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APPETITE, OBESITY AND METABOLISM

Docosahexaenoic acid affects insulin deficiency- and insulin resistance-induced alterations in cardiac mitochondria

Lipides Membranaires et Fonctions Cardiovasculaires, Institut National de la Recherche Agronomique, Unité de Recherche 1154, Faculté de Pharmacie, Université Paris-Sud, 92290 Châtenay-Malabry, France

Submitted 2 June 2003 ; accepted in final form 3 November 2003

	ABSTRACT

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The effect of docosahexaenoic acid (DHA) intake on cardiac mitochondrial function was evaluated in permeabilized fibers in insulin deficiency and insulin resistance in rats. The insulin-deficient state was obtained by streptozotocin injection 2 mo before investigations. Insulin resistance was obtained by feeding a 62% fructose diet for 3 mo. DHA was incorporated in the diet to modify the fatty acid composition of cardiac membranes, including mitochondria. Insulin deficiency decreased mitochondrial creatine kinase (mi-CK) activity and mitochondrial sensitivity to ADP. DHA intake prevented these alterations. Moreover, the insulin-deficient state significantly decreased n-3 polyunsaturated fatty acids (PUFA) and slightly increased n-6 PUFA in both cardiac and mitochondrial membranes, inducing a significant increase in the n-6-to-n-3 ratio. DHA intake maintained high myocardial and mitochondrial DHA content. Insulin deficiency also decreased glutamate- and palmitoylcarnitine-supported mitochondrial respiration, but DHA intake did not prevent these effects. In contrast, insulin resistance did not affect mi-CK activity or sensitivity to ADP. However, insulin resistance influenced the myocardial fatty acid composition with decreased n-6 and n-3 PUFA contents and increased monounsaturated fatty acid content. Only slight alterations were observed in mitochondrial fatty acid composition, and they were corrected by DHA intake. Moreover, insulin resistance decreased the glutamate-supported respiration, and DHA intake did not influence this effect. In conclusion, the impairment of cardiac mitochondrial function was more pronounced in the insulin-deficient state than in insulin resistance. The modification of fatty acid composition of cardiac and mitochondrial membranes by DHA partially prevented the mitochondrial alterations induced in the two models.

mitochondrial respiration; myocardium; polyunsaturated fatty acid

	MATERIAL AND METHODS

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Animals and models. Insulin deficiency was induced in male Wistar rats (150-200 g) with STZ (55 mg/kg im, freshly dissolved in 1 mM sodium citrate buffer, pH 7.4). The sham rats received citrate buffer. The development of the pathology was confirmed by determination of blood glucose concentration, 1 wk after the injection. Any rats in which the hyperglycemia was not confirmed after 1 wk were given a second dose of STZ. The mean blood glucose concentration was 84.9 ± 2.3 mg/dl (n = 15) and 266.1 ± 31.8 mg/dl (n = 15) in the sham group and the STZ-treated group (STZ group), respectively. All animals were individually housed for 8 wk and weighed one time a week. At the end of experiment, the fasting plasma insulin level was 66.2 ± 6.95 mU/l and 17.5 ± 3.50 mU/l in sham and STZ-treated rats, respectively. Insulin resistance was induced in male Wistar rats (100-125 g) with a custom-made fructose diet (HI group), as previously described (22). Nonhyperinsulinemic rats (NHI group) were fed a standard diet that contained starch and sucrose. The rats received these diets for 12 wk and were weighed one time a week. At the end of experiment, the fasting plasma insulin level was 135.1 ± 11.35 mU/l in HI rats.

