Metabolic Adaptation to High-frequent Hypoglycaemia in Type 1 Diabetes

Hyperinsulinemic glucose clamp studies require that insulin is administered at a steady continuous rate to achieve stable levels of hyperinsulinemia. To reach this, insulin needs to be infused intravenously using a standard intravenous pump system. The insulin dose will be adjusted according to the body surface area, aiming for insulin levels of \ 170 mIU/l, which is within the physiological range. Thus, for a subject with a bodyweight of 70 kg, body length of 180 cm and - consequently - a body surface area of 1.936 m2, the required insulin infusion can be calculated as: 1.936 x 60 x 60 ÷ 1000 = 7.0 units per hour

Epinephrine are prepared in 100 ml isotone saline solution according to weight and infused in 3 different infusion rates: 10 ng∙kg-1∙min-1, 25 ng∙kg-1∙min-1 and 50 ng∙kg-1∙min-1, for 20 minutes each. After each adrenaline infusion, substrate response will be measured by blood samples of glucose, lactate, free fatty acids, alanine, β-hydroxybutyrate, glycerol and insulin. Furthermore, cardiovascular measurements such as pulse and blood pressure are monitored as well.

With the study subject resting in the supine position, the skin is disinfected on the lateral side of the thigh around 15 cm above the knee, with chlorhexidine alcohol. Then 3-4 mL of local anaesthetic (lidocaine 20 mg/mL) is injected into the skin, subcutaneous tissue and in the upper part of the muscle with a very thin needle. When the anaesthetic effect has set in after a couple of minutes an insertion is made in the skin and the subcutaneous tissue through which the biopsy cannula is inserted into the muscle. A small piece (around 150 mg) of the muscle is collected, which may be experienced as somewhat unpleasant, but will last for a very short while ( \ 1-2 seconds). The needle is removed, a sterile Band-Aid is applied, and the study subject can leave the site after termination of the trial. The biopsy may cause some muscular tenderness for 2-3 days corresponding to minor muscular trauma.

With the study subject resting in the supine position, the skin is disinfected on one side of the abdomen around 5-10 cm lateral from the umbilicus to the knee, with chlorhexidine alcohol. Then 3-4 mL of local anaesthetic (lidocaine 20 mg/mL) is injected into the skin, subcutaneous tissue and in the upper part of the adipose tissue with a very thin needle. When the anaesthetic effect has set in after a couple of minutes an insertion is made in the skin and the subcutaneous tissue through which the biopsy cannula is inserted into the adipose tissue. A small piece (around 1 gram) of the adipose tissue is collected, which may be experienced as somewhat unpleasant, but will last for a very short while ( \ 1-2 seconds). The needle is removed, a sterile Band-Aid is applied, and the study subject can leave the site after termination of the trial. The biopsy may cause some tenderness for 2-3 days corresponding to minor trauma.

Glucagon is prepared in doses of 10 µg, 25 µg, and 50 µg and intravenously injected with intervals of 2 hours. After each glucagon injection, blood samples will be drawn to measure plasma glucose, glucagon, lactate, free fatty acids, alanine, amino-acids, β-hydroxybutyrate, glycerol and insulin. Furthermore, cardiovascular measurements such as pulse and blood pressure are monitored as well.

All potential subjects will receive a blinded continuous glucose sensor at Visit 1. At the following visits, the continuous glucose monitor (CGM) will be reviewed for hypoglycaemia episodes and replaced at the same time. At Visit 2 a final screening of the inclusion criteria will take place, which involves the CGM data of the first week. A blinded CGM will be installed a week before every visit.

Subjects will undergo a hyperinsulinemic euglycaemic glucose clamp, as mentioned above, in the MRI scanning room. After 30 minutes of stable normoglycaemia, subjects are taken into the MRI scanner (Philips Achieva 7.0 T) where brain, liver, thigh and calf muscle are scanned. After every anatomically different area, the subjects must be taken out of the scanner, while scanning coils are replaced. All subjects are advised to lie still and press the alarm button if necessary.

Resting metabolic rate will be estimated, after reaching stable plasma glucose level, via a hyperinsulinemic euglycaemic clamp, as mentioned above. This will be done by indirect calorimetry, using a ventilated hood system (Jaeger Oxycon Champion, software version 4.3, Jaeger, Mijnhardt). Subjects are instructed to lie down and rest for a period of 30 minutes. Subjects are also instructed not to move, talk or sleep unless necessary during the period of measurement. The recorded measurement after 5 minutes to 30 minutes will be used for analysis.

Thermography (Thermovision SC645, FLIR Systems, Wilsonville, OR, USA) is used to determine cutaneous vascular perfusion. Data is analogue-digital converted and sampled at 100 Hz (Powerlab, ADInstruments, Colorado Springs, CO, USA).

All potential subjects will receive a continuous glucose sensor at Visit 1. At the following visits, the CGM will be reviewed for hypoglycaemia episodes and replaced at the same time. At Visit 2 a final screening of the inclusion criteria will take place, which involves the CGM data of the first week. A CGM will be installed a week before every visit.