Bilirubin increases insulin sensitivity in leptin-receptor deficient and diet-induced obese mice through suppression of ER stress and chronic inflammation - PubMed (original) (raw)

. 2014 Mar;155(3):818-28.

doi: 10.1210/en.2013-1667. Epub 2014 Jan 1.

Hu Huang, Xinxu Yun, Do-sung Kim, Yinan Yue, Hongju Wu, Alton Sutter, Kenneth D Chavin, Leo E Otterbein, David B Adams, Young-Bum Kim, Hongjun Wang

Affiliations

Bilirubin increases insulin sensitivity in leptin-receptor deficient and diet-induced obese mice through suppression of ER stress and chronic inflammation

Huansheng Dong et al. Endocrinology. 2014 Mar.

Abstract

Obesity-induced endoplasmic reticulum (ER) stress causes chronic inflammation in adipose tissue and steatosis in the liver, and eventually leads to insulin resistance and type 2 diabetes (T2D). The goal of this study was to understand the mechanisms by which administration of bilirubin, a powerful antioxidant, reduces hyperglycemia and ameliorates obesity in leptin-receptor-deficient (db/db) and diet-induced obese (DIO) mouse models. db/db or DIO mice were injected with bilirubin or vehicle ip. Blood glucose and body weight were measured. Activation of insulin-signaling pathways, expression of inflammatory cytokines, and ER stress markers were measured in skeletal muscle, adipose tissue, and liver of mice. Bilirubin administration significantly reduced hyperglycemia and increased insulin sensitivity in db/db mice. Bilirubin treatment increased protein kinase B (PKB/Akt) phosphorylation in skeletal muscle and suppressed expression of ER stress markers, including the 78-kDa glucose-regulated protein (GRP78), CCAAT/enhancer-binding protein (C/EBP) homologous protein, X box binding protein (XBP-1), and activating transcription factor 4 in db/db mice. In DIO mice, bilirubin treatment significantly reduced body weight and increased insulin sensitivity. Moreover, bilirubin suppressed macrophage infiltration and proinflammatory cytokine expression, including TNF-α, IL-1β, and monocyte chemoattractant protein-1, in adipose tissue. In liver and adipose tissue of DIO mice, bilirubin ameliorated hepatic steatosis and reduced expression of GRP78 and C/EBP homologous protein. These results demonstrate that bilirubin administration improves hyperglycemia and obesity by increasing insulin sensitivity in both genetically engineered and DIO mice models. Bilirubin or bilirubin-increasing drugs might be useful as an insulin sensitizer for the treatment of obesity-induced insulin resistance and type 2 diabetes based on its profound anti-ER stress and antiinflammatory properties.

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Figures

Figure 1.

Figure 1.

Systemic induction of HO-1 by CoPP injection reduces hyperglycemia and increases insulin sensitivity in db/db mice. A, HO-1 induction by CoPP at 20 mg/kg (defined as CoPP20) reduced blood glucose levels in db/db mice compared with mice that received the vehicle (defined as CTR). B, Blocking HO-1 activity by ZnPP (20 mg/kg) diminished the effect of CoPP (20 mg/kg) in lowering hyperglycemia. C, Treatment with CoPP increased insulin sensitivity as demonstrated by blood glucose levels measured by ITT and the areas under the curve (insets, mg/dL · h). D, No differences in the amount of food consumed within a 24-hour period of time were observed in db/db mice that received CoPP, ZnPP, or vehicle (defined as CTR). At least 8 mice were included in each treatment group. Experiments have been repeated independently at least 3 times; **, P < .01 and *, P < .05 as analyzed by ANOVA and Student's t test.

Figure 2.

Figure 2.

Detection of molecules involved in insulin signaling in the skeletal muscles and livers of db/db mice. A, Expression of p308–p473- and total Akt and HO-1, and membrane presence of Glut4 were detected in skeletal muscle from mice treated with saline or insulin by Western blotting. CTR refers to db/db mice that received vehicle only. B, Ratios of phosphorylated Akt in total Akt, expression of Glut4 in each sample relative to the loading control were quantified in muscles. C, Expression of p308-, p473-, total Akt, HO-1, and p-GSK in liver tissues. D, Ratios of phosphorylated Akt in total Akt, phosphorylated GSK (p-GSK)/GAPDH were quantified in livers from CTR, CoPP10, and CoPP20 groups; *, P < .05, saline vs insulin.

