Cyclooxygenase-2 (PTGS2) inhibitors augment the rate of hexose transport in L6 myotubes in an insulin- and AMPKα-independent manner
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- Alpert, E., Gruzman, A., Lardi-Studler, B. et al. Diabetologia (2006) 49: 562. doi:10.1007/s00125-005-0122-2
Some cyclooxygenase-2 (COX2, also known as prostaglandin-endoperoxide synthase 2 [PTGS2]) inhibitors have been shown to increase insulin sensitivity in man or induce hypoglycaemic episodes when overconsumed or taken in combination with oral hypoglycaemic drugs. These side-effects and their impact on patients are not always recognised in routine clinical practice. We investigated whether these side-effects of COX2 (PTGS2) inhibitors result from stimulation of the glucose transport system in skeletal muscle cells.
Materials and methods
L6 myotube cultures were used to study effects of COX2 (PTGS2) inhibitors on the glucose transport system and their relationship to PTGS2 expression, insulin action and AMP-activated protein kinase α (AMPKα) activity.
The inhibitors niflumic acid, nimesulide and rofecoxib increased the rate of hexose uptake in L6 myotubes in the absence of insulin and in a dose- and time-dependent manner. They did this by increasing the total cell content of member 4 of the solute carrier family 2 (SCLC2A4, previously known as glucose transporter 4 [GLUT4]) (but not SCLC2A1 [previously known as GLUT1]) mRNA and protein and the amount of it in the plasma membrane. AMPKα was not involved in the latter effect since the inhibitors did not activate it. In addition, none of the inhibitors modulated the rate of hexose transport in vascular endothelial and smooth muscle cells expressing PTGS2 and SCLC2A1. Prostaglandin-endoperoxide synthase 1 (also known as cyclooxygenase 1) inhibitors (acetylsalicylic acid and indomethacin) did not alter the rate of hexose uptake and SCLC2A4 subcellular distribution in L6 myotubes.
This study suggests that certain COX2 (PTGS2) inhibitors can alter glucose homeostasis in vivo by stimulating glucose uptake in skeletal muscles that express PTGS2.
KeywordsCyclooxygenase inhibitors Diabetes Drug interaction Glucose transport Hypoglycaemia Niflumic acid Nimesulide Rofecoxib Skeletal muscle
AMP-activated protein kinase α
IκB kinase β
solute carrier family 2 (facilitated glucose transporter) member 1
solute carrier family 2 (facilitated glucose transporter) member 4
α-minimal essential medium
vascular endothelial cells
vascular smooth muscle cells
Inhibitors of prostaglandin endoperoxide synthase 2 (PTGS2, also known as cyclooxygenase 2 [COX2]) are potent anti-inflammatory drugs with reduced gastrointestinal side-effects. However, some of these drugs have serious adverse effects [1–4]. Among the less acknowledged side-effects are those on glucose homeostasis. For instance, inhibition of PTGS2 has been shown to improve insulin sensitivity in healthy subjects , whereas some case reports show that such inhibitors induce hypoglycaemic episodes when consumed in excess or in a combined therapy with oral hypoglycaemic drugs [6–10]. Other reports have shown that inhibition of PTGS2 prevented lipopolysaccharide (LPS)-induced impairment of glucose metabolism and insulin action in rat cardiac muscle . Interestingly, a polymorphic form of the gene encoding PTGS2 has been associated with susceptibility to type 2 diabetes in Pima Indians . Moreover, hyperglycaemia in itself induces PTGS2 expression in certain cells and tissues [13–15]. Yet it is not clear whether such PTGS2 induction occurs in insulin-sensitive tissues, such as skeletal muscles.
