A fungal protein organizes both glycogen and cell wall glucans.

Glycogen is a glucose storage molecule composed of branched α-1,4-glucan chains, best known as an energy reserve that can be broken down to fuel central metabolism. Because fungal cells have a specialized need for glucose in building cell wall glucans, we investigated whether glycogen is used for this process. For these studies, we focused on the pathogenic yeast Cryptococcus neoformans, which causes ~150,000 deaths per year worldwide. We identified two proteins that influence formation of both glycogen and the cell wall: glycogenin (Glg1), which initiates glycogen synthesis, and a protein that we call Glucan organizing enzyme 1 (Goe1). We found that cells missing Glg1 lack α-1,4-glucan in their walls, indicating that this material is derived from glycogen. Without Goe1, glycogen rosettes are mislocalized and ß-1,3-glucan in the cell wall is reduced. Altogether, our results provide mechanisms for a close association between glycogen and cell wall.

Microbiome structure and function in parallel full-scale aerobic granular sludge and activated sludge processes.

Aerobic granular sludge (AGS) and conventional activated sludge (CAS) are two different biological wastewater treatment processes. AGS consists of self-immobilised microorganisms that are transformed into spherical biofilms, whereas CAS has floccular sludge of lower density. In this study, we investigated the treatment performance and microbiome dynamics of two full-scale AGS reactors and a parallel CAS system at a municipal WWTP in Sweden. Both systems produced low effluent concentrations, with some fluctuations in phosphate and nitrate mainly due to variations in organic substrate availability. The microbial diversity was slightly higher in the AGS, with different dynamics in the microbiome over time. Seasonal periodicity was observed in both sludge types, with a larger shift in the CAS microbiome compared to the AGS. Groups important for reactor function, such as ammonia-oxidising bacteria (AOB), nitrite-oxidising bacteria (NOB), polyphosphate-accumulating organisms (PAOs) and glycogen-accumulating organisms (GAOs), followed similar trends in both systems, with higher relative abundances of PAOs and GAOs in the AGS. However, microbial composition and dynamics differed between the two systems at the genus level. For instance, among PAOs, Tetrasphaera was more prevalent in the AGS, while Dechloromonas was more common in the CAS. Among NOB, Ca. Nitrotoga had a higher relative abundance in the AGS, while Nitrospira was the main nitrifier in the CAS. Furthermore, network analysis revealed the clustering of the various genera within the guilds to modules with different temporal patterns, suggesting functional redundancy in both AGS and CAS. KEY POINTS: • Microbial community succession in parallel full-scale aerobic granular sludge (AGS) and conventional activated sludge (CAS) processes. • Higher periodicity in microbial community structure in CAS compared to in AGS. • Similar functional groups between AGS and CAS but different composition and dynamics at genus level.

Glycogen synthesis is required for adaptive thermogenesis in beige adipose tissue and affects diet-induced obesity.

Glycogen is a form of energy storage for glucose in different tissues such as liver and skeletal muscle. It remains incompletely understood how glycogen impacts on adipose tissue functionality. Cold exposure elevated the expression of Gys1 that encodes glycogen synthase 1 in brown adipose tissue (BAT) and inguinal white adipose tissue (iWAT). The in vivo function of Gys1 was analyzed using a mouse model in which Gys1 was deleted specifically in adipose tissues. Under normal chow conditions, Gys1 deletion caused little changes to body weight and glucose metabolism. Deletion of Gys1 abrogated upregulation of UCP1 and other thermogenesis-related genes in iWAT upon prolonged cold exposure or treatment with ß3-adrenergic receptor agonist CL-316,243. Stimulation of UCP1 by CL-316,243 in adipose-derived stromal cells (stromal vascular fractions, SVFs) was also reduced by Gys1 deletion. Both the basal glycogen content and CL-316,243-stimulated glycogen accumulation in adipose tissues were reduced by Gys1 deletion. High-fat diet-induced obesity and insulin resistance were aggravated in Gys1-deleted mice. The loss of body weight upon CL-316,243 treatment was also abrogated by the loss of Gys1. In conclusion, our results underscore the pivotal role of glycogen synthesis in adaptive thermogenesis in beige adipose tissue and its impact on diet-induced obesity in mice.NEW & NOTEWORTHY Glycogen is one of major types of fuel reserve in the body and its classical function is to maintain blood glucose level. This study uncovers that glycogen synthesis is required for beige fat tissue to generate heat upon cold exposure. Such a function of glycogen is linked to development of high-fat diet-induced obesity, thus extending our understanding about the physiological functions of glycogen.

