A Study to Identify Biomarkers of Hypoglycaemia in Patients With Type 2 Diabetes
A Study to Identify Biomarkers of Hypoglycaemia in Patients With Type 2 Diabetes
Glucose is a sugar carried in the blood stream that body uses for energy. If someone has
 diabetes, blood glucose level can be erratic, sometimes becoming very low this is called
 Hypoglycaemia (or a "hypo"), and can happen when blood glucose levels drop below 4 mmol/l.

 So far in order to prove that a hypo happened for a patient, blood glucose level can only be
 measured at time of the hypo and not after it.

 In this study we are trying to identify certain chemical substances (biomarkers) in diabetic
 patients that may be measured in blood tests of the patient up to after 24 hours of the hypo
 and if we could prove that a hypo has happened we could adjust tablets and or insulin dosage
 in a way to prevent further hypos.

 The study will be conducted in the Diabetes Centre in Hull Royal Infirmary and will involve
 three visits to the diabetes centre. The study can finish in a week time after the first
 visit.

 Visit 1 is the screening visit to identify eligibility to take part in the study. Visit 2
 insulin infusion will be given to make participants blood sugar level fall lower than normal
 for a short time and corrected quickly afterward. This is a stress for participant's body and
 should stimulate certain chemicals that we are trying to identify during hypo.

 In Visit 3, the main purpose of this visit which is done 24 hours after insulin infusion is
 to take a blood sample and check how participants is after visit 2.

 We will recruit 25 Type 2 Diabetic patients and 25 none diabetics to compare both results.

 Both groups should not have ischemic heart disease, underactive thyroid or seizures and on
 stable dosage of medications.
Background information It is well recognised that patients with both type 1 and type 2
 diabetes suffering repeated hypoglycaemia are at risk of death and significant morbidity (1).
 This is of major clinical importance as currently, only the recording of a low blood sugar at
 the time of hypoglycaemia gives confirmation that a hypoglycaemic event has taken place.
 There are many instances when the confirmation or exclusion of a hypoglycaemic event would be
 beneficial.

 Landmark studies such as the Diabetes Control and Complications Trial (DCCT) )(2) in type 1
 diabetes and the in numerous large studies (UKPDS, ACCORD, ADVANCE, VADT(1, 3-7)) designed to
 look at whether intensive diabetes control in type 2 diabetes reduced microvascular and
 macrovascular cardiovascular events, have helped in establishing glycaemic targets which will
 contribute to preventing the development or worsening of retinopathy, nephropathy and
 neuropathy. What these studies exemplified was that as glycaemic control became stricter, the
 risk of hypoglycaemia increased (defined as a blood glucose fall to less than 3.9mmol/l
 according to American Diabetes Association guidelines)(1, 6, 7). For many patients, the main
 barrier to achieving optimal glycaemic targets to prevent complications are their increased
 risks of hypoglycaemia that remains the most feared complication of intensifying diabetes
 therapy for both patients and their health care professional.

 Although hypoglycaemia has traditionally been considered a complication of the treatment for
 type 1 diabetes, it has recently been recognised as a problem in people with type 2 diabetes
 particularly those on intensive insulin therapy (8). In type 1 diabetes, patients on average
 experience two symptomatic events per week and one severe event, the latter of which is
 responsible for 2-4% of deaths. In type 2 diabetes the risk of a severe hypoglycaemic event
 is about 7% in the first few years following diagnosis rising to 25% thereafter. There are
 multiple concerns on the impact that a hypoglycaemic event may have leading to pathological,
 social and economic consequences. In the ACCORD study, the risk of death was significantly
 increased in those with one or more episode of severe hypoglycaemia in both the intensive and
 standard study treatment arms (1, 9). As plasma glucose falls to below 4.0mmol/L, a series of
 defensive response mechanisms occur, at individualised glycaemic thresholds, to reverse
 hypoglycaemia including a rise in catecholamine, cortisol and glucagon levels (10). This may
 lead to hypokalemia, prolonged QT interval, and cardiac arrhythmias (11). It may also lead to
 increased oxidative stress (12); impaired cardiovascular autonomic function for up to 16
 hours afterwards (13); increased inflammatory markers (14); platelet activation (15) and
 promote vascular damage. These changes might be clinically relevant as hypoglycaemia was
 associated with cardiac ischemia (chest pain) when 72-h continuous glucose monitoring along
 with simultaneous cardiac Holter monitoring were performed in people with ischemic heart
 disease and type 2 diabetes treated with insulin (16). A few studies, in people with type 1
 diabetes and healthy controls, suggested an increase in platelet activation with
 hypoglycaemia (15, 17) emphasising the increased risk that hypoglycaemia may have on
 cardiovascular events.

