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J. Biol. Chem., Vol. 279, Issue 34, 35313-35319, August 20, 2004
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From the Hexos, Inc., Woodinville, Washington 98072
Glucose and glucosamine (GlcN) cause insulin resistance over several hours by increasing metabolite flux through the hexosamine biosynthesis pathway (HBP). To elucidate the early events underlying glucose-induced desensitization, we treated isolated adipocytes with either glucose or GlcN and then measured intracellular levels of glucose-6-P (G-6-P), GlcN-6-P, UDP-Glc-NAc, and ATP. Glucose treatment rapidly increased G-6-P levels (t
< 1 min), which plateaued by 15 min and remained elevated for up to 4 h (glucose ED50 = 4mM). In glucose-treated cells, GlcN-6-P was undetectable; however, GlcN treatment (2 mM) caused a rapid and massive accumulation of GlcN-6-P. Levels increased by 5 min (
400 nmol/g) and continued to rise over 2 h (t
20 min) before reaching a plateau at >1,400 nmol/g (ED50 = 900 µM). Thus, at high GlcN concentrations, unrestricted flux into the HBP greatly exceeds the biosynthetic capacity of the pathway leading to a rapid buildup of GlcN-6-P. The GlcN-induced rise in GlcN-6-P levels was correlated with ATP depletion, suggesting that ATP loss is caused by phosphate sequestration (with the formation of GlcN-6-P) or the energy demands of phosphorylation. As expected, GlcN and glucose increased UDP-GlcNAc levels (t
1418 min), but greater levels were obtained with GlcN (45-fold for GlcN, 2-fold for glucose). Importantly, we found that low doses of GlcN (<250 µM, ED50 = 80 µM) could markedly elevate UDP-GlcNAc levels without increasing GlcN-6-P levels or depleting ATP levels. These studies on the dynamic actions of glucose and GlcN on hexosamine levels should be useful in exploring the functional role of the HBP and in avoiding the potential pitfalls in the pharmacological use of GlcN.
Received for publication, April 14, 2004 , and in revised form, June 7, 2004.
* The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Current address: Dept. of Biochemistry & Molecular Biology, Kansas University Medical Center, 3901 Rainbow Blvd., Kansas City, KS 64110.
¶ Current address: School of Medicine, University of Tsukuba, Tsukuba, Ibaraki 305, Japan.
To whom correspondence should be addressed: Hexos, Inc., 18304 NE 153rd St., Woodinville, WA 98072. Tel.: 425-844-2527; E-mail: Hexos{at}comcast.net.
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