INTRODUCTION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

PREVIOUS WORK AT OUR LABORATORY demonstrated that the impaired glucose homeostasis and insulin insensitivity that accompany the hypertriglyceridemia that develops in normal Wistar rats fed a sucrose-rich diet (SRD) depend on both the amount of carbohydrate and the length of time the diet is administered (3, 14, 22). Thus plasma glucose and insulin evolve from normoglycemia and hyperinsulinemia after a short-term (3-5 wk) to moderate hyperglycemia with normoinsulinemia during a long-term (15 wk) feeding on a SRD, respectively. Moreover, plasma free fatty acid (FFA) levels significantly increase during a long-term feeding of a SRD (22). This is particularly interesting because there is accumulated evidence that the increased availability of FFA in muscle and liver (6, 19) is associated with a reduced ability of insulin to stimulate the utilization and storage of glucose (insulin resistance). Plasma FFA levels are affected by fat cell lipolysis, which is regulated by hormone-dependent and -independent lipolytic activities (26). Insulin has several major effects on fat metabolism, including inhibition of lipolysis and stimulation of glucose transport.

Studies on the mechanisms underlying the long-term metabolic consequences of high sucrose or fructose intake are limited (5, 7, 22, 31). Only scarce information is available, particularly regarding the role of the adipose tissue (a target tissue in the regulation of plasma FFA) in the development of dyslipidemia and insulin insensitivity induced by the long-term feeding of either a fructose or a sucrose diet. In this regard, Blakely et al. (5) showed that moderate (15-20% wt/wt) fructose feeding to Wistar rats for 9 mo resulted in increased epididymal fat padlipogenic enzyme activities, whereas plasma triglyceride and cholesterol levels remained unchanged. The activities of the pentose shunt and NADP-malate dehydrogenase enzymes increase significantly in the adipose tissue of rats fed a SRD for 12 mo (9). In the case of Wistar rats fed a SRD (63% wt/wt) for up to 3 mo, preliminary results of Soria et al. (37) showed a significant increase of the epididymal fat pad mass either as total tissue weight or relative to rat body weight. It is well known that the adipose tissue mass changes under different conditions, including age, endocrine status, and energy balance (15). Differences in body fat mass could be due to differences in either the number or the size of the cells. Moreover, increased fat cell size correlated with increased lipolysis (1, 41).

At present, we are unaware of any report concerning the role of the adipose tissue on the development and worsening of impaired glucose homeostasis and insulin sensitivity present in normal rats fed a SRD for a long time. This is particularly interesting because, as mentioned above, a different hormonal and metabolic milieu evolves from the early (3-5 wk) to the late (15 wk) stages of hypertriglyceridemia.

Therefore, the present study was conducted in rats kept for either 3 or 15 wk on a SRD to explore its effect on morphological and metabolic changes of adipose tissue that could contribute to the development of insulin resistance. To achieve this goal, we investigated in epididymal fat pads 1) the number, size, and distribution of cells; 2) the enzymatic activity of both hormone-sensitive lipase (HSL) and lipoprotein lipase (LPL); and 3) the effect of insulin on lipolysis. Also, the "in vivo" whole body peripheral insulin sensitivity was estimated by the euglycemic-hyperinsulinemic clamp technique after both SRD feeding periods.

	MATERIALS AND METHODS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Animals and diets. Male Wistar rats, weighing 180-200 g and purchased form the National Institute of Pharmacology (Buenos Aires, Argentina), were maintained in an animal room under controlled temperature (22 ± 1°C), humidity, and airflow conditions, with a fixed 12:12-h light-dark cycle (light 7:00 AM-7:00 PM). They were initially fed a standard laboratory chow (Ralston Purina, St. Louis, MO). After a week of acclimatization, rats were randomly divided into two groups: control and experimental. The experimental group received a semisynthetic SRD containing by weight (g/100 g): 63% sucrose, 17% casein free of vitamins, 5% corn oil, 10% cellulose, 3.5% salt mixture (AIN-93AM-MX), 1% vitamin mixture (AIN-93M-VX), 0.2% choline chloride, and 0.3% methionine. The composition of both the vitamin and salt mixtures added to the SRD complied with the recommendations made by the Final Report of the American Institute of Nutrition Ad Hoc Writing Committee on the Reformulation of the AIN-76 A Rodent Diet (25). Details of the methodology used have been described elsewhere (25). The control group received the same semisynthetic diet but with sucrose replaced by starch [high-starch diet (CD)]. The animals had free access to food and water and were maintained on their respective diets for a period of 15 wk. Both diets provided ~15.28 kJ/g of food. The weight of each animal was recorded twice a week during the experimental period. In a separate experiment, the individual caloric intake and weight gain of eight animals in each group were assessed twice a week.

