Faculty Bibliography

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.

Osteocytes can remove and remodel small amounts of their surrounding bone matrix through osteocytic osteolysis, which results in increased volume occupied by lacunar and canalicular space (LCS). It is well established that cortical bone stiffness and strength are strongly and inversely correlated with vascular porosity, but whether changes in LCS volume caused by osteocytic osteolysis are large enough to affect bone mechanical properties is not known. In the current studies we tested the hypotheses that i) lactation and post-lactation recovery in mice alter the elastic modulus of bone tissue, and ii) such local changes in mechanical properties are related predominantly to alterations in lacunar and canalicular volume rather than bone matrix composition. Mechanical testing was performed using microindentation to measure modulus in regions containing solely osteocytes and no vascular porosity. Lactation caused a significant ( approximately 13%) reduction in bone tissue-level elastic modulus (p < 0.001). After 1 week post-weaning (recovery), bone modulus levels returned to control levels and did not change further after four weeks of recovery. LCS porosity tracked inversely with changes in cortical bone modulus. Lacunar and canalicular void space increased 7% and 15% with lactation, respectively (p < 0.05), then returned to control levels at 1 week after weaning. Neither bone mineralization (assessed by high resolution Backscattered Scanning Electron Microscopy) nor mineral/matrix ratio or crystallinity (assessed by Raman microspectroscopy) changed with lactation. Thus, changes in bone mechanical properties induced by lactation and recovery appear to depend predominantly on changes in osteocyte LCS dimensions. Moreover, this study demonstrates that tissue-level cortical bone mechanical properties are rapidly and reversibly modulated by osteocytes in response to physiological challenge. These data point to a hitherto unappreciated role for osteocytes in modulating and maintaining local bone mechanical properties

Background: Hepatic osteodystrophy (HO) refers to metabolic bone disease (MBD) seen in patients with chronic liver disease. The duration and severity of the liver disease relates directly to MBD, such that patients with less sever liver disease manifest osteoporosis mainly loosing trabecular bone, and as liver disease progresses patients develop osteomalacia (softening of the bone tissue). The pathogenesis of HO is multifactorial and includes deficiency in IGF-1, subnormal vitamin D levels, and reduced osteoprotegrin (OPG) levels. The main goal of the present study was to define the contribution of growth hormone (GH) receptor in liver to HO. Methods: Liver specific GH receptor inactivation (Li-GHRKO) results in nonalcoholic fatty liver disease (NFLD) and dyslipidemia, reductions in serum insulin-like growth factor-1 (IGF-1), and severe osteopenia. To rule out the contribution of serum IGF-1 to osteopenia in the Li-GHRKO, we restored liver IGF-1 gene expression via hepatic IGF-1 transgene (HIT). Results: Li-GHRKO-HIT mice show normal levels of IGF-1 in serum, but still display NFLD and osteopenia (Figure 1). Using osteocyte-like cell line, primary osteocytes and primary osteoblast cultures we show that the dyslipidemia in the Li-GHRKO mice contributed to reduced GHR signaling in bone and increased osteocyte apoptosis, likely leading to increased bone resorption. Conclusions: We conclude that liver GHR leads to HO via its effects on overall lipid metabolism

Background: In humans low levels of growth hormone (GH) and its mediator, insulin-like growth factor-1 (IGF-1), associate with hepatic lipid accumulation. In mice, congenital liver-specific ablation of the GH receptor (GHR) results in reductions in circulating IGF-1 and hepatic steatosis, associated with systemic insulin-resistance. Due to the intricate relationship between GH and IGF-1, the relative contribution of each hormone to the development of hepatic steatosis is unclear. Our goal was to dissect the mechanisms by which hepatic GH resistance leads to steatosis and overall insulin resistance, independent of IGF-1. Methods: We have generated a combined mouse model with liver-specific ablation of GHR in which we restored liver IGF-1 expression via hepatic IGF-1 transgene. Results: We found that liver-GHR ablation leads to increases in lipid uptake, de novo lipogenesis, hyperinsulinemia and hyperglycemia accompanied with severe insulin resistance, and increased body adiposity and serum lipids. Restoration of IGF-1 improved overall insulin sensitivity, lipid profile in serum, reduced body adiposity, but was insufficient to protect against steatosis-induced hepatic inflammation or oxidative stress. Conclusions: We conclude that the impaired metabolism in states of GH resistance results from direct actions of GH on lipid uptake and de novo lipogenesis, while its actions on extrahepatic tissues are mediated by IGF-1

