Rats of the JCR:LA-cp strain, which are homozygous for the cp gene (cp/cp), are obese, insulin-resistant, and hyperinsulinemic. They exhibit associated micro- and macrovascular disease and end-stage ischemic myocardial lesions and are highly stress sensitive. We subjected male cp/cp rats to pair feeding (providing the rats each day with the amount of food eaten by matched freely fed animals), a procedure that alters the diurnal feeding pattern, leading to a state of intermittent caloric restriction. Effects on insulin, glucose, and lipid metabolism, response to restraint stress, aortic contractile/relaxant response, and myocardial lesion frequency were investigated. Pair-fed young (12-wk-old) cp/cp rats had lower insulin and glucose levels (basal and following restraint), consistent with increased insulin sensitivity, but a greater increase in plasma nonesterified fatty acids in response to restraint. These effects were unrelated to lipolytic rates in adipose tissue but may be related to reduced fatty acid oxidation in skeletal muscle. Older (24-wk-old) pair-fed cp/cp rats had significantly reduced plasma triglyceride levels, improved micro- and macrovascular function, and reduced severity of ischemic myocardial lesions. These changes indicate a significant amelioration of end-stage disease processes in this animal model and the complexity of metabolic/physiological responses in studies involving alterations in food intake. The effects illustrate the sensitivity of the JCR:LA-cp rat, an animal model for the metabolic syndrome and associated cardiovascular disease, to the environmental and experimental milieu. Similar stress-related mechanisms may play a role in metabolically induced cardiovascular disease in susceptible human beings.
- metabolic syndrome
- fatty acids
- vascular function
- myocardial lesions
the metabolic syndrome, strongly associated with obesity, is a major and increasing cause of cardiovascular morbidity and mortality worldwide. A crucial feature of this disorder is the development of insulin resistance and consequent hyperinsulinemia, which appears to be a major determinant of the associated vasculopathy, atherosclerosis, and ischemic cardiovascular disease (16, 27). The underlying origins of the metabolic syndrome remain the subject of debate, and relatively little attention has been paid to the contribution of stress in its various forms. These vary from potentially highly deleterious oxidative stress at a cellular level to neurendocrine and behavioral-environmental stress, all potentially leading to increased cardiovascular disease (6). In contrast, there is evidence that the progression to cardiovascular disease can be alleviated by beneficial effects of exercise and reduced or intermittent food intake (24, 25).
Because of the fundamental role of hyperinsulinemia in the metabolic syndrome, reduction of insulin resistance and consequent hyperinsulinemia offer a unique target for decreasing cardiovascular disease and mortality. Treatment at a clinical level has included changes in diet, food intake, and physical activity, but these have proven difficult to implement and maintain in the human population, leading to significant efforts to develop effective pharmaceutical treatments. An essential element in this research effort has been the use of animal models that mimic the metabolic and pathophysiological aspects of the metabolic syndrome (32). Many putative pharmaceutical agents induce reduced food intake (8, 9, 36, 44), leading to uncertainty as to whether the effects seen were due to direct pharmacological actions of the drugs or were simply secondary effects related to anorexia. A common approach to this problem has been to include control (non-drug-treated) animals in the experimental design that are on a daily basis provided with a single portion of food equal to that consumed by drug-treated animals (so-called pair feeding) (29, 57). However, in the course of a pharmaceutical study, in which one of the agents had no effect on food intake, we observed that the pair-fed rats showed significant differences in metabolism and pathophysiology compared with the freely fed control animals. On investigation, we also noted that the pair-feeding procedure alone significantly altered the diurnal pattern of food intake in the rats. The animals consume the daily allocation of food within 10 h and are then food deprived for the balance of the 24-h cycle. Our serendipidous observations thus indicate that pair-fed rats are under a fixed feeding schedule that creates an intermittent fasting or caloric deprivation and a different physiological response than feeding ad libitum independent of any change in total food intake.
There is limited scientific literature on schedule feeding going back more than 35 years. However, all reports describe studies that involved significant reduction of food intake compared with ad libitum-fed animals, with typical restriction to 2–3 h of food availability per day. This kind of significant food deprivation causes metabolic and physiological changes (14, 28). For instance, Rupp et al. (35) recently reported that food restriction induced potentially deleterious impairment of Ca+2 cycling in cardiomyocytes, which they attributed to psychological stress. In contrast, Mattson et al. (25) and Wan et al. (54) have shown beneficial cardiovascular effects through intermittent fasting or intermittent metabolic deprivation through inhibition of glycolysis. We have also reported that metabolic deprivation, through administration of the glucose analog 2-deoxy-d-glucose (2-DG), improves cardiovascular function and reduces end-stage myocardial lesions in an animal model of the metabolic syndrome (49).
The JCR:LA-cp rat is a unique strain that has been used in the study of the metabolic syndrome and putative pharmacological interventions (32). If homozygous for the autosomal recessive cp gene (cp/cp), the rats are obese and spontaneously develop important elements of the pathophysiological status associated with the metabolic syndrome in humans (27, 43), including advanced intimal (atherosclerotic) lesions, myocardial ischemic lesions, and renal microvascular dysfunction and glomerular sclerosis (34, 48). The atherosclerosis is associated with a vasculopathy that includes increased vascular contractility and reduced vascular relaxation. Heterozygous (cp/+) or homozygous normal (+/+) rats are lean and metabolically normal.
