Faculty Bibliography

Studies linking insulin-like growth factor-1 (IGF-1) to age-related bone loss in humans have been reported but remain only correlative. In this investigation, we characterized the bone phenotype of aged WT C57BL/6J male mice in comparison to that of C57BL/6J mice with reduced serum IGF-1 levels arising from an igfals gene deletion (ALS knockout (ALSKO)). During the aging process, WT mice showed an increase in fat mass and decrease lean mass while ALSKO mice had stable lean and fat mass values. Skeletal analyses of femora from WT mice revealed an expansion of the marrow area and a significant accumulation of intracortical porosity associated with increased intracortical remodeling. In contrast, ALSKO mice showed only small age-related declines in the amount of cortical bone tissue and minimal intracortical porosity, at 2 years of age. Accordingly, mechanical tests of femora from 2-year-old WT mice revealed reduced stiffness and maximal load when compared to bones from ALSKO mice. We show here that lifelong reductions in serum IGF-1 compromise skeletal size in development leading to slender bones; they are also associated with decreased intracortical bone remodeling and preservation of bone strength during aging.

In this study, we investigated whether loss of GH receptor (GHR) signaling in postnatal skeletal muscle alters muscle mass and regenerative ability in adult mice and whether this was dependent on IGF-1 receptor (IGF-1R) signaling. To do so, we used mouse models with skeletal muscle-specific loss of GHR signaling (mGHRKO), IGF-1R and insulin receptor signaling (MKR), or both GHR and IGF-1R/insulin receptor signaling (mGHRKO/MKR). We did not find a reduction in muscle cross-sectional area, fiber type composition, or response to pathological muscle injury in male mGHRKO and mGHRKO/MKR mice when compared with control and MKR mice, respectively. This could potentially be explained by unchanged skeletal muscle Igf-1 expression in mGHRKO and mGHRKO/MKR mice relative to control and MKR mice, respectively. Furthermore, MKR and mGHRKO/MKR mice, but not mGHRKO mice, demonstrated reduced fiber fusion after cardiotoxin injection, suggesting that IGF-1, and not GH, promotes fiber fusion in adult mice. In summary, our data suggest that GHR signaling in postnatal skeletal muscle does not play a significant role in regulating muscle mass or muscle regeneration. Additionally, in our model, muscle Igf-1 expression is not dependent on GHR signaling in postnatal skeletal muscle.

States of growth hormone (GH) resistance, such those observed in Laron's dwarf patients, are characterized by mutations in the GH receptor (GHR), decreased serum and tissue IGF-1 levels, impaired glucose tolerance, and impaired skeletal acquisition. IGF-1 replacement therapy in such patients increases growth velocity but does not normalize growth. Herein we combined the GH-resistant (GHR knockout, GHRKO) mouse model with mice expressing the hepatic Igf-1 transgene (HIT) to generate the GHRKO-HIT mouse model. In GHRKO-HIT mice, serum IGF-1 levels were restored via transgenic expression of Igf-1, allowing us to study how endocrine IGF-1 affects growth, metabolic homeostasis, and skeletal integrity. We show that in a GH-resistant state, normalization of serum IGF-1 improved body adiposity and restored glucose tolerance but was insufficient to support normal skeletal growth, resulting in an osteopenic skeletal phenotype. The inability of serum IGF-1 to restore skeletal integrity in the total absence of GHR likely resulted from reduced skeletal Igf-1 gene expression, blunted GH-mediated effects on the skeleton that are independent of serum or tissue IGF-1, and from poor delivery of IGF-1 to the tissues. These findings are consistent with clinical data showing that IGF-I replacement therapy in patients with Laron's syndrome does not achieve full skeletal growth. (c) 2013 American Society for Bone and Mineral Research.

