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1
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Fenton AW, Hoffpauir ZA, Martin TA, Harris RA, Lamb AL. Are Allosteric Mechanisms Conserved Among Homologues? J Mol Biol 2025:169176. [PMID: 40306405 DOI: 10.1016/j.jmb.2025.169176] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/23/2025] [Revised: 04/10/2025] [Accepted: 04/24/2025] [Indexed: 05/02/2025]
Abstract
Conservation of allosteric mechanisms among homologues is often assumed but seldom tested. This assumption underpins key concepts like coevolution of residues involved in allosteric mechanisms and the comparison of structures of two different homologues to gain insights into allosteric mechanisms. As an initial assessment of whether allosteric mechanisms are conserved among homologues, this work reviews what is known about the allosteric mechanisms of liver pyruvate kinase (LPYK) vs. skeletal muscle pyruvate kinase (M1PYK), framed within a two-ligand allosteric energy cycle description of allosteric regulation. Selective observations from other PYK homologues are included when relevant. The primary focus of this review is on functional data, while expressing caution regarding the interpretation of allosteric mechanisms based solely on available X-ray crystallographic structures. Additionally, this review considers types of data that are currently lacking for these two PYK homologues, highlighting potential techniques that could be valuable for evaluating the conservation of allosteric mechanisms among homologues. In particular, a hybrid tetramer technique that has been used to study bacterial phosphofructokinases 1 is summarized. Interestingly, despite a high degree of similarity (66.5% sequence identity) between the LPYK and rM1PYK proteins, the available functional comparisons do not provide strong evidence for conserved allosteric mechanisms. Lastly, we consider whether insights into native allosteric mechanisms are relevant to allosteric mechanisms associated with allosteric drug designs.
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Affiliation(s)
- Aron W Fenton
- Department of Biochemistry and Molecular Biology, University of Kansas Medical Center, Kansas City, KS 66160, USA.
| | - Zoe A Hoffpauir
- Department of Chemistry, University of Texas at San Antonio, San Antonio, TX 78249, USA
| | - Tyler A Martin
- San Antonio Uniformed Services Health Education Consortium, Fort Sam Houston, TX 78234, USA
| | - Robert A Harris
- Department of Biochemistry and Molecular Biology, Indiana University School of Medicine, Indianapolis, IN 46202, USA
| | - Audrey L Lamb
- Department of Chemistry, University of Texas at San Antonio, San Antonio, TX 78249, USA
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2
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Rojas-Pirela M, Andrade-Alviárez D, Rojas V, Marcos M, Salete-Granado D, Chacón-Arnaude M, Pérez-Nieto MÁ, Kemmerling U, Concepción JL, Michels PAM, Quiñones W. Exploring glycolytic enzymes in disease: potential biomarkers and therapeutic targets in neurodegeneration, cancer and parasitic infections. Open Biol 2025; 15:240239. [PMID: 39904372 PMCID: PMC11793985 DOI: 10.1098/rsob.240239] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/26/2024] [Revised: 12/11/2024] [Accepted: 12/16/2024] [Indexed: 02/06/2025] Open
Abstract
Glycolysis, present in most organisms, is evolutionarily one of the oldest metabolic pathways. It has great relevance at a physiological level because it is responsible for generating ATP in the cell through the conversion of glucose into pyruvate and reducing nicotinamide adenine dinucleotide (NADH) (that may be fed into the electron chain in the mitochondria to produce additional ATP by oxidative phosphorylation), as well as for producing intermediates that can serve as substrates for other metabolic processes. Glycolysis takes place through 10 consecutive chemical reactions, each of which is catalysed by a specific enzyme. Although energy transduction by glucose metabolism is the main function of this pathway, involvement in virulence, growth, pathogen-host interactions, immunomodulation and adaptation to environmental conditions are other functions attributed to this metabolic pathway. In humans, where glycolysis occurs mainly in the cytosol, the mislocalization of some glycolytic enzymes in various other subcellular locations, as well as alterations in their expression and regulation, has been associated with the development and progression of various diseases. In this review, we describe the role of glycolytic enzymes in the pathogenesis of diseases of clinical interest. In addition, the potential role of these enzymes as targets for drug development and their potential for use as diagnostic and prognostic markers of some pathologies are also discussed.
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Affiliation(s)
- Maura Rojas-Pirela
- Instituto de Investigación Biomédica de Salamanca (IBSAL), Salamanca37007, Spain
- Unidad de Medicina Molecular, Departamento de Medicina, Universidad de Salamanca, Salamanca37007, Spain
- Servicio de Medicina Interna, Hospital Universitario de Salamanca, Salamanca37007, Spain
| | - Diego Andrade-Alviárez
- Laboratorio de Enzimología de Parásitos, Departamento de Biología, Facultad de Ciencias, Universidad de Los Andes, Mérida5101, Venezuela
| | - Verónica Rojas
- Instituto de Biología, Facultad de Ciencias, Pontificia Universidad Católica de Valparaíso, Valparaíso2373223, Chile
| | - Miguel Marcos
- Instituto de Investigación Biomédica de Salamanca (IBSAL), Salamanca37007, Spain
- Unidad de Medicina Molecular, Departamento de Medicina, Universidad de Salamanca, Salamanca37007, Spain
- Servicio de Medicina Interna, Hospital Universitario de Salamanca, Salamanca37007, Spain
| | - Daniel Salete-Granado
- Instituto de Investigación Biomédica de Salamanca (IBSAL), Salamanca37007, Spain
- Unidad de Medicina Molecular, Departamento de Medicina, Universidad de Salamanca, Salamanca37007, Spain
| | - Marirene Chacón-Arnaude
- Laboratorio de Enzimología de Parásitos, Departamento de Biología, Facultad de Ciencias, Universidad de Los Andes, Mérida5101, Venezuela
| | - María Á. Pérez-Nieto
- Unidad de Medicina Molecular, Departamento de Medicina, Universidad de Salamanca, Salamanca37007, Spain
- Fundación Instituto de Estudios de Ciencias de la Salud de Castilla y León, Soria42002, Spain
| | - Ulrike Kemmerling
- Instituto de Ciencias Biomédicas, Universidad de Chile, Facultad de Medicina, Santiago de Chile8380453, Chile
| | - Juan Luis Concepción
- Laboratorio de Enzimología de Parásitos, Departamento de Biología, Facultad de Ciencias, Universidad de Los Andes, Mérida5101, Venezuela
| | - Paul A. M. Michels
- School of Biological Sciences, University of Edinburgh, The King’s Buildings, EdinburghEH9 3FL, UK
| | - Wilfredo Quiñones
- Laboratorio de Enzimología de Parásitos, Departamento de Biología, Facultad de Ciencias, Universidad de Los Andes, Mérida5101, Venezuela
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3
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Sahoo B, Srivastava M, Katiyar A, Ecelbarger C, Tiwari S. Liver or kidney: Who has the oar in the gluconeogenesis boat and when? World J Diabetes 2023; 14:1049-1056. [PMID: 37547592 PMCID: PMC10401452 DOI: 10.4239/wjd.v14.i7.1049] [Citation(s) in RCA: 4] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 01/16/2023] [Revised: 02/20/2023] [Accepted: 04/11/2023] [Indexed: 07/12/2023] Open
Abstract
Gluconeogenesis is an endogenous process of glucose production from non-carbohydrate carbon substrates. Both the liver and kidneys express the key enzymes necessary for endogenous glucose production and its export into circulation. We would be remiss to add that more recently gluconeogenesis has been described in the small intestine, especially under high-protein, low-carbohydrate diets. The contribution of the liver glucose release, the net glucose flux, towards systemic glucose is already well known. The liver is, in most instances, the primary bulk contributor due to the sheer size of the organ (on average, over 1 kg). The contribution of the kidney (at just over 100 g each) to endogenous glucose production is often under-appreciated, especially on a weight basis. Glucose is released from the liver through the process of glycogenolysis and gluconeogenesis. Renal glucose release is almost exclusively due to gluconeogenesis, which occurs in only a fraction of the cells in that organ (proximal tubule cells). Thus, the efficiency of glucose production from other carbon sources may be superior in the kidney relative to the liver or at least on the level. In both these tissues, gluconeogenesis regulation is under tight hormonal control and depends on the availability of substrates. Liver and renal gluconeogenesis are differentially regulated under various pathological conditions. The impact of one source vs the other changes, based on post-prandial state, acid-base balance, hormonal status, and other less understood factors. Which organ has the oar (is more influential) in driving systemic glucose homeostasis is still in-conclusive and likely changes with the daily rhythms of life. We reviewed the literature on the differences in gluconeogenesis regulation between the kidneys and the liver to gain an insight into who drives the systemic glucose levels under various physiological and pathological conditions.
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Affiliation(s)
- Biswajit Sahoo
- Department of Molecular Medicine and Biotechnology, Sanjay Gandhi Postgraduate Institute of Medical Sciences, Lucknow 226014, India
| | - Medha Srivastava
- Department of Molecular Medicine and Biotechnology, Sanjay Gandhi Postgraduate Institute of Medical Sciences, Lucknow 226014, India
| | - Arpit Katiyar
- Department of Molecular Medicine and Biotechnology, Sanjay Gandhi Postgraduate Institute of Medical Sciences, Lucknow 226014, India
| | - Carolyn Ecelbarger
- Department of Medicine, Georgetown University, Washington, DC 20057, United States
| | - Swasti Tiwari
- Department of Molecular Medicine and Biotechnology, Sanjay Gandhi Postgraduate Institute of Medical Sciences, Lucknow 226014, India
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4
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Rodgers RL. Glucagon, cyclic AMP, and hepatic glucose mobilization: A half‐century of uncertainty. Physiol Rep 2022; 10:e15263. [PMID: 35569125 PMCID: PMC9107925 DOI: 10.14814/phy2.15263] [Citation(s) in RCA: 10] [Impact Index Per Article: 3.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/27/2022] [Revised: 03/16/2022] [Accepted: 03/18/2022] [Indexed: 12/14/2022] Open
Abstract
For at least 50 years, the prevailing view has been that the adenylate cyclase (AC)/cyclic AMP (cAMP)/protein kinase A pathway is the predominant signal mediating the hepatic glucose‐mobilizing actions of glucagon. A wealth of evidence, however, supports the alternative, that the operative signal most of the time is the phospholipase C (PLC)/inositol‐phosphate (IP3)/calcium/calmodulin pathway. The evidence can be summarized as follows: (1) The consensus threshold glucagon concentration for activating AC ex vivo is 100 pM, but the statistical hepatic portal plasma glucagon concentration range, measured by RIA, is between 28 and 60 pM; (2) Within that physiological concentration range, glucagon stimulates the PLC/IP3 pathway and robustly increases glucose output without affecting the AC/cAMP pathway; (3) Activation of a latent, amplified AC/cAMP pathway at concentrations below 60 pM is very unlikely; and (4) Activation of the PLC/IP3 pathway at physiological concentrations produces intracellular effects that are similar to those produced by activation of the AC/cAMP pathway at concentrations above 100 pM, including elevated intracellular calcium and altered activities and expressions of key enzymes involved in glycogenolysis, gluconeogenesis, and glycogen synthesis. Under metabolically stressful conditions, as in the early neonate or exercising adult, plasma glucagon concentrations often exceed 100 pM, recruiting the AC/cAMP pathway and enhancing the activation of PLC/IP3 pathway to boost glucose output, adaptively meeting the elevated systemic glucose demand. Whether the AC/cAMP pathway is consistently activated in starvation or diabetes is not clear. Because the importance of glucagon in the pathogenesis of diabetes is becoming increasingly evident, it is even more urgent now to resolve lingering uncertainties and definitively establish glucagon’s true mechanism of glycemia regulation in health and disease.
