title: Development of microdialysis methodology for interstitial insulin measurement in rodents.
abstract: BACKGROUND
  Accurate assessment of muscle insulin sensitivity requires measurement of insulin concentration in interstitial fluid (ISF), but has proved difficult. We aimed to optimise measurement of ISF insulin concentrations in rat muscles in vivo using microdialysis.
  
  
  METHODS
  Factorial experimental design experiments were performed in vitro to determine optimal conditions for insulin recovery with microdialysis probes. These conditions were tested in vivo, adjusted appropriately and used in lean and obese Zucker rats to compare ISF insulin concentrations basally and during hyperinsulinaemic-euglycaemic (HE) clamp.
  
  
  RESULTS
  Optimal conditions in vivo were: a 100kDa microdialysis probe inserted in muscle, perfused with 1% BSA, 1.5mM glucose in 0.9% sodium chloride at 1μl/min. Samples were collected into siliconised glass microvials. As a reference for insulin, we established a protocol of inulin infusion, beginning at -80min and reaching equilibrium within 60min. HE clamp, beginning at 0min, increased ISF insulin concentration from 122±56 basally to 429±180pmol/l (P<0.05) in lean rats and from 643±165 to 1087±243pmol/l (P=0.07) in obese rats; ISF insulin concentrations were significantly higher throughout in obese rats. The difference between ISF and plasma insulin concentration (ISF:plasma ratio) was substantially higher in obese rats, but fell to similar values in obese and lean rats during HE clamp.
  
  
  CONCLUSIONS
  Optimising insulin recovery with microdialysis allowed accurate measurement of basal ISF insulin in muscle of lean and obese Zucker rats and indicates insulin transport across capillaries is impaired in obese rats, basally and during hyperinsulinaemia.
authors.affiliation: School of Clinical and Experimental Medicine, The Medical School, University of Birmingham, Birmingham B15 2TT, United Kingdom.
authors.forename: Ann E
authors.initials: AE
authors.lastname: Griffin
authors.affiliation: Cardiovascular and Gastro-intestinal Research Department, AstraZeneca Pharmaceuticals, Mereside, Alderley Park, Macclesfield, Cheshire, SK10 4TG, United Kingdom.
authors.forename: Ruth
authors.initials: R
authors.lastname: Macdonald
authors.affiliation: School of Sport and Exercise Sciences, University of Birmingham, Birmingham B15 2TT, United Kingdom.
authors.forename: Anton J M
authors.initials: AJM
authors.lastname: Wagenmakers
authors.affiliation: School of Clinical and Experimental Medicine, The Medical School, University of Birmingham, Birmingham B15 2TT, United Kingdom. Electronic address: j.m.marshall@bham.ac.uk.
authors.forename: Janice M
authors.initials: JM
authors.lastname: Marshall
authors.affiliation: Cardiovascular and Gastro-intestinal Research Department, AstraZeneca Pharmaceuticals, Mereside, Alderley Park, Macclesfield, Cheshire, SK10 4TG, United Kingdom.
authors.forename: Simon M
authors.initials: SM
authors.lastname: Poucher
mesh_terms: D000818:Animals; D002196:Capillaries / Q000378:metabolism; D045604:Extracellular Fluid / Q000737:chemistry*; D015309:Glucose Clamp Technique; D006946:Hyperinsulinism / Q000097:blood / Q000378:metabolism; D007328:Insulin / Q000032:analysis* / Q000097:blood; D007333:Insulin Resistance; D007444:Inulin / Q000494:pharmacology; D008297:Male; D017551:Microdialysis / Q000379:methods*; D018482:Muscle, Skeletal / Q000737:chemistry*; D051381:Rats; D017208:Rats, Wistar; D011924:Rats, Zucker; D015203:Reproducibility of Results
chemical_list: D007328:Insulin; D007444:Inulin
keywords: External reference technique; Hyperinsulinaemic-euglycaemic clamp; Insulin; Interstitial fluid; Methods; Microdialysis; Zucker rat