Octopamine integrates the status of internal energy supply into the formation of food-related memories.

The brain regulates food intake in response to internal energy demands and food availability. However, can internal energy storage influence the type of memory that is formed? We show that the duration of starvation determines whether Drosophila melanogaster forms appetitive short-term or longer-lasting intermediate memories. The internal glycogen storage in the muscles and adipose tissue influences how intensely sucrose-associated information is stored. Insulin-like signaling in octopaminergic reward neurons integrates internal energy storage into memory formation. Octopamine, in turn, suppresses the formation of long-term memory. Octopamine is not required for short-term memory because octopamine-deficient mutants can form appetitive short-term memory for sucrose and to other nutrients depending on the internal energy status. The reduced positive reinforcing effect of sucrose at high internal glycogen levels, combined with the increased stability of food-related memories due to prolonged periods of starvation, could lead to increased food intake.

Deciding what and how much to eat is a complex biological process which involves balancing many types of information such as the levels of internal energy storage, the amount of food previously available in the environment, the perceived value of certain food items, and how these are remembered. At the molecular level, food contains carbohydrates that are broken down to produce glucose, which is then delivered to cells under the control of a hormone called insulin. There, glucose molecules are either immediately used or stored as glycogen until needed. Insulin signalling is also known to interact with the brain's decision-making systems that control eating behaviors; however, how our brains balance food intake with energy storage is poorly understood. Berger et al. set out to investigate this question using fruit flies as an experimental model. These insects also produce insulin-like molecules which help to relay information about glycogen levels to the brain's decision-making system. In particular, these signals reach a population of neurons that produce a messenger known as octopamine similar to the human noradrenaline, which helps regulate how much the flies find consuming certain types of foods rewarding. Berger et al. were able to investigate the role of octopamine in helping to integrate information about internal and external resource levels, memory formation and the evaluation of different food types. When the insects were fed normally, increased glycogen levels led to foods rich in carbohydrates being rated as less rewarding by the decision-making cells, and therefore being consumed less. Memories related to food intake were also short-lived ­ in other words, long-term 'food memory' was suppressed, re-setting the whole system after every meal. In contrast, long periods of starvation in insects with high carbohydrates resources produced a stable, long-term memory of food and hunger which persisted even after the flies had fed again. This experience also changed their food rating system, with highly nutritious foods no longer being perceived as sufficiently rewarding. As a result, the flies overate. This study sheds new light on the mechanisms our bodies may use to maintain energy reserves when food is limited. The persistence of 'food memory' after long periods of starvation may also explain why losing weight is difficult, especially during restrictive diets. In the future, Berger et al. hope that this knowledge will contribute to better strategies for weight management.

The sweetest polymer nanoparticles: opportunities ahead for glycogen in nanomedicine.

Most cells take simple sugar (α-D-glucose) and assemble it into highly dense polysaccharide nanoparticles called glycogen. This is achieved through the action of multiple coupled-enzymatic reactions, yielding the cellular store of polymerised glucose to be degraded in times of metabolic need. These nanoparticles can be readily isolated from various animal tissues and plants, and are commercially available on a large scale. Importantly, glycogen is highly water soluble, non-toxic, low-fouling, and biodegradable, making it an attractive nanoparticle for use in nanomedicine, for both diagnosing and treating disease. This concept has been pursued actively recently, with exciting results on a variety of fronts, especially for targeting specific tissues and delivering nucleic acid and peptide cargo. In this perspective, the role of glycogen in nanomedicine going forward is discussed, with opportunities highlighted of where these sugary nanoparticles fit into the problem of treating disease.