This study investigated potential effects of three COX2 (PTGS2) inhibitors, niflumic acid, nimesulide and rofecoxib, on the glucose transport system in cultured L6 myotubes. The general mechanism of PTGS2 inhibition by its selective inhibitors is complex: they interact readily and reversibly with the enzyme and induce semi-stable enzyme-inhibitor complexes. Once the drug dissociates from the complex, the recovery of the enzyme to its fully active form is slow . In contrast, these compounds inhibit the constitutively expressed prostaglandin endoperoxide synthase 1 (PTGS1, also known as COX1) in a classical competitive manner, albeit with a significantly lower affinity than that required for PTGS2 inhibition . Niflumic acid is a prototype COX2 (PTGS2) inhibitor that served for the development of other COX2 (PTGS2) inhibitors. It is considered a preferential inhibitor of PTGS2, but there are reports claiming equal selectivity for both PTGS1 and PTGS2 . Nimesulide was one of the early generation of prescribed COX2 (PTGS2) inhibitors, but it was withdrawn from the market in some countries, or its application was restricted, because of severe renal and hepatotoxic complications [1, 18–21]. Several case reports showed that overconsumption of nimesulide or its combination with oral hypoglycaemic drugs was associated with hypoglycaemic episodes in infants and elderly individuals [6, 7, 9]. Other trials did not document similar interactions between nimesulide and oral hypoglycaemic drugs [6, 22, 23]. Rofecoxib, one of the new generation of COX2 (PTGS2) inhibitors, was, at the start of this study, one of the biggest selling COX2 (PTGS2) inhibitors of its kind. It has recently been withdrawn from markets worldwide for reassessment of its safety because of reported incidents of increased rates of cardiovascular complications [3, 24].
Materials and methods
Tissue culture media and sera were from Biological Industries (Beth–Haemek, Israel). [α-32P]dCTP (111 TBq/mmol) was from Amersham Pharmacia Biotech (Little Chalfont, Buckinghamshire, UK). American Radiolabeled Chemicals (St Louis, MO, USA) supplied 2-[1,2-3H(N)]deoxy-d-glucose (2.22 TBq/mmol). Acetylsalicylic acid, 5-aminoimidazole-4-carboxamide-1-β-ribofuranosyl 5’-monophosphate (AICAR), 2-deoxy-d-glucose (dGlc), indomethacin, LPS (E. coli serotype 055.B), niflumic acid, nimesulide and Tri Reagent were purchased from Sigma-Aldrich Chemicals (St Louis, MO, USA). Rofecoxib and Compound 1229 were kindly donated by Merck Research Laboratories (Rahway, NJ, USA) and Aventis Pharma (Frankfurt, Germany), respectively. Upstate Biotechnology (Lake Placid, NY, USA) supplied rabbit anti-COX2 (PTGS2) polyclonal antibody. Rabbit anti-AMPKα and anti-pT172-AMPKα antibodies were from Cell Signaling Technology (Beverly, MA, USA). Enzymes, buffers and reagents for RT-PCR were purchased from Promega (Madison, WI, USA).
Male Goto–Kakizaki (GK) hyperglycaemic rats (180–200 g), from a colony originally established at Karolinska Institute (Stockholm, Sweden), were used in accordance with the guidelines of the Hebrew University of Jerusalem and our experiments were approved by the university ethics committee. The animals were kept in the Animal Facility Unit (23°C, two rats per cage, subjected to a 12-h dark/12-h light cycle) and provided with free access to standard chow and water.
L6 skeletal myocytes were grown and allowed to differentiate to multinuclear myotubes (85–90% efficiency) in α-minimal essential medium (αMEM) supplemented with 2% (vol/vol) FCS, as described . RAW 264.7 murine macrophages (courtesy of R. Gallily, Department of Immunology, Faculty of Medicine, The Hebrew University, Jerusalem, Israel) were grown and maintained as described . Primary cultures of bovine aortic endothelial and smooth muscle cells were prepared, grown and characterised as described previously .
Hexose uptake assay
The rate of [3H]dGlc uptake in myotubes was measured as described at 37°C [28, 29]. The effect of insulin (100 nmol/l, 20 min) was measured following a 2-h incubation period in serum-free αMEM supplemented with 0.5% (wt/vol) BSA. The inhibitors were diluted in culture medium 1:1000 from DMSO stock solutions. Uptake of dGlc was calculated on the basis of protein content per plate (L6 myotubes) or cell number (all other cells).
PCR analyses of Sclc2a1 (Glut1), Sclc2a4 (Glut4) and Ptgs2 mRNA
Total RNA was extracted from cell cultures using Tri Reagent according to the manufacturer’s protocol. Sense and antisense primers for RT-PCR reactions of rat Sclc2a1 and Sclc2a4 and bovine Ptgs2 were synthesised according to published sequences [30–32] by Sigma-Aldrich (Rehovot, Israel). Synthesis of cDNA and PCR was carried out in a PTC-100 programmable thermal controller (MJ Research, Waltman, MA, USA) as described [29–32]. The nucleotide sequence of each PCR product was identical to the predicted sequences. Sequence analyses were performed at the DNA Analysis Unit of the Hebrew University, Jerusalem.