Background/Purpose: We aimed to understand the mechanism by which membrane-type 1 matrix metalloproteinase (MT1-MMP, MMP-14) controls bone and cartilage homeostasis. MT1-MMP, a cell-membrane-bound proteinase with an extracellular catalytic site and a 20-amino acid cytoplasmic tail, plays a key role in postnatal bone formation. The genetic deficiency of MT1-MMP in the mouse causes dwarfism, osteopenia and severe arthritis. Deletion of MT1-MMP in bone marrow-derived mesenchymal progenitor cells (BM-MSC) recapitulates this phenotype, showing that MT1-MMP controls osteogenic differentiation in MSC. The phenotype of MT1-MMP-/- mice has been proposed to result from lack of MT1-MMP proteolytic activity. However, mounting evidence shows a variety of proteolysis-independent signaling functions of MT1-MMP. The unique tyrosine (Y573) in the MT1-MMP cytoplasmic tail is fundamental for the control of intracellular signaling. Methods: We generated a mouse with the Y573D mutation in MT1-MMP (MT1-MMP Y573D) and characterized its skeletal phenotype by histological and microCT analyses. Isolated BM-MSC were induced to differentiate into osteoblasts, chondrocytes and adipocytes, using qRT-PCR to analyze gene expression. Mouse C3H10T1/2 MSC were transfected with MT1-MMP cDNA and analyzed for Wnt signaling by luciferase reporter assays. Results: MT1-MMP Y573D mice had increased trabecular bone relative to wt littermates, marked thinning of articular cartilage with disorganized tissue architecture, clustering and cloning of chondrocytes, and pronounced decrease in bone marrow-associated and total body fat. We induced BM-MSC from wt and MT1-MMP Y573D littermates to differentiate into osteoblast and chondrocytes, and myeloid precursors into osteoclasts. The Y573D mutation dramatically increased MSC expression of osteoblast markers and strongly downregulated chondrocyte and osteoclast markers. These findings indicated that Wnt signaling is upregulated in MT1-MMP Y573D-expressing MSC. Therefore, we analyzed Wnt signaling. We transiently transfected C3H10T1/2 MSC cells in osteoblast medium with the cDNAs for wt MT1-MMP and MT1-MMP Y573D. As controls the cells were transfected with the empty vector (pcDNA) or with MT1-MMP E240A, a mutant devoid of proteolytic activity. MT1-MMP Y573D dramatically upregulated Wnt signaling relative to wt MT1-MMP and MT1-MMP E240A. Conclusion: MT1-MMP controls Wnt signaling by a mechanism independent of extracellular proteolysis and mediated by its cytoplasmic tail. MT1-MMP is a bifunctional protein, with an extracellular proteolytic activity that promotes bone formation through ECM remodeling and a cytoplasmic tail that controls osteogenesis by interacting with a key pro-osteogenic signaling pathway

Prostate cancer (PCa) is unique in its tendency to produce osteoblastic (OB) bone metastases. There are no existing therapies that specifically target the OB phase that affects 90% of men with bone metastatic disease. Prostatic acid phosphatase (PAP) is secreted by PCa cells in OB metastases and increases OB growth, differentiation, and bone mineralization. The purpose of this study was to investigate whether PAP effects on OB bone metastases are mediated by autocrine and/or paracrine alterations in the receptor activator of nuclear factor kappa-B (RANK)/RANK ligand (RANKL)/osteoprotegerin (OPG) system. To investigate whether PAP modulated these factors and altered the bone reaction, we knocked down PAP expression in VCaP cells and stably overexpressed PAP in PC3M cells, both derived from human PCa bone metastases. We show that knockdown of PAP in VCaP cells decreased OPG while increasing RANK/RANKL expression. Forced overexpression of PAP in PC3M cells had the inverse effect, increasing OPG while decreasing RANK/RANKL expression. Coculture of PCa cells with MC3T3 preosteoblasts also revealed a role for secretory PAP in OB-PCa cross talk. Reduced PAP expression in VCaP cells decreased MC3T3 proliferation and differentiation and reduced their OPG expression. PAP overexpression in PC3M cells altered the bone phenotype creating OB rather than osteolytic lesions in vivo using an intratibial model. These findings demonstrate that PAP secreted by PCa cells in OB bone metastases increases OPG and plays a critical role in the vicious cross talk between cancer and bone cells. These data suggest that inhibition of secretory PAP may be an effective strategy for PCa OB bone lesions.

CONTEXT: Pregnancy-associated plasma protein-A2 (PAPP-A2) is a metalloproteinase that specifically cleaves IGFBP-3 and IGFBP-5. Mutations in the PAPP-A2 gene have recently been shown to cause postnatal growth failure in humans, with specific skeletal features, due to the resulting decrease in IGF-1 bioavailability. However, a pharmacological treatment of this entity is yet to be established. CASE DESCRIPTION: A 10.5-year-old girl and a 6-year-old boy, siblings from a Spanish family, with short stature due to a homozygous loss-of-function mutation in the PAPP-A2 gene (p.D643fs25*) and undetectable PAPP-A2 activity, were treated with progressive doses (40, 80, 100, and 120 mug/kg) of recombinant human IGF-1 (rhIGF-1) twice daily for 1 year. There was a clear increase in growth velocity and height in both siblings. Bioactive IGF-1 was increased, and spontaneous GH secretion was diminished after acute administration of rhIGF-1, whereas serum total IGF-1 and IGFBP-3 levels remained elevated. No episodes of hypoglycemia or any other secondary effects were observed during treatment. CONCLUSION: Short-term treatment with rhIGF-1 improves growth in patients with PAPP-A2 deficiency.

