Dynamic Actions of Glucose and Glucosamine on Hexosamine Biosynthesis in Isolated Adipocytes

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 (t½ < 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 h (t½ a = GlcN flux the the of the pathway to a rapid of GlcN-6-P. rise GlcN-6-P levels was with caused by the of or the of GlcN and glucose increased levels (t½ min), levels with GlcN for for we of GlcN ED50 = levels increasing GlcN-6-P levels or the of glucose and GlcN hexosamine levels the of the and the the of 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 (t½ < 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 h (t½ a = GlcN flux the the of the pathway to a rapid of GlcN-6-P. rise GlcN-6-P levels was with caused by the of or the of GlcN and glucose increased levels (t½ min), levels with GlcN for for we of GlcN ED50 = levels increasing GlcN-6-P levels or the of glucose and GlcN hexosamine levels the of the and the the of of of insulin to a insulin To the of we a to isolated adipocytes and we to the of insulin resistance In we the of a pathway glucose-induced of the glucose glucose hexosamine biosynthesis glucose hexosamine biosynthesis a of pathway the of hexosamine we flux of glucose through the hexosamine biosynthesis pathway the of insulin resistance the hexosamine and the of the to and flux the with glucose and the rapid of glucose to glucose G-6-P then to the biosynthesis to the or to and through the the by the of a of glucose to glucosamine by the and of the GlcN-6-P a of rapid to the of the of insulin resistance by the for of and GlcN glucose GlcN a for the the of GlcN-6-P. a glucose the we the and of glucose and GlcN the of hexosamine with to elucidate the early events glucose-induced of hexosamine and to the of of and and or and of adipocytes the of by of 1 and then to a of 5 of 1 of a of cells, of adipocytes of of insulin was and the to the and the of glucose or GlcN was and adipocytes for either 4 h or for of adipocytes to and the with the the was to and of was to adipocytes for was then min 4 and the metabolite was to a the was by a of and of was measured by by the through a was and was by and levels a of to was of and G-6-P a by and of In glucose and to the to and In the G-6-P to and G-6-P was to which we of 5 of was then measured and 5 min the of G-6-P a with of and a to a by of a by of for and Levels of G-6-P measured of G-6-P of GlcN-6-P a to GlcN-6-P levels the to G-6-P we G-6-P GlcN-6-P and of to was the of G-6-P for GlcN-6-P for the of G-6-P GlcN-6-P the by the for G-6-P with a of G-6-P by the of a of G-6-P of GlcN-6-P by or and To we adipocytes for 4 h with of metabolite and then measured GlcN-6-P levels was the of GlcN-6-P with by the was and for the of a of of the adipocytes to and for 5 h the or of of with the and to of glucose by adipocytes with insulin for min of glucose measured by of of and of the of the was by by through To the for by and for of the we glucose the of Glucose was the of of Glucose GlcN adipocytes of for min the of insulin and of of GlcN measured by of of the of the was by the by through To for and for of the we GlcN the of which through the glucose of Glucose G-6-P and the of the of glucose to rapidly elevated intracellular G-6-P levels (t½ < 1 15 was and levels remained elevated for up to 4 h 4 h of treatment a glucose ED50 of 4 the rapid rise G-6-P levels was by a (t½ = 1 a and remained for up to 4 levels a treatment of we ED50 of to GlcN-6-P was glucose-treated glucose adipocytes through the and rapidly to a of glucose to the the rise through the rapid of early 5 min the of glucose rise over the h by or a of to of GlcN-6-P and cells, GlcN rapidly and the the of GlcN-6-P. To the and of metabolite we the of GlcN to hexosamine GlcN-6-P and GlcN a rapid and massive accumulation of GlcN-6-P GlcN-6-P levels increased by 5 min (∼400 nmol/g) and continued to rise over h (t½ min), a GlcN-6-P levels for 4 h with GlcN levels GlcN-6-P levels elevated GlcN = In the of a of GlcN was = of glucosamine to intracellular levels of GlcN-6-P and treated for min with insulin and then with GlcN for the and or for 4 h with the of GlcN and the of the treatment with and the was of GlcN-6-P and measured the of and the of GlcN levels a of treatment and levels over we GlcN rapidly increased the h (t½ Levels remained elevated for up to 4 4 of GlcN treatment of GlcN to increased levels by of GlcN mm) levels of the rise GlcN-6-P levels with the levels of GlcN levels GlcN-6-P we of GlcN rapidly up by to GlcN-6-P, and then through the a levels of GlcN we and intracellular of GlcN the of the which to a accumulation of of the of hexosamine we adipocytes for 1 h with GlcN and then to we intracellular GlcN-6-P levels to 1 h (t½ = In levels increased