Preparation of isolated adipocytes. The epididymal fat pads were removed via midline abdominal incision, weighed, and rinsed in isotonic saline solution a 37°C. Adipocytes were isolated according to the method of Rodbell (29), with minor modifications. Briefly, the fat pads were minced with scissors, placed in plastic flask, and incubated at 37°C in a Dubnoff shaking water bath (Precision Scientific Group, Chicago, IL) at 60 cycles/min for 1 h in a Krebs-Henseleit phosphate buffer (pH 7.4), containing 1.25 mM Ca²⁺, 4% bovine serum albumin essentially free of fatty acids, 5.5 mM glucose, and crude collagenase (1-2 mg/g of tissue) from Clostridium histolyticum. Subsequently, adipocytes were gently filtered through a ~200-mm-diameter nylon mesh to remove stroma and blood vessels, washed three times in fresh collagenase-free Krebs-Henseleit phosphate buffer, and allowed to separate from the infranatant by low-speed centrifugation (600 rpm). Isolated cells were then resuspended in collagenase-free buffer at 37°C.

Determination of fat cell size and fat cell number. The microscopic method of Di Girolamo et al. (11) was used to measure cell diameters. The sizing of 100 adipocytes was conducted by the same operator throughout the present study to improve precision as suggested by Khan et al. (18). For each experimental group of animals, the number of fat cells in similar class intervals of 2.5 µm was treated as a single variable, and the average value obtained for each class interval was used to construct a histogram representing the group. The mean diameter and volume for the entire fat cell population were calculated from the histogram according to Di Girolamo et al. (11). For the estimation of the fat pad cell number, the lipid content of 100-200 mg of fat tissues was extracted by the method of Folch et al. (13). Total cell number in the fat pad was calculated dividing the fat pad lipid content by the mean cell lipid weight. The lipid weight of the average fat cell was calculated from the mean cell volume assuming a lipid density of 0.915 (density of triolein).

Adipocyte HSL activity and glycerol release. For the study of the HSL activity, aliquots of diluted fat cells in the same buffer as described in the preparation of adipocytes were placed into plastic vials (1 × 10⁵ cell/ml), and the incubation was performed in a shaking Dubnoff water bath (60 cycles/min) at 37°C for 1 h under an atmosphere of 95% O₂- 5% CO₂. Under these conditions, there was a time-dependent increase in lipolysis for 60 min. Therefore, this time period was chosen for the incubation. A pure -agonist, isoproterenol, was used for the stimulation of lipolysis. In a preliminary study, the isoproterenol (0-10⁵ M) dose-dependent increase in lipolysis was evaluated. No statistically significant differences in the maximal lipolytic response were found at an isoproterenol concentration of 10⁵ or 10⁶ M (data not shown). Thus, to measure the HSL activity, the glycerol release was measured over 1 h at 15-min intervals, as described by Rodbell (29), in both the basal state and in the presence of isoproterenol (10⁶ M), so the maximal HSL responsiveness could be examined. Because the adipose tissue has a very low level of glycerol kinase, only a very small fraction of the glycerol produced by intracellular lipolysis can be utilized and converted to -glycerophosphate for use in triglyceride synthesis (17, 23). Glycerol release is therefore a valid index of lipolysis and thus of the HSL activity. At the end of the incubation, three aliquots of infranatant (200 µl) were removed from each incubation mixture for the measurement of glycerol by the enzymatic method of Wieland (44).

