Novel roles of miR-199b in regulating fat and bone metabolism

Public Health Problem: The incidence of obesity has reached epidemic proportions worldwide and has contributed to an increase in the risk of numerous chronic disorders-type 2 diabetes-liver pathologies, dyslipidemia, and cardiovascular diseases. Obesity can have negative effects on bone remodeling-reduced mineral density-osteoporosis Imbalance between food intake and energy expenditure-obesity-accumulation of fat mass and energy storage in white adipose tissue (WAT).https://knowledgeconnection.mainehealth.org/lambrew-retreat-2021/1036/thumbnail.jp

IGFBP-2 Directly Stimulates Osteoblast Differentiation: IGFBP-2 DIRECTLY STIMULATES OSTEOBLAST DIFFERENTIATION

Insulin like growth factor binding protein two (IGFBP-2) is important for acquisition of normal bone mass in mice; however, the mechanism by which IGFBP-2 functions is not defined. These studies investigated the role of IGFBP-2 in stimulating osteoblast differentiation. MC-3T3 preosteoblasts expressed IGFBP-2, and IGFBP-2 knockdown resulted in a substantial delay in osteoblast differentiation, reduced osteocalcin expression and Alizarin red staining. These findings were replicated in primary calvarial osteoblasts obtained from IGFBP-2 −/− mice and addition of IGFBP-2 rescued the differentiation program. In contrast, overexpression of IGFBP-2 accelerated the time course of differentiation as well as increasing the total number of differentiating cells. By day 6 IGFBP-2 overexpressing cells expressed twice as much osteocalcin as control cultures and this difference persisted. To determine the mechanism by which IGFBP-2 functions, the interaction between IGFBP-2 and receptor tyrosine phosphatase β (RPTPβ) was examined. Disruption of this interaction inhibited the ability of IGFBP-2 to stimulate AKT activation and osteoblast differentiation. Knockdown of RPTPβ enhanced osteoblast differentiation whereas overexpression of RPTPβ was inhibitory. Adding back IGFBP-2 to RPTPβ overexpressing cells was able to rescue cell differentiation via enhancement of AKT activation. To determine the region of IGFBP-2 that mediated this effect an IGFBP-2 mutant that contained substitutions of key amino acids in the heparin binding domain-1 (HBD-1) was prepared. This mutant had a major reduction in its ability to stimulate differentiation of calvarial osteoblasts from IGFBP-2 −/− mice. Addition of a synthetic peptide that contained the HBD-1 sequence to calvarial osteoblasts from IGFBP-2 −/− mice rescued differentiation and osteocalcin expression. In summary, the results clearly demonstrate that IGFBP-2 stimulates osteoblast differentiation and that this effect is mediated through its heparin binding domain-1 interacting with RPTPβ. The results suggest that stimulation of differentiation is an important mechanism by which IGFBP-2 regulates the acquisition of normal bone mass in mice

Erratum: Insulin-like growth factor-binding protein-2 is required for osteoclast differentiation

Global deletion of the Igfbp2 gene results in the suppression of bone turnover. To investigate the role of IGFBP-2 in regulating osteoclast differentiation we cultured Igfbp2−/− bone marrow cells and found a reduction in the number of osteoclasts and impaired resorption. Addition of full length IGFBP-2 restored osteoclast differentiation, fusion and resorption. To determine the molecular domains of IGFBP-2 that were required for this effect to be manifest, Igfbp2−/− bone marrow cells mice were transfected with constructs in which the heparin binding (HBD) or the IGF- binding domains of IGFBP-2 were mutated. We found that both domains were necessary for osteoclastogenesis since expression of the mutated forms of either domain failed to support the formation of functionally mature osteoclasts. To discern the mechanism by which IGFBP-2 regulates osteoclast formation, PTEN abundance and phosphorylation status as well as AKT responsiveness to IGF-I were analyzed. Igfbp2−/− cells had elevated levels of PTEN and phospho-PTEN compared with controls. Expression of wild-type IGFBP-2 reduced the level of PTEN to that of wild-type cells. Cells expressing the IGF binding mutant showed suppression of PTEN and phospho-PTEN equivalent to the wild type protein, whereas those expressing the IGFBP-2 HBD mutant showed no PTEN suppression. When the ability of IGF-I to stimulate AKT activation, measured by Thr308 and Ser473 phosphorylation, was analyzed, stimulation of Ser473 in response to IGF-I in pre-osteoclasts required the presence of intact IGFBP-2. This effect was duplicated by the addition of a CK2 inhibitor that prevents the phosphorylation of PTEN. In contrast, in fully differentiated osteoclasts stimulation of Thr308 phosphorylation required the presence of intact IGFBP-2. We conclude that IGFBP-2 is an important regulator of osteoclastogenesis and that both the heparin and the IGF binding domains of IGFBP-2 are essential for the formation of fully differentiated and functional osteoclasts

