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Articles

-Linolenic Acid Restores Renal Medullary Thick Ascending Limb Na⁺,K⁺-ATPase Activity in Diabetic Rats^{1 ,2}

UPRES EA 21–93, Faculté de Médecine Timone, 13385 Marseille cedex 05, France and ^* INSERM U 476, 13009 Marseille, France

³To whom correspondence should be addressed. E-mail: mtsima{at}mageos.com

	ABSTRACT

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

 
In diabetes, the activity of -6 desaturase, which converts linoleic acid (LA) into -linolenic acid (GLA), the first step of arachidonic acid (AA) synthesis, is decreased, leading to alterations in membrane phospholipid composition. On the other hand, 12 wk after the onset of diabetes, Na⁺,K⁺-ATPase activity is reduced in many organs, including the kidney. The medullary thick ascending limb (MTAL) reduced Na⁺,K⁺-ATPase activity, whereas the sodium load secondary to glomerular hyperfiltration was increased. The aim of our study was to examine whether the changes in membrane fatty acid composition resulting from the inhibition of -6 desaturase may be involved in the decreased Na⁺,K⁺-ATPase activity observed in the outer MTAL after 12 wk of diabetes. GLA is a fatty acid that by-passes the -6 desaturase step. We measured the membrane fatty acid composition and the Na⁺,K⁺-ATPase activity in the renal outer medulla of control and streptozotocin (STZ)-induced diabetic rats 12 wk after the induction of diabetes. Measurements were performed after supplementation of control rats with sunflower oil (SO) or GLA for 12 wk, and supplementation of 12 wk diabetic rats with SO for 12 wk or with GLA for 6 or 12 wk. Supplementation with GLA not only prevented the decrease in Na⁺,K⁺-ATPase activity observed after 12 wk of diabetes but also time dependently stimulated Na⁺,K⁺-ATPase activity in the outer medulla. The changes in Na⁺,K⁺-ATPase activity were related to parallel changes in the amount of Na⁺,K⁺-ATPase ₁ subunit protein. In addition, in diabetic rats only, Na⁺,K⁺-ATPase activity was positively correlated with the amount of AA present in cell membranes (r = 0.92, P < 0.05). Our results indicate that nutritional GLA supplementation increases Na⁺,K⁺-ATPase activity and expression in diabetic rats. In addition, the positive correlation between AA content and Na⁺,K⁺-ATPase activity suggests that in diabetic rats, alterations in membrane fatty acid composition contribute to the decreased Na⁺,K⁺-ATPase activity in outer medulla.

KEY WORDS: • arachidonic acid • -linolenic acid • Na⁺,K⁺-ATPase • rats • medullary thick ascending limb

	INTRODUCTION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

 
Diabetes mellitus is a metabolic disorder characterized by hyperglycemia and long-term microvascular complications in various organs including the kidney. In diabetes, -6 desaturase, which converts linoleic acid (LA)⁴ into -linolenic acid (GLA), is impaired, resulting in membrane fatty acid composition alterations in both animal models and humans (1 ). GLA is the precursor of arachidonic acid (AA), and ultimately several vasoactive prostanoids (1 ). Because prostanoids are not stored in cells, and because the basal concentration of unesterified AA is close to zero, the limiting step for their synthesis is the release of AA from membrane phospholipids through the activation of phospholipase A₂ (2 ). Consequently, the levels of membrane di-homo--linolenic acid (DGLA) and ultimately AA are decreased during experimental diabetes, which results in a decreased production of prostanoids, prostacyclin and prostaglandins (3 ).

The Na⁺,K⁺-ATPase is a ubiquitous membrane-bound enzyme complex that plays a fundamental role in cellular function. The basic function of the Na⁺,K⁺-ATPase is to maintain the high Na⁺ and K⁺ gradient across the plasma membrane of animal cells, at the expense of ATP hydrolysis (4 ,5 ). The Na⁺,K⁺-ATPase consists of two subunits ( and ß), surrounded by a closely associated ring of lipids that interact functionally with the Na⁺,K⁺-ATPase (6 ). The activity of the Na⁺,K⁺-ATPase is regulated by membrane fatty acids and among them, AA metabolites (7 –10 ).

