Diabetes Publish Ahead of Print, published online November 18, 2008 Small Decrements in Systemic Glucose Provoke Increases in Hypothalamic Blood Flow Prior to the Release of Counterregulatory Hormones Kathleen A. Page1, Jagriti Arora2, Maolin Qiu2, Rachna Relwani1, R. Todd Constable2 , and Robert S. Sherwin1 Departments of Internal Medicine, Division of Endocrinology 1 and Diagnostic Radiology2, Yale University School of Medicine, New Haven, CT Address for correspondence: Kathleen A. Page, MD Department of Internal Medicine, Division of Endocrinology Yale University School of Medicine New Haven, CT 06520 [email protected] Submitted 3 September 2008 and accepted 11 November 2008. This is an uncopyedited electronic version of an article accepted for publication in Diabetes. The American Diabetes Association, publisher of Diabetes, is not responsible for any errors or omissions in this version of the manuscript or any version derived from it by third parties. The definitive publisher-authenticated version will be available in a future issue of Diabetes in print and online at http://diabetes.diabetesjournals.org. Copyright American Diabetes Association, Inc., 2008 Hypothalamic Activation ABSTRACT Objective: The hypothalamus is the central brain region responsible for sensing and integrating responses to changes in circulating glucose. The aim of this study was to determine the time sequence relationship between hypothalamic activation and the initiation of the counterregulatory hormonal response to small decrements in systemic glucose. Research Design and Methods: Nine non-diabetic volunteers underwent two hyperinsulinemic clamp sessions in which pulsed arterial spin labeling (PASL) was used to measure regional cerebral blood flow (CBF) at euglycemia (~95mg/dl) on one occasion and as glucose levels were declining to a nadir of ~50mg/dl on another occasion. Plasma glucose and counterregulatory hormones were measured during both study sessions. Results: CBF to the hypothalamus significantly increased when glucose levels decreased to 77.2 + 2 mg/dl when compared to the euglycemic control session when glucose levels were 95.7 + 3 mg/dl (p=0.0009). Hypothalamic perfusion was significantly increased before there was a significant elevation in counterregulatory hormones. Conclusions: Our data suggest that the hypothalamus is exquisitely sensitive to small decrements in systemic glucose levels in healthy, non-diabetic subjects and that hypothalamic blood flow, and presumably neuronal activity, precedes the rise in counterregulatory hormones seen during hypoglycemia. Abbreviations: ACC = anterior cingulate cortex BA = Broadman’s Area PG = plasma glucose PASL = pulsed arterial spin labeling CBF = cerebral blood flow fMRI = functional magnetic resonance imaging IFG = inferior frontal gyrus ROI = region of interest STG = superior temporal gyrus 2 Hypothalamic Activation he brain relies on glucose as its reach specific tissues thereby providing a main energy substrate and small direct measure of CBF (18, 19, 20). T decrements in circulating glucose Increased CBF has long been associated provoke an elaborate counterregulatory with neuronal activation dating back to hormonal feedback response (1, 2). the first PET studies of brain function Activation of the counterregulatory (21). response requires effective detection of a Some studies, using positron emission falling glucose level. While multiple tomography (22), single photon emission glucose sensors may be involved computed tomography (23), high field MR (3,4,5,6,7) the hypothalamus has perfusion (24), and continuous arterial emerged as the dominant brain region spin labeling (25) have shown region responsible for sensing and integrating specific increases in brain CBF in responses to changes in circulating response to hypoglycemia. However, glucose levels (8,9,10,11,12). While most none of these studies specifically prior studies have relied on animal demonstrated changes in blood flow to models to study the neurophysiological the hypothalamus during hypoglycemia. response to changes in glucose, newer Moreover, in these studies CBF imaging techniques such as functional measurements were performed after magnetic resonance imaging (fMRI) and hypoglycemic levels had been achieved; positron emission tomography (PET) thus none of these studies determined provide an in vivo method to study the the CBF response to smaller decrements effect of changes in peripheral glucose in systemic glucose or the relationship levels on human brain activity. Several between regional brain activation and the fMRI studies in humans have initiation of the counterregulatory demonstrated that a rise in systemic hormonal response. glucose following glucose ingestion leads We employed PASL to determine the to an inhibition of hypothalamic activity effect of small decrements in circulating (13, 14, 15, 16). In addition, Musen et al glucose on hypothalamic blood flow in (17) recently used fMRI based on the healthy volunteers. We performed CBF Blood Oxygenation Level Dependent measurements as glucose levels were (BOLD) contrast mechanism and found declining. This approach allowed us to that insulin-induced hypoglycemia leads address the following questions: 1) Does to hypothalamic activation. However, the a decline in plasma glucose provoke fMRI-BOLD approach used in that study hypothalamic activation? 