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1.
Aging Cell ; 20(8): e13433, 2021 08.
Article En | MEDLINE | ID: mdl-34261192

Recent studies indicate a crucial role for neuronal glycogen storage and degradation in memory formation. We have previously identified alpha-amylase (α-amylase), a glycogen degradation enzyme, located within synaptic-like structures in CA1 pyramidal neurons and shown that individuals with a high copy number variation of α-amylase perform better on the episodic memory test. We reported that neuronal α-amylase was absent in patients with Alzheimer's disease (AD) and that this loss corresponded to increased AD pathology. In the current study, we verified these findings in a larger patient cohort and determined a similar reduction in α-amylase immunoreactivity in the molecular layer of hippocampus in AD patients. Next, we demonstrated reduced α-amylase concentrations in oligomer amyloid beta 42 (Aß42 ) stimulated SH-SY5Y cells and neurons derived from human-induced pluripotent stem cells (hiPSC) with PSEN1 mutation. Reduction of α-amylase production and activity, induced by siRNA and α-amylase inhibitor Tendamistat, respectively, was further shown to enhance glycogen load in SH-SY5Y cells. Both oligomer Aß42  stimulated SH-SY5Y cells and hiPSC neurons with PSEN1 mutation showed, however, reduced load of glycogen. Finally, we demonstrate the presence of α-amylase within synapses of isolated primary neurons and show that inhibition of α-amylase activity with Tendamistat alters neuronal activity measured by calcium imaging. In view of these findings, we hypothesize that α-amylase has a glycogen degrading function within synapses, potentially important in memory formation. Hence, a loss of α-amylase, which can be induced by Aß pathology, may in part underlie the disrupted memory formation seen in AD patients.


Alzheimer Disease/physiopathology , Amyloid beta-Peptides/metabolism , Glycogenolysis/genetics , alpha-Amylases/metabolism , Animals , Humans , Male , Mice
2.
Cell Metab ; 33(7): 1404-1417.e9, 2021 07 06.
Article En | MEDLINE | ID: mdl-34043942

Glycosylation defects are a hallmark of many nervous system diseases. However, the molecular and metabolic basis for this pathology is not fully understood. In this study, we found that N-linked protein glycosylation in the brain is metabolically channeled to glucosamine metabolism through glycogenolysis. We discovered that glucosamine is an abundant constituent of brain glycogen, which functions as a glucosamine reservoir for multiple glycoconjugates. We demonstrated the enzymatic incorporation of glucosamine into glycogen by glycogen synthase, and the release by glycogen phosphorylase by biochemical and structural methodologies, in primary astrocytes, and in vivo by isotopic tracing and mass spectrometry. Using two mouse models of glycogen storage diseases, we showed that disruption of brain glycogen metabolism causes global decreases in free pools of UDP-N-acetylglucosamine and N-linked protein glycosylation. These findings revealed fundamental biological roles of brain glycogen in protein glycosylation with direct relevance to multiple human diseases of the central nervous system.


Brain/metabolism , Glucosamine/metabolism , Glycogen/physiology , Protein Processing, Post-Translational , Animals , Cells, Cultured , Disease Models, Animal , Female , Glycogen/metabolism , Glycogen Synthase/genetics , Glycogen Synthase/metabolism , Glycogenolysis/genetics , Glycosylation , Lafora Disease/genetics , Lafora Disease/metabolism , Lafora Disease/pathology , Male , Mice , Mice, Inbred C57BL , Mice, Knockout , Protein Processing, Post-Translational/genetics
3.
J Clin Endocrinol Metab ; 105(2)2020 02 01.
Article En | MEDLINE | ID: mdl-31628455

CONTEXT: Glycogenin is considered to be an essential primer for glycogen biosynthesis. Nevertheless, patients with glycogenin-1 deficiency due to biallelic GYG1 (NM_004130.3) mutations can store glycogen in muscle. Glycogenin-2 has been suggested as an alternative primer for glycogen synthesis in patients with glycogenin-1 deficiency. OBJECTIVE: The objective of this article is to investigate the importance of glycogenin-1 and glycogenin-2 for glycogen synthesis in skeletal and cardiac muscle. DESIGN, SETTING, AND PATIENTS: Glycogenin-1 and glycogenin-2 expression was analyzed by Western blot, mass spectrometry, and immunohistochemistry in liver, heart, and skeletal muscle from controls and in skeletal and cardiac muscle from patients with glycogenin-1 deficiency. RESULTS: Glycogenin-1 and glycogenin-2 both were found to be expressed in the liver, but only glycogenin-1 was identified in heart and skeletal muscle from controls. In patients with truncating GYG1 mutations, neither glycogenin-1 nor glycogenin-2 was expressed in skeletal muscle. However, nonfunctional glycogenin-1 but not glycogenin-2 was identified in cardiac muscle from patients with cardiomyopathy due to GYG1 missense mutations. By immunohistochemistry, the mutated glycogenin-1 colocalized with the storage of glycogen and polyglucosan in cardiomyocytes. CONCLUSIONS: Glycogen can be synthesized in the absence of glycogenin, and glycogenin-1 deficiency is not compensated for by upregulation of functional glycogenin-2. Absence of glycogenin-1 leads to the focal accumulation of glycogen and polyglucosan in skeletal muscle fibers. Expression of mutated glycogenin-1 in the heart is deleterious, and it leads to storage of abnormal glycogen and cardiomyopathy.


