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Department of Biochemistry, School of Biochemistry, University of Litoral, Santa Fe, Argentina and * Endocrinology Research Center, Hospital Ricardo Gutierrez, Buenos Aires, Argentina
3To whom correspondence should be addressed. E-mail: ylombard{at}fbcb.unl.edu.ar.
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ABSTRACT |
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 TOP ABSTRACT MATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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KEY WORDS: • dyslipemia • ß cell function • fish oil • sucrose-rich diet
The impairment of insulin action (insulin resistance) is a major metabolic abnormality in individuals with noninsulin dependent diabetes mellitus. Experimentally, it is possible to induce a similar metabolic state by dietary manipulation. In vivo and in vitro studies in rats from our laboratory and others (1–7) have shown that diets high in sucrose lead to abnormal insulin sensitivity in the liver and peripheral tissues, including skeletal muscle. Our laboratory has shown that the sucrose-rich diet (SRD)3 rat model of dyslipidemia and insulin resistance is characterized by early hyperinsulinemia with normoglycemia after a short time (3–5 wk), and by normoinsulinemia with moderate hyperglycemia when the consumption of an SRD is extended for up to 15–40 wk (4,8). Plasma free fatty acid (FFA) levels increase throughout this time reaching the highest levels after 30–40 wk. At this point, the sucrose-fed rats were slightly overweight (9). In addition, we have recently reported that the biphasic patterns of glucose-stimulated insulin secretion from perifused islets deteriorate with the length of time the diet is consumed, showing an absence of the first peak with an increase in the second phase of hormone secretion at 30–40 wk (4,10).
Recent studies have demonstrated that the effect of lipids on ß cell function is complex. Glucose stimulation of insulin secretion is acutely potentiated by short-term exposure to fatty acids (11). In contrast, a chronic exposure to high levels of FFA impairs glucose-stimulated insulin secretion in vivo and in vitro, and could negatively influence ß cells through lipotoxicity leading to ß cell dysfunction (11,12). On the other hand, chronic hyperglycemia has been shown to have a deleterious effect on both insulin secretion and action, a concept termed glucotoxicity (13,14). The biochemical and molecular bases within the ß cell for either gluco- or lipotoxicity are still unknown. Several mechanisms have been proposed that could contribute to the dysfunction of the ß cells (15–19) such as changes in glucose oxidation, increase of triglyceride content within the islets and downregulation of several genes including Glut2 and glucokinase among others.
In addition to dislypidemia, a substantial increase of fat storage in nonadipose tissues (e.g., liver, skeletal and heart muscle) was shown in SRD-fed rats (4). Although we are unaware of any results on the triglyceride levels within the ß cells in these rats, we cannot exclude the possibility that an increase in islet fat content, as shown in the Zucker diabetic fatty rats (20), and/or an impaired glucose oxidation could be involved in the ß cell dysfunction.
The dietary intake of marine PUFA [fish oil (FO) rich in (n-3) fatty acids] has proven to be effective in lowering both plasma triglyceride and VLDL concentrations in experimental animals and in normal and hypertriglyceridemic men (21,22). It has been shown in rats that FO decreases the mRNA encoding of several enzyme proteins involved in de novo hepatic lipogenesis and enhances fatty acid oxidation (a peroxisome proliferator activity receptor 
(PPAR)-stimulated process) (23). In addition, FO prevents the onset of insulin resistance and dyslipidemia when fed to rats with high fat (24) or high sucrose diets (25,26). Few experimental studies have examined the effectiveness of FO in reversing or improving a preexisting state of dyslipidemia and insulin resistance (22,27–29). Moreover, there have been no studies designed to evaluate the effect of dietary FO on both insulin secretion patterns from isolated islets and insulin resistance in rats, in which a marked decline of the ß cell function is present after 30–40 wk of SRD consumption.
