Faculty Bibliography

The breast cancer-associated gene-1 (BRCA1) plays many important functions in multiple biological processes/pathways. Mice homozygous for a targeted deletion of full-length BRCA1 (Brca1Delta11/Delta11) display both increased tumorigenesis and premature aging, yet molecular mechanisms underlying these defects remain elusive. Here, we show that Brca1 deficiency leads to increased expression of several insulin-like growth factor (IGF) signaling axis members in multiple experimental systems, including BRCA1-deficient mice, primary mammary tumors, and cultured human cells. Furthermore, we provide evidence that activation of IGF signaling by BRCA1 deficiency can also occur in a p53-independent fashion. Our data indicate that BRCA1 interacts with the IRS-1 promoter and inhibits its activity that is associated with epigenetic modification of histone H3 and histone H4 to a transcriptional repression chromatin configuration. We further show that BRCA1-deficient mammary tumor cells exhibit high levels of IRS-1, and acute suppression of Irs-1 using RNA interference significantly inhibits growth of these cells. Those observations provide a molecular insight in understanding both fundamental and therapeutic BRCA1-associated tumorigenesis and aging.

IGF-I and insulin are structurally related polypeptides that mediate a similar pattern of biological effects via receptors that display considerably homology. Administration of recombinant human IGF-I (rhIGF-I) has been proven to improve glucose control and liver and muscle insulin sensitivity in patients with type 2 diabetes mellitus (DM). The effect of rhIGF-I treatment was evaluated in a mouse model of type 2 DM (MKR mouse), which expresses a dominant-negative form of the human IGF-I receptor under the control of the muscle creatine kinase promoter specifically in skeletal muscle. MKR mice have impaired IGF-I and insulin signaling in skeletal muscle, leading to severe insulin resistance in muscle, liver, and fat, developing type 2 DM at 5 wk of age. Six-week-old MKR mice were treated with either saline or rhIGF-I for 3 wk. Blood glucose levels were decreased in response to rhIGF-I treatment in MKR mice. rhIGF-I treatment also increased body weight in MKR with concomitant changes in body composition such as a decrease in fat mass and an increase in lean body mass. Insulin, fatty acid, and triglyceride levels were not affected by rhIGF-I, nor were insulin or glucose tolerance in MKR mice. Hyperinsulinemic-euglycemic clamp analysis demonstrated no improvement in overall insulin sensitivity. Pyruvate and glutamine tolerance tests proved that there was a decrease in the rate of glucose appearance in MKR mice treated with rhIGF-I, suggesting a reduction in the gluconeogenic capacity of liver, kidney, and small intestine. Taken together these results demonstrate that the improvement of the hyperglycemia was achieved by inhibition of gluconeogenesis rather than an improvement in insulin sensitivity. Also, these results suggest that a functional IGF-I receptor in skeletal muscle is required for IGF-I to improve insulin sensitivity in this mouse model of type 2 DM.

The role of circulating IGF-I in skeletal acquisition and the anabolic response to PTH is not well understood. We generated IGF-I-deficient mice by gene deletions of IGF ternary complex components including: (1) liver-specific deletion of the IGF-I gene (LID), (2) global deletion of the acid-labile (ALS) gene (ALSKO), and (3) both liver IGF-I and ALS inactivated genes (LA). Twelve-week-old male control (CTL), LID, ALSKO, and LA mice were treated with vehicle (VEH) or human PTH(1-34) for 4 weeks. VEH-treated IGF-I-deficient mice (i.e. LID, ALSKO and LA mice) exhibited reduced cortical cross-sectional area (P = 0.001) compared with CTL mice; in contrast, femoral trabecular bone volume fractions (BV/TV) of the IGF-I-deficient mice were consistently greater than CTL (P < 0.01). ALSKO mice exhibited markedly reduced osteoblast number and surface (P < 0.05), as well as mineral apposition rate compared with other IGF-I-deficient and CTL mice. Adherent bone marrow stromal cells, cultured in beta-glycerol phosphate and ascorbic acid, showed no strain differences in secreted IGF-I. In response to PTH, there were both compartment- and strain-specific effects. Cortical bone area was increased by PTH in CTL and ALSKO mice, but not in LID or LA mice. In the trabecular compartment, PTH increased femoral and vertebral BV/TV in LID, but not in ALSKO or LA mice. In conclusion, we demonstrated that the presentation of IGF-I as a circulating complex is essential for skeletal remodeling and the anabolic response to PTH. We postulate that the ternary complex itself, rather than IGF-I alone, influences bone acquisition in a compartment-specific manner (i.e. cortical vs trabecular bone).

