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Am J Physiol Regul Integr Comp Physiol 274: R840-R848, 1998;
0363-6119/98 $5.00
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Vol. 274, Issue 3, R840-R848, March 1998

Menhaden oil prevents but does not reverse sucrose-induced insulin resistance in rats

Deborah A. Podolin, Ellis C. Gayles, Yuren Wei, Jeffrey S. Thresher, and Michael J. Pagliassotti

Department of Pediatrics and Center for Human Nutrition, University of Colorado Health Sciences Center, Denver, Colorado 80262

    ABSTRACT
Top
Abstract
Introduction
Methods
Results
Discussion
References

Although fish oil supplementation may prevent the onset of diet-induced insulin resistance in rats, it appears to worsen glycemic control in humans with existing insulin resistance. In the present study, the euglycemic, hyperinsulinemic (4× basal) clamp technique with [3-3H]glucose and 2-deoxy-[1-14C]glucose was used to directly compare the ability of fish oil to prevent and reverse sucrose-induced insulin resistance. In study 1 (prevention study), male Wistar rats were fed a purified high-starch diet (68% of total energy), high-sucrose diet (68% of total energy), or high-sucrose diet in which 6% of the fat content was replaced by menhaden oil for 5 wk. In study 2 (reversal study), animals were fed the high-starch or high-sucrose diets for 5 wk and then the sucrose animals were assigned to one of the following groups for an additional 5 wk: high starch, high sucrose, or high sucrose with 6% menhaden oil. Rats fed the high-starch diet for 10 wk served as controls. In study 3 (2nd reversal study), animals followed a similar diet protocol as in study 2; however, the reversal period was extended to 15 wk. In study 1, the presence of the fish oil in the high-sucrose diet prevented the development of insulin resistance. Glucose infusion rates (GIR, mg · kg-1 · min-1) were 17.0 ± 0.9 in starch, 10.6 ± 1.7 in sucrose, and 15.1 ± 1.5 in sucrose with fish oil animals. However, in study 2, this same diet was unable to reverse sucrose-induced insulin resistance (GIR, 16.7 ± 1.4 in starch, 7.1 ± 1.5 in sucrose, and 4.8 ± 0.9 in sucrose with fish oil animals). Sucrose-induced insulin resistance was reversed in rats that were switched back to the starch diet (GIR, 18.6 ± 3.0). Results from study 3 were similar to those observed in study 2. In summary, fish oil was effective in preventing diet-induced insulin resistance but not able to reverse it. A preexisting insulin-resistant environment interferes with the positive effects of menhaden oil on insulin action.

liver; triglycerides; fish oil

    INTRODUCTION
Top
Abstract
Introduction
Methods
Results
Discussion
References

INSULIN RESISTANCE is a characteristic feature of non-insulin-dependent diabetes mellitus (NIDDM) (4). The development of insulin resistance in humans involves both genetic and environmental influences. Several studies have demonstrated that the macronutrient composition of the diet is an important environmental determinant of the quality of insulin action (6, 9, 17, 19, 21, 23, 25). Although most studies examining the relationship between diet and insulin resistance have focused on the development of the impairment, relatively few studies have examined the effectiveness of dietary nutrients in reversing diet-induced insulin resistance (e.g., Ref. 12).

An association between elevated triglyceride levels and impaired insulin action has been demonstrated in both humans and rats (2, 16, 21, 24). Rats fed high-fat or high-sucrose diets exhibit impaired insulin action in conjunction with elevated muscle (21) or liver and plasma triglycerides (12, 17, 23), respectively. Dietary (6, 22) and pharmacological (11) interventions that reduce triglyceride concentration ameliorate insulin resistance. Long-chain n-3 fatty acids (i.e., omega -3 fatty acids) have been shown to prevent the onset of insulin resistance when given to rats concomitantly with high-sucrose and high-fat diets (8, 22, 26). However, omega -3 fatty acid administration in humans with NIDDM results, in many cases, in negative or no effect on glycemic control (for review, see Ref. 13). It is possible that the incongruity found in the effectiveness of fish oils is related to the existing environment in which they are administered. Therefore the aim of the present study was to investigate the effectiveness of 6% menhaden oil in both preventing and reversing whole body and tissue-specific insulin resistance in rats. In addition, we sought to further examine the relationship between insulin action and triglyceride levels (plasma, liver, and muscle) in all studies.

