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Muscle phosphorylase b kinase (PHK) deficiency is a rare mild metabolic disorder caused by mutations of the PHKA1 gene encoding the αM subunit of PHK. A 16-year-old boy experienced myalgia during the maximal multistage 20-m shuttle run test targeting the maximal oxygen consumption. Although an ischemic forearm exercise test was normal, a muscle biopsy revealed subsarcolemmal glycogen accumulation. He harbored a novel insertion mutation in the PHKA1 gene that resulted in premature termination of the αM subunit close to the C-terminus. Compared with previously reported cases, his reduction in PHK activity was relatively mild.

Tumor metastasis is a series of complicated biological events. Hematogenous metastasis mediated by von Willebrand factor (vWF) is critical in tumor metastasis. However, the source of vWF and its role in tumor metastasis are controversial, and the further mechanism involved in mediating tumor metastasis is still unclear. In this study, we first demonstrated that lung adenocarcinoma cells could express vWF de novo and promotes tumor metastasis. Through the analysis of transcriptome sequencing, the metastasis promotion effect of vWF may be related to phosphorylase kinase subunit G1 (PHKG1), a catalytic subtype of phosphorylase kinase (PhK). PHKG1 was highly expressed in lung adenocarcinoma patients and led to poor prognosis. Further experiments found that lung adenocarcinoma-derived vWF induced the upregulation of PHKG1 through the PI3K/AKT pathway to promote glycogenolysis. Glycogen was funneled into glycolysis, leading to increased metastasis. Tumor metastasis assayed in vitro and in vivo showed that knockdown of PHKG1 or synergistic injection of phosphorylase inhibition based on the overexpression of vWF could inhibit metastasis. In summary, our research proved that lung adenocarcinoma-derived vWF may mediate tumor metastasis by regulating PHKG1 to promote glycogen metabolism and suggested potential targets for inhibition of lung adenocarcinoma metastasis.

Glycogen storage disease type IX (GSD-IX) constitutes nearly a quarter of all GSDs. This ketotic form of GSD is caused by mutations in phosphorylase kinase (PhK), which is composed of four subunits (α, β, γ, δ). PhK is required for the activation of the liver isoform of glycogen phosphorylase (PYGL), which generates free glucose-1-phosphate monomers to be used as energy via cleavage of the α -(1,4) glycosidic linkages in glycogen chains. Mutations in any of the PhK subunits can negatively affect the regulatory and catalytic activity of PhK during glycogenolysis. To understand the pathogenesis of GSD-IX-beta, we characterized a newly created PHKB knockout (

We report a case of a 17-month-old male with a history of developmental delay with poor muscle control, hepatomegaly, and transaminitis. Ultrasound of abdomen revealed hepatomegaly with a liver span of 13 cm, homogeneous parenchyma, and normal spleen size. Liver and muscle biopsies were obtained: the liver biopsy revealed distended hepatocytes with excessive glycogen accumulation and fine septate fibrosis. Biopsy of the right vastus lateralis muscle showed focal swollen glycogen containing mitochondria. For the developmental delay, a chromosomal microrarray was ordered. The chromosomal microarray revealed the patient to have 1q21 duplication syndrome and 16p11.2 deletion syndrome. Given the liver and muscle biopsy findings, a glycogen storage disease panel was sent which identified the patient to be hemizygous for a variant of uncertain significance denoted as p.Gly 131Val, c.392G > T in the PHKA2 gene. PKHA2 gene encodes the alpha subunit of hepatic phosphorylase kinase. This change in the PHKA2 gene was in a highly conserved region and had been reported in another patient with decreased enzymatic activity of the phosphorylase kinase and who had symptoms of GSD IX. Based on this, the patient was started on treatment for GSD IX, and his family met with a dietician.

With increasing societal development and the concurrent improvement in people's quality of life, meat consumption has gradually changed from a focus on “quantity” to “quality”. Broiler production is increasingly used as a means to improve meat quality by altering various characteristics, especially its genetic factors. However, until now, little has been known about the genetic variants related to meat quality traits in Chinese purebred chicken populations. To better understand these genetic underpinnings, a total of 17 traits related to meat quality and carcass were measured in 325 Chinese Ningdu yellow chickens. We performed DNA sequencing to detect nucleotide mutations, after which we conducted association studies between PHKG1 gene polymorphisms and traits related to meat quality and carcass. Results indicated a large phenotypic variation in meat quality traits. More specifically, the single nucleotide polymorphism (SNP) rs15845448 was significantly associated with drip loss at 24 h (P = 8.04 × 10−6) and 48 h (P = 5.47 × 10−6), pH (P = 2.39 × 10−3), and meat color L* (P = 9.88 × 10−3). Moreover, the SNP rs15845448 reduced 24 h and 48 h drip loss by 3.62% and 5.97%, respectively. However, no significant associations were found between rs15845448 and carcass traits (P > 0.05). Furthermore, a haplotype block containing two adjacent SNPs (rs15845448 and rs15845450) was identified. This block displayed four distinct haplotypes that had significant association with drip loss at 24 h and 48 h, pH, and meat color L*. Collectively, these results provide new insights into the genetic basis of meat quality in Chinese Ningdu yellow chickens. Moreover, the significance of SNP rs15845448 could be incorporated into the selection programs involving this breed.

