Impaired glucose metabolism underlies articular cartilage degeneration in osteoarthritis.

Osteoarthritis (OA) is the leading joint disease characterized by cartilage destruction and loss of mobility. Accumulating evidence indicates that the incidence and severity of OA increases with diabetes, implicating systemic glucose metabolism in joint health. However, a definitive link between cellular metabolism in articular cartilage and OA pathogenesis is not yet established. Here, we report that in mice surgically induced to develop knee OA through destabilization of medial meniscus (DMM), expression of the main glucose transporter Glut1 is notably reduced in joint cartilage. Inducible deletion of Glut1 specifically in the Prg4-expressing articular cartilage accelerates cartilage loss in DMM-induced OA. Conversely, forced expression of Glut1 protects against cartilage destruction following DMM. Moreover, in mice with type I diabetes, both Glut1 expression and the rate of glycolysis are diminished in the articular cartilage, and the diabetic mice exhibit more severe cartilage destruction than their nondiabetic counterparts following DMM. The results provide proof of concept that boosting glucose metabolism in articular chondrocytes may ameliorate cartilage degeneration in OA.

Germline specification and axis determination in viviparous and oviparous pea aphids: conserved and divergent features.

Aphids are hemimetabolous insects that undergo incomplete metamorphosis without pupation. The annual life cycle of most aphids includes both an asexual (viviparous) and a sexual (oviparous) phase. Sexual reproduction only occurs once per year and is followed by many generations of asexual reproduction, during which aphids propagate exponentially with telescopic development. Here, we discuss the potential links between viviparous embryogenesis and derived developmental features in the pea aphid Acyrthosiphon pisum, particularly focusing on germline specification and axis determination, both of which are key events of early development in insects. We also discuss potential evolutionary paths through which both viviparous and oviparous females might have come to utilize maternal germ plasm to drive germline specification. This developmental strategy, as defined by germline markers, has not been reported in other hemimetabolous insects. In viviparous females, furthermore, we discuss whether molecules that in other insects characterize germ plasm, like Vasa, also participate in posterior determination and how the anterior localization of the hunchback orthologue Ap-hb establishes the anterior-posterior axis. We propose that the linked chain of developing oocytes and embryos within each ovariole and the special morphology of early embryos might have driven the formation of evolutionary novelties in germline specification and axis determination in the viviparous aphids. Moreover, based upon the finding that the endosymbiont Buchnera aphidicola is closely associated with germ cells throughout embryogenesis, we propose presumptive roles for B. aphidicola in aphid development, discussing how it might regulate germline migration in both reproductive modes of pea aphids. In summary, we expect that this review will shed light on viviparous as well as oviparous development in aphids.

Ultra-low frequency labeling of single cell lineages in Drosophila.

We describe a new methodology for genetically labeling single cell lineages in Drosophila called DMARCM. The system offers ultra-low frequency labeling, linear induction, consistent labeling among individuals and virtually no background signal. We compare this technique to an existing approach, which has been widely adopted. We demonstrate how application of DMARCM in the gastrointestinal epithelium permits the effects of labeling frequency on tumorigenic stem cell growth to be distinguished in an established tumor model.

Both aerobic glycolysis and mitochondrial respiration are required for osteoclast differentiation.

Excessive bone resorption over bone formation is the root cause for bone loss leading to osteoporotic fractures. Development of new antiresorptive therapies calls for a holistic understanding of osteoclast differentiation and function. Although much has been learned about the molecular regulation of osteoclast biology, little is known about the metabolic requirement and bioenergetics during osteoclastogenesis. Here, we report that glucose metabolism through oxidative phosphorylation (OXPHOS) is the predominant bioenergetic pathway to support osteoclast differentiation. Meanwhile, increased lactate production from glucose, known as aerobic glycolysis when oxygen is abundant, is also critical for osteoclastogenesis. Genetic deletion of Glut1 in osteoclast progenitors reduces aerobic glycolysis without compromising OXPHOS, but nonetheless diminishes osteoclast differentiation in vitro. Glut1 deficiency in the progenitors leads to osteopetrosis due to fewer osteoclasts specifically in the female mice. Thus, Glut1-mediated glucose metabolism through both lactate production and OXPHOS is necessary for normal osteoclastogenesis.

Targeting HR Repair as a Synthetic Lethal Approach to Increase DNA Damage Sensitivity by a RAD52 Inhibitor in BRCA2-Deficient Cancer Cells.