The Lipid Handling Capacity of Subcutaneous Fat Is Programmed by mTORC2 during Development

Overweight and obesity are associated with type 2 diabetes, non-alcoholic fatty liver disease, cardiovascular disease and cancer, but all fat is not equal, as storing excess lipid in subcutaneous white adipose tissue (SWAT) is more metabolically favorable than in visceral fat. Here, we uncover a critical role for mTORC2 in setting SWAT lipid handling capacity. We find that subcutaneous white preadipocytes differentiating without the essential mTORC2 subunit Rictor upregulate mature adipocyte markers but develop a striking lipid storage defect resulting in smaller adipocytes, reduced tissue size, lipid re-distribution to visceral and brown fat, and sex-distinct effects on systemic metabolic fitness. Mechanistically, mTORC2 promotes transcriptional upregulation of select lipid metabolism genes controlled by PPARγ and ChREBP, including genes that control lipid uptake, synthesis, and degradation pathways as well as Akt2, which encodes a major mTORC2 substrate and insulin effector. Further exploring this pathway may uncover new strategies to improve insulin sensitivity.Fil: Hsiao, Wen Yu. University Of Massachussets. Medical School; Estados UnidosFil: Jung, Su Myung. University Of Massachussets. Medical School; Estados UnidosFil: Tang, Yuefeng. University Of Massachussets. Medical School; Estados UnidosFil: Haley, John A.. University Of Massachussets. Medical School; Estados UnidosFil: Li, Rui. University Of Massachussets. Medical School; Estados UnidosFil: Li, Huawei. University Of Massachussets. Medical School; Estados UnidosFil: Martinez Calejman, Camila. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Centro de Estudios Farmacológicos y Botánicos. Universidad de Buenos Aires. Facultad de Medicina. Centro de Estudios Farmacológicos y Botánicos; Argentina. University Of Massachussets. Medical School; Estados UnidosFil: Sanchez Gurmaches, Joan. University Of Massachussets. Medical School; Estados Unidos. University of Cincinnati; Estados UnidosFil: Hung, Chien-Min. University Of Massachussets. Medical School; Estados UnidosFil: Luciano, Amelia K.. University Of Massachussets. Medical School; Estados UnidosFil: DeMambro, Victoria. University of Maine; Estados UnidosFil: Wellen, Kathryn E.. University of Pennsylvania; Estados UnidosFil: Rosen, Clifford J.. University of Maine; Estados UnidosFil: Zhu, Lihua Julie. University Of Massachussets. Medical School; Estados UnidosFil: Guertin, David A.. University Of Massachussets. Medical School; Estados Unido

The Lipid Handling Capacity of Subcutaneous Fat Is Programmed by mTORC2 during Development

