1 Department of Nephrology, Centre Hospitalier Universitaire Vaudois (CHUV), Lausanne, Switzerland and 2 John Walls Renal Unit, Leicester University Hospitals, Leicester, UK
Correspondence and offprint requests to: Dr Daniel Teta, MD, Department of Nephrology, Centre Hospitalier Universitaire Vaudois (CHUV), Bugnon 17, 1011 Lausanne, Switzerland. Email: Daniel.Teta{at}chuv.hospvd.ch
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Abstract |
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Methods. 3T3-L1 adipocytes were exposed to a 50:50 mixture of dialysis solutions and medium M199 containing 10% serum for 48 h. Leptin secretion in culture cell supernatants was measured by enzyme-linked immunosorbent assay and leptin mRNA content by northern blot analysis.
Results. The high glucose-based commercial dialysate PD4 produced a higher leptin secretion compared with an identical laboratory-manufactured dialysate (Lab-D), but with a physiological glucose concentration of 5 mM (P<0.05). Raising glucose concentration from 2.75 to 40 mM in Lab-D induced a dose-dependent increase in leptin secretion of 110±12% at 48 h (P<0.001) and leptin mRNA (P<0.05; glucose 2.75 vs 40 mM). Inhibition of UDP-N-acetylglucosamine biosynthesis, with 6-diazo-5-oxo-norleucine added to Lab-D, abolished most of the glucose-stimulated leptin release and downregulated leptin gene expression. Furthermore, glucose-free Lab-D supplemented with 1 mM glucosamine, an intermediate product in UDP-N-acetylglucosamine biosynthesis, increased leptin secretion by 28±11% over control (P<0.05), although without effect on leptin mRNA, after 48 h of culture.
Conclusions. These results suggest that the PD-induced hyperleptinaemia could, in part, be mediated by the effect of glucose-based dialysis fluids on leptin production by adipocytes via activation of the hexosamine biosynthetic pathway.
Keywords: adipocyte cultures; glucose; glucosamine; hexosamine pathway; leptin secretion; peritoneal dialysis fluids
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Introduction |
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Leptin circulates in proportion to fat mass. However, patients with end-stage renal disease (ESRD) commonly have serum leptin levels several times higher than would be expected for their adipose mass. The major cause of uraemic hyperleptinaemia is reduced renal clearance, although other factors associated with ESRD may contribute to regulate leptin production in this setting. Among patients undergoing renal replacement therapy, those treated by peritoneal dialysis (PD) have extraordinarily raised serum leptin, clearly out of proportion to the fat accumulation observed in these patients [2]. In addition, serum leptin increases by 189% within 1 month after starting PD treatment, in spite of significant leptin removal by the peritoneal route [3]. It is therefore probable that factors other than fat mass stimulate leptin production in PD.
There is abundant evidence that glucose metabolism is the major regulator of leptin production [4]. In PD patients, most dialysis solutions contain high glucose concentrations. It is established that, during the dwell, solutes in PD fluids (especially glucose) are transferred by passive diffusion through the peritoneal barrier [5] and, hence, come into contact with omental adipocytes. Leptin is thought to be actively synthesized by these cells, possibly through stimulatory effects by the high glucose environment to which they are exposed [6].
The aim of the present study was, therefore, to investigate the effect of glucose-based PD solutions on leptin secretion and leptin mRNA in cultured 3T3-L1 adipocytes, to determine whether the previously reported strong stimulation of leptin secretion by glucose is still important in the context of dialysis fluids.
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Subjects and methods |
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Choice of in vitro model
In the design of this study, four in vitro models of adipose tissue were tested. Human omental adipocytes were isolated by collagenase digestion from omental biopsies from PD patients undergoing catheter insertion. The viable non-adherent adipocyte suspensions obtained were unsuitable, however, as viability declined after only 12 h in suspension. In contrast, pre-adipocytes isolated from the biopsies above were adherent to plastic flasks, proliferated and differentiated in culture. However, the small number of cells available from each tissue donor precluded their use in routine experiments. Omental adipose tissue explants (25 mm in diameter) obtained from further donors were also cultured for 48 h and showed significant, but widely varying, leptin secretion, owing to wide variations in the ratio of adipose to connective tissue in the samples. The present study was, therefore, performed in the 3T3-L1 pre-adipocyte culture model that has been characterized previously for studies of leptin secretion in this laboratory [7,8].
3T3-L1 fibroblasts were grown to confluence and differentiation to adipocytes was stimulated by incubation with IBMX, dexamethasone and insulin, as described previously [7,8]. At this stage, phase-contrast microscopy showed that all the cells exhibited typical adipocyte morphology without any apparent fibroblast contamination. For leptin mRNA studies, cells were treated as reported previously [8].
Leptin protein quantitation
Leptin concentrations in cell culture media were determined using a sandwich enzyme-linked immunosorbent assay for mouse leptin (Quantikine M; R&D Systems, Minneapolis, MN, USA), as described previously [7].
Analysis of leptin mRNA
Total RNA was extracted as described in an earlier study [8]. Leptin mRNA was determined by northern analysis according to standard methods used in this laboratory [9].