BRCA mutation, one of the most common types of mutations in breast and ovarian cancer, has been suggested to be synthetically lethal with depletion of RAD52. Pharmacologically inhibiting RAD52 specifically eradicates BRCA-deficient cancer cells. In this study, we demonstrated that curcumin, a plant polyphenol, sensitizes BRCA2-deficient cells to CPT-11 by impairing RAD52 recombinase in MCF7 cells. More specifically, in MCF7-siBRCA2 cells, curcumin reduced homologous recombination, resulting in tumor growth suppression. Furthermore, a BRCA2-deficient cell line, Capan1, became resistant to CPT-11 when BRCA2 was reintroduced. In vivo, xenograft model studies showed that curcumin combined with CPT-11 reduced the growth of BRCA2-knockout MCF7 tumors but not MCF7 tumors. In conclusion, our data indicate that curcumin, which has RAD52 inhibitor activity, is a promising candidate for sensitizing BRCA2-deficient cells to DNA damage-based cancer therapies.

Increased glycolysis mediates Wnt7b-induced bone formation.

Wingless/integrated (Wnt) signaling has emerged as a major mechanism for promoting bone formation and a target pathway for developing bone anabolic agents against osteoporosis. However, the downstream events mediating the potential therapeutic effect of Wnt proteins are not fully understood. Previous studies have indicated that increased glycolysis is associated with osteoblast differentiation in response to Wnt signaling, but direct genetic evidence for the importance of glucose metabolism in Wnt-induced bone formation is lacking. Here, we have generated compound transgenic mice to overexpress Wnt family member 7B (Wnt7b) transiently in the osteoblast lineage of postnatal mice, with or without concurrent deletion of the glucose transporter 1 (Glut1), also known as solute carrier family 2, facilitated glucose transporter member 1. Overexpression of Wnt7b in 1-mo-old mice for 1 wk markedly stimulated bone formation, but the effect was essentially abolished without Glut1, even though transient deletion of Glut1 itself did not affect normal bone accrual. Consistent with the in vivo results, Wnt7b increased Glut1 expression and glucose consumption in the primary culture of osteoblast lineage cells, and deletion of Glut1 diminished osteoblast differentiation in vitro. Thus, Wnt7b promotes bone formation in part through stimulating glucose metabolism in osteoblast lineage cells.-Chen, H., Ji, X., Lee, W.-C., Shi, Y., Li, B., Abel, E. D., Jiang, D., Huang, W., Long, F. Increased glycolysis mediates Wnt7b-induced bone formation.

Dual function of Bmpr1a signaling in restricting preosteoblast proliferation and stimulating osteoblast activity in mouse.

Exogenous bone morphogenetic proteins (Bmp) are well known to induce ectopic bone formation, but the physiological effect of Bmp signaling on normal bone is not completely understood. By deleting the receptor Bmpr1a in osteoblast lineage cells with Dmp1-Cre, we observed a dramatic increase in trabecular bone mass in postnatal mice, which was due to a marked increase in osteoblast number that was likely to be driven by hyperproliferation of Sp7(+) preosteoblasts. Similarly, inducible deletion of Bmpr1a in Sp7(+) cells specifically in postnatal mice increased trabecular bone mass. However, deletion of Smad4 by the same approaches had only a minor effect, indicating that Bmpr1a signaling suppresses trabecular bone formation through effectors beyond Smad4. Besides increasing osteoblast number in the trabecular bone, deletion of Bmpr1a by Dmp1-Cre also notably reduced osteoblast activity, resulting in attenuation of periosteal bone growth. The impairment in osteoblast activity correlated with reduced mTORC1 signaling in vivo, whereas inhibition of mTORC1 activity abolished the induction of protein anabolism genes by BMP2 treatment in vitro. Thus, physiological Bmpr1a signaling in bone exerts a dual function in both restricting preosteoblast proliferation and promoting osteoblast activity.

Reducing body fat with altitude hypoxia training in swimmers: role of blood perfusion to skeletal muscles.