 All of these changes may therefore result in the generation of biomarkers that may be able to
 identify the occurrence of a hypoglycaemia event after the blood glucose has reversed to
 normal or rebound hyperglycaemic levels have resulted. This will be of particular value in
 hypoglycaemic unawareness, nocturnal hypoglycaemia and events of uncertain aetiology where
 hypoglycaemia is part of the differential diagnosis.

 An ability to determine whether a hypoglycaemic event has occurred through measurement of a
 biomarker will be of major impact for the confidence of patients and their healthcare
 providers allowing glycaemic control to be optimised and glycaemic targets to be reached
 through more stringent diabetes therapeutic treatment regimens.

 The ability to identify nocturnal hypoglycaemia will be of particular value in all patients
 and will circumvent the need for continuous glucose monitoring or enforced waking at 3am to
 do blood glucose measurements that we currently have to rely on. Identification of biomarkers
 for hypoglycaemia will be of great value in the elderly population where it is often
 difficult to determine causes of, for example, feeling non specifically unwell, poor
 concentration, falls or faints, particularly as there are often multiple morbidities that are
 being treated at the same time (18).

 From an economic perspective it is recognised that hypoglycaemia directly accounts for 95% of
 endocrine related emergency hospitalisations (19)and it is recognised that severe
 hypoglycaemia is associated with a total cost of around £2,152 (20) It can then be seen that
 a new method to determine hypoglycaemia through biomarker determination may help prevent
 severe hypoglycaemia by earlier recognition and prevent death and morbidity.

 One promising biomarker is that of endothelial Microparticles which are heterogeneous
 population of vesicles playing a relevant role in the pathogenesis of vascular diseases,
 cancer and metabolic diseases such as diabetes mellitus.

 They are released by virtually all cell types by shedding during cell growth, proliferation,
 activation, apoptosis or senescence processes.(21) Microparticles are consistently present in
 blood stream (22) they are the main regulators of cell to cell interactions, by carrying
 specific membrane antigens from their host cells, thereby acting as diffusible vectors in the
 trancellular exchange of biological information(23).

 Microparticles expose membrane proteins of their mother cell for example CD31 is for
 endothelial cells and a proportion of platelets.(24) CD105,CD106, CD142, CD54, CD62E all
 Endothelial cells in nature.(25-28) An increased level of circulating Microparticles has been
 suggested to be one of the procoagulant determinants in patients with type 2 diabetes
 mellitus (29)Circulating levels of CD31+ (PECAM-1), CD105+, CD106+ and 62E+
 endothelial-derived MPs as well as increased concentrations of fibrinogen+, tissue factor,
 P-selectin+ platelet-derived MPs have been shown to be significantly increased in T2DM
 patients as compared with non-diabetic controls (25, 26)Indeed, platelets of patients with
 T2DM are characterised by an hyperreactive phenotype with enhanced adhesion, aggregation, and
 activation(30), that may contribute to the pathogenesis of atherothrombotic
 complications(31-33). Therefore, high levels of specific endothelial and platelets
 Microparticles circulate in the blood of patients with atherosclerotic diseases, and can be
 used as biomarkers of vascular injury and potential predictors of cardiovascular outcome,
 especially in diabetic patients(34-37).

