Sexual dimorphism of osteoclast reliance on mitochondrial oxidation of energy substrates in the mouse.

Osteoclasts specialize in bone resorption and are critical for bone remodeling. Previous studies have shown that osteoclasts possess abundant mitochondria and derive most energy through oxidative phosphorylation (OXPHOS). However, the energy substrates fueling OXPHOS in osteoclasts remain to be fully defined. Here, we showed that osteoclast differentiation was coupled with increased oxidation of glucose, glutamine, and oleate. Transcriptomic analyses with RNA sequencing revealed marked upregulation of genes participating in OXPHOS and mitochondrial fatty acid oxidation, during osteoclast differentiation. Increased mitochondrial oxidation of long-chain fatty acids was required for osteoclast differentiation in vitro. However, blocking fatty acid oxidation in vivo, by deletion of carnitine palmitoyltransferase 1a (Cpt1a) in osteoclast progenitors, impaired osteoclast formation only in the female mice. The Cpt1a-deficient females were further protected from osteoclast activation by a high-fat diet. The males, on the contrary, exhibited normal bone resorption despite Cpt1a deletion, regardless of the dietary fat content. Moreover, concurrent deletion of mitochondrial pyruvate carrier 1 and Cpt1a, blocking mitochondrial oxidation of both glucose and fatty acids in the osteoclast lineage, failed to impede bone resorption in the males. The study therefore uncovers a female-specific dependence on mitochondrial oxidation of fatty acids and glucose in osteoclasts in vivo.

Genetic activation of glycolysis in osteoblasts preserves bone mass in type I diabetes.

Type I diabetes (T1D) impairs bone accrual in patients, but the mechanism is unclear. Here in a murine monogenic model for T1D, we demonstrate that diabetes suppresses bone formation resulting in a rapid loss of both cortical and trabecular bone. Single-cell RNA sequencing uncovers metabolic dysregulation in bone marrow osteogenic cells of diabetic mice. In vivo stable isotope tracing reveals impaired glycolysis in diabetic bone that is highly responsive to insulin stimulation. Remarkably, deletion of the insulin receptor reduces cortical but not trabecular bone. Increasing glucose uptake by overexpressing Glut1 in osteoblasts exacerbates bone defects in T1D mice. Conversely, activation of glycolysis by Pfkfb3 overexpression preserves both trabecular and cortical bone mass in the face of diabetes. The study identifies defective glucose metabolism in osteoblasts as a pathogenic mechanism for osteopenia in T1D, and furthermore implicates boosting osteoblast glycolysis as a potential bone anabolic therapy.

Assessing Energy Substrate Oxidation In Vitro with 14CO2 Trapping.

Mitochondria host the machinery for the tricarboxylic acid (TCA) cycle and electron transport chain (ETC), which generate adenosine triphosphate (ATP) to maintain energy homeostasis. Glucose, fatty acids, and amino acids are the major energy substrates fueling mitochondrial respiration in most somatic cells. Evidence shows that different cell types may have a distinct preference for certain substrates. However, substrate utilization by various cells in the skeleton has not been studied in detail. Moreover, as cellular metabolism is attuned to physiological and pathophysiological changes, direct assessments of substrate dependence in skeletal cells may provide important insights into the pathogenesis of bone diseases. The following protocol is based on the principle of carbon dioxide release from substrate molecules following oxidative phosphorylation. By using substrates containing radioactively labeled carbon atoms (14C), the method provides a sensitive and easy-to-use assay for the rate of substrate oxidation in cell culture. A case study with primary calvarial preosteoblasts versus bone marrow-derived macrophages (BMMs) demonstrates different utilization of the main substrates between the two cell types.