Swimmers tend to have greater body fat than athletes from other sports. The purpose of the study was to examine changes in body composition after altitude hypoxia exposure and the role of blood distribution to the skeletal muscle in swimmers. With a constant training volume of 12.3 km/day, young male swimmers (N = 10, 14.8 ± 0.5 years) moved from sea-level to a higher altitude of 2,300 meters. Body composition was measured before and after translocation to altitude using dual-energy X-ray absorptiometry (DXA) along with 8 control male subjects who resided at sea level for the same period of time. To determine the effects of hypoxia on muscle blood perfusion, total hemoglobin concentration (THC) was traced by near-infrared spectroscopy (NIRS) in the triceps and quadriceps muscles under glucose-ingested and insulin-secreted conditions during hypoxia exposure (16% O2) after training. While no change in body composition was found in the control group, subjects who trained at altitude had unequivocally decreased fat mass (-1.7 ± 0.3 kg, -11.4%) with increased lean mass (+0.8 ± 0.2 kg, +1.5%). Arterial oxygen saturation significantly decreased with increased plasma lactate during hypoxia recovery mimicking 2,300 meters at altitude (~93% versus ~97%). Intriguingly, hypoxia resulted in elevated muscle THC, and sympathetic nervous activities occurred in parallel with greater-percent oxygen saturation in both muscle groups. In conclusion, the present study provides evidence that increased blood distribution to the skeletal muscle under postprandial condition may contribute to the reciprocally increased muscle mass and decreased body mass after a 3-week altitude exposure in swimmers.

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.

Zebrafish models for glucocorticoid-induced osteoporosis.

Glucocorticoid-induced osteoporosis (GIOP) is the most common form of secondary osteoporosis due to excessive or long-term glucocorticoid administration, disturbing the homeostasis between bone formation and bone resorption. The bone biology of zebrafish shares a high degree of similarities with mammals. In terms of molecular level, genes and signaling pathways related to skeletogenesis are also highly correlated between zebrafish and humans. Therefore, zebrafish have been utilized to develop multiple GIOP models. Taking advantage of the transparency of zebrafish larvae, their skeletal development and bone mineralization can be readily visualized through in vivo staining without invasive experimental handlings. Moreover, the feasibility of using scales or fin rays to study bone remodeling makes adult zebrafish an ideal model for GIOP research. Here, we reviewed current zebrafish models for GIOP research, focused on the tools and methods established for examining bone homeostasis. As an in vivo, convenient, and robust model, zebrafish have an advantage in performing high-throughput drug screening and could be used to investigate the action mechanisms of therapeutic drugs.

The flavonoid corylin exhibits lifespan extension properties in mouse.

In the long history of traditional Chinese medicine, single herbs and complex formulas have been suggested to increase lifespan. However, the identification of single molecules responsible for lifespan extension has been challenging. Here, we collected a list of traditional Chinese medicines with potential longevity properties from pharmacopeias. By utilizing the mother enrichment program, we systematically screened these traditional Chinese medicines and identified a single herb, Psoralea corylifolia, that increases lifespan in Saccharomyces cerevisiae. Next, twenty-two pure compounds were isolated from Psoralea corylifolia. One of the compounds, corylin, was found to extend the replicative lifespan in yeast by targeting the Gtr1 protein. In human umbilical vein endothelial cells, RNA sequencing data showed that corylin ameliorates cellular senescence. We also examined an in vivo mammalian model, and found that corylin extends lifespan in mice fed a high-fat diet. Taken together, these findings suggest that corylin may promote longevity.

JAK/STAT signaling coordinates stem cell proliferation and multilineage differentiation in the Drosophila intestinal stem cell lineage.

Adult stem cells are the most primitive cells of a lineage and are distinguished by the properties of self-renewal and multipotency. Coordinated control of stem cell proliferation and multilineage differentiation is essential to ensure a steady output of differentiated daughter cells necessary to maintain tissue homeostasis. However, little is known about the signals that coordinate stem cell proliferation and daughter cell differentiation. Here we investigate the role of the conserved JAK/STAT signaling pathway in the Drosophila intestinal stem cell (ISC) lineage. We show first, that JAK/STAT signaling is normally active in both ISCs and their newly formed daughters, but not in terminally differentiated enteroendocrine (ee) cells or enterocyte (EC) cells. Second, analysis of ISC lineages shows that JAK/STAT signaling is necessary but not sufficient for daughter cell differentiation, indicating that competence to undergo multilineage differentiation depends upon JAK/STAT. Finally, our analysis reveals JAK/STAT signaling to be a potent regulator of ISC proliferation, but not ISC self-renewal. On the basis of these findings, we suggest a model in which JAK/STAT signaling coordinates the processes of stem cell proliferation with the competence of daughter cells to undergo multilineage differentiation, ensuring a robust cellular output in the lineage.

Breast cancer-derived GM-CSF regulates arginase 1 in myeloid cells to promote an immunosuppressive microenvironment.