In the present study, rats fed an SRD up to 270 d were used as a model of ß cell dysfunction. In these rats the following questions were addressed: a) whether changes in the triglyceride content and/or altered pyruvate dehydrogenase complex (PDHc) activity, a key enzyme in the control of glucose oxidation, within the islet could contribute to the deterioration of ß cell function; and b) whether the replacement of corn oil by cod liver oil (FO) in the diet would be effective in reversing or improving the altered patterns of insulin secretion from isolated islets and their association with peripheral insulin sensitivity. To achieve these goals we studied the following: 1) the triglyceride content and the activity of PDHc in isolated ß cells; 2) the insulin secretion patterns from in vitro perifused isolated islets under the stimulation of different secretagogues (glucose, palmitate, L-arginine); and 3) the in vivo peripheral insulin sensitivity (euglycemic-hyperinsulinemic clamp).
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MATERIALS AND METHODS |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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Male Wistar rats initially weighing 170–185 g and purchased from the National Institute of Pharmacology (Buenos Aires, Argentina), were maintained under controlled temperature (22 ± 1°C), humidity and air flow condition, with a fixed 12-h light:dark cycle (light 0700 to 1900). They were initially fed a standard nonpurified diet containing by weight (g/100 g): 63 starch (corn, sorghum, wheat, oats, barley), 22 protein, 3.5 fat, 6 fiber, 1 vitamin mixture and 4 salt mixtures (Ralston Purina, St. Louis, MO). After 1 wk of acclimation, the rats were randomly divided into two groups. The experimental group (n = 78) received a purified SRD (62.5 g/100 g), while the control group (n = 36) received the same purified diet but with sucrose replaced by cornstarch (62.5 g/100 g) [high starch diet (CD)]. The experimental group received the SRD for 210 d after which the rats were randomly subdivided into two groups. The rats of the first subgroup continued the SRD up to 270 d of feeding. The second subgroup (SRD + FO) received the SRD in which the source of fat (corn oil, 8 g/100 g) had been replaced by FO (7 g/100 g plus 1 g/100 g of corn oil) from d 210 to 270. The control group received the CD throughout the experiment. The SRD without the addition of FO used from d 210 to 270 and the CD were balanced for the cholesterol and vitamins D and A present in the FO (Table 1). Diets were isoenergetic, providing 
16.3 kJ/g of food and were consumed by the rats ad libitum. Diets were prepared every day by adding the oils and base mixture containing the other nutrients. The oils and base mixture were separately stored at 4°C until preparation of the diet. Dietary fats were analyzed by capillary GC as previously described (22) (Table 2). Fatty acids constituting <0.4% of the total were not included. The weight of each rat was recorded twice each week during the experimental period. In a separate experiment, the individual energy intakes and weight gains of eight rats in each group and subgroup were assessed twice each week. At the end of the 270-d feeding period, except as otherwise indicated, food was removed at the end of the dark period (0700 h) and experiments were performed between 0900 and 1200 h. The experimental protocol was approved by the Human and Animal Research Committee of the School of Biochemistry, University of Litoral, Santa Fe, Argentina.
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Six rats from each dietary group were anesthetized with an intraperitoneal injection of pentobarbital (60 mg/kg body weight). The blood samples obtained from the jugular vein were rapidly centrifuged at 3500 x g for 15 min at 4°C and the plasma either immediately assayed or stored at -20°C and examined within 3 d. Plasma triglyceride, FFA and glucose levels were determined by spectrophotometric methods and insulin was measured by an immunoreactive assay as previously described (4). The immunoreactive insulin assays were calibrated against rat insulin standard (Novo Nordisk, Copenhagen, Denmark). The epididymal and retroperitoneal adipose tissues were removed and weighed.
Euglycemic clamp studies.
Whole body peripheral insulin sensitivity was measured using the euglycemic hyperinsulinemic clamp technique as previously described (10). Briefly, after 5 h of food deprivation 6 rats from each dietary group were anesthetized, a blood sample taken and glucose and insulin levels assessed. Afterwards, an infusion of highly purified porcine neutral insulin (Actrapid; Novo Nordisk) was administered at 5.69 nmol/(kg · h) for 2 h. Glycemia was maintained at a euglycemic level by infusing 200 g/L of glucose at a variable rate. The glucose infusion began 5 min after the insulin infusion started. The blood glucose concentration was measured using a Glucomether Analyzer (Boehringer Mannheim, Indianapolis, IN) within 2 min after the samples were obtained. The glucose infusion rate (GIR) during h 2 of the clamp study was taken as the net steady state of the whole-body glucose. In all studies, blood samples (0.3 mL) for insulin determination (10) were obtained at 60, 90 and 120 min. Details of the methodology have been previously described (10).