In various organs damaged by long-term diabetic degenerative complications, Na⁺,K⁺-ATPase activity is decreased (11 –18 ). In the kidney, diabetes-induced alteration of the Na⁺,K⁺-ATPase activity is only partially restored by insulin therapy (19 ,20 ), indicating that in addition to insulinopenia, other factors chronically influence the Na⁺,K⁺-ATPase activity. A direct relationship between membrane fatty acid composition and a defect in the phosphoinositide signaling pathway resulting in decreased Na⁺,K⁺-ATPase activity may contribute to the pathogenesis of diabetic degenerative complications, as shown for diabetic neuropathy (21 ). Furthermore, membrane fatty acid composition, which regulates membrane fluidity, is correlated with the diabetes-induced decrease in Na⁺,K⁺-ATPase activity in the sciatic nerve (22 ).

The reduced availability of GLA and its metabolites secondary to the inhibition of -6 desaturase, can be by-passed by dietary supplementation with a GLA-rich oil. In streptozotocin (STZ)-treated rats, dietary supplementation with GLA resulted in partial prevention of the diabetes-induced decrease in Na⁺,K⁺-ATPase activity in the sciatic nerve after 12 wk of diabetes. The effect of GLA was dependent on the duration of the supplementation and was correlated with changes in membrane fatty acid composition (9 ).

The medullary thick ascending limb (MTAL) plays a major role in sodium reabsorption and production of concentration gradients that ultimately concentrate the urine. The driving force for sodium reabsorption is generated by the Na⁺,K⁺-ATPase activity (23 ). Diabetic nephropathy is a disease affecting the entire nephron. During early diabetes, the tubular cells undergo increased sodium reabsorption work as a consequence of the increased sodium content in the glomerular effluent. In long-term diabetic rats, after an initial stimulation, Na⁺,K⁺-ATPase activity is decreased in the MTAL after 12 wk duration of STZ-induced diabetes, despite a continuous high filtered load of Na⁺ (19 ,24 ).

To investigate the consequences of the diabetes-induced -6-desaturase inhibition and the role of lipid membrane composition in the control of the Na⁺,K⁺-ATPase in MTAL, we studied the effect of GLA supplementation.

	MATERIALS AND METHODS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

 
Rats.

The study was done according to the guidelines of the French Department of Agriculture, Fishing and Diet on the experimental use of laboratory rats with agreement number A 13823. The principles of laboratory animal care (NIH) were followed. Male Sprague-Dawley rats (n = 30; Iffa Credo, Saint-Germain de l’Arbresle, France) weighing 200 g were randomly assigned to five age-matched groups (n = 6). In the diabetic groups, diabetes was induced by a single intravenous injection of STZ (65 mg/kg). For these injections, STZ (Sigma, St. Louis, MO) was freshly dissolved in sodium citrate buffer, 0.01 mol/L, pH 5.5, and was used within 5 min of its preparation. Control rats were injected with buffer only. All diabetic rats were maintained without insulin. Diabetes was checked 3 d after the STZ-induction and on the last day of the study by the presence of hyperglycemia (>25 mmol/L) in blood samples collected from the tip of the tail (Reflolux, Boehringer Mannheim, Mannheim, Germany). Rats consumed food and water ad libitum. The food was standard nonpurified rodent diet (AO4, UAR, Epinay-sur-Orge, France). Gavage was started on the day of STZ or buffer administration. Two oils were chosen for supplementation, i.e., di-linolein-mono--linolenate (DLMG 45) containing 245 g/kg GLA, and sunflower oil (SO), an inert oil with no GLA (Scotia Pharmaceutical, Guildford, Surrey, UK). All groups treated with DLMG 45 received 260 mg of GLA (1 mL of DLMG 45) by gavage in a single daily dose, as previously described (9 ). The same volume of SO was given to the other groups to avoid differences in lipid intake because DLMG and SO have similar energy values. No differences were observed in food intake between groups supplemented with the two oils. The nondiabetic (control) groups were treated with SO or GLA for 12 wk. Diabetic rats were divided into three groups treated as follows: 1) SO for 12 wk; 2) GLA for 6 wk; and 3) GLA for 12 wk. All rats were anesthetized with ethyl ether, and both kidneys were collected.