    METHODS
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Abstract
Introduction
Methods
Results
Discussion
References

Animals

Male Wistar rats (body weight ~150 g on arrival) were housed individually in stainless steel wire-bottom cages in a temperature-, humidity-, and light-controlled (12:12 h light-dark cycle) animal facility that met guidelines of the American Association for the Accreditation of Laboratory Animal Care. Animals had access to water ad libitum. The protocol was approved by the University of Colorado Health Sciences Center Animal Care Committee.

Diet

On arrival, animals were placed on a purified high-starch diet (Table 1; 68% of energy from corn starch, 20% protein, and 12% corn oil) for a 2-wk baseline period. Food intake was measured 3 days/wk, and body weight was measured 1 day/wk throughout all studies.

                              
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  		Table 1.   Composition of starch, sucrose, and sucrose plus menhaden oil diets

Dietary Protocol

The dietary protocol used is outlined in Fig. 1.


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  		Fig. 1.   Dietary protocol for animals fed their respective diets for 5, 10, or 20 wk. ST5, animals fed a 68% starch diet for 5 wk; SU5, animals fed a 68% sucrose diet for 5 wk; FO5, animals fed a diet of 68% sucrose + 6% of dietary fat as menhaden oil for 5 wk; ST10, animals fed a 68% starch diet for 10 wk; SU5/SU5, animals fed a 68% sucrose diet for 10 wk; SU5/ST5, animals fed a 68% sucrose diet for 5 wk then switched to a 68% starch diet for 5 wk; SU5/FO5, animals fed a 68% sucrose diet for 5 wk then switched to a 68% sucrose + 6% menhaden oil diet for 5 wk; ST20, animals fed a 68% starch diet for 20 wk; SU5/SU15, animals fed a 68% sucrose diet for 20 wk; SU5/ST15, animals fed a 68% sucrose diet for 5 wk then switched to a 68% starch diet for 15 wk; SU5/FO15, animals fed a 68% sucrose diet for 5 wk then switched to a 68% sucrose + 6% menhaden oil diet for 15 wk.

Prevention study (study 1). After the baseline period, animals were randomly assigned to one of three groups: high-starch diet, high-sucrose diet (Table 1; 68% of energy from sucrose, 20% protein, and 12% corn oil), high-sucrose diet containing 6% menhaden oil (fish oil) and 6% corn oil (Table 1). All animals were match fed for energy intake. Only the fish oil diet contained the antioxidant tert-butylhydroquinone, since results from the reversal study demonstrated that its inclusion in the starch or sucrose diets did not produce effects on any of the parameters measured. After 5 wk, euglycemic, hyperinsulinemic clamps were performed on animals fed high starch (ST5, n = 7), high sucrose (SU5, n = 8), or high sucrose plus fish oil (FO5, n = 7). These groups were used to examine whether the presence of fish oil in the diet could prevent sucrose-induced insulin resistance.

Reversal study (study 2). The reversal study comprised a 10-wk dietary period: 5 wk to produce insulin resistance and then another 5 wk for reversal of insulin resistance. After the baseline period, animals were randomly assigned to one of two groups: a high-starch (ST10) or high-sucrose diet. After this 5-wk period, sucrose-fed animals were switched to one of the following diets (which all contained antioxidant tert-butylhydroquinone to control for its inclusion in the fish oil diet) for 5 wk: high sucrose (SU5/SU5), high starch (SU5/ST5), or high sucrose containing 6% fish oil and 6% corn oil (SU5/FO5). All animals were match fed for energy intake. These groups were used to determine whether sucrose-induced insulin resistance could be reversed. After 10 wk, euglycemic, hyperinsulinemic clamps were performed on ST10 (n = 6), SU5/SU5 (n = 7), SU5/ST5 (n = 9), and SU5/FO5 (n = 7) animals.

The present series of studies sought to examine whether 1) fish oil in the context of a high-sucrose diet (i.e., a diet that produces insulin resistance) could prevent or reverse insulin resistance and 2) sucrose-induced insulin resistance could be reversed. Based on these aims, a starch diet containing fish oil would not be necessary.

