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FJ
EXPRESS SUMMARY ARTICLE The Full-length version of this article is also available, published online March 26, 2002 as doi:10.1096/fj.01-0816fje. |
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,2
* Roche Vitamins Ltd, Research and Development, Department of Human Nutrition and Health, 4070 Basel, Switzerland;
F. Hoffmann-La Roche Ltd., Pharma Research, 4070 Basel, Switzerland; and
University Freiburg, Institute of Cell Biology, Biology II, 79104 Freiburg, Germany
3Correspondence: Roche Vitamins Ltd., Human Nutrition and Health, 4070 Basel, Switzerland. E-mail. beat.fluehmann{at}roche.com
SPECIFIC AIMS
The promiscuity of the peroxisome proliferator-activated receptors (PPAR) toward a broad range of exogenous and endogenous ligands suggests a pivotal role between food intake and the transcriptional regulation of glucose and lipid metabolism. In this study, we aimed to identify and elucidate the underling molecular mechanisms of possible beneficial health effects of the natural diet-derived PPAR ligand phytanic acid, with a special emphasis on non-insulin-dependent diabetes mellitus prevention.
PRINCIPAL FINDINGS
1. Transcriptional activation of PPAR isoforms by phytanic acid in CV-1 cells
We investigated the trans-activational effect of phytanic acid and its natural stereomers on the acyl CoA oxidase PPAR-responsive element (ACO-PPRE), using a luciferase reporter gene construct (ACO-PPRE)4-tk-luc cotransfected with the human PPAR isoforms 
, ß, and 
in CV-1 cells. Endogenous ACO gene expression and expression of comparable reporter constructs have been shown to be ligand-dependently activated by PPARs. With up to 300 µM phytanic acid for 24 h, no effect on cell viability was observed in any cell line used.
A 24 h stimulation of CV-1 cells with 30 µM phytanic acid revealed a significant stimulation of luciferase activity in cells cotransfected with either PPAR, -ß, or - whereas the phytanic acid precursors trans- and cis-phytol had no or very low trans-activational potential (Fig. 1
). Phytanic acid showed a stronger selectivity toward PPAR and - vs. PPARß.
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Results of the (3RS,7R,11R) phytanic acid treatment were compared with those obtained from treatments with palmitic acid and cervonic acid (DHA) as well as ligands Wy-14643, prostaglandin A-1 (PGA-1), or ciglitazone, specific for receptor isoforms PPAR, -ß, and -, respectively. (Fig. 1)
.
A dose-dependent stimulation of PPAR isoforms with (3R,7R,11R) or (3S, 7R,11R) phytanic acid revealed no significant difference in trans-activation compared with the mixture.
2. Glucose uptake
Measurement in cultures of primary rat hepatocyte revealed a substantial increase of 2-deoxy-D-glucose uptake in hepatocytes treated with 100 µM (3RS,7R,11R) phytanic acid compared with the vehicle control, 100 µM palmitic acid, or DHA (Fig. 2
A).
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To see whether genes involved in hepatic glucose uptake were regulated by phytanic acid, mRNA expression for glucose transporter (GLUT) 1, -2, and glucokinase was investigated in primary rat hepatocytes using TaqManTM real-time RT-PCR. As mRNA levels of ß-actin and 18S ribosomal RNA were not affected by phytanic acid, all results were normalized to ß-actin mRNA or 18S rRNA levels. mRNA levels of GLUT 2 and glucokinase were found to be maximal after 24 h stimulation with (3RS,7R,11R) phytanic acid. A 2.2 (1.6–2.9) -fold and 2.4 (1.7–3.3) -fold induction of mRNA levels for GLUT 1 was observed in hepatocytes stimulated for 24 h with 100 µM (3RS,7R,11R) phytanic and palmitic acid, respectively (Fig. 2B
). Lower (3RS,7R,11R) phytanic and palmitic acid concentrations as well as DHA concentrations of up to 100 µM did not affect GLUT 1 mRNA levels. Treatment of the cells for 24 h with 100 µM (3RS,7R,11R) phytanic acid resulted in a 3.2 (2.7–3.8) -fold increase in GLUT 2 mRNA levels (Fig. 2B
). In comparison, palmitic acid and DHA did not affect GLUT 2 mRNA expression. mRNA level of glucokinase was induced by 100 µM (3RS,7R,11R) phytanic acid 3.0 (2.5–3.4) -fold, but was slightly reduced by 100 µM palmitic acid and DHA.
3. Adipocyte differentiation
To investigate whether phytanic acid is able to differentiate preadipocytic precursor cells due to its PPAR activity in lipid accumulating adipocytes, mouse embryo fibroblast cells C3H10 T1/2 were used. Cells were treated with insulin in the absence or presence of 50 µM ciglitazone, 50 µM Wy-14643, or 50 µM (3RS,7R,11R) phytanic acid. In cultures treated with ciglitazone, a large number of cells changed morphology toward adipocytes with lipid accumulating droplets. In contrast, in cultures treated with (3RS,7R,11R) phytanic acid, very few adipocytes were found. Moreover, these adipocytes were observed to be markedly smaller in size than the adipocytes differentiated by ciglitazone. Wy-14643-treated cells were indistinguishable from vehicle-stimulated cells.
