Glycogenolysis Pathways Research Articles

Hydrogen Sulfide Impairs Glucose Utilization and Increases Gluconeogenesis in Hepatocytes

Mounting evidence has established hydrogen sulfide (H(2)S) as an important gasotransmitter with multifaceted physiological functions. The aim of the present study was to investigate the role of H(2)S on glucose utilization, glycogen synthesis, as well as gluconeogenesis in both HepG(2) cells and primary mouse hepatocytes. Incubation with NaHS (a H(2)S donor) impaired glucose uptake and glycogen storage in HepG(2) cells via decreasing glucokinase activity. Adenovirus-mediated cystathionine γ-lyase (CSE) overexpression increased endogenous H(2)S production and lowered glycogen content in HepG(2) cells. Glycogen content was significantly higher in liver tissues from CSE knockout (KO) mice compared to that from wild type (WT) mice in fed condition. Glucose consumption was less in primarily cultured hepatocytes isolated from WT mice than those from CSE KO mice, but more glucose was produced by hepatocytes via gluconeogenesis and glycogenolysis pathways in WT mice than in CSE KO mice. NaHS treatment reduced the phosphorylation of AMP-activated protein kinase, whereas stimulation of AMP-activated protein kinase by 5-aminoimidazole-4-carboxamide-1-β-d-ribofuranoside reversed H(2)S-impaired glucose uptake. H(2)S-increased glucose production was likely through increased phosphoenolpyruvate carboxykinase activity. In addition, insulin at the physiological range inhibited CSE expression, and H(2)S decreased insulin-stimulated phosphorylation of Akt in HepG(2) cells. CSE expression was increased, however, in insulin-resistant state induced by exposing cells to high levels of insulin (500 nm) and glucose (33 mm) for 24 h. Taken together, these data suggest that the interaction of H(2)S and insulin in liver plays a pivotal role in regulating insulin sensitivity and glucose metabolism.

Neonatal Gene Therapy of Glycogen Storage Disease Type Ia Using a Feline Immunodeficiency Virus–based Vector

Glycogen storage disease type Ia (GSD-Ia), also known as von Gierke disease, is caused by a deficiency of glucose-6-phosphatase-alpha (G6Pase), a key enzyme in glucose homeostasis. From birth, affected individuals cannot maintain normal blood glucose levels and suffer from a variety of metabolic disorders, leading to life-threatening complications. Gene therapy has been proposed as a possible option for treatment of this illness. Vectors have been constructed from feline immunodeficiency virus (FIV), a nonprimate lentivirus, because the wild-type virus does not cause disease in humans. Previously, we have shown that these vectors are capable of integrating stably into hepatocyte cell lines and adult murine livers and lead to long-term transgene expression. In the current work, we have assessed the ability to attenuate disease symptoms in a murine model of GSD-Ia. Single administration of FIV vectors containing the human G6Pase gene to G6Pase-alpha(-/-) mice did not change the biochemical and pathological phenotype. However, a double neonatal administration protocol led to normalized blood glucose levels, significantly extended survival, improved body weight, and decreased accumulation of liver glycogen associated with the disease. This approach shows a promising paradigm for treating GSD-Ia patients early in life thereby avoiding long-term consequences.

An Integrated Study of Acute Effects of Valproic Acid in the Liver Using Metabonomics, Proteomics, and Transcriptomics Platforms

An integrated omics approach was undertaken in order to elucidate a systems biology level understanding of the acute hepatotoxcity of valproic acid (VPA). Metabonomics, proteomics and gene expression microarray platforms were employed in this systems biology study. CD-1 female pregnant mice were injected subcutaneously with 600 mg/kg VPA or vehicle control. Urine, serum, and liver tissue were collected at 6, 12, and 24 h after dosing. Principal component analysis (PCA) of the metabonomics data showed clustering of the dosed groups away from the controls for the urine samples. Looser clustering was seen in the other sample sets investigated. However, VPA administration resulted in altered glucose concentrations in urine samples at 12 and 24 h and in aqueous liver tissue extracts at 12 h after VPA administration. Proteomics studies identified two proteins, glycogen phosphorylase and amylo-1,6-glucosidase, which were increased in dosed animals relative to control. Both of these proteins are involved in converting glycogen to glucose. Examination of the expression of 20,000 liver genes did not reveal significantly altered expression at 6, 12, or 24 h after VPA exposure. The combined studies indicated a perturbation in the glycogenolysis pathway following administration of VPA.