the intracellular levels of GlcN-6-P by the continued flux of GlcN-6-P through the a which to glucose and then to we G-6-P levels rapidly (t½ = 15 the of glucose GlcN-6-P and levels of treated for min with insulin and then for 1 h with then and for the of GlcN-6-P and measured the of of by of to levels To the of GlcN levels we the or of was GlcN to levels GlcN with GlcN to accumulation of GlcN-6-P we of or the with massive by the rapid levels to GlcN (t½ = to the rapid accumulation of GlcN-6-P by 5 min to with the levels GlcN by of or the of glucosamine levels and of insulin adipocytes treated for min with insulin and then for 4 h with the of treated for min with insulin and then for the with of measured the of adipocytes for 5 h with insulin and the of the of the and of glucose measured the of of the and and treatment of isolated adipocytes for 5 h with of GlcN to of the insulin resistance was and was was or levels and GlcN-6-P levels to the of with the levels with the through the by levels of and the of of to of 5 5 the of 5 glucose the intracellular levels of GlcN-6-P and the and we of GlcN to GlcN-6-P the of glucose with treatment with GlcN In was a the GlcN ED50 of GlcN to ED50 of with glucose the of GlcN-6-P the levels of GlcN GlcN-6-P levels to levels to the of glucose and GlcN of glucose and glucosamine GlcN-6-P, and adipocytes treated for min with insulin and then with GlcN and 5 glucose for the or for 4 h with 5 glucose the of GlcN the of isolated adipocytes for min with insulin and then for min with the of was then by for min and then the of GlcN the of 5 increased levels with ED50 of a the GlcN with treated the of glucose caused by of GlcN the of glucose glucose a for glucose and GlcN glucose was to the levels of GlcN-6-P by was levels In the of glucose and GlcN increased levels by with of GlcN with the and levels GlcN-6-P of GlcN to GlcN-6-P, increased a of the of the to accumulation of of GlcN-6-P and a with 5 glucose and GlcN and the of hexosamine by the of the of for accumulation of GlcN-6-P the (t½ of and of glucose (t½ of a of min was with GlcN with a of min with glucose and GlcN 5 to levels a to the of glucose to the of GlcN and the of glucose to to of the to GlcN levels GlcN we intracellular of isolated adipocytes with either glucose or GlcN insulin resistance over several glucose through and the of hexosamine the of insulin resistance we the early events desensitization, the of hexosamine was to adipocytes for min with insulin the and then to glucose or GlcN for 1 min to 4 h over 4 h of glucose or adipocytes and and to intracellular levels of GlcN-6-P, and glucose was to cells, we intracellular G-6-P levels rapidly levels early (t½ < 1 rapid through the the 15 G-6-P levels to a glucose and the rapid of G-6-P to Levels of G-6-P remained elevated for 4 h and to glucose of 4 early 5 glucose and through the Levels of continued to rise over min (t½ a was 1 to 4 the levels 5 of to the of the was the of to GlcN-6-P, through the of GlcN (2 mm) to a rapid rise levels (t½ plateaued a glucose-treated for for for to the of to the and of the levels flux the through of a of GlcN the a to of of of hexosamine of the of GlcN to levels = increasing GlcN-6-P. In GlcN the of glucose the of hexosamine levels of GlcN = we a rapid and massive accumulation of GlcN-6-P. a GlcN-6-P was by 5 min (∼400 nmol/g) and continued to rise over h (t½ a for rapid and accumulation to the of GlcN to the of the flux the rapidly the of the to a rapid of of and accumulation of GlcN-6-P to the of ATP. 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To the and of metabolite we the of GlcN to hexosamine GlcN-6-P and GlcN a rapid and massive accumulation of GlcN-6-P GlcN-6-P levels increased by 5 min (∼400 nmol/g) and continued to rise over h (t½ min), a GlcN-6-P levels for 4 h with GlcN levels GlcN-6-P levels elevated GlcN = In the of a of GlcN was = and the of GlcN levels a of treatment and levels over we GlcN rapidly increased the h (t½ Levels remained elevated for up to 4 4 of GlcN treatment of GlcN to increased levels by of GlcN mm) levels of the rise GlcN-6-P levels with the levels of GlcN levels GlcN-6-P we of GlcN rapidly up by to GlcN-6-P, and then through the a levels of GlcN we and intracellular of GlcN the of the which to a accumulation of GlcN-6-P. of the of hexosamine we adipocytes for 1 h with GlcN and then to we intracellular GlcN-6-P levels to 1 h (t½ = In levels increased the intracellular levels of GlcN-6-P by the continued flux of GlcN-6-P through the a which to glucose and then to we G-6-P levels rapidly (t½ = 15 the of glucose of by of to levels To the of GlcN levels we the or of was GlcN to levels GlcN with GlcN to accumulation of GlcN-6-P we of or the with massive by the