Perifusion of isolated islets.
Rats were decapitated and the islets were isolated by collagenase digestion and collected under a stereoscopic microscope as previously described (4,10). After the islets were washed twice with a Krebs-Ringer bicarbonate (KRB) buffer, groups of 30 to 40 islets isolated from each rat were loaded in a 13-mm chamber containing a 5-µm nylon membrane filter. Islets were perifused with KRB containing 3 mmol/L glucose, 250 mg/L essentially fatty acid–free bovine serum albumin, 40 mg/L dextran 70, pH = 7.4 at 37°C (constantly gassed with 95% O2, 5% CO2) at a flow rate of 0.9–1.2 mL/min. After a prewash period of 30 min, two basal samples were obtained. Then the KRB containing a high glucose concentration (16.5 mmol/L) was used until the end of the perifusion period (40 min). Aliquots from the effluent were collected at 1-min intervals until min 15, and then at 5-min intervals until min 40, and were stored at -20°C until insulin analysis. To compare the effect of different nutrients (insulin secretagogues) on insulin secretion (IS), the perifusion was performed for 40 min with KRB buffer as described above with 16.5 mmol/L glucose plus 0.5 mmol/L palmitic acid (as the sodium salt) or 5 mmol/L glucose plus 20 mmol/L arginine. In all experiments, aliquots from the effluent for insulin assays were collected as described above. Six rats from each dietary group were used in each experiment. Complete details of the methodology used have been previously described (4,10).
Islet lipid extractions.
Islets isolated from each experimental group as described above were used to determine the triglyceride content according to the methodology described by Briaud et al. (15). Briefly, 75 to 100 islets were washed with an ice-cold phosphate buffer and the pellets were resuspended in chloroform:methanol (2:1, vol:vol) and stored overnight at 4°C under N2. After washing with distilled water, the lower phase was evaporated under N2. The samples were resuspended in chloroform, and an aliquot quickly transferred to glass tubes in duplicate and air dried. The dry lipid was resuspended in Thesit (polyoxyethylene 9 lauryl ether) and dried as described above. Fifty µL of water was added and the tubes were vortexed and incubated in a 37°C shaking water bath for 10 min as described by Van Veldhoven et al. (31). A Sigma triglyceride (GPO-trinder; Sigma Chemical) reagent was added to the tubes that were gently mixed and incubated at 37°C for 5 min. The absorbance was read at 540 nm. The triglyceride content of the samples was located on a standard curve established in each assay with 1 to 50 µg of triolein.
Extraction and assay of PDHc activity from pancreatic islets.
The PDHc was extracted as described by Zhou et al. (32). Briefly, 300 islets were homogenized in a 300-µL homogenization buffer composed of 50 mmol/L HEPES (pH = 7.5), 0.2 mmol/L KCl, 3 mmol/L N-p-tosyl-L-lysine chlorometyl ketone, 0.1 g/L trypsin inhibitor from egg white, 200000 KIU/L aprotinin, 2% rat serum and 0.25% (vol/vol) Triton X-100. The PDHc was extracted by freezing/thawing (liquid nitrogen/room temperature) three times and passed through a 0.5-mL insulin syringe 10 times. The samples were centrifuged at 5000 x g for 10 min at 4°C and maintained on ice. An aliquot of the sample was mixed with ice cold potassium phosphate and NaF to a final concentration of 25 mmol/L each, and was used to determine the active form (PDHa) of PDHc. The total PDH activity was assayed after conversion of inactive PDH into active PDH complex with broad specificity phosphoprotein phosphatase, as we have previously described (28). The aliquots of islets extracts (150 µL) were incubated with 25 mmol/L MgCl2, 1 mmol/L CaCl2 and phosphoprotein phosphatase for 20 min at 30°C followed by the immediate assay of PDHc activity. PDHa and PDHt activities were spectrophotometrically determined at 30°C by measuring the reduction of NAD+ as we have previously described in detail (28). The enzyme activities were expressed as µU/islets.
