13C MRS Studies of the Control of Hepatic Glycogen Metabolism at High Magnetic Fields

Abstract

Introduction: Glycogen is the primary intracellular storage form of carbohydrates. In contrast to most tissues where stored glycogen can only supply the local tissue with energy, hepatic glycogen is mobilized and released into the blood to maintain appropriate circulating glucose levels, and is delivered to other tissues as glucose in response to energetic demands. Insulin and glucagon, two current targets of high interest in the pharmaceutical industry, are well-known glucose-regulating hormones whose primary effect in liver is to modulate glycogen synthesis and breakdown. The purpose of these studies was to develop methods to measure glycogen metabolism in real time non-invasively both in isolated mouse livers, and in non-human primates (NHPs) using 13 C MRS.Methods: Livers were harvested from C57/Bl6 mice and perfused with [1-13 C] Glucose. To demonstrate the ability to measure acute changes in glycogen metabolism ex-vivo, fructose, glucagon, and insulin were administered to the liver ex-vivo. The C1 resonance of glycogen was measured in real time with 13 C MRS using an 11.7T (500 MHz) NMR spectrometer. To demonstrate the translatability of this approach, NHPs (male rhesus monkeys) were studied in a 7 T Philips MRI using a partial volume 1 H/ 13 C imaging coil. NPHs were subjected to a variable IV infusion of [1-13 C] glucose (to maintain blood glucose at 3-4x basal), along with a constant 1 mg/kg/min infusion of fructose. The C1 resonance of glycogen was again measured in real time with 13 C MRS. To demonstrate the ability to measure changes in glycogen metabolism in vivo, animals received a glucagon infusion (1 µg/kg bolus followed by 40 ng/kg/min constant infusion) half way through the study on the second study session.Results: In both perfused mouse livers and in NHPs, hepatic 13 C-glycogen synthesis (i.e., monotonic increases in the 13 C-glycogen NMR signal) was readily detected. In both paradigms, addition of glucagon resulted in cessation of glycogen synthesis and induction of glycogen breakdown. In the perfused liver, inclusion of insulin was able to dose-dependently block the effect of glucagon.Miller et al. C MRS of Hepatic GlycogenConclusion: Hepatic glycogen synthesis, as well as acute hormonally-induced changes thereof, can be measured using 13 C MRS at high magnetic fields both ex-vivo and in vivo. Measurements of this process represent novel, translatable biomarkers of glucagon action, and additionally may be useful for pharmacological targets which modulate glycogen metabolism.

Probing Hepatic Glucose Metabolism via 13C NMR Spectroscopy in Perfused Livers—Applications to Drug Development

“…*All other perfusion lines are Tygon tubing of inner diameter 1/32" to 1/16". Reprinted from reference [11].…”

“…As can easily be seen, the rate of glycogen synthesis (i.e., the slope of the line in Figure 4B) increases with increasing [1- 13 C] glucose concentration, thus recapitulating the known glucose dependence of hepatic glycogen synthesis in vivo. Reprinted from reference [11].…”

NMR visibility of deuterium‐labeled liver glycogen in vivo

“…2,3 Despite the large molecular size, the structure of glycogen provides significant mobility for the individual glucose units to allow the detection of glycogen in vivo using 13 C NMR, both at natural abundance or following administration of 13 glucose. [4][5][6][7][8] Similarly, 1 H MRS has been described to detect glycogen in rat liver in vivo using an nOe-based editing technique to subtract overlapping lipid peaks. 9 Both 13 C and 1 H MRS focus on peaks of glycogen (C1, H1, respectively), that have a different chemical shift than the equivalent atom position in glucose.…”

“…Glycogen is a large branched polymer of 110‐290‐nm diameter, harboring up to 50,000 glucose molecules, and reaching levels of up to 400 g in human liver 2,3 . Despite the large molecular size, the structure of glycogen provides significant mobility for the individual glucose units to allow the detection of glycogen in vivo using 13 C NMR, both at natural abundance or following administration of 13 C‐labeled glucose 4‐8 . Similarly, 1 H MRS has been described to detect glycogen in rat liver in vivo using an nOe‐based editing technique to subtract overlapping lipid peaks 9 …”

“…glucose. [4][5][6][7][8] Similarly, 1 H MRS has been described to detect glycogen in rat liver in vivo using an nOe-based editing technique to subtract overlapping lipid peaks. 9 Both 13 C and 1 H MRS focus on peaks of glycogen (C1, H1, respectively), that have a different chemical shift than the equivalent atom position in glucose.…”