Membranes were hybridized with a [32P]dCTP-labelled cDNA probe for mouse leptin, generated as described previously [8]. RNA loading was normalized using a cDNA probe for cyclophilin, which was generously supplied by the Institute of Pharmacology and Toxicology, University of Lausanne, Switzerland.
Experimental design
For leptin secretion studies, 3T3-L1 adipocytes were exposed for 48 h to a 50:50 mixture of the PD dialysis solution and M199 with 10% FBS containing 500 IU/ml penicillin, 500 µg/ml streptomycin and 0.5 mmol/l sodium pyruvate. The dilution of PD fluid in this way was used to mimic the equilibration of dialysis solutions that occurs early after their infusion into the peritoneal cavity, i.e. a fall in glucose and lactate concentrations and osmolality and an increase in pH [10]. A commercial dialysate with a high glucose concentration (1.36%, PD4; Baxter Healthcare Ltd) or a laboratory-manufactured, filter-sterilized dialysate (Lab-D) of identical electrolyte composition and pH to PD4 were used. The use of the laboratory-made solution enabled the D-glucose concentration of the final test medium to be varied. A 1.36% glucose solution contains 76 mM D-glucose and, hence, after dilution 50:50 with M199 (glucose concentration: 5.5 mM), the test medium has a final glucose concentration of 40.75 mM. Glucose concentrations in Lab-D were varied between 2.5 and 40 mM, allowing assessment of the effect of glucose over a pathophysiological concentration range. Mannitol was used in place of glucose in Lab-D as an osmotic control (Mann) to match PD4's osmolality.
Aliquots of cell supernatants were collected at 24 and 48 h. The samples were frozen at 20°C for subsequent leptin assays. Cell monolayers were lysed by scraping the cells from the culture plate into 0.5 M sodium hydroxide. Total protein was measured using a modified Lowry technique (BioRad DC protein assay; BioRad).
For leptin mRNA experiments, the cells were treated as above for 12, 24 and 48 h. After each of these time points, 1.5 ml phenol guanidine isothiocyanate reagent (Trizol®; Invitrogen) was added. The flasks were subsequently stored at 20°C prior to RNA extraction.
Investigation of the hexosamine pathway
The hexosamine biosynthetic pathway, which is known to use the inwards intracellular glucose flux to regulate leptin production [11], was investigated in two ways: (a) the addition of DON, an inhibitor of the regulatory enzyme glutamine:fructose-6-phosphate amidotransferase (GFAT), to glucose-based Lab-D in order to reduce the flux through this pathway; and (b) the use of D-glucosamine (2 amino-2-deoxy-D-glucose) that, after transport into the cell and phosphorylation, acts downstream of GFAT and, hence, increases the flux through this pathway. Glucosamine is supplied as hydrochloride salt, which caused a dose-dependent fall in pH in preliminary experiments. As acidosis per se decreases leptin production in this cell line [7,8], the pH of the glucosamine-supplemented media was adjusted by the addition of NaHCO3. A further problem is that glucose and glucosamine may compete for the same plasma membrane transporter (GLUT-4), potentially blunting effects of glucosamine. Experiments were, therefore, conducted in glucose-free dialysis solutions with 1 mM pyruvate as an alternative energy source. Under these conditions, the percentage of lactate dehydrogenase (LDH) released was unaffected (see below) and leptin concentration in the medium was greater at 48 h than at 24 h, demonstrating the continued viability of the cells.
Assessment of cell viability
Cell viability was assessed by measurement of LDH activity released into the cell culture supernatant using a commercial spectrophotometric assay (DG1340-K; Sigma). Cytotoxicity was expressed as the LDH activity in culture supernatant as a percentage of total LDH activity in the cells.
Statistical analyses
Data were expressed as means±SEM. For comparison of means between two groups, an unpaired t-test was used. Comparison of means between multiple groups was by analysis of variance with Duncan's multiple range test. Statistical significance was defined as P<0.05.
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Results |
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Modulation of the hexosamine biosynthetic pathway
The hexosamine biosynthetic pathway has been proposed as a nutrient sensor linking the incoming glucose flux with leptin gene expression and secretion [11]. Whether this pathway is still involved in the presence of unphysiological PD fluids was therefore determined.
DON (20 µmol/l), added to Lab-D with a high glucose concentration of 20 mM, significantly reduced leptin secretion at 48 h, abolishing most of the leptin-stimulating effect of glucose (Figure 4). Under identical conditions, the addition of DON to Lab-D also decreased leptin mRNA over a 48 h time course (Figure 5). This effect could not be accounted for by cell toxicity, since LDH release in the cell supernatants was insignificant.
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Discussion |
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This study provides the first evidence that glucose-based dialysis solutions do, indeed, stimulate leptin production from cultured adipocytes (Figure 1), an effect that is attributable to glucose (Figure 2) but not to hyperosmolarity (Figure 1) or to agents generated by heat sterilization of the dialysate. This marked effect of glucose on leptin secretion was obtained in spite of the known inhibitory effect of the low pH [7,8] that occurs in PD solutions.