The insulin-like growth factor 1 receptor (IGF-1R) is a multifunctional receptor that mediates signals for cell proliferation, differentiation, and survival. Genetic experiments showed that IGF-1R inactivation in skin results in a disrupted epidermis. However, because IGF-1R-null mice die at birth, it is difficult to study the effects of IGF-1R on skin. By using a combined approach of conditional gene ablation and a three-dimensional organotypic model, we demonstrate that IGF-1R-deficient skin cocultures show abnormal maturation and differentiation patterns. Furthermore, IGF-1R-null keratinocytes exhibit accelerated differentiation and decreased proliferation. Investigating the signaling pathway downstream of IGF-1R reveals that insulin receptor substrate 2 (IRS-2) overexpression compensates for the lack of IGF-1R, whereas IRS-1 overexpression does not. We also demonstrate that phosphatidylinositol 3-kinase and extracellular signal-regulated kinase 1 and 2 are involved in the regulation of skin keratinocyte differentiation and take some part in mediating the inhibitory signal of IGF-1R on differentiation. In addition, we show that mammalian target of rapamycin plays a specific role in mediating IGF-1R impedance of action on keratinocyte differentiation. In conclusion, these results reveal that IGF-1R plays an inhibitory role in the regulation of skin development and differentiation.

Insulin resistance is a cardinal feature of normal pregnancy and excess growth hormone (GH) states, but its underlying mechanism remains enigmatic. We previously found a significant increase in the p85 regulatory subunit of phosphatidylinositol kinase (PI 3-kinase) and striking decrease in IRS-1-associated PI 3-kinase activity in the skeletal muscle of transgenic animals overexpressing human placental growth hormone. Herein, using transgenic mice bearing deletions in p85alpha, p85beta, or insulin-like growth factor-1, we provide novel evidence suggesting that overexpression of p85alpha is a primary mechanism for skeletal muscle insulin resistance in response to GH. We found that the excess in total p85 was entirely accounted for by an increase in the free p85alpha-specific isoform. In mice with a liver-specific deletion in insulin-like growth factor-1, excess GH caused insulin resistance and an increase in skeletal muscle p85alpha, which was completely reversible using a GH-releasing hormone antagonist. To understand the role of p85alpha in GH-induced insulin resistance, we used mice bearing deletions of the genes coding for p85alpha or p85beta, respectively (p85alpha (+/-) and p85beta(-/-)). Wild type and p85beta(-/-) mice developed in vivo insulin resistance and demonstrated overexpression of p85alpha and reduced insulin-stimulated PI 3-kinase activity in skeletal muscle in response to GH. In contrast, p85alpha(+/-)mice retained global insulin sensitivity and PI 3-kinase activity associated with reduced p85alpha expression. These findings demonstrated the importance of increased p85alpha in mediating skeletal muscle insulin resistance in response to GH and suggested a potential role for reducing p85alpha as a therapeutic strategy for enhancing insulin sensitivity in skeletal muscle.

Leptin has metabolic effects on peripheral tissues including muscle, liver, and pancreas, and it has been successfully used to treat lipodystrophic diabetes, a leptin-deficient state. To study whether leptin therapy can be used for treatment of more common cases of type 2 diabetes, we used a mouse model of type 2 diabetes (MKR mice) that show normal leptin levels and are diabetic due to a primary defect in both IGF-I and insulin receptors signaling in skeletal muscle. Here we show that leptin administration to the MKR mice resulted in improvement of diabetes, an effect that was independent of the reduced food intake. The main effect of leptin therapy was enhanced hepatic insulin responsiveness possibly through decreasing gluconeogenesis. In addition, the reduction of lipid stores in liver and muscle induced by enhancing fatty acid oxidation and inhibiting lipogenesis led to an improvement of the lipotoxic condition. Our data suggest that leptin could be a potent antidiabetic drug in cases of type 2 diabetes that are not leptin resistant.

