Parameters were measured after 4weeks of treatment with carbenoxolone or vehicle (50 mg/kg body weight/day). Empty bars indicate lean phenotype where as filled bars indicate obese phenotype. Relative gene expression was measured using calnexin gene as internal control. Relative expression of gene of interest in lean control was taken as 1. Values are means ± SEM for 4 animals for group. . #<i>p</i><0.05, ##<i>p</i><0.01 and ### <i>p</i><0.001 comparing vehicle-treated lean and obese rats. *<i>p</i><0.05, **<i>p</i><0.01 and *** <i>p</i><0.001 comparing carbenoxolone-treated animals with vehicle-treated animals of the same phenotype.</p

Effect of carbenoxolone on hepatic triglyceride and glycogen content.

<p>(A). Hepatic triglyceride content. (B). Hepatic glycogen content. (C). Photographs of liver tissue sections stained with haematoxylin and eosin (to study fat accumulation). (D). Photographs of liver tissue sections stained with oil Red O (to study fat accumulation). (E). Photographs of liver tissue sections stained with periodic acid-Schiff's stain (to study glycogen content). (F). Photographs of liver tissues sections stained with Masson's trichrome stain (to observe fibrosis). Parameters were measured after 4weeks of treatment with carbenoxolone or vehicle (50 mg/kg body weight/day). Empty bars indicate lean phenotype where as filled bars indicate obese phenotype. LC, Lean control; LT, Lean- treated; OC, Obese control; OT, Obese -treated. Images were taken at Magnification of 10X. Values are means ± SEM for 6 animals for group. #<i>p</i><0.05, ##<i>p</i><0.01 and ### <i>p</i><0.001 comparing vehicle-treated lean and obese rats. *<i>p</i><0.05, **<i>p</i><0.01 and *** <i>p</i><0.001 comparing carbenoxolone-treated animals with vehicle-treated animals of the same phenotype.</p

Effect of carbenoxolone on 11β-HSD1 activity in WNIN/Ob lean and obese rats.

<p>(A). Liver. (B). Subcutaneous adipose tissue. (C). Omental adipose tissue. (D). Skeletal muscle. 11β-HSD1 activity was measured after 1 h of subcutaneous injection of carbenoxolone or vehicle (50 mg/kg body weight/day). Empty bars indicate lean phenotype where as filled bars indicate obese phenotype. Values are means ± SEM for 6 animals for group. #<i>p</i><0.05, ##<i>p</i><0.01 and ### <i>p</i><0.001 comparing vehicle-treated lean and obese rats. *<i>p</i><0.05, **<i>p</i><0.01 and *** <i>p</i><0.001 comparing carbenoxolone-treated animals with vehicle-treated animals of the same phenotype.</p

Effect of carbenoxolone on body composition of WNIN/Ob lean and obese rats.

<p>(A). fat percentage. (B). Lean body mass. (C). Fat free mass. Body composition was measured by TOBEC after 4weeks of treatment with carbenoxolone or vehicle (50 mg/kg body weight/day). Empty bars indicate lean phenotype where as filled bars indicate obese phenotype. Values are means ± SEM for 6 animals for group. #<i>p</i><0.05, ##<i>p</i><0.01 and ### <i>p</i><0.001 comparing vehicle-treated lean and obese rats. *<i>p</i><0.05, **<i>p</i><0.01 and *** <i>p</i><0.001 comparing carbenoxolone-treated animals with vehicle-treated animals of the same phenotype.</p

Effect of carbenoxolone on plasma biochemical parameters in WNIN/Ob lean and obese rats.

<p>(A). Fed-state corticosterone. (B). Fasting corticosterone. (C). Fed-state triglycerides. (D). Fasting triglycerides. (E). Fed-state total cholesterol. (F). Fasting total cholesterol. (G). Fed-state HDL cholesterol. (H). Fasting HDL cholesterol. Plasma parameters were measured after 4weeks of treatment with carbenoxolone or vehicle (50 mg/kg bodyweight/day). Empty bars indicate lean phenotype where as filled bars indicate obese phenotype. Values are means ± SEM for 6 animals for group. #<i>p</i><0.05, ##<i>p</i><0.01 and ### <i>p</i><0.001 comparing vehicle-treated lean and obese rats. *<i>p</i><0.05, **<i>p</i><0.01 and *** <i>p</i><0.001 comparing carbenoxolone-treated animals with vehicle-treated animals of the same phenotype.</p

Effect of carbenoxolone on adipose tissue morphology and glycogen content.

<p>(A). Adipocyte size (number of cells/16sq.mm). (B). Photographs of adipose tissue sections stained with haematoxylin and eosin (to observe adipocyte cell size). (C). Photographs of adipose tissue sections with Haematoxylin and Eosin (to observe adipose tissue inflammation characterized by presence of ‘crown like structures’-indicated by arrows). (D). Photographs of adipose tissue sections stained with Masson's trichrome stain (to observe fibrosis-indicated by arrows). (E). Tissue glycogen content. Parameters were measured after 4weeks of treatment with carbenoxolone or vehicle (50 mg/kg body weight/day). Empty bars indicate lean phenotype, where as filled bars indicate obese phenotype. OC, Obese control; OT, Obese- treated. Images were taken at Magnification of 10X. Values are means ± SEM for 6 animals for group. . #<i>p</i><0.05, ##<i>p</i><0.01 and ### <i>p</i><0.001 comparing vehicle-treated lean and obese rats. *<i>p</i><0.05, **<i>p</i><0.01 and *** <i>p</i><0.001 comparing carbenoxolone-treated animals with vehicle-treated animals of the same phenotype.</p

Effect of carbenoxolone on insulin signaling in gastrocnemius muscle of WNIN/Ob lean and obese rats.

<p>(A). Representative western blot for AKT. (B). Representative western blot for pAKT (Ser 473). (C). Representative western blot for PTP1B. (C). Representative western blot for β-actin. Parameters were measured after 4weeks of treatment with carbenoxolone or vehicle (50 mg/kg body weight/day). Relative protein levels were measured using β-actin as internal control. LC, Lean Control; LT, Lean Treated; OC, Obese Control; OT, Obese control. Relative expression of protein of interest in lean control was taken as 1. Values are means ± SEM for 4 animals for group. . #<i>p</i><0.05 comparing vehicle-treated lean and obese rats. *<i>p</i><0.05 comparing carbenoxolone-treated animals with vehicle-treated animals of the same phenotype.</p

Effect of carbenoxolone on food intake, body weight and organ weights in WNIN/Ob lean and obese rats.

<p>Values represent means ± SEM of 6 rats per group. LC, Lean control; OC, Obese control; LT, Lean- treated; OT, Obese- treated. Parameters were measured after four weeks of treatment with carbenoxolone (50 mg/kg/body weight/day) or vehicle (same volume of phosphate buffer saline).</p>#<p><i>p</i><0.05,</p>##<p><i>p</i><0.01 and ### <i>p</i><0.001 comparing vehicle-treated lean and obese rats.</p>*<p><i>p</i><0.05,</p>**<p><i>p</i><0.01 and *** <i>p</i><0.001 comparing carbenoxolone- treated animals with vehicle-treated animals of the same phenotype. </p