Faculty Bibliography

Laron syndrome (LS), or primary growth hormone (GH) insensitivity, is the best-characterized entity among the congenital insulin-like growth factor 1 (IGF1) deficiencies. Life-long exposure to minute endogenous IGF1 levels is linked to low stature as well as a number of endocrine and metabolic abnormalities. While elevated IGF1 is correlated with increased cancer incidence, epidemiological studies revealed that patients with LS do not develop tumors. The mechanisms associated with cancer protection in LS are yet to be discovered. Recent genomic analyses identified a series of metabolic genes that are overrepresented in patients with LS. Given the augmented expression of these genes in a low IGF1 milieu, we hypothesized that they may constitute targets for IGF1 action. Thioredoxin-interacting protein (TXNIP) plays a critical role in cellular redox control by thioredoxin. TXNIP serves as a glucose and oxidative stress sensor, being commonly silenced by genetic or epigenetic events in cancer cells. Consistent with its enhanced expression in LS, we provide evidence that TXNIP gene expression is negatively regulated by IGF1. These results were corroborated in animal studies. In addition, we show that oxidative and glucose stresses led to marked increases in TXNIP expression. Supplementation of IGF1 attenuated TXNIP levels, suggesting that IGF1 exerts its antiapoptotic effect via inhibition of TXNIP Augmented TXNIP expression in LS may account for cancer protection in this condition. Finally, TXNIP levels could be potentially useful in the clinic as a predictive or diagnostic biomarker for IGF1R-targeted therapies.

Hepatic osteodystrophy is multifactorial in its pathogenesis. Numerous studies have shown that impairments of the hepatic growth hormone/insulin-like growth factor-1 axis (GH/IGF-1) are common in patients with non-alcoholic fatty liver disease, chronic viral hepatitis, liver cirrhosis, and chronic cholestatic liver disease. Moreover these conditions are also associated with low bone mineral density (BMD) and greater fracture risk, particularly in cortical bone sites. Hence, we addressed whether disruptions in the GH/IGF-1 axis were causally related to the low bone mass in states of chronic liver disease using a mouse model of liver-specific GH-receptor (GHR) gene deletion (Li-GHRKO). These mice exhibit chronic hepatic steatosis, local inflammation, and reduced BMD. We then employed a crossing strategy to restore liver production of IGF-1 via hepatic IGF-1 transgene (HIT). The resultant Li-GHRKO-HIT mouse model allowed us to dissect the roles of liver-derived IGF-1 in the pathogenesis of osteodystrophy during liver disease. We found that hepatic IGF-1 restored cortical bone acquisition, microarchitecture, and mechanical properties during growth in Li-GHRKO-HIT mice, which was maintained during aging. However, trabecular bone volume was not restored in the Li-GHRKO-HIT mice. We found increased bone resorption indices in vivo as well as increased basal reactive oxygen species and increased mitochondrial stress in osteoblast cultures from Li-GHRKO and the Li-GHRKO-HIT as compared to control mice. Changes in systemic markers such as inflammatory cytokines, osteoprotegrin, osteopontin, parathyroid hormone, osteocalcin, or carboxy-terminal collagen crosslinks, could not fully account for the diminished trabecular bone in the Li-GHRKO-HIT mice. Thus, the reduced serum IGF-1 associated with hepatic osteodystrophy is a main determinant of low cortical but not trabecular bone mass.

Aging is associated with dysregulation of glucose and lipid metabolism. Various factors that contribute to the dysregulation include both modifiable (e.g. obesity, insulin resistance) and non-modifiable risk factors (age-associated physiologic changes). Although there is no linear relationship between aging and prevalence of non-alcoholic fatty liver disease, current data strongly suggests that advanced age leads to more severe histological changes and poorer clinical outcomes. Hepatic lipid accumulation could lead to significant hepatic and systemic consequences including steatohepatitis, cirrhosis, impairment of systemic glucose metabolism and metabolic syndrome, thereby contributing to age-related diseases. Insulin, leptin and adiponectin are key regulators of the various physiologic processes that regulate hepatic lipid metabolism. Recent advances have expanded our understanding in this field, highlighting the role of novel mediators such as FGF 21, and mitochondria derived peptides. In this review, we will summarize the mediators of hepatic lipid metabolism and how they are altered in aging.