Glucosyltransferases/genetics , Glycogen Storage Disease/genetics , Glycoproteins/genetics , Muscle, Skeletal/metabolism , Myocardium/metabolism , Adult , Aged , Aged, 80 and over , Child , Female , Glucans/metabolism , Glycogenolysis/genetics , Humans , Male , Mutation , Mutation, Missense
4.
Food Chem Toxicol ; 135: 110894, 2020 Jan.
Article En | MEDLINE | ID: mdl-31644924

Acrylamide (AA), a food contaminant, caused islet remodeling and increased hepatic glycogen content in male rats, but the effect of AA on glucose homeostasis in female rats remains unclear. In this study, female SD rats were orally treated with 0, 15, or 30 mg/kg·bw AA for 3 weeks. The levels of fasting blood glucose (FBG), blood glucose after oral administration of glucose, plasma insulin and hepatic glycogen were measured. The histology of the pancreas was observed, and the transcription of key genes involved in glucose metabolism and insulin signaling in liver were determined. Compared with the control, exposure to 30 mg/kg·bw of AA significantly increased FBG level, reduced hepatic glycogen content and impaired glucose tolerance. Moreover, damaged islets were observed at 15 and 30 mg/kg·bw AA-exposed groups. In addition, AA exposure significantly promoted gluconeogenesis and glycogenolysis (up-regulation of pc, g6p and gp) and decreased glycolysis (down-regulation of gck and pfk). Alternations in these processes may be associated with decreased plasma insulin levels and inhibited insulin-regulated IRS/PI3K/Akt/Foxo1 signaling transduction under AA exposure. Overall, our findings demonstrated that AA disrupted glucose homeostasis and elevated FBG level in female rats possibly by interfering with glucose metabolism and hampering the physiological effect of insulin.


Acrylamide/adverse effects , Blood Glucose/metabolism , Homeostasis/drug effects , Animals , Female , Gene Expression/drug effects , Gluconeogenesis/genetics , Glucose Intolerance/chemically induced , Glycogenolysis/genetics , Glycolysis/genetics , Insulin/metabolism , Islets of Langerhans/drug effects , Islets of Langerhans/pathology , Liver/drug effects , Liver Glycogen/metabolism , Rats, Sprague-Dawley , Signal Transduction/genetics
5.
Cell Metab ; 30(5): 903-916.e7, 2019 11 05.
Article En | MEDLINE | ID: mdl-31523006

Nuclear glycogen was first documented in the early 1940s, but its role in cellular physiology remained elusive. In this study, we utilized pure nuclei preparations and stable isotope tracers to define the origin and metabolic fate of nuclear glycogen. Herein, we describe a key function for nuclear glycogen in epigenetic regulation through compartmentalized pyruvate production and histone acetylation. This pathway is altered in human non-small cell lung cancers, as surgical specimens accumulate glycogen in the nucleus. We demonstrate that the decreased abundance of malin, an E3 ubiquitin ligase, impaired nuclear glycogenolysis by preventing the nuclear translocation of glycogen phosphorylase and causing nuclear glycogen accumulation. Re-introduction of malin in lung cancer cells restored nuclear glycogenolysis, increased histone acetylation, and decreased growth of cancer cells transplanted into mice. This study uncovers a previously unknown role for glycogen metabolism in the nucleus and elucidates another mechanism by which cellular metabolites control epigenetic regulation.


Carcinoma, Non-Small-Cell Lung/metabolism , Cell Nucleus/metabolism , Glycogenolysis/genetics , Histones/metabolism , Lung Neoplasms/metabolism , A549 Cells , Acetylation , Animals , Carbon/metabolism , Carcinoma, Non-Small-Cell Lung/pathology , Glycogen/biosynthesis , Glycogen Phosphorylase/metabolism , HEK293 Cells , Humans , Lung Neoplasms/pathology , Mice , Mice, Knockout , Mice, Nude , Transfection , Ubiquitin-Protein Ligases/genetics , Ubiquitin-Protein Ligases/metabolism
6.
PLoS One ; 13(12): e0209647, 2018.
Article En | MEDLINE | ID: mdl-30576384