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Affiliation(s)
- Robert L. Rodgers
- Department of Biomedical and Pharmaceutical Sciences College of Pharmacy University of Rhode Island Kingston Rhode Island USA
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5
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Characterizing the regulation of pyruvate kinase in response to hibernation in ground squirrel liver (Urocitellus richardsonii). Comp Biochem Physiol B Biochem Mol Biol 2020; 248-249:110466. [DOI: 10.1016/j.cbpb.2020.110466] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.2] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/08/2020] [Revised: 06/15/2020] [Accepted: 06/22/2020] [Indexed: 01/24/2023]
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6
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Gassaway BM, Cardone RL, Padyana AK, Petersen MC, Judd ET, Hayes S, Tong S, Barber KW, Apostolidi M, Abulizi A, Sheetz JB, Kshitiz, Aerni HR, Gross S, Kung C, Samuel VT, Shulman GI, Kibbey RG, Rinehart J. Distinct Hepatic PKA and CDK Signaling Pathways Control Activity-Independent Pyruvate Kinase Phosphorylation and Hepatic Glucose Production. Cell Rep 2020; 29:3394-3404.e9. [PMID: 31825824 DOI: 10.1016/j.celrep.2019.11.009] [Citation(s) in RCA: 5] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/26/2018] [Revised: 07/31/2019] [Accepted: 11/04/2019] [Indexed: 12/11/2022] Open
Abstract
Pyruvate kinase is an important enzyme in glycolysis and a key metabolic control point. We recently observed a pyruvate kinase liver isoform (PKL) phosphorylation site at S113 that correlates with insulin resistance in rats on a 3 day high-fat diet (HFD) and suggests additional control points for PKL activity. However, in contrast to the classical model of PKL regulation, neither authentically phosphorylated PKL at S12 nor S113 alone is sufficient to alter enzyme kinetics or structure. Instead, we show that cyclin-dependent kinases (CDKs) are activated by the HFD and responsible for PKL phosphorylation at position S113 in addition to other targets. These CDKs control PKL nuclear retention, alter cytosolic PKL activity, and ultimately influence glucose production. These results change our view of PKL regulation and highlight a previously unrecognized pathway of hepatic CDK activity and metabolic control points that may be important in insulin resistance and type 2 diabetes.
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Affiliation(s)
- Brandon M Gassaway
- Department of Cellular and Molecular Physiology, Yale University, New Haven, CT, USA; Department of Systems Biology Institute, Yale University, New Haven, CT, USA
| | - Rebecca L Cardone
- Department of Internal Medicine, Yale University, New Haven, CT, USA
| | | | - Max C Petersen
- Department of Cellular and Molecular Physiology, Yale University, New Haven, CT, USA; Department of Internal Medicine, Yale University, New Haven, CT, USA
| | | | | | | | - Karl W Barber
- Department of Cellular and Molecular Physiology, Yale University, New Haven, CT, USA; Department of Systems Biology Institute, Yale University, New Haven, CT, USA
| | - Maria Apostolidi
- Department of Cellular and Molecular Physiology, Yale University, New Haven, CT, USA; Department of Systems Biology Institute, Yale University, New Haven, CT, USA
| | | | - Joshua B Sheetz
- Department of Cellular and Molecular Physiology, Yale University, New Haven, CT, USA
| | - Kshitiz
- Department of Systems Biology Institute, Yale University, New Haven, CT, USA; Department of Biomedical Engineering, Yale University, New Haven, CT, USA
| | - Hans R Aerni
- Department of Cellular and Molecular Physiology, Yale University, New Haven, CT, USA; Department of Systems Biology Institute, Yale University, New Haven, CT, USA
| | | | | | - Varman T Samuel
- Department of Internal Medicine, Yale University, New Haven, CT, USA; Veterans Affairs Medical Center, West Haven, CT, USA
| | - Gerald I Shulman
- Department of Cellular and Molecular Physiology, Yale University, New Haven, CT, USA; Department of Internal Medicine, Yale University, New Haven, CT, USA
| | - Richard G Kibbey
- Department of Cellular and Molecular Physiology, Yale University, New Haven, CT, USA; Department of Internal Medicine, Yale University, New Haven, CT, USA
| | - Jesse Rinehart
- Department of Cellular and Molecular Physiology, Yale University, New Haven, CT, USA; Department of Systems Biology Institute, Yale University, New Haven, CT, USA.
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7
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Hunter RW, Hughey CC, Lantier L, Sundelin EI, Peggie M, Zeqiraj E, Sicheri F, Jessen N, Wasserman DH, Sakamoto K. Metformin reduces liver glucose production by inhibition of fructose-1-6-bisphosphatase. Nat Med 2018; 24:1395-1406. [PMID: 30150719 PMCID: PMC6207338 DOI: 10.1038/s41591-018-0159-7] [Citation(s) in RCA: 217] [Impact Index Per Article: 31.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/08/2016] [Accepted: 07/24/2018] [Indexed: 01/03/2023]
Abstract
Metformin is a first-line drug for the treatment of individuals with type 2 diabetes, yet its precise mechanism of action remains unclear. Metformin exerts its antihyperglycemic action primarily through lowering hepatic glucose production (HGP). This suppression is thought to be mediated through inhibition of mitochondrial respiratory complex I, and thus elevation of 5'-adenosine monophosphate (AMP) levels and the activation of AMP-activated protein kinase (AMPK), though this proposition has been challenged given results in mice lacking hepatic AMPK. Here we report that the AMP-inhibited enzyme fructose-1,6-bisphosphatase-1 (FBP1), a rate-controlling enzyme in gluconeogenesis, functions as a major contributor to the therapeutic action of metformin. We identified a point mutation in FBP1 that renders it insensitive to AMP while sparing regulation by fructose-2,6-bisphosphate (F-2,6-P2), and knock-in (KI) of this mutant in mice significantly reduces their response to metformin treatment. We observe this during a metformin tolerance test and in a metformin-euglycemic clamp that we have developed. The antihyperglycemic effect of metformin in high-fat diet-fed diabetic FBP1-KI mice was also significantly blunted compared to wild-type controls. Collectively, we show a new mechanism of action for metformin and provide further evidence that molecular targeting of FBP1 can have antihyperglycemic effects.
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Affiliation(s)
- Roger W Hunter
- Nestlé Institute of Health Sciences SA, Lausanne, Switzerland
- School of Physiology, Pharmacology & Neuroscience, University of Bristol, Bristol, UK
| | - Curtis C Hughey
- Department of Molecular Physiology and Biophysics and the Vanderbilt Mouse Metabolic Phenotyping Center, Vanderbilt University, Nashville, TN, USA
| | - Louise Lantier
- Department of Molecular Physiology and Biophysics and the Vanderbilt Mouse Metabolic Phenotyping Center, Vanderbilt University, Nashville, TN, USA
| | - Elias I Sundelin
- Departments of Clinical Medicine and Biomedicine, Aarhus University, Aarhus, Denmark
| | - Mark Peggie
- MRC Protein Phosphorylation and Ubiquitylation Unit, College of Life Sciences, University of Dundee, Dundee, UK
| | - Elton Zeqiraj
- Lunenfeld-Tanenbaum Research Institute, Mount Sinai Hospital, Toronto, ON, Canada
- Astbury Centre for Structural Molecular Biology, School of Molecular and Cellular Biology, Faculty of Biological Sciences, University of Leeds, Leeds, UK
| | - Frank Sicheri
- Lunenfeld-Tanenbaum Research Institute, Mount Sinai Hospital, Toronto, ON, Canada
- Departments of Biochemistry and Molecular Genetics, University of Toronto, Toronto, ON, Canada
| | - Niels Jessen
- Departments of Clinical Medicine and Biomedicine, Aarhus University, Aarhus, Denmark
| | - David H Wasserman
- Department of Molecular Physiology and Biophysics and the Vanderbilt Mouse Metabolic Phenotyping Center, Vanderbilt University, Nashville, TN, USA
| | - Kei Sakamoto
- Nestlé Institute of Health Sciences SA, Lausanne, Switzerland.
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Abstract
The liver is crucial for the maintenance of normal glucose homeostasis - it produces glucose during fasting and stores glucose postprandially. However, these hepatic processes are dysregulated in type 1 and type 2 diabetes mellitus, and this imbalance contributes to hyperglycaemia in the fasted and postprandial states. Net hepatic glucose production is the summation of glucose fluxes from gluconeogenesis, glycogenolysis, glycogen synthesis, glycolysis and other pathways. In this Review, we discuss the in vivo regulation of these hepatic glucose fluxes. In particular, we highlight the importance of indirect (extrahepatic) control of hepatic gluconeogenesis and direct (hepatic) control of hepatic glycogen metabolism. We also propose a mechanism for the progression of subclinical hepatic insulin resistance to overt fasting hyperglycaemia in type 2 diabetes mellitus. Insights into the control of hepatic gluconeogenesis by metformin and insulin and into the role of lipid-induced hepatic insulin resistance in modifying gluconeogenic and net hepatic glycogen synthetic flux are also discussed. Finally, we consider the therapeutic potential of strategies that target hepatosteatosis, hyperglucagonaemia and adipose lipolysis.
