Lipolysis supports bone formation by providing osteoblasts with endogenous fatty acid substrates to maintain bioenergetic status.

Bone formation is a highly energy-demanding process that can be impacted by metabolic disorders. Glucose has been considered the principal substrate for osteoblasts, although fatty acids are also important for osteoblast function. Here, we report that osteoblasts can derive energy from endogenous fatty acids stored in lipid droplets via lipolysis and that this process is critical for bone formation. As such, we demonstrate that osteoblasts accumulate lipid droplets that are highly dynamic and provide the molecular mechanism by which they serve as a fuel source for energy generation during osteoblast maturation. Inhibiting cytoplasmic lipolysis leads to both an increase in lipid droplet size in osteoblasts and an impairment in osteoblast function. The fatty acids released by lipolysis from these lipid droplets become critical for cellular energy production as cellular energetics shifts towards oxidative phosphorylation during nutrient-depleted conditions. In vivo, conditional deletion of the ATGL-encoding gene Pnpla2 in osteoblast progenitor cells reduces cortical and trabecular bone parameters and alters skeletal lipid metabolism. Collectively, our data demonstrate that osteoblasts store fatty acids in the form of lipid droplets, which are released via lipolysis to support cellular bioenergetic status when nutrients are limited. Perturbations in this process result in impairment of bone formation, specifically reducing ATP production and overall osteoblast function.

Altered Osteoblast Metabolism with Aging Results in Lipid Accumulation and Oxidative Stress Mediated Bone Loss.

Cellular aging is associated with dysfunction of numerous tissues affecting multiple organ systems. A striking example of this is related to age-related bone loss, or osteoporosis, increasing fracture incidence. Interestingly, the two compartments of bone, cortical and cancellous or trabecular, rely on different mechanisms for development and maintenance during 'normal' aging. At a cellular level, the aging process disturbs a multitude of intracellular pathways. In particular, alterations in cellular metabolic functions thereby impacting cellular bioenergetics have been implicated in multiple tissues. Therefore, this study aimed to characterize how metabolic processes were altered in bone forming osteoblasts in aged mice compared to young mice. Metabolic flux analyses demonstrated both stromal cells and mature, matrix secreting osteoblasts from aged mice exhibited mitochondrial dysfunction. This was also accompanied by a lack of adaptability or metabolic flexibility to utilize exogenous substrates compared to osteoblasts cultured from young mice. Additionally, lipid droplets accumulated in both early stromal cells and mature osteoblasts from aged mice, which was further depicted as increased lipid content within the bone cortex of aged mice. Global transcriptomic analysis of the bone further supported these metabolic data as enhanced oxidative stress genes were up-regulated in aged mice, while osteoblast-related genes were down-regulated when compared to the young mice. Collectively, these data suggest that aging results in altered osteoblast metabolic handling of both exogenous and endogenous substrates which could contribute to age-related osteoporosis.

Endocrinal metabolic regulation on the skeletal system in post-menopausal women.

Osteoporosis is a common endocrinologic disorder characterized as a chronic bone loss condition. Sexual dimorphism is ubiquitous in the incidence of osteoporosis with post-menopausal women being acutely affected. Gonadal sex hormones including estrogen act as crucial regulators of bone mass; therefore, loss of such hormones leads to an imbalance in skeletal turnover leading to osteoporosis. Estrogen can influence both bone formation as well as resorption by reducing osteoblast activity and enhancing osteoclastogenesis. Additionally, estrogen is a potent regulator of systemic metabolism. Recent studies have provided clues that estrogenic effect on bone might also involve alterations in bone cell metabolism and bioenergetic potential. While direct effects of gonadal hormones ability to alter intracellular metabolism of bone cells has not been studied, there is precedence within the literature that this is occurring and contributing to post-menopausal bone loss. This review aims to serve as a perspective piece detailing the prospective role of gonadal hormones regulating bone cell metabolic potential.

Using Real-Time Cell Metabolic Flux Analyzer to Monitor Osteoblast Bioenergetics.

Bone formation by osteoblasts is an essential process for proper bone acquisition and bone turnover to maintain skeletal homeostasis, and ultimately, prevent fracture. In the interest to both optimize peak bone mass and combat various musculoskeletal diseases (i.e., post-menopausal osteoporosis, anorexia nervosa, type 1 and 2 diabetes mellitus), incredible efforts have been made in the field of bone biology to fully characterize osteoblasts throughout their differentiation process. Given the primary role of mature osteoblasts to secrete matrix proteins and mineralization vesicles, it has been noted that these processes take an incredible amount of cellular energy, or adenosine triphosphate (ATP). The overall cellular energy status is often referred to as cellular bioenergetics, and it includes a series of metabolic reactions that sense substrate availability to derive ATP to meet cellular needs. Therefore, the current method details the process of isolating primary, murine bone marrow stromal cells (BMSCs) and monitoring their bioenergetic status using the Real-time cell metabolic flux analyzer at various stages in osteoblast differentiation. Importantly, these data have demonstrated that the metabolic profile changes dramatically throughout osteoblast differentiation. Thus, using this physiologically relevant cell type is required to fully appreciate how a cell's bioenergetic status can regulate the overall function.

