INTRODUCTION

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DIETARY NUTRIENTS HAVE A PROFOUND influence on insulin action, and therefore, may contribute to the development of glucose intolerance and type II diabetes (10). The ability of dietary nutrients to influence insulin action is dependent on a number of factors including the amount of the nutrient in the diet, the duration of exposure to the nutrient, and other lifestyle factors (e.g., physical activity). For example, Harris and Kor (12) demonstrated that a reduction in dietary fat from 40 to 30% of total energy produced rapid improvement in insulin action in rats. Similarly, variations in the amount of sucrose in diets containing similar total amounts of carbohydrate influenced the magnitude of increase in fasting levels of insulin, glucose, and triglycerides in humans (21, 32) and the tissue distribution of insulin resistance in rats (26). It also appears that the duration of exposure to either a high-fat (19) or high-sucrose (27) diet will influence the tissue distribution of insulin resistance. Finally, exercise training appears to influence the ability of a high-fat diet to produce insulin resistance (13, 16, 18). Clearly, to understand diet-induced insulin resistance, consideration must be given to these factors.

In addition, it is currently unclear if age can modify the ability of dietary nutrients to influence insulin action. High-sucrose diet-induced increases in glucose-stimulated pancreatic insulin secretion appear to be greater in older (>12 mo) compared with younger (<4 mo) rats (11). Chevalier et al. (4) demonstrated that a high-sucrose diet (70% by weight) increased serum triglycerides in older (body wt 500 g at start of dietary period) compared with weanling rats. This difference in the triglyceride response to a high-sucrose diet at different points in the life span may have implications for sucrose-induced changes in insulin action (27, 33). These data suggest that dietary nutrient-induced changes in insulin secretion, triglyceride concentration, and, potentially, insulin action may be significantly influenced by the age at which a given dietary nutrient is provided. The aim of the present study was to investigate the effects of age on diet-induced insulin resistance. Toward this end, the effects of a high-starch (control diet), high-sucrose diet (HSD), and high-fat diet (HFD) on whole body and tissue-specific insulin action were determined in rats at four different points in the life span.

	METHODS

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Animals

Diet and Feeding Protocol

After the baseline period, animals were randomly assigned to one of three dietary groups: STD, HSD (Table 1; 68% of energy from sucrose, 20% protein, 12% corn oil), or HFD (Table 1; 35% of energy from corn starch, 20% protein, 45% corn oil). These groups underwent a matched-energy feeding paradigm for an additional 5 wk. The feeding paradigm consisted of matching energy intake in the HSD and HFD groups to the ad libitum energy intake of the STD group. Some of the data for the STD groups have been published previously (23) and are used here for comparison purposes with the HSD and HFD groups.

Basal and Euglycemic, Hyperinsulinemic Clamp Studies

On the day of the experiment, extensions were added to catheters of 6- to 8-h fasted animals. The extensions allowed access to catheters for infusion and sampling without disturbing the animal. After placement of the extensions, animals were allowed to rest for ~20 min. After this, a baseline blood sample was taken followed by measurement of arterial blood pressure. This latter measurement was obtained 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 90 min. Euglycemic, hyperinsulinemic clamps consisted of a primed, continuous infusion of insulin (4 mU · kg¹ · min¹) 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 (~125 mg/dl). 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. Plasma insulin concentrations during the experiment were determined from the final blood sample taken. No more than 12% of the animals' blood volume was taken (total blood volume assumed to be 8% of body wt). After the final blood sample, the animal was anesthetized with pentobarbital sodium (50 mg/kg iv). The following tissues were removed, immediately frozen with precooled clamps, and then placed in liquid nitrogen for subsequent tracer and metabolite analyses: 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

Metabolites, hormones, and enzymes. Plasma glucose levels were determined by the glucose oxidase method (15), using a Beckman glucose analyzer (Fullerton, CA). 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 (20). Glycogen concentration was measured according to the procedures of Chan and Exton (3). Glycogen synthase was measured according to the procedures of Nuttal and Gannon (25). Total protein was measured according to the methods of Lowry et al. (22).

Glucose kinetics. Basal rates of glucose disappearance (Rd) were estimated by isotope dilution (6). Rd during the euglycemic, hyperinsulinemic clamp was determined as previously described (8).

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 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 (17).

