Specific ATPases drive compartmentalized glycogen utilization in rat skeletal muscle

Glycogen is a key energy substrate in excitable tissue and especially in skeletal muscle fibers it contributes with a substantial, but also local energy production. A heterogenic subcellular distribution of three distinct glycogen pools in skeletal muscle is proved by transmission electron microscopy (TEM), which is thought to represent the requirements for local energy stores at the subcellular level. Here, we show that the three main energy-consuming ATPases in skeletal muscles (Ca2+-, Na+,K+-, and myosin ATPases) utilize different local pools of glycogen. These results clearly demonstrate compartmentalized glycogen metabolism and emphasize that spatially distinct pools of glycogen particles act as energy substrate for separated energy requiring processes, suggesting a new paradigm for understanding glycogen metabolism in working muscles, muscle fatigue and metabolic disorders.

#85: Nutrient Availability Drives Community Dynamics in the Vaginal Microbiota

Background Nutrient utilization is both critical for niche occupation and is the driver of competitive and cooperative interactions in microbial communities. The FRT is replete with host-associated glycans in the form of glycoproteins, epithelial glycogen stores, and the breakdown products of these glycans. I hypothesized that host-associated glycans drive environment, microbe–microbe and host–microbe interactions in the FRT. Methods We have developed robust, scalable, high-throughput culturing systems to empirically define the substrate utilization traits from more than 60 unique bacterial species capable of colonizing the vagina. In addition, we are using batch and continuous culture in vitro cultivation of multispecies communities to study vaginal bacteria within the complex community, that closely recapitulate key dynamics observed in vivo. Results Demonstrating the power of these in vitro models, I have defined the carbohydrate utilization profiles of hundreds of unique FRT isolates, identifying species and strain-level variation in utilization of host-derived carbohydrates. Given the known abundance of glycogen in the vaginal epithelium, I hypothesized that the utilization of host-associated glycogen represents an adaptation to the vaginal environment. Indeed, we identify glycogen degradation enzymes in diverse species resident in the reproductive tract, and find enrichment in genes encoding glycogen-degrading enzymes in L. crispatus strains derived from vaginal as opposed to intestinal sites. Metatranscriptomic analyses from human samples demonstrate that bacterial glycogen and maltose (a breakdown product of glycogen) utilization genes are highly expressed in the vagina and elucidate patterns of gene expression suggestive of context-dependent competition and cooperation for glycogen utilization in vivo. To empirically investigate the impact of glycogen availability and glycogen utilization in FRT microbiota communities, I assembled type strains or co-resident consortia into model, polymicrobial communities in vitro. These studies demonstrate that among health-associated L. crispatus strains, those that use glycogen have a competitive advantage during growth in a complex community. However, preliminary results suggest that some strains may benefit from cross-fed nutrients liberated by other members of the consortium. Conclusions Taken together, these data establish that strain-level variability in glycan utilization contributes to competitive fitness during growth in community, and suggest that these traits may influence community stability or persistence in vivo. Moreover, the methods we have developed provide a scalable system in which to empirically study ecological dynamics within complex community ex vivo.

Frequent Carbohydrate Ingestion Reduces Muscle Glycogen Depletion and Postpones Fatigue Relative to a Single Bolus

The timing of carbohydrate ingestion and how this influences net muscle glycogen utilization and fatigue has only been investigated in prolonged cycling. Past findings may not translate to running because each exercise mode is distinct both in the metabolic response to carbohydrate ingestion and in the practicalities of carbohydrate ingestion. To this end, a randomized, cross-over design was employed to contrast ingestion of the same sucrose dose either at frequent intervals (15 × 5 g every 5 min) or at a late bolus (1 × 75 g after 75 min) during prolonged treadmill running to exhaustion in six well-trained runners ( 61 ± 4 ml·kg−1·min−1). The muscle glycogen utilization rate was lower in every participant over the first 75 min of running (Δ 0.51 mmol·kg dm−1·min−1; 95% confidence interval [−0.02, 1.04] mmol·kg dm−1·min−1) and, subsequently, all were able to run for longer when carbohydrate had been ingested frequently from the start of exercise compared with when carbohydrate was ingested as a single bolus toward the end of exercise (105.6 ± 3.0 vs. 96.4 ± 5.0 min, respectively; Δ 9.3 min, 95% confidence interval [2.8, 15.8] min). A moderate positive correlation was apparent between the magnitude of glycogen sparing over the first 75 min and the improvement in running capacity (r = .58), with no significant difference in muscle glycogen concentrations at the point of exhaustion. This study indicates that failure to ingest carbohydrates from the outset of prolonged running increases reliance on limited endogenous muscle glycogen stores—the ergolytic effects of which cannot be rectified by subsequent carbohydrate ingestion late in exercise.