Adenosine signaling is involved in glucose metabolism in hepatocytes and myocytes in vitro. However, no information is available regarding the effect of adenosine on glucose metabolism in vivo. Thus, we examined how extracellular adenosine acts on glucose metabolism using mice. Subcutaneous injections of adenosine (10, 25, and 50 mg/kg bodyweight) dose-dependently increased blood glucose levels, with the peak occurring at 30 min post injection. At 30 min after adenosine injection (25 mg/kg bodyweight), glycogen content in the liver, but not the skeletal muscle, was significantly decreased. Hepatic glycogen depletion by fasting for 12 h suppressed the increase of blood glucose levels at 30 min after adenosine injection. These results suggest that adenosine increases blood glucose levels by stimulating hepatic glycogenolysis. To investigate the effect of adenosine on the adrenal gland, we studied the glycogenolysis signal in adrenalectomized (ADX) mice. Adenosine significantly increased the blood glucose levels in sham mice but not in the ADX mice. The decrease in hepatic glycogen content induced by adenosine in the sham mice was partially suppressed in the ADX mice. The level of plasma corticosterone, the main glucocorticoid in mice, was significantly increased in the sham mice by adenosine but its levels were low in ADX mice injected with either PBS or adenosine. These results suggest that adenosine promotes secretion of corticosterone from the adrenal glands, which causes hepatic glycogenolysis and subsequently the elevation of blood glucose levels. Our findings are useful for clarifying the physiological functions of adenosine in glucose metabolism in vivo.


Adenosine/metabolism , Adrenal Glands/metabolism , Corticosterone/blood , Liver/metabolism , Adrenal Glands/pathology , Adrenal Glands/surgery , Adrenalectomy , Animals , Fasting , Glucose/metabolism , Glycogenolysis/genetics , Hepatocytes/metabolism , Hepatocytes/pathology , Insulin/metabolism , Liver/pathology , Liver Glycogen/metabolism , Mice
7.
Free Radic Res ; 52(5): 568-582, 2018 May.
Article En | MEDLINE | ID: mdl-29544378

Oxidative stress due to enhanced production or reduced scavenging of reactive oxygen species (ROS) has been associated with diet (dyslipidemia) induced obesity and insulin resistance (IR). The present study was undertaken to assess the role of p47phox in IR using wild type (WT) and p47phox-/- mice, fed with different diets (HFD, LFD or Chow). Augmented body weight, glucose intolerance and reduced insulin sensitivity were observed in p47phox-/- mice fed with 45% HFD and 10% LFD. Further, body fat and circulating lipids were increased significantly with 5 weeks LFD feeding in p47phox-/- mice, while parameters of energy homeostasis were reduced as compared with WT mice. LFD fed knockout (KO) mice showed an enhanced hepatic glycogenolysis, and reduced insulin signalling in liver and adipose tissue, while skeletal muscle tissue remained unaffected. A significant increase in hepatic lipids, adiposity, as well as expression of genes regulating lipid synthesis, breakdown and efflux were observed in LFD fed p47phox-/- mice after 5 weeks. On the other hand, mice lacking p47phox demonstrated altered glucose tolerance and tissue insulin sensitivity after 5 weeks chow feeding, while changes in body weight, respiratory exchange ratio (RER) and heat production are non-significant. Our data demonstrate that lack of p47phox is sufficient to induce IR through altered glucose and lipid utilization by the liver and adipose tissue.


Adipose Tissue/metabolism , Dyslipidemias/metabolism , Glucose/metabolism , Insulin Resistance , Liver/metabolism , NADPH Oxidases/genetics , Obesity/metabolism , Adipose Tissue/pathology , Animals , Cytokines/genetics , Cytokines/metabolism , Diet, Fat-Restricted , Diet, High-Fat , Dyslipidemias/etiology , Dyslipidemias/genetics , Dyslipidemias/pathology , Gene Expression Regulation , Glycogenolysis/genetics , Insulin Receptor Substrate Proteins/genetics , Insulin Receptor Substrate Proteins/metabolism , Lipid Metabolism/genetics , Liver/pathology , Male , Mice , Mice, Knockout , Muscle, Skeletal/metabolism , NADPH Oxidases/deficiency , Obesity/etiology , Obesity/genetics , Obesity/pathology , Oxidative Stress , Proto-Oncogene Proteins c-akt/genetics , Proto-Oncogene Proteins c-akt/metabolism , Reactive Oxygen Species/metabolism , Signal Transduction
8.
Mol Cell Biol ; 37(14)2017 07 15.
Article En | MEDLINE | ID: mdl-28461393

Cyclic AMP-responsive element binding protein, hepatocyte specific (CREBH), is a liver-enriched, endoplasmic reticulum-tethered transcription factor known to regulate the hepatic acute-phase response and lipid homeostasis. In this study, we demonstrate that CREBH functions as a circadian transcriptional regulator that plays major roles in maintaining glucose homeostasis. The proteolytic cleavage and posttranslational acetylation modification of CREBH are regulated by the circadian clock. Functionally, CREBH is required in order to maintain circadian homeostasis of hepatic glycogen storage and blood glucose levels. CREBH regulates the rhythmic expression of the genes encoding the rate-limiting enzymes for glycogenolysis and gluconeogenesis, including liver glycogen phosphorylase (PYGL), phosphoenolpyruvate carboxykinase 1 (PCK1), and the glucose-6-phosphatase catalytic subunit (G6PC). CREBH interacts with peroxisome proliferator-activated receptor α (PPARα) to synergize its transcriptional activities in hepatic gluconeogenesis. The acetylation of CREBH at lysine residue 294 controls CREBH-PPARα interaction and synergy in regulating hepatic glucose metabolism in mice. CREBH deficiency leads to reduced blood glucose levels but increases hepatic glycogen levels during the daytime or upon fasting. In summary, our studies revealed that CREBH functions as a key metabolic regulator that controls glucose homeostasis across the circadian cycle or under metabolic stress.