Assay of the antilipolytic action of insulin. To study the antilipolytic action of insulin, isoproterenol (10⁷ M) and adenosine deaminase (1 U/ml) were added to the fat cell suspensions (1 × 10⁵ cells/ml), and incubations were conducted during 1 h at 37°C under an atmosphere of 95% O₂-5% CO₂ both in the absence or presence of insulin (purified porcine insulin, Novo Laboratory) at a final concentration of 2.0 nM. A preliminary dose-dependence (0.5-6.0 nM) study showed that the maximal antilipolytic action of the hormone reached a plateau between 1.7 and 6.0 nM. At the end of the incubation, three aliquots of infranatant (200 µl) were removed from each incubation mixture and the glycerol release measured as described above (44). The antilipolytic action of insulin was expressed as the ratio of the value of the insulin-inhibited lipolysis to that of the isoproterenol-stimulated lipolysis in the absence of the hormone as a percent.

Euglycemic clamp studies. The whole body peripheral insulin sensitivity was measured using the euglycemic-hyperinsulinemic clamp technique as previously described elsewhere (7). The glucose infusion rate (GIR) during the second hour of the clamp was taken as the net steady state of whole body glucose. In all studies, blood samples (0.3 ml) for insulin determination (16) were obtained at 60, 90, and 120 min.

Analytic methods. The rats were anesthetized with an intraperitoneal injection of pentobarbital sodium (60 mg/kg body wt). Blood samples were obtained from the jugular vein and immediately centrifuged at 4°C. The plasma samples obtained were either immediately assayed or stored at 20°C and examined within the following 3 days. Plasma glucose (2), triglyceride (21) and FFA (12) were determined by spectrophotometric methods. Immunoreactive insulin was measured by the method of Herbert et al. (16). The immunoreactive insulin assay was calibrated against the rat insulin standard (Novo Nordisk, Copenhagen, Denmark). The epididymal adipose tissue was removed as mentioned above, and the LPL activity was measured as previously described (38) and was expressed as picomoles of substrate transformed per second (pkat) per gram of fresh tissue.

Statistical analysis. Results are expressed as means ± SE. The statistical significance between groups was determined by Student's t-test, or, when appropriate, data were subjected to a two-way ANOVA (BMPD, University of California, Los Angeles, CA) with diet and time as the main effects. When significance was found, Scheffé's post hoc comparison tests were made. The statistical significance was accepted at P < 0.05 (36).

	RESULTS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Food consumption and body weight gain. Comparable weight gain and food intake, expressed as caloric intake in kilojoules per day, were observed in SRD and CD rats during the 15-wk period, indicating that the SRD was readily consumed and utilized by the experimental animals. The values obtained were as follows (means ± SE; n = 8): weight gain: 2.13 ± 0.10 g/day in SRD vs. 2.08 ± 0.09 g/day in CD; caloric intake: 302.1 ± 10.3 kJ/day in SRD vs. 300.5 ± 13.1 kJ/day in CD. Total liver weight is not significantly changed in the SRD-fed rats compared with rats fed a CD for the same period of time. Values were as follows (means ± SE; n = 8): 11.55 ± 0.97 g in SRD vs. 10.91 ± 0.50 g at 3 wk and 14.50 ± 0.70 g in SRD vs. 13.00 ± 0.50 g in CD after 15 wk on diet.

Plasma metabolites level. As previously reported by our laboratory (22) and confirmed by the present findings, Table 1 shows that plasma triglyceride and FFA were significantly higher in the SRD fed animals compared with age-matched controls fed the CD. A more pronounced difference in both triglyceride and FFA levels was observed after 15 wk on the diet. Plasma glucose levels showed a significant increase in the SRD group only in the long-term feeding (15 wk). On the other hand, whereas plasma insulin levels were statistically higher in the short-term (3 wk) feeding on the SRD, comparable values were recorded in both dietary groups at 15 wk on the diet.

