Purification of functional mouse skeletal muscle mitochondria using percoll density gradient centrifugation.

Objective: Our goal was to isolate purified mitochondria from mouse skeletal muscle using a Percoll density gradient and to assess bioenergetic function and purity via Seahorse Extracellular Flux (XF) Analyses and mass spectrometry.

Results: Mitochondria isolated from murine quadriceps femoris skeletal muscle using a Percoll density gradient method allowed for minimally contaminated preparations with time from tissue harvest to mitochondrial isolation and quantification in about 3-4 h. Percoll purification from 100 to 200 mg fresh tissue yielded ~ 200-400 ug protein.

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.

A Novel ENU-Induced Mutation Causes Motor Deficits in Mice without Causing Peripheral Neuropathy.

Mitochondrial fission and fusion are required for maintaining functional mitochondria. The mitofusins (MFN1 and MFN2) are known for their roles in mediating mitochondrial fusion. Recently, MFN2 has been implicated in other important cellular functions, such as mitophagy, mitochondrial motility, and coordinating endoplasmic reticulum-mitochondria communication.

Purification of functional mouse skeletal muscle mitochondria using Percoll density gradient centrifugation.

Objective: Our goal was to isolate purified mitochondria from mouse skeletal muscle using a Percoll density gradient and to assess bioenergetic function and purity via Seahorse Extracellular Flux (XF) Analyses and mass spectrometry.

Results: Mitochondria isolated from murine quadriceps femoris skeletal muscle using a Percoll density gradient method allowed for minimally contaminated preparations with time from tissue harvest to mitochondrial isolation and quantification in about 3-4 hours. Percoll purification from 100-200 mg fresh tissue yielded ∼200-400 ug protein.

The mitophagy receptor BNIP3 is critical for the regulation of metabolic homeostasis and mitochondrial function in the nucleus pulposus cells of the intervertebral disc.

The contribution of mitochondria to the metabolic function of hypoxic NP cells has been overlooked. We have shown that NP cells contain networked mitochondria and that mitochondrial translocation of BNIP3 mediates hypoxia-induced mitophagy. However, whether BNIP3 also plays a role in governing mitochondrial function and metabolism in hypoxic NP cells is not known.

Spatial transcriptomics reveals metabolic changes underly age-dependent declines in digit regeneration.

De novo limb regeneration after amputation is restricted in mammals to the distal digit tip. Central to this regenerative process is the blastema, a heterogeneous population of lineage-restricted, dedifferentiated cells that ultimately orchestrates regeneration of the amputated bone and surrounding soft tissue. To investigate skeletal regeneration, we made use of spatial transcriptomics to characterize the transcriptional profile specifically within the blastema.

Propranolol Promotes Bone Formation and Limits Resorption Through Novel Mechanisms During Anabolic Parathyroid Hormone Treatment in Female C57BL/6J Mice.

Although the nonselective β-blocker, propranolol, improves bone density with parathyroid hormone (PTH) treatment in mice, the mechanism of this effect is unclear. To address this, we used a combination of in vitro and in vivo approaches to address how propranolol influences bone remodeling in the context of PTH treatment. In female C57BL/6J mice, intermittent PTH and propranolol administration had complementary effects in the trabecular bone of the distal femur and fifth lumbar vertebra (L ), with combination treatment achieving microarchitectural parameters beyond that of PTH alone.

Energy Metabolism of Osteocytes.

Purpose Of Review: In this review, we provide a recent update on bioenergetic pathways in osteocytes and identify potential future areas of research interest. Studies have identified a role for regulation of bone formation and bone resorption through osteocyte mechanosensing and osteocyte secreted factors. Nevertheless, there is a paucity of studies on the bioenergetics and energy metabolism of osteocytes, which are required for the regulation of bone remodeling.

A regulatory variant at 3q21.1 confers an increased pleiotropic risk for hyperglycemia and altered bone mineral density.

Skeletal and glycemic traits have shared etiology, but the underlying genetic factors remain largely unknown. To identify genetic loci that may have pleiotropic effects, we studied Genome-wide association studies (GWASs) for bone mineral density and glycemic traits and identified a bivariate risk locus at 3q21. Using sequence and epigenetic modeling, we prioritized an adenylate cyclase 5 (ADCY5) intronic causal variant, rs56371916.

Role of autophagy in intervertebral disc and cartilage function: implications in health and disease.

The intervertebral disc and cartilage are specialized, extracellular matrix-rich tissues critical for absorbing mechanical loads, providing flexibility to the joints, and longitudinal growth in the case of growth plate cartilage. Specialized niche conditions in these tissues, such as hypoxia, are critical in regulating cellular activities including autophagy, a lysosomal degradation pathway that promotes cell survival. Mounting evidence suggests that dysregulation of autophagic pathways underscores many skeletal pathologies affecting the spinal column, articular and growth plate cartilages.

Mitochondrial Function and Metabolism of Cultured Skeletal Cells.

Measuring cellular metabolism accurately is necessary to understand bioenergetic pathways in cells. The major ATP generating pathways in cells are oxidative phosphorylation and glycolysis. We have recently analyzed and published bioenergetic pathways active in osteoblasts undergoing differentiation in response to various substrates.