Circadian Rhythm/physiology , Cyclic AMP Response Element-Binding Protein/metabolism , Gluconeogenesis/physiology , Glucose/metabolism , Glycogenolysis/genetics , Homeostasis/physiology , Animals , Cyclic AMP Response Element-Binding Protein/genetics , Endoplasmic Reticulum/metabolism , Gene Expression Regulation/physiology , Hepatocytes/metabolism , Liver/metabolism , Male , Mice, Inbred C57BL , Mice, Knockout
9.
Nat Rev Neurol ; 12(7): 393-402, 2016 07.
Article En | MEDLINE | ID: mdl-27231184

Skeletal muscle disorders of glycogenolysis and glycolysis account for most of the conditions collectively termed glycogen storage diseases (GSDs). These disorders are rare (incidence 1 in 20,000-43,000 live births), and are caused by autosomal or X-linked recessive mutations that result in a specific enzyme deficiency, leading to the inability to utilize muscle glycogen as an energy substrate. McArdle disease (GSD V) is the most common of these disorders, and is caused by mutations in the gene encoding muscle glycogen phosphorylase. Symptoms of McArdle disease and most other related GSDs include exercise intolerance, muscle contracture, acute rhabdomyolysis, and risk of acute renal failure. Older patients may exhibit muscle wasting and weakness involving the paraspinal muscles and shoulder girdle. For patients with these conditions, engaging with exercise is likely to be beneficial. Diagnosis is frequently delayed owing to the rarity of the conditions and lack of access to appropriate investigations. A few randomized clinical trials have been conducted, some focusing on dietary modification, although the quality of the evidence is low and no specific recommendations can yet be made. The development of EUROMAC, an international registry for these disorders, should improve our knowledge of their natural histories and provide a platform for future clinical trials.


Glycogen Storage Disease , Glycogenolysis , Glycolysis , Muscular Diseases , Glycogen Storage Disease/complications , Glycogen Storage Disease/enzymology , Glycogen Storage Disease/genetics , Glycogen Storage Disease/physiopathology , Glycogenolysis/genetics , Glycolysis/genetics , Humans , Muscular Diseases/complications , Muscular Diseases/enzymology , Muscular Diseases/genetics , Muscular Diseases/physiopathology
11.
PLoS One ; 10(6): e0131476, 2015.
Article En | MEDLINE | ID: mdl-26114292

Protein phosphatase 1 (PP1) is one of the major protein phosphatases in eukaryotic cells. It plays a key role in regulating glycogen synthesis, by dephosphorylating crucial enzymes involved in glycogen homeostasis such as glycogen synthase (GS) and glycogen phosphorylase (GP). To play this role, PP1 binds to specific glycogen targeting subunits that, on one hand recognize the substrates to be dephosphorylated and on the other hand recruit PP1 to glycogen particles. In this work we have analyzed the functionality of the different protein binding domains of one of these glycogen targeting subunits, namely PPP1R3D (R6) and studied how binding properties of different domains affect its glycogenic properties. We have found that the PP1 binding domain of R6 comprises a conserved RVXF motif (R102VRF) located at the N-terminus of the protein. We have also identified a region located at the C-terminus of R6 (W267DNND) that is involved in binding to the PP1 glycogenic substrates. Our results indicate that although binding to PP1 and glycogenic substrates are independent processes, impairment of any of them results in lack of glycogenic activity of R6. In addition, we have characterized a novel site of regulation in R6 that is involved in binding to 14-3-3 proteins (RARS74LP). We present evidence indicating that when binding of R6 to 14-3-3 proteins is prevented, R6 displays hyper-glycogenic activity although is rapidly degraded by the lysosomal pathway. These results define binding to 14-3-3 proteins as an additional pathway in the control of the glycogenic properties of R6.


14-3-3 Proteins/metabolism , Glycogen/metabolism , Protein Interaction Domains and Motifs , Protein Phosphatase 1/metabolism , Amino Acid Sequence , Animals , Binding Sites/genetics , Glycogenolysis/genetics , HEK293 Cells , Humans , Mice , Models, Molecular , Molecular Sequence Data , Protein Binding , Protein Interaction Domains and Motifs/genetics , Protein Phosphatase 1/chemistry , Protein Phosphatase 1/genetics , Protein Subunits , Structure-Activity Relationship
12.
Biosci Rep ; 34(4)2014 Jul 01.
Article En | MEDLINE | ID: mdl-24837458