Glycogen storage diseases (GSDs) result from the deficiency of enzymes involved in glycogen synthesis and breakdown into glucose. Mutations in the gene PHKA2 encoding phosphorylase kinase regulatory subunit alpha 2 have been linked to GSD type IXa. We describe a family with two adult brothers with neonatal hepatosplenomegaly and later onset of hearing loss, cognitive impairment, and cerebellar involvement. Whole-exome sequencing was performed on both subjects and revealed a shared hemizygous missense variant (c.A1561G; p.T521A) in exon 15 of PHKA2. The phenotype broadens the clinical and magnetic resonance imaging spectrum of GSD type IXa to include later onset neurological manifestations.

Background Pathogenic mutations in the PHKG2 are associated with a very rare disease—glycogen storage disease IXc (GSD-IXc)—and are characterized by severe liver disease. Case presentation Here, we report a patient with jaundice, hypoglycaemia, growth retardation, progressive increase in liver transaminase and prominent hepatomegaly from the neonatal period. Genetic testing revealed two novel, previously unreported PHKG2 mutations (F233S and R320DfsX5). Functional experiments indicated that both F223S and R320DfsX5 lead to a decrease in key phosphorylase b kinase enzyme activity. With raw cornstarch therapy, hypoglycaemia and lactic acidosis were ameliorated and serum aminotransferases decreased. Conclusion These findings expand the gene spectrum and contribute to the interpretation of clinical presentations of these two novel PHKG2 mutations.

Background. Circular RNA phosphorylase kinase regulatory subunit alpha 2 (circPHKA2; hsa_circ_0090002) has a significantly, specifically different expression in acute ischemic stroke (AIS) patients’ blood. Here, we intended to investigate the role and mechanism of circPHKA2 in oxygen-glucose deprivation- (OGD-) induced stoke model in human brain microvascular endothelial cells (HBMEC). Methods. Expression of circPHKA2, microRNA- (miR-) 574-5p, and superoxide dismutase-2 (SOD2) was detected by quantitative PCR and western blotting. Cell injury was measured by detecting cell proliferation (EdU assay and CCK-8 assay), migration (transwell assay), neovascularization (tube formation assay), apoptosis (flow cytometry and western blotting), endoplasmic reticulum stress (western blotting), and oxidative stress (assay kits). Direct intermolecular interaction was determined by bioinformatics algorithms, dual-luciferase reporter assay, biotin-labelled miRNA capture, and argonaute 2 RNA immunoprecipitation. Results. circPHKA2 was downregulated in AIS patients’ blood in SOD2-correlated manner. Reexpressing circPHKA2 rescued EdU incorporation, cell viability and migration, tube formation, B cell lymphoma-2 (Bcl-2) expression, and SOD activity of OGD-induced HBMEC and alleviate apoptotic rate and levels of Bcl-2-associated protein (Bax), glucose-regulated protein 78 kD (GRP78), C/EBP-homologous protein (CHOP), caspase-12, reactive oxygen species (ROS), and malondialdehyde (MDA). Additionally, blocking SOD2 partially attenuated these roles of circPHKA2 overexpression. Molecularly, circPHKA2 upregulated SOD2 expression via interacting with miR-574-5p, and miR-574-5p could target SOD2. Similarly, allied to neurovascular protection of circPHKA2 was the downregulation of miR-574-5p. Conclusion. circPHKA2 could protect HBMEC against OGD-induced cerebral stroke model via the miR-574-5p/SOD2 axis, suggesting circPHKA2 as a novel and promising candidate in ischemic brain injury.

Idiopathic ketotic hypoglycemia (IKH) is a diagnosis of exclusion with glycogen storage diseases (GSDs) as a differential diagnosis. GSD IXa presents with ketotic hypoglycemia (KH), hepatomegaly, and growth retardation due to PHKA2 variants. In our multicenter study, 12 children from eight families were diagnosed or suspected of IKH. Whole‐exome sequencing or targeted next‐generation sequencing panels were performed. We identified two known and three novel (likely) pathogenic PHKA2 variants, such as p.(Pro869Arg), p.(Pro498Leu), p.(Arg2Gly), p.(Arg860Trp), and p.(Val135Leu), respectively. Erythrocyte phosphorylase kinase activity in three patients with the novel variants p.(Arg2Gly) and p.(Arg860Trp) were 15%–20% of mean normal. One patient had short stature and intermittent mildly elevated aspartate aminotransferase, but no hepatomegaly. Family testing identified two asymptomatic children and 18 adult family members with one of the PHKA2 variants, of which 10 had KH symptoms in childhood and 8 had mild symptoms in adulthood. Our study expands the classical GSD IXa phenotype of PHKA2 missense variants to a continuum from seemingly asymptomatic carriers, over KH‐only with phosphorylase B kinase deficiency, to more or less complete classical GSD IXa. In contrast to typical IKH, which is confined to young children, KH may persist into adulthood in the KH‐only phenotype of PHKA2.