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Affiliation(s)
- Max C Petersen
- Department of Internal Medicine, Yale School of Medicine
- Department of Cellular &Molecular Physiology, Yale School of Medicine
| | | | - Gerald I Shulman
- Department of Internal Medicine, Yale School of Medicine
- Department of Cellular &Molecular Physiology, Yale School of Medicine
- Howard Hughes Medical Institute, Yale School of Medicine, New Haven, Connecticut 06520, USA
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9
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Fenton AW. Are all regions of folded proteins that undergo ligand-dependent order-disorder transitions targets for allosteric peptide mimetics? Biopolymers 2016; 100:553-7. [PMID: 23520021 DOI: 10.1002/bip.22239] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/11/2012] [Revised: 03/11/2013] [Accepted: 03/17/2013] [Indexed: 02/06/2023]
Abstract
Although the classical view of how proteins function relied on well folded structures, it is now recognized that the functions of many proteins are dependent on being intrinsically disordered. The primary consideration in this work is the intermediate group of proteins that are overall well folded, but which contain small regions that undergo order-disorder transitions. In particular, the current focus is on those order-disorder transitions that are energetically coupled to ligand binding. As exemplified by the case of human liver pyruvate kinase (hL-PYK), peptides that mimic the sequence of the order-disorder region can be used as allosteric regulators of the enzyme. On the basis of this example and others reported in the literature, we propose that a similar use of peptides that mimic protein regions that experience ligand-dependent order-disorder transitions can be a generalized initiation point for the development of allosteric drugs.
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Affiliation(s)
- Aron W Fenton
- Department of Biochemistry and Molecular Biology, The University of Kansas Medical Center, MS 3030, 3901 Rainbow Boulevard, Kansas City, KS, 66160
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10
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Röder PV, Wu B, Liu Y, Han W. Pancreatic regulation of glucose homeostasis. Exp Mol Med 2016; 48:e219. [PMID: 26964835 PMCID: PMC4892884 DOI: 10.1038/emm.2016.6] [Citation(s) in RCA: 544] [Impact Index Per Article: 60.4] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/20/2015] [Revised: 12/03/2015] [Accepted: 12/06/2015] [Indexed: 12/11/2022] Open
Abstract
In order to ensure normal body function, the human body is dependent on a tight control of its blood glucose levels. This is accomplished by a highly sophisticated network of various hormones and neuropeptides released mainly from the brain, pancreas, liver, intestine as well as adipose and muscle tissue. Within this network, the pancreas represents a key player by secreting the blood sugar-lowering hormone insulin and its opponent glucagon. However, disturbances in the interplay of the hormones and peptides involved may lead to metabolic disorders such as type 2 diabetes mellitus (T2DM) whose prevalence, comorbidities and medical costs take on a dramatic scale. Therefore, it is of utmost importance to uncover and understand the mechanisms underlying the various interactions to improve existing anti-diabetic therapies and drugs on the one hand and to develop new therapeutic approaches on the other. This review summarizes the interplay of the pancreas with various other organs and tissues that maintain glucose homeostasis. Furthermore, anti-diabetic drugs and their impact on signaling pathways underlying the network will be discussed.
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Affiliation(s)
- Pia V Röder
- Metabolism in Human Diseases Unit, Institute of Molecular and Cell Biology, Agency for Science, Technology and Research (A*STAR), Singapore, Singapore
- Metabolism in Human Diseases Unit, Institute of Molecular and Cell Biology, Agency for Science, Technology and Research (A*STAR), Singapore 138673, Singapore. E-mail: or
| | - Bingbing Wu
- Laboratory of Metabolic Medicine, Singapore Bioimaging Consortium, A*STAR, Singapore, Singapore
| | - Yixian Liu
- Laboratory of Metabolic Medicine, Singapore Bioimaging Consortium, A*STAR, Singapore, Singapore
| | - Weiping Han
- Metabolism in Human Diseases Unit, Institute of Molecular and Cell Biology, Agency for Science, Technology and Research (A*STAR), Singapore, Singapore
- Laboratory of Metabolic Medicine, Singapore Bioimaging Consortium, A*STAR, Singapore, Singapore
- Metabolism in Human Diseases Unit, Institute of Molecular and Cell Biology, Agency for Science, Technology and Research (A*STAR), Singapore 138673, Singapore. E-mail: or
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11
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Hasenour CM, Wall ML, Ridley DE, Hughey CC, James FD, Wasserman DH, Young JD. Mass spectrometry-based microassay of (2)H and (13)C plasma glucose labeling to quantify liver metabolic fluxes in vivo. Am J Physiol Endocrinol Metab 2015; 309:E191-203. [PMID: 25991647 PMCID: PMC4504936 DOI: 10.1152/ajpendo.00003.2015] [Citation(s) in RCA: 64] [Impact Index Per Article: 6.4] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 01/05/2015] [Accepted: 05/14/2015] [Indexed: 01/06/2023]
Abstract
Mouse models designed to examine hepatic metabolism are critical to diabetes and obesity research. Thus, a microscale method to quantitatively assess hepatic glucose and intermediary metabolism in conscious, unrestrained mice was developed. [(13)C3]propionate, [(2)H2]water, and [6,6-(2)H2]glucose isotopes were delivered intravenously in short- (9 h) and long-term-fasted (19 h) C57BL/6J mice. GC-MS and mass isotopomer distribution (MID) analysis were performed on three 40-μl arterial plasma glucose samples obtained during the euglycemic isotopic steady state. Model-based regression of hepatic glucose and citric acid cycle (CAC)-related fluxes was performed using a comprehensive isotopomer model to track carbon and hydrogen atom transitions through the network and thereby simulate the MIDs of measured fragment ions. Glucose-6-phosphate production from glycogen diminished, and endogenous glucose production was exclusively gluconeogenic with prolonged fasting. Gluconeogenic flux from phosphoenolpyruvate (PEP) remained stable, whereas that from glycerol modestly increased from short- to long-term fasting. CAC flux [i.e., citrate synthase (VCS)] was reduced with long-term fasting. Interestingly, anaplerosis and cataplerosis increased with fast duration; accordingly, pyruvate carboxylation and the conversion of oxaloacetate to PEP were severalfold higher than VCS in long-term fasted mice. This method utilizes state-of-the-art in vivo methodology and comprehensive isotopomer modeling to quantify hepatic glucose and intermediary fluxes during physiological stress in mice. The small plasma requirements permit serial sampling without stress and the affirmation of steady-state glucose kinetics. Furthermore, the approach can accommodate a broad range of modeling assumptions, isotope tracers, and measurement inputs without the need to introduce ad hoc mathematical approximations.
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Affiliation(s)
| | - Martha L Wall
- Department of Chemical and Biomolecular Engineering, and
| | | | | | - Freyja D James
- Mouse Metabolic Phenotyping Center, Vanderbilt University, Nashville, Tennessee
| | - David H Wasserman
- Department of Molecular Physiology and Biophysics, Mouse Metabolic Phenotyping Center, Vanderbilt University, Nashville, Tennessee
| | - Jamey D Young
- Department of Molecular Physiology and Biophysics, Department of Chemical and Biomolecular Engineering, and
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12
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Nagaraju R, Joshi AKR, Rajini PS. Organophosphorus insecticide, monocrotophos, possesses the propensity to induce insulin resistance in rats on chronic exposure. J Diabetes 2015; 7:47-59. [PMID: 24698518 DOI: 10.1111/1753-0407.12158] [Citation(s) in RCA: 23] [Impact Index Per Article: 2.3] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 01/23/2014] [Revised: 03/14/2014] [Accepted: 03/31/2014] [Indexed: 01/09/2023] Open
Abstract
BACKGROUND Our earlier studies had shown that monocrotophos (MCP), an organophosphorus insecticide (OPI), has the propensity to augment the secondary complications associated with type-1 diabetes. The present study investigates whether rats exposed for prolonged periods to monocrotophos would develop insulin resistance mediated by alteration in glucose homeostasis. METHODS Male rats were administered sublethal doses of monocrotophos daily for 180 days. Interim blood samples were collected to measure alteration in blood glucose and lipid profile. Rats were also subjected to glucose and insulin tolerance test and fasting blood glucose and insulin levels were measured to calculate insulin resistance by HOMA-IR method. After 180 days, the rats were also evaluated for pancreatic histology and activities of hepatic gluconeogenetic enzymes. RESULTS Monocrotophos elicited a gradual and sustained increase in blood glucose and insulin resistance in rats with concomitant glucose intolerance and reduced insulin sensitivity. MCP exposure was also associated with increase in weights of key white adipose pads, activities of gluconeogenesis enzymes and increase in pancreatic islet diameter, all of which led to hyperglycemia, hyperinsulinemia and dyslipidaemia. CONCLUSION Long-term exposure of rats to MCP resulted in glucose intolerance with hyperinsulinemia, a hallmark of insulin resistance. Our data suggest that chronic exposure to low doses of monocrotophos, might lead to development of insulin resistance by altering lipid profile and glucose homeostasis.
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Affiliation(s)
- Raju Nagaraju
- Food Protectants and Infestation Control Department, Council of Scientific and Industrial Research, Central Food Technological Research Institute, Mysore, India
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13
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Abstract
Glycolysis, a central metabolic pathway, harbors evolutionary conserved enzymes that modulate and potentially shift the cellular metabolism on requirement. Pyruvate kinase, which catalyzes the last but rate-limiting step of glycolysis, is expressed in four isozymic forms, depending on the tissue requirement. M2 isoform (PKM2) is exclusively expressed in embryonic and adult dividing/tumor cells. This tetrameric allosterically regulated isoform is intrinsically designed to downregulate its activity by subunit dissociation (into dimer), which results in partial inhibition of glycolysis at the last step. This accumulates all upstream glycolytic intermediates as an anabolic feed for synthesis of lipids and nucleic acids, whereas reassociation of PKM2 into active tetramer replenishes the normal catabolism as a feedback after cell division. In addition, involvement of this enzyme in a variety of pathways, protein-protein interactions, and nuclear transport suggests its potential to perform multiple nonglycolytic functions with diverse implications, although multidimensional role of this protein is as yet not fully explored. This review aims to provide an overview of the involvement of PKM2 in various physiological pathways with possible functional implications.
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Affiliation(s)
- Vibhor Gupta
- National Centre of Applied Human Genetics, School of Life Sciences, Jawaharlal Nehru University, New Delhi 110067, India.
