Glucosamine-induced endoplasmic reticulum stress affects GLUT4 expression via activating transcription factor 6 in rat and human skeletal muscle cells.
OBJECTIVE
Glucosamine, generated during hyperglycaemia, causes insulin resistance in different cells. Here we sought to evaluate the possible role of endoplasmic reticulum (ER) stress in the induction of insulin resistance by glucosamine in skeletal muscle cells.


METHODS
Real-time RT-PCR analysis, 2-deoxy-D: -glucose (2-DG) uptake and western blot analysis were carried out in rat and human muscle cell lines.


RESULTS
In both rat and human myotubes, glucosamine treatment caused a significant increase in the expression of the ER stress markers immunoglobulin heavy chain-binding protein/glucose-regulated protein 78 kDa (BIP/GRP78 [also known as HSPA5]), X-box binding protein-1 (XBP1) and activating transcription factor 6 (ATF6). In addition, glucosamine impaired insulin-stimulated 2-DG uptake in both rat and human myotubes. Interestingly, pretreatment of both rat and human myotubes with the chemical chaperones 4-phenylbutyric acid (PBA) or tauroursodeoxycholic acid (TUDCA), completely prevented the effect of glucosamine on both ER stress induction and insulin-induced glucose uptake. In both rat and human myotubes, glucosamine treatment reduced mRNA and protein levels of the gene encoding GLUT4 and mRNA levels of the main regulators of the gene encoding GLUT4 (myocyte enhancer factor 2 a [MEF2A] and peroxisome proliferator-activated receptor-gamma coactivator 1alpha [PGC1alpha]). Again, PBA or TUDCA pretreatment prevented glucosamine-induced inhibition of GLUT4 (also known as SLC2A4), MEF2A and PGC1alpha (also known as PPARGC1A). Finally, we showed that overproduction of ATF6 is sufficient to inhibit the expression of genes GLUT4, MEF2A and PGC1alpha and that ATF6 silencing with a specific small interfering RNA is sufficient to completely prevent glucosamine-induced inhibition of GLUT4, MEF2A and PGC1alpha in skeletal muscle cells.


CONCLUSIONS
In this work we show that glucosamine-induced ER stress causes insulin resistance in both human and rat myotubes and impairs GLUT4 production and insulin-induced glucose uptake via an ATF6-dependent decrease of the GLUT4 regulators MEF2A and PGC1alpha.
Raciti
G A
GA
Dipartimento di Biologia e Patologia Cellulare e Molecolare, Università di Napoli Federico II, Via Sergio Pansini, 5, Naples 80131, Italy.
Iadicicco
C
C
Ulianich
L
L
Vind
B F
BF
Gaster
M
M
Andreozzi
F
F
Longo
M
M
Teperino
R
R
Ungaro
P
P
Di Jeso
B
B
Formisano
P
P
Beguinot
F
F
Miele
C
C
D051702:Activating Transcription Factor 6 / Q000235:genetics / Q000378:metabolism*; D000704:Analysis of Variance; D000818:Animals; D015153:Blotting, Western; D002460:Cell Line; D002478:Cells, Cultured; D047369:Chromatin Immunoprecipitation; D004305:Dose-Response Relationship, Drug; D004721:Endoplasmic Reticulum / Q000187:drug effects / Q000378:metabolism*; D000091342:Endoplasmic Reticulum Chaperone BiP; D005944:Glucosamine / Q000378:metabolism* / Q000494:pharmacology; D005947:Glucose / Q000378:metabolism / Q000494:pharmacology; D051275:Glucose Transporter Type 4 / Q000235:genetics / Q000378:metabolism*; D006360:Heat-Shock Proteins / Q000235:genetics / Q000378:metabolism; D006801:Humans; D007328:Insulin / Q000378:metabolism / Q000494:pharmacology; D007333:Insulin Resistance / Q000502:physiology; D026161:MADS Domain Proteins / Q000235:genetics / Q000378:metabolism; D064326:MEF2 Transcription Factors; D008875:Middle Aged; D018832:Molecular Chaperones / Q000235:genetics / Q000378:metabolism; D018485:Muscle Fibers, Skeletal / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D018006:Myogenic Regulatory Factors / Q000235:genetics / Q000378:metabolism; D000071248:Peroxisome Proliferator-Activated Receptor Gamma Coactivator 1-alpha; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D034741:RNA, Small Interfering / Q000235:genetics / Q000378:metabolism; D016601:RNA-Binding Proteins / Q000235:genetics / Q000378:metabolism; D051381:Rats; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014157:Transcription Factors / Q000235:genetics / Q000378:metabolism
D051702:Activating Transcription Factor 6; D000091342:Endoplasmic Reticulum Chaperone BiP; D051275:Glucose Transporter Type 4; C000717247:HSPA5 protein, human; D006360:Heat-Shock Proteins; D007328:Insulin; D026161:MADS Domain Proteins; D064326:MEF2 Transcription Factors; C578037:MEF2A protein, rat; D018832:Molecular Chaperones; D018006:Myogenic Regulatory Factors; D000071248:Peroxisome Proliferator-Activated Receptor Gamma Coactivator 1-alpha; C501429:Ppargc1a protein, rat; D012333:RNA, Messenger; D034741:RNA, Small Interfering; D016601:RNA-Binding Proteins; D014157:Transcription Factors; D005947:Glucose; D005944:Glucosamine