STBD1 (starch-binding domain-containing protein 1) belongs to the CBM20 (family 20 carbohydrate binding module) group of proteins, and is implicated in glycogen metabolism and autophagy. However, very little is known about its regulation or interacting partners. Here, we show that the CBM20 of STBD1 is crucial for its stability and ability to interact with glycogen-associated proteins. Mutation of a conserved tryptophan residue (W293) in this domain abolished the ability of STBD1 to bind to the carbohydrate amylose. Compared with the WT (wild-type) protein, this mutant exhibited rapid degradation that was rescued upon inhibition of the proteasome. Furthermore, STBD1 undergoes ubiquitination when expressed in COS cells, and requires the N-terminus for this process. In contrast, inhibition of autophagy did not significantly affect protein stability. In overexpression experiments, we discovered that STBD1 interacts with several glycogen-associated proteins, such as GS (glycogen synthase), GDE (glycogen debranching enzyme) and Laforin. Importantly, the W293 mutant of STBD1 was unable to do so, suggesting an additional role for the CBM20 domain in protein-protein interactions. In HepG2 hepatoma cells, overexpressed STBD1 could associate with endogenous GS. This binding increased during glycogenolysis, suggesting that glycogen is not required to bridge this interaction. Taken together, our results have uncovered new insights into the regulation and binding partners of STBD1.


Carbohydrates/genetics , Membrane Proteins/genetics , Membrane Proteins/metabolism , Protein Interaction Domains and Motifs/genetics , Animals , Autophagy/genetics , COS Cells , Carcinoma, Hepatocellular/genetics , Carcinoma, Hepatocellular/metabolism , Cell Line , Cell Line, Tumor , Chlorocebus aethiops , Glycogen/genetics , Glycogen/metabolism , Glycogenolysis/genetics , HEK293 Cells , HeLa Cells , Hep G2 Cells , Humans , Liver Neoplasms/genetics , Liver Neoplasms/metabolism , Mutation/genetics , Proteasome Endopeptidase Complex/genetics , Proteasome Endopeptidase Complex/metabolism , Tryptophan/genetics , Tryptophan/metabolism , Ubiquitination/genetics
13.
FEBS J ; 281(9): 2136-47, 2014 May.
Article En | MEDLINE | ID: mdl-24593051

Insulin resistance is a metabolic disorder associated with type 2 diabetes. Recent reports have shown that fibroblast growth factor-21 (FGF-21) plays an important role in the progression of insulin resistance. However, the biochemical and molecular mechanisms by which changes in FGF-21 activation result in changes in the rates of hepatic gluconeogenesis and glycogenolysis remain to be elucidated. In this study, we developed adenovirus-mediated shRNA against FGF-21 to inhibit FGF-21 expression in ApoE knockout mice. Using this mouse model, we determined the effects of FGF-21 knockdown in vivo on hepatic glucose production, gluconeogenesis and glycogenolysis, and their relationship with the signal transducer and activator of transcription 3 (STAT3)/suppressor of cytokine signaling 3 (SOCS3) signal pathways. We show that liver-specific knockdown of FGF-21 in high-fat diet-fed ApoE knockout mice resulted in a 39% increase in glycogenolysis and a 75% increase in gluconeogenesis, accompanied by increased hepatic expression of glucose-6-phosphatase and phosphoenolpyruvate carboxykinase. Furthermore, FGF-21 knockdown decreased phosphorylation of STAT3 and SOCS3 expression in high-fat diet-fed mice. Our data suggest that hepatic FGF-21 knockdown increases gluconeogenesis and glycogenolysis by activation of glucose-6-phosphatase and phosphoenolpyruvate carboxykinase via the STAT3/SOCS3 pathway, ultimately leading to exacerbation of hepatic insulin resistance.


Fibroblast Growth Factors/physiology , Gene Silencing , Gluconeogenesis/genetics , Glycogenolysis/genetics , Liver/metabolism , STAT3 Transcription Factor/metabolism , Signal Transduction , Suppressor of Cytokine Signaling Proteins/metabolism , Animals , Fibroblast Growth Factors/blood , Fibroblast Growth Factors/genetics , Homeostasis , Insulin Resistance , Mice , Mice, Knockout , RNA Interference , Suppressor of Cytokine Signaling 3 Protein
14.
J Mol Evol ; 78(1): 66-74, 2014 Jan.
Article En | MEDLINE | ID: mdl-24258790

Frugivorous and nectarivorous bats rely largely on hepatic glycogenesis and glycogenolysis for postprandial blood glucose disposal and maintenance of glucose homeostasis during short time starvation, respectively. The glycogen synthase 2 encoded by the Gys2 gene plays a critical role in liver glycogen synthesis. To test whether the Gys2 gene has undergone adaptive evolution in bats with carbohydrate-rich diets in relation to their insect-eating sister taxa, we sequenced the coding region of the Gys2 gene in a number of bat species, including three Old World fruit bats (OWFBs) (Pteropodidae) and two New World fruit bats (NWFBs) (Phyllostomidae). Our results showed that the Gys2 coding sequences are highly conserved across all bat species we examined, and no evidence of positive selection was detected in the ancestral branches leading to OWFBs and NWFBs. Our explicit convergence test showed that posterior probabilities of convergence between several branches of OWFBs, and the NWFBs were markedly higher than that of divergence. Three parallel amino acid substitutions (Q72H, K371Q, and E666D) were detected among branches of OWFBs and NWFBs. Tests for parallel evolution showed that two parallel substitutions (Q72H and E666D) were driven by natural selection, while the K371Q was more likely to be fixed randomly. Thus, our results suggested that the Gys2 gene has undergone parallel evolution on amino acid level between OWFBs and NWFBs in relation to their carbohydrate metabolism.