2) If so, what is assesses only relative changes in the plasma glucose level that correlates oxygenated hemoglobin in specific brain with hypothalamic activation? 3) How regions and does not directly measure does hypothalamic activation temporally tissue perfusion. MRI pulsed arterial spin relate to the counterregulatory hormone labeling (PASL) provides a method for response to hypoglycemia? measuring absolute blood flow responses An understanding of how the throughout the brain to changes in hypothalamus, a key brain glucose circulating glucose levels. PASL sensing region, responds to decrements magnetically tags the arterial blood prior in circulating glucose levels in healthy to entering the brain and then examines humans provides critical information that the transit time for the tagged blood to can be used to determine how metabolic 3 Hypothalamic Activation disorders, such as diabetes, may alter minutes during the euglycemic session. this response. PG levels were measured at 5 minute intervals and additional plasma samples Research Methods and Design were drawn at -20, 0, 30, 60, 120, and Subjects. Nine (8 male, 1 female) 135 minutes for measurement of insulin, healthy, non-diabetic subjects glucagon, catecholamines, cortisol, and participated in this study. Subject growth hormone. C-peptide was participants had a mean age (+SD) of measured at -20, 0, 30, and 60 minute 28+5 and a mean BMI of 23.6+2. time points. The two study sessions were Subjects underwent a screening history, carried out in a single-blind fashion, in physical examination, and laboratory variable order across subjects. testing and only individuals with no fMRI acquisition methods. Imaging history of significant disease, including Protocol. MR Imaging was performed on diabetes, were included in the study. a 3T Siemens Trio whole-body scanner Exclusion criteria also included any (Siemens Medical Systems, Erlangen, contraindications for MRI including Germany) with a circularly polarized head pregnancy and metal implants. Before coil. Pulsed arterial spin labeling (PASL) each study session, female subjects were utilizing the EPISTAR QUIPSS PASL MRI required to have a negative urine technique was used to measure CBF. pregnancy test. The Yale University The PASL acquisition parameters were: School of Medicine Human Investigation field of view = 256 × 256 mm2; matrix = Committee approved this study and all 60 × 64; bandwidth = 2298 Hz/pixel; slice subjects provided informed, written thickness = 6 mm; inter-slice spacing = 3 consent before participation in the study. mm. Ten AC-PC aligned slices were General Experimental Protocol. acquired from inferior to superior in an Subjects participated in two study ascending order.The whole imaging slab sessions which were separated by a was positioned on the upper part of the minimum of 7 days. On the morning of brain, with the lowest slice passing the study, an IV catheter was inserted through AC-PC to acquire the top part of into a distal arm or hand vein; this arm the brain; the imaging slab positioned on was gently heated, allowing for sampling the lower part of the brain with the 7th of arterialized venous blood. A second IV slice from the bottom passing through catheter was established for the AC-PC to acquire the bottom part of the administration of insulin and glucose. brain. Acquisition of each slice took During the 135 minute study sessions, a approximately 54 ms. The repetition time primed continuous infusion of IV insulin at was TR = 3000 ms; the echo time was 2mu/kg/min was initiated, with a variable TE = 26 ms. During each EPI acquisition, infusion of 20% glucose adjusted to fat was suppressed and the phase- achieve euglycemia (plasma glucose correction echoes were collected and (PG) ~95mg/dl) on one occasion and applied. A bipolar gradient of encoding hypoglycemia (PG ~ 50mg/dl) on the velocity Venc = 20 mm/s was applied to the other occasion. Regional CBF imaging slices for intra-vascular signal measurements were performed using suppression. PASL at approximately 30 minutes after To quantify regional CBF for both the start of the plasma glucose decline upper and lower parts of the brain,