Insulin and insulin-like growth factors (IGFs) belong to the most biologically characterized family of peptides involved in metabolism, growth and development. The cellular responses to the IGFs are mediated primarily by the IGF-I receptor. The IGF-I receptor is a member of the family of tyrosine kinase growth factor receptors, and is highly homologous (70%) to the insulin receptor, especially in the tyrosine kinase domain (84%) ADDIN. Upon ligand binding to the extracellular region, the intrinsic tyrosine kinase domain of the receptor is activated. In the past it was believed that insulin activates primarily metabolic processes while IGFs promote cell growth and differentiation. However, in the last two decades many animal models of IGFI deficiency and excess revealed the importance of IGF-I in carbohydrate and lipid metabolism and now it is clear that these peptide hormones together with growth hormone (GH) work in a coordinate and interdependent manner. In the circulation, IGFs are bound in a binary complex with a family of high affinity IGF-binding proteins (IGFBPs) ADDIN. However, most of the circulating IGF-I associates with a high molecular weight complex approximately 150 KDa consisting of IGFBP-3 and the acid labile subunit (ALS) ADDIN. Once the ternary complex dissociates, the binary complexes of IGFBP-IGFs are removed from the circulation and by crossing the endothelium to reach the target tissues and to interact with cell surface receptors. In the present review we will summarize the role of GH and IGF in somatic growth and focus on the metabolic effects of IGF-I deficiency as assessed in various mouse models.

The tumor suppressor phosphatase and tensin homologue (PTEN) is involved in cell proliferation, adhesion, and apoptosis. PTEN overexpression in mammary epithelium leads to reduced cell number and impaired differentiation and secretion. In contrast, overexpression of the proto-oncogene Wnt-1 in mammary epithelium leads to mammary hyperplasia and subsequently focal mammary tumors. To explore the possibility that PTEN intersects with Wnt-induced tumorigenesis, mice that ectopically express PTEN and Wnt-1 in mammary epithelium were generated. PTEN overexpression resulted in an 11% reduction of Wnt-1-induced tumors within a 12-month period and the onset of tumors was delayed from an average of 5.9 to 7.7 months. The rate of tumor growth, measured from 0.5 cm diameter until the tumors reached 1.0 cm diameter, was increased from 8.4 days in Wnt-1 mice to 17.7 days in Wnt-1 mice overexpressing PTEN. Here we show for the first time in vivo that overexpression of PTEN in the Wnt-1 transgenic mice resulted in a marked decrease in the insulin-like growth factor (IGF)-I receptor levels leading to a reduced IGF-I-mediated mitogenesis. Moreover, the percentage of BrdUrd-positive epithelial nuclei was decreased by 48%. beta-Catenin immunoreactivity was significantly decreased and the percentage of signal transducer and activator of transcription 5a (stat5a)-positive mammary epithelial cells was increased by 2-fold in Wnt-1 mice overexpressing PTEN. The present study shows that PTEN can partially inhibit the Wnt-1-induced mammary tumorigenesis in early neoplastic stages by blocking the AKT pathway and by reducing the IGF-I receptor levels in mammary gland. This study identifies the PTEN as a therapeutic target for the treatment of mammary cancer and presumably other types of cancer.

Over the past two decades it has become widely appreciated that a relationship exists between the insulin-like growth factors (IGFs) and cancer. Many cancers have been shown to overexpress the IGF-I receptor and produce the ligands (IGF-I or IGF-II) and some combinations of the six IGF-binding proteins. With the recent demonstration by epidemiological studies that an elevated serum IGF-I level is associated with an increased relative risk of developing a number of epithelial cancers, interest has been sparked in this area of research with the possibility of targeting the IGF-I receptor in cancer treatment protocols. This review highlights many of the most relevant studies in this exciting area of research, focusing in particular on lessons learned from animal models of cancer.

GH and IGF-I are potent regulators of muscle growth and function. Although IGF-I is known to mediate many of the effects of GH, it is not yet clear whether all effects of GH are completely dependent on the IGF-I system. To evaluate the biological effects of the GH/IGF-I axis on muscle growth, we administrated recombinant human GH to mice, which lack IGF-I function specifically in skeletal muscle, due to the overexpression of a dominant-negative IGF-I receptor in this tissue (MKR mice). GH treatment significantly increased the levels of hepatic IGF-I mRNA and serum IGF-I levels in both wild-type (WT) and MKR mice. These GH-induced effects were paralleled by increases in body weight and in the weights of most GH-responsive organs in both groups of mice. Interestingly, unlike WT mice, GH treatment had no effect on skeletal muscle weight in MKR mice. GH treatment failed to reverse the impaired muscle function in MKR mice. Furthermore, MKR mice exhibited no effects of GH on the cross-sectional area of myofibers and the proliferation of satellite cells. Taken together, these data suggest that the in vivo effects of GH on muscle mass and strength are primarily mediated by activation of the IGF-I receptor.