Metabolic Adaptation to High-frequent Hypoglycaemia in Type 1 Diabetes

Study rationale The risk of severe hypoglycaemia is a major daily concern for people with diabetes treated with insulin. Severe hypoglycaemia is the main barrier in achieving the recommended glycaemic targets and may indirectly be the main driver for late diabetic complications and related morbidity, mortality and health care costs. In people with diabetes, recurrent exposure to insulin-induced mild hypoglycaemia leads to significant adaptive physiologic responses. While the metabolism of the brain and hormonal responses to hypoglycaemia have been studied extensively, this study will as the first, systematically investigate the chronic adaptation of peripheral metabolism to recurrent hypoglycaemia in diabetes. Knowledge about such responses can lead to interventions that attenuate the devastating effects of acute hypoglycaemia induced by insulin in people with diabetes. Thereby, the risk of developing severe hypoglycaemia can be reduced which ultimately will improve long-term diabetes outcomes and reduce health care costs.

Hypothesis Patients with type 1 diabetes that are exposed to high-frequent recurrent hypoglycaemia will adapt their metabolism in a way, which supports the preservation of brain fuelling.

Objectives

Primary objective The overall objective is to gain new knowledge about mechanisms involved in adaptation to recurrent hypoglycaemia in diabetes by investigating patients with type 1 diabetes and healthy controls. The knowledge to be obtained may feed into experimental hypoglycaemic clamp studies to further elucidate the effect of the adaptations during acute hypoglycaemia. Ultimately, it can lead to intervention studies aiming at the maintenance of functional capability during hypoglycaemia in patients with type 1 diabetes to reduce their risk of severe hypoglycaemia.

Secondary objectives

* To study the metabolic consequences of recurrent hypoglycaemia in the brain, liver, muscle and adipose tissues
* To study the consequences of recurrent hypoglycaemia on resting metabolic rest
* To study the consequences of recurrent hypoglycaemia on glucagon and adrenaline sensitivity
* To study the consequences of recurrent hypoglycaemia on epigenetic profiles
* To study the consequences of recurrent hypoglycaemia on oxidative stress
* To study the psychological factors associated with recurrent hypoglycaemia

• insulin human (Drug), also known as Actrapid: 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
• Epinephrin (Drug), also known as Adrenalin: 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.
• Muscle biopsy (Procedure): 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.
• Adipose tissue biopsy (Procedure): 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 (Drug), also known as GlucaGen: 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.
• IPRO 2 Medtronic Minimed (Device), also known as Blinded Continuous Glucose Monitoring: 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.
• 7 Tesla (7T) Magnetic Resonance Imaging (Procedure), also known as 7T MRI: 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.
• Indirect Calorimetry using Jaeger Oxycon Champion (Procedure): 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.
• Core temperature and thermography using Thermovision SC645 (Procedure): 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).
• Freestyle Libre 2 (Device), also known as Intermittently scanned continuous glucose monitor: 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.

Primary outcomes

• Metabolite- and lipid profiling [5 minutes]: Metabolite- and lipid profiling of blood samples using metabolomics profiling platforms during euglycaemia
• Brain lactate concentration [20 minutes]: Brain lactate concentration using non-invasive magnetic resonance (MR) spectroscopy during euglycaemia
• Brain adenosine triphosphate (ATP) concentration [20 minutes]: Brain ATP concentration using non-invasive MR spectroscopy during euglycaemia
• Glycogen in muscle and adipose tissue [5 minutes]: Glycogen in muscle and adipose tissue biopsies during euglycaemia
• Non-specific proteins in muscle and adipose tissue [5 minutes]: Non-specific proteins in muscle and adipose tissue biopsies during euglycaemia
• Glycogen concentration [40 minutes]: Glycogen in liver and muscle tissue using non-invasive MR spectroscopy during euglycaemia.

