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OR11-2 Growth Hormone regulates the circulating levels of the mitochondria-derived peptide humanin
Oral Presentations / Growth Hormone & IGF Research 22 (2012) S1–S31
isolated from serum or expression of unglycosylated protein in E. coli. To rectify this and explore their properties, we produced O-glycosylated human IGF-II isoforms. These isoforms bound the IGF-GR and insulin receptor isoform A (IR-A) with affinities similar to mature IGF-II. They also formed binary complexes with IGFBP-2, IGFBP-3 and IGFBP-5. However, there were marked differences in their ability to induce phosphorylation of the IGF-GR, IR-A and IR-B receptors in vitro, with the IGF-II isoforms inducing different levels of receptor activation and saturation of activation compared to mature IGF-II. Despite these differences, in vitro assays using cells individually expressing IGF-GR, IR-A or IR-B demonstrated that very low levels of all ligands were sufficient to induce a proliferative response. Finally, compared to mature IGF-II, in vitro IGFBP-3/ALS and IGFBP-5/ALS ternary complex formation with the IGF-II isoforms was severely retarded, an effect reliant on the presence of the O-glycosylated sugars within the E-domain. The big-IGF-II (1–87) species also displayed a severe loss of binding to IGF-IIR. These findings provide the first insights into the unique individual biological properties associated with each IGF-II isoform. Reference(s) [1] Dransfield, D. et al. Mol Cancer Ther 9:1809–1819 (2010).
OR11-2 Growth Hormone regulates the circulating levels of the mitochondria-derived peptide humanin C. Lee1 , A. Bartke2 , Y. Fang2 , J. Wan1 , P. Cohen1 . 1 University of Southern California, Los Angeles, United States; 2 Southern Illinois University, Springfield, United States Humanin (HN) is a novel 24 amino acid peptide encoded within the 16S ribosomal RNA region of the mitochondrial DNA. It was cloned independently by three separate groups, including ours, and described to be a potent cytoprotective and metaboloprotective peptide in vitro and in vivo. HN has intracellular functions, acting as a partner for IGFBP-3 and BAX, as well as extracellular actions via 2 different types of membrane receptors, to regulate cellular survival and metabolism. We have developed an ELISA assay that accurately measures humanin in plasma of humans and mice and have previously published that humanin levels fall dramatically with age, suggesting that in might be linked to the aging process as well as diseases of aging. Mitochondria have been heavily implicated in the aging process, with much emphasis on their metabolic roles. While multiple signals are known to be sent to the mitochondria, very little is understood on how retrograde mitochondrial signaling contributes to their age-regulatory effects. Studies using various model organisms from yeast to mice established GH as a potent regulator of lifespan. GH-deficient Ames dwarf mice, which live over 50% longer compared to littermates, have several distinguished metabolic signatures including enhanced mitochondrial function. GH transgenic mice, are large in size and short-lived, and have been reported to have increased oxidative stress. We examined whether humanin levels are altered in models of altered GH activity in these mice. Our findings show that GH regulates HN expression. HN levels are ~70% higher in Ames homozygous df/df mice (n = 10) compared to controls (n = 10), p < 0.01, whereas a ~50% decrease is observed in mice that overexpress GH under the control of the PEPCK promotor (n = 12) compared to age matched control mice (n = 14), p < 0.01. To further assess a potential role for GH in the regulation of humanin in humans, we compared humanin levels in the plasma of short children (n = 10) before and after initiation of growth hormone treatment for 1–2 months. Humanin levels in these GH-deficient or -insufficient children were high and fell to less than half of the initial values in response to GH treatment. Our data indicate that GH is a key regulator of circulating humanin levels (in addition to age), and suggest that the GH-IGF-IGFBP3
S29