Diets. The rats were fed ad libitum a semipurified jellied diet (21) in accordance with the AIN-93 recommendations. The meal was prepared to form a jellied mass cut into cubes for feeding, stored at -20°C, and fed daily to maintain moisture content and food intake. For the insulin deficiency model involving the sham and STZ groups, we prepared two diets, which differed only in lipid composition. Different fats (80 g/kg) were incorporated into a standard fabricated diet composed of starch (526.2 g/kg), sucrose (100 g/kg), cellulose (50 g/kg), soy protein isolate (140 g/kg, ICN 905456), L-cystine (1.8 g/kg), gelatin to jellify (50 g/kg), salt mixture (40 g/kg, ICN 960401), vitamin mixture (10 g/kg, ICN 960402), and choline bitartrate (2 g/kg). For the dietary control groups, sham-control (Ctrl), and STZ-Ctrl, the control lipid part was composed of 40 g/kg cocoa butter (Cacao Barry) + 40 g/kg sunflower seed oil (Fruidor). For the DHA-enriched diet groups, sham-DHA and STZ-DHA, the lipid part was composed of 40 g/kg cocoa butter + 30.4 g/kg sunflower seed oil + 9.6 g/kg DHA (Ropufa "60," Hoffmann-Laroche). For the insulin-resistant model (NHI and HI rats), we prepared four diets that differed in their carbohydrate and lipid composition. The NHI-Ctrl group received the same diet as the sham-Ctrl group, with carbohydrate comprised of starch (526.2 g/kg) and sucrose (100 g/kg) and lipid comprised of 40 g/kg cocoa butter + 40 g/kg sunflower seed oil. NHI-DHA rats received the same diet as sham-DHA, composed of starch (526.2 g/kg) and sucrose (100 g/kg) as the carbohydrate and 40 g/kg cocoa butter + 30.4 g/kg sunflower seed oil and 9.6 g/kg DHA as the lipid. For the HI groups, fructose (626.2 g/kg) replaced starch and sucrose as the carbohydrate. For HI-Ctrl, the lipid comprised 40 g/kg cocoa butter + 40 g/kg sunflower seed oil, and for HI-DHA, the lipid comprised 40 g/kg cocoa butter + 30.4 g/kg sunflower seed oil + 9.6 g/kg DHA. Independently of the carbohydrate composition, the fatty acid composition of the Ctrl and DHA diets was determined by gas chromatography, after lipid extraction. As shown in Table 1, the diets were quantitatively similar in their saturated fatty acid, monounsaturated fatty acid, and PUFA but differed qualitatively in their PUFA composition.

Biochemical investigations. At the end of the diet period, the rats were anesthetized after a 12-h fast with pentobarbital sodium (60 mg/kg), and blood was collected for serum biochemistry. Plasma triglycerides were determined using the spectrophotometric Triglycerides-INT kit (Sigma). Insulin was determined by RIA using the Insulin-CT 100 (CIS-BIO International) in vitro test, and glucose was measured by the glucose oxidase method. The kidneys were collected and weighted, and the hearts were used for permeabilized muscle fiber investigations, isolated mitochondria investigations, and cardiac fatty acid determination.

Mitochondria functional properties. The respiratory parameters of the mitochondria populations were investigated in situ in saponin-permeabilized fibers (31). Thin fiber bundles (100-250 µm in diameter) were excised from the subendocardial surface of the left ventricle. The bundles were incubated with intense shaking for 30 min in solution S supplemented with 50 µg/ml saponin to selectively destroy the integrity of the sarcolemma. The bundles were then transferred to solution R for 10 min to wash out adenine nucleotides and phosphocreatine. All procedures were carried out at 4°C. The respiratory rates were determined with a Clark electrode (Hansatech, Eurosep Instruments) in an oxygraphic cell containing 7-10 fiber bundles in 1 ml of solution R at 22°C with continuous stirring. After measurement, the bundles were removed and dried, and the respiratory rates were expressed as millimoles O₂ per minute per gram (dry weight), on the basis of an oxygen solubility of 230 mmol O₂/l. To measure the mitochondrial respiratory characteristics, cardiac fibers were exposed to increasing ADP concentrations with glutamate (5 mM) and malate (2 mM) in the absence or presence of creatine (20 mM). The ADP-stimulated respiration above basal oxygen consumption was plotted to determine V_max and the apparent Michaelis constant for ADP (K_m). K_m represents the mitochondrial sensitivity to ADP. The K_m ratio in the absence and in the presence of creatine (K_m - Cr/K_m + Cr) was taken as an index of functional activity of mitochondrial creatine kinase (mi-CK). To evaluate the functional activity of pyruvate dehydrogenase and -oxidation, basal- and ADP-stimulated respiration (V_o and V_max, respectively) were measured in the presence of malate (2 mM) and either pyruvate (200 µM), octanoate (100 µM), or palmitoylcarnitine (100 µM). For each substrate, mitochondrial oxidation/phosphorylation coupling was assessed through the value of the acceptor control ratio (= V_max/V_o).