Second reversal study (study 3). To determine whether the duration of exposure to fish oil was a factor in reversing sucrose-induced insulin resistance, the reversal period was extended 10 wk more. After the baseline period, animals were randomly assigned to one of two groups: a high-starch (ST20) or high-sucrose diet. After this 5-wk period, sucrose-fed animals were switched to one of the following diets (which all contained antioxidant tert-butylhydroquinone to control for its inclusion in fish oil diet) for 15 wk: high sucrose (SU5/SU15), high starch (SU5/ST15), or high sucrose containing 6% fish oil and 6% corn oil (SU5/FO15). All animals were match fed for energy intake. After 20 wk, euglycemic, hyperinsulinemic clamps were performed on ST20 (n = 9), SU5/SU15 (n = 8), SU5/ST15 (n = 9), and SU5/FO15 (n = 9) animals.

The fish oil diet was stored at -20°C and replaced every other day. All other diets were stored in a cool room out of direct light. Diets were based on American Institute of Nutrition recommendations (18) and prepared by Research Diets (New Brunswick, NJ).

Basal study. To obtain basal (i.e., without exogenous insulin infusion) measures of glucose kinetics and tissue glucose uptake, additional animals in each of the dietary groups were studied after 5, 10, or 20 wk.

Basal and Euglycemic, Hyperinsulinemic Clamps

After the specified dietary period (5, 10, or 20 wk), basal (saline infusion) or euglycemic, hyperinsulinemic clamps were performed. In preparation for the clamps, animals' carotid arteries and jugular veins were cannulated (23). Briefly, animals were anesthetized (im, 5 mg/kg acepromazine, 10 mg/kg xylazine, and 50 mg/kg ketamine), and cannulas (PE-50, Intramedic Clay Adams polyethylene tubing) were inserted in the carotid artery up to the aortic arch and into the jugular vein up to the vena cava, sutured to the respective vessel, and exteriorized through the back of the neck. Animals were allowed 4 days to recover and were at >= 92.5% of presurgery body weight on the day of study.

On the day of the experiment, extensions were added to catheters of 6- to 8-h-fasted animals for ease of sampling, and animals were allowed to rest for 20 min. After this, a baseline blood sample (for preexperiment plasma glucose and insulin concentrations) was taken along with blood pressure and hematocrit. Blood pressure was obtained from the artery using a calibrated electronic blood pressure unit (Stoelting, Wood Dale, IL). Then either basal or euglycemic, hyperinsulinemic clamps were initiated. The basal study consisted of a primed (12 µCi), continuous (0.1 µCi/min) infusion of high-performance liquid chromatography-purified [3-3H]glucose in saline for a 90-min period. Euglycemic, hyperinsulinemic clamps consisted of a primed, continuous infusion of insulin (4 mU · kg-1 · min-1) and [3-3H]glucose. A variable glucose infusion (10 or 20% dextrose) was used to maintain plasma glucose at baseline values. The glucose infusate was spiked with [3-3H]glucose to a specific activity similar to the plasma specific activity that would occur from the continuous infusion alone. This was done to minimize changes in glucose specific activity. The total experiment time was ~90 min during which arterial blood was sampled at ~5-min intervals, and the glucose infusion rate (GIR) was adjusted accordingly to maintain euglycemia (~7 mM). In both basal and clamp studies, a bolus injection of 2-deoxy-D-[1-14C]glucose (2-DG, 40 µCi) was administered via the carotid cannula at ~45 min (steady-state glucose levels during clamp). Blood samples were then taken at 2.5, 5, 10, 15, 20, 30, 35, 40, and 45 min. Circulating insulin concentration during the experiment was determined from the final blood sample taken. No more than 12% of the animals' blood volume (assumed to be 8% of body weight) was taken. After the last blood sample, the animal was anesthetized with pentobarbital sodium (70 mg/kg iv). The following tissues were removed, immediately frozen with precooled clamps, and then placed in liquid nitrogen for subsequent tracer, triglyceride, and metabolite analyses: liver, gastrocnemius, soleus, and biceps femoris. The epididymal, retroperitoneal, and mesenteric fat pads were removed, weighed, and frozen. A portion of subcutaneous fat, from the region above the biceps femoris, was also removed and frozen.