axis is involved in the regulation of mitochondrial function through the regulation of humanin levels (via GH) and bioavailability (via IGFBP-3 binding). We speculate that this relationship may have important implications for the role of humanin in aging and longevity and opens the possibility of its therapeutic potential in diseases of aging and as a co-therapy with GH. OR11-3 IGF-II and neurogenesis: in vitro and in vivo analyses A.N. Ziegler1 , M. Constancia2 , I. Sandovici2 , T.L. Wood1 , S.W. Levison1 . 1 UMDNJ-NJMS, Dept of Neurology and Neurosciences, Graduate School of Biomedical Sciences, Newark, United States; 2 University of Cambridge, Dept. Obstetrics & Gynaecology, Cambridge, United Kingdom The insulin-like growth factor (IGF) system plays a critical role in brain development and growth. IGF-I and IGF-II both activate the IGF-1R. In contrast, IGF-II, but not IGF-I activates a splice variant of the insulin receptor (IR) known as IR-A. We have recently shown that IGF-II promotes self-renewal of neural stem cells (NSCs) via the IR-A in vitro (Ziegler et al Stem Cells 30:1265, 2012). We also showed that transplanted neural precursors cultured in IGF-II honed to the subventricular zone (SVZ), a proliferative niche for NSCs in the brain. To test the hypothesis that IGF-II acts on the NSCs as opposed to the later multipotential progenitors in culture, we used multimarker flow cytometry to analyze the relative proportions of NSCs and multiple types of progenitors. Flow cytometry revealed that IGF-II increases the proportion of NCSs and early multipotential progenitors while decreasing later stage progenitors in the NSC lineage. IGF-II exists along a gradient within the SVZ, thus we hypothesized that decreasing IGF-II levels in vivo would compromise adult neurogenesis. Using an inducible IGF-II knockout we disrupted IGF-II expression at one month of age. The mice were then given two thymidine analogs (CldU and IdU) to evaluate label-retaining cells over time. Two months after inducing igf2 gene deletion we found reduced neurogenesis in the olfactory bulb, visualized by a dramatic decrease in the numbers of doublecortin-positive cells. Using an analysis of olfaction, mice lacking IGF-II are behaviorally impaired. Altogether these results support an essential role for IGF-II in NSC self-renewal both in vitro and in vivo. Supported by a Dean’s Grant from NJMS awarded to SWL and TLW and F31NS065607 awarded to ANZ. OR11-4 Prevalence of posttraumatic growth hormone deficiency is highly dependent on the diagnostic set-up – results from The Danish National study on posttraumatic hypopituitarism M. Klose1 , K. Stockholm2 , J. Janukonyte´ 2 , L. Frederiksen3 , M. Andersen3 , P. Laurberg4 , J. Sandahl Christiansen2 , U. FeldtRasmussen5 . 1 Copenhagen University Hospital, Rigshospitalet, Dept of Endocrinology, Copenhagen, Denmark; 2 Aarhus University Hospital, Aarhus, Denmark; 3 Odense University Hospital, Odense, Denmark; 4 Aalborg University Hospital, Aalborg, Denmark; 5 Copenhagen University Hospital, Rigshospitalet, Copenhagen, Denmark Introduction and aim: Pituitary function screening has been suggested in traumatic brain injury (TBI), based on reports of high risk of growth hormone (GH) deficiency in particular. We aimed to describe the prevalence of GH deficiency in a national TBI population, including patients admitted to a Danish hospital in 2008, as compared to healthy controls. Patients and methods: Patients were identified with a head trauma diagnosis from Danish Board of Health diagnostic code registry. Based on retrospective chart review, 856 of 2014 patients were eligible for inclusion, meeting the following criteria: 1) loss of consciousness, amnesia, or cranial/cerebral imaging abnormalities; 2) no history of alcohol or substance abuse, active malignancy or chronic disease that could interfere with the diagnosis or study
isolated from serum or expression of unglycosylated protein in E. coli. To rectify this and explore their properties, we produced O-glycosylated human IGF-II isoforms. These isoforms bound the IGF-GR and insulin receptor isoform A (IR-A) with affinities similar to mature IGF-II. They also formed binary complexes with IGFBP-2, IGFBP-3 and IGFBP-5. However, there were marked differences in their ability to induce phosphorylation of the IGF-GR, IR-A and IR-B receptors in vitro, with the IGF-II isoforms inducing different levels of receptor activation and saturation of activation compared to mature IGF-II. Despite these differences, in vitro assays using cells individually expressing IGF-GR, IR-A or IR-B demonstrated that very low levels of all ligands were sufficient to induce a proliferative response. Finally, compared to mature IGF-II, in vitro IGFBP-3/ALS and IGFBP-5/ALS ternary complex formation with the IGF-II isoforms was severely retarded, an effect reliant on the presence of the O-glycosylated sugars within the E-domain. The big-IGF-II (1–87) species also displayed a severe loss of binding to IGF-IIR. These findings provide the first insights into the unique individual biological properties associated with each IGF-II isoform. Reference(s) [1] Dransfield, D. et al. Mol Cancer Ther 9:1809–1819 (2010).