Bone marrow adipocytes - Good, bad, or just different?

Skeletal remodeling is essential for proper maintenance of adult bone mass, and due to its heavy energetic demands this process is closely tied to whole body metabolic. Thus, bone formation by the osteoblast, bone resorption by the osteoclast, and mechano-sensing by the osteocyte, are highly coupled processes that are essential for bone turnover. When one experiences a disruption in these processes, over time increased skeletal fragility and fracture can result. In addition to these primary cells, secondary cells within the skeletal niche are suspected to directly coordinate bone health as well. The bone marrow compartment provides a unique microenvironment in which communication occurs between white blood cells, red blood cells, platelets, and immune cells, in addition to classic bone cells (osteoblasts, osteoclasts, and osteocytes) that can both directly and indirectly impact skeletal homeostasis. One such cell population that has attracted much attention and scientific inquiry in the past decade are bone marrow adipocytes (BMAdipo) which can be found interspersed throughout the marrow compartment, and collectively are often referred to as bone marrow adipose tissue (BMAT). Although our understanding of BMAT has advanced significantly in the past decade, many questions remain relative to their lineage and function. The current collection presents recent studies and the up-to-date understanding of bone marrow adipocytes, along with future clinical perspectives related to this unique depot.

High throughput detection and genetic epidemiology of SARS-CoV-2 using COVIDSeq next-generation sequencing.

The rapid emergence of coronavirus disease 2019 (COVID-19) as a global pandemic affecting millions of individuals globally has necessitated sensitive and high-throughput approaches for the diagnosis, surveillance, and determining the genetic epidemiology of SARS-CoV-2. In the present study, we used the COVIDSeq protocol, which involves multiplex-PCR, barcoding, and sequencing of samples for high-throughput detection and deciphering the genetic epidemiology of SARS-CoV-2. We used the approach on 752 clinical samples in duplicates, amounting to a total of 1536 samples which could be sequenced on a single S4 sequencing flow cell on NovaSeq 6000. Our analysis suggests a high concordance between technical duplicates and a high concordance of detection of SARS-CoV-2 between the COVIDSeq as well as RT-PCR approaches. An in-depth analysis revealed a total of six samples in which COVIDSeq detected SARS-CoV-2 in high confidence which were negative in RT-PCR. Additionally, the assay could detect SARS-CoV-2 in 21 samples and 16 samples which were classified inconclusive and pan-sarbeco positive respectively suggesting that COVIDSeq could be used as a confirmatory test. The sequencing approach also enabled insights into the evolution and genetic epidemiology of the SARS-CoV-2 samples. The samples were classified into a total of 3 clades. This study reports two lineages B.1.112 and B.1.99 for the first time in India. This study also revealed 1,143 unique single nucleotide variants and added a total of 73 novel variants identified for the first time. To the best of our knowledge, this is the first report of the COVIDSeq approach for detection and genetic epidemiology of SARS-CoV-2. Our analysis suggests that COVIDSeq could be a potential high sensitivity assay for the detection of SARS-CoV-2, with an additional advantage of enabling the genetic epidemiology of SARS-CoV-2.

Quantitative Lipid Droplet Proteomics Reveals Mycobacterium tuberculosis Induced Alterations in Macrophage Response to Infection.

Growing evidence suggests the importance of lipid metabolism in pathogenesis of tuberculosis. Neutral lipids form the majority of lipids in a caseous granuloma, a pathology characteristic of tuberculosis. Cytosolic lipid droplets (LDs) of macrophages form the store house of these lipids and have been demonstrated to contribute to the inflammatory response to infection. The proteome of lipid droplets reflects the mechanisms of lipid metabolism active under a condition. However, infection induced changes in the proteome of these dynamic organelles remains elusive. Here, we employed quantitative proteomics to identify alterations induced upon infection with live Mycobacterium tuberculosis (Mtb) in comparison with heat killed bacilli or uninfected macrophages. We found increased abundance of proteins coupled with lipid metabolism, protein synthesis, and vesicular transport function in LDs upon infection with live Mtb. Using biochemical methods and microscopy, we validated ADP-ribosyltransferase (Arf)-like 8 (ARL8B) to be increased on the lipid droplet surface of live Mtb infected macrophages and that ARL8B is a bonafide LD protein. This study provides the first proteomic evidence that the dynamic responses to infection also encompass changes at the level of LDs. This information will be important in understanding how Mtb manipulates lipid metabolism and defense mechanisms of the host macrophage.