Disruption of Glycogen Utilization Markedly Improves the Efficacy of Carboplatin against Preclinical Models of Clear Cell Ovarian Carcinoma

High stage and recurrent ovarian clear cell carcinoma (OCC) are associated with poor prognosis and resistance to chemotherapy. A distinguishing histological feature of OCC is abundant cytoplasmic stores of glucose, in the form of glycogen, that can be mobilized for cellular metabolism. Here, we report the effect on preclinical models of OCC of disrupting glycogen utilization using the glucose analogue 2-deoxy-D-glucose (2DG). At concentrations significantly lower than previously reported for other cancers, 2DG markedly improves the efficacy in vitro of carboplatin chemotherapy against chemo-sensitive TOV21G and chemo-resistant OVTOKO OCC cell lines, and this is accompanied by the depletion of glycogen. Of note, 2DG doses—of more than 10-fold lower than previously reported for other cancers—significantly improve the efficacy of carboplatin against cell line and patient-derived xenograft models in mice that mimic the chemo-responsiveness of OCC. These findings are encouraging, in that 2DG doses, which are substantially lower than previously reported to cause adverse events in cancer patients, can safely and significantly improve the efficacy of carboplatin against OCC. Our results thus justify clinical trials to evaluate whether low dose 2DG improves the efficacy of carboplatin in OCC patients.

Bacterial Glycogen Provides Short-Term Benefits in Changing Environments

ABSTRACT Changing nutritional conditions challenge microbes and shape their evolutionary optimization. Here, we used real-time metabolomics to investigate the role of glycogen in the dynamic physiological adaptation of Escherichia coli to fluctuating nutrients following carbon starvation. After the depletion of environmental glucose, we found significant metabolic activity remaining, which was linked to rapid utilization of intracellular glycogen. Glycogen was depleted by 80% within minutes of glucose starvation and was similarly replenished within minutes of glucose availability. These fast time scales of glycogen utilization correspond to the short-term benefits that glycogen provided to cells undergoing various physiological transitions. Cells capable of utilizing glycogen exhibited shorter lag times than glycogen mutants when starved between periods of exposure to different carbon sources. The ability to utilize glycogen was also important for the transition between planktonic and biofilm lifestyles and enabled increased glucose uptake during pulses of limited glucose availability. While wild-type and mutant strains exhibited comparable growth rates in steady environments, mutants deficient in glycogen utilization grew more poorly in environments that fluctuated on minute scales between carbon availability and starvation. Taken together, these results highlight an underappreciated role of glycogen in rapidly providing carbon and energy in changing environments, thereby increasing survival and competition capabilities under fluctuating and nutrient-poor conditions. IMPORTANCE Nothing is constant in life, and microbes in particular have to adapt to frequent and rapid environmental changes. Here, we used real-time metabolomics and single-cell imaging to demonstrate that the internal storage polymer glycogen plays a crucial role in such dynamic adaptations. Glycogen is depleted within minutes of glucose starvation and similarly is replenished within minutes of glucose availability. Cells capable of utilizing glycogen exhibited shorter lag times than glycogen mutants when starved between periods of exposure to different carbon sources. While wild-type and mutant strains exhibited comparable growth rates in steady environments, mutants deficient in glycogen utilization grew more poorly in environments that fluctuated on minute scales between carbon availability and starvation. These results highlight an underappreciated role of glycogen in rapidly providing carbon and energy in changing environments, thereby increasing survival and competition capabilities under fluctuating and nutrient-poor conditions.