Zi-Jian Lan

Introduction: Adenosine, 5’-Se-methyl-5’-seleno-,2’,3’-diacetate (NPC43) is a recently identified small, non-peptidyl molecule which restores normal insulin signaling in a mouse model of type 2 diabetes (Lan et al). The present study investigated the ability of NPC43 as an oral and injectable insulin-replacing agent to activate insulin receptor (INSR) and counter hyperglycemia in streptozotocin (STZ)-induced type 1 diabetic (T1D) mice. Research design and methods: In this study, STZ was intraperitoneally injected into wild-type mice to induce hyperglycemia and hypoinsulinemia, the main features of T1D. These STZ-induced T1D mice were given NPC43 orally or intraperitoneally and blood glucose levels were measured using a glucometer. Protein levels of phosphorylated and total Insrβ, protein kinase B (Akt) and AS160 (critical for glucose uptake) in the skeletal muscle and liver of STZ-induced T1D mice following oral NPC43 treatment were determined by western blot analysis. In addition, hepatic expression of activated Insr in STZ-induced T1D mice after intraperitoneal NPC43 treatment was measured by ELISA. Student’s t-test was used for statistical analysis. Results: Oral administration of NPC43 at a dose of 5.4 or 10.8 mg/kg body weight (mpk) effectively lowered blood glucose levels in STZ-induced T1D mice at ≥1 hour post-treatment and the glucose-lowering activity of oral NPC43 persisted for 5 hours. Blood glucose levels were also reduced in STZ-induced T1D mice following intraperitoneal NPC43 (5.4 mpk) treatment. Protein levels of phosphorylated Insrβ, Akt and AS160 were significantly increased in the skeletal muscle and liver of STZ-induced T1D mice after oral NPC43 (5.4 mpk) treatment. In addition, activation of hepatic Insr was observed in STZ-induced T1D mice following intraperitoneal NPC43 (5.4 mpk) treatment. Conclusions: We conclude that NPC43 is a de facto fast-acting oral and injectable insulin mimetic which activates Insr and mitigates hyperglycemia in a mouse model of T1D.

The pathophysiology of type 2 diabetes mellitus (T2D) is characterized by reduced or absent insulin receptor (INSR) responsiveness to its ligand, elevated hepatic glucose output and impaired glucose uptake in peripheral tissues, particularly skeletal muscle. Treatments to reduce hyperglycemia and reestablish normal insulin signaling are much sought after. Any agent which could be orally administered to restore INSR function, in an insulin-independent manner, would have major implications for the management of this global disease. We have discovered a non-peptidyl small molecule, adenosine, 5′-Se-methyl-5′-seleno-, 2′,3′-diacetate [referred to as non-peptidyl compound #43 (NPC43)], which restores INSR signaling in the complete absence of insulin. Initial screening of numerous compounds in human HepG2 liver cells revealed that NPC43 significantly inhibited glucose production. The compound was potently anti-hyperglycemic and anti-hyperinsulinemic in vivo, in insulin-resistant T2D Lepr^db/db mice, following either acute or chronic treatment by oral gavage and intraperitoneal injection, respectively. The compound acted at the level of INSR and activated it in both liver and skeletal muscle of Lepr^db/db mice. In cell culture, the compound activated INSR in both liver and skeletal muscle cells; furthermore, it cooperated with insulin to depress glucose-6-phosphatase catalytic subunit (G6pc) expression and stimulate glucose uptake, respectively. Our results indicated that the compound directly interacted with INSRα, triggering appropriate phosphorylation and activation of the receptor and its downstream targets. Unlike insulin, NPC43 did not activate insulin-like growth factor 1 receptor in either liver or skeletal muscle. We believe this compound represents a potential oral and/or injectable insulin replacement therapy for diabetes and diseases associated with insulin resistance.

Gametogenetin (GGN) binding protein 2 (GGNBP2) is a zinc finger protein expressed abundantly in spermatocytes and spermatids. We previously discovered that Ggnbp2 resection caused metamorphotic defects during spermatid differentiation and resulted in an absence of mature spermatozoa in mice. However, whether GGNBP2 affects meiotic progression of spermatocytes remains to be established. In this study, flow cytometric analyses showed a decrease in haploid, while an increase in tetraploid spermatogenic cells in both 30‐ and 60‐day‐old Ggnbp2 knockout testes. In spread spermatocyte nuclei, Ggnbp2 loss increased DNA double‐strand breaks (DSB), compromised DSB repair and reduced crossovers. Further investigations demonstrated that GGNBP2 co‐immunoprecipitated with a testis‐enriched protein GGN1. Immunofluorescent staining revealed that both GGNBP2 and GGN1 had the same subcellular localizations in spermatocyte, spermatid and spermatozoa. Ggnbp2 loss suppressed Ggn expression and nuclear accumulation. Furthermore, deletion of either Ggnbp2 or Ggn in GC‐2spd cells inhibited their differentiation into haploid cells in vitro. Overexpression of Ggnbp2 in Ggnbp2 null but not in Ggn null GC‐2spd cells partially rescued the defect coinciding with a restoration of Ggn expression. Together, these data suggest that GGNBP2, likely mediated by its interaction with GGN1, plays a role in DSB repair during meiotic progression of spermatocytes.