Necrosis Driven Triglyceride Synthesis Primes Macrophages for Inflammation During Mycobacterium tuberculosis Infection.

Pulmonary tuberculosis (TB) exhibits granulomatous inflammation, a site of controlling bacterial dissemination at the cost of host tissue damage. Intrigued by the granuloma type-dependent expression of inflammatory markers in TB, we sought to investigate underlying metabolic changes that drive amplification of inflammation in TB. Here, we show an association of higher inflammation in necrotic granulomas with the presence of triglyceride (TG)-rich foamy macrophages. The conspicuous absence of these macrophages in solid granulomas identified a link between the ensuing pathology and the metabolic programming of foamy macrophages. Consistent with in vivo findings, in vitro infection of macrophages with Mycobacterium tuberculosis (Mtb) led to increase in TG synthesis only under conditions of ~60% necrosis. Genetic and pharmacologic intervention that reduced necrosis prevented this bystander response. We further demonstrate that necrosis independent of Mtb also elicits the same bystander response in human macrophages. We identified a role for the human enzyme involved in TG synthesis, diacylglycerol O-acyltransferase (DGAT1), in this phenomenon. The increased TG levels in necrosis-associated foamy macrophages promoted the pro-inflammatory state of macrophages to infection while silencing expression of diacylglycerol O-acyltransferase (DGAT1) suppressed expression of pro-inflammatory genes. Our data thus invoke a role for storage lipids in the heightened host inflammatory response during infection-associated necrosis. Our data provide a functional role to macrophage lipid droplets in host defense and open new avenues for developing host-directed therapies against TB.

Phospholipid homeostasis, membrane tenacity and survival of Mtb in lipid rich conditions is determined by MmpL11 function.

The mycobacterial cell wall is a chemically complex array of molecular entities that dictate the pathogenesis of Mycobacterium tuberculosis. Biosynthesis and maintenance of this dynamic entity in mycobacterial physiology is still poorly understood. Here we demonstrate a requirement for M. tuberculosis MmpL11 in the maintenance of the cell wall architecture and stability in response to surface stress. In the presence of a detergent like Tyloxapol, a mmpL11 deletion mutant suffered from a severe growth attenuation as a result of altered membrane polarity, permeability and severe architectural damages. This mutant failed to tolerate permissible concentrations of cis-fatty acids suggesting its increased sensitivity to surface stress, evident as smaller colonies of the mutant outgrown from lipid rich macrophage cultures. Additionally, loss of MmpL11 led to an altered cellular fatty acid flux in the mutant: reduced incorporation into membrane cardiolipin was associated with an increased flux into the cellular triglyceride pool. This increase in storage lipids like triacyl glycerol (TAG) was associated with the altered metabolic state of higher dormancy-associated gene expression and decreased sensitivity to frontline TB drugs. This study provides a detailed mechanistic insight into the function of mmpL11 in stress adaptation of mycobacteria.

Adipocyte Model of Mycobacterium tuberculosis Infection Reveals Differential Availability of Iron to Bacilli in the Lipid-Rich Caseous Environment.

Mycobacterium tuberculosis, a successful human pathogen, utilizes multiple carbon sources from the host but adapts to a fatty-acid-rich environment in vivo We sought to delineate the physiologic response of M. tuberculosis to a lipid-rich environment by using differentiated adipocytes as a model system. Global transcriptome profiling based on RNA sequencing was performed for bacilli from infected adipocytes and preadipocytes. Genes involved in de novo fatty acid synthesis were downregulated, while those predicted to be involved in triglyceride biosynthesis were upregulated, in bacilli isolated from adipocytes, indicating reliance on host-derived fatty acids. Transcription factor network analysis indicated suppression of IdeR-regulated genes, suggesting decreased iron uptake by M. tuberculosis in the adipocyte model. This suppression of iron uptake coincided with higher ferritin and iron levels in adipocytes than in preadipocytes. In accord with the role of iron in mediating oxidative stress, we observed upregulation of genes involved in mitigating oxidative stress in M. tuberculosis isolated from adipocytes. We provide evidence that oleic acid, a major host-derived fatty acid, helps reduce the bacterial cytoplasm, thereby providing a safe haven for an M. tuberculosis mutant that is sensitive to iron-mediated oxidative stress. Via an independent mechanism, host ferritin is also able to rescue the growth of this mutant. Our work highlights the inherent synergy between macronutrients and micronutrients of the host environment that converge to provide resilience to the pathogen. This complex synergy afforded by the adipocyte model of infection will aid in the identification of genes required by M. tuberculosis in a caseous host environment.