Oral post-exercise garlic extract supplementation enhances glycogen replenishment but does not up-regulate mitochondria biogenesis mRNA expression in human-exercised skeletal muscle.

PURPOSE: Garlic extract (GA) is purported to enhance antioxidant and anti-inflammatory activity and glucose regulation in humans. The present study investigated the effects of post-exercise GA supplementation on GLUT4 expression, glycogen replenishment, and the transcript factors involved with mitochondrial biosynthesis in exercised human skeletal muscle. METHODS: The single-blinded crossover counterbalanced study was completed by 12 participants. Participants were randomly divided into either GA (2000 mg of GA) or placebo trials immediately after completing a single bout of cycling exercise at 75% Maximal oxygen uptake (VO2max) for 60 minutes. Participants consumed either GA (2000 mg) or placebo capsules with a high glycemic index carbohydrate meal (2 g carb/body weight) immediately after exercise. Muscle samples were collected at 0-h and 3-h post-exercise. Muscle samples were used to measure glycogen levels, GLUT4 protein expression, as well as transcription factors for glucose uptake, and mitochondria biogenesis. Plasma glucose, insulin, glycerol, non-esterified fatty acid (NEFA) concentrations, and respiratory exchange ratio (RER) were also analyzed during the post-exercise recovery periods. RESULTS: Skeletal muscle glycogen replenishment was significantly elevated during the 3-h recovery period for GA concurrent with no difference in GLUT4 protein expression between the garlic and placebo trials. PGC1-α gene expression was up-regulated for both GA and placebo after exercise (p < 0.05). Transcript factors corresponding to muscle mitochondrial biosynthesis were significantly enhanced under acute garlic supplementation as demonstrated by TFAM and FIS1. However, the gene expression of SIRT1, ERRα, NFR1, NFR2, MFN1, MFN2, OPA1, Beclin-1, DRP1 were not enhanced, nor were there any improvements in GLUT4 expression, following post-exercise garlic supplementation. CONCLUSION: Acute post-exercise garlic supplementation may improve the replenishment of muscle glycogen, but this appears to be unrelated to the gene expression for glucose uptake and mitochondrial biosynthesis in exercised human skeletal muscle.

Dietary Variation Effect on Life History Traits and Energy Storage in Neotropical Species of Drosophila (Diptera; Drosophilidae).

The ability of an organism to respond to nutritional stress can be a plastic character under the action of natural selection, affecting several characteristics, including life history and energy storage. The genus Drosophila (Diptera; Drosophilidae) presents high variability regarding natural resource exploration. However, most works on this theme have studied the model species D. melanogaster Meigen, 1830 and little is known about Neotropical drosophilids. Here we evaluate the effects of three diets, with different carbohydrate-to-protein ratios, on life history (viability and development time) and metabolic pools (triglycerides, glycogen, and total soluble protein contents) of three Neotropical species of Drosophila: D. maculifrons Duda, 1927; D. ornatifrons Duda, 1927, both of the subgenus Drosophila Sturtevant, 1939, and D. willistoni Sturtevant, 1916 of the subgenus Sophophora Sturtevant, 1939. Our results showed that only D. willistoni was viable on all diets, D. maculifrons was not viable on the sugary diet, while D. ornatifrons was barely viable on this diet. The sugary diet increased the development time of D. willistoni and D. ornatifrons, and D. willistoni glycogen content. Thus, the viability of D. maculifrons and D. ornatifrons seems to depend on a certain amount of protein and/or a low concentration of carbohydrate in the diet. A more evident effect of the diets on triglyceride and protein pools was detected in D. ornatifrons, which could be related to the adult attraction to dung and carrion baited pitfall as food resource tested in nature. Our results demonstrated that the evolutionary history and differential adaptations to natural macronutrient resources are important to define the amplitude of response that a species can present when faced with dietary variation.