Blood Glucose/physiology , Chiroptera/genetics , Glycogen Synthase/genetics , Liver Glycogen/biosynthesis , Nucleic Acid Amplification Techniques/veterinary , Amino Acid Sequence , Amino Acid Substitution/genetics , Animals , Base Sequence , Biological Evolution , Blood Glucose/genetics , Blood Glucose/metabolism , Chiroptera/classification , Evolution, Molecular , Glycogenolysis/genetics , Liver Glycogen/genetics , Phylogeny , Sequence Analysis, DNA
15.
Nat Commun ; 4: 2316, 2013.
Article En | MEDLINE | ID: mdl-23939267

During fasting, animals maintain their energy balance by shifting their energy source from carbohydrates to triglycerides. However, the trigger for this switch has not yet been entirely elucidated. Here we show that a selective hepatic vagotomy slows the speed of fat consumption by attenuating sympathetic nerve-mediated lipolysis in adipose tissue. Hepatic glycogen pre-loading by the adenoviral overexpression of glycogen synthase or the transcription factor TFE3 abolished this liver-brain-adipose axis activation. Moreover, the blockade of glycogenolysis [corrected] through the knockdown of the glycogen phosphorylase gene and the resulting elevation in the glycogen content abolished the lipolytic signal from the liver, indicating that glycogen is the key to triggering this neurocircuitry. These results demonstrate that liver glycogen shortage activates a liver-brain-adipose neural axis that has an important role in switching the fuel source from glycogen to triglycerides under prolonged fasting conditions.


Adipose Tissue/innervation , Fasting/metabolism , Liver Glycogen/metabolism , Sympathetic Nervous System/metabolism , Triglycerides/metabolism , Adipose Tissue/metabolism , Animals , Basic Helix-Loop-Helix Leucine Zipper Transcription Factors/biosynthesis , Basic Helix-Loop-Helix Leucine Zipper Transcription Factors/genetics , Basic Helix-Loop-Helix Leucine Zipper Transcription Factors/metabolism , Brain/metabolism , Energy Metabolism , Glycogen Phosphorylase/genetics , Glycogen Phosphorylase/metabolism , Glycogen Synthase/biosynthesis , Glycogen Synthase/genetics , Glycogen Synthase/metabolism , Glycogenolysis/genetics , Guanethidine/pharmacology , Lipolysis/physiology , Liver/innervation , Liver/metabolism , Male , Mice , Mice, Inbred ICR , Nerve Block , Sympathetic Nervous System/drug effects , Sympatholytics/pharmacology , Vagus Nerve/surgery
16.
J Proteome Res ; 12(8): 3801-8, 2013 Aug 02.
Article En | MEDLINE | ID: mdl-23827011

Enhanced green fluorescent protein (EGFP) is a widely used biological reporter. However, the effects of EGFP expression in vivo are still unclear. To investigate the effects of EGFP transgenic expression in vivo, we employed an NMR-based metabonomics method to analyze the metabonome of EGFP transgenic mice. The results show that the metabonomes of urine, liver, and kidney of the EGFP transgenic mice are different from their wild-type counterparts. The EGFP mice expressed high levels of urinary 3-ureidopropionate, which is due to the down-regulated transcriptional level of ß-ureidopropionase. The expression of EGFP in vivo also affects other metabolic pathways, including nucleic acid metabolism, energy utilization, and amino acids catabolism. These findings indicate that EGFP transgenic expression is not as inert as has been considered. Our investigation provides a holistic view on the effect of EGFP expression in vivo, which is useful when EGFP is employed as a functional biological indicator. Our work also highlights the potential of a metabonomics strategy in studying the association between molecular phenotypes and gene function.


Gene Expression Regulation , Green Fluorescent Proteins/genetics , Kidney/metabolism , Liver/metabolism , Mice, Transgenic/urine , Amidohydrolases/genetics , Amidohydrolases/metabolism , Amino Acids/genetics , Amino Acids/metabolism , Animals , Citric Acid Cycle/genetics , Female , Genes, Reporter , Genome-Wide Association Study , Glycogenolysis/genetics , Green Fluorescent Proteins/metabolism , Mice , Mice, Transgenic/genetics , Nucleic Acids/genetics , Nucleic Acids/metabolism , beta-Alanine/analogs & derivatives , beta-Alanine/urine
17.
Biochim Biophys Acta ; 1830(3): 2574-82, 2013 Mar.
Article En | MEDLINE | ID: mdl-23274741