Cell sizing and number. Fat cell size was determined by measuring the diameter of 50 collagenase-treated cells under a microscope (7). Fat cell number (FCN) was determined using the following formula: number of cells/g = average cell size (pl) × 0.95 (ng lipid/1 pl) × g/10⁹ ng. This product was then multiplied by the number of grams of tissue to get FCN. We have previously found that this method correlates strongly (r > 0.9) with FCN determinations based on directly measured lipid content (unpublished data).

Data analysis. Data are expressed as means ± SE. Data were analyzed by two-way ANOVA with age and diet as grouping factors. Significant (P < 0.05) diet, age, or diet and age effects were determined from the two-way ANOVA F statistic. Diet and age effect is used here and in RESULTS to denote a diet and age interaction. When significant differences (P < 0.05) were found among groups, pairwise multiple comparisons were made using the Newman-Keuls method.

	RESULTS

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Energy intake, body weight, and fat pad weight. Body weight [pooled average of diet (STD, HSD, HFD) and experimental (basal and clamp) groups] after the baseline period was 143 ± 5 g in W, 256 ± 5 g in Y, 442 ± 10 g in M, and 802 ± 19 g in O. During the 5-wk dietary period, there was a significant age effect on energy intake, body weight gain, and fat pad weight in both the basal and clamp groups (Table 2).

Blood pressure. There were no significant age or diet effects on arterial blood pressure. Pooled values were (in mmHg) 122 ± 6 in W, 118 ± 6 in Y, 114 ± 7 in M, and 125 ± 8 in O.

Circulating substrates and hormones. There were no significant age or diet effects on plasma glucose from basal studies (Table 3) or prior to euglycemic, hyperinsulinemic clamps (preclamp, Table 4). There was a significant diet effect on plasma triglycerides from basal studies (Table 3) and preclamp samples (Table 4). The diet effect was due to a significant increase in triglyceride concentrations in HSD. A significant age effect was observed on plasma free fatty acids (FFA) from basal studies (Table 3) and preclamp samples (Table 4). This age effect was due to a significant increase in FFA concentrations in O rats. Significant diet and age effects were observed on plasma insulin from basal (Table 3) and preclamp samples (Table 4). A significant increase in insulin levels was observed in Y and M rats fed the HSD, whereas with the HFD they were increased in W, Y, and M. 

There were no significant diet or age effects on plasma glucose levels during clamps (Table 4). There were significant age and diet effects on plasma FFA levels during clamps (Table 4). Age effects were primarily due to a significant increase in O rats, and diet effects were observed in Y and M rats. There was a significant diet effect on plasma triglycerides during clamps (Table 4). The diet effect was observed in Y and M rats fed the HSD. There was a significant age effect on plasma insulin levels during clamps (Table 4). Insulin levels were significantly increased in M and O rats compared with W and Y rats.

Basal and clamp Rd. A significant age effect was observed on basal Rd (Fig. 1). This age effect was due to a significant reduction in O rats. A significant age and diet effect was observed for clamp Rd (Fig. 1). Clamp Rd was significantly reduced in M and O rats compared with W and Y rats. The HFD significantly reduced clamp Rd in W, Y, and M rats, whereas in HSD it was reduced in Y and M rats only.

View larger version (27K): [in this window] [in a new window]   Fig. 1.   Basal (A) and insulin-stimulated (B) glucose disappearance in weanling (W), young (Y), mature (M), and older (O) rats fed a high-starch, high-sucrose, or high-fat diet for 5 wk. Values are means ± SE for n = 6-9 animals per group. * Significant age effect based on F statistic from two-way ANOVA (P < 0.05). + Significant diet effect based on F statistic from two-way ANOVA (P < 0.05).

Muscle R'g. There were significant diet and age effects on basal and clamp muscle R'g (Table 5). In the basal state, age effects were due to increased R'g in HFD. The HSD reduced clamp muscle R'g in Y and M rats, whereas in HFD R'g was reduced in W, Y, and M rats.

View this table: [in this window] [in a new window]   Table 5.   Muscle R'g (µg · g1 · min1 ) from basal and clamp studies

Gastrocnemius muscle metabolites and glycogen synthase. There were no significant diet or age effects on glycogen concentration, G-6-P concentration, or the percent active form of glycogen synthase under basal conditions (Table 6). There were significant age and diet effects on glycogen concentration and G-6-P concentrations during clamps (Table 6). There was a significant age effect on glycogen synthase activity during clamps (Table 6). Age effects were primarily due to significant reductions in glycogen concentration, G-6-P, and glycogen synthase in O rats. Specific diet effects were similar to those described for R'g. Total glycogen synthase activity (nmol · min¹ · mg protein¹) was not significantly different among age groups: 53 ± 5 in W, 58 ± 6 in Y, 50 ± 5 in M, and 46 ± 6 in O. There were no significant diet effects on total glycogen synthase (preceding data represent pooled averages within each age group).

