Sebastien Elis

Elevated serum levels of IGF‐1 are sufficient to establish normal body size and skeletal properties even in the absence of tissue IGF‐1

Use of recombinant insulin‐like growth factor 1 (IGF‐1) as a treatment for primary IGF‐1 deficiency in children has become increasingly common. When untreated, primary IGF‐1 deficiency may lead to a range of metabolic disorders, including lipid abnormalities, insulin resistance, and decreased bone density. To date, results of this therapy are considered encouraging; however, our understanding of the role played by IGF‐1 during development remains limited. Studies on long‐term treatment with recombinant IGF‐1 in both children and animals are few. Here, we used two novel transgenic mouse strains to test the long‐term effects of elevated circulating IGF‐1 on body size and skeletal development. Overexpression of the rat igf1 transgene in livers of mice with otherwise normal IGF‐1 expression (HIT mice) resulted in approximately threefold increases in serum IGF‐1 levels throughout growth, as well as greater body mass and enhanced skeletal size, architecture, and mechanical properties. When the igf1 transgene was overexpressed in livers of igf1 null mice (KO‐HIT), the comparably elevated serum IGF‐1 failed to overcome growth and skeletal deficiencies during neonatal and early postnatal growth. However, between 4 and 16 weeks of age, increased serum IGF‐1 fully compensated for the absence of locally produced IGF‐1 because body weights and lengths of KO‐HIT mice became comparable with controls. Furthermore, micro‐computed tomography (µCT) analysis revealed that early deficits in skeletal structure of KO‐HIT mice were restored to control levels by adulthood. Our data indicate that in the absence of tissue igf1 gene expression, maintaining long‐term elevations in serum IGF‐1 is sufficient to establish normal body size, body composition, and both skeletal architecture and mechanical function.

Serum IGF-1 Affects Skeletal Acquisition in a Temporal and Compartment-Specific Manner

Insulin-like growth factor-1 (IGF-1) plays a critical role in the development of the growing skeleton by establishing both longitudinal and transverse bone accrual. IGF-1 has also been implicated in the maintenance of bone mass during late adulthood and aging, as decreases in serum IGF-1 levels appear to correlate with decreases in bone mineral density (BMD). Although informative, mouse models to date have been unable to separate the temporal effects of IGF-1 depletion on skeletal development. To address this problem, we performed a skeletal characterization of the inducible LID mouse (iLID), in which serum IGF-1 levels are depleted at selected ages. We found that depletion of serum IGF-1 in male iLID mice prior to adulthood (4 weeks) decreased trabecular bone architecture and significantly reduced transverse cortical bone properties (Ct.Ar, Ct.Th) by 16 weeks (adulthood). Likewise, depletion of serum IGF-1 in iLID males at 8 weeks of age, resulted in significantly reduced transverse cortical bone properties (Ct.Ar, Ct.Th) by 32 weeks (late adulthood), but had no effect on trabecular bone architecture. In contrast, depletion of serum IGF-1 after peak bone acquisition (at 16 weeks) resulted in enhancement of trabecular bone architecture, but no significant changes in cortical bone properties by 32 weeks as compared to controls. These results indicate that while serum IGF-1 is essential for bone accrual during the postnatal growth phase, depletion of IGF-1 after peak bone acquisition (16 weeks) is compartment-specific and does not have a detrimental effect on cortical bone mass in the older adult mouse.

Elevated serum IGF‐1 levels synergize PTH action on the skeleton only when the tissue IGF‐1 axis is intact