BACKGROUND: Tick embryogenesis is a metabolically intensive process developed under tightly controlled conditions and whose components are poorly understood. METHODS: In order to characterize the role of AKT (protein kinase B) in glycogen metabolism and cell viability, glycogen determination, identification and cloning of an AKT from Rhipicephalus microplus were carried out, in parallel with experiments using RNA interference (RNAi) and chemical inhibition. RESULTS: A decrease in glycogen content was observed when AKT was chemically inhibited by 10-DEBC treatment, while GSK3 inhibition by alsterpaullone had an opposing effect. RmAKT ORF is 1584-bp long and encodes a polypeptide chain of 60.1 kDa. Phylogenetic and sequence analyses showed significant differences between vertebrate and tick AKTs. Either AKT or GSK3 knocked down cells showed a 70% reduction in target transcript levels, but decrease in AKT also reduced glycogen content, cell viability and altered cell membrane permeability. However, the GSK3 reduction promoted an increase in glycogen content. Additionally, either GSK3 inhibition or gene silencing had a protective effect on BME26 viability after exposure to ultraviolet radiation. R. microplus AKT and GSK3 were widely expressed during embryo development. Taken together, our data support an antagonistic role for AKT and GSK3, and strongly suggest that such a signaling axis is conserved in tick embryos, with AKT located upstream of GSK3. GENERAL SIGNIFICANCE: The AKT/GSK3 axis is conserved in tick in a way that integrates glycogen metabolism and cell survival, and exhibits phylogenic differences that could be important for the development of novel control methods.


Arthropod Proteins/genetics , Glycogen Synthase Kinase 3/genetics , Glycogen/metabolism , Glycogenolysis/genetics , Proto-Oncogene Proteins c-akt/genetics , Rhipicephalus/genetics , Animals , Arthropod Proteins/antagonists & inhibitors , Arthropod Proteins/metabolism , Benzazepines/pharmacology , Cell Line , Cell Membrane Permeability/radiation effects , Cell Survival/radiation effects , Cloning, Molecular , Embryo, Nonmammalian , Gene Expression Regulation/radiation effects , Glycogen/genetics , Glycogen Synthase Kinase 3/antagonists & inhibitors , Glycogen Synthase Kinase 3/metabolism , Glycogenolysis/radiation effects , Indoles/pharmacology , Open Reading Frames , Oxazines/pharmacology , Phylogeny , Proto-Oncogene Proteins c-akt/antagonists & inhibitors , Proto-Oncogene Proteins c-akt/metabolism , RNA, Small Interfering/genetics , Rhipicephalus/embryology , Rhipicephalus/metabolism , Sequence Homology, Amino Acid , Signal Transduction/radiation effects , Species Specificity , Ultraviolet Rays
18.
Obesity (Silver Spring) ; 18(10): 1881-7, 2010 Oct.
Article En | MEDLINE | ID: mdl-20203631

Modulation of the expression of the protein phosphatase-1 (PP1) glycogen-targeting subunit PTG exerts profound effects on cellular glycogen metabolism in vitro and in vivo. PTG contains three distinct binding domains for glycogen, PP1, and a common site for glycogen synthase and phosphorylase. The impact of disrupting the PP1-binding domain on PTG function was examined in 3T3-L1 adipocytes. A full-length PTG mutant was generated as an adenoviral construct in which the valine and phenylalanine residues in the conserved PP1-binding domain were mutated to alanine (PTG-VF). Infection of fully differentiated 3T3-L1 adipocytes with the PTG-VF adenovirus reduced glycogen stores by over 50%. In vitro, PTG-VF competitively interfered with wild-type PTG action, suggesting that the mutant construct acted as a dominant-negative molecule. The reduction in cellular glycogen storage was due to a significantly increased rate of glycogen turnover. Interestingly, acute basal and insulin-stimulated glucose uptake and glycogen synthesis rates were enhanced in PTG-VF expressing cells vs. control 3T3-L1 adipocytes, likely as a compensatory response to the loss of glycogen stores. These results indicate that the mutation of the PP1-binding domain on PTG resulted in the generation of a dominant-negative molecule that impeded endogenous PTG action and reduced cellular glycogen levels, through enhancement of glycogenolysis rather than impairment of glycogen synthesis.


Adipocytes/metabolism , Glucose/metabolism , Glycogen Synthase/metabolism , Glycogen/metabolism , Glycogenolysis/genetics , Intracellular Signaling Peptides and Proteins/metabolism , Protein Phosphatase 1/metabolism , 3T3-L1 Cells , Adenoviridae , Animals , Binding Sites , Genetic Vectors , Glycogenolysis/physiology , Intracellular Signaling Peptides and Proteins/genetics , Mice , Mutation, Missense
19.
Physiol Genomics ; 40(1): 34-47, 2009 Dec 30.
Article En | MEDLINE | ID: mdl-19808840