Adipose tissue R'g. There was a significant age effect on basal adipose tissue R'g when expressed relative to cell number or total fat pad mass (Table 7). This was due to a significant increase in O rats. There were significant age and diet effects on clamp adipose tissue R'g when expressed per gram and total fat pad mass (Table 8). Although age was associated with a significant decrease in R'g in O rats when expressed per gram, R'g when expressed per pad was significantly increased in M and O rats.

	DISCUSSION

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In the present study, the effects of age on diet-induced peripheral insulin resistance were examined. The data suggest that age-associated peripheral insulin resistance occurs in both younger (Y vs. M rats) and older rats (M vs. O rats). Age-associated peripheral insulin resistance was accompanied by increased fat pad mass and circulating FFAs. Of particular interest, was the inability of the HSD to induce insulin resistance in weanling rats. Furthermore, an age-associated increase in basal skeletal muscle glucose uptake was observed in high-fat-fed rats.

Several studies have investigated insulin action on glucose metabolism in aging rat models (2, 9, 13, 24). Most recently, Barzilai and Rossetti (2) demonstrated that the reduction in insulin action on glucose metabolism typically observed between 2 and 4 mo of age in the rat was accompanied by a disproportionate increase in fat mass relative to lean body mass. In addition, a further increase in fat mass in 14-mo-old rats was not accompanied by a significant change in insulin responsiveness. In the present study, a significant decrease in insulin-stimulated glucose disappearance and skeletal muscle R'g was observed in starch-fed M vs. Y and W rats. This reduction was accompanied by a significant increase in fat pad weight. In contrast to the data from Barzilai and Rossetti (2), a significant reduction in insulin-stimulated skeletal muscle R'g was observed between starch-fed O vs. M rats. The most obvious explanation for this discrepancy may lie in the fact that the older rats in the present study (O group) were ~50% heavier than those used by Barzilai and Rossetti (2). In addition, the present study used a lower insulin infusion rate during the clamp procedure, thus circulating FFA concentrations were significantly increased in O rats compared with the other groups. These data suggest that competition between FFA and glucose may contribute to the quality of insulin action throughout the life span and emphasize the important link between fat pad mass and glucose metabolism.

Consistent with previous studies (4), sucrose-induced insulin resistance and hypertriglyceridemia were not observed in W rats. Thus it is possible that the lack of sucrose-induced insulin resistance in W rats resulted from the absence of hypertriglyceridemia. Although, previous studies have demonstrated a direct correlation between circulating triglycerides and insulin resistance (27, 33), no direct evidence has been provided to demonstrate that this relationship is causative. More likely, the lack of any sucrose-induced effects on insulin action and triglyceride levels relates to the differential handling of fructose and glucose (i.e., the monosaccharide moieties of sucrose) in W rats. Lipogenic enzyme activities in adipose tissue increase at weaning on exposure to a high-carbohydrate diet (5). It has been proposed that a high capacity for muscle glycogen synthesis (and insulin action on muscle glucose metabolism) during the peripubertal period may be required to sustain metabolic requirements associated with rapid growth (1, 30). Although, the active form of glycogen synthase in the gastrocnemius muscle was not significantly different among weanling, young, and mature rats in the present study, we did not determine the rate of glycogen synthesis. Future work is therefore required to determine whether requirements for muscle glycogen synthesis in weanling rats interfere with sucrose's ability to impair insulin action. Thus one difference between weanling and older rats may be in the partitioning of carbohydrate among tissues and intracellular pathways. This difference may be characterized by a greater extrahepatic disposition of carbohydrate in weanling rats. It is important to note that the HFD produced insulin resistance in weanling rats. These results suggest that simple sugars and polyunsaturated fatty acids produce insulin resistance via distinct mechanisms.

Basal skeletal muscle glucose uptake increased as a function of age in the HFD group. Previous studies have demonstrated a paradoxical increase in skeletal muscle glucose uptake in response to high lipid concentrations (14). Thus the age-associated increase in basal skeletal muscle glucose uptake in the HFD group may be a consequence of the combined high dietary lipid and the age-associated increase in circulating FFA (Table 4). If true, the lack of change in basal glucose uptake in the other two dietary groups may be a consequence of the low-fat diet. It is also possible that this adaptation may be required to maintain normoglycemia and/or to overcome the low carbohydrate content of the diet. Future studies are required to investigate these possibilities and the cellular sites responsible for the increase in skeletal muscle glucose uptake under basal conditions.