Isolation of heart mitochondria. A fragment of the heart (400 mg) was placed immediately in ice-cold isolation medium and chopped with scissors. Trypsin 250 (Difco Laboratories) was added (0.125 mg/ml), and the samples were mixed thoroughly and left for 15 min on ice. The preparations were diluted two times with isolation medium containing trypsin inhibitor (0.65 mg/ml) and BSA (1 mg/ml). The suspension was stirred and decanted, and the supernatant was then homogenized carefully in a glass potter. The samples were centrifuged at 600 g for 10 min at 4°C. The supernatant was centrifuged again at 8,000 g for 15 min at 4°C. The pellet was resuspended in isolation medium containing BSA (1 mg/ml) and centrifuged again at 8,000 g for 15 min at 4°C. This operation was repeated two times. The pellet was finally suspended in 1 ml of serum albumin-free isolation medium. The protein concentration was measured using the bicinchoninic acid protein assay kit (Sigma).

Analysis of lipids. The lipids were analyzed as previously described (22). Briefly, the lipids were extracted from heart or mitochondria according to Folch et al. (7) in 2:1 chloroform-methanol. For the heart, phospholipids were separated from nonphosphorous lipids on silicic acid cartridges (11), and the fatty acids were trans-methylated with BF3-methanol (17). The methyl esters were analyzed by gas chromatography on a EC-WAX capillary column (0.32 x 30 m) with FID, using C17:0 as internal standard.

Solution and reagents. Solutions S and R both contained 10 mM EGTA-CaEGTA buffer (free Ca²⁺ concentration, 100 nM), 1 mM free Mg²⁺, 20 mM taurine, 0.5 mM dithiothreitol, and 20 mM imidazole. The ionic strength was adjusted to 0.16 M by addition of potassium methanesulfonate. Solution S (pH 7.1) also contained MgATP (5 mM) and phosphocreatine (15 mM). Solution R (pH 7.1) contained 3 mM potassium phosphate and 2 mg/ml fatty acid-free BSA instead of high energy phosphates. Malate (2 mM) + either glutamate (5 mM), pyruvate (200 µM), octanoate (100 µM), or palmitoylcarnitine (100 µM) were used as respiration substrates. The isolation medium (pH 7.2) used for mitochondrial isolation contained 0.3 M sucrose, 10 mM Na-HEPES, and 0.2 mM EDTA. All reagents were purchased from Sigma.

Statistical analysis. Nonlinear fits to Michaelis-Menten kinetics were computed by a nonlinear least-square routine. The data were expressed as means ± SE and submitted to ANOVA with two fixed factors among diet and diabetes. When significantly different, the means were further compared by the Neuman-Keuls test (3).

	RESULTS

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Characteristics of the rats. The STZ treatment resulted in a diabetic state with polyuria, polydipsia, and a polyphagia. The evolution of the body weight in the sham and STZ groups is shown on Fig. 1A. From the 1st wk after the injection of STZ, the body weight gain was significantly lower in STZ-Ctrl rats than in sham-Ctrl rats, in spite of polyphagia. This difference kept increasing throughout the experiment. The addition of DHA to the diet did not affect growth in the sham group but significantly limited the negative effect of STZ on weight gain. There was a significant cross-interaction between the pathology and the DHA diet from the 6th wk. The values of blood glucose and triglycerides and the weight of heart and kidneys are shown in Table 2. In both STZ-treated groups, the rats displayed significantly higher blood glucose levels and similar triglyceride levels. The DHA supplement reduced the mean blood glucose levels in both sham and STZ groups. Compared with the sham-Ctrl group, the kidney weight was significantly increased and the heart weight significantly decreased in the STZ-Ctrl group (+35 and -13%, respectively), but DHA did not influence these differences. Conversely, the rats of the STZ-Ctrl group displayed an increased heart-to-body weight ratio because of the reduced weight gain in the STZ-Ctrl group. For this parameter, the effect of DHA was significant, although it could be related to the positive effect of this diet on weight gain (see above). The time course of the body weights in the NHI and HI groups is shown in Fig. 1B. From the 1st wk, the body weight gain was significantly lower in HI rats than in NHI rats, independent of the diet. This difference remained constant throughout the experiment. The heart and kidney weights and blood levels of glucose and triglycerides are shown in Table 2. The heart weight was not affected by insulin resistance nor by dietary DHA. In contrast, the kidney weight increased moderately (+6%) in the HI-Ctrl group, but DHA had no effect on this parameter. The development of insulin resistance did not affect blood glucose levels but significantly increased plasma triglycerides. The DHA intake significantly decreased glycemia in both NHI and HI groups but did not affect triglycerides.