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Anand P, Murali K, Tandon V, Murthy P, Chandra R. Insulinotropic effect of cinnamaldehyde on transcriptional regulation of pyruvate kinase, phosphoenolpyruvate carboxykinase, and GLUT4 translocation in experimental diabetic rats. Chem Biol Interact 2010; 186:72-81. [DOI: 10.1016/j.cbi.2010.03.044] [Citation(s) in RCA: 93] [Impact Index Per Article: 6.2] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/01/2010] [Revised: 03/22/2010] [Accepted: 03/25/2010] [Indexed: 10/19/2022]
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15
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Tejwani GA. Regulation of fructose-bisphosphatase activity. ADVANCES IN ENZYMOLOGY AND RELATED AREAS OF MOLECULAR BIOLOGY 2006; 54:121-94. [PMID: 6303063 DOI: 10.1002/9780470122990.ch3] [Citation(s) in RCA: 41] [Impact Index Per Article: 2.2] [Reference Citation Analysis] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 01/19/2023]
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16
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Hue L. The role of futile cycles in the regulation of carbohydrate metabolism in the liver. ADVANCES IN ENZYMOLOGY AND RELATED AREAS OF MOLECULAR BIOLOGY 2006; 52:247-331. [PMID: 6261536 DOI: 10.1002/9780470122976.ch4] [Citation(s) in RCA: 41] [Impact Index Per Article: 2.2] [Reference Citation Analysis] [MESH Headings] [Grants] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 01/19/2023]
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17
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Mohammad S, Taha A, Akhtar K, Bamezai RNK, Baquer NZ. In vivo effect ofTrigonella foenum graecumon the expression of pyruvate kinase, phosphoenolpyruvate carboxykinase, and distribution of glucose transporter (GLUT4) in alloxan-diabetic rats. Can J Physiol Pharmacol 2006; 84:647-54. [PMID: 16900249 DOI: 10.1139/y05-164] [Citation(s) in RCA: 35] [Impact Index Per Article: 1.8] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/22/2022]
Abstract
Plasma glucose levels are maintained by a precise balance between glucose production and its use. Liver pyruvate kinase (PK) and phosphoenolpyruvate carboxykinase (PEPCK), 2 key enzymes of glycolysis and gluconeogenesis, respectively, play a crucial role in this glucose homeostasis along with skeletal muscle glucose transporter (GLUT4). In the diabetic state, this balance is disturbed owing to the absence of insulin, the principal factor controlling this regulation. In the present study, alloxan-diabetic animals having high glucose levels of more than 300 mmol/L have been taken and the administration of Trigonella seed powder (TSP) to the diabetic animals was assessed for its effect on the expression of PK and PEPCK in liver and GLUT4 distribution in skeletal muscle of alloxan-diabetic rats. TSP treatment to the diabetic animals resulted in a marked decrease in the plasma glucose levels. Trigonella treatment partially restored the altered expression of PK and PEPCK. TSP treatment also corrected the alterations in the distribution of GLUT4 in the skeletal muscle.
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Affiliation(s)
- Sameer Mohammad
- Hormone and Drug Research Laboratory, School of Life Sciences, Jawaharlal Nehru University, New Delhi 110067, India
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Mueller AS, Pallauf J, Rafael J. The chemical form of selenium affects insulinomimetic properties of the trace element: investigations in type II diabetic dbdb mice. J Nutr Biochem 2003; 14:637-47. [PMID: 14629895 DOI: 10.1016/j.jnutbio.2003.08.001] [Citation(s) in RCA: 35] [Impact Index Per Article: 1.6] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/23/2022]
Abstract
The objective of the present study was to investigate the effects of oral selenate application in comparison to selenium deficiency and selenite treatment on the development of the diabetic status (glucose tolerance, insulin resistance and activities of glycolytic and gluconeogenic marker enzymes) in dbdb mice, representing a type II diabetic animal model. Therefore 21 adult male dbdb mice were assigned to 3 experimental groups of 7 animals each and put on a selenium deficient diet (< 0.03 mg/kg diet) based on torula yeast. Group 0Se was kept on selenium deficiency for 10 weeks while the mice of the groups SeIV and SeVI were supplemented daily with 15% of their individual LD(50) of sodium selenite or sodium selenate in addition to the diet. After 10 weeks a distinct melioration of the diabetic status indicated by a corrected glucose tolerance and a lowered insulin resistance was measured in selenate treated mice (group SeVI) in comparison to their selenium deficient and selenite treated companions and to their initial status. Activities of the glycolytic marker enzymes hexokinase, phosphofructokinase and pyruvate kinase were increased 1.7 to 3-fold in liver and/or adipose tissue by selenate treatment as compared to mice on selenium deficiency and mice with selenite administration. In contrast selenate treatment (SeVI) repressed the activity of liver pyruvate carboxylase the first enzyme in gluconeogenesis by about 33% in comparison to the selenium deficient (0Se) and selenite treated mice (SeIV). However the current study revealed an insulinomimetic role for selenate (selenium VI) also in type II diabetic animals due to a melioration of insulin resistance. In contrast selenium deficiency and especially selenite (selenium IV) impaired the diabetic status of dbdb mice, demonstrating the need for investigations on the insulinomimetic action of selenium due to the metabolism of different selenium compounds.
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Affiliation(s)
- Andreas S Mueller
- Biochemie Zentrum Heidelberg, Im Neuenheimer Feld 328, D-69120, Heidelberg, Germany.
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Becker DJ, Reul B, Ozcelikay AT, Buchet JP, Henquin JC, Brichard SM. Oral selenate improves glucose homeostasis and partly reverses abnormal expression of liver glycolytic and gluconeogenic enzymes in diabetic rats. Diabetologia 1996; 39:3-11. [PMID: 8720597 DOI: 10.1007/bf00400407] [Citation(s) in RCA: 99] [Impact Index Per Article: 3.4] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 02/01/2023]
Abstract
Selenium is a trace element that exerts certain insulin-like actions in vitro. In this study, we evaluated its in vivo effects on the glucose homeostasis of rats made diabetic and insulin-deficient by streptozotocin. Na2SeO4 was administered ad libitum in drinking water and/or food for 10 weeks. The elevated plasma glucose levels (approximately 25 mmol/l) and glucosuria (approximately 85 mmol/day) of untreated rats were decreased by 50 and 80%, respectively, by selenate treatment. The beneficial effect of selenate was also evident during oral and intravenous glucose tolerance tests: the integrated glucose responses were decreased by 40-50% as compared to those in untreated rats. These effects were not due to an increase in plasma insulin levels. Compared to non-diabetic rats, pancreatic insulin reserves were reduced by more than 90% in treated and untreated diabetic rats. The hepatic activities and mRNA levels of two key glycolytic enzymes, glucokinase and L-type pyruvate kinase were blunted in diabetic rats. They increased approximately two- to threefold after selenate treatment, to reach 40-75% of the values in non-diabetic rats. In contrast, elevated activity and mRNA levels of the gluconeogenic enzyme, phosphoenolpyruvate carboxykinase, were reduced by 40-65% after selenate administration. Since selenate induced a moderate decrease in body weight due to an anorexigenic effect, we checked that there was no improvement of glucose homeostasis or hepatic glucose metabolism in an additional group of calorie-restricted diabetic rats, which was weight-matched with the selenate group. In addition, no obvious toxic side-effects on the kidney or liver were observed in the rats receiving selenate. In conclusion, selenate induces a sustained improvement of glucose homeostasis in streptozotocin-diabetic rats by an insulin-like action, which involves partial correction of altered pretranslational regulatory mechanisms in liver metabolism.
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Affiliation(s)
- D J Becker
- Endocrinology and Metabolism Unit, University of Louvain, Brussels, Belgium
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20
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Petersen KF, Cline GW, Blair JB, Shulman GI. Substrate cycling between pyruvate and oxaloacetate in awake normal and 3,3'-5-triiodo-L-thyronine-treated rats. THE AMERICAN JOURNAL OF PHYSIOLOGY 1994; 267:E273-7. [PMID: 8074207 DOI: 10.1152/ajpendo.1994.267.2.e273] [Citation(s) in RCA: 21] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 01/28/2023]
Abstract
Substrate cycling between pyruvate and oxaloacetate was assessed in awake 24-h fasted normal and triiodothyronine (T3)-treated rats. After a 20- or 60-min infusion of [3-13C]alanine (99% enriched, 12 mg/min) the 13C enrichments of liver glucose and alanine carbons were analyzed by 13C and 1H nuclear magnetic resonance spectroscopy and gas chromatography-mass spectrometry. Substrate cycling from phosphoenolpyruvate to pyruvate [via pyruvate kinase (PK)] and from oxaloacetate to pyruvate [via malic enzyme (ME)] relative to the pyruvate carboxylase (PC) flux [i.e., (PK+ME)/PC] was assessed by the ratio of the 13C enrichment of C-2 alanine relative to that in C-5 glucose. In the normal rats (PK+ME)/PC was 0.26 +/- 0.07 (n = 7, t = 20 min) and 0.37 +/- 0.08 (n = 4, t = 60 min). In the T3-treated rats the (PK+ME)/PC increased four- to fivefold to 1.03 +/- 0.19 (n = 8, t = 20 min) and to 1.83 +/- 0.19 (n = 3, t = 60 min) (P < 0.05 vs. normal rats). The liver enzyme activity of PK did not change with T3 treatment (normal 14.22 +/- 5.25 U/g liver vs. T3 treated 13.40 +/- 1.10 U/g liver), whereas both the enzyme activity ratio of PK (normal 0.47 +/- 0.15 vs. T3 treated 0.77 +/- 0.03, P < 0.05) and the activity of ME (normal 0.89 +/- 0.30 U/g liver vs. T3 treated 4.25 +/- 0.60 U/g liver, P < 0.05) increased with T3 treatment.(ABSTRACT TRUNCATED AT 250 WORDS)
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Affiliation(s)
- K F Petersen
- Department of Internal Medicine, Yale University School of Medicine, New Haven, Connecticut 06520-8020
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21
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Katz J, Wals P, Lee WN. Isotopomer studies of gluconeogenesis and the Krebs cycle with 13C-labeled lactate. J Biol Chem 1993. [DOI: 10.1016/s0021-9258(19)74421-4] [Citation(s) in RCA: 99] [Impact Index Per Article: 3.1] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/16/2022] Open
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22
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Brichard SM, Henquin JC, Girard J. Phlorizin treatment of diabetic rats partially reverses the abnormal expression of genes involved in hepatic glucose metabolism. Diabetologia 1993; 36:292-8. [PMID: 8477872 DOI: 10.1007/bf00400230] [Citation(s) in RCA: 42] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 01/31/2023]
Abstract
Liver insulin resistance and glucagon-stimulated hepatic glucose production are characteristics of the diabetic state. To determine the potential role of glucose toxicity in these abnormalities, we examined whether phlorizin treatment of streptozotocin-diabetic rats resulted in altered expression of genes involved in key steps of hepatic glucose metabolism. By inhibiting renal tubular glucose reabsorption, phlorizin infusion to diabetic rats induced normoglycaemia, did not significantly alter low circulating insulinaemia, but caused a marked decrease in hyperglucagonaemia. Glucokinase and L-type pyruvate kinase mRNA levels were reduced respectively by 90% and 70% in fed diabetic rats, in close correlation with changes in enzyme activities. Eighteen days of phlorizin infusion partially restored glucokinase mRNA and activity (40% of control levels), but had no effect on L-type pyruvate kinase mRNA and activity. In contrast to the glycolytic enzymes, mRNA and activity of the gluconeogenic enzyme, phosphoenolpyruvate carboxykinase were increased (10- and 2.2-fold, respectively) in fed diabetic rats. Phlorizin administration decreased phosphoenolpyruvate carboxykinase mRNA to values not different from those in control rats, while phosphoenolpyruvate carboxykinase activity remained 50% higher than that in control rats. The 50% rise in liver glucose transporter (GLUT 2) mRNA and protein, produced by diabetes, was also corrected by phlorizin treatment. In conclusion, we propose that phlorizin treatment of diabetic rats may induce a partial shift of the predominating gluconeogenesis, associated with hepatic glucose overproduction, into glycolysis, by correction of impaired pre-translational regulatory mechanisms. This could be essentially mediated through improved pancreatic alpha-cell function and subsequent lowering of hyperglucagonaemia. These observations suggest that glucagon-stimulated hepatic glucose production may result, in part, from glucose toxicity.