Analytic Methods

Plasma and tissue radioactivity. Plasma tracer samples were deproteinized with Ba(OH)2 and ZnSO4 and stored at 0°C overnight. An aliquot was dried to eliminate 3H2O and reconstituted with distilled water, and 3H and 14C disintegrations per minute were determined by liquid scintillation spectrometry (Beckman Instruments, Fullerton, CA). Skeletal muscle and adipose tissue 14C-phosphorylated 2-DG was determined on homogenates using ion-exchange chromatography and liquid scintillation counting (9).

Metabolites and hormones. Plasma glucose levels were determined by the glucose oxidase method (7) using a Beckman glucose analyzer (Fullerton, CA). Tissue triglycerides were extracted (16, 21), and plasma and tissue concentrations were determined with assay kit 320-A (Sigma, St. Louis, MO). Plasma insulin was measured by radioimmunoassay (Linco Research, St. Louis, MO). Gastrocnemius glucose 6-phosphate (G-6-P) concentrations were measured by the method of Lang and Michal (10).

Glucose kinetics. Basal rates of glucose appearance (Ra) and disappearance (Rd) were estimated by isotope dilution (3). Rates of endogenous glucose Ra (Endo Ra) and Rd during the euglycemic, hyperinsulinemic clamp were determined as previously described (5). Samples were collected under steady-state conditions to avoid underestimation of Endo Ra. Values for glucose and insulin concentration, glucose specific activity, Endo Ra, and Rd are the average of three samples taken over the final 10-min steady-state period.

Tissue-specific glucose uptake. The estimation of glucose uptake in individual tissues (R'g) using accumulation of phosphorylated 2-DG is based on the fact that 2-DG is trapped in most tissues, except for liver, and undergoes negligible further reaction. The decay curve of plasma 2-DG after a bolus injection was determined over a 45-min period, and specific activity was integrated. The integrated specific activity was divided into the tissue phosphorylated 2-DG level to yield R'g. The use of R'g as a relative index of glucose uptake in individual tissues is based on the assumption that any difference between 2-DG and glucose is unaffected by the experimental conditions (9, 23).

Data Analyses

Glucose kinetic data presented are the average of three time points taken under steady-state conditions. Steady-state conditions were defined as <1.0%/min change in glucose specific activity. Experiments were excluded if these steady-state conditions did not exist or if the coefficient of variation (CV) of the plasma glucose level during the last 45 min of the clamp was >10%. The CV of the plasma glucose level over this time period for included experiments was 5.6 ± 0.4% and was not significantly different among groups. Data are reported as means ± SE. Determination of statistical significance was performed by analysis of variance (ANOVA) with Newman-Keuls post hoc test for determination of specific differences (P < 0.05).

    RESULTS
Top
Abstract
Introduction
Methods
Results
Discussion
References

Basal Study

Total body weight gain, energy intake, and final body weight were not different between basal (data not shown) and clamp animals for each dietary period. The basal rate Ra (9.2 ± 0.2 mg · kg-1 · min-1) and Rd (9.1 ± 0.1 mg · kg-1 · min-1) were not significantly different among the different dietary groups (range, 7.6-10.9 mg · kg-1 · min-1, n = 5-7/group). This was also the case for basal rates of skeletal muscle R'g (average of gastrocnemius, soleus, and biceps femoris, 1.4 ± 0.2 µg · g-1 · min-1) and adipose tissue R'g (average of epididymal, retroperitoneal, and mesenteric fat pads and a portion of subcutaneous fat from the region above the biceps femoris, 1.0 ± 0.1 µg · g-1 · min-1). These data were therefore used as representative basal values. Gastrocnemius G-6-P levels were not significantly different among groups (0.4 ± 0.2 µmol/g).

Prevention Study (Study 1)

Energy- and clamp-related data. Energy intake and body weight gain during the dietary period were not significantly different among groups (Table 2). The summed weight of the epididymal, retroperitoneal, and mesenteric fat pads was not significantly different among groups (pooled average, 18.9 ± 1.1 g). Before the clamp, plasma glucose levels were not significantly different among groups (Table 2). Plasma insulin levels were significantly elevated in SU5 compared with ST5 and FO5. The steady-state plasma glucose and insulin levels during the clamp were not significantly different among groups (Table 2). Arterial blood pressure was not significantly different among groups (Table 2).