View larger version (18K): [in this window] [in a new window]   Fig. 1. Time course of the body weight (mean ± SE) in the insulin-deficiency experiment (A) and insulin-resistance experiment (B). Results of the 2-way ANOVA shown in inset, including streptozotocin {STZ [or hyperinsulinemia (HI)]} and docosahexaenoic acid (DHA) as fixed factors. CI, cross interaction; Sh, sham; Ctrl, control; NS, not significant.

ADP-stimulated mitochondrial respiration. In the presence of glutamate + malate, NADH-generating substrates, the basal oxygen consumption rate was accelerated by stepwise additions of ADP. Figure 2A shows the mean of oxygraphic traces of mitochondrial respiration in the presence of creatine, in sham-Ctrl, STZ-Ctrl, and STZ-DHA groups. The curves show a decrease in mitochondria sensitivity to ADP in the STZ-Ctrl group, whereas for the STZ-DHA, the respiration rate curve was in between the curve of the sham and STZ-Ctrl rats, suggesting that the alteration in affinity for ADP induced by insulin deficiency was limited by dietary DHA. The mean oxygraphic traces of mitochondrial respiration in the presence of creatine, in NHI-Ctrl, HI-Ctrl, and HI-DHA groups are shown in Fig. 2B. The mean oxygen consumption curves of NHI-Ctrl and HI-Ctrl rats are exactly superimposed, suggesting that insulin resistance did not affect the mitochondrial affinity for ADP. The mean curve of the HI-DHA group was slightly shifted, suggesting a moderate increase of ADP affinity by dietary DHA.

View larger version (17K): [in this window] [in a new window]   Fig. 2. Time course of oxygen consumption (mean ± SE). A: sham and STZ-treated groups. The sham-DHA curve was exactly superimposed on the sham-Ctrl curve and was omitted for clarity. B: NHI and HI groups. The NHI-DHA curve was exactly superimposed on the NHI-Ctrl curve and was omitted for clarity.

Mitochondrial affinity for ADP. From the oxygraphic traces, the respiration rate was calculated for each ADP concentration and plotted as a function of ADP concentration, and the K_m for ADP was determined. Figure 3 shows the K_m values for the STZ-treated rats. Without creatine, the STZ treatment had no effect on K_m (STZ-Ctrl vs. sham-Ctrl and STZ-DHA vs. sham-DHA). However, the DHA groups (sham-DHA and STZ-DHA) showed an increased K_m compared with the control groups (sham-Ctrl and STZ-Ctrl). The addition of creatine to the medium significantly decreased the K_m for ADP resulting from ADP regeneration catalyzed by mi-CK. In the presence of creatine, the K_m increased in the STZ-Ctrl group compared with the sham-Ctrl group, indicating a decrease of the mitochondrial sensitivity to ADP. However, the effect of dietary DHA was significantly different in the STZ-DHA group compared with the sham-DHA group, demonstrated by an increased mitochondrial sensitivity to ADP in the STZ-DHA group. As shown in Fig. 2B, neither insulin resistance nor dietary DHA changed the ADP sensitivity of cardiac mitochondria. In the absence of creatine, the K_m values were 385 ± 36.8 and 392 ± 22.5 µM in the NHI group and the HI group, respectively. In the presence of creatine, the K_m values were 94 ± 12.3 and 99 ± 8.7 µM in the NHI group and the HI group, respectively (data not shown).

View larger version (32K): [in this window] [in a new window]   Fig. 3. Effect of STZ-treatment on the Michaelis constant for ADP (Km) in the absence and in the presence of 20 mM creatine (mean ± SE). STZ effect, STZ-treated groups vs. sham group; DHA effect, control group vs. DHA group.

mi-CK functional activity. Figure 4 shows the K_m ratio in the absence and in the presence of creatine (K_m - Cr/K_m + Cr), in the insulin-deficient model. In the STZ-Ctrl group, the activity of mi-CK was significantly decreased compared with the sham-Ctrl group. Dietary DHA had no effect on mi-CK in the sham rats but significantly increased the K_m ratio in the STZ-DHA group. In contrast, as stated above for the mitochondrial affinity for ADP, neither insulin resistance nor dietary DHA affected mi-CK activity in either the NHI group (4.50 ± 0.90) or the HI group (4.05 ± 0.33).