PHOSPHO1 is a skeletal regulator of insulin resistance and obesity.

Background: The classical functions of the skeleton encompass locomotion, protection and mineral homeostasis. However, cell-specific gene deletions in the mouse and human genetic studies have identified the skeleton as a key endocrine regulator of metabolism. The bone-specific phosphatase, Phosphatase, Orphan 1 (PHOSPHO1), which is indispensable for bone mineralisation, has been recently implicated in the regulation of energy metabolism in humans, but its role in systemic metabolism remains unclear.

Hypoxic Regulation of Mitochondrial Metabolism and Mitophagy in Nucleus Pulposus Cells Is Dependent on HIF-1α-BNIP3 Axis.

Nucleus pulposus (NP) cells reside in an avascular and hypoxic microenvironment of the intervertebral disc and are predominantly glycolytic due to robust HIF-1 activity. It is generally thought that NP cells contain few functional mitochondria compared with cells that rely on oxidative metabolism. Consequently, the contribution of mitochondria to NP cell metabolism and the role of hypoxia and HIF-1 in mitochondrial homeostasis is poorly understood.

Exploring mechanisms of increased cardiovascular disease risk with antipsychotic medications: Risperidone alters the cardiac proteomic signature in mice.

Atypical antipsychotic (AA) medications including risperidone (RIS) and olanzapine (OLAN) are FDA approved for the treatment of psychiatric disorders including schizophrenia, bipolar disorder and depression. Clinical side effects of AA medications include obesity, insulin resistance, dyslipidemia, hypertension and increased cardiovascular disease risk. Despite the known pharmacology of these AA medications, the mechanisms contributing to adverse metabolic side-effects are not well understood.

Sirtuin 3 deficiency does not impede digit regeneration in mice.

The mitochondrial deacetylase sirtuin 3 (SIRT3) is thought to be one of the main contributors to metabolic flexibility-promoting mitochondrial energy production and maintaining homeostasis. In bone, metabolic profiles are tightly regulated and the loss of SIRT3 has deleterious effects on bone volume in vivo and on osteoblast differentiation in vitro. Despite the prominent role of this protein in bone stem cell proliferation, metabolic activity, and differentiation, the importance of SIRT3 for regeneration after bone injury has never been reported.

Reproducibility of CRISPR-Cas9 methods for generation of conditional mouse alleles: a multi-center evaluation.

Background: CRISPR-Cas9 gene-editing technology has facilitated the generation of knockout mice, providing an alternative to cumbersome and time-consuming traditional embryonic stem cell-based methods. An earlier study reported up to 16% efficiency in generating conditional knockout (cKO or floxed) alleles by microinjection of 2 single guide RNAs (sgRNA) and 2 single-stranded oligonucleotides as donors (referred herein as "two-donor floxing" method).

Results: We re-evaluate the two-donor method from a consortium of 20 laboratories across the world.

The mitophagy receptor Bcl-2-like protein 13 stimulates adipogenesis by regulating mitochondrial oxidative phosphorylation and apoptosis in mice.

Metabolic programming of bone marrow stromal cells (BMSCs) could influence the function of progenitor osteoblasts or adipocytes and hence determine skeletal phenotypes. Adipocytes predominantly utilize oxidative phosphorylation, whereas osteoblasts use glycolysis to meet ATP demand. Here, we compared progenitor differentiation from the marrow of two inbred mouse strains, C3H/HeJ (C3H) and C57BL6J (B6).

A novel mouse model overexpressing Nocturnin results in decreased fat mass in male mice.

Nocturnin (NOCT) belongs to the Mg dependent Exonucleases, Endonucleases, Phosphatase (EEP) family of enzymes that exhibit various functions in vitro and in vivo. NOCT is known to function as a deadenylase, cleaving poly-A tails from mRNA (messenger RNA) transcripts. Previously, we reported a role for NOCT in regulating bone marrow stromal cell differentiation through its interactions with PPARγ.

A Renewable Source of Human Beige Adipocytes for Development of Therapies to Treat Metabolic Syndrome.

Molecular- and cellular-based therapies have the potential to reduce obesity-associated disease. In response to cold, beige adipocytes form in subcutaneous white adipose tissue and convert energy stored in metabolic substrates to heat, making them an attractive therapeutic target. We developed a robust method to generate a renewable source of human beige adipocytes from induced pluripotent stem cells (iPSCs).

Progenitor recruitment and adipogenic lipolysis contribute to the anabolic actions of parathyroid hormone on the skeleton.

Intermittent administration of parathyroid hormone (PTH) stimulates bone formation in vivo and also suppresses the volume of bone marrow adipose tissue (BMAT). In contrast, a calorie-restricted (CR) diet causes bone loss and induces BMAT in both mice and humans. We used the CR model to test whether PTH would reduce BMAT in mice by both altering cell fate and inducing lipolysis of marrow adipocytes.

Energy Metabolism of Bone.

Biological processes utilize energy and therefore must be prioritized based on fuel availability. Bone is no exception to this, and the benefit of remodeling when necessary outweighs the energy costs. Bone remodeling is important for maintaining blood calcium homeostasis, repairing micro cracks and fractures, and modifying bone structure so that it is better suited to withstand loading demands.

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.