There is growing evidence that insulin‐like growth factor 1 (IGF‐1) and parathyroid hormone (PTH) have synergistic actions on bone and that part of the anabolic effects of PTH is mediated by local production of IGF‐1. In this study we analyzed the skeletal response to PTH in mouse models with manipulated endocrine or autocrine/paracrine IGF‐1. We used mice carrying a hepatic IGF‐1 transgene (HIT), which results in a threefold increase in serum IGF‐1 levels and normal tissue IGF‐1 expression, and Igf1 null mice with blunted IGF‐1 expression in tissues but threefold increases in serum IGF‐1 levels (KO‐HIT). Evaluation of skeletal growth showed that elevations in serum IGF‐1 in mice with Igf1 gene ablation in all tissues except the liver (KO‐HIT) resulted in a restoration of skeletal morphology and mechanical properties by adulthood. Intermittent PTH treatment of adult HIT mice resulted in increases in serum osteocalcin levels, femoral total cross‐sectional area, cortical bone area and cortical bone thickness, as well as bone mechanical properties. We found that the skeletal response of HIT mice to PTH was significantly higher than that of control mice, suggesting synergy between IGF‐1 and PTH on bone. In sharp contrast, although PTH‐treated KO‐HIT mice demonstrated an anabolic response in cortical and trabecular bone compartments compared with vehicle‐treated KO‐HIT mice, their response was identical to that of PTH‐treated control mice. We conclude that (1) in the presence of elevated serum IGF‐1 levels, PTH can exert an anabolic response in bone even in the total absence of tissue IGF‐1, and (2) elevations in serum IGF‐1 levels synergize PTH action on bone only if the tissue IGF‐1 axis is intact. Thus enhancement of PTH anabolic actions depends on tissue IGF‐1.

Growth hormone mediates pubertal skeletal development independent of hepatic IGF-1 production.

Deficiencies in either growth hormone (GH) or insulin‐like growth factor 1 (IGF‐1) are associated with reductions in bone size during growth in humans and animal models. Liver‐specific IGF‐1‐deficient (LID) mice, which have 75% reductions in serum IGF‐1, were created previously to separate the effects of endocrine (serum) IGF‐1 from autocrine/paracrine IGF‐1. However, LID mice also have two‐ to threefold increases in GH, and this may contribute to the observed pubertal skeletal phenotype. To clarify the role of GH in skeletal development under conditions of significantly reduced serum IGF‐1 levels (but normal tissue IGF‐1 levels), we studied the skeletal response of male LID and control mice to GH inhibition by pegvisomant from 4 to 8 weeks of age. Treatment of LID mice with pegvisomant resulted in significant reductions in body weight, femur length (Le), and femur total area (Tt.Ar), as well as further reductions in serum IGF‐1 levels by 8 weeks of age, compared with the mean values of vehicle‐treated LID mice. Reductions in both Tt.Ar and Le were proportional after treatment with pegvisomant. On the other hand, the relative amount of cortical tissue formed (RCA) in LID mice treated with pegvisomant was significantly less than that in both vehicle‐treated LID and control mice, indicating that antagonizing GH action, either directly (through GH receptor signaling inhibition) or indirectly (through further reductions in serum/tissue IGF‐1 levels), results in disproportionate reductions in the amount of cortical bone formed. This resulted in bones with significantly reduced mechanical properties (femoral whole‐bone stiffness and work to failure were markedly decreased), suggesting that compensatory increases of GH in states of IGF‐1 deficiency (LID mice) act to protect against a severe inhibition of bone modeling during growth, which otherwise would result in bones that are too weak for normal and/or extreme loading conditions.

Sex-specific regulation of body size and bone slenderness by the acid labile subunit.

Insulin‐like growth factor 1 (IGF‐1) is a crucial mediator of body size and bone mass during growth and development. In serum, IGF‐1 is stabilized by several IGF‐1‐binding proteins (IGFBPs) and the acid labile subunit (ALS). Previous research using ALS knockout (ALSKO) mice indicated a growth retardation phenotype, and clinical reports of humans have indicated short stature and low bone mineral density (BMD) in patients with ALS deficiency. To determine the temporal and sex‐specific effects of ALS deficiency on body size and skeletal development during growth, we characterized control and ALSKO mice from 4 to 16 weeks of age. We found that female ALSKO mice had an earlier‐onset reduction in body size (4 weeks) but that both female and male ALSKO mice were consistently smaller than control mice. Interestingly, skeletal analyses at multiple ages showed increased slenderness of ALSKO femurs that was more severe in females than in males. Both male and female ALSKO mice appeared to compensate for their more slender bones through increased bone formation on their endosteal surfaces during growth, but ALSKO females had increased endosteal bone formation compared with ALSKO males. This study revealed age‐ and sex‐specific dependencies of body size and bone size on the ALS. These findings may explain the heterogeneity in growth and BMD measurements reported in human ALS‐deficient patients.

