Oral Gavage Delivery of Stable Isotope Tracer for In Vivo Metabolomics

Abstract

Stable isotope-resolved metabolomics (SIRM) is a powerful tool for understanding disease. Advances in SIRM techniques have improved isotopic delivery and expanded the workflow from exclusively in vitro applications to in vivo methodologies to study systemic metabolism. Here, we report a simple, minimally-invasive and cost-effective method of tracer delivery to study SIRM in vivo in laboratory mice. Following a brief fasting period, we orally administered a solution of [U-13C] glucose through a blunt gavage needle without anesthesia, at a physiological dose commonly used for glucose tolerance tests (2 g/kg bodyweight). We defined isotopic enrichment in plasma and tissue at 15, 30, 120, and 240 min post-gavage. 13C-labeled glucose peaked in plasma around 15 min post-gavage, followed by period of metabolic decay and clearance until 4 h. We demonstrate robust enrichment of a variety of central carbon metabolites in the plasma, brain and liver of C57/BL6 mice, including amino acids, neurotransmitters, and glycolytic and tricarboxylic acid (TCA) cycle intermediates. We then applied this method to study in vivo metabolism in two distinct mouse models of diseases known to involve dysregulation of glucose metabolism: Alzheimer’s disease and type II diabetes. By delivering [U-13C] glucose via oral gavage to the 5XFAD Alzheimer’s disease model and the Lepob/ob type II diabetes model, we were able to resolve significant differences in multiple central carbon pathways in both model systems, thus providing evidence of the utility of this method to study diseases with metabolic components. Together, these data clearly demonstrate the efficacy and efficiency of an oral gavage delivery method, and present a clear time course for 13C enrichment in plasma, liver and brain of mice following oral gavage of [U-13C] glucose—data we hope will aid other researchers in their own 13C-glucose metabolomics study design.

In Situ Microwave Fixation to Define the Terminal Rodent Brain Metabolome

“…Stable isotope tracing is a powerful technique to study metabolic flux and enzyme activity in vivo and is frequently used to study brain metabolism 21,57 . We performed an isotopic tracing experiment using an in vivo oral delivery method developed previously 44 to test the effects of in situ fixation by focused microwave on stable isotope enrichment of central carbon metabolites. In line with pooled metabolomics analysis, we observed evidence of increased glucose and glycogen utilization demonstrated by decreased M6 isotopologue enrichment of glucose and glycogen, and increased M3 isotopologue enrichment of 3-PG during cryopreservation.…”

“…1A). Brain and other tissues from both cohorts of animals were pulverized while in liquid nitrogen by a cryomill and prepped for pooled metabolomics analysis by gas chromatography mass spectrometry (GCMS) for both polar metabolites and biomass [43][44][45] . For this study, we focused on the neocortex (CTX), hippocampus (HIPP), and the hindbrain dorsal vagal complex (DVC) which contains glucose sensing neurons that undergo functional neuroplasticity after prolonged hyperglycemia 46 .…”

Stable Isotope-Resolved Metabolomics Reveals the Abnormal Brain Glucose Catabolism in Depression Based on Chronic Unpredictable Mild Stress Rats

“…Moreover, the customized diet undoubtedly increases the cost of the experiment. 37,38 The jugular vein injection method has the strengths of long-term infusion and depth of labeling, but it has high technical requirements and causes injury to the organism. 39,40 The tail vein injection method has the superiority of long-term infusion, depth of pathway coverage, and constant isotopic enrichment, but it restricts animal activities as the intravenous infusion in clinical practice.…”

“…The oral administration method has the advantages of being noninvasive and less stressful, but ensuring the consistency of introduction time and food intake is difficult. Moreover, the customized diet undoubtedly increases the cost of the experiment. , The jugular vein injection method has the strengths of long-term infusion and depth of labeling, but it has high technical requirements and causes injury to the organism. , The tail vein injection method has the superiority of long-term infusion, depth of pathway coverage, and constant isotopic enrichment, but it restricts animal activities as the intravenous infusion in clinical practice. Above all, no administration method is ideal, and each method has its advantages and disadvantages.…”

Physiological impact of in vivo stable isotope tracing on cancer metabolism

“…Furthermore, it minimizes the metabolic response to stress induced by animal handling. Although this tracer delivery method is emerging as a promising strategy to perform in vivo stable isotope tracing, it is not often used because of limitations such as the strict control of the animal feeding behavior, long tracing periods, and a substantial economic outlay [ 20 , 21 ]. To date, the most commonly used procedures to deliver labeled nutrients are constant intravenous infusions and single or multiple discrete boluses (i.v, i. p, or gavage) (please refer to Table 1 ).…”