Eph receptors belong to a subfamily of receptor tyrosine kinases that are activated by membrane-spanning ligands called ephrins. Previously, we demonstrated that the ephrinB1-EphB2 interaction regulates odontogenic/osteogenic differentiation from dental pulp cells (DPCs) in vitro. The goal of this study was to identify the molecular mechanisms regulated by the EphB2/ephrinB1 system that govern tertiary dentin formation in vitro and in vivo. During tooth development, ephrinB1, and EphB2 were expressed in preodontoblast and odontoblasts at postnatal day 4. EphrinB1 was continuously expressed in odontoblasts and odontoblastic processes until the completion of tooth eruption. In addition, ephrinB1 was expressed in odontoblastic processes 2 wk following tooth injury without pulp exposure, whereas EphB2 was expressed in the center of pulp niches but not odontoblasts. In a model of tooth injury with pulp exposure, ephrinB1 was strongly expressed in odontoblasts 4 wk postinjury. In vitro studies with human and mouse DPCs treated with calcium hydroxide (CH) or mineral trioxide aggregate (MTA) showed an increased expression of insulin-like growth factor 1 (IGF-1). Experiments using several inhibitors of IGF-1 receptor signaling revealed that inhibiting the Ras/Raf-1/MAPK pathway inhibited EphB2 expression, and inhibiting the PI3K/Akt/mTOR pathway specifically inhibited ephrinB1 gene expression. Tooth injury in mice with odontoblast-specific IGF-1 receptor ablation exhibited a reduced tertiary dentin volume, mineral density, and ephrinB1 expression 4 wk following injury. We conclude that the IGF-1/ephrinB1 axis plays significant roles in the early stages of tooth injury. Further research is needed to fully understand the potential of targeting ephrinB1 as a regenerative pulp therapy.

Growth hormone (GH) and insulin like growth factor-1 (IGF-1) are anabolic hormones that facilitate somatic and skeletal growth, and regulate metabolism via endocrine and autocrine/paracrine mechanisms. We hypothesized that excess tissue production of GH will protect skeletal growth and integrity in states of reductions in serum IGF-1 levels. To test our hypothesis we used the bovine GH (bGH) transgenic mice as a model of GH hypersecretion and ablated the liver-derived acid labile subunit (ALS), which stabilizes IGF-1 complexes with IGF-binding protein-3 (IGFBP-3) and -5 in circulation. We used a genetic approach to create bGH/ALSKO mice, and siRNA gene silencing approach to reduce als or igf-1 gene expression. We found that in both models, decreased IGF-1 levels in serum associated with decreased body and skeletal size of the bGH mice. Excess GH produced more robust bones, but compromised mechanical properties in male mice. Excess GH production in tissues did not protect from trabecular bone loss in response to reductions in serum IGF-1 (in bGH/ALSKO or bGH mice treated with siRNAs). Reduced serum IGF-1 levels in the bGH mice did not alleviate the hyperinsulinemia, and did not resolve liver or kidney pathologies that resulted from GH hypersecretion. We conclude that reduced serum IGF-1 levels decrease somatic and skeletal growth even in states of excess GH.

Mutation in the insulin-like growth factor-1 receptor (IGF1R) gene is a rare cause for intrauterine and postnatal growth disorders. Patients identified with IGF1R mutations present with either normal or impaired glucose tolerance. None of the cases described so far showed hypoglycemia. We aimed to identify the genetic basis for small for gestational age, short stature and hypoglycemia over three generations in one family. The proband, a 9-year-old male, presented in infancy with recurrent hypoglycemic episodes, symmetric intrauterine growth retardation and postnatal growth retardation. Blood DNA samples from the patient, his parents, a maternal sister and maternal grandmother underwent Sanger sequencing of the IGF1R gene. Primary skin fibroblast cultures of the patient, his mother and age- and sex-matched control donors were used for gene expression and receptor functional analyses. We found a novel heterozygous mutation (c.94 + 1g > a, D1105E) affecting the splicing site of the IGF1R mRNA in the patient, his mother and his grandmother. Primary fibroblast cultures derived from the patient and his mother showed reduced proliferation and impaired activation of the IGF1R, evident by reduced IGF1R and AKT phosphorylation upon ligand binding. In conclusion, the newly identified heterozygous missense mutation in exon 1 of IGF1R (D1105E) results in impaired IGF1R function and is associated with small for gestational age, microcephaly and abnormal glucose metabolism. Further studies are required to understand the mechanisms by which this mutation leads to hypoglycemia.

Type 1 diabetes mellitus (T1DM) is one of the most common chronic diseases diagnosed in childhood. Childhood and adolescent years are also the most important period for growth in height and acquisition of skeletal bone mineral density (BMD). The growth hormone (GH)/insulin like growth factor -1 (IGF-1) axis which regulates growth, is affected by T1DM, with studies showing increased GH and decreased IGF-1 levels in children with T1DM. There is conflicting data as to whether adolescents with TIDM are able to achieve their genetically-determined adult height. Furthermore, data support that adolescents with T1DM have decreased peak BMD, although the pathophysiology of which has not been completely defined. Various mechanisms have been proposed for the decrease in BMD including low osteocalcin levels, reflecting decreased bone formation; increased sclerostin, an inhibitor of bone anabolic pathways; and increased leptin, an adipocytokine which affects bone metabolism via central and peripheral mechanisms. Other factors implicated in the increased bone resorption in T1DM include upregulation of the osteoprotegerin/ receptor-activator of the nuclear factor-kappaB ligand pathway, elevated parathyroid hormone levels, and activation of other cytokines involved in chronic systemic inflammation. In this review, we summarize the clinical studies that address the alterations in the GH/IGF-I axis, linear growth velocity, and BMD in children and adolescents with T1DM; and we review the possible molecular mechanisms that may contribute to an attenuation of linear growth and to the reduction in the acquisition of peak bone mass in the child and adolescent with T1DM.