Tissue preparation.

After anesthesia, the two kidneys were perfused through the aorta with ice-cold saline. Then kidneys were removed, weighed and the cortex and medulla were isolated from one kidney under stereomicroscopic control. The inner stripe of the outer medulla was carefully excised from the other kidney, minced on ice, and fragments of medullary tubules were obtained by gentle pressure through nylon filters with pore size decreasing from 150 to 100 µm. As assessed using a stereomicroscope (Stemi DV4 Stereomicroscope; Carl Zeiss, Oberkochen, Germany), MTAL accounted for 90% of the tubule fragments in this preparation. Therefore, it will be referred to as MTAL suspension.

MTAL suspensions were homogenized in 2 mL of ice-cold saline containing 11 mmol/L Tris buffer, pH 7.4, at 4°C with a motorized Potter homogenizer (model 94348, Heidolph, Germany) using three 15-s bursts. During the entire procedure, samples were maintained on ice. A membrane fraction was then prepared from these homogenates with a three-step procedure. First, the homogenate was centrifuged at 5500 x g for 10 min at 4°C to eliminate connective tissue and large cellular debris. Second, the supernatant was centrifuged at 7500 x g for 15 min to eliminate intracellular organelles. Third, the supernatant was resuspended and then centrifuged at 40,000 x g for 60 min. The resulting pellets were then resuspended in the assay buffer and used as described below. Na⁺,K⁺-ATPase activity was controlled at each step of the procedure.

Membrane fatty acid phospholipid composition.

Fatty acids were analyzed as methyl esters by gas chromatography on a Perkin Elmer model Autosystem XL with a fused silica capillary column (30 m x 0.22 mm i.d.) BPX 70 (SGE, Villeneuve Saint Georges, France) equipped with a flame ionization detector and using hydrogen as the carrier gas. The temperature program ranged from 160 to 205°C at 1°C/min. Peak areas from the resulting chromatogram were measured with a Perkin Elmer 1022 S integrator. After extraction of lipids, fatty acid methyl esters were prepared according to the methanolysis method (25 ,26 ).

Measurement of Na⁺,K⁺-ATPase activity.

Na⁺,K⁺-ATPase activity was measured by spectrophotometric determination of inorganic phosphate (Pi) released from ATP, with or without ouabain, a specific Na⁺,K⁺-ATPase inhibitor, as previously described by Raccah et al. (11 ). Membrane activity assays were performed with or without 1 mmol/L ouabain (Sigma). After incubation with 4 mmol/L vanadate-free ATP (Sigma) at 37°C for 10 min, the reaction was stopped by addition of ice-cold trichloroacetic acid at a final concentration of 5%. After centrifugation at 4°C and 5500 x g for 10 min, the amount of Pi in the supernatant was determined according to the method of Hurst (27 ). Na⁺,K⁺-ATPase activity was calculated as the difference between Pi released per milligram protein per hour in the presence or absence of ouabain. Membrane protein concentration was determined using the Bio-Rad protein assay (Laboratories GmbH, Munich, Germany). All assays were performed in triplicate and blanks were included in each experiment to determine the endogenous phosphate and nonenzyme-related breakdown of ATP.

SDS-PAGE and Western blots.