Background Glycogen storage disease (GSD) type IXb is one of the rare variants of GSDs. It is a genetically heterogeneous metabolic disorder due to deficient hepatic phosphorylase kinase activity. Diagnosis of GSD can be difficult because of overlapping manifestations. Mutation analysis of the genes related to each type of GSD is supposed to be problem-solving, however, the presence of novel mutations can be confusing. In this case report, we will describe our experience with a young girl with the diagnosis of GSD and two novel mutations related to GSD type IXb. Case presentation A 3-year- old girl presented with short stature, hepatomegaly, and liver cirrhosis. No specific diagnosis was made based on laboratory data, so liver biopsy and targeted-gene sequencing (TGS) were performed to find out the specific molecular basis of her disease. It was confirmed that the patient carries two novel variants in the PHKB gene. The variant in the PHKB gene was classified as pathogenic. Conclusions This is the first reported case of a dual molecular mutation of glycogen storage disease type IXb in the same patient. Two novel variants in PHKB were identified and one of them was a pathogenic split-site mutation. In conclusion, for the first time, identification of the novel variants in this patient expands the molecular and the phenotype basis of dual variants in GSD-IXb.

INTRODUCTION: Liver Glycogen Storage Disease IX is a rare metabolic disorder of glycogen metabolism caused by deficiency of the phosphorylase kinase enzyme (PhK). Variants in the PHKG2 gene, encoding the liver-specific catalytic γ2 subunit of PhK, are associated with a liver GSD IX subtype known as PHKG2 GSD IX or GSD IX γ2. There is emerging evidence that patients with GSD IX γ2 can develop severe and progressive liver disease, yet research regarding the disease has been minimal to date. Here we characterize the first mouse model of liver GSD IX γ2. METHODS: A Phkg2(−/−) mouse model was generated via targeted removal of the Phkg2 gene. Knockout (Phkg2(−/−), KO) and wild type (Phkg2(+/+), WT) mice up to 3 months of age were compared for morphology, Phkg2 transcription, PhK enzyme activity, glycogen content, histology, serum liver markers, and urinary glucose tetrasaccharide Glcα1–6Glcα1–4Glcα1–4Glc (Glc(4)). RESULTS: When compared to WT controls, KO mice demonstrated significantly decreased liver PhK enzyme activity, increased liver: body weight ratio, and increased glycogen in the liver, with no glycogen accumulation observed in the brain, quadricep, kidney, and heart. KO mice demonstrated elevated liver blood markers as well as elevated urine Glc(4), a commonly used biomarker for glycogen storage disease. KO mice demonstrated features of liver structural damage. Hematoxylin & Eosin and Masson’s Trichrome stained KO mice liver histology slides revealed characteristic GSD hepatocyte architectural changes and early liver fibrosis, as have been reported in liver GSD patients. DISCUSSION: This study provides the first evidence of a mouse model that recapitulates the liver-specific pathology of patients with GSD IX γ2. The model will provide the first platform for further study of disease progression in GSD IX γ2 as well as for the evaluation of novel therapeutics.

Background Genetic analysis of gene expression level is a promising approach for characterizing candidate genes that are involved in complex economic traits such as meat quality. In the present study, we conducted expression quantitative trait loci (eQTL) and allele-specific expression (ASE) analyses based on RNA-sequencing (RNAseq) data from the longissimus muscle of 189 Duroc × Luchuan crossed pigs in order to identify some candidate genes for meat quality traits. Results Using a genome-wide association study based on a mixed linear model, we identified 7192 cis-eQTL corresponding to 2098 cis-genes (p ≤ 1.33e-3, FDR ≤ 0.05) and 6400 trans-eQTL corresponding to 863 trans-genes (p ≤ 1.13e-6, FDR ≤ 0.05). ASE analysis using RNAseq SNPs identified 9815 significant ASE-SNPs in 2253 unique genes. Integrative analysis between the cis-eQTL and ASE target genes identified 540 common genes, including 33 genes with expression levels that were correlated with at least one meat quality trait. Among these 540 common genes, 63 have been reported previously as candidate genes for meat quality traits, such as PHKG1 (q-value = 1.67e-6 for the leading SNP in the cis-eQTL analysis), NUDT7 (q-value = 5.67e-13), FADS2 (q-value = 8.44e-5), and DGAT2 (q-value = 1.24e-3). Conclusions The present study confirmed several previously published candidate genes and identified some novel candidate genes for meat quality traits via eQTL and ASE analyses, which will be useful to prioritize candidate genes in further studies.

