Consequences of Hypoglycaemia on Cardiovascular and Inflammatory Responses

Study Overview
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Brief Summary
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People with Type 1 diabetes (T1DM), type 2 diabetes (T2DM) and healthy volunteers will undergo a hypoglycaemic clamp to to investigate the effect of hypoglycaemia on cardiovascular and inflammatory responses.

Detailed Description
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Objectives: The overall aim of the present study is to investigate the effect of hypoglycaemia on cardiovascular and inflammatory responses, molecular mechanisms and epigenetic profiles in various groups of people with diabetes type 1, type 2 and healthy volunteers. Study design: Intervention study Intervention: All subjects will undergo a hyperinsulinemic normoglycaemic-hypoglycaemic glucose clamp (nadir 2.8 mmol/L), during and after which blood and urine will be sampled for further examination for up to one week.

Official Title
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Consequences of Hypoglycaemia on Cardiovascular and Inflammatory Responses in Patients With Diabetes Mellitus Type 1, Type 2 and Healthy Volunteers

Conditions
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Hypoglycemia, Inflammatory Response, Diabetes Mellitus, Type 1, Diabetes Mellitus, Type 2

Intervention / Treatment
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* Procedure: hyperinsulinemic normoglycaemic-hypoglycaemic glucose clamp


Participation Criteria
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Eligibility Criteria
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Inclusion Criteria: Overall inclusion criteria Ability to provide written informed consent Must be able to speak and read Danish (for Hillerød-site) and Dutch (for Nijmegen-site) Insulin treatment according to basal-bolus insulin regimen (injections or insulin pump) (except for group 5) Body-Mass Index: 19-40 kg/m2 Age ≥18 years, ≤ 80 years Blood pressure: <140/90 mmHg Duration of diabetes > 1 year (except for group 5) HbA1c < 100 mmol/mol Group specific Group 1: HbA1c >64 mmol/mol Group 2: impaired awareness of hypoglycaemia (IAH) as assessed by a score of ≥3 on the modified Clarke questionnaire, ≥4 on the Gold questionnaire and a positive score on the Pedersen-Bjergaard questionnaire. Group 3: normal awareness of hypoglycaemia (NAH) as assessed by a score of <3 on the modified Clarke questionnaire, <4 on the Gold questionnaire and a negative score on the Pedersen-Bjergaard. Group 4: Insulin treatment for at least 1 year Group 5/6: HbA1c <42 mmol/mol Exclusion Criteria: - Severe medical or psychological conditions interfering with the perception of hypoglycaemia other than IAH such as brain injuries, epilepsy, a major cardiovascular disease event or anxiety disorders Use of immune-modifying drugs or antibiotics Treatment with glucose-modifying (other than insulin, SGLT-2 inhibitors and metformin) agents (e.g. prednisolon) Use of anti-depressive drugs Pregnancy or breastfeeding or unwillingness to undertake measures for birth control Use of statins (e.g. stop statins >2 weeks before performing blood sampling. This can be safely done in the context of primary prevention) Any event of cardiovascular disease in the past 5 years (e.g. myocardial infarction, stroke, heart failure, symptomatic peripheral arterial disease) Auto-inflammatory or auto-immune diseases Any infection in past three months Previous vaccination in the past three months Laser coagulation for proliferative retinopathy in the past six months Proliferative retinopathy Diabetic nephropathy as reflected by an albumin-creatinine ratio ˃ 30 mg/gor an estimated glomerular filtration rate (by MDRD) ˂60ml/min/1.73m2 History of pancreatitis (acute or chronic) or pancreatic cancer

Ages Eligible for Study
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Minimum Age: 18 Years
Maximum Age: 80 Years

Sexes Eligible for Study
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All

Accepts Healthy Volunteers
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Accepts Healthy Volunteers

Study Plan
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How is the study designed?
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Design Details

Primary Purpose: Diagnostic
Allocation: Non-Randomized
Intervention Model: Parallel Assignment
Masking: None (Open Label)