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Affiliation(s)
- S M Brichard
- Centre de Recherche sur l'Endocrinologie Moléculaire et le Développement, CNRS, Meudon, France
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Brichard SM, Desbuquois B, Girard J. Vanadate treatment of diabetic rats reverses the impaired expression of genes involved in hepatic glucose metabolism: effects on glycolytic and gluconeogenic enzymes, and on glucose transporter GLUT2. Mol Cell Endocrinol 1993; 91:91-7. [PMID: 8472858 DOI: 10.1016/0303-7207(93)90259-m] [Citation(s) in RCA: 57] [Impact Index Per Article: 1.8] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 01/31/2023]
Abstract
The trace element vanadium is a potent insulinomimetic agent in vitro. Oral administration of vanadate to rats made diabetic by streptozotocin (45 mg/kg i.v.) caused a 65% fall in plasma glucose levels without modifying low insulinemia. We studied whether the hypoglycemic effect of vanadate was associated with altered expression of genes involved in key steps of hepatic glucose metabolism. Glucokinase (GK) and L-type pyruvate kinase (L-PK) mRNA levels were decreased respectively by 90% and 70% in fed diabetic rats, in close correlation with changes in enzyme activities. Eighteen days of vanadate treatment partially restored GK mRNA and activity (40% of control levels), and totally restored L-PK parameters. In contrast to the glycolytic enzymes, mRNA levels and activity of the gluconeogenic enzyme, phosphoenolpyruvate carboxykinase (PEPCK) were increased (15- and 2-fold, respectively) in fed diabetic rats. Vanadate treatment normalized both PEPCK mRNA and activity in diabetic rat liver. The 2-fold increase in liver glucose transporter (GLUT2) mRNA and protein, produced by diabetes, was also corrected by this treatment. In conclusion, oral vanadate given to diabetic rats induces a shift of the predominating gluconeogenic flux, with subsequent high hepatic glucose production, into a glycolytic flux by pretranslational regulatory mechanisms.
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Rashed HM, Nair BG, Patel TB. Regulation of hepatic glycolysis and gluconeogenesis by atrial natriuretic peptide. Arch Biochem Biophys 1992; 298:640-5. [PMID: 1329663 DOI: 10.1016/0003-9861(92)90460-e] [Citation(s) in RCA: 20] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/26/2022]
Abstract
Recently we reported the presence of both the guanylyl cyclase-linked (116 kDa) and the ANF-C (66 kDa) atrial natriuretic peptide receptors in the rat liver. Since ANF 103-125 (atriopeptin II) stimulates cGMP production in livers and because cGMP has previously been shown to mimic the actions of cAMP in regulating hepatic carbohydrate metabolism, studies were performed to investigate the effects of atriopeptin II on hepatic glycolysis and gluconeogenesis. Additionally, employing analogs of atrial natriuretic hormone [des-(Q116, S117, G118, L119, G120) ANF 102-121 (C-ANF) and des-(C105,121) ANF 104-126 (analog I)] which bind only the ANF-C receptors, the role of the ANF-C receptors in the hepatic actions of atriopeptin II was evaluated. In perfused livers of fed rats atriopeptin II, but not C-ANF and analog I, inhibited hepatic glycolysis and stimulated glucose production. Moreover, analog I did not alter the ability of atriopeptin II to inhibit hepatic glycolysis. Atriopeptin II, but not C-ANF and analog I, also stimulated cGMP production in perfused rat livers. Furthermore, while atriopeptin II inhibited the activity ratio of pyruvate kinase by 30%, C-ANF did not alter hepatic pyruvate kinase activity. Finally, in rat hepatocytes, atriopeptin II stimulated the synthesis of [14C]glucose from [2-14C]pyruvate by 50% and this effect of atriopeptin II was mimicked by the exogenously supplied cGMP analog, 8-bromo cGMP. Thus atriopeptin II increases hepatic gluconeogenesis and inhibits glycolysis, in part by inhibiting pyruvate kinase activity, and the effects of atriopeptin II are mediated via activation of guanylyl cyclase-linked ANF receptors which elevate cGMP production.
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Affiliation(s)
- H M Rashed
- Department of Pharmacology, University of Tennessee, Memphis 38163
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25
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Roselino JE, De Castro-e-Silva Júnior O, Ceneviva R. Lack of control of liver gluconeogenesis in cholestatic rats with reduced portal blood flow. Hepatology 1992; 16:1055-60. [PMID: 1328007 DOI: 10.1002/hep.1840160432] [Citation(s) in RCA: 9] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 12/26/2022]
Abstract
Previous studies indicated a role for ischemia in the metabolic changes induced by cholestasis. Liver pyruvate kinase is a key enzyme for the concurrent control of glycolysis and gluconeogenesis. In this experiment the control of pyruvate kinase activity was investigated in cholestatic rats. Pyruvate kinase kinetics changed from a sigmoidal type in sham-operated rats to a hyperbolic type in obstructed rats. The change in the enzymatic kinetics paralleled the reduction in the portal blood flow, which reached 50% of the control value 7 days after obstruction. Dibutyryl cyclic AMP (5 mumol/kg body wt) plus theophylline 0.1 mmol/L failed to inactivate the enzyme when injected into the portal veins of rats whose livers were obstructed 7 days before. Both the kinetics changes and the lack of phosphorylation control are compatible with ischemia.
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Affiliation(s)
- J E Roselino
- Department of Biochemistry, School of Medicine of Ribeirão Preto SP, University of São Paulo, Brazil
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26
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Rashed SM, Patel TB. Regulation of hepatic energy metabolism by epidermal growth factor. EUROPEAN JOURNAL OF BIOCHEMISTRY 1991; 197:805-13. [PMID: 1903108 DOI: 10.1111/j.1432-1033.1991.tb15975.x] [Citation(s) in RCA: 20] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 12/29/2022]
Abstract
Employing the non-recirculating perfused rat liver preparation, we have investigated the regulation of hepatic gluconeogenesis, and metabolic fluxes through the tricarboxylic acid cycle and 2-oxoglutarate dehydrogenase reaction by epidermal growth factor (EGF) which mimics the actions of both insulin and Ca(2+)-mobilizing hormones (e.g. vasopressin). As monitored by the rate of 14CO2 production from [2-14C]pyruvate (0.5 mM), EGF (10 nM) transiently stimulated the activity of the tricarboxylic acid cycle. EGF also transiently stimulated hepatic gluconeogenesis from pyruvate. The transient stimulation of tricarboxylic acid cycle activity and gluconeogenesis were accompanied by an increase in perfusate Ca2+ content indicating that EGF also altered hepatic Ca2+ fluxes. EGF-elicited stimulation of gluconeogenesis was, at least in part, the result of a transient (50%) inhibition of pyruvate kinase activity. Likewise, EGF-mediated stimulation of tricarboxylic acid cycle activity can, in part, be attributed to EGF-elicited stimulation of metabolic flux through the mitochondrial, Ca(2+)-sensitive, 2-oxoglutarate dehydrogenase reaction. The regulation of hepatic metabolism by EGF appears to be the manifestation of alteration in cellular Ca2+ content since in experiments performed under conditions known to abolish the ability of EGF to alter cytosolic free-Ca2+ concentrations, i.e. in livers of pertussis-toxin-treated rats, EGF did not alter either perfusate Ca2+ content or any of the metabolic parameters monitored. Additionally, experiments involving pulsatile infusion of either EGF or phenylephrine into livers demonstrated that, unlike the alpha 1-adrenergic receptor, homologous desensitization of the EGF receptor occurs. Such a homologous desensitization of the EGF receptor can explain the transient nature of EGF-elicited stimulation of various metabolic processes. Since protein kinase C activation by EGF can lead to receptor desensitization, experiments were performed with phorbol esters which either activate or do not alter protein kinase C activity. While the inactive phorbol ester 4 alpha-phorbol 12,13-didecanoate did not modulate the hepatic actions of EGF, activation of protein kinase C by 4 beta-phorbol 12-myristate 13-acetate (70 nM) abolished the ability of EGF to stimulate gluconeogenesis, tricarboxylic acid cycle activity and metabolic flux through the 2-oxoglutarate dehydrogenase complex.