Adipokinetic Hormones in Fifth Instar Romalea guttata (Orthoptera: Acrididae): Activation of Glycogen Phosphorylase Does Not Produce Hypertrehalosemia

Romalea guttata Houttuyn (= R. microptera Beavois) is flightless, lethargic, aposematic, and chemically defended. R. guttata stores large quantities of two adipokinetic hormone (AKH) family peptides in its corpora cardiaca. In adults, these peptides (Rom-CC-I and Grb-AKH) activate fat body glycogen phosphorylase but are not hypertrehalosemic. Because juvenile R. guttata contain sufficient peptide to be bioactive, we sought to determine whether these peptides are hypertrehalosemic, phosphorylase activating, or hyperlipemic in juveniles. Late fifth (= last) instar and adult R. guttata activated phosphorylase in response to Rom-CC-I injections. These same individuals showed no hypertrehalosemia in response to Rom-CC-I. We hypothesize that the glycogenolysis pathway is not started by activation of glycogen phosphorylase in response to Rom-CC-I. From fourth instar through third week adult, R. guttata showed a slight, statistically insignificant hypolipemia, but clearly no hyperlipemia. R. guttata differs from Locusta migratoria in that it appears to show neither hypertrehalosemia nor hyperlipemia at any developmental stage.

Enzymatic control of glycogenolysis during anoxic submergence in the freshwater turtle Trachemys scripta

Freshwater turtles Trachemys scripta elegans endure prolonged severe hypoxia, and even complete anoxia, while diving or hibernating underwater. Metabolic adaptations supporting survival include the activation of glycogenolysis and glucose output from liver, as well as strong metabolic rate depression. The present study analyzes the enzymes of both the phosphorolytic (glycogen phosphorylase, phosphorylase b kinase, cAMP-dependent protein kinase) and glucosidic (α-glucosidase) pathways of glycogenolysis in turtle organs. Turtles were subjected to 5 hr of submergence in N 2-bubbled water at 7°C and then activities of phosphorolytic and glucosidic enzymes were assayed in liver, heart, brain, and red and white skeletal muscle, and compared with aerobic controls. In vitro incubations also assessed protein kinase A control of phosphorolytic enzymes. A functional enzyme cascade system for the activation of glycogen phosphorylase was found in all organs, and both phosphorylase and phosphorylase kinase were stimulated by in vitro incubation with the catalytic subunit of cAMP-dependent protein kinase. Anoxic submergence led to significant increases in phosphorylase activities in liver and heart (phosphorylase a rose 2- and 2.5-fold, respectively) but phosphorylase kinase and protein kinase A activities in liver were reduced after 5 hr exposure. Both acidic (pH 4) and neutral (pH 7) forms of α-glucosidase were detected in all five organs with highest activities in liver. Activity of acid α-glucosidase, which degrades lysosomal glycogen, increased by 2-fold in liver during anoxic submergence. The data show that glycogen breakdown in turtle liver during anoxic submergence may result from coordinated activations of both the cytoplasmic phosphorolytic and the lysosomal glucosidic pathways of glycogenolysis.

Calcium control of muscle phosphorylase kinase through the combined action of calmodulin and troponin.

Although it has been believed for several years that Ca2+ are the means by which glycogenolysis and muscle contraction are synchronized, it is only over the past two years that this concept has started to be placed on a firm molecular basis. The current evidence suggests that the regulation of phosphorylase kinase by Ca2+ in vivo is achieved through the interaction of this divalent cation with calmodulin (the delta subunit) and troponin C, and that the relative importance of these two calcium binding proteins depends on the state of phosphorylation of the enzyme [FIGURE 1]. In the low-activity dephosphorylated b form, increasing Ca2+ from 0.1 microM to concentrations in the microM range produces a 5-10-fold activation through the binding of Ca2+ to the delta subunit, and a further 15-25-fold activation through the binding of Ca2+ to troponin C [TABLE 1]. Troponin C rather than the delta subunit is therefore the dominant calcium dependent regulator of the b form, providing an attractive mechanism for coupling glycogenolysis and muscle contraction. On the other hand, the high-activity phosphorylated a form is only activated very slightly by troponin [Section 7]. The delta subunit is therefore the dominant calcium dependent regulator of the hormonally activated state of the enzyme. It has recently become clear that phosphorylase kinase not only phosphorylates and activates phosphorylase, but also phosphorylates glycogen synthase, decreasing its activity. The regulation of phosphorylase kinase by Ca2+ may therefore also provide a mechanism for achieving synchronous control of the pathways of glycogenolysis and glycogen synthesis.