Introduction Liver Glycogen Storage Disease Type IX (GSD IX) is one of the most common forms of GSD. It is caused by a deficiency in enzyme phosphorylase kinase (PhK), a complex, hetero-tetrameric enzyme comprised of four subunits - α, β, γ, and δ - each with tissue specific isoforms encoded by different genes. Until the recent availability of gene panels and exome sequencing, the diagnosis of liver GSD IX did not allow for differentiation of these subtypes. This study presents the first comprehensive literature review for liver GSD IX subtypes - GSD IX α2, β, and γ2. We aim to better characterize the natural history of liver GSD IX and further investigate if there are subtype-specific differences in clinical presentation. Methods A comprehensive literature review was performed with the help of a medical librarian at Duke University Medical Center to gather all published patients of liver GSD IX. Our refined search yielded 74 articles total. Available patient data were compiled into an excel spreadsheet. Data were analyzed via descriptive statistics. The number of patients with specific symptoms were individually summed and reported as a percentage of the total number of patients for which data were available or were averaged and reported as a mean numerical value. Published pathology reports were scored using the International Association of the Study of the Liver Scale. Results There were a total of 183 GSD IX α2 patients, 17 GSD IX β patients, and 30 GSD IX γ2 patients. Average age at diagnosis was 4 years for GSD IX α2 patients, 2.34 years for GSD IX β patients, and 1.81 years for GSD IX γ2 patients. Hepatomegaly was reported in 164/176 (93.2%) of GSD IX α2 patients, 16/17 (94.1%) of GSD IX β patients, and 30/30 (100%) of GSD IX γ2 patients. Fasting hypoglycemia was reported in 53/121 (43.8%) of GSD IX α2 patients, 8/16 (50%) of GSD IX β patients, and 18/19 (94.7%) of GSD IX γ2 patients. Liver biopsy pathology reports were available and interpreted for 46 GSD IX α2 patients, 3 GSD IX β patients, and 24 GSD IX γ2 patients. 22/46 (47.8%) GSD IX α2 patients, 1/3 (33.3%) GSD IX β patients, and 23/24 (95.8%) GSD IX γ2 patients with available pathology reports documented either some degree of fibrosis or cirrhosis. Conclusion Our comprehensive review demonstrates quantitatively that the clinical presentation of GSD IX γ2 patients is more severe than that of GSD IX α2 or β patients. However, our study also shows the existence of a severe phenotype in GSD IX α2, evidenced by early onset liver pathology in conjunction with clinical symptoms. There is need for a more robust natural history study to better understand the variability in liver pathophysiology within liver GSD IX; in addition, further study of mutations and gene mapping could bring a better understanding of the relationship between genotype and clinical presentation.

Background Glycogen storage disease (GSD) is a relatively rare inborn metabolic disorder, our study aims to investigate the genotypic and clinical feature of hepatic GSDs in China. Methods The clinical and genotypic data of 49 patients with hepatic GSDs were collected retrospectively and analyzed. Results After gene sequencing, 49 patients were diagnosed as GSDs, including GSD Ia (24 cases), GSD IIIa (11 cases), GSD IXa (8 cases), GSD VI (3 cases) and GSD Ib (3 cases). About 45 gene variants of G6PC, AGL, PHKA2, PYGL, and SLC37A4 were detected; among which, 22 variants were unreported previously. c.648G>T (p. Leu216Leu) of G6PC exon 5 is the most common variant for GSD Ia patients (20/24,83.33%）, splice variant c.1735+1G>T of AGL exon 13 is relatively common among GSD IIIa, while novel variant accounts for the majority of GSD IXa and GSD VI patients. As for clinical features, there was no significant difference in the onset age among group GSD Ia, GSD IIIa, and GSD IXa, but the age at diagnosis and average disease duration from diagnosis of GSD Ia were significantly higher than GSD IIIa and GSD IXa. Body weight of GSD patients was basically normal, but growth retardation was relatively common among them, especially for GSD Ia patients; and renomegaly was only found in GSD Ia. Besides, serum cholesterol, triglyceride, lactic acid, and uric acid in GSD Ia were significantly higher than those with GSD IIIa and IXa (p, Glycogen storage disease (GSD) is a relatively rare inborn metabolic disorder. In order to further understand the genotypic and clinical characteristics of hepatic GSDs, so as to facilitate early diagnosis and treatment, 49 Chinese cases with hepatic GSDs were recruited retrospectively and their clinical features were analyzed.

Most glucose is processed in muscle, for energy or glycogen stores. Malignant Hyperthermia Susceptibility (MHS) exemplifies muscle conditions that increase [Ca2+]cytosol. 42% of MHS patients have hyperglycemia. We show that phosphorylated glycogen phosphorylase (GPa), glycogen synthase (GSa) – respectively activated and inactivated by phosphorylation – and their Ca2+-dependent kinase (PhK), are elevated in microsomal extracts from MHS patients’ muscle. Glycogen and glucose transporter GLUT4 are decreased. [Ca2+]cytosol, increased to MHS levels, promoted GP phosphorylation. Imaging at ~100 nm resolution located GPa at sarcoplasmic reticulum (SR) junctional cisternae, and apo-GP at Z disk. MHS muscle therefore has a wide-ranging alteration in glucose metabolism: high [Ca2+]cytosol activates PhK, which inhibits GS, activates GP and moves it toward the SR, favoring glycogenolysis. The alterations probably cause these patients’ hyperglycemia. For basic studies, MHS emerges as a variable stressor, which forces glucose pathways from the normal to the diseased range, thereby exposing novel metabolic links., eLife digest Animals and humans move by contracting the skeletal muscles attached to their bones. These muscles take up a type of sugar called glucose from food and use it to fuel contractions or store it for later in the form of glycogen. If muscles fail to use glucose it can lead to excessive sugar levels in the blood and a condition called diabetes. Within muscle cells are stores of calcium that signal the muscle to contract. Changes in calcium levels enhance the uptake of glucose that fuel these contractions. However, variations in calcium have also been linked to diabetes, and it remained unclear when and how these ‘signals’ become harmful. People with a condition called malignant hyperthermia susceptibility (MHS for short) have genetic mutations that allow calcium to leak out from these stores. This condition may result in excessive contractions causing the muscle to over-heat, become rigid and break down, which can lead to death if left untreated. A clinical study in 2019 found that out of hundreds of patients who had MHS, nearly half had high blood sugar and were likely to develop diabetes. Now, Tammineni et al. – including some of the researchers involved in the 2019 study – have set out to find why calcium leaks lead to elevated blood sugar levels. The experiments showed that enzymes that help convert glycogen to glucose are more active in patients with MHS, and found in different locations inside muscle cells. Whereas the enzymes that change glucose into glycogen are less active. This slows down the conversion of glucose into glycogen for storage and speeds up the breakdown of glycogen into glucose. Patients with MHS also had fewer molecules that transport glucose into muscle cells and stored less glycogen. These changes imply that less glucose is being removed from the blood. Next, Tammineni et al. used a microscopy technique that is able to distinguish finely separated objects with a precision not reached before in living muscle. This revealed that when the activity of the enzyme that breaks down glycogen increased, it moved next to the calcium store. This effect was also observed in the muscle cells of MHS patients that leaked calcium from their stores. Taken together, these observations may explain why patients with MHS have high levels of sugar in their blood. These findings suggest that MHS may start decades before developing diabetes and blood sugar levels in these patients should be regularly monitored. Future studies should investigate whether drugs that block calcium from leaking may help prevent high blood sugar in patients with MHS or other conditions that cause a similar calcium leak.