                              
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  		Table 2.   Energy- and clamp-related data from animals fed their respective diets for 5 wk

GIR, glucose kinetics, and tissue R'g. The GIR (mg · kg-1 · min-1) necessary to maintain euglycemia was 17.0 ± 0.9 in ST5, 10.6 ± 1.7 in SU5, and 15.1 ± 1.5 in FO5. Endo Ra was significantly higher (66%) and glucose Rd significantly lower in SU5 compared with ST5 and FO5 (Fig. 2). Muscle and adipose tissue R'g were significantly reduced in SU5 compared with ST5 and FO5 (Table 3).

G-6-P and triglycerides. Gastrocnemius G-6-P levels were significantly reduced in SU5 compared with ST5 and FO5 (Table 3). Plasma triglyceride levels were significantly elevated in SU5 compared with ST5 and FO5 (Fig. 3). Liver triglyceride levels were significantly elevated in both SU5 and FO5 (Fig. 3). Muscle triglyceride levels were significantly elevated in FO5 compared with SU5 (Fig. 3).


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  		Fig. 2.   Endogenous glucose appearance (top) and glucose disappearance (bottom) during euglycemic, hyperinsulinemic clamps in animals fed high starch (ST5; n = 7), high sucrose (SU5; n = 8), or high sucrose with 6% menhaden oil (FO5; n = 7) for 5 wk. * Significantly different from all other groups (P < 0.05). ddager  Significantly different from starch control animals (P < 0.05).

                              
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  		Table 3.   Tissue R'g and G-6-P concentration after euglycemic, hyperinsulinemic clamps in animals fed their respective diets for 5 wk

Reversal Study (Study 2)

Energy- and clamp-related data. Energy intake and body weight gain during the dietary period were not significantly different among groups (Table 4). The summed weight of the epididymal, retroperitoneal, and mesenteric fat pads was not significantly different among groups (pooled average, 34.4 ± 2.2 g). Before the clamp, plasma glucose levels were not significantly different among groups (Tables 4). Plasma insulin levels were significantly elevated in SU5/SU5 and SU5/FO5 compared with ST10 and ST5/ST5. The steady-state plasma glucose and insulin levels during the clamp were not significantly different among groups (Table 4). Arterial blood pressure was not significantly different among groups (Table 4).


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  		Fig. 3.   Triglyceride concentration in plasma (top), liver (middle), and gastrocnemius muscle (bottom) in animals fed high starch (ST5; n = 7), high sucrose (SU5; n = 8), or high sucrose with 6% menhaden oil (FO5; n = 7) for 5 wk. * Significantly different from all other groups (P < 0.05).

                              
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  		Table 4.   Energy- and clamp-related data from animals fed their respective diets for 10 wk

GIR, glucose kinetics, and tissue R'g. GIR (mg · kg-1 · min-1) was 16.7 ± 1.4 in ST10, 7.1 ± 1.5 in SU5/SU5 (P < 0.05 vs. ST10 and SU5/ST5), 4.8 ± 0.9 in SU5/FO5 (P < 0.05 vs. ST10 and SU5/ST5), and 18.6 ± 3.0 in SU5/ST5. Endo Ra was significantly higher and glucose Rd significantly lower in SU5/SU5 and SU5/FO5 compared with ST10 and SU5/ST5 (Fig. 4). Skeletal muscle and adipose tissue R'g were significantly reduced in SU5/SU5 and SU5/FO5 compared with ST10 and SU5/ST5 (Table 5).


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  		Fig. 4.   Endogenous glucose appearance (top) and glucose disappearance (bottom) during euglycemic, hyperinsulinemic clamps in animals fed high starch (ST10; n = 6), high sucrose (SU5/SU5; n = 7), high sucrose followed by high starch (SU5/ST5; n = 9), or high sucrose followed by high sucrose with 6% menhaden oil (SU5/FO5; n = 7) for 10 wk. * Significantly different from all other groups (P < 0.05).

                              
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  		Table 5.   Tissue R'g and G-6-P concentration after euglycemic, hyperinsulinemic clamps in animals fed their respective diets for 10 wk

G-6-P and triglycerides. Gastrocnemius G-6-P levels were significantly reduced in SU5/SU5 and SU5/FO5 compared with ST10 (Table 5). Plasma triglyceride levels were significantly elevated in SU5/SU5 compared with ST10 and SU5/ST5 (Fig. 5). No significant differences were evident in liver or muscle triglyceride concentration among groups (Fig. 5).