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Affiliation(s)
- S M Rashed
- Department of Pharmacology, University of Tennessee, Memphis
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Harris RA, Yamanouchi K, Roach PJ, Yen TT, Dominianni SJ, Stephens TW. Stabilization of Glycogen Stores and Stimulation of Glycogen Synthesis in Hepatocytes by Phenacyl Imidazolium Compounds. J Biol Chem 1989. [DOI: 10.1016/s0021-9258(18)63750-0] [Citation(s) in RCA: 15] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/26/2022] Open
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Belfiore F, Rabuazzo AM, Iannello S, Campione R, Castorina S, Urzi F. Extra-pancreatic action of glibenclamide in man: reduction in vitro of the inhibitory effect of glucagon and epinephrine on the hepatic key glycolytic enzymes phosphofructokinase (PFK) and pyruvate kinase (PK). Eur J Clin Invest 1989; 19:367-71. [PMID: 2528458 DOI: 10.1111/j.1365-2362.1989.tb00243.x] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.1] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 01/01/2023]
Abstract
Human liver slices (surgery biopsies) were preincubated with glucagon or epinephrine for 10 min at 37 degrees C in Krebs-Henseleit solution at pH 7.4, in the absence or presence of glibenclamide, and then homogenized and assayed for phosphofructokinase (PFK) and pyruvate kinase (PK) activity at subsaturating, near physiological, substrate concentrations (suitable for detecting regulatory effects). Preincubation with 10 microM glucagon (n = 7) or 10 microM epinephrine (n = 7) resulted in a reduction of PFK activity of 25% (P less than 0.02) and 29% (P less than 0.05), respectively. Addition of 2 microM glibenclamide in the preincubation mixture reduced the inhibitory effect of glucagon by 99% (P less than 0.05) and that of epinephrine by 70% (P less than 0.01). Likewise, 10 microM glucagon (n = 6) or 10 microM epinephrine (n = 4) reduced PK activity by 40% (P less than 0.01) and 46% (P less than 0.01), respectively. Addition of 2 microM glibenclamide significantly reduced the inhibitory effect of glucagon by 77% (P less than 0.05) and that of epinephrine by 33% (P less than 0.05). In the absence of the hormones, glibenclamide was without effect. Thus, glibenclamide opposes the inhibitory effect of glucagon and epinephrine on two key hepatic glycolytic enzymes. Since the inhibition of key glycolytic enzymes favours gluconeogenesis, the observed action of glibenclamide, if it occurs also in vivo, might reduce the glucagon- and epinephrine-stimulated gluconeogenesis, and could be regarded as an insulin-like action.
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Affiliation(s)
- F Belfiore
- Istituto di Clinica Medica III, University of Catania Medical School, Italy
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Mohan C, Geiger PJ, Bessman SP. The intracellular site of action of insulin: the mitochondrial Krebs cycle. CURRENT TOPICS IN CELLULAR REGULATION 1989; 30:105-42. [PMID: 2515941 DOI: 10.1016/b978-0-12-152830-0.50006-2] [Citation(s) in RCA: 22] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 01/01/2023]
Affiliation(s)
- C Mohan
- Department of Pharmacology and Nutrition, University of Southern California School of Medicine, Los Angeles 90033
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30
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Carlsen BD, Lambeth DO, Ray PD. Synthesis of malate from phosphoenolpyruvate by rabbit liver mitochondria: implications for lipogenesis. BIOCHIMICA ET BIOPHYSICA ACTA 1988; 965:1-8. [PMID: 2831992 DOI: 10.1016/0304-4165(88)90143-2] [Citation(s) in RCA: 11] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 01/02/2023]
Abstract
(1) Rabbit liver mitochondria can convert exogenous phosphoenolpyruvate to malate. (2) Malate production is dependent on phosphoenolpyruvate and HCO3- and is stimulated by CN- or malonate alone and especially in combination. (3) Malate production is inhibited 70% by 3-mercaptopicolinate, a specific inhibitor of phosphoenolpyruvate carboxykinase, and 50-60% by 1,2,3-benzenetricarboxylate, an inhibitor of the tricarboxylate transporter. (4) Rat liver mitochondria incubated with phosphoenolpyruvate under identical conditions do not produce malate. (5) Malate production from phosphoenolpyruvate is stimulated by exogenous GDP or IDP but not by ADP. (6) Data support the conclusion that malate is being produced from oxalacetate generated by reversal of mitochondrial phosphoenolpyruvate carboxykinase. A possible role for this enzyme in hepatic lipogenesis is suggested.
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Affiliation(s)
- B D Carlsen
- Department of Biochemistry and Molecular Biology, University of North Dakota School of Medicine, Grand Forks 58202
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31
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Meienhofer MC, De Medicis E, Cognet M, Kahn A. Regulation of genes for glycolytic enzymes in cultured rat hepatoma cell lines. EUROPEAN JOURNAL OF BIOCHEMISTRY 1987; 169:237-43. [PMID: 3691493 DOI: 10.1111/j.1432-1033.1987.tb13603.x] [Citation(s) in RCA: 29] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 01/07/2023]
Abstract
We examined the control by hormones and culture conditions of the expression of pyruvate kinase L, aldolase B, and a liver-specific 5.4-kb mRNA species [Pichard, A. L. et al. (1985) Biochem. J. 226, 637-644] in three rat hepatoma cell lines, MH1C1, Fao and Faza. The expression level of these markers ranges from 2% (for pyruvate kinase L mRNA) to 10-12% (for 5.4-kb mRNA species) of the glucose-induced mRNA values found in rat liver. The mRNAs of the three liver-specific genes strongly decrease after treatment of the hepatoma cells with cyclic 8-bromo-AMP, cyclic dibutyryl-AMP or forkolin, pyruvate kinase L mRNA being the most sensitive to this inhibiting effect. In contrast, the concentration of pyruvate kinase L mRNA nuclear precursors is not modified by the cyclic AMP analogues, indicating that these agents do not act at the transcriptional level but, instead, probably destabilize the transcripts. Glucose or fructose does not modify the expression of these three marker genes in any of the studied cell lines. Insulin is inefficient in modifying concentrations of the mRNAs for pyruvate kinase L and aldolase B, alone or in the presence of carbohydrates. In contrast, it stimulates about fivefold the expression of the 5.4-kb mRNA species in the MH1C1 cell line; this stimulation is carbohydrate-independent. The hepatoma cell lines mimic, therefore, the effect of cyclic AMP on the inhibition in vivo of the expression of genes encoding glycolytic or lipogenic enzymes [Vaulont, S. et al. (1984) Biochem. Biophys. Res. Commun. 125, 135-147]. In contrast, the effect of carbohydrates [Munnich, A. et al. (1984) J. Biol. Chem. 259, 10228-10231] is undetectable. The insulin sensitivity of the liver-specific genes is conserved for the 5.4-kb mRNA species only, especially in the MH1C1 cell line, but not for the other investigated mRNAs, which seems to reflect a fundamental difference in the in vivo effect of insulin on these genes. Finally, S1 nuclease mapping of the start-site of pyruvate kinase L gene transcription shows that the normal site used in vivo is also used in the Fao and Faza lines while, in the MH1C1 line, it coexists with multiple aberrant upstream initiation sites.
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Affiliation(s)
- M C Meienhofer
- Laboratoire de Recherche en Génétique et Pathologie Moléculaires, Institut National de la Santé et de la Recherche Médicale Unité 129, Centre Hospitalier Universitaire, Cochin, Paris
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Petersen TDP, Hochachka PW, Suarez RK. Hormonal control of gluconeogenesis in rainbow trout hepatocytes: Regulatory role of pyruvate kinase. ACTA ACUST UNITED AC 1987. [DOI: 10.1002/jez.1402430202] [Citation(s) in RCA: 38] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/07/2022]
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33
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Rafter GW, Blair JB. Reaction of liver pyruvate kinase with sulfhydryl reagents: effect on its activity. BIOCHIMICA ET BIOPHYSICA ACTA 1987; 913:195-9. [PMID: 3593739 DOI: 10.1016/0167-4838(87)90330-x] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.1] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 01/06/2023]
Abstract
Homogeneous liver pyruvate kinase was reacted with different sulfhydryl reagents, which included o-iodosobenzoate, 5',5'-dithiobis(2-nitrobenzoic acid) and N-ethylmaleimide. Activity determinations of the treated enzyme made with and without Fru(1,6)P2 indicate that the protein contains two sulfhydryl groups per subunit important to its properties, one more accessible than the other. Fru(1,6)P2 added to mixtures prevented loss of activity obtained with o-iodosobenzoate and 5',5'-dithiobis(2-nitrobenzoic acid). It appears that Fru(1,6)P2 does not interfere with the reaction of the reagent with the sulfhydryl group, but prevents an ensuing conformational change, which leads to changes in the enzyme's properties.
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34
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Abstract
The metabolic effects of 24 hours of neonatal fasting in unanesthetized dogs were compared to fasting for three hours during the first day of life. Blood glucose, lactate, and ketones were unaltered while FFA (0.94 +/- 0.07 v 0.70 +/- 0.04 mmol/L, P less than .01), glycerol (0.21 +/- 0.01 v 0.12 +/- 0.01 mmol/L, P less than .01), and triglycerides (0.41 +/- 0.03 v 0.23 +/- 0.03 mmol/L, P less than .01) were lower at 24 hours. Glucose production and lactate and alanine turnover were unaffected while palmitate turnover declined (8.8 +/- 0.7 v 5.1 +/- 0.5 mumol/kg/min, P less than .01). Oxygen consumption decreased (6.9 +/- 0.4 v 6.0 +/- 0.3 mL/kg/min, P less than .02) while RQ increased (0.79 +/- 0.02 v 0.86 +/- 0.03, P less than 0.05) at 24 hours. Hepatic glycogen content declined (575 +/- 37 to 266 +/- 32 mumol/g, P less than .001) and could account for a GP of 12 mumol/kg/min between 3 and 24 hours of age. Both gluconeogenesis from lactate and alanine increased, together accounting for 7% and 21% of glucose production at 3 and 24 hours. The increment in gluconeogenesis may be facilitated by augmented hepatic cytosolic phosphoenolpyruvate carboxykinase at 24 hours (1.8 +/- 0.2 v 14.1 +/- 0.8 nmol/min mg protein, P less than .01). Despite the decline in VO2, hepatic ATP and energy charge were unaltered by 24 hours of fasting. These data suggest that FFA availability diminishes during a prolonged neonatal canine fast resulting in lower VO2. Furthermore, as FFA availability declines, glucose utilization becomes the predominant precursor for energy production.(ABSTRACT TRUNCATED AT 250 WORDS)
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Laville M, Khalfallah Y, Vidal H, Beylot M, Comte B, Riou JP. Hormonal control of glucose production and pyruvate kinase activity in isolated rat liver cells: influence of hypothyroidism. Mol Cell Endocrinol 1987; 50:247-53. [PMID: 3569654 DOI: 10.1016/0303-7207(87)90023-2] [Citation(s) in RCA: 5] [Impact Index Per Article: 0.1] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 01/06/2023]
Abstract
Hormonal control of glucose production and of L-pyruvate kinase activity has been measured in isolated liver cells from fed control and thyroidectomized rats. In hypothyroid rats, sensitivity to isoproterenol as measured by these parameters was increased: the apparent K0.5 for isoproterenol-induced stimulation of glucose production decreased from 8.0 +/- 3 X 10(-6) M in control rats to 2.0 +/- 0.2 X 10(-8) M in hypothyroid rats (P less than 0.001) and the apparent K0.5 for inhibition of L-pyruvate kinase was 5 +/- 2 X 10(-7) M vs. 7 +/- 2 X 10(-9) M (P less than 0.001) in control and thyroidectomized rats, respectively. Utilisation of specific adrenergic antagonists confirmed increased beta-adrenergic responsiveness in hypothyroid rats. This phenomenon was not reversed by 3 days of T3 treatment (10 micrograms/100 g body weight). Sensitivity to the alpha-agonist was unchanged by thyroid status. Stimulation of glucose production and inhibition of L-pyruvate kinase activity by glucagon and their reversal by insulin were not affected by hypothyroidism. The dose-response curve to vasopressin and its maximal effect measured on stimulation of glucose production were unchanged in thyroidectomized rats. Thus, hypothyroidism produces a specific enhancement of liver beta-adrenergic responsiveness without affecting sensitivity to glucagon, insulin and vasopressin.