Inhibition of pyruvate dehydrogenase accelerates anaerobic glycolysis under postmortem simulating conditions.

This research aimed to explore the potential influence of mitochondria on the rate of anaerobic glycolysis. We hypothesized that mitochondria could reduce the rate of anaerobic glycolysis and pH decline by metabolizing a portion of glycolytic pyruvate. We utilized an in vitro model and incorporated CPI-613 and Avidin to inhibit pyruvate dehydrogenase (PDH) and pyruvate carboxylase (PC), respectively. Four treatments were tested: 400 µM CPI-613, 1.5 U/ml Avidin, 400 µM CPI-613 + 1.5 U/ml Avidin, or control. Glycolytic metabolites and pH of the in vitro model were evaluated throughout a 1440-min incubation period. CPI-613-containing treatments, with or without Avidin, decreased pH levels and increased glycogen degradation and lactate accumulation compared to the control and Avidin treatments (P < 0.05), indicating increased glycolytic flux. In a different experiment, two treatments, 400 µM CPI-613 or control, were employed to track the fates of pyruvate using [13C6]glucose. CPI-613 reduced the contribution of glucose carbon to tricarboxylic acid cycle intermediates compared to control (P < 0.05). To test whether the acceleration of acidification in reactions containing CPI-613 was due to an increase in the activity of key enzymes of glycogenolysis and glycolysis, we evaluated the activities of glycogen phosphorylase, phosphofructokinase, and pyruvate kinase in the presence or absence of 400 µM CPI-613. The CPI-613 treatment did not elicit an alteration in the activity of these three enzymes. These findings indicate that inhibiting PDH increases the rate of anaerobic glycolysis and pH decline, suggesting that mitochondria are potential regulators of postmortem metabolism.

A bout of aerobic exercise in the heat increases carbohydrate use but does not enhance the disposal of an oral glucose load, in healthy active individuals.

We investigated if a bout of exercise in a hot environment (HEAT) would reduce the postprandial hyperglycemia induced by glucose ingestion. The hypothesis was that HEAT stimulating carbohydrate oxidation and glycogen use would increase the disposal of an ingested glucose load [i.e., oral glucose tolerance test (OGTT); 75 g of glucose]. Separated by at least 1 wk, nine young healthy individuals underwent three trials after an overnight fast in a randomized order. Two trials included 50 min of pedaling at 58 ± 5% V̇o2max either in a thermoneutral (21 ± 1°C; NEUTRAL) or in a hot environment (33 ± 1°C; HEAT) eliciting similar energy expenditure (503 ± 101 kcal). These two trials were compared with a no-exercise trial (NO EXER). Twenty minutes after exercise (or rest), subjects underwent an OGTT, while carbohydrate oxidation (CHOxid, using indirect calorimetry) plasma blood glucose, insulin concentrations (i.e., [glucose], [insulin]), and double tracer glucose kinetics ([U-13C] glucose ingestion and [6,6-2H2] glucose infusion) were monitored for 120 min. At rest, [glucose], [insulin], and rates of appearance/disappearance of glucose in plasma (glucose Ra/Rd) were similar among trials. During exercise, heart rate, tympanic temperature, [glucose], glycogen oxidation, and total CHOxid were higher during HEAT than NEUTRAL (i.e., 149 ± 35 vs. 124 ± 31 µmol·kg-1·min-1, P = 0.010). However, during the following OGTT, glucose Rd was similar in HEAT and NEUTRAL trials (i.e., 25.1 ± 3.6 vs. 25.2 ± 5.3 µmol·kg-1·min-1, P = 0.981). Insulin sensitivity (i.e., ISIndexMATSUDA) only improved in NEUTRAL compared with NO EXER (10.1 ± 4.6 vs. 8.8 ± 3.7 au; P = 0.044). In summary, stimulating carbohydrate use with exercise in a hot environment does not improve postprandial plasma glucose disposal or insulin sensitivity in a subsequent OGTT.NEW & NOTEWORTHY Exercise in the heat increases estimated muscle glycogen use. Reduced muscle glycogen after exercise in the heat could increase insulin-mediated glucose uptake during a subsequent oral glucose tolerance test (OGTT). However, plasma glucose kinetics are not improved during the OGTT in response to a bout of exercise in the heat, and insulin sensitivity worsens. Heat stress activates glucose counterregulatory hormones whose actions may linger during the OGTT, preventing increased glucose uptake.