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  		Fig. 5.   Triglyceride concentration in plasma (top), liver (middle), and gastrocnemius muscle (bottom) in animals fed high starch (ST10, n = 6), high sucrose (SU5/SU5, n = 7), high sucrose followed by high starch (SU5/ST5, n = 9), or high sucrose followed by high sucrose with 6% menhaden oil (SU5/FO5, n = 7) for 10 wk. dagger  Significantly different from ST10 and SU5/ST5 (P < 0.05).

Second Reversal Study (Study 3)

Energy- and clamp-related data. Energy intake and body weight gain during the dietary period were not significantly different among groups (Table 6). The sum of fat pads (pooled average, 50.2 ± 3.9 g) was not significantly different among groups. Preglucose, clamp glucose, and clamp insulin levels as well as arterial blood pressure (pooled average, 115.6 ± 8.1 mmHg) were not significantly different among groups (Table 6). Preinsulin levels were significantly elevated in SU5/SU15 and SU5/FO15 compared with ST20 and SU5/ST15 (Table 6).

                              
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  		Table 6.   Energy- and clamp-related data from animals fed their respective diets for 20 wk

GIR, glucose kinetics, and tissue R'g. GIR was decreased ~30% in SU5/SU15 and SU5/FO15 compared with ST20 and SU5/ST15. Endo Ra was significantly elevated in SU5/SU15 and SU5/FO15 compared with ST20 and SU5/ST15 (Fig. 6). Gucose Rd was significantly decreased in SU5/SU15 and SU5/FO15 compared with ST20 and SU5/ST15 (Fig. 6). Muscle R'g (µg · g-1 · min-1) was significantly reduced in SU5/SU15 (2.4 ± 0.5) and SU5/FO15 (4.2 ± 1.3) compared with ST20 (10.6 ± 1.7) and SU5/ST15 (10.3 ± 1.6). Adipose tissue R'g was significantly reduced in SU5/SU15 (0.6 ± 0.1) and SU5/FO15 (1.9 ± 0.7) compared with ST20 (3.9 ± 0.9) and SU5/ST15 (4.9 ± 1.2).


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  		Fig. 6.   Endogenous glucose appearance (top) and glucose disappearance (bottom) during euglycemic, hyperinsulinemic clamps in animals fed high starch (ST20; n = 9), high sucrose (SU5/SU15; n = 8), high sucrose followed by high starch (SU5/ST15; n = 9), or high sucrose followed by high sucrose with 6% menhaden oil (SU5/FO15; n = 9) for 20 wk. * Significantly different from all other groups (P < 0.05).

G-6-P and triglycerides. Gastrocnemius G-6-P levels (µmol/g) were significantly reduced in SU5/SU15 (0.4 ± 0.2) and SU5/FO15 (0.3 ± 0.1) compared with ST20 (0.8 ± 0.1) and SU5/ST15 (0.8 ± 0.2). Plasma triglyceride levels were significantly elevated in SU5/SU15 compared with the other groups (Fig. 7). Although not statistically significant, liver triglyceride levels were increased ~35% in SU5/SU15 (Fig. 7). Muscle triglyceride concentrations were not significantly different among groups (Fig. 7).


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  		Fig. 7.   Triglyceride concentration in plasma (top), liver (middle), and gastrocnemius muscle (bottom) in animals fed high starch (ST20, n = 9), high sucrose (SU5/SU15, n = 8), high sucrose followed by high starch (SU5/ST15, n = 9), or high sucrose followed by high sucrose with 6% menhaden oil (SU5/FO15, n = 9) for 20 wk. * Significantly different from all other groups (P < 0.05).

    DISCUSSION
Top
Abstract
Introduction
Methods
Results
Discussion
References

In the present study, a high-sucrose diet produced insulin resistance over a 5-wk period. Inclusion of 6% menhaden oil in the high-sucrose diet, without a change in total fat content, prevented the sucrose-induced impairment in insulin action. When the sucrose diet containing menhaden oil was given to insulin-resistant animals for 5 wk, insulin action on glucose metabolism remained impaired. Thus the presence of a preexisting insulin-resistant state interfered with the beneficial effects of menhaden oil on insulin action. This was not due to an inability to reverse sucrose-induced insulin resistance, because switching the insulin-resistant animals back to the control diet effectively reversed the insulin-resistant state.