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36
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2 Pyruvate Kinase. ACTA ACUST UNITED AC 1987. [DOI: 10.1016/s1874-6047(08)60253-x] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register]
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37
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Exton JH. Mechanisms of hormonal regulation of hepatic glucose metabolism. DIABETES/METABOLISM REVIEWS 1987; 3:163-83. [PMID: 3032541 DOI: 10.1002/dmr.5610030108] [Citation(s) in RCA: 118] [Impact Index Per Article: 3.1] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 01/03/2023]
Abstract
Acute hormonal regulation of liver carbohydrate metabolism mainly involves changes in the cytosolic levels of cAMP and Ca2+. Epinephrine, acting through beta 2-adrenergic receptors, and glucagon activate adenylate cyclase in the liver plasma membrane through a mechanism involving a guanine nucleotide-binding protein that is stimulatory to the enzyme. The resulting accumulation of cAMP leads to activation of cAMP-dependent protein kinase, which, in turn, phosphorylates many intracellular enzymes involved in the regulation of glycogen metabolism, gluconeogenesis, and glycolysis. These are (1) phosphorylase b kinase, which is activated and, in turn, phosphorylates and activates phosphorylase, the rate-limiting enzyme for glycogen breakdown; (2) glycogen synthase, which is inactivated and is rate-controlling for glycogen synthesis; (3) pyruvate kinase, which is inactivated and is an important regulatory enzyme for glycolysis; and (4) the 6-phosphofructo-2-kinase/fructose 2,6-bisphosphatase bifunctional enzyme, phosphorylation of which leads to decreased formation of fructose 2,6-P2, which is an activator of 6-phosphofructo-1-kinase and an inhibitor of fructose 1,6-bisphosphatase, both of which are important regulatory enzymes for glycolysis and gluconeogenesis. In addition to rapid effects of glucagon and beta-adrenergic agonists to increase hepatic glucose output by stimulating glycogenolysis and gluconeogenesis and inhibiting glycogen synthesis and glycolysis, these agents produce longer-term stimulatory effects on gluconeogenesis through altered synthesis of certain enzymes of gluconeogenesis/glycolysis and amino acid metabolism. For example, P-enolpyruvate carboxykinase is induced through an effect at the level of transcription mediated by cAMP-dependent protein kinase. Tyrosine amino-transferase, serine dehydratase, tryptophan oxygenase, and glucokinase are also regulated by cAMP, in part at the level of specific messenger RNA synthesis. The sympathetic nervous system and its neurohumoral agonists epinephrine and norepinephrine also rapidly alter hepatic glycogen metabolism and gluconeogenesis acting through alpha 1-adrenergic receptors. The primary response to these agonists is the phosphodiesterase-mediated breakdown of the plasma membrane polyphosphoinositide phosphatidylinositol 4,5-P2 to inositol 1,4,5-P3 and 1,2-diacylglycerol. This involves a guanine nucleotide-binding protein that is different from those involved in the regulation of adenylate cyclase. Inositol 1,4,5-P3 acts as an intracellular messenger for Ca2+ mobilization by releasing Ca2+ from the endoplasmic reticulum.(ABSTRACT TRUNCATED AT 400 WORDS)
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Mohan C, Bessman SP. Effect of insulin on the metabolic distribution of carbons 1, 2, and 3 of pyruvate. Arch Biochem Biophys 1986; 248:190-9. [PMID: 3089162 DOI: 10.1016/0003-9861(86)90416-9] [Citation(s) in RCA: 12] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/04/2023]
Abstract
It has long been known that the carbons of pyruvate are converted to CO2 at different points in the metabolic process. This report deals with the observation that insulin affects the oxidation of carbons 2 and 3 primarily and has little effect on the oxidation of the carboxyl carbon. Oxidation of different carbons of pyruvate and their incorporation into various metabolic components was studied in isolated rat hepatocytes. Insulin stimulated the 14CO2 production from [2-14C]- and [3-14C]pyruvate and from [U-14C]alanine. However, it had little or no effect on the activity of the pyruvate dehydrogenase complex as measured by the evolution of 14CO2 from [1-14C]pyruvate or [1-14C] alanine. Insulin also stimulated the incorporation of carbons 2 and 3 of pyruvate into protein but had no effect on the incorporation of carbon 1. Incorporation of [1-14C]- and [U-14C]alanine into protein was differentially enhanced by insulin in a manner similar to that of the pyruvate carbons. The fact that insulin stimulates the incorporation of [1-14C]alanine into protein but not [1-14C]pyruvate suggests the possibility of a compartmentation of pyruvate metabolism in the isolated hepatocytes. These studies show that the stimulation of [2-14C]- and [3-14C]pyruvate incorporation into protein involves the stimulatory effect of insulin on the activity of the Krebs cycle which is evident from the fact that insulin did not stimulate the pyruvate carbons to enter protein via alanine but the incorporation via glutamate was increased by about 40%.
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39
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Blair JB, Sattsangi S, Hartwell R. Regulation of pyruvate kinase in cultured rat hepatocytes. Influence of glucose, ethanol, glucagon, and dexamethasone. J Biol Chem 1986. [DOI: 10.1016/s0021-9258(17)35953-7] [Citation(s) in RCA: 9] [Impact Index Per Article: 0.2] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/30/2022] Open
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40
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Wilkes GE, Janssens PA. Physiological and metabolic changes associated with weaning in the tammar wallaby, Macropus eugenii. J Comp Physiol B 1986; 156:829-37. [PMID: 3794017 DOI: 10.1007/bf00694258] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.1] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/07/2023]
Abstract
The tammar wallaby (Macropus eugenii) is a small macropodid marsupial in which the major part of weaning occupies the period between 28 and 36 weeks of pouch life. Before weaning the diet of the tammar is high in carbohydrate and low in lipid/volatile fatty acid whereas the reverse applies after weaning. The adult tammar is a forestomach fermenter. The aim of this study was to elucidate some of the physiological and metabolic changes associated with this major change in the diet. Hepatic glycogen content increased gradually early in development to a maximum of 7% of liver weight at 28-30 weeks of pouch life. It then fell precipitously to less than 1% of liver weight at 36 weeks before recovering to the adult level of about 3% liver weight. Plasma glucose levels were maintained at about 10 mM until 36 weeks, after which they fell gradually to adult values of about 4 mM. Hepatic hexokinase activity increased several-fold between 18 and 30 weeks of pouch life, remained high until 42 weeks, and then fell to the adult level. The hepatic activities of fructose-bisphosphatase and particulate phosphoenolpyruvate carboxykinase (PEPCK) were unchanged during development but soluble hepatic PEPCK activity, which was low until 28 weeks of pouch life, increased 3-4 fold between 30 and 36 weeks and then fell slightly to the adult level. Hepatic pyruvate kinase increased in activity up to 28 weeks and then fell to about half peak values at 36 weeks and 20% of peak activity in the adult.(ABSTRACT TRUNCATED AT 250 WORDS)
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41
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Mohan C, Bessman SP. Anabolic regulation of gluconeogenesis by insulin in isolated rat hepatocytes. Arch Biochem Biophys 1985; 242:563-73. [PMID: 3904628 DOI: 10.1016/0003-9861(85)90244-9] [Citation(s) in RCA: 14] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/07/2023]
Abstract
The role of substrate availability in the regulation of gluconeogenesis in isolated rat hepatocytes was studied using [U-14C]alanine as a tracer in the presence of different concentrations of L-alanine in the incubation medium. At low alanine concentrations (0.5 mM) insulin decreased the 14C incorporation into the glucose pool and increased the incorporation of tracer carbons into the protein and lipid pools and into CO2. The net radioactivity lost from the glucose pool was only a small percentage of the total increase in the activity of the protein, lipid, CO2, or glycogen pools, supporting the notion that the effect of insulin in diminishing gluconeogenesis is secondary to its effects on pathways using pyruvate. At higher concentrations of alanine (2.5, 5.0, and 10.0 mM) in the incubation medium insulin increased the movement of alanine carbons into protein and glucose. This suggests that at higher substrate concentrations the ability of the liver to synthesize proteins is overwhelmed and the pyruvate carbons are forced into the gluconeogenesis pathway. These results were further confirmed by using [U-14C]lactate. The increases in observed specific activity of glucose following insulin administration would not be possible if insulin acted by affecting the activity of any enzyme directly involved in the formation or utilization of pyruvate, most of which have been proposed as sites of insulin action. Data presented show that insulin "inhibits" gluconeogenesis by affecting a change in substrate availability.