Differential utilisation of subcellular skeletal muscle glycogen pools: a comparative analysis between 1 and 15 min of maximal exercise.

In skeletal muscle, glycogen particles are distributed both within and between myofibrils, as well as just beneath the sarcolemma. Their precise localisation may influence their degradation rate. Here, we investigated how exercise at different intensities and durations (1- and 15-min maximal exercise) with known variations in glycogenolytic rate and contribution from anaerobic metabolism affects utilisation of the distinct pools. Furthermore, we investigated how decreased glycogen availability achieved through lowering carbohydrate and energy intake after glycogen-depleting exercise affect the storage of glycogen particles (size, numerical density, localisation). Twenty participants were divided into two groups performing either a 1-min (n = 10) or a 15-min (n = 10) maximal cycling exercise test. In a randomised, counterbalanced, cross-over design, the exercise tests were performed following short-term consumption of two distinct diets with either high or moderate carbohydrate content (10 vs. 4 g kg-1 body mass (BM) day-1) mediating a difference in total energy consumption (240 vs. 138 g kg-1 BM day-1). Muscle biopsies from m. vastus lateralis were obtained before and after the exercise tests. Intermyofibrillar glycogen was preferentially utilised during the 1-min test, whereas intramyofibrillar glycogen was preferentially utilised during the 15-min test. Lowering carbohydrate and energy intake after glycogen-depleting exercise reduced glycogen availability by decreasing particle size across all pools and diminishing numerical density in the intramyofibrillar and subsarcolemmal pools. In conclusion, distinct subcellular glycogen pools were differentially utilised during 1-min and 15-min maximal cycling exercise. Additionally, lowered carbohydrate and energy consumption after glycogen-depleting exercise altered glycogen storage by reducing particle size and numerical density, depending on subcellular localisation. KEY POINTS: In human skeletal muscle, glycogen particles are localised in distinct subcellular compartments, referred to as intermyofibrillar, intramyofibrillar and subsarcolemmal pools. The intermyofibrillar and subsarcolemmal pools are close to mitochondria, while the intramyofibrillar pool is at a distance from mitochondria. We show that 1 min of maximal exercise is associated with a preferential utilisation of intermyofibrillar glycogen, and, on the other hand, that 15 min of maximal exercise is associated with a preferential utilisation of intramyofibrillar glycogen. Furthermore, we demonstrate that reduced glycogen availability achieved through lowering carbohydrate and energy intake after glycogen-depleting exercise is characterised by a decreased glycogen particle size across all compartments, with the numerical density only diminished in the intramyofibrillar and subsarcolemmal compartments. These results suggest that exercise intensity influences the subcellular pools of glycogen differently and that the dietary content of carbohydrates and energy is linked to the size and subcellular distribution of glycogen particles.

Glycogen Granules Are Degraded by Non-Selective Autophagy in Nitrogen-Starved Komagataella phaffii.