Glycogen phosphorylase kinase β-subunit (PHKB) is a regulatory subunit of phosphorylase kinase (PHK), involving in the activation of glycogen phosphorylase (GP) and the regulation of glycogen breakdown. Emerging evidence suggests that PHKB plays a role in tumor progression. However, the function of PHKB in HCC progression remains elusive. Here, our study revealed that the expression of PHKB significantly decreased in HCC tissues, and the low expression of PHKB could serve as an independent indicator for predicting poor prognosis in HCC. Functional experiments showed that PHKB knockdown significantly promoted cell proliferation both in vitro and in vivo, whereas PHKB overexpression resulted in opposing effects. Additionally, in vitro assays revealed that the over (or high) expression of PHKB greatly hindered HCC cell invasion and increased apoptosis rates. Also, we found that the over (or high) expression of PHKB effectively suppressed the epithelial-mesenchymal transition, which was further confirmed by our clinical data. Intriguingly, the biological function of PHKB in HCC was independent of glycogen metabolism. Mechanically, PHKB could inhibit AKT and STAT3 signaling pathway activation in HCC. Collectively, our data demonstrate that PHKB acts as a novel prognostic indicator for HCC, which exerts its suppression function via inactivating AKT and STAT3. Our data might provide novel insights into progression and facilitate the development of a new therapeutic strategy for HCC.

Small GTPases, together with their regulatory and effector molecules, are key intermediaries in the complex signalling pathways that control almost all cellular processes, working as molecular switches to transduce extracellular cues into cellular responses that drive vital functions, such as intracellular transport, biomolecule synthesis, gene activation and cell survival. How all of these networks are linked to metabolic pathways is a subject of intensive study. Because any response to cellular action requires some form of energy input, elucidating how cells coordinate the signals that lead to a tangible response involving metabolism is central to understand cellular activities. In this review, we summarize recent advances in our understanding of the molecular basis of the crosstalk between small GTPases of the Ras superfamily, specifically Rac1 and Ras/Rap1, and glycogen phosphorylase in T lymphocytes. Abbreviations: ADCY: adenylyl cyclase; ADCY6: adenylyl cyclase 6; BCR: B cell receptor; cAMP: 3',5'-cyclic adenosine monophosphate; CRIB: Cdc42/Rac binding domain; DLPFC: dysfunction of the dorsolateral prefrontal cortex; EGFR: epidermal growth factor receptor; Epac2: exchange protein directly activated by cAMP; GDP: guanodine-5'-diphosphate; GPCRs: G protein-coupled receptors; GTP: guanodin-5'-triphosphate; IL2: interleukin 2; IL2-R: interleukin 2 receptor; JAK: janus kinases; MAPK: mitogen-activated protein kinase; O-GlcNAc: O-glycosylation; PAK1: p21 activated kinase 1; PI3K: phosphatidylinositol 3-kinase; PK: phosphorylase kinase; PKA: cAMP-dependent protein kinase A; PKCθ: protein kinase Cθ; PLCγ: phospholipase Cγ; Src: proto-oncogene tyrosine-protein kinase c; STAT: signal transducer and activator of transcription proteins.