rapid levels to GlcN (t½ = to the rapid accumulation of GlcN-6-P by 5 min to with the levels GlcN by of or the of the and and treatment of isolated adipocytes for 5 h with of GlcN to of the insulin resistance was and was was or levels and GlcN-6-P levels to the of with the levels with the through the by levels of and the of of to of 5 5 the of 5 glucose the intracellular levels of GlcN-6-P and the and we of GlcN to GlcN-6-P the of glucose with treatment with GlcN In was a the GlcN ED50 of GlcN to ED50 of with glucose the of GlcN-6-P the levels of GlcN GlcN-6-P levels to levels to the of glucose and GlcN GlcN the of 5 increased levels with ED50 of a the GlcN with treated the of glucose caused by of GlcN the of glucose glucose a for glucose and GlcN glucose was to the levels of GlcN-6-P by was levels In the of glucose and GlcN increased levels by with of GlcN with the and levels GlcN-6-P of GlcN to GlcN-6-P, increased a of the of the to accumulation of GlcN-6-P. of GlcN-6-P and a with 5 glucose and GlcN and the of hexosamine by the of the of for accumulation of GlcN-6-P the (t½ of and of glucose (t½ of a of min was with GlcN with a of min with glucose and GlcN 5 to levels a to the of glucose to the of GlcN and the of glucose to to of the to GlcN levels GlcN we intracellular of isolated adipocytes with either glucose or GlcN insulin resistance over several glucose through and the of hexosamine the of insulin resistance we the early events desensitization, the of hexosamine was to adipocytes for min with insulin the and then to glucose or GlcN for 1 min to 4 h over 4 h of glucose or adipocytes and and to intracellular levels of GlcN-6-P, and glucose was to cells, we intracellular G-6-P levels rapidly levels early (t½ < 1 rapid through the the 15 G-6-P levels to a glucose and the rapid of G-6-P to Levels of G-6-P remained elevated for 4 h and to glucose of 4 early 5 glucose and through the Levels of continued to rise over min (t½ a was 1 to 4 the levels 5 of to the of the was the of to GlcN-6-P, through the of GlcN (2 mm) to a rapid rise levels (t½ plateaued a glucose-treated for for for to the of to the and of the levels flux the through of a of GlcN the a to of of of hexosamine of the of GlcN to levels = increasing GlcN-6-P. In GlcN the of glucose the of hexosamine levels of GlcN = we a rapid and massive accumulation of GlcN-6-P. a GlcN-6-P was by 5 min (∼400 nmol/g) and continued to rise over h (t½ a for rapid and accumulation to the of GlcN to the of the flux the rapidly the of the to a rapid of of and accumulation of GlcN-6-P to the of ATP. GlcN GlcN-6-P levels to 4 was a rise GlcN-6-P by a GlcN a rapid GlcN-6-P by 5 min and a rapid (t½ by of or the of of elevated GlcN-6-P levels a levels to or of GlcN-6-P or the to GlcN hexosamine levels and insulin resistance insulin resistance min of and over Levels of increased to GlcN-6-P to levels to GlcN the of the To we the of GlcN and glucose isolated we a and a massive accumulation of GlcN-6-P to we to the of metabolite of the of we levels rapidly (t½ of min for of min for we of GlcN levels to levels GlcN-6-P to the underlying glucose-induced insulin resistance of to the to the of GlcN of glucose to the flux of GlcN the glucose several glucose through the of the glucose and GlcN to GlcN the of the massive accumulation of GlcN-6-P. rise GlcN-6-P to through of and of GlcN a of levels of glucose and insulin GlcN of of glucose and insulin glucose of of levels and of GlcN-6-P of GlcN of insulin of of GlcN to and the of which insulin resistance caused by of intracellular ATP. was a of GlcN by the of GlcN and glucose insulin resistance by adipocytes and of we GlcN by rapidly increasing flux through the elevated levels of and increased of several of the In the of GlcN and glucose glucose of GlcN cause a massive intracellular accumulation of GlcN-6-P and ATP. glucose the of GlcN treatment the by a of GlcN hexosamine and insulin resistance of the with or GlcN-6-P levels and with of ATP. the and of to the the of GlcN and the and of GlcN and a of events insulin and insulin Glucose the of which and and a of a and the the the of insulin of hexosamine a the of to the of events of glucose to the of In the we isolated adipocytes to the and early of glucose and GlcN hexosamine flux and the of GlcN-6-P and adipocytes a the underlying glucose-induced insulin resistance and through the of isolated adipocytes with either glucose or GlcN insulin resistance over several glucose through and the of hexosamine the of insulin resistance we the early events desensitization, the of hexosamine was to adipocytes for min with insulin the and then to glucose or GlcN for 1 min to 4 h over 4 h of glucose or adipocytes and and to intracellular levels of GlcN-6-P, and ATP.