Fat pad morphology and triglyceride content. As can be seen in Table 2, after 3 wk on the SRD, epididymal tissue weight as well as adipose cell volume, triglyceride contents (µmol/cell), and cell number were comparable in both dietary groups. At the end of the experimental period (15 wk), we observed in the CD-fed rats that the epididymal fat depot grew accordingly with the increase of body weight. Similar results were reported in an aging study of male Wistar rats by Newby et al. (27). Compared with short-term feeding (3 wk), epididymal fat cell number expressed per gram of tissue was moderately (27%) lower, whereas cell volume was significantly higher. Regarding the SRD-fed rats, there was also a significant increase of the fat pad weight when the diet was extended up to 15 wk. However, the adipocytes of SRD-fed animals were 76% more voluminous than their respective age-matched controls fed a CD. A significant reduction of the adipose cell number, expressed per gram of tissue, was observed. Epididymal cells number reached only 61% of the value recorded in their respective controls fed a CD. This shows a hypertrophy of the fat cells at this particular point in time in the SRD-fed rats. On the other hand, considering both the type of diet and the duration of feeding, no differences in total cell number were observed when expressed as total epididymal fat weight. At 15 wk of diet, triglyceride content (µmol/cell) in fat tissue of rats fed a SRD was significantly higher (P < 0.05) than in CD-fed animals (Table 2).

Adipose cell size distribution. Figure 1 shows the histograms of adipose cell size distribution (at 2.5-µm intervals) at the end of both dietary periods. After 3 wk, the SRD rats (Fig. 1A) exhibited no differences in cell size distribution with the age-matched control rats fed a CD. At 15 wk on the diet (Fig. 1B), there was a clear differentiation in the cell size distribution with a significant increase (36%) of the mean cell diameter in the SRD group compared with the CD-fed animals.

View larger version (28K): [in this window] [in a new window]   Fig. 1.   Representative histograms showing distribution of adipocytes cell mean diameters isolated from the epididymal depots of rats fed a control diet (CD) or sucrose-rich diet (SRD) for different periods of time. The histograms were constructed by sizing, at intervals of 2.5 µm, 100 adipocytes from each individual rat. Eight animals were included in each experimental group. Bars represent the mean of the cell measured (percent) that falls within a given size indicated. A: histogram of animals during 3 wk on a CD (open bars) or a SRD (shaded bars). B: histogram of animals during 15 wk on a CD (open bars) or a SRD (shaded bars).

Euglycemic-hyperinsulinemic clamp studies. To assess the effect of dietary substitution on whole body peripheral insulin sensitivity (insulin resistance levels), euglycemic-hyperinsulinemic clamp studies were performed at the end of the two experimental periods. Blood glucose was clamped at 5.5-6.0 mM. Values of 5-h postprandial blood glucose before the clamp were as follows (means ± SE; n = 6): 5.68 ± 0.35 mM in SRD and 5.55 ± 0.13 mM in CD at 3 wk on diet, and 7.93 ± 0.14 mM in SRD and 5.59 ± 0.14 mM in CD at 15 wk. The steady-state values of blood glucose and insulin concentration measured over the last 60 min of the clamp are illustrated in the table included in Fig. 2. The GIR, which measured insulin action in vivo, was significantly low (P < 0.01) in the SRD group at the end of both experimental periods compared with rats fed the CD. However, a more pronounced difference was recorded in SRD-fed rats after the long-term feeding (Fig. 2).

View larger version (16K): [in this window] [in a new window]   Fig. 2.   Glucose infusion rate (GIR) during euglycemic-hyperinsulinemic clamp in rats fed a CD (open bars) or SRD (solid bars) for different periods of time. Values are means ± SE; 6 animals were included in each experimental group. Glucose and insulin, steady state of blood glucose and insulin concentration, respectively, during the last 60 min of the clamp. *P < 0.05 SRD vs. CD at the same experimental period. **P < 0.05 SRD at 15 wk vs. SRD at 3 wk on diet.

Adipocyte HSL activity and glycerol release. As shown in Fig. 3, the basal activity for HSL in epididymal adipose cells of rats fed a SRD for 3 wk was higher than in their age-matched controls fed a CD. Moreover, basal HSL activity increases up to fourfold in the adipocytes of rats fed a SRD during 15 wk. In all experiments performed with isoproterenol, the stimulated rate of lipolysis was significantly greater than the basal rate (P < 0.05). The stimulated rate for HSL activity in the SRD adipocytes at 15 wk was 20% higher than the HSL activity recorded in the adipocytes of rats fed a CD. Basal and stimulated HSL activities remained unchanged throughout the 15-wk experimental period in the CD rats.