Preparation of cell lysates and western blot analyses
Soluble protein fractions were prepared from Triton X-100-treated cells in the presence of protease and phosphatase inhibitors, as described . Protein samples (10–60 μg) were electrophoresed (6–12% SDS-PAGE). Western blot analyses of SCLC2A1 and SCLC2A4 were performed as described , and those for PTGS2, AMPKα and pT172-AMPKα were performed according to the antibody supplier’s protocol.
Cell surface biotinylation and measurement of cell surface SCLC2A1 (GLUT1) and SCLC2A4 (GLUT4)
Data are given as mean±SEM; Student’s two-tailed t test was used for group comparisons. A p value of <0.05 was taken to indicate statistical significance.
Regulation of PTGS2 (COX2) expression in L6 myotubes and in skeletal muscles
Niflumic acid, nimesulide and rofecoxib augmented hexose uptake in PTGS2-expressing L6 myotubes
Potential effects of COX1 (PTGS1) inhibitors on the hexose transport system in L6 myotubes were also investigated. The non-competitive inhibitor acetylsalicylic acid (0.1–5.0 mmol/l; 2–24 h) and the competitive inhibitor indomethacin (0.1–1.0 mmol/l; 2–24 h) had no noticeable effect on the rate of dGlc uptake in L6 myotubes maintained at 5.5 or 23.0 mmol/l glucose (data not shown). In addition, Compound 1229, an inhibitor of IκB kinase β (IKKβ) , was used (0.2–1.0 μmol/l, 16 h) to determine whether IKKβ was involved in COX2 (PTGS2) inhibitor-dependent effects in a manner similar to that reported for salicylates in adipocytes . This inhibitor had no apparent effect on basal or COX2 (PTGS2) inhibitor-stimulated dGlc uptake in L6 myotubes (data not shown).
COX2 (PTGS2) inhibitors increased total SCLC2A4 (GLUT4), but not SCLC2A1 (GLUT1), content and its plasma membrane localisation in L6 myotubes
COX2 (PTGS2) inhibitors did not activate AMPKα in L6 myotubes
COX2 (PTGS2) inhibitors failed to alter the rate of hexose transport in PTGS2–expressing vascular cells
This study shows that COX2 (PTGS2) inhibitors augment the rate of hexose transport in L6 myotubes in a dose- and time-dependent manner, and that this effect is more prominent in myotubes exposed to high glucose levels, in which PTGS2 expression is increased, than in myotubes exposed to 5.5 mmol/l glucose. The major effect of the inhibitors is the recruitment of SCLC2A4 (GLUT4), but not SCLC2A1 (GLUT1), to the plasma membrane. A very low level of SCLC2A3 (GLUT3) expression in the L6 cell line used in this study precluded reliable determinations of the total and plasma membrane abundance of this transporter. The failure of COX2 (PTGS2) inhibitors to alter SCLC2A1 abundance in the plasma membrane of L6 myocytes or to modulate the rate of hexose transport in PTGS2-expressing vascular endothelial and smooth muscle cells indicates that these inhibitors affect predominantly SCLC2A4-mediated glucose transport. The observation that COX2 (PTGS2) inhibitors themselves increased the level of PTGS2 expression in skeletal muscle cells is interesting. It is unclear whether such induction of PTGS2 occurs in vivo.
The present data show that the level of COX2 (PTGS2) inhibitor-dependent stimulation of hexose uptake (approximately three-fold; Fig. 2) was higher than the corresponding relative recruitment of SCLC2A4 to the plasma membrane (approximately two-fold; Fig. 3a). Thus, it remains to be investigated whether COX2 (PTGS2) inhibitors increase the intrinsic activity of GLUT4 in the plasma membrane of myotubes, in addition to their augmenting effect on GLUT4 abundance in this compartment.