Markers for monitoring clearance of Mycobacterium tuberculosis (Mtb) infection during anti-TB drug treatment could facilitate management of tuberculosis (TB) treatment, but are lacking. We aimed to screen for Mtb clearance markers from in-vitro-infected leucocytes and to evaluate these markers in followed-up active TB (ATB) patients and latent TB (LTBI) cases after anti-TB drug treatment. Extracellular proteins from primary leucocytes infected with each of the Mtb lineages (East-Asian, Indo-Oceanic, Euro-American and the laboratory strain H37Rv) were screened as possible clearance markers. Leucocytes infected with Staphylococcus aureus acted as controls. The proteomic analysis was performed using GeLC-MS/MS. Several quantitative and qualitative candidate clearance markers were found. These proteins were suppressed during the infection stage of all Mtb lineages and re-expressed after bacillary clearance. PSTK, FKBP8 and MGMT were common clearance markers among the four Mtb lineages in our model. Only PSTK was a potential clearance marker based on western blot validation analysis from culture supernatants. The PSTK marker was further validated with western blot analysis using serum samples (n = 6) from ATB patients and LTBI cases during anti-TB drug treatment, and from healthy controls (n = 3). Time-dependent increase of PSTK was found both in ATB and LTBI patients during the course of anti-TB drug treatment, but not in healthy controls. We have demonstrated that PSTK is a potential treatment-monitoring marker for active and latent TB.

The key regulatory enzymes of glycogenolysis are phosphorylase kinase, a hetero-oligomer with four different types of subunits, and glycogen phosphorylase, a homodimer. Both enzymes are activated by phosphorylation and small ligands, and both enzymes have distinct isoforms that are predominantly expressed in muscle, liver, or brain; however, whole-transcriptome high-throughput sequencing analyses show that in brain both of these enzymes are likely composed of subunit isoforms representing all three tissues. This Minireview examines the regulatory properties of the isoforms of these two enzymes expressed in the three tissues, focusing on their potential regulatory similarities and differences. Additionally, the activity, structure, and regulation of the remaining enzyme necessary for glycogenolysis, glycogen-debranching enzyme, are also reviewed.

In the tightly regulated glycogenolysis cascade, the breakdown of glycogen to glucose-1-phosphate, phosphorylase kinase (PhK) plays a key role in regulating the activity of glycogen phosphorylase. PhK is a 1.3 MDa hexadecamer, with four copies each of four different subunits (α, β, γ and δ), making the study of its structure challenging. Using hydrogen-deuterium exchange, we have analyzed the regulatory β subunit and the catalytic γ subunit in the context of the intact non-activated PhK complex to study the structure of these subunits and identify regions of surface exposure. Our data suggest that within the non-activated complex the γ subunit assumes an activated conformation and are consistent with a previous docking model of the β subunit within the cryoelectron microscopy envelope of PhK.

Phosphorylase kinase (PhK), a 1.3 MDa regulatory enzyme complex in the glycogenolysis cascade, has four copies each of four subunits, (αβγδ)4 , and 325 kDa of unique sequence (the mass of an αβγδ protomer). The α, β and δ subunits are regulatory, and contain allosteric activation sites that stimulate the activity of the catalytic γ subunit in response to diverse signaling molecules. Due to its size and complexity, no high resolution structures have been solved for the intact complex or its regulatory α and β subunits. Of PhK's four subunits, the least is known about the structure and function of its largest subunit, α. Here, we have modeled the full-length α subunit, compared that structure against previously predicted domains within this subunit, and performed hydrogen-deuterium exchange on the intact subunit within the PhK complex. Our modeling results show α to comprise two major domains: an N-terminal glycoside hydrolase domain and a large C-terminal importin α/β-like domain. This structure is similar to our previously published model for the homologous β subunit, although clear structural differences are present. The overall highly helical structure with several intervening hinge regions is consistent with our hydrogen-deuterium exchange results obtained for this subunit as part of the (αβγδ)4 PhK complex. Several low exchanging regions predicted to lack ordered secondary structure are consistent with inter-subunit contact sites for α in the quaternary structure of PhK; of particular interest is a low-exchanging region in the C-terminus of α that is known to bind the regulatory domain of the catalytic γ subunit.

Background and Purpose Glycogen phosphorylase (GP) is the key enzyme for glycogen degradation. GP inhibitors (GPi-s) are glucose lowering agents that cause the accumulation of glucose in the liver as glycogen. Glycogen metabolism has implications in beta cell function. Glycogen degradation can maintain cellular glucose levels, which feeds into catabolism to maintain insulin secretion, and elevated glycogen degradation levels contribute to glucotoxicity. The purpose of this study was to assess whether influencing glycogen metabolism in beta cells by GPi-s affects the function of these cells. Experimental Approach The effects of structurally different GPi-s were investigated on MIN6 insulinoma cells and in a mouse model of diabetes. Key Results GPi treatment increased glycogen content and, consequently, the surface area of glycogen in MIN6 cells. Furthermore, GPi treatment induced insulin receptor β (InsRβ), Akt and p70S6K phosphorylation, as well as pancreatic and duodenal homeobox 1(PDX1) and insulin expression. In line with these findings, GPi-s enhanced non-stimulated and glucose-stimulated insulin secretion in MIN6 cells. The InsRβ was shown to co-localize with glycogen particles as confirmed by in silico screening, where components of InsR signalling were identified as glycogen-bound proteins. GPi-s also activated the pathway of insulin secretion, indicated by enhanced glycolysis, mitochondrial oxidation and calcium signalling. Finally, GPi-s increased the size of islets of Langerhans and improved glucose-induced insulin release in mice. Conclusion and Implications These data suggest that GPi-s also target beta cells and can be repurposed as agents to preserve beta cell function or even ameliorate beta cell dysfunction in different forms of diabetes. Linked Articles This article is part of a themed section on Inventing New Therapies Without Reinventing the Wheel: The Power of Drug Repurposing. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v175.2/issuetoc