Higher fat and lower carbohydrate and amino acid oxidation are observed in women compared with men during endurance exercise. We hypothesized that the observed sex difference is due to estrogen and that menstrual cycle phase or supplementation of men with 17beta-estradiol (E(2)) would coordinately influence the mRNA content of genes involved in lipid and/or carbohydrate metabolism in skeletal muscle. Twelve men and twelve women had muscle biopsies taken before and immediately after 90 min of cycling at 65% peak oxygen consumption (Vo(2peak)). Women were studied in the midfollicular (Fol) and midluteal (Lut) phases, and men were studied after 8 days of E(2) or placebo supplementation. Targeted RT-PCR was used to compare mRNA content for genes involved in transcriptional regulation and lipid, carbohydrate, and amino acid metabolism. Sex was the greatest predictor of substrate metabolism gene content. Sex affected the mRNA content of FATm, FABPc, SREBP-1c, mtGPAT, PPARdelta, PPARalpha, CPTI, TFP-alpha, GLUT4, HKII, PFK, and BCOADK (P < 0.05). E(2) administration significantly (P < 0.05) affected the mRNA content of PGC-1alpha, PPARalpha, PPARdelta, TFP-alpha, CPTI, SREBP-1c, mtGPAT, GLUT4, GS-1, and AST. Acute exercise increased the mRNA abundance for PGC-1alpha, HSL, FABPc, CPTI, GLUT4, HKII, and AST (P < 0.05). Menstrual cycle had a small effect on PPARdelta, GP, and glycogenin mRNA content. Overall, women have greater mRNA content for several genes involved in lipid metabolism, which is partially due to an effect of E(2).


Estradiol/pharmacology , Exercise/physiology , Gene Expression Regulation/drug effects , Menstrual Cycle/physiology , Muscle, Skeletal/drug effects , Muscle, Skeletal/metabolism , Sex Characteristics , Amino Acids/metabolism , Biological Transport/drug effects , Biological Transport/genetics , Fatty Acids/genetics , Female , Follicular Phase/drug effects , Follicular Phase/genetics , Glucose/metabolism , Glycogenolysis/drug effects , Glycogenolysis/genetics , Glycolysis/drug effects , Glycolysis/genetics , Heat-Shock Proteins/genetics , Heat-Shock Proteins/metabolism , Humans , Hydrolysis/drug effects , Lipid Metabolism/drug effects , Lipid Metabolism/genetics , Luteal Phase/drug effects , Luteal Phase/genetics , Male , Menstrual Cycle/drug effects , Mitochondria/drug effects , Mitochondria/genetics , Oxidation-Reduction/drug effects , Peroxisome Proliferator-Activated Receptor Gamma Coactivator 1-alpha , Phosphorylation/drug effects , Sarcolemma/drug effects , Sarcolemma/metabolism , Transcription Factors/genetics , Transcription Factors/metabolism , Young Adult
20.
Neurology ; 70(20): 1876-82, 2008 May 13.
Article En | MEDLINE | ID: mdl-18401027

OBJECTIVE: It is unclear to what extent muscle phosphorylase b kinase (PHK) deficiency is associated with exercise-related symptoms and impaired muscle metabolism, because 1) only four patients have been characterized at the molecular level, 2) reported symptoms have been nonspecific, and 3) lactate responses to ischemic handgrip exercise have been normal. METHODS: We studied a 50-year-old man with X-linked PHK deficiency using ischemic forearm and cycle ergometry exercise tests to define the derangement of muscle metabolism. We compared our findings with those in patients with McArdle disease and in healthy subjects. RESULTS: Sequencing of PHKA1 showed a novel pathogenic mutation (c.831G>A) in exon 7. There was a normal increase of plasma lactate during forearm ischemic exercise, but lactate did not change during dynamic, submaximal exercise in contrast to the fourfold increase in healthy subjects. Constant workload elicited a second wind in all patients with McArdle disease, but not in the patient with PHK deficiency. IV glucose administration appeared to improve exercise tolerance in the patient with PHK deficiency, but not to the same extent as in the patients with McArdle disease. Lipolysis was higher in the patient with PHK deficiency than in controls. CONCLUSION: These findings demonstrate that X-linked PHK deficiency causes a mild metabolic myopathy with blunted muscle glycogen breakdown and impaired lactate production during dynamic exercise, which impairs oxidative capacity only marginally. The different response of lactate to submaximal and maximal exercise is likely related to differential activation mechanisms for myophosphorylase.


Chromosomes, Human, X , Glycogen Storage Disease Type VIII/genetics , Glycogenolysis/genetics , Phosphorylase Kinase/genetics , Point Mutation , Exercise Test , Glycogen/metabolism , Glycogen Storage Disease Type V/genetics , Glycogen Storage Disease Type V/metabolism , Glycogen Storage Disease Type VIII/metabolism , Humans , Lactic Acid/metabolism , Male , Middle Aged , Muscle Weakness/genetics , Muscle Weakness/metabolism , Muscle, Skeletal/enzymology , Oxidative Stress/genetics , Phosphorylase Kinase/deficiency , Phosphorylase Kinase/metabolism , Physical Exertion/physiology , Protein Subunits/genetics , Protein Subunits/metabolism
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