 Aims and Objectives

 Objectives :

 Identification and comparison of Microparticles (CD105, CD106, CD31, CD54, CD62E, CD142)
 following a hypoglycemic event in patients with type 2 diabetes and in subjects without type
 2 diabetes.

 Study Design Prospective parallel study performed in the Diabetes research centre, Hull Royal
 Infirmary involving patients with type 2 diabetes and a control group without diabetes
Inclusion Criteria for diabetic patients.

 1. Men and women who are white British or white Europeans between 40 - 70 years of age
 with type 2 diabetes.

 2. Duration of diabetes less than 10 years.

 3. Stable dose of medication over the last 3 months.

 4. No history of severe hypoglycemic in the preceding 3 months.

 5. No hypoglycemic unawareness

 6. Able to give informed consent.

 Exclusion Criteria for diabetic patients.

 1. Type 1 diabetes.

 2. Duration of diabetes of more than 10 years.

 3. Patients who are on insulin.

 4. HbA1c of ≥ 10%.

 5. BMI <18 or >50 kg/m2

 6. Alcohol excess defined as 14 units per week for a man or a woman. Binge drinking is
 defined 6 units for a man or women in a single drinking session.

 7. Renal impairment with estimated Glomerular Filtration Rate (eGFR<60 ml/min/1.73 m2).

 8. Impaired Liver function tests defined as ALT or AST more than or equal 2.5 times upper
 limit of the normal.

 9. History or presence of malignant neoplasms within the last 5 years( except basal and
 squamous cell skin cancer and in-situ carcinoma).

 10. Psychiatric illness.

 11. Any form of gastrointestinal tract surgery.

 12. Any medications that can mask Hypoglycaemia such as B-Blockers or Clonidine.

 13. Medication that may cause change in glucose metabolism.

 14. Patients on any form of steroids in the last six months.

 15. Female who is pregnant, breast feeding or intended to become pregnant or of child
 bearing potential not using adequate contraceptive methods.

 16. History of pancreatitis (Acute or Chronic).

 17. History of severe hypoglycaemia or hypoglycaemic unawareness.

 18. Any disorder which in the opinion of the investigator might jeopardize subject's
 safety.

 19. Contraindications to insulin infusion aiming for hypoglycaemia:

 -  Ischaemic heart disease.

 -  Epilepsy or previous history of seizures.

 -  Drop attacks.

 -  History of adrenal insufficiency.

 -  Untreated hypothyroidism.

 Inclusion Criteria for the control group.

 1. Men and women who are white British or white Europeans between 40 - 70 years of age.

 2. Stable dose of medications in last 3 months.

 3. Able to give informed consent.

 Exclusion Criteria for the control group

 1. History of diabetes

 2. BMI <18 or >50 kg/m2

 3. HbA1C>6% (42mmol/mol)

 4. Alcohol excess defined as 14 units per week for a man or a women. Binge drinking is
 defined 6 units for a man or women in a single drinking session.

 5. Renal impairment with estimated Glomerular Filtration Rate (eGFR<60 ml/min/1.73 m2).

 6. Impaired Liver function tests defined as ALT or AST more than or equal 2.5 times upper
 limit of the normal.

 7. History or presence of malignant neoplasms within the last 5 years( except basal and
 squamous cell skin cancer and in-situ carcinoma).

 8. Psychiatric illness

 9. Female who is pregnant, breast feeding or intended to become pregnant or of child
 bearing potential not using adequate contraceptive methods.

 10. Medication that may cause change in glucose metabolism

 11. Any form of gastrointestinal tract surgery.

 12. Patient on any form of steroids in the last 6 months.

 13. Any disorder which in the opinion of the investigator might jeopardize subject's
 safety.

 14. Contraindications to insulin infusion aiming for hypoglycaemia:

 -  Ischaemic heart disease.

 -  Epilepsy or previous history of seizures.

 -  Drop attacks.

 -  History of adrenal insufficiency.

 -  Untreated hypothyroidism.