View larger version (31K): [in this window] [in a new window]   Fig. 4. Effect of STZ-treatment on mitochondrial creatine kinase (mi-CK) activity (mean ± SE).

Substrates used for mitochondrial respiration. To investigate the effect of fatty acid/carbohydrate balance on energy production, the mitochondrial respiration was measured in the presence of several substrates. Figure 5 shows the respiration rate for each substrate, in the absence and the presence of 1 mM ADP (V_o and V_max, respectively), in the insulin deficiency model. For both V_o and V_max, the STZ treatment induced a significant decrease in glutamate- and palmitoylcarnitine-supported respiration (Fig. 5, A and D, respectively). In contrast, the STZ treatment had no effect on the pyruvate and octanoate-supported respiration (Fig. 5, B and C, respectively). Irrespective of the substrate provided, dietary DHA did not affect mitochondrial oxygen consumption nor was the acceptor control ratio altered by STZ treatment or by dietary DHA (data not shown). The same parameters were investigated in insulin resistance, and Fig. 6 shows the V_o and V_max respiration rates for each substrate. Neither insulin resistance nor DHA intake affected the V_o respiration rate. In contrast, the V_max of glutamate-supported respiration (Fig. 6A) was significantly lower in the HI groups (both HI-Ctrl and HI-DHA) than in the corresponding NHI groups. Again, dietary DHA had no effect. Pyruvate-supported respiration slightly increased in the HI-Ctrl group compared with the NHI-Ctrl group (Fig. 6B). In the HI-DHA group, respiration was significantly decreased compared with NHI-DHA and HI-Ctrl. The effect of dietary DHA in the HI group was opposite to that in the NHI group (Fig. 6B). The octanoate-supported respiration was not affected by hyperinsulinemia (HI-Ctrl vs. NHI-Ctrl, Fig. 6C), but, in the HI-DHA group, the respiration was significantly decreased compared with NHI-DHA and HI-Ctrl. The palmitoylcarnitine-supported respiration (Fig. 6D) was not affected by hyperinsulinemia (HI-Ctrl group vs. NHI-Ctrl group and HI-DHA group vs. NHI-DHA group), but in both the NHI-DHA and HI-DHA groups respiration was significantly increased compared with the NHI-Ctrl and HI-Ctrl groups. Whatever the substrate, neither insulin resistance nor dietary DHA affected the mitochondrial acceptor control ratio (data not shown).

View larger version (39K): [in this window] [in a new window]   Fig. 5. Effect of STZ treatment on the use of substrates in mitochondrial respiration (mean ± SE). A: glutamate (5 mM) + malate (2 mM); B: pyruvate (200 µM) + malate (2 mM); C: octanoate (100 µM) + malate (2 mM); D: palmitoylcarnitine (100 µM) + malate (2 mM). Vo, respiration rate without ADP; Vmax, respiration rate with 1 mM ADP.

View larger version (41K): [in this window] [in a new window]   Fig. 6. Effect of hyperinsulinemia on the use of substrates in mitochondrial respiration (mean ± SE). A: glutamate (5 mM) + malate (2 mM); B: pyruvate (200 µM) + malate (2 mM); C: octanoate (100 µM) + malate (2 mM); D: palmitoylcarnitine (100 µM) + malate (2 mM). NHI, nonhyperinsulinemic rats; HI effect, hyperinsulinemic group vs. nonhyperinsulinemic group.

