Research Design and Methods
We enrolled eight healthy nondiabetic volunteers and nine patients with T1DM. Data from healthy volunteers have partly been reported before but were reanalyzed for this study. Patients with T1DM were excluded if they had a history of repeated severe hypoglycemia, a severe hypoglycemic incident in the past 6 months, or evidence of hypoglycemia unawareness on the Clarke's questionnaire. Patients with signs of autonomic neuropathy, peripheral neuropathy, proliferative retinopathy, or micro- or macroalbuminuria by review of medical records or physical examination were also excluded from participating. The study protocol was approved by the institutional review board of the Radboud University Nijmegen Medical Centre, and all volunteers gave written informed consent. For subjects participating in both euglycemic and hypoglycemic study protocols, experiments were scheduled in random order and at least 2 weeks apart. In females, a 4- or 8-week interval was chosen to avoid influences from the menstrual cycle. All nondiabetic volunteers and three T1DM patients had data available from both experiments. For six patients, data were only available from either the hypoglycemic clamp (n = 2) or the euglycemic clamp (n = 4).
Hyperinsulinemic Glucose Clamps
Hyperinsulinemic (60 mU/min/m), euglycemic (5.0 mmol/L), or hypoglycemic (3.0 mmol/L) glucose clamps were conducted, as described previously. Briefly, the brachial artery was cannulated for blood sampling, and a contralateral antecubital vein was cannulated for administration of insulin and glucose 20% to maintain plasma glucose at the predetermined level for at least 50 min. Exogenous glucose was given in the form of [1-C]glucose 20% weight for weight at variable enrichments as described earlier to increase plasma C enrichment to stable levels during both euglycemic and hypoglycemic experiments. Arterial blood was sampled every 5 min for immediate determination of plasma glucose levels and for later determination of C isotopic enrichment of glucose by nuclear magnetic resonance (H-NMR).
Magnetic Resonance Spectroscopy
All data were acquired on a 3T MR system (Magnetom Trio, Siemens, Erlangen, Germany). A C coil was placed in a birdcage H coil, and an ISIS-DEPT sequence was used for localization and polarization transfer to increase the signal-to-noise ratio of C signals. A voxel of ~125 mL was placed in occipital brain tissue. Data were acquired dynamically with a time resolution of 2.5 min, starting at least 10 min after the glycemic target was reached.
C MRS Data Processing and Quantification
C MR spectra acquired during the final 50 min of the clamps, during which plasma glucose values were stable, were averaged. In the resulting spectra, peaks of glucose and myo-inositol (mI) were fitted with the advanced magnetic resonance algorithm in jMRUI. The natural abundance signal of mI was used to quantify the C-labeled glucose concentration, based on the premise that mI has a stable concentration of 6 μmol/g. We assumed that mI was not labeled by exogenous [1-C]glucose in the time frame of the experiment.C MR spectra measured from a phantom were used to eliminate effects of the pulse sequence profile on the experimental spectra. Absolute quantification of the total glucose concentration in the brain was achieved by correcting the C glucose concentration with the C enrichment of plasma glucose as measured by H-NMR. Data were also corrected for the presence of blood vessels in the voxel, assuming that the voxel contained 5% vessel volume.
MM kinetic parameters were derived from the data using reversible MM kinetics, as described by Gruetter et al., assuming a linear relationship between plasma glucose (Glcpl) and brain glucose (Glcbr). In this model, Kt denotes the MM constant for substrate concentration at half maximal transport, Tmax the maximum transport rate, CMRglc the consumption rate of glucose, and Vd the physical distribution of glucose (0.77 mL/g).
The data in this study were fitted by linear regression analysis. From this linear relationship, Tmax/CMRglc and Kt were calculated. The kinetic parameters were determined using a bootstrapping method implemented in Matlab (Mathworks, Natick, MA). From the original dataset, the same amount of data points is selected randomly, and this is repeated 10,000 times.
All data are expressed as means ± SD, unless mentioned otherwise. Differences in means were tested by two-tailed Student t tests; a P value <0.05 was considered statistically significant. Statistical analyses were performed with GraphPad Prism 4 (GraphPad, La Jolla, CA) and SPSS 16.0 (SPSS Inc., Chicago, IL).