The hexosamine pathway and glucose dialysate
As the flux through the hexosamine pathway of glucose metabolism has been suggested previously as a mediator of the effect of glucose on leptin synthesis and secretion [11], we tested whether modulation of this pathway could still affect leptin protein release and leptin mRNA in the context of dialysis fluid.
DON, which reduces hexosamine pathway flux by inhibition of GFAT, was shown nearly to abolish the leptin-stimulating effect induced by high glucose-based dialysates. This was associated with a decrease in leptin mRNA, suggesting that GFAT modulates leptin production through effects on transcription or stability of leptin mRNA. To test the hypothesis that increasing the metabolic flux into the hexosamine pathway would result in increased leptin production, we incubated cultured adipocytes in Lab-D supplemented with glucosamine. Under conditions designed to avoid potential interference through artefactual glucosamine actions (fall of pH and ATP depletion), glucosamine significantly increased leptin secretion over the control value at 48 h.
Our data in part confirm previous studies that investigated this issue [15,16]. However, the current study has examined these effects specifically in the context of PD fluids. Low pH, high lactate and high osmolarity in dialysates inhibit cell function in many cell types [10]. Therefore, the maintenance of leptin responses to subtle modulations of the hexosamine pathway (Figures 47) in the presence of dialysate is noteworthy. The only discrepancy was that glucosamine had no effect on leptin mRNA, which is not in agreement with Zhang et al. [16], who demonstrated that glucosamine increased leptin gene promoter activity in 3T3-L1 adipocytes. It should be emphasized that, to avoid unwanted side effects, glucosamine was used in the present study only at a fairly low dose yielding a stimulation of leptin secretion considerably smaller than that observed with high concentrations of glucose. We have shown previously in studies with glucose transport inhibitors in these cells [8] that large changes in glucose influx lead to decreases in both leptin secretion and leptin mRNA, whereas more moderate inhibition of glucose influx decreases leptin secretion without detectable effect on mRNA levels. In response to moderate changes in glucose or glucosamine flux, a non-transcriptional mechanism may be responsible for changes in leptin secretion, possibly through effects at the level of translation. Reports that the hexosamine pathway and its resulting O-Glc-NAc glycosylation of the eukaryotic initiation factor-2 binding protein p67 can initiate translation [17] support this view.
Limitations of the present culture model
Incubation conditions. In clinical practice, PD fluids have unphysiologically high glucose concentrations; however, these concentrations are not sustained in the peritoneal cavity since they rapidly fall to 38% of initial values at 4 h [10]. For these reasons, our experiments were restricted to the pathophysiological range of 2.540 mM final glucose concentration. The stimulating effects of glucose-based dialysates were seen at 24 h (leptin mRNA) and 48 h (leptin protein release) and these incubation times are longer than the dwell times in PD. It must be emphasized that even the best study design employing cultured cells in vitro may not exactly mimic clinical practice. However, potential effects seen in vitro at 24 and 48 h may be relevant, since patients on PD repeatedly replace the dialysate with a fresh solution.
Choice of cell line. As a clonal cell line, 3T3-L1 cells have the advantage of a homogeneous cell population allowing precise comparisons of defined treatments, without the confounding factor of variation between different donors. Nevertheless, the 3T3-L1 pre-adipocyte model (like all in vitro models) has limitations in studying the response of omental adipocytes to PD fluids. This is a mouse rather than human cell line and has been used previously as a model of subcutaneous rather than visceral (omental) adipocytes [18]. However, the endocrine effects of cell lines (including 3T3-L1) in vivo, when implanted in various fat depots, vary considerably depending on the location of implantation [19]. This means that the in vivo environment rather than the nature of the adipocyte cell line per se is the crucial factor. It is not certain, therefore, that adipocytes from other sources (e.g. visceral adipocytes from PD patients), if grown in vitro in isolation, would have provided a more accurate model than 3T3-L1 adipocytes. Furthermore, omental adipocytes are more leptin-responsive to nutrient stimuli in vivo than are subcutaneous adipocytes [20]. Therefore, even if 3T3-L1 do behave more like subcutaneous adipocytes than visceral adipocytes [18], this may mean that the glucose effect observed simply underestimates the effect that would be seen in visceral adipocytes. It has also been shown that leptin responses to changes in glucose availability are qualitatively similar in rat [4], human subcutaneous [15] and 3T3-L1 adipocytes [16], suggesting that marked qualitative differences between cell lines in the glucose response of leptin are unlikely.
It should be emphasized, however, that results obtained with any single cell line must be interpreted with caution and it will, therefore, be interesting to see whether results like those in the present study are also observed in future studies with human adipocyte lines.
In summary, this study provides evidence that glucose contained in dialysis fluids stimulates leptin production from cultured adipocytes and that this effect is at least partly mediated through the hexosamine biosynthetic pathway. The data support the notion that leptin is actively synthesized by intra-abdominal adipose tissue of patients undergoing PD, thus, contributing to sustained hyperleptinaemia associated with this treatment.
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Acknowledgments |
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Conflict of interest statement. None declared.
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References |
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