Secondary outcomes

• Estimated glucose production during glucagon stimulation [Every 5 minutes up to 5 hours]: Area under the curve (AUC) for plasma glucose during glucagon injections. Plasma glucose measurement
• Estimated glucose production during epinephrine stimulation [Every 5 minutes up to 90 minutes]: Area under the curve (AUC) for plasma glucose during epinephrine infusion. Plasma glucose measurement
• Indirect calorimetry [60 minutes]: Estimating resting metabolic rate, before and during hyperinsulinemic-hypoglycemic clamp
• Thermography [5 minutes]: Estimating skin temperature, before and during hyperinsulinemic-hypoglycemic clamp
• Plasma lactate during glucagon injections. [Every 40 minutes up to 5 hours]: Plasma lactate during glucagon injections.
• Plasma free fatty acids during glucagon injections. [Every 40 minutes up to 5 hours]: Plasma free fatty acids during glucagon injections.
• Plasma glycerol during glucagon injections. [Every 40 minutes up to 5 hours]: Plasma glycerol during glucagon injections.
• Plasma alanine during glucagon injections. [Every 40 minutes up to 5 hours]: Plasma alanine during glucagon injections.
• Plasma β-hydroxybutyrate during glucagon injections. [Every 40 minutes up to 5 hours]: Plasma β-hydroxybutyrate during glucagon injections.
• Plasma insulin during glucagon injections. [Every 40 minutes up to 5 hours]: Plasma insulin during glucagon injections.
• Plasma glucagon during glucagon injections. [Every 40 minutes up to 5 hours]: Plasma glucagon during glucagon injections.
• Plasma metabolomics during glucagon injections. [Every 40 minutes up to 5 hours]: Plasma metabolomics during glucagon injections.
• Plasma lactate during epinephrine infusion [Every 20 minutes up to 90 minutes]: Plasma lactate during epinephrine infusion
• Plasma free fatty acids during epinephrine infusion [Every 20 minutes up to 90 minutes]: Plasma free fatty acids during epinephrine infusion
• Plasma glycerol during epinephrine infusion [Every 20 minutes up to 90 minutes]: Plasma glycerol during epinephrine infusion
• Plasma alanine during epinephrine infusion [Every 20 minutes up to 90 minutes]: Plasma alanine during epinephrine infusion
• Plasma β-hydroxybutyrate during epinephrine infusion [Every 20 minutes up to 90 minutes]: Plasma β-hydroxybutyrate during epinephrine infusion
• Plasma insulin during epinephrine infusion [Every 20 minutes up to 90 minutes]: Plasma insulin during epinephrine infusion
• Plasma glucagon during epinephrine infusion [Every 20 minutes up to 90 minutes]: Plasma glucagon during epinephrine infusion
• Plasma epinephrine during epinephrine infusion [Every 20 minutes up to 90 minutes]: Plasma catecholamines during epinephrine infusion
• Plasma norepinephrine during epinephrine infusion [Every 20 minutes up to 90 minutes]: Plasma catecholamines during epinephrine infusion
• Plasma metabolomics during epinephrine infusion [Every 20 minutes up to 90 minutes]: Plasma metabolomics during epinephrine infusion
• Personality traits using the psychometry questionnaire Type D Scale-14 (DS-14) [30 minutes]: Personality traits using the psychometry questionnaire DS-14, score between 0-28, the higher, the more likely they have type D personality
• Personality traits using the psychometry questionnaire Toronto Alexithymia Scale (TAS-20) [30 minutes]: Personality traits using the psychometry questionnaire TAS-20, score 20-100, the higher score the more likely they are alexithymia
• Diabetes and hypoglycaemia status using psychometry questionnaire Hypoglycemia Fear Survey - Worry (HFS-W) [30 minutes]: Diabetes and hypoglycaemia status using psychometry questionnaire HFS-W, score 0-72, the higher score the higher fear for hypoglycemia
• Diabetes and hypoglycaemia status using psychometry questionnaire Hypoglycemia Attitudes and Behavior Scale (HABS) [30 minutes]: Diabetes and hypoglycaemia status using psychometry questionnaire HABS, score from 14-45, higher score more fear of hypoglycemia
• Diabetes and hypoglycaemia status using psychometry questionnaire Problem Areas in Diabetes (PAID) [30 minutes]: Diabetes and hypoglycaemia status using psychometry questionnaire PAID, 0-80, the higher score, the more problems with diabetes
• Food consumption [30 minutes]: Using Food Frequency Questionnaire to analyze food consumption
• Hypoglycemia awareness status [10 minutes]: Using hypoglycemia awareness status questionnaire , 0-7, higher score indicate hypoglycemia unawareness

Sex: All, ages: 18 Years to 70 Years, accepts healthy volunteers

Inclusion Criteria:

1. Ability to provide written informed consent
2. Male or female aged 18-70 years
3. Must be able to speak and read Danish
4. Type 1 diabetes patients or healthy individuals (control goup)
5. A documented clinically relevant history of type 1 diabetes
6. In insulin treatment regimen
7. The subject must be willing and able to comply with trial protocol

Exclusion Criteria:

1. History of severe psychological condition
2. History of severe heart disease
3. History of epilepsy, former apoplexies and dementia
4. History of muscle diseases
5. History of liver disease
6. History of malignancy unless a disease-free period exceeding 5 years
7. Implants not compatible for MRI scans
8. History of alcohol or drug abuse
9. Pregnant or lactating woman

• Results publication: She R, Suvitaival T, Andersen HU, Hommel E, Norgaard K, Wojtaszewski JFP, Legido-Quigley C, Pedersen-Bjergaard U. Metabolic effect of adrenaline infusion in people with type 1 diabetes and healthy individuals. Diabetologia. 2024 Jun;67(6):1095-1106. doi: 10.1007/s00125-024-06116-5. Epub 2024 Mar 1. PMID 38427076

• Ulrik Pedersen-Bjergaard, MD,PhD,Prof, Principal investigator, Nordsjaellands Hospital

NIH grants: 2019