Gametogenetin-binding protein 2 (GGNBP2) is encoded in human chromosome 17q12-q23, a region known as a breast and ovarian cancer susceptibility locus. GGNBP2, also referred to ZFP403, has a single C2H2 zinc finger and a consensus LxxLL nuclear receptor-binding motif. Here, we demonstrate that GGNBP2 expression is reduced in primary human breast tumors and in breast cancer cell lines, including T47D, MCF-7, LCC9, LY2, and MDA-MB-231 compared with normal, immortalized estrogen receptor α (ERα) negative MCF-10A and MCF10F breast epithelial cells. Overexpression of GGNBP2 inhibits the proliferation of T47D and MCF-7 ERα positive breast cancer cells without affecting MCF-10A and MCF10F. Stable GGNBP2 overexpression in T47D cells inhibits 17β-estradiol (E₂)-stimulated proliferation as well as migration, invasion, anchorage-independent growth in vitro, and xenograft tumor growth in mice. We further demonstrate that GGNBP2 protein physically interacts with ERα, inhibits E₂-induced activation of estrogen response element-driven reporter activity, and attenuates ER target gene expression in T47D cells. In summary, our in vitro and in vivo findings suggest that GGNBP2 is a novel breast cancer tumor suppressor functioning as a nuclear receptor corepressor to inhibit ERα activity and tumorigenesis.

E12009 Background: Gametogenetin binding protein 2 (GGNBP2) is encoded in human chromosome 17q12-q23, a region known as a breast and ovarian cancer susceptibility locus. GGNBP2 has a single C2H2 zinc finger and a consensus LxxLL nuclear receptor (NR) binding motif. It has been reported that GGNBP2 being remarkably down-regulated in several human cancer cell lines. The similar NR binding motifs are also found in some other known breast cancer susceptibility genes such asBRCA1, suggesting that GGNBP2 may function as a candidate of breast cancer susceptibility gene. Methods: A cancer expression array containing 50 pair-wise human normal and breast tumor tissues were utilized to examine GGNBP2 expression in breast cancer samples. To address the potential anti-breast tumor activity of GGNBP2 in vitro, we expressed exogenous GGNBP2 in T47D, MCF-7, MCF-10A and MCF-10F cells. Cell proliferation, migration and invasion were assessed by cell growth curve, wound healing assays and colony formation assays, respectively. In vitro protein-protein interaction and luciferase reporter assays were performed to determine whether GGNBP2 interacts with ERα and modulates its transcriptional activity. qPCR and Western blot were carried out to evaluate the effects of GGNBP2 overexpression on ERa target genes, CCND1 and TFF1 expression. The anti-oncogenic activity of GGNBP2 was further verified by xenograft in mice. Results: GGNBP2 expression is reduced in primary human breast tumors. Overexpression of GGNBP2 selectively inhibits the proliferation of ERα positive breast cancer cells. Stable GGNBP2 overexpression in T47D cells inhibits 17β-estradiol (E2)–stimulated proliferation as well as migration, invasion, anchorageindependent growth in vitro and xenograft tumor growth in mice. We further demonstrate that GGNBP2 protein physically interacts with ERα, inhibits E2-induced activation of estrogen response element-driven reporter activity and attenuates ER target gene expression in T47D cells. Conclusions: GGNBP2 is a novel breast cancer tumor suppressor functioning as a nuclear receptor co-repressor to restrain ERα activity and tumorigenesis in human breast cancer.

The Ggnbp2 null mutant embryos died in utero between Embryonic Days 13.5 to 15.5 with dysmorphic placentae, characterized by excessive nonvascular cell nests consisting of proliferative trophoblastic tissue and abundant trophoblast stem cells (TSCs) in the labyrinth. Lethality of Ggnbp2 null embryos was caused by insufficient placental perfusion as a result of remarkable decreases in both fetal and maternal blood vessels in the labyrinth. These defects were accompanied by a significant elevation of c-Met expression and phosphorylation and its downstream effector Stat3 activation. Knockdown of Ggnbp2 in wild-type TSCs in vitro provoked the proliferation but delayed the differentiation with an upregulation of c-Met expression and an enhanced phosphorylation of c-Met and Stat3. In contrast, overexpression of Ggnbp2 in wild-type TSCs exhibited completely opposite effects compared to knockdown TSCs. These results suggest that loss of GGNBP2 in the placenta aberrantly overactivates c-Met-Stat3 signaling, alters TSC proliferation and differentiation, and ultimately compromises the structure of placental vascular labyrinth. Our studies for the first time demonstrate that GGNBP2 is an essential factor for pregnancy success acting through the maintenance of a balance of TSC proliferation and differentiation during placental development.