Tumor-infiltrating myeloid cells contribute to the development of the immunosuppressive tumor microenvironment. Myeloid cell expression of arginase 1 (ARG1) promotes a protumor phenotype by inhibiting T cell function and depleting extracellular l-arginine, but the mechanism underlying this expression, especially in breast cancer, is poorly understood. In breast cancer clinical samples and in our mouse models, we identified tumor-derived GM-CSF as the primary regulator of myeloid cell ARG1 expression and local immune suppression through a gene-KO screen of breast tumor cell-produced factors. The induction of myeloid cell ARG1 required GM-CSF and a low pH environment. GM-CSF signaling through STAT3 and p38 MAPK and acid signaling through cAMP were required to activate myeloid cell ARG1 expression in a STAT6-independent manner. Importantly, breast tumor cell-derived GM-CSF promoted tumor progression by inhibiting host antitumor immunity, driving a significant accumulation of ARG1-expressing myeloid cells compared with lung and melanoma tumors with minimal GM-CSF expression. Blockade of tumoral GM-CSF enhanced the efficacy of tumor-specific adoptive T cell therapy and immune checkpoint blockade. Taken together, we show that breast tumor cell-derived GM-CSF contributes to the development of the immunosuppressive breast cancer microenvironment by regulating myeloid cell ARG1 expression and can be targeted to enhance breast cancer immunotherapy.

Malic Enzyme Couples Mitochondria with Aerobic Glycolysis in Osteoblasts.

The metabolic program of osteoblasts, the chief bone-making cells, remains incompletely understood. Here in murine calvarial cells, we establish that osteoblast differentiation under aerobic conditions is coupled with a marked increase in glucose consumption and lactate production but reduced oxygen consumption. As a result, aerobic glycolysis accounts for approximately 80% of the ATP production in mature osteoblasts. In vivo tracing with 13C-labeled glucose in the mouse shows that glucose in bone is readily metabolized to lactate but not organic acids in the TCA cycle. Glucose tracing in osteoblast cultures reveals that pyruvate is carboxylated to form malate integral to the malate-aspartate shuttle. RNA sequencing (RNA-seq) identifies Me2, encoding the mitochondrial NAD-dependent isoform of malic enzyme, as being specifically upregulated during osteoblast differentiation. Knockdown of Me2 markedly reduces the glycolytic flux and impairs osteoblast proliferation and differentiation. Thus, the mitochondrial malic enzyme functionally couples the mitochondria with aerobic glycolysis in osteoblasts.

Germ-plasm specification and germline development in the parthenogenetic pea aphid Acyrthosiphon pisum: Vasa and Nanos as markers.

The germarium, oocytes and embryos of the parthenogenetic viviparous pea aphid Acyrthosiphon pisum are contained within a single ovariole. This species provides an excellent model for studying how maternally-inherited germ plasm is specified and how it is transferred to primordial germ cells. Previous studies have shown that germ cells are first segregated at the embryonic posterior after formation of the blastoderm. We used two cross-reacting antibodies against the conserved germline markers Vasa and Nanos, which specifically identified these presumptive germ cells, to investigate whether germ cells were determined during early development. We observed randomly-distributed weak expression of Vasa signals in the developing oocyte but no localization in the oocyte segregated from the germarium. Localized Vasa was not apparent until it was detected at the posterior in the embryo undergoing the second nuclear division. Nanos, on the other hand, was localized to a nuage-like structure surrounding the nucleus in the developing and segregated oocytes. At the beginning of the oocyte maturation division, Nanos localization shifted to the posterior and could be identified in successive stages until it was incorporated into the germ cells. Taken together, our results suggest that germ plasm is specified in the developing oocyte and that Nanos is an earlier germline marker than Vasa. Germ cells stained for Vasa remained at a dorsal location in the egg during mid-development and then were guided into abdominal segments A1 to A6 during germ-band retraction. We infer that germ cells coalesce with segmented gonadal mesoderm during this period.

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.

Gli1 identifies osteogenic progenitors for bone formation and fracture repair.

Bone formation in mammals requires continuous production of osteoblasts throughout life. A common molecular marker for all osteogenic mesenchymal progenitors has not been identified. Here, by lineage-tracing experiments in fetal or postnatal mice, we discover that Gli1+ cells progressively produce osteoblasts in all skeletal sites. Most notably, in postnatal growing mice, the Gli1+ cells residing immediately beneath the growth plate, termed here "metaphyseal mesenchymal progenitors" (MMPs), are essential for cancellous bone formation. Besides osteoblasts, MMPs also give rise to bone marrow adipocytes and stromal cells in vivo. RNA-seq reveals that MMPs express a number of marker genes previously assigned to mesenchymal stem/progenitor cells, including CD146/Mcam, CD44, CD106/Vcam1, Pdgfra, and Lepr. Genetic disruption of Hh signaling impairs proliferation and osteoblast differentiation of MMPs. Removal of ß-catenin causes MMPs to favor adipogenesis, resulting in osteopenia coupled with increased marrow adiposity. Finally, postnatal Gli1+ cells contribute to both chondrocytes and osteoblasts during bone fracture healing. Thus Gli1 marks mesenchymal progenitors responsible for both normal bone formation and fracture repair.