Unbound (bioavailable) IGF1 enhances somatic growth

SUMMARY Understanding insulin-like growth factor-1 (IGF1) biology is of particular importance because, apart from its role in mediating growth, it plays key roles in cellular transformation, organ regeneration, immune function, development of the musculoskeletal system and aging. IGF1 bioactivity is modulated by its binding to IGF-binding proteins (IGFBPs) and the acid labile subunit (ALS), which are present in serum and tissues. To determine whether IGF1 binding to IGFBPs is necessary to facilitate normal growth and development, we used a gene-targeting approach and generated two novel knock-in mouse models of mutated IGF1, in which the native Igf1 gene was replaced by Des-Igf1 (KID mice) or R3-Igf1 (KIR mice). The KID and KIR mutant proteins have reduced affinity for the IGFBPs, and therefore present as unbound IGF1, or ‘free IGF1’. We found that both KID and KIR mice have reduced serum IGF1 levels and a concomitant increase in serum growth hormone levels. Ternary complex formation of IGF1 with the IGFBPs and the ALS was markedly reduced in sera from KID and KIR mice compared with wild type. Both mutant mice showed increased body weight, body and bone lengths, and relative lean mass. We found selective organomegaly of the spleen, kidneys and uterus, enhanced mammary gland complexity, and increased skeletal acquisition. The KID and KIR models show unequivocally that IGF1-complex formation with the IGFBPs is fundamental for establishing normal body and organ size, and that uncontrolled IGF bioactivity could lead to pathological conditions.

Increased serum IGF-1 levels protect the musculoskeletal system but are associated with elevated oxidative stress markers and increased mortality independent of tissue igf1 gene expression

Although the literature suggests a protective (anabolic) effect of insulin‐like growth factor‐1 (IGF‐1) on the musculoskeletal system during growth and aging, there is evidence that reductions in IGF‐1 signaling are advantageous for promoting an increase in life span through reduction in oxidative stress‐induced tissue damage. To better understand this paradox, we utilized the hepatocyte‐specific IGF‐1 transgenic (HIT) mice, which exhibit 3‐fold increases in serum IGF‐1, with normal IGF‐1 expression in other tissues, and mice with an IGF‐1 null background that exclusively express IGF‐1 in the liver, which thereby deliver IGF‐1 by the endocrine route only (KO‐HIT mice). We found that in the total absence of tissue igf1 gene expression (KO‐HIT), increases in serum IGF‐1 levels were associated with increased levels of lipid peroxidation products in serum and increased mortality rate at 18 months of age in both genders. Surprisingly, however, we found that in female mice, tissue IGF‐1 plays an important role in preserving trabecular bone architecture as KO‐HIT mice show bone loss in the femoral distal metaphysis. Additionally, in male KO‐HIT mice, increases in serum IGF‐1 levels were insufficient to protect against age‐related muscle loss.