Fatty acid composition. The fatty acid composition of cardiac membrane phospholipids and mitochondrial membranes in STZ-treated rats is shown in Table 3. The fatty acid composition was strongly affected by insulin deficiency. Interestingly, the STZ-Ctrl group was characterized by an increase in n-6 PUFA and a decrease in n-3 PUFA, including eicosapentaenoic acid (EPA, 20:5 n-3), docosapentaenoic acid (DPA, 22:5 n-3), and DHA. Next, the n-6-to-n-3 ratio was strongly increased in the STZ-Ctrl group. As expected, DHA supplementation strongly altered the PUFA composition of cardiac phospholipids with a significant increase in n-3 PUFA, including mainly DHA, but also EPA and DPA. The increase in n-3 PUFA was balanced by a significant decrease in arachidonic acid. Dietary DHA prevented the increase in the n-6-to-n-3 ratio in the STZ-treated rats. The ANOVA revealed a significant cross-interaction between STZ treatment and DHA supplementation. Moreover, as shown in Table 3, the STZ treatment also strongly affected the fatty acid composition of cardiac mitochondrial membranes. The linoleic acid content significantly increased in the STZ-Ctrl group, whereas arachidonic acid content significantly decreased. Moreover, the n-6-to-n-3 ratio increased in the STZ-Ctrl group because of a significant decrease in n-3 PUFA. The DHA intake limited any further decrease of arachidonic acid content in STZ-treated rats and prevented the increase of the n-6-to-n-3 ratio. The fatty acid composition of phospholipids of heart membranes in the insulin resistance model is shown in Table 4. The fatty acid composition was weakly affected by insulin resistance. Interestingly, insulin resistance increased the saturated fatty acid and monounsaturated fatty acid content but decreased the n-6 PUFA content, mainly at the level of the arachidonic acid precursors, 20:2 n-6 and 18:2 n-6 linoleic acid. Moreover, the n-3 PUFA content was not significantly affected. The DHA supplementation induced the same modifications in the fatty acid composition as those described above for the STZ groups. After 3 mo, the development of insulin resistance did not significantly affect the cardiac phospholipid fatty acid composition, although a significant cross-interaction between insulin resistance and dietary DHA was observed for some fatty acids. Insulin resistance weakly affected the fatty acid composition of mitochondrial phospholipids (Table 4). Although the n-6-ton-3 ratio was slightly increased by insulin resistance, saturated fatty acid, monounsaturated fatty acid, and PUFA were not significantly affected by the pathology. Moreover, the fatty acid alteration induced by DHA supplementation was roughly similar in mitochondrial membranes and total cardiac phospholipids. The cross-interaction between insulin resistance and dietary DHA was significantly different only for the n-6-to-n-3 ratio.

	DISCUSSION

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An imbalance between fatty acid and glucose utilization in energy synthesis is known to occur in the insulin-deficient state. Several studies using isolated cardiac mitochondria (2, 6, 20, 25, 26, 28, 29) or permeabilized cardiac fibers (32) have shown that mitochondrial function is strongly affected in insulin deficiency. In this study, we evaluated the effects of STZ-induced insulin deficiency on mitochondrial function in cardiac permeabilized fibers. This animal model is known to develop many of the features reported in human subjects with uncontrolled diabetes mellitus, including hyperglycemia, polydipsia, polyuria, and weight loss (30). In this study, the rats treated with STZ developed hyperglycemia and hypoinsulinemia after 2 mo, and the body weight gain was lower than in sham rats. In this experiment, dietary DHA elicited a slight reduction in glycemia, which was observed in the two models. A similar result was observed in a previous study using the same insulin-resistance model (22). This subject is not well documented in the literature. Specific investigations would be necessary to understand the possible hypoglycemic effect of DHA. Moreover the insulin-deficient rats developed renal hypertrophy and an increased heart weight-to-body weight ratio compared with sham rats. The functional state of the total cardiac mitochondrial population was investigated in saponin-permeabilized fibers. In this model of insulin deficiency, the mitochondrial sensitivity to ADP was slightly decreased in the presence of creatine. The activity of mi-CK, which catalyzed the local ADP regeneration, was decreased. Using the same model, Savabi (25) observed a decrease in mi-CK activity and a decrease in the ability of creatine to stimulate oxidative phosphorylation. Another study reported a significant decrease in total creatine kinase activity and a diminution of the various creatine kinase isoenzymes in both atria and ventricles (26). In this study, dietary DHA compensated for the diminution of creatine kinase activity associated with insulin deficiency, suggesting a relationship between the decrease in activity and the modifications of mitochondrial membrane fatty acid composition. In STZ-induced insulin deficiency, an increase in linoleic acid together with a decrease in arachidonic acid content of cardiac phospholipids was previously reported (9, 12). In this study, the same effect was observed in both cardiac phospholipid fatty acid and mitochondrial membrane fatty acid composition. The decrease in long-chain PUFA and the linoleic acid accumulation, observed in both cardiac and mitochondrial membranes, supports the previously reported decrease in 6-desaturase (5), the key enzyme in the conversion of linoleic acid to long-chain PUFA. Moreover, the insulin-deficient state decreased the membrane n-3 PUFA content (mainly DHA), balanced by an increased n-6 PUFA content, resulting in a significant increase in the n-6-to-n-3 ratio. This model of insulin deficiency was reported to put myocardial n-3 fatty acid status at risk in the absence of dietary n-3 PUFA supplementation (16). Liautaud et al. (13) pointed out the poor capacity of the cardiac myocyte to achieve the last desaturation step of long-chain PUFA, from the close precursor DPA (22:5 n-3) to DHA (22:6 n-3) (13). The occurrence of a 4-desaturase is not clearly documented, and the conversion of DPA to DHA was shown to require two successive elongation steps, a 6 desaturation step and one cycle of -oxidation on a 24-carbone PUFA, which may be the inefficient step in the heart (33). This may explain why the insulin deficiency-induced 6-desaturase inhibition may contribute to hampered DHA incorporation in membrane lipids. The present study showed that dietary DHA can compensate for this trend by promoting increased incorporation of DHA in cardiac membranes, including mitochondria. The decrease in creatine kinase activity may be related to the alterations in mitochondrial membrane fatty acid composition observed in this study. This enzyme is located on the internal mitochondrial membrane surface, and the altered composition of this membrane may influence the membrane-bound enzymes, perhaps resulting in altered creatine kinase activity and/or mitochondrial affinity for ADP. Interestingly, the two PUFA that were affected by insulin deficiency in this study are linoleic acid, which is the main component of cardiolipin, a major mitochondrial phospholipid, and DHA, a fatty acid that affects oxygen consumption by the heart (19).