View larger version (32K): [in this window] [in a new window]   Fig. 3.   Basal and isoproterenol-stimulated hormone-sensitive lipase (HSL) activity in isolated adipocyte from epididymal fat tissue of rats fed a CD or SRD diet for different periods of time. Values are means ± SE; 6 animals were included in each experimental group. Open bars, CD basal; solid bars, SRD basal; hatched bars, CD isoproterenol (106 M); crosshatched bars, SRD isoproterenol (106 M). HSL was estimated as the glycerol release from the isolated adipocytes. For further details on the methodology, see MATERIALS AND METHODS. *P < 0.05 isoproterenol (106 M) vs. basal. **P < 0.05 SRD basal vs. CD basal. *** P < 0.05 SRD isoproterenol (106 M) vs. CD isoproterenol (106 M) at 15 wk on the diet.

Insulin-mediated inhibition of lipolysis. Insulin-mediated suppression of isoproterenol-stimulated lipolysis is shown in Fig. 4. Although insulin inhibited to the same extent the lipolysis elicited by isoproterenol at 3 and 15 wk in CD adipocytes, it failed to inhibit -agonist-stimulated lipolysis in epididymal fat cell isolated from rats fed either 3 or 15 wk on a SRD. Animals fed a SRD showed a decreased adipocyte sensitivity to the antilipolytic action of the hormone compared with the CD fed rats. This was more noticeable when the diet was extended up to 15 wk.

View larger version (14K): [in this window] [in a new window]   Fig. 4.   Relative maximal antilipolytic action of insulin in isolated epididymal adipocytes of rats fed a CD (open bars) or a SRD (solid bars) for different periods of time. Values are means ± SE; 6 animals were used in each experimental group. Adipocytes were incubated in triplicate with 107 M isoproterenol in the presence or absence of 2 nM insulin. The results are expressed as the ratio of the value of the insulin inhibited-lipolysis to that of the isoproterenol-stimulated lipolysis in the absence of insulin. CD and SRD. *P < 0.05 SRD vs. CD at the same experimental period.

	DISCUSSION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

In this work using isolated adipocytes we have studied the effect of both a short-term (3 wk) and a long-term (15 wk) administration of a SRD on the morphological aspects and metabolic function of the epididymal adipose tissue in a nongenetic model of diet-induced dyslipidemia and insulin resistance. The new information provided by the present study showed the following. 1) There was both a moderate increase of basal lipolysis and a decrease of the antilipolytic action of insulin in the adipocytes of rats fed a SRD for 3 wk. At this point, whole body insulin resistance is not associated with a measurable increase of adiposity (neither size alterations nor increases in adipose tissue mass were recorded during this period). 2) There was an increase of fat pad mass, mainly due to a hypertrophy of fat cells, with a clear alteration in the cell size distribution that develops after a long-term (15 wk) on a SRD. This was accompanied by a significant increase of basal and stimulated lipolysis and by a marked decrease of the antilipolytic action of insulin. Moreover, the development of fat cell hypertrophy appears together with a worsening of whole body insulin resistance. Our results also indicate that the administration of a SRD for short time induces metabolic changes in the epididymal fat tissue of Wistar rats that precede the detection of increased adiposity, which is observed only after 15 wk on this diet.

Insulin and catecholamines play an important role controlling the activity of HSL. The present results suggest that the hyperinsulinemia induced by short-term SRD feeding does not downregulate HSL activity because the lipolytic activity from the adipocytes of rats fed this diet was significantly increased compared with rats fed a CD. Furthermore, a reduced antilipolytic action of insulin was observed. In agreement with the present results, Vrána et al. (42, 43) showed a diminished sensitivity to insulin and increased fatty acid release from adipocytes of Wistar rats fed a fructose-rich diet for 3-4 wk. Also, an increase of adipocyte lipolysis and lack of its response to insulin has been observed in humans fed a sucrose diet (34). When the diet duration was prolonged up to 15 wk, a moderate hyperglycemia with normoinsulinemia was observed in SRD-fed animals. At that time, the antilipolytic action of insulin of the enlarged adipocytes was reduced. Moreover, both basal and stimulated lipolytic activities were significantly higher compared with rats fed a CD. Recently, Morimoto et al. (26) showed an elevated basal lipolysis in the enlarged epididymal fat cells of obese rats.

