In Vivo Study of Glycogen Metabolism in 3T3-L1 Adipocytes Under Perfusion by 13C-NMR Spectroscopy
W. Pawlina, M.L. Buszko1, R. A. Zaldivar and S.C. Frost
University of Florida College of Medicine, Gainesville, FL 32610, and
1
Department of Microbiology & Cell Science, Gainesville, Florida 32611, USA.
Over the past decade, United States’ population of overweight people has increased from 25 % to 33 %. In obesity, the adipose tissue mass expands to maintain rates of fatty acid oxidation commensurate on average with fat intake. The expansion of the adipose tissue is influenced not only by the diet’s fat content but also by tissue glycogen concentration that supports its ability to utilize fatty acids for triglyceride biosynthesis. The adipose pool of glycogen changes little in response to the normal daily pattern of feeding and fasting which contrasts to the glycogen pool in liver. However, after extended fast, these levels can increase 100-fold upon refeeding with significant correlation between the size of this pool and the rates of fatty acid synthesis. This led investigators some fifty years ago to suggest that glycogen serves as a "metabolic intermediate" in adipose tissue lipogenesis.
Glycogen metabolism has been studied by a number of investigators using NMR, particularly in muscle [1]. However, as far as we are aware, this is the first time that 13C-NMR has been used to analyze glycogen metabolism in adipose tissue. Adipose tissue in some aspects is similar to muscle tissue - they both express insulin-dependent glucose transporter, GLUT4, and neither tissue can release free glucose, as does the liver. Our studies show that glucose transport controls the synthesis of glycogen in adipocytes - Fig 1. We studied 3T3-L1 adipocytes, which exhibit high rate of insulin-sensitive glucose transport, using perfusion system as a model for in vivo study of glycogen degradation and synthesis.
In order to examine the regulation of glycogen metabolism, we have devised a technique to embed 3T3-L1 adipocytes in low melt agarose. This was adapted from a procedure published by Soma et al. [2]. Briefly, cells were scraped from plates and suspended in 2% ultra-low temperature gelling agarose in a 1:1 ratio. This suspension was drawn into a 5-ml syringe and injected into Teflon tubing of 0.5-mm diameter. The tubing was immersed into an ice bath for 2-3 min to solidify the mixture. The gelled agarose was then extruded as threads into culture medium. These embedded cells exhibit glucose transport activity which is both inhibited by cytochalasin B (proving specificity) and stimulated by insulin (proving sensitivity). In addition, these cells extract glucose from the medium for lactate production as measured by 13C-NMR spectrometry. Notably, placing fat cells in threads overcomes the propensity of these cells to "float" enabling us to perform single pass perfusion studies.
The perfusion system has been described by Pawlina et al. [3]. The agarose-embedded cells, pre-labeled with 13C-glucose incorporated into the glycogen pool, were perfused with either glucose- or fructose-containing medium. The isotopically labeled glycogen pool was then analyzed by 13C-NMR spectrometry. The spectra of immobilized cells were collected in a 7 Tesla magnetic field using a modified [4] NT-300 spectrometer. Typically, 1000 signal-averaged scans were recorded every hour with a 25
m s excitation pulse and a 3 s repetition delay - Fig. 2. These studies showed, first, that 13C-glucose is used for both glycogen and fatty acids biosynthes [3]. Second, the glycogen pool decreases when cells are perfused in the presence of either glucose or fructose [3]. However, the total glycogen content does not change in the presence of glucose (when measured spectrophotometrically), demonstrating that glucose is exchanging within the glycogen pool. This is in contrast with fructose perfusion, where the glycogen content decreases [5] at the same rate as that when the cells are perfused with glucose-free medium.
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