Diabetes mellitus Practicals – experimental diabetes mellitus in laboratory animal Animal models of type 1 diabetes • 1889 – pancreas removal – Minkowski and Von Mering • diabetic syndrome in dog • Banting and Best – insulin discovery and testing • exp. diabetes induced in various species – β-cells toxins and viruses • specific strains in which insulindeficient diabetes develops spontaneously Chemically induced insulin-deficient diabetes • alloxan – first known diabetogenic chemical agent (1943) • islet-cells necrosis in rabbit – high doses • β-cells necrosis – acts on membrane and interior • inhibits insulin release • is taken up into the β-cell – glucokinase, PARP – free radicals – practical problems in vivo • instability at physiological pH • dosage variation with age and species • toxicity on other organs • streptozotocin (STZ) – induces severe diabetes • i.v. or i.p. – most commonly used model – β-cell necrosis within 1-2 days – insulin falls to 10-30% – hyperglycemia 20-30 mmol/l – at dosage 50 mg/kg severe ketosis does not develop • survival without insulin replacement – may share cytotoxic mechanisms with alloxan Mechanisms of alloxan and STZ action Animal models with spontaneous T1DM • BB rat (BioBreeding) – defects in immunity • inflitration of islets with T lymphocytes • autoantibodies against GAD (glutamic acid decarboxylase) are present • severe hypoinsulinemia and hyperglycemia – two main T1DM susceptibility genes • 8 additional loci • NOD mouse – non-obese diabetic – autoimmunie diabetes – diabetes develops as a result of insulitis – NOD mice will develop spontaneous diabetes when left in a sterile environment – affects 80 % of females and 20 % of males Summary of rodent models of T1DM Summary of rodent models of T2DM Practicals i.p. ANESTHESIA 3) repeated measurement of glycemia on glucometer in 30 a 90 min time intervals 4) determination of glucosuria in urine sample 1 week before 1/2 animals ALLOXAN i.v. 30 mg/kg results: • graph FPG - 30mPG - 90mPG • comparison of DM x non-DM 1) blood sample from a tail vein 2) measurement of FPG on glucometer application of 20% glucose 1ml/100g i.p. Oral glucose tolerance test (oGTT) • tool used for diagnosis of – diabetes mellitus • presence of diabetes in the family, • in obese patients and in hypertesion, • patients with glycemia 6.1 – 7.0 mmol/l twice in the row – gestational diabetes • early (< 12th week of pregnancy in women with at least 2 risk factors – age > 30 years – presence of diabetes in the family – macrosomia – obesity – diabetes mellitus in previous pregnancy – glycosuria – hypertension in previous pregnancy – repeated abortions – “prediabetes” • impaired glucose tolerance (IGT) – 2-h PPG 7.8 - <11.1 mmol/l during oGTT • impaired fasting glucose (IFG) – FPG 5.6 – <7 mmol/l • procedure – FPG, drinking glucose solution (75 g + 250 ml water) within 5 – 10 min, glycemia measurement after 60 and 120 min Definition of DM • DM is a group of metabolic disorders characterized by hyperglycemia as a reason of impaired effect of insulin – absolute – relative • insulin resistance • impaired insulin secretion (gluco- and lipotoxicity) • chronic hyperglycemia leads to cell & tissue damage (complications) – retina – kidney – nerves Diabetes prevalence Diagnosis of DM • classical symptoms of diabetes + random plasma glycemia 11.1 mmol/l – any time of the day – symptoms include polyuria, polydipsia and rapid loose of weight • FPG (fasting plasma glucose) 7.0 mmol/l – fasting means at least 8 h from the last meal • 2-h PG (postprandial glucose) 11.1 mmol/l during oGTT – according to WHO standard load of 75 g of glucose Interpretation of glycemia • FPG: – <6.1 mmol/l = normal glycemia – 6.1-7.0 mmol/l = IFG (impaired fasting glucose) – 7.0 mmol/l = diabetes • oGTT – 2h PG: – <7.8 mmol/l = normal glucose tolerance – 7.8-11.1 mmol/l = IGT (impaired glucose tolerance) – 11.1 mmol/l = diabetes oGTT interpretation 11,1 11,1 7,0 5,6 7,8 7,8 3 4 5 6 7 8 9 10 11 12 FPG 60 min 120 min glycemia(mmol/L) diabetes IGT IFG normal Regulation of glycemia • humoral – principal • insulin • glucagon – auxiliary • glucocorticoids • adrenalin • growth hormone • neural – sympaticus • hyperglycemia – parasympaticus • hypoglycemia plasma (glycaemia 3–6 mmol/l) FOOD PRODUCTION of GLUCOSE by LIVER CNS A OTHER TISSUES MUSCLE, ADIPOSE TISSUE glykogenolysis inzulin-dependent utilisation non-inzulindependent utilisation glukoneogeneze - pyruvát - laktát - aminokyseliny - glycerol INSULIN GLUCAGON Normal glucose homeostasis Mutual interchange of substrates in intermediate metabolism GLUCOSE glucose-6-P pyruvate ATP lactate ATP acetyl-CoA citrate cycle respiratory chain and oxidative phosphorylation CO2 H2O lactate cycle in liver glycolysis GLYCOGEN glucose-1- phosphate liver, muscle glycogenesis, glycogenolysis glycerol glucogennic aminoacids gluconeogenesis liver, kidney, intestine free fatty acids -oxidation keton bodies ATP Question – how does glucose enter the cell??? GlucoseNa+ Glucose Insulin • preproinsulin  proinsulin  insulin + C-peptide • exocytosis into portal circulation – 50% degraded during first pass through liver • total daily production 20 - 40 U – 1/2 basal secretion, 1/2 stimulated • basal secretion pulsatile – 5 - 15 min intervals • stimulated – glucose, amino acids, FFA, GIT hormones – early phase (ready insulin) – late phase (synthesis de novo) Synthesis of insulin endoplasmic reticulum Golgi apparatus microvesicles PREPROINSULIN (11.5 kDa) PROINSULIN (9 kDa) secretory granules 11 INSULIN + C-PEPTIDE prohormon-konvertáza 3prohormon-konvertáza 2 karboxypeptidáza PROINSULIN INSULIN + C-PEPTIDE Relationship glycemia – insulin secretion Arg+ Lys+ His+ Ca2+ channel K+ glucose GLUT-2 glucokinase ATP ATP-sensitive K+ channel closing glucose-6-P depolarization Ca2+ translocation and exocytosis of insulin granules Leu Gln regulace SACHARIDY regulace AMINOKYSELINAMI Islet β-cell metabolic activation Intracellular cascade of insulin receptor IRS GLUT4 glukóza     insulin P P PIP2 PIP3 P PI3-K PKB stimulation of glycogen synthesis stimulation of lipogenesis gene expression “lipid raft” Intracellular insulin signalling Classification of tissues according to insulin action: • insulin-sensitive – muscle, adipose tissue • facilitated diffusion by GLUT4 • integration into cytoplasmic membrane regulated by insulin – liver • stimulation of glycogenolysis • inhibition of gluconeogenesis • insulin-non-sensitive – others (incl. muscle, adipose tissue, liver) – transport of glucose depends on • concentration gradient • density of transporters (GLUT1- 4,8-10) • rate of glycolysis Insulin action in the muscle Insulin action in adipose tissue Insulin action in the liver Diabetes mellitus • heterogeneous syndrome characterized by hyperglycemia due to deficiency of insulin action (as a result of complete depletion or peripheral resistance) • prevalence of DM in general population 5%, over the age of 65 already 25% Causes of insulin deficiency • absolute – destruction of the -cells of the islets of Langerhan´s • relative – insulin • abnormal molecule of insulin (mutation) • defective conversion of preproinsulin to insulin • circulating antibodies against insulin or receptor – insulin resistance in peripheral tissue • receptor defect • post-receptor defect Classification of DM I. DIABETES MELLITUS Diabetes mellitus of type 1 (T1DM) Diabetes mellitus of type 2 (T2DM) Gestational diabetes mellitus Other specific types - genetic defects of β cell function (MODY) - genetic abnormalities of insulin receptor - exocrine pancreas disorders - endocrinopathies - iatrogenic - rare genetic syndromes II. IMPAIRED GLUCOSE TOLERANCE (IGT) - with obesity - without obesity Type 1 DM (formerly IDDM) • selective destruction of  cells of LO in genetically predisposed individuals – chrom. 