Statistical analysis.
Results are expressed as means ± SEM. The significance between groups was determined by ANOVA followed by the inspection of all differences between pairs of means by the Newman Keul's test (33). When appropriate, differences within the group were determined by the Student's t test. Differences with P-values < 0.05 were considered significant.
Reagents.
Enzymes for assays, substrate and coenzyme were purchased from Sigma Chemical or from Boehringer Mannheim Biochemical. Cod liver oil was purchased from ICN (Costa Mesa, CA). All other chemicals were of reagent grade.
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RESULTS |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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Energy intakes and body weights were carefully monitored in all groups of rats throughout the experimental period. As we have previously shown (10), the increase (P < 0.05) in body weights (18%) and energy intakes in rats chronically fed an SRD for 210 d were still present when the SRD was fed for 270 d. Rats fed an SRD+FO from d 210 to 270 had a moderate reduction of weight gain compared with both SRD and CD groups (Table 3).
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At the end of the dark period (0700 h) plasma triglyceride, FFA and glucose concentrations were higher in rats fed the SRD for 270 d compared with age-matched controls. However, plasma insulin levels did not differ (Table 5). Confirming recent reports from our laboratory (10) plasma levels of the above metabolites were comparable in rats fed an SRD for 210 d (data not shown). A complete normalization of all these variables occurred in rats fed SRD+FO from d 210 to 270. Furthermore, the enhanced liver triglyceride concentration in the SRD-fed rats did not differ from the controls in those fed SRD+FO (Table 5).
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The postprandial blood glucose concentrations 5 h before the clamp were as follows: (mmol/L, mean ± SEM, n = 6) 5.25 ± 0.10 in CD-fed rats, 7.95 ± 0.18 in SRD-fed rats and 5.30 ± 0.15 in SRD+FO-fed rats. Plasma insulin levels similar to those recorded at the end of the dark period were obtained in all dietary groups (data not shown). The GIR, which measures insulin action in vivo, was lower (P < 0.01) in the SRD group (24.17 ± 4.34 µmol/(kg · min), n = 6) compared with CD-fed rats (64.45 ± 5.00 µmol/(kg · min), n = 6). However, in the SRD+FO group, the GIR did not differ from the controls (68.10 ± 6.30 µmol/(kg · min), n = 6). There were no changes in hematocrit from the start to the end of the clamp in any of the groups (data not shown).
Perifusion of isolated islets.
Rats fed the CD showed the classic biphasic pattern of glucose (16.5 mmol/L) stimulated IS. Perifused islets from SRD-fed rats showed an alteration of the biphasic patterns with an absence of the first peak and an increase in the second phase of hormone secretion compared with CD-fed rats (Fig. 1A). On the other hand, in the SRD+FO group the glucose-induced IS showed a pattern comparable with CD-fed rats (Fig. 1A).
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Under the stimulus of L-arginine (20 mmol/L plus 5 mmol/L glucose) the first peak and second phase of insulin release were lower in SRD-fed rats compared with age-matched CD-fed rats (Fig. 1C). The islets of SRD+FO-fed rats improved (P < 0.05) the hormone secretion patterns, although the insulin values did not reach those observed in the CD-fed rats.
Triglyceride concentrations and PDHc activity in isolated islets.
Rats fed an SRD showed an increase (P < 0.05) of the islet triglycerides content that was accompanied by a decrease (P < 0.05) in PDHa, the active form of PDHc, activity compared with CD-fed rats (Fig. 2A and B). However, in the SRD+FO-fed group neither variable differed from the CD group. Moreover, the total PDHc activity (PDHt) did not differ among the groups (µU/islet, mean ± SEM, n = 6) 8.50 ± 0.45 in the CD group, 9.90 ± 0.52 in the SRD group and 8.13 ± 0.70 in the SRD+FO group.
