**2.3 Fem1b-KO mice development**

In this study, the gene targeting by homologous recombination has been used to generate Fem1b knockout (Fem1b-KO) mice with inactivation of the Fem1b gene. It was performed with a deletion of Fem1b coding exon 1, which contains the translation initiation codon and the first two ankyrin repeats [9]. The results show that these mice display abnormal glucose homeostasis, with abnormal glucose tolerance tests and defective glucose-stimulated insulin secretion. These findings indicate that Fem1b is involved in pancreatic islet β-cell function and provide further evidence for involvement of a pathway resembling nematode sex determination in mammalian glucose homeostasis. This approach utilized standard methodology (Figure 1) and the basic elements of the targeting vector and screening strategy by Southern blot and PCR genotyping. Figure 2 was generated by Zeiss AvioVision with the immunohistochemical analysis demonstrates a loss of specific Fem1b staining in islets of Fem1b-KO homozygotes.

Fig. 1. General strategy, with expected KpnI digestion products along with 5\_ and 3\_ probes for Southern blot. The boxes labeled 1 and 2 represent exons 1 and 2, respectively. Exon 1 is replaced by the PGK-Neo gene (labeled arrow) in the targeted allele.

Fig. 2. Immunostaining of homozygous Fem1b-KO pancreas with anti-Fem1b C-terminus antibody Li-51, demonstrating the absence of specific staining for Fem1b.

### **2.4 Glucose homeostasis in Fem1b-KO mice**

As noted above, mammalian homologues of nematode sex determination genes have recently been shown to be involved in glucose homeostasis and type 2 diabetes mellitus. Based on this logic, glucose homeostasis was evaluated in the Fem1b-KO mice by using established experimental methods. As a first-line screen, these mice were between 3 and 4 months of age. The iP-ITT showed minimally abnormal results (Figure 3), suggesting that insulin resistance is not the primary defect in homozygotes, although it could be contributing.

Fig. 3. Intraperitoneal insulin tolerance test (males and females).

Fig. 2. Immunostaining of homozygous Fem1b-KO pancreas with anti-Fem1b C-terminus

As noted above, mammalian homologues of nematode sex determination genes have recently been shown to be involved in glucose homeostasis and type 2 diabetes mellitus. Based on this logic, glucose homeostasis was evaluated in the Fem1b-KO mice by using established experimental methods. As a first-line screen, these mice were between 3 and 4 months of age. The iP-ITT showed minimally abnormal results (Figure 3), suggesting that insulin resistance is not the primary defect in homozygotes, although it could be

antibody Li-51, demonstrating the absence of specific staining for Fem1b.

Fig. 3. Intraperitoneal insulin tolerance test (males and females).

**2.4 Glucose homeostasis in Fem1b-KO mice** 

contributing.

To evaluate whether the defective acute-phase insulin secretion is related to a defect in secretion per se as opposed to a defect in insulin production, the insulin content was measured o in these mice (Figure 4), which demonstrates that Fem1b-KO homozygotes have increased insulin content compared to that of wild-type controls.

Fig. 4. Pancreatic insulin content in fasted mice (four Fem1b-KO homozygous mice and four wild-type mice).