Autophagy was initially recognized as a bulk degradation process that randomly sequesters and degrades cytoplasmic material in lysosomes (vacuoles in yeast). In recent years, various types of selective autophagy have been discovered. Glycophagy, the selective autophagy of glycogen granules, is one of them. While autophagy of glycogen is an important contributor to Pompe disease, which is characterized by the lysosomal accumulation of glycogen, its selectivity is still a matter of debate. Here, we developed the Komagataella phaffii yeast as a simple model of glycogen autophagy under nitrogen starvation conditions to address the question of its selectivity. For this, we turned the self-glucosylating initiator of glycogen synthesis, Glg1, which is covalently bound to glycogen, into the Glg1-GFP autophagic reporter. Our results revealed that vacuolar delivery of Glg1-GFP and its processing to free GFP were strictly dependent on autophagic machinery and vacuolar proteolysis. Notably, this process was independent of Atg11, the scaffold protein common for many selective autophagy pathways. Importantly, the non-mutated Glg1-GFP (which synthesizes and marks glycogen) and mutated Glg1Y212F-GFP (which does not synthesize glycogen and is degraded by non-selective autophagy as cytosolic Pgk1-GFP) were equally well delivered to the vacuole and had similar levels of released GFP. Therefore, we concluded that glycogen autophagy is a non-selective process in K. phaffii yeast under nitrogen starvation conditions.

Neuron-astrocyte metabolic coupling facilitates spinal plasticity and maintenance of inflammatory pain.

Long-lasting pain stimuli can trigger maladaptive changes in the spinal cord, reminiscent of plasticity associated with memory formation. Metabolic coupling between astrocytes and neurons has been implicated in neuronal plasticity and memory formation in the central nervous system, but neither its involvement in pathological pain nor in spinal plasticity has been tested. Here we report a form of neuroglia signalling involving spinal astrocytic glycogen dynamics triggered by persistent noxious stimulation via upregulation of the Protein Targeting to Glycogen (PTG) in spinal astrocytes. PTG drove glycogen build-up in astrocytes, and blunting glycogen accumulation and turnover by Ptg gene deletion reduced pain-related behaviours and promoted faster recovery by shortening pain maintenance in mice. Furthermore, mechanistic analyses revealed that glycogen dynamics is a critically required process for maintenance of pain by facilitating neuronal plasticity in spinal lamina 1 neurons. In summary, our study describes a previously unappreciated mechanism of astrocyte-neuron metabolic communication through glycogen breakdown in the spinal cord that fuels spinal neuron hyperexcitability.

Myocardial glycophagy flux dysregulation and glycogen accumulation characterize diabetic cardiomyopathy.

Diabetic heart disease morbidity and mortality is escalating. No specific therapeutics exist and mechanistic understanding of diabetic cardiomyopathy etiology is lacking. While lipid accumulation is a recognized cardiomyocyte phenotype of diabetes, less is known about glycolytic fuel handling and storage. Based on in vitro studies, we postulated the operation of an autophagy pathway in the myocardium specific for glycogen homeostasis - glycophagy. Here we visualize occurrence of cardiac glycophagy and show that the diabetic myocardium is characterized by marked glycogen elevation and altered cardiomyocyte glycogen localization. We establish that cardiac glycophagy flux is disturbed in diabetes. Glycophagy may represent a potential therapeutic target for alleviating the myocardial impacts of metabolic disruption in diabetic heart disease.

The costs of overwintering in paper wasps (Polistes dominula and Polistes gallicus): the use of energy stores.

