Evaluation of the Effect of Hypoglycemia With PET and a Norepinephrine Transporter Ligand

Hypoglycemia elicits a multifaceted hormonal response that aims to restore glycemic levels to
normal. As blood glucose levels start to fall, there is a cessation of insulin secretion. At
the top of this hierarchy of counterregulatory responses are glucagon and epinephrine, which
are the two principal circulating hormones that increase glucose production and inhibit
glucose utilization to raise plasma glucose levels back to normal. In conjunction with these
circulating hormones there is activation of the sympathetic nervous system, which acts to
stimulate hepatic glucose production and lipolysis and suppress peripheral glucose uptake. In
cases of prolonged and/or more severe hypoglycemia, growth hormone and cortisol are mobilized
to stimulate the synthesis of gluconeogenic enzymes and inhibit glucose utilization.In
non-diabetic individuals, glucagon and epinephrine are usually very effective and the latter
responses are rarely required in the acute situation. In contrast, impaired glucose
counterregulation presents itself in longstanding diabetes and with antecedent
hypoglycemia.Within the first five years after the onset of type 1 diabetes, the primary
defense against hypoglycemia, the release of glucagon, either becomes significantly
attenuated or is completely absent and this impairment appears to be specific for the
stimulus of hypoglycemia. Hence, patients with diabetes primarily depend on the release of
catecholamines as their main defense against hypoglycemia. Unfortunately, with longer
duration of diabetes and especially with poor glycemic control, epinephrine secretion and
sympathetic activation are also compromised, making these patients even more vulnerable to
the threat of hypoglycemia. In patients with diabetes, hypoglycemia arises from the interplay
of a relative excess of exogenous insulin and defective glucose counterregulation and it
remains a limiting factor in attaining proper glycemic management. Both the Diabetes Control
and Complications Trial (DCCT) conducted in type 1 patients and the United Kingdom
Prospective Diabetes Study (UKPDS) conducted in type 2 patients have established the
importance of maintaining good glucose control over a lifetime of diabetes to avoid
ophthalmologic, renal and neurological complications. However, lowering glycemic goals for
patients with diabetes increases their risk for hypoglycemia exposure. According to the DCCT,
type 1 patients put on intensive insulin therapy, though having improved outcomes for
diabetic complications, are at a 3-fold higher risk of experiencing severe hypoglycemia
compared to those on conventional insulin therapy9. Moreover, recent antecedent hypoglycemia
reduces autonomic response (catecholamines) and development of symptoms (which normally
prompts behavioral defenses such as eating) to subsequent hypoglycemia10-13. Thus begins the
vicious cycle of recurrent hypoglycemia where hypoglycemia leads to further impairment of
counterregulatory responses which in turn, begets more hypoglycemia and so forth. Because of
the imperfections of current insulin therapies, those patients attempting to achieve tight
glycemic control suffer an untold number of asymptomatic hypoglycemic episodes. Current
estimates of symptomatic hypoglycemic episodes range from 2-3 incidences per week on average
and severe, debilitating episodes occur once or twice each year. Therefore, understanding how
the body senses falling blood glucose levels and initiates counterregulatory mechanisms will
be crucial if we are to prevent or eliminate hypoglycemia. Sensors that detect changes in
blood glucose levels and initiate glucose counterregulatory responses have been identified in
the hepatic portal vein, the carotid body and most importantly in the brain. In the brain,
the predominant sensors are located in the VMH and they are crucial for detecting falling
blood glucose levels and for initiating counterregulatory responses. Although the VMH has
been implicated as the primary glucose sensor in rodents, no human data are available.
Moreover, the exact mechanism leading to VMH activation is not well understood. It was
proposed that during hypoglycemia, a rise in VMH norepinephrine (NE) levels improves the
counterregulatory response to hypoglycemia27. While these studies highlight the importance of
the local NE elevation in the VMH, no one has examined the mechanisms that regulate local NE
levels during hypoglycemia. NETs limit the action of NE through reuptake into the cytoplasm,
regulating the extent of time that NE remains in the synapse28. Studies in rats showed that
chronic elevations of intracerebral insulin can significantly decrease NET mRNA expression in
the locus coeruleus, while hypoinsulinemia resulting from streptozotocin-induced diabetes
significantly elevates NET mRNA levels. These data suggest that endogenous insulin may be one
factor that regulates the synthesis and re-uptake of NE in the CNS. This hypothesis has been
confirmed and showed that treating hippocampal tissue and cervical ganglion neurons cells
with insulin led to a decrease in NET surface expression. However, the direct effect of
insulin on NET levels in humans has never been studied.

We have developed a novel approach to measure noradrenergic function using PET scanning and a
highly selective norepinephrine transporter (NET) ligand, (S,S)-[11C]O-methylreboxetine
([11C]MRB). Measuring changes in brain NET concentration is now possible with the use of
[11C]MRB and a high resolution HRRT PET system.

Inclusion Criteria:

1. Males or females between 18 and 55 years of age

2. Who are able to give voluntary written informed consent

3. Able to tolerate PET and MR imaging

4. Have clinical laboratory test results within normal reference range for the population
or investigator site, or results with acceptable deviations that are judged to be not
clinically significant by the investigator.

5. Have no current uncontrolled medical condition such as neurological, cardiovascular,
endocrine, renal, liver, or thyroid pathology

6. Have no history of a neurological or psychiatric disorder

7. No history of previous allergic reactions to drugs

8. Do not suffer from claustrophobia or any MRI contradictions

Exclusion Criteria:

1. History of liver disease

2. Pregnancy/breast feeding (as documented by pregnancy testing at screening and on days
of the imaging studies).

3. Anemia (Hct <37 in women and < 40 in men)

4. Presence of acute or unstable medical or neurological illness. Subjects will be
excluded from the study if they present with any history of serious medical or
neurological illness or if they show signs of a major medical or neurological illness
on examination or lab testing including history of seizures, head injury, brain tumor,
heart, liver or kidney disease, eating disorder, diabetes.

5. Drug abuse (except nicotine)(Nicotine dependence will be permitted in all groups but
controlled for in the analysis).

6. Use of antidepressants.

7. Clotting disorders or recent anticoagulant therapy.

8. MRI-incompatible implants and other contraindications for MRI, such as pace-maker,
artificial joints, non-removable body piercings, tattoos larger than 1 cm in diameter,
claustrophobia, etc

9. Clinically significant pulmonary, renal, cardiac or hepatic impairment or cancer, have
clinically significant infectious disease, including AIDS or HIV infection, or
previous positive test for hepatitis B, hepatitis C, HIV-1, or HIV-2; subjects will be
asked about this. No testing will be performed.

10. Have received a diagnostic or therapeutic radiopharmaceutical within 7 days prior to
participation in this study.

11. Blood donation during the 8-week period preceding the PET scan.

12. Participation in other research studies involving ionizing radiation within one year
of the PET scans that would cause the subject to exceed the yearly dose limits for
normal volunteers.

13. Unable to fast overnight prior to the PET scan.