Elevated Circulating IGF-I Promotes Mammary Gland Development and Proliferation

Animal studies have shown that IGF-I is essential for mammary gland development. Previous studies have suggested that local IGF-I rather than circulating IGF-I is the major mediator of mammary gland development. In the present study we used the hepatic IGF-I transgenic (HIT) and IGF-I knockout/HIT (KO-HIT) mouse models to examine the effects of enhanced circulating IGF-I on mammary development in the presence and absence of local IGF-I. HIT mice express the rat IGF-I transgene under the transthyretin promoter in the liver and have elevated circulating IGF-I and normal tissue IGF-I levels. The KO-HIT mice have no tissue IGF-I and increased circulating IGF-I. Analysis of mammary gland development reveals a greater degree of complexity in HIT mice as compared to control and KO-HIT mice, which demonstrate similar degrees of mammary gland complexity. Immunohistochemical evaluation of glands of HIT mice also suggests an enhanced degree of proliferation of the mammary gland, whereas KO-HIT mice exhibit mammary gland proliferation similar to control mice. In addition, HIT mice have a higher percentage of proliferating myoepithelial and luminal cells than control mice, whereas KO-HIT mice have an equivalent percentage of proliferating myoepithelial and luminal cells as control mice. Thus, our findings show that elevated circulating IGF-I levels are sufficient to promote normal pubertal mammary epithelial development. However, HIT mice demonstrate more pronounced mammary gland development when compared to control and KO-HIT mice. This suggests that both local and endocrine IGF-I play roles in mammary gland development and that elevated circulating IGF-I accelerates mammary epithelial proliferation.

The Insulin-like Growth Factor-1 Binding Protein Acid-labile Subunit Alters Mesenchymal Stromal Cell Fate

Age-related osteoporosis is accompanied by an increase in marrow adiposity and a reduction in serum insulin-like growth factor-1 (IGF-1) and the binding proteins that stabilize IGF-1. To determine the relationship between these proteins and bone marrow adiposity, we evaluated the adipogenic potential of marrow-derived mesenchymal stromal cells (MSCs) from mice with decreased serum IGF-1 due to knockdown of IGF-1 production by the liver or knock-out of the binding proteins. We employed 10–16-week-old, liver-specific IGF-1-deficient, IGFBP-3 knock-out (BP3KO) and acid-labile subunit knock-out (ALSKO) mice. We found that expression of the late adipocyte differentiation marker peroxisome proliferator-activated receptor γ was increased in marrow isolated from ALSKO mice. When induced with adipogenic media, MSC cultures from ALSKO mice revealed a significantly greater number of differentiated adipocytes compared with controls. MSCs from ALSKO mice also exhibited decreased alkaline-phosphatase positive colony size in cultures that were stimulated with osteoblast differentiation media. These osteoblast-like cells from ALSKO mice failed to induce osteoclastogenesis of control cells in co-culture assays, indicating that impairment of IGF-1 complex formation with ALS in bone marrow alters cell fate, leading to increased adipogenesis.

Resistin in Dairy Cows: Plasma Concentrations during Early Lactation, Expression and Potential Role in Adipose Tissue

Resistin is an adipokine that has been implicated in energy metabolism regulation in rodents but has been little studied in dairy cows. We determined plasma resistin concentrations in early lactation in dairy cows and investigated the levels of resistin mRNA and protein in adipose tissue and the phosphorylation of several components of insulin signaling pathways one week post partum (1 WPP) and at five months of gestation (5 MG). We detected resistin in mature bovine adipocytes and investigated the effect of recombinant bovine resistin on lipolysis in bovine adipose tissue explants. ELISA showed that plasma resistin concentration was low before calving, subsequently increasing and reaching a peak at 1 WPP, decreasing steadily thereafter to reach pre-calving levels at 6 WPP. Plasma resistin concentration was significantly positively correlated with plasma non esterified fatty acid (NEFA) levels and negatively with milk yield, dry matter intake and energy balance between WPP1 to WPP22. We showed, by quantitative RT-PCR and western blotting, that resistin mRNA and protein levels in adipose tissue were higher at WPP1 than at 5 MG. The level of phosphorylation of several early and downstream insulin signaling components (IRβ, IRS-1, IRS-2, Akt, MAPK ERK1/2, P70S6K and S6) in adipose tissue was also lower at 1 WPP than at 5 MG. Finally, we showed that recombinant bovine resistin increased the release of glycerol and mRNA levels for ATGL (adipose triglyceride lipase) and HSL (hormone-sensitive lipase) in adipose tissue explants. Overall, resistin levels were high in the plasma and adipose tissue and were positively correlated with NEFA levels after calving. Resistin is expressed in bovine mature adipocytes and promotes lipid mobilization in adipose explants in vitro.