Overwintering insects are facing energetic challenges because of food shortage, low temperature, and desiccation stress. Paper wasps of the genus Polistes overwinter as mated adults (gynes) in hibernacula protecting them from predation, snow, and rain but barely from low environmental temperature. In different climates, they face differing overwintering temperature regimes, and therefore they may differ in their energy use. We investigated how much of energy resources built up until autumn is used during diapause dormancy in natural hibernacula by measuring lipid, glycogen, and free carbohydrate content in autumn and early spring in Polistes dominula from temperate European (Austrian) and warm Mediterranean (Italian) climate and Polistes gallicus from Mediterranean climate. Winter energy consumption amounted to ~ 339 and ~ 310 J per wasp in the Austrian and Italian Polistes dominula populations. The smaller Italian Polistes gallicus consumed ~ 247 J. This amounts to 2.62, 2.35, and 1.79 J per day. Of this, the energy demand was mainly fuelled by lipids (84%, 93%, and 90%, respectively), but glycogen stores contributed also considerably (16%, 6%, and 9%). Free carbohydrates decreased only by 0.7%, 1%, and 0.8%. While fat stores seem still sufficient in spring, the wasps depleted most of their carbohydrates. The energy reserves of 396, 400, and 147 J per wasp remaining in spring in the three populations seem sufficient to fuel rest or simple brood care activities for a whole summer but restrict foraging flights to a few hours (~ 3.5-6 h). Results suggest that energy supply might become challenging in expected future climate scenarios.

Enteric and systemic postprandial lactate shuttle phases and dietary carbohydrate carbon flow in humans.

Dietary glucose in excess is stored in the liver in the form of glycogen. As opposed to direct conversion of glucose into glycogen, the hypothesis of the postprandial lactate shuttle (PLS) proposes that dietary glucose uptake is metabolized to lactate in the gut, thereby being transferred to the liver for glycogen storage. In the present study, we provide evidence of a PLS in young healthy men and women. Overnight fasted participants underwent an oral glucose tolerance test, and arterialized lactate concentration and rate of appearance were determined. The concentration of lactate in the blood rose before the concentration of glucose, thus providing evidence of an enteric PLS. Secondary increments in the concentration of lactate in the blood and its rate of appearance coincided with those of glucose, which indicates the presence of a larger, secondary, systemic PLS phase driven by hepatic glucose release. The present study challenges the notion that lactate production is the result of hypoxia in skeletal muscles, because our work indicates that glycolysis proceeds to lactate in fully aerobic tissues and dietary carbohydrate is processed via lactate shuttling. Our study proposes that, in humans, lactate is a major vehicle for carbohydrate carbon distribution and metabolism.

Treatment of infantile-onset Pompe disease in a rat model with muscle-directed AAV gene therapy.

OBJECTIVE: Pompe disease (PD) is caused by deficiency of the lysosomal enzyme acid α-glucosidase (GAA), leading to progressive glycogen accumulation and severe myopathy with progressive muscle weakness. In the Infantile-Onset PD (IOPD), death generally occurs <1 year of age. There is no cure for IOPD. Mouse models of PD do not completely reproduce human IOPD severity. Our main objective was to generate the first IOPD rat model to assess an innovative muscle-directed adeno-associated viral (AAV) vector-mediated gene therapy. METHODS: PD rats were generated by CRISPR/Cas9 technology. The novel highly myotropic bioengineered capsid AAVMYO3 and an optimized muscle-specific promoter in conjunction with a transcriptional cis-regulatory element were used to achieve robust Gaa expression in the entire muscular system. Several metabolic, molecular, histopathological, and functional parameters were measured. RESULTS: PD rats showed early-onset widespread glycogen accumulation, hepato- and cardiomegaly, decreased body and tissue weight, severe impaired muscle function and decreased survival, closely resembling human IOPD. Treatment with AAVMYO3-Gaa vectors resulted in widespread expression of Gaa in muscle throughout the body, normalizing glycogen storage pathology, restoring muscle mass and strength, counteracting cardiomegaly and normalizing survival rate. CONCLUSIONS: This gene therapy holds great potential to treat glycogen metabolism alterations in IOPD. Moreover, the AAV-mediated approach may be exploited for other inherited muscle diseases, which also are limited by the inefficient widespread delivery of therapeutic transgenes throughout the muscular system.

Stratification of tumour cell radiation response and metabolic signatures visualization with Raman spectroscopy and explainable convolutional neural network.