Overweight and obesity are associated with type 2 diabetes, non-alcoholic fatty liver disease, cardiovascular disease and cancer, but all fat is not equal, as storing excess lipid in subcutaneous white adipose tissue (SWAT) is more metabolically favorable than in visceral fat. Here, we uncover a critical role for mTORC2 in setting SWAT lipid handling capacity. We find that subcutaneous white preadipocytes differentiating without the essential mTORC2 subunit Rictor upregulate mature adipocyte markers but develop a striking lipid storage defect resulting in smaller adipocytes, reduced tissue size, lipid re-distribution to visceral and brown fat, and sex-distinct effects on systemic metabolic fitness. Mechanistically, mTORC2 promotes transcriptional upregulation of select lipid metabolism genes controlled by PPARgamma and ChREBP, including genes that control lipid uptake, synthesis, and degradation pathways as well as Akt2, which encodes a major mTORC2 substrate and insulin effector. Further exploring this pathway may uncover new strategies to improve insulin sensitivity

Novel Insights into the Insulin-Like Growth Factor 1 System Revealed by Studies of New Mutant Mouse Models

The Insulin-like growth factor system (IGF) is a major growth regulatory signaling network comprised of two-ligands IGF-I and IGF-II, two receptors IGF type I and II (IGF-IR and IGFII-R), and regulatory proteins consisting of the acid-labile subunit (ALS) and six binding proteins (IGFBP-1-6). IGF-I binding stimulates autophosphorylation of the receptor, creating binding sites for the insulin like substrates (IRS-1-4) that act as docking proteins transducing signals down a number of pathways most notably the RAS/Raf-1/MAPK and PI3K/PDK/AKT pathways. The molecular and phenotypic investigations of two mouse models with genetic mutations in IGFBP-2 and IRS-1 are reported here revealing novel insights into the IGF-I pathway. The first model studied, Igfbp2 null (Igfbp2- -) mice have been reported previously to have no major physiological defects. Developmental data reported herein revealed that male Igfbp2- - were heavier, had shorter femurs, and smaller spleens than controls. Serum IGF-I was increased in Igfbp2- - with two fold increases in hepatic Igfbp3 and Igfbp5 mRNA transcripts. The skeletal phenotype of the null was gender specific; female Igfbp2- - had increased cortical bone mass, while male Igfbp2- - had reductions in both cortical and trabecular bone mass compared to controls. Serum osteocalcin was reduced in male Igfbp2- -. In vitro and in vivo, osteoblasts, osteoclasts, and bone formation rates were reduced in male Igfbp2- -. The phosphatase and tensin homolog (PTEN) protein levels were increased in the null mice pointing to a potential mechanism for decreased IGF activity and bone formation. The second model investigated, a spontaneous mouse mutant, designated \u27small\u27 (sml), genetically mapped to the Chromosome 1 region containing the Irsl gene. Sequencing this mutant gene revealed a single nucleotide deletion resulting in a premature stop codon. Despite normal mRNA levels, Western blot analysis revealed no detectable protein in mutant liver lysates. The Irslsml/Irslsml (Irslsml sml) mice are small, lean, hearing impaired, have 20% less serum IGF-I, are hyperinsulinemic and are mildly insulin resistant compared to control littermates. Irslsml sml mice have low bone mineral density, reduced trabecular and cortical thicknesses and low bone formation rates, while osteoblast and osteoclast numbers were increased in the females but not different in the males compared to controls. Irslsmlsml bone marrow stromal cell cultures showed decreased pre-osteoblasts and normal numbers of osteoclasts. Irslsml sml stromal cells treated with IGF-I exhibited a 50% decrease in AKT phosphorylation, and increased IRS-2 phosphorylation. Similarities between engineered knockouts and the spontaneous mutation of Irsl were identified as well as significant differences with respect to heterozygosity and gender. Thus, studies with these two mouse mutant models suggest a new role for IGFBP-2 in bone development modified by gender having implications for the gender-biased progression of osteoporosis in humans. Furthermore, the demonstration of an unanticipated heterozygous phenotype in the Irslsml mice suggests that similar mutations in IRS-1 in humans may be responsible for short stature and/or osteoporosis. In summary, systematic investigation of the Igfbp2- - and Irslsml sml mice demonstrate the power of mouse mutant gene models to reveal novel insights into the biological actions of the IGF-I system

Prrx1-CreAlplfl/flmice, a model for further investigations into the pathophysiological changes in Hypophosphatemia

