Non Invasive Assessment of Liver Glycogen Kinetics and ATP Synthesis in Type1 Diabetics
Non Invasive Assessment of Liver Glycogen Kinetics and ATP Synthesis in Type1 Diabetics
Patients with Type 1 diabetes (T1DM) suffer from impaired postprandial hepatic glycogen
 storage and breakdown, if they are under poor glycaemic control. Poor glycogen storage in the
 liver puts these patients at risk of fasting hypoglycaemia. Amelioration of glycaemic control
 could improve these abnormalities and thereby reduce the risk of hypoglycaemia in these
 patients. The "gold standard" technique for the assessment of hepatic glycogen metabolism in
 humans, 13 C magnetic resonance spectroscopy (13C-MRS), is expensive and limited to a few
 centers worldwide. Furthermore, treated type 1 diabetic patients exhibit skeletal muscle
 insulin resistance when treated insufficiently. This condition can also be reversed by
 improvement of glycaemic control. Recent studies link skeletal muscle insulin resistance to
 impaired mitochondrial function. Up to date, the impact of glycaemic control on skeletal
 muscle mitochondrial function has not yet been assessed.

 Aim 1 of our project is to establish a new assessment method for glycogen metabolism. This
 new method is based on oral administration of 2H2O and acetaminophen.

 Our second aim is to examine the impact of improvements of glycaemic control on skeletal
 muscle mitochondrial function in type 1 diabetic patients.

 Our third aim is to assess the ATP-synthesis in T1DM.

 We will conduct a prospective study on 14 patients with type 1 diabetes and 14 healthy
 controls.

 On the respective study day, participants will be served three standardized meals, blood
 sugar will be controlled hourly and blood samples will be drawn at timed intervals to
 determine glucoregulatory hormones, metabolites and enrichments of [6,6-2H2]glucose.

 During the night, four 13C-MRS-measurements will be performed in combination with
 [6,6-2H2]glucose infusion to assess glucose production, glycogen breakdown and
 gluconeogenesis.

 In addition, patients will drink 3g/kg bodyweight 2H2O and acetaminophen will be
 administered. Thus the new 2H2O-acetaminophen method will be applied simultaneously with the
 "gold standard" method.

 The following morning, mitochondrial function will be assessed in skeletal muscle from
 unidirectional flux through ATP synthase by 31P MRS.

 TIDM patients will be studied twice. First, under conditions of insufficient glycaemic
 control and the second time after three months of intensified insulin treatment using CSII
 pumps aiming at optimized metabolic control. Healthy controls will be studied only once.

 To assess muscular mitochondrial function in T1DM we will measure ATP synthesis in a calf
 muscle with magnetic resonance spectroscopy. First, we will conduct a basal measurement.
 Thereafter, we will start a hyperinsulinaemic euglycemic calmp to stimulate the ATP synthesis
 and measure again.

 This study will provide information on rates of post absorptive glycogen breakdown,
 gluconeogenesis, and postprandial glycogen storage in the liver and on the skeletal muscle
 mitochondrial function under conditions of optimized glycaemic control for 3 months.

 Finally, this study will demonstrate whether or not poorly controlled type 1 diabetic
 patients exhibit abnormalities in muscle mitochondrial function and to what extent those
 alterations can be reversed by optimized glycaemic control. We expect to validate the
 2H2O-acetaminophen method, which will provide justification for a broad scale in clinical
 studies.
Non-Invasive Assessment of Liver Glycogen-Kinetics in Type1 Diabetics

 Background:

 Hepatic glycogen is the principal short-term reserve for circulating glucose in humans. Up to
 50-60% of endogenous glucose production is derived from hepatic glycogenolysis during
 overnight fasting. In healthy subjects, deprivation of hepatic glycogen by prolonged fasting
 (60-65 hours) depresses fasting glucose production and plasma glucose levels approach the
 hypoglycemic range. T1DM were shown to have lower rates of hepatic glycogen synthesis during
 feeding and lower rates of glycogenolysis during fasting. Thus, this dangerous condition may
 develop during overnight fasting.

 Importantly, defective hepatic glycogen metabolism in T1D can be therapeutically restored,
 suggesting that measurements of glycogen kinetics could be useful for evaluating both new and
 existing therapies of glycemic control.

 The accepted "gold standard" for hepatic glycogenolysis measurements in humans involves a
 direct measurement of the natural abundance 13C hepatic glycogen signal using localized 13C
 NMR on a high-field clinical whole body magnetic resonance system. This method is only
 available in a handful of clinical research centers around the world.

 Our proposed measurement is highly practical and relatively inexpensive since it involves
 oral administration of a small amount of deuterated water (2H2O) tracer and a standard dose
 of Acetaminophen. This new method is based on the analysis of deuterium enrichment of urinary
 glucuronide, which is derived from the glucose moiety of hepatic UDP-glucose, the immediate
 hexose precursor pool of glycogen synthesis.

 To date, there have been no direct comparisons of the 2H2O measurement and clinical 13C MR
 methods for quantifying rates of fasting glycogenolysis in T1D subjects.