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42
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Abstract
Rabbit pancreatic islet cytosol catalyzes the calcium-activated phosphorylation by [gamma 32P]ATP of a protein with a molecular weight of 57,000 that is precipitated with antipyruvate kinase antibodies. We were unable to demonstrate that phosphorylation in the presence of calcium or cAMP had any immediate effect on rat pancreatic islet pyruvate kinase activity. This finding is consistent with our inability to confirm the finding of others that pancreatic islets contain phosphoenolpyruvate carboxykinase activity (Diabetes, 34:246, 1985). Since the carboxykinase catalyzes phosphoenolpyruvate formation and pyruvate kinase catalyzes essentially the opposite reaction, if the carboxykinase were present in the beta cell, pyruvate kinase would need to be inhibited to prevent recycling of phosphoenolpyruvate.
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43
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Abstract
In isolated rat liver cells, the inhibition of L-pyruvate kinase (L-PK) by a cyclic AMP-dependent phosphorylation mechanism is involved in the hormonal control of glycolysis and gluconeogenesis. The aim of this study was to ascertain whether or not the in vivo phosphorylation state of the enzyme was maintained during the liver perfusion used to prepare isolated liver cells. When the L-PK phosphorylation state was studied indirectly in liver extracts by kinetic measurement, it was found that, during the perfusion, the S0.5 of phosphoenol pyruvate (PEP) for L-PK was decreased in a time-dependent manner from 1 +/- 0.08 to 0.64 +/- 0.1 mM (P less than 0.01) and 0.58 +/- 0.06 mM in liver cells. This shift was prevented only by the addition of glucagon to the perfusion medium. The extent of phosphorylation of L-PK was also estimated by incubation of the liver extract with [gamma-32P]ATP, protein kinase, and cyclic AMP, and measurement of 32Pi incorporated in L-PK by specific immunoprecipitation. In liver extracts removed at the beginning of the perfusion, 0.4 mol Pi/mol L-PK was incorporated and there was no stimulation by cyclic AMP. In contrast, in the liver extracts removed after 30 min of perfusion, cyclic AMP stimulated 32P incorporation two to threefold, and 1.6 mol Pi/mol L-PK was incorporated. These data suggest that L-PK was activated by a dephosphorylation mechanism during rat liver perfusion. This phenomenon could be involved in the classical inactivation of gluconeogenesis observed in the perfused rat liver model.
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44
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Lytton J, Lin JC, Guidotti G. Identification of two molecular forms of (Na+,K+)-ATPase in rat adipocytes. Relation to insulin stimulation of the enzyme. J Biol Chem 1985. [DOI: 10.1016/s0021-9258(20)71224-x] [Citation(s) in RCA: 160] [Impact Index Per Article: 4.0] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/15/2022] Open
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45
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Feuers RJ, Casciano DA. Insulin and glucagon's reciprocal mediation of a dual regulation mechanism for pyruvate kinase. Biochem Biophys Res Commun 1984; 124:651-6. [PMID: 6388579 DOI: 10.1016/0006-291x(84)91604-8] [Citation(s) in RCA: 6] [Impact Index Per Article: 0.1] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/20/2023]
Abstract
The addition of glucagon to hepatocytes in primary culture produced a rapid and sustained increase in the Km (1.27 mM phosphoenol pyruvate) of pyruvate kinase. The low Km (0.4 mM) form of the enzyme was seen when cells were retreated with insulin, demonstrating a short-term regulation mechanism. Injections of insulin, glucagon or glucagon followed by insulin demonstrated that a similar mechanism occurs in vivo. Results from longer times after injection indicated that another mechanism occurs when altered activity was the result of changes in V max and not Km. Thus, a dual mechanism for regulation of pyruvate kinase occurs. A rapid responding system functions by modification of the enzyme, while a long-term system functions by altering the rate of synthesis, thus changing the amount of enzyme present.
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46
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Fleig WE, Geerling I, Röben H, Ditschuneit H. Effects of insulin, glucagon and dexamethasone on pyruvate kinase in cultured hepatocytes. BIOCHIMICA ET BIOPHYSICA ACTA 1984; 805:165-73. [PMID: 6386057 DOI: 10.1016/0167-4889(84)90164-2] [Citation(s) in RCA: 13] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 01/19/2023]
Abstract
Long-term (24-48 h) and short-term (10-30 min) regulation by hormones of hepatic pyruvate kinase activity was investigated in adult rat hepatocytes cultured under serum-free conditions. In the absence of hormones, pyruvate kinase total activity decreased to 83%, 67% and 39% of the initial level at 24, 48 and 72 h of culture. Insulin (100 nM) maintained total activity significantly above control levels throughout this period. In contrast, glucagon (100 nM) and dexamethasone (100 nM) accelerated the gradual decrease within 24 h (glucagon) or 48 h (dexamethasone) of culture. In these long-term experiments, activity at non-saturating concentrations of phosphoenolpyruvate was decreased by glucagon and dexamethasone but not directly modulated by insulin. However, insulin increased the cellular content of the pyruvate kinase activator fructose-1,6-diphosphate. In short-term experiments on cells cultured under serum- and hormone-free conditions for 48 h, both glucagon and dexamethasone independently caused a rapid, dose-dependent increase of the K0.5 for phosphoenolpyruvate within 10 min, while Vmax was not affected. Insulin inhibited this action of glucagon and dexamethasone and, in their absence, significantly increased substrate affinity for phosphoenolpyruvate within 30 min. Cellular fructose-1,6-diphosphate contents remained unchanged under these conditions. The data identify glucocorticoids and insulin - in addition to glucagon - as short-term regulators of the catalytic properties of pyruvate kinase. All three hormones are effective in the long-term control of total enzyme activity.
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47
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Blair JB, Walker RG. Rat liver pyruvate kinase: influence of ligands on activity and fructose 1,6-bisphosphate binding. Arch Biochem Biophys 1984; 232:202-13. [PMID: 6742850 DOI: 10.1016/0003-9861(84)90536-8] [Citation(s) in RCA: 17] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/21/2023]
Abstract
The ability for various ligands to modulate the binding of fructose 1,6-bisphosphate (Fru-1,6-P2) with purified rat liver pyruvate kinase was examined. Binding of Fru-1,6-P2 with pyruvate kinase exhibits positive cooperativity, with maximum binding of 4 mol Fru-1,6-P2 per enzyme tetramer. The Hill coefficient (nH), and the concentration of Fru-1,6-P2 giving half-maximal binding [FBP]1/2, are influenced by several factors. In 150 mM Tris-HCl, 70 mM KCl, 11 mM MgSO4 at pH 7.4, [FBP]1/2 is 2.6 microM and nH is 2.7. Phosphoenolpyruvate and pyruvate enhance the binding of Fru-1,6-P2 by decreasing [FBP]1/2. ADP and ATP alone had little influence on Fru-1,6-P2 binding. However, the nucleotides antagonize the response elicited by pyruvate or phosphoenolpyruvate, suggesting that the competent enzyme substrate complex does not favor Fru-1,6-P2 binding. Phosphorylation of pyruvate kinase or the inclusion of alanine in the medium, two actions which inhibit the enzyme activity, result in diminished binding of low concentrations of Fru-1,6-P2 with the enzyme. These effectors do not alter the maximum binding capacity of the enzyme but rather they raise the concentrations of Fru-1,6-P2 needed for maximum binding. Phosphorylation also decreased the nH for Fru-1,6-P2 binding from 2.7 to 1.7. Pyruvate kinase activity is dependent on a divalent metal ion. Substituting Mn2+ for Mg2+ results in a 60% decrease in the maximum catalytic activity for the enzyme and decreases the concentration of phosphoenolpyruvate needed for half-maximal activity from 1 to 0.1 mM. As a consequence, Mn2+ stimulates activity at subsaturating concentrations of phosphoenolpyruvate, but inhibits at saturating concentrations of the substrate or in the presence of Fru-1,6-P2. Both Mg2+ and Mn2+ diminish binding of low concentrations of Fru-1,6-P2; however, the concentrations of the metal ions needed to influence Fru-1,6-P2 binding exceed those needed to support catalytic activity.
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48
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The permissive effects of glucocorticoid on hepatic gluconeogenesis. Glucagon stimulation of glucose-suppressed gluconeogenesis and inhibition of 6-phosphofructo-1-kinase in hepatocytes from fasted rats. J Biol Chem 1984. [DOI: 10.1016/s0021-9258(17)42808-0] [Citation(s) in RCA: 34] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/18/2022] Open
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49
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Riquelme PT, Wernette-Hammond ME, Kneer NM, Lardy HA. Mechanism of action of 2,5-anhydro-D-mannitol in hepatocytes. Effects of phosphorylated metabolites on enzymes of carbohydrate metabolism. J Biol Chem 1984. [DOI: 10.1016/s0021-9258(17)42964-4] [Citation(s) in RCA: 34] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 10/22/2022] Open
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50
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Belfiore F, Rabuazzo AM, Iannello S, Vasta D, Campione R. Insulin resistance in the obese hyperglycemic (ob/ob) mouse. Failure of hyperinsulinemia to activate hepatic pyruvate kinase (PK). Metabolism 1984; 33:104-6. [PMID: 6694552 DOI: 10.1016/0026-0495(84)90119-7] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 01/21/2023]
Abstract
In obese hyperglycemic (ob/ob) mice, as compared to controls, hepatic pyruvate kinase (PK) activity was enhanced by 35.63% (214.75 +/- 13.60 nmol/min/mg protein v 158.33 +/- 10.47, P less than 0.01) when measured at saturating (6.6 mmol/L) concentration of the substrate phosphoenolpyruvate (total activity), but the activity recorded at subsaturating (1.3 mmol/L) substrate concentration (active fraction) was unchanged (86.37 +/- 6.42 v 85.66 +/- 13.59) or even decreased if expressed as percent of the total activity (40.21 +/- 2.56% v 54.10 +/- 5.07, P less than 0.05). Since insulin induces the synthesis of hepatic PK and favors the conversion of the inactive (phosphorylated) to the active (dephosphorylated) form, these findings suggest that in ob/ob mice the striking hyperinsulinemia, although it is able to increase the hepatic content of PK, fails to activate this enzyme. This may favor gluconeogenesis in these animals. The hepatic concentration of PK effectors (fructose-1,6-P2 and phosphoenolpyruvate) was unchanged in ob/ob mice, and the in vitro effect of the activator fructose-1,6-P2 (15 mumol/L), which would favor the activation (dephosphorylation) of PK, was preserved. It is suggested that hepatic PK in ob/ob mice is resistant to activation by insulin.
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