To determine whether the changes in Na⁺,K⁺-ATPase activity were secondary to an increased expression of Na⁺,K⁺-ATPase subunits, we performed an immunodetection of ₁ subunits by Western blotting. Microsomal preparations (3 µg), representative of the enzymological study, were diluted in 3 volumes of sample buffer containing 0.5 mol/L Tris-HCl, pH 6.8, 0.1% glycerol, 10% SDS and 1% bromophenol blue supplemented with 1% ß-mercaptoethanol. Electrophoresis was carried out with a Miniprotean II Cell Apparatus by SDS-PAGE on 4–15% gradient ready gels (Bio-Rad, Ivry sur Seine, France) for 90 min at 100 V. Proteins were then transferred to nitrocellulose membranes (Hybond, Amersham, Les Ulis, France) in a transfer buffer containing 192 mmol/L glycine, 24 mmol/L Tris, 0.1% SDS and 10% methanol at 4°C for 45 min at 200 mA constant current. After incubation in PBS (80 mmol/L Na₂HPO₄, 20 mmol/L NaH₂PO₄, and 100 mmol/L NaCl, pH 7.5, supplemented with 3% low fat milk) for 20 min to minimize nonspecific binding, the resulting nitrocellulose blots were probed overnight at 4°C with antibodies specific for the Na⁺,K⁺-ATPase, ₁ subunit (UBI, Lake Placid, NY). Membranes were then washed four times with PBS supplemented with 0.1% Tween 20 and incubated with peroxidase-conjugated anti-mouse immunoglobulin G (Amersham) for 15 min at 37°C. Then, membranes were washed once in PBS supplemented with 0.1% Tween 20 for 15 min, and rinsed 4 times with water. Antigen-antibody complexes were detected by chemoluminescence according to the manufacturers instructions. Results were quantified with an AGFA ARCUS scanning densitometer (Agfa-Gevaert, AG, Köln, Germany) in transparency mode at a resolution of 300 pixels per inch (ppi). The scan was processed on a Macintosh 7500 running the commercial software, Kodak Digital Science 1D (Kodak, New Haven, CT).

Statistical analysis.

Before assessing the different variables using a parametric or a nonparametric test, we performed a Kolmogorov-Smirnov test for normality and a Bartlett test for homogeneous variance. Fatty acid composition and Na⁺,K⁺-ATPase activities were investigated by a Kruskal-Wallis test. Differences between groups were identified by the Mann-Whitney U test and were considered significant at P < 0.05. One-way ANOVA was used to analyze differences in plasma glucose, body, kidney and MTAL weights among the different groups. Differences among groups were identified by the Scheffé test. P-values of < 0.05 were considered significant. All analyses were performed by StatView software (Abacus Concepts, Berkeley, CA). Results are expressed as means ± SEM.

	RESULTS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

 
Biological variables.

Control rats had normal blood glucose at the end of the study, whereas diabetic rats exhibited blood glucose >25 mmol/L from 2 d to 12 wk after STZ injection (Table 1 ). In addition, body weight was reduced by 35% in diabetic rats, (P < 0.0001). Treatment of diabetic rats with GLA did not significantly alter these variables.

View this table: [in this window] [in a new window]   Table 1. Effect of supplementation of the diet of control and diabetic rats with sunflower oil (SO) or -linolenic acid (GLA) on blood glucose, body weight and relative kidney weights12

Effect of diabetes and GLA supplementation on membrane fatty acid composition.

GLA did not affect membrane fatty acid composition in control rats (Fig. 1 ). On the other hand, the percentage of MTAL cell membrane polyunsaturated fatty acids (PUFA) was lower in MTAL from 12 wk SO diabetic rats compared with control SO rats (37.7 ± 1.3% vs. 48.5 ± 0.1%, P < 0.003). After 6–12 wk GLA supplementation, the PUFA of MTAL membranes from diabetic rats did not differ from values in diabetic rats fed SO. Analysis of specific PUFA showed that the percentage of LA (Fig. 1A ) was greater in MTAL cell membranes from diabetic rats fed SO compared with the control SO group. In the 12 wk SO group, the increase of LA was associated with decreases in the GLA (Fig. 1 B) and AA (Fig. 1 C) contents of MTAL cell membranes compared with the control SO group. Arachidonic acid content in the 12 wk SO group was 50% of the control SO group value. When diabetic rats were fed the GLA-rich oil, the percentages of membrane GLA and AA increased. The amounts of membrane-bound GLA of the 6–12 wk GLA and 0–12 wk GLA groups were greater than in the control SO and GLA rats (Fig. 1 B). The AA levels in diabetic 6–12 wk GLA and 0–12 wk GLA groups increased with the duration of GLA supplementation, but did not reach the levels of control GLA rats (Fig. 1 C).