 Mitochondrial dysfunction assessed by impaired myocellular ATP synthesis, is associated with
 insulin resistance in relatives of T2DM, in patients with overt T2DM and T1DM with poor
 glycemic control. However it is yet unknown to what extend alterations in hyperglycemia
 contribute to this abnormality. Our hypothesis is that improvement of hyperglycemia in type 1
 diabetic patients who do dot suffer from genetically induced insulin resistance, will
 increase myocellular ATP synthesis. Thus, this study will examine basal myocellular ATP
 synthetic flux in patients with type 1 diabetes mellitus before and after improvement of
 glycemic control. In addition, we will perform hyperinsulinaemic euglycemic clamp tests to
 stimulate mitochondrial ATP synthesis.

 Clinical Protocols:

 Simultaneous in vivo 13C NMR and 2H2O-glucuronide measurements of hepatic glycogenolysis
 (Vienna):

 A total of 24 subjects consisting of 12 healthy controls and 12 TID patients, first, in
 insufficient metabolic control (HbA1c 8.5-10.0%) and again after 3 months of intensified
 insulin treatment using continuous subcutaneous insulin infusion (CSII pump) aiming at
 optimized metabolic control (HbA1c <7.5%) will be studied following informed consent at the
 MR Centre-of-Excellence, Medical University of Vienna.

 All measurements will either take place in the Hanusch Hospital (Heinrich Collin Straße 30,
 A-1140 Vienna) or the MR Center-of-Excellence at the General Hospital of Vienna(Lazerettgasse
 14, A-1090 Vienna).

 For a 24 hour period before the study, T1D patients will be instructed to omit NPH or
 Zn-insulin and only use regular insulin to control blood glucose concentrations.

 On day 1, staring in the Hanusch Hospital, subjects will ingest 3 standard mixed meals (60%
 CHO, 20% protein and 20% fat; 720kcal, 710kcal and 800kcal) at 08:00, 13:00 and 18.40. The
 last meal will be served after transferring to the MR-Centre-of-Excellence and the first
 MR-measurement.

 Blood sugar will be controlled hourly and blood samples will be drawn at timed intervals to
 determine glucoregulatory hormones and metabolites.

 Subjects will be transferred periodically to the magnetic resonance spectroscopy unit, where
 in vivo 13C NMR spectra lasting 1 hour will be performed at 17.30-18.30 (before dinner),
 23:30-0:30, 02:00-03:00 and 06:50-07:50. There will be performed an additional 31P NMR
 measurement to assess the intramyocellular ATP synthesis of the right leg between
 05.30-06.30.

 At 22:30, a 8-hour primed infusion of [6,6-2H2]glucose will be started. The priming dose of 5
 mg/kg will be adjusted according to fasting blood glucose levels and will be followed by a
 constant infusion of 0.05 mg/kg/min. Plasma samples will be collected twice before the
 infusion starts and then from 0:30 -0:50, 3:10 - 3:30 and 6:30-6:50 in ten minutes intervals
 respectively, to quantify enrichment of plasma [6,6-2H2]glucose.

 At 23.00, subjects will ingest 2H2O to 0.3% body water and at 03:00, they will ingest 1000 mg
 Acetaminophen (Paracetamol).

 At 6:00 the participants are instructed to void. This Urine will be collected as Urine 1.
 Between 06:00 and 08:00, Urine will be collected for recovery of Acetaminophen glucuronide
 (Urine2) at which point the study will finish. The urine will be evaporated, frozen and sent
 to Coimbra for analysis.

 After day one, intensified insulin treatment using continuous subcutaneous insulin infusion
 (CSII pump) will start. Patients will be re-measured after three month according to the same
 protocol.

 Healthy controls will be examined only once.

 ATP-synthesis will be measured on a separate study day. Patients and healthy controls will be
 admitted to the MR-Centre-of-Excellence at 6:00 a.m. First, there will be a basal measurement
 of ATP-synthesis. Thereafter, the clamp will be started and conducted for 4 hours. Then, the
 second 31P NMR measurement will be performed to assess whether ATP synthesis can be
 stimulated in T1DM patients.

 Participants will be released after a meal at 15:00
Inclusion Criteria:

 -  Type 1 Diabetes Mellitus

 -  HbA1c at the beginning of the trial between 8,5% and 10%

 -  Age: 18-50 years

 -  BMI <30 kg/m2 (due to limited MR diameter)

 -  Normal routine lab tests (blood cell count, kidney, liver, pancreas, thyroid and
 neuromuscular function)

 -  Availability within the local area throughout the study

 -  Ability to understand and sign the consent forms

 Exclusion Criteria:

 -  Current smoking

 -  Present drug treatment

 -  Contraindications for MRS studies: claustrophobia and metalliferous implants

 -  Pregnancy

 -  HIV or Hepatitis

 -  acute disease 2 weeks previous to the examination

 -  Heart disease

 -  Hypertension (RR>140/95)

 -  Liver disease

 -  Kidney disease

 -  Pulmonary disease

 -  Thyroid disease