A Randomized Double Blinded Study to Examine the Use of N-acetyl Cysteine for the Prevention and Treatment of HAAF in Patients With Type 1 Diabetes

This study will consist of a screening visit and two 2-day intervention visits separated by
approximately 8 weeks.

At the screening visit, informed consent will be obtained, a standardized form will be used
to ensure subjects meet inclusion/exclusion criteria, and baseline hemoglobin A1c will be
obtained. On arrival, two IVs will be placed and they will be randomized to receive NAC or
saline infusion. Baseline blood samples will be drawn for glucose, epinephrine,
norepinephrine, cortisol, cysteine and glutathione measurements. Subjects will then be given
25 mg IV diphenhydramine (Benadryl) followed by a 60 minute infusion of NAC (150 mg) or a
similar volume of saline between 8 am and 9 am, followed by a four hour infusion of 50 mg of
NAC. Thirty minutes after the start of the NAC infusion, a hyperinsulinemic (2.0 mu/kg/min)
hypoglycemic (target = 50 mg/dl) clamp protocol will be started. During the morning study,
blood samples will be collected every 5 minutes for monitoring of blood glucose levels and
every 15 minutes for later measurement of serum epinephrine, norepinephrine, cortisol. Plasma
and red blood cells samples will also be collected every 15 minutes for later measurement of
NAC, cysteine, glutathione, and GSH/GSSG ratios (redox status). During the final 15 minutes
of the morning clamp, subjects will be asked to quantitate their symptoms using a
standardized method (13). At the completion of the morning clamp, glucose will be given to
return the participant to euglycemia. 2 hours after the end of the morning clamp, a second
hyperinsulinemic (2.0 mu/kg/min) hypoglycemic (target = 50 mg/dl) clamp protocol will be
followed over two hours. . The afternoon clamp study will proceed as in the morning except
that no serum or plasma will be collected except for the monitoring of glucose and collection
of samples for subsequent measurement of NAC, cysteine, glutathione, and GSH/GSSG ratios.
After the completion of the afternoon clamps, subjects will be returned to euglycemia and fed
a meal. They will be discharged home.

At 7 AM the following morning they will under a single 2 hour hyperinsulinemic hypoglycemic
stepped clamp (75, 65, 55,45 mg/dl targets) during which blood samples will be collected as
on the morning of day 1. Symptom scores will be collected in the final 15 minutes of each
step in the clamp.

At the completion of the study on day 2 of Part 1, they will be scheduled to return for Part
2 in 8 weeks. This timing is selected to ensure that female participants are studied at the
same phase of the menstrual cycle. During Part 2 they will receive the treatment not provided
during Part 1 in a blinded fashion. The rest of the study protocol will be the same as in
Part 1.

Inclusion Criteria:

- Healthy controls

- Age 18 - 65 years

- Baseline hemoglobin A1C <6.0%

Exclusion Criteria:

- History of stroke, seizures (other than those related to hypoglycemia), arrhythmias,
active cardiac disease (all of which could be associated with adverse cardiac or
neurological events during hypoglycemia)

- Pregnancy or plan to become pregnant during the study period

- Diagnosis of asthma (increases risk of hypersensitivity reactions to NAC)

- Use of anti-oxidants or drugs that can alter glucose metabolism

- Concomitant medical problems that may prevent the subject from successfully completing
the protocol

- Unwillingness to avoid exercise during the 7 days before each part of the study