This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Developments in in vivo functional MRI and MR spectroscopy provide important new avenues to evaluate the metabolic dynamics of functional activation. This application proposes to assess the pathophysiology of functional activation in euglycemia and hypoglycemia in humans, and to examine the effects of ketones. Importantly, although this application suggests an emphasis towards a possible 'therapeutic' approach towards the cognitive dysfunction often seen in hypoglycemia in T1DM patients, we believe this work may also provide insight to how the brain uses fuels (glucose, ketones) with activation, a topic that continues to be in debate. We pursue this issue especially given the RFA NS 02-008 ('This RFA solicits applications for studies designed to elucidate the effects of hypoglycemia on glial and neuronal cells...define the effect of varying glycemic levels on cerebral metabolism [and] pathological consequences of hypoglycemic insult') because we believe that ketones are preferentially oxidized by neurons compared to glia17,18 and the problem of cerebral hypoglycemia relates in part to how the brain is able (or not) to draw on alternate fuels in activation. This proposal examines the dynamics of perfusion, BOLD activation and lactate generation during euglycemia and hypoglycemia, and then when ketones are available. We will do this using functional MRI, MR spectroscopy and models of cerebral physiology to test how the brain responds when provided the option of alternate fuels during hypoglycemia. Considerable debate remains as to the metabolic needs of functional activity. While in resting human brain oxygen and glucose use are believed to be coupled, functional activation provokes an increase in glucose use (measured by fluorodeoxyglucose, FDG) that is disproportionately high relative to changes in oxygen use.3,19 However, it is possible that the methods used to measure cerebral fuel use may be contributing. Collins et al20 compared [14C]deoxyglucose (DG) and [6-14C]glucose use by quantitative autoradiography of stimulated rat brain, finding that the DG method showed a ~80% rise in uptake while the glucose method showed a ~30% increase. This difference, also seen by other workers,21 has been explained as activation-induced differences in glucose use (glycolysis, anaplerosis) vs. glucose oxidation. However, with neurophysiologic data showing extensive astrocytic networks (e.g., microinjection of biocytin into an astrocyte demonstrates rapid spread into 50-100+ other cells, 8,9), another explanation is raised. While activation-induced spread of [6-14C]glucose is possible, phosphorylated-[14C]DG may not be able to redistribute. Therefore functionally provoked glucose use, which may normally distribute through such a network, would-in a DG study-appear to be spatially restricted. From this view, glucose use may in fact correlate with oxygen consumption, distinct from the more prevalent hypothesis (i.e., brain activation is mostly glycolytic). The pertinence of this for hypoglycemia is emphasized, since the view of 'coupled' function would suggest that alternate fuels could readily support functional activity. Important supporting data for the Fox and Raichle view is the observation of a ~50-250% increase in tissue lactate with functional stimulation,22-24 interpreted to result from anaerobic glycolysis fueling activation. However, such an increase in lactate primarily means a transient mismatch between production and clearance, and may be seen either in obligate glycolysis, increased anaplerosis or oxidation. If the fate of glucose is oxidation, then a role for ketones in hypoglycemia becomes a much stronger possibility. Experimentally, the availability of ketones in hypoglycemia may potentially strongly influence the dynamics of activity-induced lactate and regions of activation-which can be studied using MR spectroscopy and functional MRI (fMRI). Thus in aim 1, we will test the hypothesis that the dynamics of perfusion, BOLD fMRI and lactate are significantly influenced by hypoglycemia. In normal subjects, we will examine three conditions of altered substrate availability: euglycemia (EG, baseline), mild insulin-induced hypoglycemia (IHG, reaching a plasma level of 60mg/dl or 3.45mM) and fasting-induced hypoglycemia (FHG). We anticipate that compared to the EG state, the extent of perfusion increase will be less in the IHG state, and the least in the FHG state, reflecting the insufficiency of glucose in the IHG state. Similarly, we anticipate that BOLD activation and lactate generation will be less in the IHG states. In contrast, we anticipate that the fMRI detected areas of activation will be similar in the baseline (non-fasted) and FHG state while it is less under IHG conditions, reflecting the availability of total oxidative fuel (includes glucose and ketones). Importantly, if the FHG condition results in equal or greater lactate than the EG and IHG condition, this suggests that glycolysis is necessary for functional activation. The paradigm of elementary activation will be a visual alternating radial checkerboard.
In aim 2, we will more stringently test the hypothesis of ketone contributions towards activation in control and patient subjects. We will do this by initially acquiring hypoglycemic data, followed by infusions of ketones with activation. In both control and type 1 diabetic patients, we will test the hypothesis that in hypoglycemia, ketone infusions can result in changes in MR-detected (perfusion, BOLD and lactate) measures consistent with improved neural activity. Patient subjects will be studied in the EG, acquiring measurements of cerebral blood flow and lactate under baseline and functional activation. IHG will be induced followed by repeat MR measurements with/without functional activity. We anticipate that the dynamics of flow and lactate will be different in the IHG state than under EG, similar to that in aim 1. In a second study evaluating both patients and control subjects, IHG will be induced initially, and MR measurements acquired with/without functional activity. We will then infuse ketones (IHK), and acquire MR data with/without functional activity. We anticipate that the IHK studies will display a return to EG levels of BOLD activity and perfusion, while displaying a lower activation induced lactate change. We anticipate also that the diabetic patients may demonstrate greater sensitivity to the ketone infusion, reflecting a greater chronic reliance on alternate fuels for brain function.
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