The inclusion of 6% menhaden oil in the sucrose diet prevented the development of insulin resistance. This finding is consistent with previous work in which fish oil was included in either a high-sucrose (8) or a high-fat (22) diet. Prevention of insulin resistance in the present study cannot be attributed to a change in the percentage of fat in the diet, since both the control and sucrose plus fish oil diets consisted of 12% fat (% of total energy).

The mechanism or mechanisms responsible for the apparent fish oil-induced prevention of sucrose-induced insulin resistance are unknown. Previous studies (16, 21, 24) have demonstrated a strong association between elevated triglyceride concentration and diet-induced insulin resistance. Specifically, sucrose-induced hepatic insulin resistance was significantly correlated with hepatic triglyceride levels, whereas peripheral insulin resistance was significantly correlated with plasma triglyceride levels (16). Thus one explanation for the prevention of sucrose-induced insulin resistance may be related to the hypolipidemic actions of fish oil. However, in the prevention study (study 1), the presence of fish oil prevented the increase in plasma but not liver triglyceride levels previously observed with high-sucrose feeding. These data suggest that the prevention of peripheral but not hepatic insulin resistance may be related to the ability of fish oil to prevent plasma hypertriglyceridemia.

The prevention of hepatic insulin resistance appears to be independent of hepatic triglyceride concentration. We have recently observed that fluidity was decreased and the saturated fatty acid content in phospholipids and triglycerides increased in the sinusoidal plasma membrane of hepatocytes isolated from sucrose-fed animals (17a). Thus the effectiveness of menhaden oil in preventing the development of sucrose-induced insulin resistance may be due to the amelioration of sucrose-induced changes in membrane fluidity and/or membrane fatty acid composition. The laborious analyses that were required in the present series of studies made analysis of membrane lipids and fluidity impossible in the present study; however, this possibility is presently being investigated. Alternatively, prevention of sucrose-induced insulin resistance when fish oil is present in the diet may be related to other unidentified mechanisms.

We hypothesized that if the inclusion of 6% fish oil in the high-sucrose diet was able to prevent insulin resistance, the same diet when given to insulin-resistant animals for 5 wk would reverse the impaired insulin action. However, this did not occur. In fact, providing this diet for as long as 15 wk did not result in significant improvement in sucrose-induced insulin resistance.

A possible explanation for the ability of the fish oil diet to prevent but not reverse insulin resistance may involve the presence of the insulin-resistant environment. It is possible that the insulin-resistant state alters the partitioning and/or metabolism of omega -3 fatty acids, rendering them ineffective with respect to insulin action. Nagy et al. (15) have reported that a diet high in one of the components of fish oil, docosahexaenoic acid (DHA), resulted in reduced glucose uptake in adipocytes under basal and insulin-stimulated conditions. Thus, if the insulin-resistant state resulted in greater utilization and/or accumulation of DHA, there might be no improvement in insulin action. In addition, the insulin-resistant state, once established, may be maintained by factors different from those involved in its development. For example, the presence of hyperinsulinemia or changes in the hepatocyte plasma membrane (17a) may impair the efficacy of fish oil to reverse insulin resistance.

When the prevention and reversal studies are considered together, there appears to be a dissociation between triglyceride concentration and insulin resistance. This dissociation was observed in the prevention study in which sucrose-induced hepatic insulin resistance was prevented despite the presence of significantly elevated hepatic triglyceride concentrations. In study 2, insulin-resistant animals fed the sucrose and fish oil diet exhibited impaired insulin action in the periphery despite normal plasma triglyceride concentration. It seems likely that if the triglyceride pool contributes to changes in insulin action (22), it does so through its turnover or metabolism and/or composition rather than simply via its concentration. It should be emphasized that we did not observe a significant increase in liver triglyceride concentrations in either the SU5/SU5 or SU5/SU15 groups. This suggests that the above statements be interpreted with caution.