To study the expression and intracellular localization of phosphorylase kinase β (PHKβ) protein in colorectal cancers (CRCs), analyze its correlation with clinicopathological features and prognosis, and study the biological roles and mechanism-of-action of PHKβ in CRC cell lines. Quantitative polymerase chain reaction (qPCR) and western blot assays were performed to compare the expressions of PHKβ mRNA and protein in CRC tissues and matched normal mucosa. Tissue microarrays and immunohistochemical staining were performed to detect the expression and intracellular location of PHKβ protein and analyze its correlation with the clinicopathological characteristics and prognosis in CRC patients. Proliferation, cell cycle, wound healing, and xenograft models were used to elucidate the potential role of PHKβ in vitro and in vivo. PHKβ mRNA and protein were found to be overexpressed in CRC tissue compared to the levels in normal mucosa. Positive expression of PHKβ was significantly correlated with TNM stage and distal metastasis, and elevated expression of PHKβ was an independent prognostic factor in patients with CRC. PHKβ knockdown impaired proliferation of CRC in vitro and in vivo and induced cell cycle arrest. PHKβ affects CRC cell growth and represents a novel prognostic biomarker.

Introduction: Glycogen storage disease (GSD) type IX, characterized by liver enlargement and elevated aminotransferase levels, is the most frequent type of GSD. The global incidence of GSD type IXa is only about 1/100,000 individuals. Case reports of GSD type IX are rare in China. We present the first case report of GSD type IXa in Northeast China caused by mutation of PHKA2. Patient concerns: An 11-year-old boy was referred to our hospital because of liver enlargement with consistently elevated transaminase levels over 6 months. Diagnosis: Histopathological results following an ultrasound-guided liver biopsy confirmed a diagnosis of GSD. Further genetic testing showed that the patient had GSD type IXa caused by the c.133C>T mutation in PHAK2. Interventions: We placed the patient on a high-protein and high-starch diet and provided hepatoprotective and supportive therapy. Outcomes: The patient's transaminase levels decreased significantly and were nearly normal at 10-month follow-up. Conclusion: This is the first reported case of GSD type IXa in Northeast China. We hope that the detailed and complete report of this case will provide a reference for the diagnosis of liver enlargement of unknown etiology in future clinical practice.

Summary Astrocytic glycogen is an important energy reserve in the brain and is believed to supply fuel during energy crisis. However, the pattern of glycogen metabolism in ischemic stroke and its potential therapeutic impact on neurological outcomes are still unknown. Here, we found extensive brain glycogen accumulation after reperfusion in ischemic stroke patients and primates. Glycogenolytic dysfunction in astrocytes is responsible for glycogen accumulation, caused by inactivation of the protein kinase A (PKA)-glycogen phosphorylase kinase (PhK)-glycogen phosphorylase (GP) cascade accompanied by the activation of glycogen synthase kinase-3β (GSK3β). Genetic or pharmacological augmentation of astrocytic GP could promote astrocyte and neuron survival and improve neurological behaviors. In addition, we found that insulin exerted a neuroprotective effect, at least in part by rescuing the PKA-PhK-GP cascade to maintain homeostasis of glycogen metabolism during reperfusion. Together, our findings suggest a promising intervention for undesirable outcomes in ischemic stroke., Graphical Abstract, Highlights • Glycogen accumulates upon cerebral reperfusion in humans, primates, and rodents • Impaired glycogenolysis underlies excess glycogen during cerebral reperfusion • Activating glycogenolysis protects against acute and subacute reperfusion insult • Insulin mediates neuroprotection partly by rescuing glycogenolysis upon reperfusion, Neuroscience; Molecular Neuroscience; Cellular Neuroscience

Glycogen storage disease type IX (GSD IX) is caused by a deficiency of hepatic phosphorylase kinase. The aim of this study was to clarify the clinical features, long term outcomes, and genetic analysis of GSD IX in Korea. A GSD gene panel was created and hybridization capture-based next-generation sequencing was performed. We investigated clinical laboratory data, results of molecular genetic analysis, liver biopsy findings, and long-term outcomes. Ten children were diagnosed with GSD IX at Seoul National University Children's Hospital. Hypoglycemia, hyperlactacidemia, hypertriglyceridemia, hyperuricemia, liver fibrosis on liver biopsy, and short stature was found in 30%, 56%, 100%, 60%, 80% and 50% of the children, respectively. Seven PHKA2 variants were identified in eight children with GSD IXa—one nonsense (c.2268dupT; p.(Asp757Ter)), two splicing (c.918+1G > A, c.718-2A > G), one frameshift (c.405_419delinsTCCTGGCC; p.(Asp136ProfsTer11)), and three missense variants (c.3628G > A; p.(Gly1210Arg), c.1245G > T and c.2746C > T; p.(Arg916Trp)). Two variants of PHKG2 were identified in two children with GSD IXc—one frameshift (c.783delC; p.(Ser262AlafsTer6)) and one missense (c.661G > A; p.(Val221Met)). Elevated liver enzymes and hypertriglyceridemia in children with GSD IXa tended to improve with age. For the first time, we report hepatocellular carcinoma in a patient with GSD IXc. The GSD gene panel is a useful diagnostic tool to confirm GSD IX. The clinical phenotype of GSD IXc is severe and monitoring for the development of hepatocellular carcinoma should be implemented.