View larger version (30K): [in this window] [in a new window]   Figure 1. Percentage of linoleic (panel A), -linolenic (GLA; panel B) and arachidonic (panel C) acids in medullary thick ascending limb (MTAL) cell membranes of control and diabetic rats fed sunflower oil (SO) or GLA. Results are mean g/100 g of total fatty acids ± SEM, n = 6. Values without a common letter differ, P < 0.05.

Na⁺,K⁺-ATPase activity [µmol P_i/(mg protein·h)] was significantly decreased to 20% of the SO control value in the diabetic 12 wk SO group (7.3 ± 2.8 vs. 32.5 ± 10.5, P < 0.001, Fig. 2 ). In normal rats, GLA supplementation did not affect Na⁺,K⁺-ATPase activity. In contrast, diabetic rats fed GLA for 12 wk had greater Na⁺,K⁺-ATPase activity than diabetic rats fed SO (115.8 ± 23.4 vs. 7.5 ± 1.9, P < 0.001). The increase in Na⁺,K⁺-ATPase activity was time dependent and was already present (63.2 ± 21.2 vs. 7.5 ± 1.9, P < 0.001) when GLA was given for 6 wk. In diabetic rats, Na⁺,K⁺-ATPase activities measured in 12 wk SO, 6–12 wk GLA and 0–12 wk GLA groups correlated with the percentage of membrane AA in cell membranes (r = 0.92; P < 0.0001; Fig. 3 ).

View larger version (31K): [in this window] [in a new window]   Figure 2. Na+,K+-ATPase activity in medullary thick ascending limb (MTAL) suspensions from control and diabetic rats fed sunflower oil (SO) or -linolenic acid (GLA). Values are means ± SEM, n = 6. Values without a common letter differ, P < 0.05.

View larger version (17K): [in this window] [in a new window]   Figure 3. Correlation between the membrane arachidonic acid (AA) content and the Na+,K+-ATPase activity in the medullary thick ascending limb (MTAL) of diabetic rats fed sunflower oil (SO) or -linolenic acid (GLA). Na+,K+-ATPase activity and AA content were determined in the same MTAL samples. Each point represents one rat.

GLA did not alter Na⁺,K⁺-ATPase ₁ subunit expression in nondiabetic rats (control GLA, data not shown). In 12 wk diabetic rats (0–12 wk SO, 6–12 wk GLA, 0–12 wk GLA), ₁ subunit expression was dependent on the duration of nutritional supplementation (Fig. 4 ) and was closely correlated with the Na⁺,K⁺-ATPase activity (r = 0.96, P < 0.05)

View larger version (40K): [in this window] [in a new window]   Figure 4. Medullary thick ascending limb (MTAL) Na+,K+-ATPase 1 subunit Western blots from diabetic rats fed sunflower oil (SO) or -linolenic acid (GLA). Values are mean ± SEM percentages of the control rats fed SO, n = 6. Values without a common letter differ, P < 0.05.

	DISCUSSION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

In physiologic situations, unesterified AA is released after activation of receptor-dependent phospholipase A₂ from membrane phospholipids and can be metabolized through the cyclooxygenase, the lipoxygenase and the cytochrome P₄₅₀ monooxygenase pathways, which are involved in ion pump regulation (28 –33 ). In MTAL under physiologic conditions, the AA metabolites inhibit NaCl reabsorption (34 ). The decreased -5 and -6 desaturase activities during diabetes influence the amount of PUFA in membranes of several organs, including kidney (1 ,35 ). Because GLA production is reduced in diabetes, the levels of DGLA, a product of GLA elongation, and ultimately AA, resulting from -5 desaturation of DGLA, are reduced, and the percentage of LA increases (3 ). Subsequently, the membrane fatty acid composition is abnormal and the local production of prostanoids is reduced (36 –39 ). This situation has been shown to influence several enzymes, including the Na⁺,K⁺-ATPase (9 ,40 ). A previous study from our group showed that changes in sciatic nerve Na⁺,K⁺-ATPase activity observed 8 wk after induction of diabetes were related in part to altered membrane fatty acid composition. GLA supplementation partially restored the membrane fatty acid composition and allowed the recovery of Na⁺,K⁺-ATPase activity in STZ-induced diabetic rats (9 ). The present study extends these findings to the kidney. During diabetes, effects of GLA supplementation did not appear to be mediated through the action of prostanoids synthesized by the cyclooxygenase pathway (41 ). The changes in membrane fatty acid composition may influence Na⁺,K⁺-ATPase activity through the improvement of nitric oxide synthesis (42 ). Furthermore, Na⁺,K⁺-ATPase displays changes in ouabain affinity in relation to the cell membrane fatty acid composition (43 ,44 ). Thus, the mechanisms by which PUFA modulate Na⁺,K⁺-ATPase activity may rely on the following: 1) fatty acid–dependent effects on the conformational properties of the protein complex; 2) the enzyme abundance; 3) synthesis of several metabolites of AA; or 4) changes in the thickness and fluidity of the phospholipid bilayer (45 ,46 ).