We measured gastrocnemius muscle G-6-P in the present study to determine whether sucrose-induced insulin resistance involved changes in the transport and/or phosphorylation steps of glucose metabolism. In the present study, animals exhibiting insulin resistance had decreased rates of glucose uptake and gastrocnemius G-6-P levels compared with controls. We interpret this to mean that there is impairment in glucose transport and/or phosphorylation in the gastrocnemius muscle of the insulin-resistant animals. We have previously demonstrated that sucrose-induced impairments in the glucose metabolic pathway also occur distal to G-6-P (16, 17). Whole body glycolysis was not different in sucrose-induced insulin-resistant animals; however, tracer-estimated muscle glycogen synthesis and glycogen synthase activity were (16, 17). It appears that sucrose-induced insulin resistance involves several steps in glucose metabolism.

Switching the sucrose-induced insulin-resistant animals back to a high-starch diet reversed sucrose-induced insulin resistance. Whether the improvements in insulin action observed were due to a reversal of the sucrose-induced impairments and not merely a compensatory mechanism is unclear. It is noteworthy that although 5 wk on the high-starch diet was sufficient to reverse sucrose-induced insulin resistance, the presence of fish oil in the sucrose diet was unable to do so over a period of even 15 wk.

It may be observed that some differences in clamp insulin levels existed among dietary groups. It may be suggested that the slightly lower (nonsignificant) clamp insulin levels contributed to the inability of the fish oil diet to reverse insulin resistance. Previous data on the dose-response relationship between insulin concentration and Ra, Rd, and GIR suggest that the differences in clamp insulin levels observed in the present study would result in little difference in any of these parameters (maximal contribution of 0.4 mg · kg-1 · min-1) (17).

It should be noted that although 6% menhaden oil was effective in preventing insulin resistance, it is possible that a higher concentration is needed to reverse it. Lombardo et al. (12) have reported that the glucose intolerance resulting from a high-sucrose diet (64% of total energy) could be reversed with the inclusion of 16% cod liver oil (% of total energy). It may also be possible that difference in the polyunsaturated-to-saturated ratio between the two fish oils (1.23 for cod liver oil vs. 0.88 for menhaden oil) may contribute to differences in the effectiveness of fish oil in reversing insulin resistance. Clearly, more investigation in this area is warranted.

Insulin resistance and hypertension are characteristic features of syndrome X. A number of studies examining diet and insulin resistance have found blood pressure and insulin resistance to be tightly associated (e.g., Ref. 20). However, in the present study and previous studies in our laboratory (e.g., Ref. 16), we have found no relationship between diet-induced insulin resistance (high sucrose and high fat) and hypertension. In fact, none of the groups in this study exhibited any significant elevations in arterial blood pressure. It appears that elevated blood pressure is involved in some forms of insulin resistance but not in others.

Results from the present study indicate that sucrose-induced insulin resistance is prevented by replacing half of the corn oil (6% of total energy) with menhaden oil. However, once insulin action was impaired, fish oil feeding was not able to reverse it. These data imply that the beneficial effects of fish oil consumption are offset by the insulin-resistant state. They also suggest that fish oil administration should be used with caution. Furthermore, the present study has shown that sucrose-induced insulin resistance is reversible. This finding may have implications for the role of dietary carbohydrates in the treatment of insulin-resistant states.

    ACKNOWLEDGEMENTS

This work was supported by National Institute of Diabetes and Digestive and Kidney Diseases Grant DK-47416 and Bristol-Meyers Squibb/Mead Johnson. D. A. Podolin was funded by National Institute of Diabetes and Digestive and Kidney Diseases Institutional Training Grant DK-07658. We acknowledge the metabolic core of the Colorado Clinical Nutrition Research Unit (National Institute of Diabetes and Digestive and Kidney Diseases Grant P30-DK-48520-01) for assistance with insulin measurements.

    FOOTNOTES

Address for reprint requests: M. J. Pagliassotti, Dept. of Pediatrics, 4200 E. Ninth Ave., Campus Box C225, University of Colorado Health Sciences Center, Denver, CO 80262.

Received 22 August 1997; accepted in final form 5 December 1997.

    REFERENCES
Top
Abstract
Introduction
Methods
Results
Discussion
References

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AJP Regul Integr Compar Physiol 274(3):R840-R848
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