Figure 3.

Figure 3.

Administration of bilirubin reduces the blood glucose level and increases insulin sensitivity in db/db mice. A, Treatment of db/db mice with bilirubin (BR) significantly reduced blood glucose levels in db/db mice compared with those that received vehicle (defined as CTR). B, Mice that received BR showed significantly lower blood glucose levels at most time points and smaller area under the curve (AUC) compared with control after ITT test. C, Expression of p308 and total Akt, and membrane expression of Glut4 were observed in muscles of mice that received BR compared with controls after insulin stimulation. D, Quantification of p-308Akt in total Akt; and E, relative expression of membrane Glut4 in muscles from db/db mice treated with or without BR. At least 8 mice were included in each treatment group; *, P < .05 and **, P < .01, ANOVA or Student's t test.

Figure 4.

Figure 4.

Bilirubin treatment suppresses expression of ER stress markers in db/db mice. A, Relative expression of BiP, Chop, XBP-1, and ATF4 mRNA in muscles of db/db mice treated with vehicle (db/db) or bilirubin (db/db+BR). Relative expression of BiP, Chop, XBP-1, and ATF4 mRNA in livers of db/db mice treated with vehicle (db/db) or bilirubin (db/db+BR). *, P < .05 vehicle vs bilirubin.

Figure 5.

Figure 5.

Bilirubin (BR) treatment reduces blood glucose levels and obesity and increases insulin sensitivity in DIO mice. A, Changes in blood glucose levels in DIO mice treated with BR (DIO+BR) compared with control mice treated with vehicle (DIO+V); *, P < .05 and **, P < .01, BR vs vehicle. B, BR treatment increases insulin sensitivity in DIO mice. Data shown are blood glucose levels at different time points as measured by ITT; inset, areas under the curves. C, Changes in body weights of DIO mice. The body weights of mice were measured daily after BR treatment; *, P < .05, BR vs vehicle. D, Average food intake per mouse per 24-hour period in DIO mice treated with BR or vehicle. At least 8 mice were included in each treatment group.

Figure 6.

Figure 6.

Bilirubin treatment promotes Akt phosphorylation in DIO mice. A, Phosphorylation of Akt at Thr308 was determined in muscle tissues from DIO mice treated with bilirubin, vehicle, or in chow controls 6 minutes after stimulation with either saline or insulin. B, Quantification of p-308Akt in total Akt. C, Expression of p308 Akt was determined in livers from DIO mice treated with bilirubin, vehicle, or in chow controls after stimulation with either saline or insulin. D, Quantification of p-308Akt in total Akt in liver. BR, bilirubin; V, vehicle.

Figure 7.

Figure 7.

Bilirubin (BR) treatment suppresses expression of ER stress markers and proinflammatory cytokines, and inhibits macrophage infiltration in adipose tissue of DIO mice. A, Relative expression of BiP, Chop, XBP-1, and ATF4 mRNA in DIO mice treated with vehicle (DIO+V) or BR (DIO+BR) and chow control (chow). B, Relative expression of TNF-α, IL-1β, and MCP-1 in mice fed with normal chow (chow), DIO treated with vehicle (DIO+V), and DIO treated with BR (DIO+BR). C, Immunohistochemical staining of epididymal tissues from BR-treated DIO mice showed significantly less macrophage infiltration compared with mice that received vehicle. Arrows point to macrophages (F4/80+ cells). *, P < .05, Student's t test.

Figure 8.

Figure 8.

Bilirubin (BR) ameliorates hepatic steatosis by suppressing ER stress in the liver. A, H&E staining of liver sections from mice fed normal chow (chow), DIO mice treated with vehicle (V), or DIO mice treated with BR (BR). B, Relative expression levels of BiP, Chop, and ATF4 mRNA in liver from mice fed normal chow (chow), DIO mice treated with V (DIO+V), or DIO mice treated with BR (DIO+BR). C, Expression of TNF-α and IL-1β in mice fed normal chow (chow), DIO mice treated with V (DIO+V), or DIO mice treated with BR (DIO+BR). *, P < .05, Student's t test.

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