Reprogramming of cellular metabolism is a driving factor of tumour progression and radiation therapy resistance. Identifying biochemical signatures associated with tumour radioresistance may assist with the development of targeted treatment strategies to improve clinical outcomes. Raman spectroscopy (RS) can monitor post-irradiation biomolecular changes and signatures of radiation response in tumour cells in a label-free manner. Convolutional Neural Networks (CNN) perform feature extraction directly from data in an end-to-end learning manner, with high classification performance. Furthermore, recently developed CNN explainability techniques help visualize the critical discriminative features captured by the model. In this work, a CNN is developed to characterize tumour response to radiotherapy based on its degree of radioresistance. The model was trained to classify Raman spectra of three human tumour cell lines as radiosensitive (LNCaP) or radioresistant (MCF7, H460) over a range of treatment doses and data collection time points. Additionally, a method based on Gradient-Weighted Class Activation Mapping (Grad-CAM) was used to determine response-specific salient Raman peaks influencing the CNN predictions. The CNN effectively classified the cell spectra, with accuracy, sensitivity, specificity, and F1 score exceeding 99.8%. Grad-CAM heatmaps of H460 and MCF7 cell spectra (radioresistant) exhibited high contributions from Raman bands tentatively assigned to glycogen, amino acids, and nucleic acids. Conversely, heatmaps of LNCaP cells (radiosensitive) revealed activations at lipid and phospholipid bands. Finally, Grad-CAM variable importance scores were derived for glycogen, asparagine, and phosphatidylcholine, and we show that their trends over cell line, dose, and acquisition time agreed with previously established models. Thus, the CNN can accurately detect biomolecular differences in the Raman spectra of tumour cells of varying radiosensitivity without requiring manual feature extraction. Finally, Grad-CAM may help identify metabolic signatures associated with the observed categories, offering the potential for automated clinical tumour radiation response characterization.

Assessments of individual fiber glycogen and mitochondrial volume percentages reveal a graded reduction in muscle oxidative power during prolonged exhaustive exercise.

During submaximal exercise, there is a heterogeneous recruitment of skeletal muscle fibers, with an ensuing heterogeneous depletion of muscle glycogen both within and between fiber types. Here, we show that the mean (95% CI) mitochondrial volume as a percentage of fiber volume of non-glycogen-depleted fibers was 2 (-10:6), 5 (-21:11), and 12 (-21:-2)% lower than all the sampled fibers after continuing exercise for 1, 2 h, and until task failure, respectively. Therefore, a glycogen-dependent fatigue of individual fibers during submaximal exercise may reduce the muscular oxidative power. These findings suggest a relationship between glycogen and mitochondrial content in individual muscle fibers, which is important for understanding fatigue during prolonged exercise.

Hyperglycemia - A culprit of podocyte pathology in the context of glycogen metabolism.

Prolonged disruption in the balance of glucose can result in metabolic disorders. The kidneys play a significant role in regulating blood glucose levels. However, when exposed to chronic hyperglycemia, the kidneys' ability to handle glucose metabolism may be impaired, leading to an accumulation of glycogen. Earlier studies have shown that there can be a significant increase in glucose storage in the form of glycogen in the kidneys in diabetes. Podocytes play a crucial role in maintaining the integrity of filtration barrier. In diabetes, exposure to elevated glucose levels can lead to significant metabolic and structural changes in podocytes, contributing to kidney damage and the development of diabetic kidney disease. The accumulation of glycogen in podocytes is not a well-established phenomenon. However, a recent study has demonstrated the presence of glycogen granules in podocytes. This review delves into the intricate connections between hyperglycemia and glycogen metabolism within the context of the kidney, with special emphasis on podocytes. The aberrant storage of glycogen has the potential to detrimentally impact podocyte functionality and perturb their structural integrity. This review provides a comprehensive analysis of the alterations in cellular signaling pathways that may potentially lead to glycogen overproduction in podocytes.