Eph receptors belong to a subfamily of receptor tyrosine kinases activated by membrane bound ligands called ephrins. Recent studies have shown that osteoblasts express the EphB4 receptor and it's ligand ephrinB2 and B1, while osteoclasts express a few members of the ephrinBs, suggesting that these proteins are involved in bone modeling during growth. Past studies have shown that parathyroid hormone (PTH) stimulates bone modeling via enhanced expression of the insulin-like growth factor-1 (IGF-I) and the ephrinB2 and EphB4 in osteoblasts. To define the interactions between PTH/IGF-1/Eph signaling pathways in bone, male mice at 4 weeks of age were injected 80 mcg/kg/day for 5 days. To our surprise we found 3 fold increase in the expression of Ephrin B1 in the femoral cortical shells from male mice, while the expression of ephrinB2 or EphB4 did not differ significantly between PTH treated and untreated groups. Thus, we used the osteoblast- and osteocyte-specific ephrinB1 knockout (KO) mice (using the osteocalcin-cre and the dentin matrix protein (DMP)-1-cre, respectively) to investigate their bone response to intermittent PTH treatment. We used micro Computer Tomography (CT) to characterize the basal morphology of femurs dissected from male mice. We found decreases in tissue mineral density in cortical bone of the mid-shaft femur in both Osteocalcin-ephrinB1 (1.45 +/- 0.05 g/cm³ , p=0.01) and DMP-1-ephrinB1 KO mice (1.465 +/- 0.05 mg/cm³ , p=0.02) as compared to controls (1.64 +/- 0.02 mg/cm³ ) . Similarly, we found decreased bone mineral density in the trabecular bone assessed at the distal femur (Osteocalcin-eprinB1 0.147+/-0.005 p=0.004, DMP-1-ephrinB1 0.175 +/- 0.02 p=0.06, and controls 0.248 +/- 0.02 mg/cm³ ) . However, cortical and trabecular bone morphology of males did not differ between the groups. Female DMP-ephrinB1 KO mice showed decreased total cross-sectional area (1.14 +/- 0.09 vs 1.27+/-0.06 mm² , p = 0.02), polar moment of inertia (0.12 +/- 0.03 vs 0.14+/- 0.01 mm⁴ , p= 0.03), and marrow area (0.76 +/- 0.03 vs 0.86 +/- 0.06 mm² , p = 0.03) as compared to control mice with no significant difference in tissue mineral density. Female DMPephrinB1 KO mice did not show any trabecular bone phenotype. Bone anabolic response to intermittent PTH treatment in Osteocalcin-eprinB1 and DMP-1-ephrinB1 KO mice is under investigation

Mitochondrial dysfunction has been recognized as a prominent feature of the diminishing cell function in aging bone. Studies of the long-lived growth hormone receptor knockout (GHRKO) mice, which show compromised skeletal growth, have suggested increased mitochondrial biogenesis and function in tissues, such as kidney, heart, and skin fibroblasts. The roles of GHR in maintaining mitochondrial function in osteocytes, the predominant population of bone cells, were not studied. Our goal was to understand the mechanisms by which GHRKO affects mitochondrial volume and function in the adult and aged bones. Using primary osteocyte cultures from 8 weeks old control and GHRKO mice we found that unlike in kidney and heart, in bone tissue of GHRKO mice despite ~40% decreases in osteocyte volume, mitochondrial volume remained the same at the level of ~20% of cell volume. Mitochondrial membrane potential (MMP) is a critical functional measure of mitochondria. Using tetramethylrhodamine ethyl ester, a potentiometric fluorescent probe, we found ~10% decreases in MMP in osteocytes from GHRKO when compared to osteocytes from control mice, suggesting decreased mitochondrial function with ablation of GHR. To further investigate the changes in mitochondrial function we measured the redox state of the mitochondrial NAD+/NADH pair. We found that GHRKO osteocytes show reduced NADH redox index compared to controls, indicating that the overall levels of NADH in the cell reduced. This suggests reduced TCA cycle activity. The impaired mitochondrial function was further confirmed by assay of cellular respiration. We found that GHRKO osteocytes show reduced oxygen consumption rate as compared to controls, and reduced mitochondrial reserve capacity. This data together with decreased MMP and NADH levels, establish that mitochondrial function is compromised in the absence of GHR in osteocytes