Arms and Interventions

| Participant Group/Arm | Intervention/Treatment |
| --- | --- |
| Active Comparator: T1DM poor glycaemic control<br>patients with type 1 diabetes and poor glycaemic control (HbA1c >8% / >64 mmol/mol will undergo hyperinsulinemic normoglycaemic-hypoglycaemic glucose clamp | Procedure: hyperinsulinemic normoglycaemic-hypoglycaemic glucose clamp<br>* For this study, a hyperinsulinemic normoglycaemic-hypoglycaemic glucose clamp will be conducted to investigate the effect of hypoglycaemia. This means that subjects will receive an intravenous insulin infusion, at a continuous rate of 60 mU∙m-2∙min-1, as well as glucose 20% w/w intravenously at a variable rate, adjusted by arterial plasma glucose levels, measured at 5 minute intervals.<br>* Other names: hypoglycaemic clamp;|
| Active Comparator: T1DM impaired awareness<br>patients with type 1 diabetes and impaired awareness of hypoglycaemia will undergo hyperinsulinemic normoglycaemic-hypoglycaemic glucose clamp | Procedure: hyperinsulinemic normoglycaemic-hypoglycaemic glucose clamp<br>* For this study, a hyperinsulinemic normoglycaemic-hypoglycaemic glucose clamp will be conducted to investigate the effect of hypoglycaemia. This means that subjects will receive an intravenous insulin infusion, at a continuous rate of 60 mU∙m-2∙min-1, as well as glucose 20% w/w intravenously at a variable rate, adjusted by arterial plasma glucose levels, measured at 5 minute intervals.<br>* Other names: hypoglycaemic clamp;|
| Active Comparator: T1DM Normal awareness<br>patients with type 1 diabetes and normal awareness of hypoglycaemia will undergo hyperinsulinemic normoglycaemic-hypoglycaemic glucose clamp | Procedure: hyperinsulinemic normoglycaemic-hypoglycaemic glucose clamp<br>* For this study, a hyperinsulinemic normoglycaemic-hypoglycaemic glucose clamp will be conducted to investigate the effect of hypoglycaemia. This means that subjects will receive an intravenous insulin infusion, at a continuous rate of 60 mU∙m-2∙min-1, as well as glucose 20% w/w intravenously at a variable rate, adjusted by arterial plasma glucose levels, measured at 5 minute intervals.<br>* Other names: hypoglycaemic clamp;|
| Active Comparator: T2DM + Insulin<br>patients with type 2 diabetes with insulin treatment for at least 1 year will undergo hyperinsulinemic normoglycaemic-hypoglycaemic glucose clamp | Procedure: hyperinsulinemic normoglycaemic-hypoglycaemic glucose clamp<br>* For this study, a hyperinsulinemic normoglycaemic-hypoglycaemic glucose clamp will be conducted to investigate the effect of hypoglycaemia. This means that subjects will receive an intravenous insulin infusion, at a continuous rate of 60 mU∙m-2∙min-1, as well as glucose 20% w/w intravenously at a variable rate, adjusted by arterial plasma glucose levels, measured at 5 minute intervals.<br>* Other names: hypoglycaemic clamp;|
| Active Comparator: Healthy control T2DM<br>healthy controls without diabetes and age, gender and BMI matched with diabetes type 2 participants will undergo hyperinsulinemic normoglycaemic-hypoglycaemic glucose clamp | Procedure: hyperinsulinemic normoglycaemic-hypoglycaemic glucose clamp<br>* For this study, a hyperinsulinemic normoglycaemic-hypoglycaemic glucose clamp will be conducted to investigate the effect of hypoglycaemia. This means that subjects will receive an intravenous insulin infusion, at a continuous rate of 60 mU∙m-2∙min-1, as well as glucose 20% w/w intravenously at a variable rate, adjusted by arterial plasma glucose levels, measured at 5 minute intervals.<br>* Other names: hypoglycaemic clamp;|
| Active Comparator: Healthy control T1DM<br>Healthy controls without diabetes and age, gender and BMI matched with diabetes type 1 participants will undergo hyperinsulinemic normoglycaemic-hypoglycaemic glucose clamp | Procedure: hyperinsulinemic normoglycaemic-hypoglycaemic glucose clamp<br>* For this study, a hyperinsulinemic normoglycaemic-hypoglycaemic glucose clamp will be conducted to investigate the effect of hypoglycaemia. This means that subjects will receive an intravenous insulin infusion, at a continuous rate of 60 mU∙m-2∙min-1, as well as glucose 20% w/w intravenously at a variable rate, adjusted by arterial plasma glucose levels, measured at 5 minute intervals.<br>* Other names: hypoglycaemic clamp;|

What is the study measuring?
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Primary Outcome Measures

| Outcome Measure | Measure Description | Time Frame |
| --- | --- | --- |
| Inflammatory responses of hypoglycaemia by measuring the cytokine production of isolated monocytes using ELISA | Cytokine production (TNF-alfa, IL-6, IL-10 and IL-1β) of isolated and stimulated monocytes | 1.5 year | 

Secondary Outcome Measures

| Outcome Measure | Measure Description | Time Frame |
| --- | --- | --- |
| Atherogenic responses of (recurrent) hypoglycaemia using foam cell formation. | Measurement of Ox-LDL uptake by measuring intracellular apolipoproteine B | 1.5 year | 
| Metabolomics profile of each group | Un-targeted metabolomics and identification of metabolites based on exact mass using metabolomics library | 1.5 year | 
| Epigenetic modifications | Epigenetic modifications due to hypoglycaemia in the promoter regions of the pro-inflammatory cytokines in monocytes | 1.5 year | 
| Cardiac function responses to hypoglycaemia using echocardiography | Cardiac function responses to hypoglycaemia using echocardiography | 1.5 year | 
| Cognitive function responses to hypoglycaemia using cognitive function tests (TAP, PASAT) | Amount of correct answers | 1.5 year | 
| Oxidative stress responses using oxidative stress marker | Excretion of guanine nucleosides in urine (ng/mL) | 1.5 year |