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DISCUSSION |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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An increase of pancreatic triglyceride concentrations and a decrease of the PDHc activity, a key enzyme in the control of glucose oxidation, was accompanied by highly deteriorated insulin secretion patterns in response to glucose stimulus. The islets acute exposure to palmitate in the perifusion medium did not improve insulin release. Moreover, hormone secretion under the stimulus of L-arginine was lower than those recorded in the isolated ß cells from CD-fed rats. These findings were accompanied by a sharp increase of plasma FFA and triglyceride levels, mild hyperglycemia and peripheral insulin resistance. An increase of epididymal and retroperitoneal fat tissues weights was accompanied by a moderate increase of body weight. The presence of FO in the diet completely normalized both the fat storage and the PDHc activity within the ß cells as well as the insulin secretion patterns stimulated by glucose. The hormone secretion under the stimulus of either palmitate or L-arginine was also improved. Dietary FO reversed the whole body peripheral insulin insensitivity, abnormal glucose homeostasis and the dyslipidemia, and decreased adipose tissue weight without detectable changes in basal plasma insulin levels.
A long-term exposure of the ß cells to excessive levels of FFA, triglyceride or both, either in vivo or in vitro, contributed to the ß cell dysfunction. Pancreatic islets from rats fed an SRD for 9 mo were chronically exposed to a metabolic milieu characterized by mild hyperglycemia with an oversupply of plasma triglyceride and FFA. Our results show that in these rats, an increase in the triglyceride storage within the islets occurred concomitantly with a reduction of the PDHc activity. A decrease flux through the PDHc is associated with lower PDHa (active form of PDHc) levels, and could involve activation of PDH kinase. The inhibition of the PDHc limits the conversion of pyruvate derived from glycolisis to acetyl CoA, diminishes the oxidative glucose metabolism, a signal for insulin secretion and synthesis (34), and therefore, could impair the glucose-induced insulin release. Zhou et al. (32) showed that PDHc activity decreases and PDH kinase activity increases in the islets of obese diabetic mice. Furthermore, the enlarged fat islets could release large amounts of fatty acids through islets lipolysis, and their acylCoA esters could also influence glucose oxidation. A marked elevation of triglyceride and a deposition of the fat droplet have been observed in islets of both OLETF rats, a model of hypertriglyceridemia associated diabetes (35) and ZDF rats (36).
As expected (37), the acute exposure of isolated islets to palmitate enhanced both the first and second phase of glucose-stimulated insulin secretion in CD-fed rats. However, an impaired insulin secretion pattern was observed in the presence of palmitate from the isolated islets of SRD-fed rats. This might also be coupled with the increased triglyceride content and low PDHc activity observed in the freshly isolated ß cells of SRD-fed rats. Chen et al. (38) recently demonstrated that perifused islets from insulin resistant rats fed a sucrose diet for 2 wk are more sensitive to the untoward effect of palmitate. Zhou et al. (16) showed that prolonged exposure to palmitate in normal rats increases triglyceride store and induces ß cell insensitivity to glucose. This was associated with a decrease of PDHc and an increase of PDH kinase activities. At present, we are unaware of studies regarding the mechanism/s behind the inhibition rather than stimulation upon acute exposure of palmitate in glucose-induced insulin release in the ß cells of the long-term SRD-fed rat.
The present results also show abnormal behavior of the ß cell response under the stimulus of L-arginine in vitro. Because L-arginine acts distally to any anticipated early metabolic signal, mechanism/s other than the increased fat storage and diminished PDHc activity may be involved in the ß cell dysfunction of rats fed a long-term SRD (18,34).
The increased fat storage content in nonadipose tissue is not limited to the ß cells in the SRD-fed rat. We have recently demonstrated a sustained increase of triglyceride and long-chain acyl CoA with a profound decrease of PDHc activity and a sharp deterioration of whole body peripheral insulin sensitivity in skeletal muscle (4). This condition may reflect the early start of type 2 diabetes, because many patients have chronically elevated plasma FFA and triglyceride levels, altered peripheral insulin sensitivity and loss of the first peak of insulin in response to glucose.