6 - HLA (DR3-DQ2 a DR4-DQ8), chrom. 11 - inzulin gene – initiation by infection (viruses) • autoimmunity mediated by T-lymphocytes (antibodies against  cells (ICA, GAD) though) • manifestation typically in childhood • absolute dependence on exogenous supplementation by insulin Type 2 DM (formerly NIDDM) • imbalance between secretion and affect of insulin • genetic predisposition – polygenic – insulin resistance – impairment of secretion • clinically manifested T2DM has concomitant insulin resistance and impairment of secretion – due to epigenetic factors – typically in older adults • 90% of subjects is obese – metabolic syndrome!!! Epidemiology of T2DM • another risk factors – sleep restriction • 57% nárůst rizika DM (10 let pozorování, 70 000 žen) – léky – environmental pollutants – Low birthweight and fetal malnutrition The ominous octet Insulin resistance • physiologic amount of insulin does not cause adequate response • compensatory hyperinsulinism • further worsening by down-regulation of insulin receptors Pathway to T2DM Natural history of T2DM Maturity-onset diabetes of the young (MODY1-6) • group of monogenic conditions with autosomal dominant inheritance • childhood, adolescence or early adulthood onset • genetically determined -cells dysfunction – but long-term measurable Cpeptide without signs of autoimmunity • 1% of diabetic patients • two subgroups – mutations in glucokinase (MODY2) • glucokinase = glucose sensor (production and releasing of insulin is slowing) • mild form without considerable risk of complications – mutations in the genes encoding transcription factors (remaining 5 types) • severe -cells defects progressively leading to diabetes with serious complications • Affected glucose-stimulated production and release of insulin and also proliferation and differentiation of - cells Main characteristics of T1DM and T2DM and MODY T1DM T2DM MODY onset childhood adults childhood genetic disposition yes (oligogenic) yes (polygenic) yes (monogenic) clinical manifestation often acute mild or none mild autoimmunity yes no no insulin resistance no yes no dependence on insulin yes no no Obesity no yes no Clinical presentation of manifest DM • due to the increase of blood osmolality, osmotic diuresis and dehydration – classical • polyuria • thirst • polydipsia • weight loss • temporary impairment of visus • cutaneous infections • acute – hyperglycemic coma • ketoacidotic • non-ketoticidotic – hyperosmolar nonketoacidotic hyperglycemia – lactate acidosis Complications of DM • microvascular – diabetic retinopathy – diabetic nephropathy – diabetic neuropathy (sensoric, motoric, autonomic) • macrovascular – atherosclerosis (CAD, peripheral and cerebrovascular vascular disease) • combined – diabetic foot (ulcerations, amputations and Charcot´s joint) • others – periodontitis – cataract – glaucoma fruktóza-6-fosfát glyceraldehyd-3-fosfát pyruvát NAD NADH+H+ GAPDH glukóza glukóza-6-fosfát POLYOLOVÁ DRÁHA TVORBA DAG HEXOZAMINOVÁ DRÁHA ROS tvorba AGE, modifikace proteinů, indukce zánětlivých změn tvorba osmoticky aktivního sorbitolu, další konzumpce NAD+ tvorba UDP-NAc-glukosaminu prohlubuje inzulinovou rezistenci aktivace proteinkinázy C a prostřednictvím PKC další tvorby ROS NEENZYMATICKÁ GLYKACE NADH / NAD+ v důsledku aktivace reparačních a antioxidačních systémů se spotřebovává NAD+