In humans low levels of growth hormone (GH) and its mediator, insulin-like growth factor-1 (IGF-1), associate with hepatic lipid accumulation. In mice, congenital liver-specific ablation of the GH receptor (GHR) results in reductions in circulating IGF-1 and hepatic steatosis, associated with systemic insulin-resistance. Due to the intricate relationship between GH and IGF-1, the relative contribution of each hormone to the development of hepatic steatosis is unclear. Our goal was to dissect the mechanisms by which hepatic GH resistance leads to steatosis and overall insulin resistance, independent of IGF-1. We have generated a combined mouse model with liver-specific ablation of GHR in which we restored liver IGF-1 expression via hepatic IGF-1 transgene. We found that liver-GHR ablation leads to increases in lipid uptake, de novo lipogenesis, hyperinsulinemia and hyperglycemia accompanied with severe insulin resistance, and increased body adiposity and serum lipids. Restoration of IGF-1 improved overall insulin sensitivity, lipid profile in serum, reduced body adiposity, but was insufficient to protect against steatosis-induced hepatic inflammation or oxidative stress. We conclude that the impaired metabolism in states of GH resistance results from direct actions of GH on lipid uptake and de novo lipogenesis, while its actions on extrahepatic tissues are mediated by IGF-1.

Mutations in multiple genes of the growth hormone/IGF-I axis have been identified in syndromes marked by growth failure. However, no pathogenic human mutations have been reported in the six high-affinity IGF-binding proteins (IGFBPs) or their regulators, such as the metalloproteinase pregnancy-associated plasma protein A2 (PAPP-A2) that is hypothesized to increase IGF-I bioactivity by specific proteolytic cleavage of IGFBP-3 and -5. Multiple members of two unrelated families presented with progressive growth failure, moderate microcephaly, thin long bones, mildly decreased bone density and elevated circulating total IGF-I, IGFBP-3, and -5, acid labile subunit, and IGF-II concentrations. Two different homozygous mutations in PAPPA2, p.D643fs25* and p.Ala1033Val, were associated with this novel syndrome of growth failure. In vitro analysis of IGFBP cleavage demonstrated that both mutations cause a complete absence of PAPP-A2 proteolytic activity. Size-exclusion chromatography showed a significant increase in IGF-I bound in its ternary complex. Free IGF-I concentrations were decreased. These patients provide important insights into the regulation of longitudinal growth in humans, documenting the critical role of PAPP-A2 in releasing IGF-I from its BPs.

Osteocyte apoptosis is essential to activate bone remodeling in response to fatigue microdamage and estrogen withdrawal, such that apoptosis inhibition in vivo prevents the onset of osteoclastic resorption. Osteocyte apoptosis has also been spatially linked to bone resorption due to disuse, but whether apoptosis plays a similar controlling role is unclear. We therefore 1) evaluated the spatial and temporal effects of disuse from hindlimb unloading (HLU) on osteocyte apoptosis, RANKL expression, bone resorption and loss in mouse femora, and 2) tested whether osteocyte apoptosis was required to activate osteoclastic activity in cortical and trabecular bone by treating animals subjected to HLU with the pan-caspase apoptosis inhibitor, QVD (quinolyl-valyl-O-methylaspartyl-[-2,6-difluorophenoxy]-methylketone). Immunohistochemistry was used to identify apoptotic and RANKL-producing osteocytes in femoral diaphysis and distal trabecular bone, and microCT was used to determine the extent of trabecular bone loss due to HLU. In both cortical and trabecular bone, 5 days of HLU increased osteocyte apoptosis significantly (3- and 4-fold, respectively, p < 0.05 vs Ctrl). At Day 14, the apoptotic osteocyte number in femoral cortices declined to near control levels but remained elevated in trabeculae (3-fold vs Ctrl, p < 0.05). The number osteocyte producing RANKL in both bone compartments was also significantly increased at Day 5 of HLU (>1.5 fold vs Ctrl, p < 0.05) and further increased by Day 14. Increases in osteocyte apoptosis and RANKL production preceded increases in bone resorption at both endocortical and trabecular surfaces. QVD completely inhibited not only the HLU-triggered increases in osteocyte apoptosis but also RANKL production and activation of bone resorption at both sites. Finally, microCT studies revealed that apoptosis inhibition completely prevented the trabecular bone loss caused by HLU. Together these data indicate that osteocyte apoptosis plays a central and controlling role in triggering osteocyte RANKL production and the activation of new resorption leading to bone loss in disuse