The Her2 oncogene is expressed in approximately 25% of human breast cancers and is associated with metastatic progression and poor outcome. Epidemiological studies report that breast cancer incidence and mortality rates are higher in women with type 2 diabetes. Here, we use a mouse model of Her2-mediated breast cancer on a background of hyperinsulinemia to determine how elevated circulating insulin levels affect Her2-mediated primary tumor growth and lung metastasis. Hyperinsulinemic (MKR(+/+)) mice were crossed with doxycycline-inducible Neu-NT (MTB/TAN) mice to produce the MTB/TAN/MKR(+/+) mouse model. Both MTB/TAN and MTB/TAN/MKR(+/+) mice were administered doxycycline in drinking water to induce Neu-NT mammary tumor formation. In tumor tissues removed at 2, 4, and 6 weeks of Neu-NT overexpression, we observed increased tumor mass and higher phosphorylation of the insulin receptor/IGF1 receptor, suggesting that activation of these receptors in conditions of hyperinsulinemia could contribute to the increased growth of mammary tumors. After 12 weeks on doxycycline, although no further increase in tumor weight was observed in MTB/TAN/MKR(+/+) compared with MTB/TAN mice, the number of lung metastases was significantly higher in MTB/TAN/MKR(+/+) mice compared with controls (MTB/TAN/MKR(+/+) 16.41+/-4.18 vs MTB/TAN 5.36+/-2.72). In tumors at the 6-week time point, we observed an increase in vimentin, a cytoskeletal protein and marker of mesenchymal cells, associated with epithelial-to-mesenchymal transition and cancer-associated fibroblasts. We conclude that hyperinsulinemia in MTB/TAN/MKR(+/+) mice resulted in larger primary tumors, with more mesenchymal cells and therefore more aggressive tumors with more numerous pulmonary metastases.

BACKGROUND & AIMS: Dysregulated glucose homeostasis and lipid accumulation characterize non-alcoholic fatty liver disease (NAFLD), but underlying mechanisms are obscure. We report here that Kruppel-like factor 6 (KLF6), a ubiquitous transcription factor that promotes adipocyte differentiation, also provokes the metabolic abnormalities of NAFLD by post-transcriptionally activating PPARalpha-signaling. METHODS: Mice with either hepatocyte-specific depletion of KLF6 ('DeltaHepKlf6') or global KLF6 heterozygosity (Klf6+/-) were fed a high fat diet (HFD) or chow for 8 or 16 weeks. Glucose and insulin tolerance tests were performed to assess insulin sensitivity. Overexpression and knockdown of KLF6 in cultured cells enabled the elucidation of underlying mechanisms. In liver samples from a cohort of 28 NAFLD patients, the expression of KLF6-related target genes was quantified. RESULTS: Mice with global- or hepatocyte-depletion of KLF6 have reduced body fat content and improved glucose and insulin tolerance, and are protected from HFD-induced steatosis. In hepatocytes, KLF6 deficiency reduces PPARalpha-regulated genes (Trb3, Pepck) with diminished PPARalpha protein but no change in Pparalpha mRNA, which is explained by the discovery that KLF6 represses miRNA 10b, which leads to induction of PPARalpha. In NAFLD patients with advanced disease and inflammation, the expression of miRNA 10b is significantly downregulated, while PEPCK mRNA is upregulated; KLF6 mRNA expression also correlates with TRB3 as well as PEPCK gene expression. CONCLUSIONS: KLF6 increases PPARalpha activity, whereas KLF6 loss leads to PPARalpha repression and attenuation of lipid and glucose abnormalities associated with a high fat diet. The findings establish KLF6 as a novel regulator of hepatic glucose and lipid metabolism in fatty liver.

Dyslipidemia has been associated with an increased risk for developing cancer. However, the implicated mechanisms are largely unknown. To explore the role of dyslipidemia in breast cancer growth and metastasis, we used the apolipoprotein E (ApoE) knockout mice (ApoE(-/-)), which exhibit marked dyslipidemia, with elevated circulating cholesterol and triglyceride levels in the setting of normal glucose homeostasis and insulin sensitivity. Non-metastatic Met-1 and metastatic Mvt-1 mammary cancer cells derived from MMTV-PyVmT/FVB-N transgenic mice and c-Myc/vegf tumor explants respectively, were injected into the mammary fat pad of ApoE(-/-) and wild-type (WT) females consuming a high-fat/high-cholesterol diet and tumor growth was evaluated. ApoE(-/-) mice exhibited increased tumor growth and displayed a greater number of spontaneous metastases to the lungs. Furthermore, intravenous injection of Mvt-1 cells resulted in a greater number of pulmonary metastases in the lungs of ApoE(-/-) mice compared with WT controls. To unravel the molecular mechanism involved in enhanced tumor growth in ApoE(-/-) mice, we studied the response of Mvt-1 cells to cholesterol in vitro. We found that cholesterol increased Akt(S473) phosphorylation in Mvt-1 cells as well as cellular proliferation, whereas cholesterol depletion in the cell membrane abrogated Akt(S473) phosphorylation induced by exogenously added cholesterol. Furthermore, in vivo administration of BKM120, a small-molecule inhibitor of phosphatidylinositol 3-kinase (PI3K), alleviated dyslipidemia-induced tumor growth and metastasis in Mvt-1 model with a concomitant decrease in PI3K/Akt signaling. Collectively, we suggest that the hypercholesterolemic milieu in the ApoE(-/-) mice is a favorable setting for mammary tumor growth and metastasis.