Background Meat production from the commercial crossbred Duroc × (Landrace × Yorkshire) (DLY) pig is predominant in the pork industry, but its meat quality is often impaired by low ultimate pH (pHu). Muscle glycogen level at slaughter is closely associated with pHu and meat technological quality, but its genetic basis remains elusive. The aim of this study was to identify genes and/or causative mutations associated with muscle glycogen level and other meat quality traits by performing a genome-wide association study (GWAS) and additional analyses in a population of 610 DLY pigs. Results Our initial GWAS identified a genome-wide significant (P = 2.54e−11) quantitative trait locus (QTL) on SSC15 (SSC for Sus scrofa chromosome) for the level of residual glycogen and glucose (RG) in the longissimus muscle at 45 min post-mortem. Then, we demonstrated that a low-frequency (minor allele frequency = 0.014) R200Q missense mutation in the PRKAG3 (RN) gene caused this major QTL effect on RG. Moreover, we showed that the 200Q (RN–) allele was introgressed from the Hampshire breed into more than one of the parental breeds of the DLY pigs. After conditioning on R200Q, re-association analysis revealed three additional QTL for RG on SSC3 and 4, and on an unmapped scaffold (AEMK02000452.1). The SSC3 QTL was most likely caused by a splice mutation (g.8283C>A) in the PHKG1 gene that we had previously identified. Based on functional annotation, the genes TMCO1 on SSC4 and CKB on the scaffold represent promising candidate genes for the other two QTL. There were significant interaction effects of the GWAS tag SNPs at those two loci with PRKAG3 R200Q on RG. In addition, a number of common variants with potentially smaller effects on RG (P A. Conclusions We found that the RN– allele segregates in the parental lines of our DLY population and strongly influences its meat quality. Our findings also indicate that the genetic basis of RG in DLY can be mainly attributed to two major genes (PRKAG3 and PHKG1), along with many minor genes. Electronic supplementary material The online version of this article (10.1186/s12711-019-0488-0) contains supplementary material, which is available to authorized users.

Equine melanocytic neoplasm (EMN) is a cutaneous neoplasm and is mostly observed in aged grey horses. This preliminary study aimed to identify potential proteins to differentiate normal, mild and severe EMN from serum proteomic profiling. Serum samples were collected from 25 grey horses assigned to three groups: normal (free of EMN, n = 10), mild (n = 6) and severe EMN (n = 9). To explore the differences in proteins between groups, proteomic profiling and analysis were employed. Accordingly, 8241 annotated proteins out of 8725 total proteins were compared between normal and EMN groups and inspected based on differentially expressed proteins (DEPs). Through DEP analysis, 95 significant DEPs differed between normal and EMN groups. Among these DEPs, 41 significant proteins were categorised according to protein functions. Based on 41 significant proteins, 10 were involved in metabolism and 31 in non-metabolism. Interestingly, phospholipid phosphatase6 (PLPP6) and ATPase subunit alpha (Na+/K+-ATPase) were considered as potential proteins uniquely expressed in mild EMN and related to lipid and energy metabolism, respectively. Non-metabolism-related proteins (BRCA1, phosphorylase B kinase regulatory subunit: PHKA1, tyrosine-protein kinase receptor: ALK and rho-associated protein kinase: ROCK1) correlated to melanoma development differed among all groups. The results of our study provide a foundation for early EMN biomonitoring and prevention.

Oximes have been studied for decades because of their significant roles as acetylcholinesterase reactivators. Over the last twenty years, a large number of oximes have been reported with useful pharmaceutical properties, including compounds with antibacterial, anticancer, anti-arthritis, and anti-stroke activities. Many oximes are kinase inhibitors and have been shown to inhibit over 40 different kinases, including AMP-activated protein kinase (AMPK), phosphatidylinositol 3-kinase (PI3K), cyclin-dependent kinase (CDK), serine/threonine kinases glycogen synthase kinase 3 α/β (GSK-3α/β), Aurora A, B-Raf, Chk1, death-associated protein-kinase-related 2 (DRAK2), phosphorylase kinase (PhK), serum and glucocorticoid-regulated kinase (SGK), Janus tyrosine kinase (JAK), and multiple receptor and non-receptor tyrosine kinases. Some oximes are inhibitors of lipoxygenase 5, human neutrophil elastase, and proteinase 3. The oxime group contains two H-bond acceptors (nitrogen and oxygen atoms) and one H-bond donor (OH group), versus only one H-bond acceptor present in carbonyl groups. This feature, together with the high polarity of oxime groups, may lead to a significantly different mode of interaction with receptor binding sites compared to corresponding carbonyl compounds, despite small changes in the total size and shape of the compound. In addition, oximes can generate nitric oxide. This review is focused on oximes as kinase inhibitors with anticancer and anti-inflammatory activities. Oximes with non-kinase targets or mechanisms of anti-inflammatory activity are also discussed.