Hypophosphatasia (HPP) is caused by loss of function mutations in the tissuenonspecific alkaline phosphatase (TNSALP) gene (Alpl) which results in rickets, osteomalicia and bone fragility with severe childhood forms resulting in lean body mass. TNSALP is a glycosylphosphatidylinositol (GPI)-anchored phosphatase localized at the plasma membrane which when cleaved can also be found in circulation. In vitro, inhibition of TNSALP leads to a significant decrease in lipid accumulation suggesting TNSALP, an enzyme known for its mineralizing properties, may be pro-adipogenic. TNSALP has been implicated in bone marrow mesenchymal stem cell (BMMSC) lineage determination between osteoblasts and adipocytes through direct interactions with lowdensity lipoprotein receptor-related protein 6 (LRP6), modulating WNT signaling by an unknown mechanism. Alpl-/- mice die postnatally, thus, to further delineate TNSALP’s role in bone homeostasis and adipogenesis we are utilizing a Prrx1-Cre driver mated with to an Alplflfl(CreAlpflfl) mouse line

Developmental thyroid hormone action on pro-opiomelanocortin-expressing cells programs hypothalamic BMPR1A depletion and brown fat activation

Thyroid hormone excess secondary to global type 3 deiodinase (DIO3) deficiency leads to increased locomotor activity and reduced adiposity, but also to concurrent alterations in parameters of the leptin-melanocortin system that would predict obesity. To distinguish the underlying contributions to the energy balance phenotype of DIO3 deficiency, we generated mice with thyroid hormone excess targeted to pro-opiomelanocortin (POMC)-expressing cells via cell-specific Dio3 inactivation. These mice exhibit a male-specific phenotype of reduced hypothalamic Pomc expression, hyperphagia, and increased activity in brown adipose tissue, with adiposity and serum levels of leptin and thyroid hormones remained normal. These male mice also manifest a marked and widespread hypothalamic reduction in the expression of bone morphogenetic receptor 1a (BMPR1A), which has been shown to cause similar phenotypes when inactivated in POMC-expressing cells. Our results indicate that developmental overexposure to thyroid hormone in POMC-expressing cells programs energy balance mechanisms in a sexually dimorphic manner by suppressing adult hypothalamic Bmpr1a expression

Effects of PTH on osteoblast bioenergetics in response to glucose

Parathyroid hormone acts through its receptor, PTHR1, expressed on osteoblasts, to control bone remodeling. Metabolic flexibility for energy generation has been demonstrated in several cell types dependent on substrate availability. Recent studies have identified a critical role for PTH in regulating glucose, fatty acid and amino acid metabolism thus stimulating both glycolysis and oxidative phosphorylation. Therefore, we postulated that PTH stimulates increased energetic output by osteoblasts either by increasing glycolysis or oxidative phosphorylation depending on substrate availability. To test this hypothesis, undifferentiated and differentiated MC3T3E1C4 calvarial pre-osteoblasts were treated with PTH to study osteoblast bioenergetics in the presence of exogenous glucose. Significant increases in glycolysis with acute ∼1 h PTH treatment with minimal effects on oxidative phosphorylation in undifferentiated MC3T3E1C4 in the presence of exogenous glucose were observed. In differentiated cells, the increased glycolysis observed with acute PTH was completely blocked by pretreatment with a Glut1 inhibitor (BAY-876) resulting in a compensatory increase in oxidative phosphorylation. We then tested the effect of PTH on the function of complexes I and II of the mitochondrial electron transport chain in the absence of glycolysis. Utilizing a novel cell plasma membrane permeability mitochondrial (PMP) assay, in combination with complex I and II specific substrates, slight but significant increases in basal and maximal oxygen consumption rates with 24 h PTH treatment in undifferentiated MC3T3E1C4 cells were noted. Taken together, our data demonstrate for the first time that PTH stimulates both increases in glycolysis and the function of the electron transport chain, particularly complexes I and II, during high energy demands in osteoblasts