Our study showed that MTAL membrane fatty acid composition changed during STZ-induced diabetes in rats and confirmed the limiting step of the -6 desaturase activity in AA production. Membrane PUFA composition of diabetic rats showed a time-dependent increase in linoleic acid content, a substrate for -6 desaturase activity, and a decrease in AA content, the product of -5 and -6 desaturase activities. When GLA, a fatty acid that by-passes the -6 desaturase step, was given for 6 or 12 wk, it restored and increased the amount of GLA in MTAL cell membranes, attesting to the efficacy of the nutritional intervention and confirming the rate-limiting step due to diabetes-induced -6 desaturase inhibition. Furthermore, the increase in GLA content of cell membranes in diabetic rats fed GLA instead of SO suggests that the -5 desaturase is also inhibited during diabetes. The GLA-rich diet also partially prevented the diabetes-induced reduction in AA, therefore confirming the rate-limiting step of both -5 and -6 desaturase, because elongase is not inhibited during diabetes (47 ). Interestingly, the amount of AA present in the membranes was dependent on the duration of GLA supplementation, whereas SO did not prevent the decrease in membrane AA, confirming the efficacy of the therapeutic intervention on the membrane fatty acid composition. At the same time, the LA content was slightly reduced as a result of the increase in GLA and AA content, whereas PUFA content in cell membranes was unaffected.

Together with changes in the membrane fatty acid composition, we confirmed the diabetes-induced long-term decrease in MTAL Na⁺,K⁺-ATPase activity. In diabetic rats fed SO for 12 wk, or GLA for 6 or 12 wk, the Na⁺,K⁺-ATPase activity was correlated with the amount of AA present in the membranes. Because the Na⁺,K⁺-ATPase activity was not determined in liposomes but in native membrane vesicles, we could not establish a direct relationship between the membrane fatty acid composition and the sodium pump activity. However, when the decrease in membrane AA of diabetic rats was prevented by the nutritional intervention, MTAL Na⁺,K⁺-ATPase activity was restored during long-term diabetes. This increase in Na⁺,K⁺-ATPase activity was associated with an increase in ₁ subunit expression, which suggests a recruitment of new pump units rather than an increase in the turnover rate of preexisting units. Thus, we suggest that in diabetic rats, the Na⁺,K⁺-ATPase activity is influenced by changes in membrane fatty acid composition. In GLA-fed diabetic rats, the Na⁺,K⁺-ATPase activity was restored together with membrane AA content compared with diabetic SO-fed rats. The increased Na⁺,K⁺-ATPase activity in diabetic GLA-fed rats was secondary to an increase of expression of the ₁ subunit. A comparable result was reported in one study describing nutritional supplementation with menhaden oil vs. beef tallow oil in BHE/cdb rats, which mimic human type 2 diabetes. This study showed that AA and Na⁺,K⁺-ATPase activity increased in renal cortex. The mechanism proposed to be involved was a change in membrane fluidity (48 ).