OR11-2 Growth Hormone regulates the circulating levels of the mitochondria-derived peptide humanin C. Lee1 , A. Bartke2 , Y. Fang2 , J. Wan1 , P. Cohen1 . 1 University of Southern California, Los Angeles, United States; 2 Southern Illinois University, Springfield, United States Humanin (HN) is a novel 24 amino acid peptide encoded within the 16S ribosomal RNA region of the mitochondrial DNA. It was cloned independently by three separate groups, including ours, and described to be a potent cytoprotective and metaboloprotective peptide in vitro and in vivo. HN has intracellular functions, acting as a partner for IGFBP-3 and BAX, as well as extracellular actions via 2 different types of membrane receptors, to regulate cellular survival and metabolism. We have developed an ELISA assay that accurately measures humanin in plasma of humans and mice and have previously published that humanin levels fall dramatically with age, suggesting that in might be linked to the aging process as well as diseases of aging. Mitochondria have been heavily implicated in the aging process, with much emphasis on their metabolic roles. While multiple signals are known to be sent to the mitochondria, very little is understood on how retrograde mitochondrial signaling contributes to their age-regulatory effects. Studies using various model organisms from yeast to mice established GH as a potent regulator of lifespan. GH-deficient Ames dwarf mice, which live over 50% longer compared to littermates, have several distinguished metabolic signatures including enhanced mitochondrial function. GH transgenic mice, are large in size and short-lived, and have been reported to have increased oxidative stress. We examined whether humanin levels are altered in models of altered GH activity in these mice. Our findings show that GH regulates HN expression. HN levels are ~70% higher in Ames homozygous df/df mice (n = 10) compared to controls (n = 10), p < 0.01, whereas a ~50% decrease is observed in mice that overexpress GH under the control of the PEPCK promotor (n = 12) compared to age matched control mice (n = 14), p < 0.01. To further assess a potential role for GH in the regulation of humanin in humans, we compared humanin levels in the plasma of short children (n = 10) before and after initiation of growth hormone treatment for 1–2 months. Humanin levels in these GH-deficient or -insufficient children were high and fell to less than half of the initial values in response to GH treatment. Our data indicate that GH is a key regulator of circulating humanin levels (in addition to age), and suggest that the GH-IGF-IGFBP3
S29
axis is involved in the regulation of mitochondrial function through the regulation of humanin levels (via GH) and bioavailability (via IGFBP-3 binding). We speculate that this relationship may have important implications for the role of humanin in aging and longevity and opens the possibility of its therapeutic potential in diseases of aging and as a co-therapy with GH. OR11-3 IGF-II and neurogenesis: in vitro and in vivo analyses A.N. Ziegler1 , M. Constancia2 , I. Sandovici2 , T.L. Wood1 , S.W. Levison1 . 1 UMDNJ-NJMS, Dept of Neurology and Neurosciences, Graduate School of Biomedical Sciences, Newark, United States; 2 University of Cambridge, Dept. Obstetrics & Gynaecology, Cambridge, United Kingdom The insulin-like growth factor (IGF) system plays a critical role in brain development and growth. IGF-I and IGF-II both activate the IGF-1R. In contrast, IGF-II, but not IGF-I activates a splice variant of the insulin receptor (IR) known as IR-A. We have recently shown that IGF-II promotes self-renewal of neural stem cells (NSCs) via the IR-A in vitro (Ziegler et al Stem Cells 30:1265, 2012). We also showed that transplanted neural precursors cultured in IGF-II honed to the subventricular zone (SVZ), a proliferative niche for NSCs in the brain. To test the hypothesis that IGF-II acts on the NSCs as opposed to the later multipotential progenitors in culture, we used multimarker flow cytometry to analyze the relative proportions of NSCs and multiple types of progenitors. Flow cytometry revealed that IGF-II increases the proportion of NCSs and early multipotential progenitors while decreasing later stage progenitors in the NSC lineage. IGF-II exists along a gradient within the SVZ, thus we hypothesized that decreasing IGF-II levels in vivo would compromise adult neurogenesis. Using an inducible IGF-II knockout we disrupted IGF-II expression at one month of age. The mice were then given two thymidine analogs (CldU and IdU) to evaluate label-retaining cells over time. Two months after inducing igf2 gene deletion we found reduced neurogenesis in the olfactory bulb, visualized by a dramatic decrease in the numbers of doublecortin-positive cells. Using an analysis of olfaction, mice lacking IGF-II are behaviorally impaired. Altogether these results support an essential role for IGF-II in NSC self-renewal both in vitro and in vivo. Supported by a Dean’s Grant from NJMS awarded to SWL and TLW and F31NS065607 awarded to ANZ. OR11-4 Prevalence of posttraumatic growth hormone deficiency is highly dependent on the diagnostic set-up – results from The Danish National study on posttraumatic hypopituitarism M. Klose1 , K. Stockholm2 , J. Janukonyte´ 2 , L. Frederiksen3 , M. Andersen3 , P. Laurberg4 , J. Sandahl Christiansen2 , U. FeldtRasmussen5 . 1 Copenhagen University Hospital, Rigshospitalet, Dept of Endocrinology, Copenhagen, Denmark; 2 Aarhus University Hospital, Aarhus, Denmark; 3 Odense University Hospital, Odense, Denmark; 4 Aalborg University Hospital, Aalborg, Denmark; 5 Copenhagen University Hospital, Rigshospitalet, Copenhagen, Denmark Introduction and aim: Pituitary function screening has been suggested in traumatic brain injury (TBI), based on reports of high risk of growth hormone (GH) deficiency in particular. We aimed to describe the prevalence of GH deficiency in a national TBI population, including patients admitted to a Danish hospital in 2008, as compared to healthy controls. Patients and methods: Patients were identified with a head trauma diagnosis from Danish Board of Health diagnostic code registry. Based on retrospective chart review, 856 of 2014 patients were eligible for inclusion, meeting the following criteria: 1) loss of consciousness, amnesia, or cranial/cerebral imaging abnormalities; 2) no history of alcohol or substance abuse, active malignancy or chronic disease that could interfere with the diagnosis or study