We investigated the mitochondrial utilization of different substrates in cardiac saponin-permeabilized fibers. In the presence of NADH-generating substrates (glutamate + malate), the insulin-deficient state strongly decreased both basal and ADP-stimulated respiration, in accordance with the previous observations by Veksler et al. (32). Dietary DHA was unable to affect this respiration decrease. In the presence of pyruvate, insulin deficiency did not affect basal respiration but slightly decreased ADP-stimulated respiration. The pyruvate dehydrogenase and tricarboxylic acid cycle were not affected by the pathology because basal oxygen consumption was similar in insulin-deficient rat mitochondria and sham rat mitochondria. The reduction of glutamate utilization in insulin-deficient rat mitochondria could be explained by decreased glutamate uptake into mitochondria or by decreased glutamate dehydrogenase activity. In cardiac muscle, insulin deficiency increases the utilization of fatty acids for energy synthesis (14). We compared the ability of mitochondria to oxidize long-chain fatty acids that require carnitine for entry into mitochondria and short-chain fatty acids that do not require carnitine. Permeabilized fibers used in this study lost their cytoplasmic content, and it was necessary to use the acyl-carnitine form of the long-chain fatty acid. The respiration rate in the presence of short-chain fatty acid was not affected by insulin deficiency, a confirmation that the tricarboxylic acid cycle was not impaired in the insulin-deficient state. Moreover, the -oxidation rate was not affected. Conversely, in the presence of long-chain fatty acyl-carnitine, basal and ADP-stimulated respiration was decreased in insulin-deficient mitochondria, but DHA supplementation failed to prevent this effect. In the conditions used here for mitochondrial respiration measurements, fatty acids and pyruvate were not in competition, and the glucose/fatty acid balance for energy production, which decreases in the insulin-deficient state, was not determined. Moreover, irrespective of the substrate used, neither STZ treatment nor DHA supplement affected the coupling between oxygen consumption and phosphorylation.

This nutritional model of insulin resistance is based on a diet in which fructose is the only carbohydrate supplied to the rats, and they develop hyperinsulinemia and insulin resistance in a short time (10). In the present study, after 3 mo of fructose diet, the rats exhibited hyperinsulinemia, normoglycemia characteristic of insulin resistance, and hypertriglyceridemia. Moreover, body weight decreased compared with control rats. The effect of the fructose-enriched diet on the body weight is a matter of debate. Some authors observed a body weight increase after up to 8 wk of fructose diet (18, 22). Others reported a body weight decrease after 13 days (10), 6 wk of fructose diet (8), or no difference compared with the control rats after 3 mo (27). Moreover, we observed renal hypertrophy but no difference in heart weight. DHA supplementation did not prevent these morphological effects of the fructose diet. The functional state of the total cardiac mitochondrial population was investigated in saponin-permeabilized fibers. The mitochondrial sensitivity for ADP was not influenced by either insulin resistance or dietary DHA, and the activity of creatine kinase was not altered. This small effect correlated with the slight effect of the fructose-diet on mitochondrial membrane fatty acids. Insulin resistance did not change the n-6 or n-3 PUFA content in the mitochondrial membrane. Nevertheless, the n-6-to-n-3 ratio was slightly increased in insulin-resistant rats, and this effect was reversed and overcompensated by dietary DHA. More alterations were observed in cardiac phospholipid fatty acid composition, including a significant decrease in both n-6 and n-3 PUFA content. Interestingly, this decrease in PUFA was balanced by an increase in monounsaturated fatty acid. The PUFA decrease was more pronounced for the n-3 than the n-6 series, producing a slight increase in the n-6-to-n-3 ratio. These results are in agreement with the previous observations by Rousseau et al. (22) using the same animal model. Moreover, dietary DHA compensated for all the changes in cardiac membrane n-3 PUFA composition induced by insulin resistance.

