👤 Roberto Gramignoli

A liver-humanized mouse model for CPS1-deficiency was generated by the high-level repopulation of the mouse liver with CPS1-deficient human hepatocytes. When compared with mice that are highly repopulated with CPS1-proficient human hepatocytes, mice that are repopulated with CPS1-deficient human hepatocytes exhibited characteristic symptoms of human CPS1 deficiency including an 80% reduction in CPS1 metabolic activity, delayed clearance of an ammonium chloride infusion, elevated glutamine and glutamate levels, and impaired metabolism of [ Show less

Glucose stimulates rodent and human β-cell replication, but the intracellular signaling mechanisms are poorly understood. Carbohydrate response element-binding protein (ChREBP) is a lipogenic glucose-sensing transcription factor with unknown functions in pancreatic β-cells. We tested the hypothesis that ChREBP is required for glucose-stimulated β-cell proliferation. The relative expression of ChREBP was determined in liver and β-cells using quantitative RT-PCR (qRT-PCR), immunoblotting, and immunohistochemistry. Loss- and gain-of-function studies were performed using small interfering RNA and genetic deletion of ChREBP and adenoviral overexpression of ChREBP in rodent and human β-cells. Proliferation was measured by 5-bromo-2'-deoxyuridine incorporation, [(3)H]thymidine incorporation, and fluorescence-activated cell sorter analysis. In addition, the expression of cell cycle regulatory genes was measured by qRT-PCR and immunoblotting. ChREBP expression was comparable with liver in mouse pancreata and in rat and human islets. Depletion of ChREBP decreased glucose-stimulated proliferation in β-cells isolated from ChREBP(-/-) mice, in INS-1-derived 832/13 cells, and in primary rat and human β-cells. Furthermore, depletion of ChREBP decreased the glucose-stimulated expression of cell cycle accelerators. Overexpression of ChREBP amplified glucose-stimulated proliferation in rat and human β-cells, with concomitant increases in cyclin gene expression. In conclusion, ChREBP mediates glucose-stimulated proliferation in pancreatic β-cells. Show less

The nuclear receptor liver X receptor (LXR) is a ligand-dependent transcription factor that plays an important role in the metabolism and homeostasis of cholesterol, lipids, bile acids, and steroid hormones. MicroRNAs (miRNAs) are recently recognized important negative regulators of gene expression. In this report, we showed that miRNA hsa-miR-613 played an important role in the autoregulation of the human LXRα gene. hsa-miR-613 targeted the endogenous LXRα through its specific miRNA response element (613MRE) within the LXRα 3'-untranslated region. Interestingly and paradoxically, the expression of hsa-miR-613 itself was induced upon the activation of LXR. However, hsa-miR-613 did not appear to be a direct LXR target gene. Instead, the positive regulation of hsa-miR-613 by LXR was mediated by the sterol regulatory element binding protein (SREBP)-1c, a known LXR target gene. Promoter analysis revealed an SREBP response element in the hsa-miR-613 gene promoter. Treatment with insulin also induced the expression of hsa-miR-613 in an SREBP-1c-dependent manner, further supporting the role of SREBP-1c in the positive regulation of this miRNA species. Finally, the autoinduction of LXRα by a LXR agonist was enhanced when hsa-miR-613 was inhibited or SREBP-1c was down-regulated. hsa-miR-613 appeared to specifically target the human LXRα. We propose that the negative regulation mediated by hsa-miR-613 and SREBP-1c and the previously reported positive regulation mediated by an LXR response element in the LXRα gene promoter constitute a ying-yang mechanism to ensure a tight regulation of this nuclear receptor of many metabolic functions. Show less