Expression profiling determined by RT-PCR after 7 days of treatment showed a strong up-regulation of adipose specific marker genes for adipocyte fatty acid binding protein (aP2): 88.0 (78.1–99.3) -fold; lipoprotein lipase (LPL): 9.4 (6.5–13.5) -fold; and PPAR2: 75.4 (57.5–98.9) -fold in ciglitazone-treated cells; phytanic acid exerted only minor effects: 4.7 (3.8–6.0); 1.08 (0.58–1.98); 1.64 (1.15–2.34). Wy-14643 treatment showed no effect in regulating PPAR2 (0.90, 0.85–0.95) and even down-regulated aP2 0.35 (0.29–0.42) and LPL 0.06 (0.03–0.12) gene expression.
CONCLUSIONS
Next to skeletal muscle and adipose tissue, liver plays a crucial role in glucose metabolism and is an important regulator of glucose levels in plasma. Data presented here demonstrate that glucose uptake in primary cultures of rat hepatocytes is markedly induced by concentrations of phytanic acid one order of magnitude higher than normal human serum levels. The enzymes involved in glucose uptake we examined are two members of the facilitative glucose transporter family and glucokinase. An up-regulation of GLUT 1 and -2 mRNA levels was observed, paralleled by an increase in 2-deoxy-D-glucose uptake. In primary rat hepatocyte cultures, mRNA for GLUT 4 (the main glucose transporter in skeletal muscle) was not detectable. The liver-type glucose transporter GLUT 2 is distinguished from other GLUT isoforms by being a low-affinity glucose transporter with a high turnover rate. The presence of a low-affinity glucose transporter ensures that, in liver cells, the glucose flux will be directly proportional to the glucose gradient across the cell membrane. Moreover, in hepatocytes, GLUT 2 is coupled with the regulated phosphorylation activity of glucokinase. Recent studies emphasize the critical role of glucokinase in the tight regulation of glucose uptake, storage, and gluconeogenesis. During states of glycogen synthesis, glucokinase is up-regulated and can increase the formation of intracellular glucose-6-phosphate, maintaining a low intracellular concentration of free glucose. Consequently, paralleled up-regulation of GLUT 2 and glucokinase must have a synergistic effect on plasma glucose clearing in the liver (Fig. 3
).
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Phytanic acid has been described as a ligand for RXR and PPAR. The results from our study show that phytanic acid also exerts a certain promiscuity in trans-activating PPARß and -. This trans-activation potential is unique compared with other fatty acids and the phytanic acid precursors used in our assays. With the finding that the GLUT 2 gene is regulated by a PPRE and that liver glucokinase mRNA is induced in Zucker diabetic fatty rats by a PPAR agonist, a PPAR-mediated mechanism may explain the glucose influx observed in hepatocytes treated with phytanic acid.
PPAR forms permissive heterodimers with RXR, meaning that either partner can regulate the transcriptional activity by interacting with its own ligand. Cotreatment of cells with ligands for PPAR and RXR results in an additive effect. It was shown that ligands selective for RXR activate PPRE driven reporter genes. In vivo sensitization to insulin was demonstrated in diabetic and obese mice in response to RXR agonists, comparable to the effects observed with thiazolidinediones. Recently it has been shown in experimental animal models for impaired glucose tolerance that compounds with dual PPAR and - activities have blood glucose- and triglyceride-lowering properties. The ability of phytanic acid to activate RXR as well as PPAR and - suggests the expression of a specific set of genes. The property of phytanic acid as a nonspecific agonist of different nuclear receptors is also reflected in the limited potential to differentiate C3H10 T1/2 cells into mature lipid-accumulating adipocytes. It is well known that highly selective PPAR agonists are extremely powerful in differentiating mesenchymal precursors into mature adipocytes.
We have shown that, in contrast to other fatty acids, phytanic acid is able to enhance glucose uptake in hepatocytes without strongly promoting adipogenic differentiation. Phytanic acid differs from palmitic acid and DHA in its potential to up-regulate glucose transporters and glucokinase, resulting in increased glucose uptake in primary rat hepatocytes. These effects may be explained by the observation that phytanic acid, at physiological concentrations, is an effective agonist for both PPAR and -, thereby activating the transcription of a distinct pattern of genes that favors glucose uptake. In conclusion, we postulate that phytanic acid, a naturally occurring compound that is part of the human diet, may have a potential role in the management of insulin resistance.
FOOTNOTES
1 To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.01-0816fje; to cite this article, use FASEB J. (March 26, 2002) 10.1096/fj.01-0816fje 
2 These authors contributed equally to this paper.
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