Exercise training exacerbates tourniquet ischemia-induced decreases in GLUT4 expression and muscle atrophy in rats.

The current study determined the interactive effects of ischemia and exercise training on glycogen storage and GLUT4 expression in skeletal muscle. For the first experiment, an acute 1-h tourniquet ischemia was applied to one hindlimb of both the 1-week exercise-trained and untrained rats. The contralateral hindlimb served as control. For the second experiment, 1-h ischemia was applied daily for 1 week to both trained (5 h post-exercise) and untrained rats. GLUT4 mRNA was not affected by acute ischemia, but exercise training lowered GLUT4 mRNA in the acute ischemic muscle. GLUT4 protein levels were elevated by exercise training, but not in the acute ischemic muscle. Exercise training elevated muscle glycogen above untrained levels, but this increase was reversed by chronic ischemia. GLUT4 mRNA and protein levels were dramatically reduced by chronic ischemia, regardless of whether the animals were exercise-trained or not. Chronic ischemia significantly reduced plantaris muscle mass, with a greater decrease found in the exercise-trained rats. In conclusion, the exercise training effect on muscle GLUT4 protein expression was prevented by acute ischemia. Furthermore, chronic ischemia-induced muscle atrophy was exacerbated by exercise training. This result implicates that exercise training could be detrimental to skeletal muscle with severely impaired microcirculation.

A possible link between exercise-training adaptation and dehydroepiandrosterone sulfate- an oldest-old female study.

The purpose of this study was to determine the association between the level of salivary dehydroepiandrosterone sulfate (DHEA-S) and the magnitude of adaptation to exercise training in insulin sensitivity for aged females. A group of 16 females, aged 80-93 years old, was divided into 2 groups according to their baseline DHEA-S levels: Lower Halves (N = 8) and Upper Halves (N = 8), and participated in a 4-month exercise intervention trial. Insulin response with an oral glucose tolerance test (OGTT), cholesterol, blood pressure (BP), motor performance, and DHEA-S were determined at baseline and 4 months after the training program. Glucose tolerance and body mass index (BMI) remained unchanged with training for both groups. Insulin, fasted cholesterol, diastolic blood pressure, reaction time, and locomotive function were significantly lowered by training only in the Upper Halves group. Changes in the area under curve of insulin (IAUC) were negatively correlated with the baseline DHEA-S level (R= - 0.60, P < 0.05). The current study provides the first evidence that oldest-old subjects with low DHEA-S level appear to be poor responders to exercise-training adaptations.

Gene duplication, gene loss and evolution of expression domains in the vertebrate nuclear receptor NR5A (Ftz-F1) family.

Fushi tarazu factor 1 (Ftz-F1, NR5A) is a zinc-finger transcription factor that belongs to the nuclear receptor superfamily and regulates genes that are involved in sterol and steroid metabolism in gonads, adrenals, liver and other tissues. To understand the evolutionary origins and developmental genetic relationships of the Ftz-F1 genes, we have cloned four homologous Ftz-f1 genes in zebrafish, called ff1a, ff1b, ff1c and ff1d. These four genes have different temporal and spatial expression patterns during development, indicating that they have distinct mechanisms of genetic regulation. Among them, the ff1a expression pattern is similar to mammalian Nr5a2, while the ff1b pattern is similar to that of mammalian Nr5a1. Genetic mapping experiments show that these four ff1 genes are located on chromosome segments conserved between the zebrafish and human genomes, indicating a common ancestral origin. Phylogenetic and conserved synteny analysis show that ff1a is the orthologue of NR5A2, and that ff1b and ff1d genes are co-orthologues of NR5A1 that arose by a gene-duplication event, probably a whole-genome duplication, in the ray-fin lineage, and each gene is located next to an NR6A1 co-orthologue as in humans, showing that the tandem duplication occurred before the divergence of human and zebrafish lineages. ff1c does not have a mammalian counterpart. Thus we have characterized the phylogenetic relationships, expression patterns and chromosomal locations of these Ftz-F1 genes, and have demonstrated their identities as NR5A genes in relation to the orthologous genes in other species.