Elevated mouse hepatic betatrophin expression does not increase human β-cell replication in the transplant setting

Abstract

The recent discovery of betatrophin, a protein secreted by the liver and white adipose tissue in conditions of insulin resistance and shown to dramatically stimulate replication of mouse insulin-producing β-cells, has raised high hopes for the rapid development of a novel therapeutic approach for the treatment of diabetes. At present, however, the effects of betatrophin on human β-cells are not known. Here we use administration of the insulin receptor antagonist S961, shown to increase betatrophin gene expression and stimulate β-cell replication in mice, to test its effect on human β-cells. Although mouse β-cells, in their normal location in the pancreas or when transplanted under the kidney capsule, respond with a dramatic increase in β-cell DNA replication, human β-cells are completely unresponsive. These results put into question whether betatrophin can be developed as a therapeutic approach for treating human diabetes.

Figure 1

Elevated betatrophin expression does not stimulate replication of human β-cells. A: Random blood glucose levels of NOD-Scid mice treated with vehicle (n = 6) or the insulin receptor antagonist S961 (n = 10). Glucose levels in S961-treated mice were significantly elevated from day 1 to 7 (P < 0.001). B: Hepatic betatrophin mRNA levels in mice treated with vehicle (n = 3) or S961 (n = 4) as determined by quantitative RT-PCR. Levels of mRNA were normalized to those of TATA-box binding protein as the internal control and are expressed as fold over vehicle. *P < 0.05. C: Gonadal white adipose tissue betatrophin mRNA levels in mice treated with vehicle (n = 8) or S961 (n = 6) as determined by quantitative RT-PCR. β-Cell replication in NOD-Scid mice treated with vehicle or S961 was determined by BrdU incorporation (red signal). β-Cells were identified by insulin immunofluorescence staining (green). D: β-Cells in the endocrine pancreas of vehicle-treated mice. E: Transplanted human islets recovered from the kidney capsule of vehicle-treated mice. F: Transplanted mouse islets recovered from the kidney capsule of vehicle-treated mice. G: β-Cells in the endocrine pancreas of S961-treated mice. Note the frequent BrdU⁺ β-cells (yellow arrows). H: Transplanted human islets recovered from the kidney capsule of S961-treated mice. Insulin/BrdU double-positive cells are very rare. I: Transplanted mouse islets recovered from the kidney capsule of S961-treated mice. Note the frequent BrdU⁺ β-cells (yellow arrows). J: β-Cell replication was quantified as the percentage of BrdU⁺/insulin (Ins)⁺ double-positive cells of the total number of insulin⁺ cells from the six conditions shown in D–I. n = 5 for vehicle and for S961. **P < 0.01. β-Cell replication in transplanted mouse islets from 2-month-old or 12-month-old donor mice was also quantified separately. K: β-Cell replication was determined as the percentage of Ki67⁺/insulin⁺ double-positive cells of the total number of insulin⁺ cells in the six conditions shown in D–I. n = 5 for vehicle and for S961. **P < 0.01.

Figure 2

No evidence for increased β-cell apoptosis in S961-treated mice. Apoptosis in NOD-Scid mice treated with vehicle or S961 was determined by TUNEL staining (red signal). β-Cells were identified by insulin immunofluorescence staining (green). A: β-Cells in the endocrine pancreas of vehicle-treated mice. B: Transplanted human islets recovered from the kidney capsule of vehicle-treated mice. C: Transplanted mouse islets recovered from the kidney capsule of vehicle-treated mice. D: β-Cells in the endocrine pancreas of S961-treated mice. E: Transplanted human islets recovered from the kidney capsule of S961-treated mice. F: Transplanted mouse islets recovered from the kidney capsule of S961-treated mice. G: Positive control for the TUNEL staining assay using DNAse-treated tissue.