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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.

Islet allografts from donor mice exposed to CO are protected from immune rejection after transplantation via the suppression of membrane trafficking/activation of TLR4 in islets/β cells. The molecular mechanisms of how CO suppresses TLR4 activation in β cells remain unclear and are the focus of this study. Cells of the insulinoma cell line, βTC3, were stably transfected with pcDNA3-TLR4-YFP and pDsRed-Monomer-Golgi plasmids and used to identify the subcellular distribution of TLR4 before and after LPS stimulation by confocal microscopy. Immunofluorescence analysis revealed that TLR4 mainly resides in the Golgi apparatus in βTC3 cells when in a quiescent state. LPS stimulation led to a rapid trafficking of TLR4 from the Golgi to the cell membrane. Physical interaction between TLR4 and myeloid differentiation factor-2 (MD-2) was confirmed by immunoprecipitation. Depleting MD-2 using small interfering RNA or blocking the N-glycosylation of cells using tunicamycin blocked membrane trafficking of TLR4. Pre-exposing cells to CO at a concentration of 250 parts per million suppressed membrane trafficking of TLR4 via inhibiting its glycosylation and the interaction between TLR4 and MD-2. In conclusion, MD-2 is required for the glycosylation of TLR4 and its consequent membrane trafficking in βTC3 cells. CO suppresses membrane activation of TLR4 via blocking its glycosylation and the physical interaction between TLR4 and MD-2.

We have previously demonstrated that bilirubin administration to the recipient induces tolerance towards islet cell transplants across a complete MHC mismatch in a mouse model. Here we assess the mechanisms of such protection. Bilirubin treatment of recipients improved function of islet allografts by suppressing expressions of proinflammatory and proapoptotic genes in those islets and by increasing Foxp3+T regulatory (Treg) cells at the site of transplanted islets at various days after transplantation. No prolongation of graft survival was observed in recipients treated with bilirubin when CD4+CD25+T cells were predepleted from those recipients, indicating that Treg cells are necessary for the protective effect of bilirubin. Adoptive transfer of Treg cells from tolerant mice into Rag1-/-recipients resulted in long-term acceptance of skin allografts in an alloantigen-specific manner, suggesting that Treg cells are sufficient to induce tolerance. In addition, bilirubin treatment promoted de novo generation of Treg cells in Rag1-/-recipients. Thus, bilirubin treatment to the recipients prolongs islet allograft survival via a Treg-dependent manner in which CD4+CD25+Treg cells are both necessary and sufficient for tolerance induction and graft acceptance. Bilirubin treatment promotes de novo generation of Treg cells that might account for the protective effects of bilirubin given to recipients.