Diabetes mellitus Practicals – experimental diabetes mellitus in laboratory animal Ústav patologické fyziologie LF MU Definition of diabetes mellitus (DM) • DM is a group of metabolic disorders characterized by hyperglycemia as a result of impaired effect of insulin • absolute • insulin is missing • relative • insulin resistance • impaired insulin secretion (gluco- and lipotoxicity) • chronic hyperglycemia leads to cell & tissue damage (complications) • retina • kidney • nerves 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 Oral glucose tolerance test • 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 oGTT interpretation Animal models of type 1 diabetes • 1889 – pancreas removal • Minkowski and Von Mering • diabetic syndrome in dog • Banting and Best • insulin discovery and testing • the Nobel Prize in Physiology or Medicine 1923 • exp. diabetes induced in various species • β-cells toxins and viruses • specific strains in which insulin-deficient diabetes develops spontaneously Sir Frederick Banting and Dr. Charles Best co-discoverers of Insulin 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 Experimental induction of DM • anesthesia induction • Narkamon/Rometar, 0.5 ml/100 g of weight • intraperitoneal administration • skin incision • i.v. administration of alloxan • In the jugular vein • suture • the wound is closed with several individual stitches • second part of the experiment follows after 1 week The experiment 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.