The second goal of the present study was to analyze the effect of FO on the reversion or improvement of the ß cell dysfunction and insulin resistance. The increased availability of plasma FFA and triglyceride was reduced because the dyslipidemia present in the SRD group was completely reversed after FO administration. In addition, in vivo peripheral insulin insensitivity was normalized with a decrease of epipidymal and retroperitoneal fat pad weights. The reduction of retroperitoneal tissue weight would indicate a reduction of body lipid (39). Moreover, the rats supplemented with FO gained very little weight relative to the other two groups, and this probably contributed to the improvement of insulin resistance.
The normalization of plasma lipid impacted the ß cell function. Triglyceride stores and PDHc activity were similar to those in the CD-fed rats, and the impaired glucose-stimulated insulin secretion from the ß cells was completely normalized. Moreover, acute exposure to palmitate or L-arginine improved insulin secretion. All of these changes were achieved by shifting the source of fat in the diet from corn oil to FO for 60 d.
De novo hepatic lipogenesis and VLDL secretion are increased in SRD-fed rats. Dietary PUFA of the (n-3) and (n-6) families suppress the transcription of hepatic genes encoding lipogenic (fatty acid biosynthesis) and glycolytic enzymes (e.g., L-pyruvate kinase). However, (n-3) but not (n-6) fatty acids suppress triglyceride synthesis, VLDL secretion and lower serum triglyceride levels (23). Moreover, Nescher et al. (40) recently showed that in rats, dietary FO, in contrast to safflower oil rich in (n-6) fatty acids, increases the fatty acid oxidative capacity of the liver through its ability to function as a ligand activator of PPAR and thereby induces the transcription of several genes encoding proteins affiliated with fatty acid oxidation. Therefore, a normalization of liver triglyceride content and a reduction of lipid flux to the nonadipose tissue could contribute to the reversal of the lipotoxicity associated with lipid overload.
In addition to the regulation of plasma lipid levels, the possible mechanisms involved in the effects of FO on insulin action are still unclear. Increasing evidence suggests that the fatty acid composition of membrane phospholipids of insulin target tissues is a critical factor that influences both the insulin secretion and its biological action (e.g., changes in membrane fluidity, diacylglycerol second messenger function) (41). We have recently observed an increase of (n-3) fatty acids [eicosapentaenoic acid 20:5(n-3) and docosahexaenoic acid 22:6(n-3)] in the phospholipids of the skeletal muscle of SRD-fed rats (unpublished results), and in agreement with the present results, peripheral insulin insensitivity is also reversed under the presence of dietary FO. Although our study did not analyze the fatty acid composition of the ß cell membrane, it is possible that changes in the type of fatty acids phospholipids could contribute to the reversion of impaired insulin secretion from the pancreatic islets of FO-treated SRD-fed rats.
In conclusion, the increased fat store and the decreased PDHc activity in the ß cells seem to play a key role in the abnormal insulin secretion of long-term SRD-fed rats. This is consistent with the reversion of these alterations after the administration of dietary FO. Finally, the present results suggest that lipotoxicity could participate in the progressive demise of the ß cell in this nutritional model of dyslipidemia and insulin resistance.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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2 Supported by Agencia Nacional de Promoción Científica y Tenológica (ANPCYT) and University of Litoral by Grants PICT 05–6960/BID 1201/OC and CAI+D 13/B 306 - 2002.
4 Abbreviations used: CD, control diet; FFA, free fatty acids; FO, fish oil (cod liver oil); GIR, glucose infusion rate; IS, insulin secretion; KRB, Krebs-Ringer bicarbonate; PPAR, peroxisome proliferator activity receptor ; PDHc, pyruvate dehydrogenase complex; SRD, sucrose-rich diet.
Manuscript received 19 June 2003. Initial review completed 7 July 2003. Revision accepted 1 October 2003.
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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