2 - TI Pharma

Experimental protocol – clamp: After an overnight fast of 12 hours, subjects were admitted
at the clinical research unit at 0730 AM. An indwelling cannula was inserted into an antecubital
vein for infusion of stable-isotope tracers, glucose and insulin. To obtain arterialized venous
blood samples, a retrograde cannula was inserted in a contralateral wrist vein and maintained
in a thermoregulated box at 50°C. 0.9% NaCl was infused to keep the sampling line patent.
[6,6-2H ]Glucose, [1,1,2,3,3,- 2 2H ]glycerol and L-[1- 5 13C]valine were used as tracers (> 99%
enriched; Cambridge Isotopes, Andover, MA) to study glucose kinetics, lipolysis and valine
turnover respectively. At T = 0 h (0800 AM), blood samples were drawn for determination of
background enrichments. Then, a primed, continuous infusion of isotopes was started: [6,6-
2H2 ]glucose (prime: 11 mmol/kg; continuous: 0.11 mmol/kg.min), [1,1,2,3,3,-2H ]glycerol
5
(prime: 1.6 mmol/kg; continuous: 0.11 mmol/kg.min), and L-[1-13C]valine (prime: 13.7
mmol/kg; continuous: 0.153 mmol,kg.min) and continued until the end of the clamp. After
a 2-h equilibrium period (14 h of fasting), 3 blood samples were drawn for determination
of basal glucose concentrations, isotope enrichments, glucoregulatory hormones and
nonesterified fatty acids (NEFA). Thereafter, a 2-step hyperinsulinemic-euglycemic clamp
was started: step 1 included an infusion of insulin at a rate of 20 mU/m2 .min (Actrapid 100
IU/mL; Novo Nordisk Farma BV, Alphen aan den Rijn, Netherlands) to assess hepatic insulin
sensitivity. Glucose 20% was started to maintain a plasma glucose concentration of 5 mmol/l.
[6,6-2H ]Glucose was added to the glucose solution to achieve glucose enrichments of 1% in
2
order to approximate the values for enrichment reached in plasma and thereby minimizing
changes in isotopic enrichment due to changes in the infusion rate of exogenous glucose.
Plasma glucose concentrations were measured every 5 min at bedside. After 2-h of insulin
infusion, 5 blood samples were drawn at 5min intervals for the measurement of glucose
concentrations and isotopic enrichments. Another blood sample was drawn for measurement
of glucoregulatory hormones and NEFA. Hereafter, insulin infusion was increased to a rate of
60 mU/m2 .min (step 2) to assess peripheral insulin sensitivity. After 2 h of insulin infusion,
blood sampling for glucose, isotope enrichments, glucoregulatory hormones and NEFA was
repeated (Supplementary Figure 1b).
Indirect calorimetry: Oxygen consumption (VO 2 ) and carbon dioxide production (VCO 2 )
were measured continuously during the final 20 min of both the basal state and during step
2 of the hyperinsulinemic-euglycemic clamp by indirect calorimetry using a ventilated hood
system (Vmax model 2900; Sensormedics, Anaheim, CA). The VO and VCO measurements
2 2
during the last 10 minutes were used for further calculations.
Study medication: Prednisolone tablets were purchased from Pfizer AB (Sollentuna, Sweden)
and matching placebo tablets were obtained from Xendo Drug Development (Groningen, The
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Chapter 8