Regarding the use of various substrates, our results suggest that insulin resistance does not influence the mitochondrial main basal respiration pathways (pyruvate dehydrogenase, tricarboxylic acid cycle, and -oxidation). However, in ADP-stimulated respiration, the mitochondria of insulin-resistant rat hearts showed a significant decrease in glutamate-supported oxygen consumption. This result suggests a deficiency of either glutamate dehydrogenase or mitochondrial glutamate uptake to supply complex I with glutamate when the respiration is stimulated by ADP. Dietary DHA was unable to affect this respiration decrease. The slight increase of pyruvate-supported respiration in insulin resistance could be explained by a weak increase in pyruvate dehydrogenase activity. Dietary DHA did not affect this pyruvate-supported respiration in non-insulin-resistant rats but totally compensated for the effects of insulin resistance. This discrepancy between the effect of DHA on basal function and pathological function was previously reported for cardiac cell electrophysiology. Although incorporation of DHA in cardiomyocyte membranes had no effect on action potential and contraction parameters in normoxia, it significantly improved the recovery of the same parameters after ischemia and reperfusion (4). Cardiomyocyte incorporation of DHA also affects active but not basal oxygen consumption in the intact working heart (19). Neither insulin resistance nor dietary DHA individually affected the respiration of octanoate; however, the cross-interaction between the two factors was statistically significant, suggesting that DHA is only effective in insulin resistance, as it is for pyruvate. The observation that insulin resistance-induced alterations in pyruvate- and octanoate-supported respiration are fully compensated when the mitochondrial membrane n-3 fatty acid composition is maintained supports the hypothesis that the metabolic changes arise as a secondary response to membrane alteration rather than from a direct effect of the pathology. On the other hand, in contrast to the three- and eight-carbon fatty acids (pyruvate, octanoate), the respiration rate in the presence of a long-chain fatty acid (16-carbon palmitoylcarnitine) was affected by DHA supplementation in both sham control and HI groups. Nevertheless, insulin resistance did not affect the coupling between oxygen consumption and phosphorylation, irrespective of the metabolic substrate.

In conclusion, the present study identified that mitochondrial function was altered in both insulin-deficient and hyperinsulinemic insulin-resistant states. Furthermore, although some alterations were similar in the two pathologies, mitochondrial function was affected more by insulin deficiency than by insulin resistance. We also observed similar modifications in both mitochondrial membrane and cardiac cell membrane phospholipid fatty acid composition, but the effects of insulin deficiency were more pronounced and were associated with alterations in mi-CK activity. However, the results of this study demonstrate that DHA supplementation during development of insulin deficiency or insulin resistance has beneficial effects on mitochondrial function. DHA intake was able to prevent the decrease in function of mitochondrial membrane-bound systems, including mi-CK activity and mitochondrial sensitivity to ADP.

	ACKNOWLEDGMENTS

 
We thank the Cacao Butter and Hoffman-LaRoche for the generous gift of pure cacao butter and DHA, respectively, We are indebted to D. Anger (Hôpital Avicenne, Bobigny, France) and D. Porquet (Hôpital Necker, Paris, France) for insulin determinations, to P. L. McLennan (Dept. of Biomedical Science, Sidney, Australia) for editing the manuscript, and to A. M. Gueugneau and Drs. C. Héliès-Toussaint and S. Tardivel for experimental assistance.

	FOOTNOTES

 

Address for reprint requests and other correspondence: A. Grynberg, Lipides Membranaires et Fonctions Cardiovasculaires, Institut National de la Recherche Agronomique, UR 1154, Faculté de Pharmacie, Université Paris-Sud, 92290 Châtenay-Malabry, France (E-mail: grynberg{at}jouy.inra.fr).

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

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