The molecular interactions of COX2 (PTGS2) inhibitor-induced augmentation of hexose uptake are not fully understood. The present results show that the effects of insulin on the glucose transport system and SCLC2A4 subcellular distribution on the one hand and of the inhibitors on the other were not additive. In fact, insulin further stimulated the rate of hexose uptake as well as SCLC2A4 translocation to the plasma membrane in myotubes that were already exposed to maximal effective concentrations of COX2 (PTGS2) inhibitors (Figs. 2c and 3a). Therefore, it is unlikely that the inhibitors utilise the insulin transduction mechanism to exert their effects. Indeed, preliminary experiments indicated that COX2 (PTGS2) inhibitors failed to mimic insulin and induce targeted tyrosine and serine phosphorylations in IRS-1 and protein kinase B/Akt (data not shown). Moreover, the alternative AMPKα-dependent pathway, which stimulates hexose transport in skeletal muscles by recruiting SCLC2A4 to the plasma membrane upon reduction in cellular energy charge and following muscle contraction [38–40], was not activated by any of the COX2 (PTGS2) inhibitors tested. More investigations, based upon preliminary observations in our laboratory linking the effects of COX2 (PTGS2) inhibitors to the activation of protein kinase Cδ , may ascertain the molecular mechanism by which COX-2 (PTGS2) inhibitors stimulate glucose transport in SCLC2A4 expressing cells and tissues.
It is not known whether COX2 (PTGS2) inhibitor-dependent stimulation of glucose transport results from direct inhibition of PTGS2. The findings on the stronger effects of the inhibitors in L6 myotubes expressing high levels of PTGS2 under hyperglycaemic conditions seem to support this notion. The specific PTGS2 product involved in this function is still unknown. Long and Pekala have suggested that SCLC2A4 expression in 3T3-L1 adipocytes is suppressed by prostaglandin (PG) E2α , while Chiou and Fong showed a PGF2α-dependent increased expression of SCLC2A1 in these cells . In the present study, PGE2α, PGF2α and PGA2α failed to modulate the rate of glucose transport when added alone or together with COX2 (PTGS2) inhibitors to L6 myotubes (data not shown). It is possible that these molecules were rapidly oxidised during the relatively long incubation period, failing to reach minimal effective concentrations in cells.
Acetylsalicylic acid has been shown to reverse insulin resistance in 3T3-L1 adipocytes by inhibiting IKKβ . In the present study both acetylsalicylic acid and indomethacin failed to modulate glucose transport in myotubes. Furthermore, in order to exclude an IKKβ-dependent interaction in myotubes we tested Compound 1229, an inhibitor of IKKβ, alone or in combination with COX2 (PTGS2) inhibitors, and found no evidence for such interactions.
The present findings seem highly relevant to case reports on COX2 (PTGS2) inhibitor-induced hypoglycaemic episodes in man. We suggest that vulnerable diabetic patients are those who receive, in addition to conventional oral hypoglycaemic drugs, COX2 (PTGS2) inhibitors to relieve symptoms of chronic or acute inflammatory processes. While some individuals from this group could benefit from COX2 (PTGS2) inhibitor therapy through having their blood glucose level reduced towards the normoglycaemic range, other susceptible patients might experience an excessive reduction in blood glucose level towards the hypoglycaemic range. We suspect that the individuals at risk are those in whom PTGS2 expression is increased excessively in skeletal muscles in the presence of inflammatory mediators. Our findings on LPS-induced expression of PTGS2 in skeletal muscles of diabetic GK rats support this hypothesis (Fig. 1b).
In conclusion, this study shows that nimesulide, niflumic acid and rofecoxib increase the expression of SCLC2A4 and its plasma membrane localisation as well as the rate of hexose uptake in L6 myotubes in a dose-, time- and non-insulin-dependent manner. It is not yet established whether such stimulatory effects are shared with all other available COX2 (PTGS2) inhibitors. However, such possible interactions of these inhibitors in susceptible individuals make it necessary for physicians to be aware of this problem and to carefully monitor patients in order to optimise drug treatment and avoid adverse interactions.
Preliminary results from this study were presented at the EASD congress in Munich, Germany, in September 2004 . S. Sasson and R. Reich are members of the David R. Bloom Center for Pharmacy, The Hebrew University of Jerusalem. We thank N. Tennagels (Aventis Pharma) for his useful comments and suggestions and R. Blejter for her technical assistance. E. Alpert and A. Gruzman received fellowships from the Hebrew University Center for Diabetes Research. This work was supported by grants from the Chief Scientist of the Israel Ministry of Health, The Yedidut Foundation Mexico and the David R. Bloom Center for Pharmacy.