We and other investigators have shown that during diabetes, alterations in Na⁺,K⁺-ATPase activity may result from parallel changes in the amounts of ₁ and ß₁ subunits (48 ). Consistent with this hypothesis, the early diabetes-induced increase in Na⁺,K⁺-ATPase activity was related to increased protein expression (14 ). The increase in Na⁺,K⁺-ATPase activity was explained as an adaptive mechanism to the sodium load increase into distal tubules (49 ). The secondary long-term diabetes-induced decrease in Na⁺,K⁺-ATPase activity was correlated with a decrease in Na⁺,K⁺-ATPase ₁ and ß₁ subunits (24 ). The latter results were specific to the MTAL and could not be explained by a decrease in sodium delivery to the distal tubule because the glomerular filtration rate remained normal. In the present study, although GLA had clear and measurable effects on the Na⁺,K⁺-ATPase activity, we did not demonstrate any significant change in the body weights of diabetic rats fed either SO or GLA, probably because the effect of MTAL Na⁺,K⁺-ATPase on renal Na handling was negligible compared with the persistent glycosuria-induced osmotic diuresis.

Thus, we suggest that the long-term effects of diabetes on Na⁺,K⁺-ATPase might be influenced by the cell membrane fatty acid composition. Nutritional supplementation with GLA during diabetes altered the cell membrane fatty acid composition after 12 wk of diabetes, and therefore allowed the physiologic response to the persistent stimulus by the diabetes-induced increase in sodium load in MTAL. Further nutritional studies in insulin-treated animals are warranted to understand the physiologic effect of GLA supplementation on renal Na handling of diabetic rats.

On the whole in diabetic rats, the amount of AA in MTAL cell membranes was dependent on the duration of GLA treatment, the MTAL Na⁺,K⁺-ATPase activity and expression after 12 wk of diabetes were also dependent on the duration of GLA supplementation and the Na⁺,K⁺-ATPase activity was correlated with the AA membrane fatty acid composition. Thus, we suggest that the membrane fatty acid composition does not directly alter Na,K-ATPase intrinsic properties. Rather, membrane AA content acts in part either through membrane fluidity or may recruit or induce synthesis of cell-specific regulatory factors that determine the functional effect of membrane composition on enzyme kinetics through regulation of the enzyme abundance, and therefore allow long term functional adaptation during diabetes in rat.

In conclusion, we found that the diabetes-induced decreased -6 desaturase activity is associated with significant changes in MTAL cell membrane fatty acid composition and Na⁺,K⁺-ATPase activity. GLA supplementation significantly affected the membrane fatty acid composition and resulted in the persistence of the adaptive increase in Na⁺,K⁺-ATPase activity, supported by similar changes in protein expression in the MTAL.

	ACKNOWLEDGMENTS

 
The authors are indebted to Eric Féraille for critical reading and scientific advise, and David F. Horrobin for technical and intellectual support.

	FOOTNOTES

 
¹ Presented in abstract form at the 36th annual meeting of EASD, Jerusalem, Israel [Tsimaratos, M., Coste, T., Djemli, A., Dufayet, D., Gerbi, A., Barone, R., Vague, P. & Raccah, D. (2000) Tubular effects of -linolenic acid supplementation during streptozotocin-induced diabetes. Diabetologia 43 (suppl. 1) A259 (abs.)] and in Société Française de Pédiatrie, Réunion annuelle du 16 au 19 mai, Reims, France [Tsimaratos, M., Coste, T., Djemli, A., Dufayet, D., Gerbi, A., Barone, R., Vague, P. & Raccah, D. (2001) Time dependent MTAL-specific Na,K-ATPase activity in streptozotocin-induced diabetes mellitus in rat. Arch. Pediatr. 8: 106.].

² Supported in part by Scotia Pharmaceuticals (Scotland).

⁴ Abbreviations used: AA, arachidonic acid; DGLA, di-homo--linolenic acid; DLMG 45, di-linolein-mono--linolenate oil; GLA, -linolenic acid; LA, linoleic acid; MTAL, medullary thick ascending limb of Henle; Pi, inorganic phosphate; SO, sunflower oil; PUFA, sum of polyunsaturated fatty acids; STZ, streptozotocin.

Manuscript received May 21, 2001. Initial review completed June 27, 2001. Revision accepted August 20, 2001.

	LITERATURE CITED

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

 

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