ABSTRACT
Failure of pancreatic ß cells to compensate for insulin resistance is a prerequisite for the development of type 2 diabetes. Sustained elevated circulating levels of free fatty acids and glucose contribute to ß-cell failure. Selective inhibition of histone deacetylase (HDAC)-3 protects pancreatic ß cells against inflammatory and metabolic insults in vitro. In the present study, we tested the ability of a selective HDAC3 inhibitor, BRD3308, to reduce hyperglycaemia and increase insulin secretion in a rat model of type 2 diabetes. At diabetes onset, an ambulatory hyperglycaemic clamp was performed. HDAC3 inhibition improved hyperglycaemia over the study period without affecting weight gain. At the end of the hyperglycaemic clamp, circulating insulin levels were significantly higher in BRD3308-treated rats. Pancreatic insulin staining and contents were also significantly higher. These findings highlight HDAC3 as a key therapeutic target for ß-cell protection in type 2 diabetes.
Subject(s)
Diabetes Mellitus, Experimental/drug therapy , Histone Deacetylase Inhibitors/metabolism , Histone Deacetylases/metabolism , Obesity/drug therapy , Animals , Blood Glucose/metabolism , Diabetes Mellitus, Experimental/blood , Diabetes Mellitus, Experimental/complications , Diabetes Mellitus, Type 2/blood , Diabetes Mellitus, Type 2/complications , Diabetes Mellitus, Type 2/drug therapy , Fatty Acids, Nonesterified/metabolism , Glucose Clamp Technique , Histone Deacetylase Inhibitors/therapeutic use , Histone Deacetylases/therapeutic use , Hyperglycemia/drug therapy , Insulin/blood , Insulin/metabolism , Insulin Resistance , Insulin Secretion , Insulin-Secreting Cells/metabolism , Male , Obesity/blood , Obesity/complications , Rats , Rats, Zucker , Weight GainABSTRACT
AIMS/HYPOTHESIS: The aim of the study was to identify surface bio-markers and corresponding antibody tools that can be used for the imaging and immunoisolation of the pancreatic beta cell and its progenitors. This may prove essential to obtain therapeutic grade human beta cells via stem cell differentiation. METHODS: Using bioinformatics-driven data mining, we generated a gene list encoding putative plasma membrane proteins specifically expressed at distinct stages of the developing pancreas and islet beta cells. In situ hybridisation and immunohistochemistry were used to further prioritise and identify candidates. RESULTS: In the developing pancreas seizure related 6 homologue like (SEZ6L2), low density lipoprotein receptor-related protein 11 (LRP11), dispatched homologue 2 (Drosophila) (DISP2) and solute carrier family 30 (zinc transporter), member 8 (SLC30A8) were found to be expressed in early islet cells, whereas discoidin domain receptor tyrosine kinase 1 (DDR1) and delta/notch-like EGF repeat containing (DNER) were expressed in early pancreatic progenitors. The expression pattern of DDR1 overlaps with the early pancreatic and duodenal homeobox 1 (PDX1)âº/NK6 homeobox 1 (NKX6-1)⺠multipotent progenitor cells from embryonic day 11, whereas DNER expression in part overlaps with neurogenin 3 (NEUROG3)⺠cells. In the adult pancreas SEZ6L2, LRP11, DISP2 and SLC30A8, but also FXYD domain containing ion transport regulator 2 (FXYD2), tetraspanin 7 (TSPAN7) and transmembrane protein 27 (TMEM27), retain an islet-specific expression, whereas DDR1 is undetectable. In contrast, DNER is expressed at low levels in peripheral mouse and human islet cells. Re-expression of DDR1 and upregulation of DNER is observed in duct-ligated pancreas. Antibodies to DNER and DISP2 have been successfully used in cell sorting. CONCLUSIONS/INTERPRETATION: Extracellular epitopes of SEZ6L2, LRP11, DISP2, DDR1 and DNER have been identified as useful tags by applying specific antibodies to visualise pancreatic cell types at specific stages of development. Furthermore, antibodies recognising DISP2 and DNER are suitable for FACS-mediated cell purification.
Subject(s)
Antigens, Surface/metabolism , Cell Separation/methods , Islets of Langerhans/metabolism , Stem Cells/metabolism , Adult , Animals , Biomarkers/metabolism , Cell Line , Computational Biology/methods , Data Mining , Flow Cytometry , Humans , Immunohistochemistry , In Situ Hybridization, Fluorescence , Islets of Langerhans/cytology , Islets of Langerhans/embryology , Mice , Mice, Inbred BALB C , Organ Culture Techniques , Stem Cells/cytologyABSTRACT
The aim of this study was to characterize two monoclonal antibodies (F6A11 and F109-D12) generated against Pdx1 (pancreatic and duodenal homeobox-1), a homeodomain transcription factor, which is critical for pancreas formation as well as for normal pancreatic beta cell function. For production of monoclonal antibodies, we immunized Robertsonian POSF (RBF)mice with a GST-Pdx1 fusion protein containing a 68-amino acid C-terminal fragment of rat Pdx1. These monoclonal antibodies detect Pdx1 by western blotting and allow immunohistochemical detection of Pdx1 in both mouse and rat tissue. F6A11 and F109-D12 produce IHC staining patterns indistinguishable from that obtained with highly specific polyclonal Pdx1 antisera raised in rabbits and goats, when applied to embryonic or adult mouse pancreatic tissue. In contrast to previously generated polyclonal anti-Pdx1 antisera, we also demonstrate that F6A11 works for intracellular fluorescence activated cell sorting (FACS) staining of Pdx1. By using F6A11, we characterize the induction of Pdx1 in the Doxycycline (DOX) inducible insulinoma cell line INSralphabeta-Pdx1 and follow the reduction of Pdx1 after removing Dox. Finally, we show that induction of exogenous Pdx1 leads to a reduction in endogenous Pdx1 levels, which suggests that a negative feedback loop is involved in maintaining correct levels of Pdx1 in the cell.