It has been shown that either high-sucrose or -fructose intake induces an elevation of the blood pressure (40). Although the mechanisms remain unclear, a possible pathway could be the activation of the sympathetic nervous system. Therefore, the possibility that through this mechanism the adipose tissue lipolysis could be increased cannot be ruled out. Recently, Shafrir (32) demonstrated that a sucrose diet induces an elevation of both plasma triiodothyronine and intracellular lipase activity of fat pad in spiny mice. Our results do not provide data concerning the levels of plasma triiodothyronine in SRD-fed rats.

After 15 wk on a SRD, the twofold increase in fat pad weight without substantial changes in body weight represents an important physiological effect of dietary sucrose in Wistar rats. In addition, a twofold increase in cell volume and an abnormal cell size distribution with reduction in cell number population (expressed per g of tissue) demonstrated that hypertrophy rather than hyperplasia accompanied the increased tissue weight after a long-term SRD feeding compared with age-matched controls fed a CD. The response to sucrose depends on the rodent species. For instance, Osborne-Mendel rats respond with fat accumulation and weight gain, whereas in control S 5B/PI rats sucrose induces weight loss (30). The spontaneously hypertensive/ N-corpulent rats gain weight with sucrose feeding (24), whereas Cohen rats decrease in weight and lose adipose tissue (9) and spiny mice lose weight on prolonged sucrose diet (32). Also, Sprague-Dawley rats fed a fructose-rich diet also exhibit an increase of adipose tissue weight, as shown by Blakely et al. (5) and Rizkalla et al. (28).

Adipose tissue metabolism is influenced by the size of the constituent cells in both rats (4, 35) and humans (33). We observed an increase of triglyceride stores within the fat pad only in long-term SRD-fed rats. LPL was significantly higher in this tissue. Therefore, this could a least partially promote the rise of triglyceride content. The present results do not allow speculation about the enzymes activities involved in the lipogenic pathways in the adipose tissue of rats fed a SRD during 15 wk. Blackely et al. (5) and Cohen et al. (9) demonstrated an enhancement in the activities of several key enzymes related to this pathway in the epididymal tissue of rats fed a sucrose or fructose diet for up to 12 mo. Thus we cannot discard the possibility that these changes could also contribute to the increased lipid stores in this tissue.

A moderate decrease of whole body peripheral insulin sensitivity (low GIR) accompanied the hypertriglyceridemia after 3 wk on an SRD. Similarly, Storlien et al. (39) found that feeding sucrose to normal rats over a 4-wk period leads to a major whole body altered insulin action. Although the impaired suppression of endogenous glucose production by insulin accounts for the major part of this effect, a smaller contribution from peripheral tissues, including epididymal pads, was also found. Adipose tissue accounts for only a small fraction of the total glucose disposal in the intact organisms (10). The worsening of GIR recorded in the SRD after 15 wk of feeding would indicate a key role of skeletal muscle in the whole body insulin insensitivity. However, adipose tissue is a target tissue to supply fatty acids to the whole body utilization. Chronic elevation of plasma FFA exerts an important modulator effect on insulin action (20). In the present study, the finding of a permanent increase and availability of plasma FFA mainly due to both an increase of basal lipolysis and a substantial reduction of antilipolytic action of insulin in the adipose tissue of the SRD-fed rats could contribute to and/or worsen the impaired insulin sensitivity and whole body glucose uptake and utilization.

Finally, the present work provides new information indicating that the length of time on a SRD plays an important role in the evolution of adiposity and metabolic changes observed in the fat pad of normal Wistar rats. Furthermore, this study clearly demonstrates that the insulin resistance present after 3 wk of feeding a SRD was not associated with increased fat pad mass. Moreover, the development of adiposity and fat cell hypertrophy together with a worsening of glucose homeostasis and insulin sensitivity were only observed after long-term feeding of a SRD.