A Novel ENU-Induced

The fission and fusion of mitochondria are important processes for maintaining mitochondrial health. One of the proteins responsible for mediating mitochondrial fusion, mitofusin 2 (MFN2), has over 100 known mutations that cause Charcot–Marie–Tooth disease type 2A (CMT2A). This disease causes the nerves that control your muscles to degenerate, leading to muscle atrophy and weakness, problems walking, and other related symptoms. In this paper, we describe a mouse line with a recessive mutation in the Mfn2 gene (Leu643Pro) that causes a similar set of symptoms, including abnormal gait, weight loss, and decreased muscular endurance. However, further analysis of these mice revealed signs of skeletal muscle dysfunction (including smaller mitochondria) and bone abnormalities, with little evidence of axon degeneration typical of CMT2A. While this makes these mice a poor model for CMT2A, they are the first reported mouse line with a mutation in the transmembrane domain, a region critical for MFN2′s role in mitochondrial fusion. For this reason, we believe these mice will be a valuable tool for scientists interested in studying the biological functions of MFN2

Guidelines for the Use and Interpretation of Assays for Monitoring Autophagy (4th edition)

In 2008, we published the first set of guidelines for standardizing research in autophagy. Since then, this topic has received increasing attention, and many scientists have entered the field. Our knowledge base and relevant new technologies have also been expanding. Thus, it is important to formulate on a regular basis updated guidelines for monitoring autophagy in different organisms. Despite numerous reviews, there continues to be confusion regarding acceptable methods to evaluate autophagy, especially in multicellular eukaryotes. Here, we present a set of guidelines for investigators to select and interpret methods to examine autophagy and related processes, and for reviewers to provide realistic and reasonable critiques of reports that are focused on these processes. These guidelines are not meant to be a dogmatic set of rules, because the appropriateness of any assay largely depends on the question being asked and the system being used. Moreover, no individual assay is perfect for every situation, calling for the use of multiple techniques to properly monitor autophagy in each experimental setting. Finally, several core components of the autophagy machinery have been implicated in distinct autophagic processes (canonical and noncanonical autophagy), implying that genetic approaches to block autophagy should rely on targeting two or more autophagy-related genes that ideally participate in distinct steps of the pathway. Along similar lines, because multiple proteins involved in autophagy also regulate other cellular pathways including apoptosis, not all of them can be used as a specific marker for bona fide autophagic responses. Here, we critically discuss current methods of assessing autophagy and the information they can, or cannot, provide. Our ultimate goal is to encourage intellectual and technical innovation in the field

IGF-1 regulation of key signaling pathways in bone.

Insulin-like growth factor 1 (IGF-1) is an unique peptide that functions in an endocrine/paracrine and autocrine manner in most tissues. Although it was postulated initially that liver-derived IGF-1 was the major source of IGF-1 (that is, the somatomedin hypothesis), it is also produced in a wide variety of tissues and can function in numerous ways as both a proliferative and differentiative factor. One such tissue is bone and all cell lineages in the skeleton have been shown to not only require IGF-1 for normal development and function but also to respond to IGF-1 via the IGF-1 receptor. Ligand-receptor activation leads to several distinct downstream signaling cascades, which have significant implications for cell survival, protein synthesis and energy utilization. The novel role of IGF-1 in regulating metabolic demands of the bone remodeling unit is currently under investigation. More studies are likely to shed new light on various aspects of skeletal physiology and potentially may lead to new therapeutics

The skeleton: a multi-functional complex organ: new insights into osteoblasts and their role in bone formation: the central role of PI3Kinase.

Studies on bone development, formation and turnover have grown exponentially over the last decade in part because of the utility of genetic models. One area that has received considerable attention has been the phosphatidylinositol 3-kinase (PI3K) signaling pathway, which has emerged as a major survival network for osteoblasts. Genetic engineering has enabled investigators to study downstream effectors of PI3K by directly overexpressing activated forms of AKT in cells of the skeletal lineage or deleting Pten that leads to a constitutively active AKT. The results from these studies have provided novel insights into bone development and remodeling, critical processes in the lifelong maintenance of skeletal health. This paper reviews those data in relation to recent advances in osteoblast biology and their potential relevance to chronic disorders of the skeleton and their treatment

Bone as an endocrine organ.