Basal rates of adipose tissue glucose uptake were significantly increased as a function of age when expressed on a per cell or per pad basis (Table 8). A significant decrease in the basal rate of glucose disappearance was also evident when older rats were compared with the three other age groups. These data suggest that the contribution of skeletal muscle and adipose tissue to basal glucose disposal increased as a function of age. Although age-associated reductions in insulin-stimulated adipose tissue R'g were observed when glucose uptake was expressed per gram of tissue, this was not the case when R'g was expressed per cell or per pad. In fact, the data from the present study suggest that the contribution of the fat pad mass to total glucose removal under hyperinsulinemic conditions increased with age. Whether this represents an adaptation that causes fat pad expansion or results from fat pad expansion requires further study (23).

The concentration of G-6-P and the percent active form of glycogen synthase under hyperinsulinemic conditions were decreased by age. These data imply that age can reduce both glucose transport/phosphorylation and glycogen synthesis (assuming that reductions in the active form translate into reductions in glycogen synthesis). Barzilai and Rossetti (2) also demonstrated a decrease in glycogen synthesis under hyperinsulinemic conditions in 14- vs. 2- and 4-mo-old male Sprague-Dawley rats (insulin levels were maximal for stimulation of glucose disposal). Both the HSD and HFD resulted in a decrease in the concentration of G-6-P under hyperinsulinemic conditions. Thus diet effects on insulin-stimulated glucose metabolism appear to be manifest at the level of transport/phosphorylation. The absence of diet effects on glycogen synthase may reflect the insulin level used in the present study. Previous studies have demonstrated a diet-induced reduction of insulin-stimulated glycogen synthesis at higher insulin concentrations (28). It is likely that the absence of diet effects in the older rats reflects the presence of significant age-related insulin resistance and the short duration of diet exposure relative to age in this group.

A significant age effect was observed on basal FFA concentrations. This observation is consistent with previous studies (2) and probably relates to changes in body composition in this animal model. In contrast to previous studies, we also observed a significant age effect on FFA concentrations under hyperinsulinemic conditions. This effect was due primarily to a significantly greater FFA concentration in the older rats. The changes in FFA concentrations are not surprising given the significant changes in body composition that occurred with aging. The sum of the retroperitoneal, epididymal, and mesenteric fat pads is highly correlated with total carcass lipid (n = 150, r = 0.93, P < 0.001; unpublished observations). Using this relationship we calculated total carcass lipid and lean body mass (by difference) in the present study. Total carcass lipid (in grams) was ~33, 56, 108, and 221 in W, Y, M, and O rats, respectively. Lean body mass (in grams) was ~267, 344, 462, and 599 in W, Y, M, and O rats, respectively. When considered as a percentage of total body weight, these values are consistent with other studies in which body composition was measured directly (2). For example, for body fat, these values would be 11% for W, 14% for Y, 19% for M, and 27% for O rats. It would appear that the large change in body fat mass in O rats likely contributed to the further reductions in insulin action observed in skeletal muscle.

The lack of diet effects on glucose disappearance, muscle R'g, and G-6-P concentrations in the older group of rats deserves further comment. These data imply that a lower limit of insulin resistance exists for these parameters. Thus the ability of dietary nutrients, body composition, and circulating lipids to effect a change in these parameters may depend on the existing quality of insulin action. However, it is interesting to note that both the HSD and the HFD appeared to reduce insulin stimulation of the active form of glycogen synthase in O compared with M rats. Thus the defined lower limit for interactive effects among dietary nutrients, body composition, and circulating lipids on insulin action may depend on the pathways and/or enzymes studied.

In summary, age significantly modified the effects of HFD and HSD on insulin-mediated glucose metabolism. The HSD produced insulin resistance in young and mature rats only. In addition, sucrose-induced hypertriglyceridemia was not observed in weanling rats. Thus weanling rats appear to handle simple sugars differently from older rats. The HFD produced insulin resistance in weanling, young, and mature rats. These diet-induced reductions in insulin action occurred independently of significant changes in body fat. Increased fat pad mass appears to contribute to age-associated changes in insulin action on glucose metabolism, but this effect can be modified by the presence of a HFD.