The GH/IGF-I axis plays a critical role in postnatal growth and development. In our previous studies we showed that hepatic IGF-1 transgene (HIT) driven by the transthyretin (TTR) promoter was able to restore postnatal growth of the IGF-1 null mice (KO-HIT mouse model), such that their body size was indistinguishable from controls. Interestingly however, despite 2 fold elevations in serum IGF-1 levels in the KO-HIT mice, GH levels remained normal. Thus, we could not rule out direct effects of GH on body size, which are tissue IGF-1-independent. We therefore developed a new mouse model where the hepatic IGF-1 transgene (HIT) driven by the transthyretin (TTR) promoter was expressed in a mouse model of GHRKO (GHRKO-HIT). Consistent with our previous results, expression of the hepatic IGF-1 transgene (HIT) under the TTR promoter led to 2 fold increase in serum IGF-1 levels (648+/-49ng/ml vs 292+/-13ng/ml, respectively). However, in GHRKO-HIT mice serum IGF-1 levels were not elevated and were comparable to those of control mice (320+/-1ng/ml vs 292+/- 13ng/ml, respectively). Likewise, serum IGFBP-3 levels reduced significantly in the GHRKO mice and were not restored by overexpression of the IGF-1 transgene. The acid labile subunit (ALS), which stabilizes IGF-1 in serum, is regulated by GH and was absent in the GHRKO as well as in the GHRKO-HIT mice. These results indicate the IGF1 ternary complex with the IGFBP-3 and the ALS is impaired in the absence of GHR. Interestingly, despite normal levels of serum IGF-1, post-natal growth of the GHRKO-HIT mice was impaired as evident by ~40% reductions in body weight and ~20% reductions in body length in both genders of two different genetic backgrounds (i.e. C57Bl/6 and FVB/N). These striking results show that in the absence of GHR activity, serum IGF-1 is insufficient to restore normal growth and development, implying that GHR is the master regulator of body size

Women with type 2 diabetes mellitus (T2DM) are at a greater risk of developing and dying from breast cancer than women without T2DM. Insulin resistance and hyperinsulinemia underlie the pathogenesis of T2DM. In the MKR mouse model of insulin resistance, we have previously shown increased activation of the phosphatidylinositol 3-kinase (PI3K)/Akt/mTOR pathway in association with accelerated mammary tumor growth. In this study, we demonstrate that inhibiting PI3K with the oral pan-class I PI3K inhibitor, NVP-BKM120 reduced the growth of Met-1 and MCNeuA mammary tumor orthografts in the MKR mouse. NVP-BKM120 treatment decreased phosphorylation of Akt and S6 ribosomal protein (S6rp); no change in Erk1/2 phosphorylation was seen. Hyperglycemia, hypertriglyceridemia and greater hyperinsulinemia developed in the MKR mice treated with NVP-BKM120. We previously reported reduced tumor growth using intraperitoneal rapamycin in the MKR mouse, with the development of hyperglycemia and hypertriglyceridemia. Therefore, we examined whether the oral PI3K/mTOR inhibitor NVP-BEZ235 augmented the tumor suppressing effects of PI3K inhibition. We also investigated the effect of targeted PI3K/mTOR inhibition on PI3K/Akt/mTOR and Erk1/2 signaling, and the potential effects on glycemia. NVP-BEZ235 suppressed the growth of Met-1 and MCNeuA tumor orthografts, and decreased Akt and S6rp phosphorylation, despite increased Erk1/2 phosphorylation in Met-1 orthografts of MKR mice. Less marked hyperglycemia and hyperinsulinemia developed with NVP-BEZ235 than NVP-BKM120. Overall, the results of this study demonstrated that inhibiting PI3K/Akt/mTOR signaling with the oral agents NVP-BKM120 and NVP-BEZ235 decreased mammary tumor growth in the hyperinsulinemic MKR mouse. Inhibiting PI3K alone led to more severe metabolic derangement than inhibiting both PI3K and mTOR. Therefore, PI3K may be an important target for the treatment of breast cancer in women with insulin resistance. Monitoring for hyperglycemia and dyslipidemia should be considered when using these agents in humans, given the metabolic changes detected in this study.

Insulin-like growth factor 1 (IGF-1) is a pleiotropic polypeptide. Its expression is tightly regulated and it plays significant roles during early development, maturation, and adulthood. This article discusses the roles of IGF-1 in determination of body size, skeletal acquisition, muscle growth, carbohydrate metabolism, and longevity, as learned from mouse models.