OBJECTIVE: To review the recent evidence that has emerged supporting the role of bone as an endocrine organ. METHODS: This review will detail how bone has emerged as a bona fide endocrine gland, and with that, the potential therapeutic implications that could be realized for this hormone-secreting tissue by detailing the evidence in the literature supporting this view. RESULTS: The recent advances point to the skeleton as an endocrine organ that modulates glucose tolerance and testosterone production by secretion of the bone-specific protein osteocalcin. CONCLUSIONS: Bone has classically been viewed as an inert structure that is necessary for mobility, calcium homeostasis, and maintenance of the hematopoietic niche. Recent advances in bone biology using complex genetic manipulations in mice have highlighted the importance of bone not only as a structural scaffold to support the human body, but also as a regulator of a number of metabolic processes that are independent of mineral metabolism

Intracellular lipid droplets support osteoblast function.

Bone formation is an osteoblast-specific process characterized by high energy demands due to the secretion of matrix proteins and mineralization vesicles. While glucose has been reported as the principle fuel source for osteoblasts, recent evidence supports the tenet that osteoblasts can utilize fatty acids as well. Although the ability to accumulate lipid droplets has been demonstrated in many cell types, there has been little evidence that osteoblasts possess this characteristic. The current study provides evidence that osteoblastogenesis is associated with lipid droplet accumulation capable of supplying energy substrates (fatty acids) required for the differentiation process. Understanding the role of fatty acids in metabolic programming of the osteoblast may lead to novel approaches to increase bone formation and ultimately bone mass

Bioenergetics during calvarial osteoblast differentiation reflect strain differences in bone mass.

Osteoblastogenesis is the process by which mesenchymal stem cells differentiate into osteoblasts that synthesize collagen and mineralize matrix. The pace and magnitude of this process are determined by multiple genetic and environmental factors. Two inbred strains of mice, C3H/HeJ and C57BL/6J, exhibit differences in peak bone mass and bone formation. Although all the heritable factors that differ between these strains have not been elucidated, a recent F1 hybrid expression panel (C3H × B6) revealed major genotypic differences in osteoblastic genes related to cellular respiration and oxidative phosphorylation. Thus, we hypothesized that the metabolic rate of energy utilization by osteoblasts differed by strain and would ultimately contribute to differences in bone formation. In order to study the bioenergetic profile of osteoblasts, we measured oxygen consumption rates (OCR) and extracellular acidification rates (ECAR) first in a preosteoblastic cell line MC3T3-E1C4 and subsequently in primary calvarial osteoblasts from C3H and B6 mice at days 7, 14, and 21 of differentiation. During osteoblast differentiation in media containing ascorbic acid and β-glycerophosphate, all 3 cell types increased their oxygen consumption and extracellular acidification rates compared with the same cells grown in regular media. These increases are sustained throughout differentiation. Importantly, C3H calvarial osteoblasts had greater oxygen consumption rates than B6 consistent with their in vivo phenotype of higher bone formation. Interestingly, osteoblasts utilized both oxidative phosphorylation and glycolysis during the differentiation process although mature osteoblasts were more dependent on glycolysis at the 21-day time point than oxidative phosphorylation. Thus, determinants of oxygen consumption reflect strain differences in bone mass and provide the first evidence that during collagen synthesis osteoblasts use both glycolysis and oxidative phosphorylation to synthesize and mineralize matrix

Energy metabolism of the osteoblast: implications for osteoporosis.

Osteoblasts, the bone-forming cells of the remodeling unit, are essential for growth and maintenance of the skeleton. Clinical disorders of substrate availability (e.g., diabetes mellitus, anorexia nervosa, and aging) cause osteoblast dysfunction, ultimately leading to skeletal fragility and osteoporotic fractures. Conversely, anabolic treatments for osteoporosis enhance the work of the osteoblast by altering osteoblast metabolism. Emerging evidence supports glycolysis as the major metabolic pathway to meet ATP demand during osteoblast differentiation. Glut1 and Glut3 are the principal transporters of glucose in osteoblasts, although Glut4 has also been implicated. Wnt signaling induces osteoblast differentiation and activates glycolysis through mammalian target of rapamycin, whereas parathyroid hormone stimulates glycolysis through induction of insulin-like growth factor-I. Glutamine is an alternate fuel source for osteogenesis via the tricarboxylic acid cycle, and fatty acids can be metabolized to generate ATP via oxidative phosphorylation although temporal specificity has not been established. More studies with new model systems are needed to fully understand how the osteoblast utilizes fuel substrates in health and disease and how that impacts metabolic bone diseases