The cp mutation results in the presence of a stop codon in the extracellular domain of the leptin receptor (ObR) (56), leading to the absence of membrane-bound ObR, of any of the isoforms in cp/cp animals. In the absence of the ObR, the animals develop a marked hyperleptinemia and hyperphagia. A profound insulin resistance develops between the ages of 4 and 7 wk with accompanying VLDL hypertriglyceridemia (40, 51). The insulin-resistant state is accompanied by an exaggerated sensitivity to brief immobilization stress that has both metabolic and neural components (21, 26). The cp/cp rats maintain euglycemia at the expense of extremely high circulating insulin concentrations (47). The hyperphagic cp/cp rats must divert glucose derived from a high-carbohydrate diet to the liver, with disposal by conversion to triglyceride (TG) and export to the plasma as VLDL (17, 46), leading to VLDL hyperlipidemia. The hypertriglyceridemia is thus not caused by an impaired clearance of lipoproteins from the circulation but by enhanced hepatic secretion and subsequent modification of the lipoproteins (40, 51). The findings suggest that the insulin resistance of the cp/cp rats, and possibly also of humans, is related to abnormal lipid metabolism, associated with decreased fatty acid (FA) oxidation, increased TG levels in tissues (particularly in muscle) (3, 4, 42), and stress-induced release of FA from adipose tissue (26). High insulin levels appear, in themselves, to have pathological effects and may be a major contributor to the atherosclerosis and vascular dysfunction (1, 27, 36). Circulating FA levels of the cp/cp rats in response to immobilization-induced stress (26), which reached those normally associated with severe diabetes, could also contribute to the vascular damage and dysfunction as well as aggravate the insulin resistance. Our earlier findings also suggest that stress may play a significant exacerbating role in the abnormal metabolism and pathophysiology of this animal model (21).
Our hypothesis is that stress, as suggested by Brindley and Rolland (10) and supported by Björntorp (6), plays a significant role in both the metabolic syndrome and associated pathophysiology. However, “stress responses” arise in a variety of ways, are not a single entity, and have proven difficult to study experimentally. We suggest that some variants of psychological stress, especially in sensitive individuals and those with the metabolic syndrome, are deleterious and exacerbate the pathophysiological processes (19). In contrast, there is evidence that intermittent food deprivation, or inhibition of oxidative metabolism and accompanying physiological changes, can have beneficial effects on metabolism and pathological sequelae (25, 49, 54). The metabolic and physiological abnormalities of the cp/cp rat develop and change as the animals develop from juveniles to mature adults, and our experimental study was designed to address effects on critical elements of the pathophysiologcal process at appropriate ages. Thus, we subjected juvenile/adolescent animals of the JCR:LA-cp strain to pair feeding and then assessed both basal metabolism and response to restraint stress when they were young adults. The metabolic end points were changes in insulin and glucose metabolism, synthesis and oxidation of FA, and rates of lipolysis by adipose tissue. In a second study, we used fully adult rats subjected to pair feeding, without reduction of total food intake, to assess effects on metabolism and end-stage pathophysiology represented by micro- and macrovascular dysfunction and ischemic myocardial lesion frequency.
Experiment 1: metabolic effects of pair feeding and immobilization stress in adolescent cp/cp rats.
Pair-fed cp/cp animals were subjected to the procedure from 8 wk of age (fully insulin resistant) to 12 wk of age (young adults). In this study, the rats were pair fed to match the food intake of freely fed cp/cp animals and compared with control (freely fed) cp/cp and +/? rats. At 12 wk of age, a restraint stress test was performed with measurement of indexes of lipid and glucose metabolism followed by the rats being killed and blood and tissue sampling for measurement of biochemical parameters.
Experiment 2: metabolic and physiological effects of pair feeding of adult cp/cp rats.
Adult rats were placed in the feeding protocol at 12 wk of age and with food intake paired to freely eating cp/cp control rats. At 24 wk of age a meal tolerance test was performed, with the rats killed 1 wk later and blood and urine sampling taken for biochemical parameters. Aortae were removed for vascular function studies and hearts and kidneys for histology.
Male JCR:LA-cp rats, cp/cp (obese) and +/? (lean; 2:1 cp/+ and +/+), were bred and maintained in our established rat colony (43). The rats were housed individually in polycarbonate cages and placed on a reversed light cycle (lights on at 1800 and off at 0600) 1 wk prior to the start of the pair-feeding protocol to facilitate metabolic studies during the active (dark) phase of their diurnal cycle. All food was Lab Diet 5001 (PMI Nutrition International, Brentwood, MO). Rats were weighed and food intake determined twice/wk throughout the experimental period. All care and treatment of the rats was in accordance with the guidelines of the Canadian Council on Animal Care and was reviewed and approved in advance by the Health Sciences Animal Policy and Welfare Committe of the University of Alberta.
Blood samples were obtained from the tip of the tail of conscious, unrestrained rats (45) during the dark phase between 0800 and 1200. At termination (either 13 or 25 wk of age), all rats were killed under anesthesia with isofluorane in oxygen during the same time period. Terminal blood samples were obtained by cardiac puncture and urine collected from the bladder. Tissues were removed for in vitro study and used immediately or flash-frozen in liquid nitrogen and stored at −80°C for later assay.
Control rats were provided with a significant excess of food in the cage top hopper at all times such that the remaining food was equivalent to 3 days consumption. Pair-fed cp/cp rats were placed on a protocol that provided, in the hopper at a fixed time of day (6 h into the dark period), the amount of food that control animals ate on the corresponding experimental day. In all other respects, the rats in the different groups were treated identically. Under these circumstances, pair-fed rats ate the food provided within 12 h and then remained without food for the remainder of each 24-h period. Occasionally (<1% of feeding periods), individual rats had small amounts of food (∼10%) remaining after 24 h.
Rats were subjected to a single stress response test with a 15-min restraint period, but without chronic vessel cannulation as in our previous work (26). The animals were studied in the fed state, postabsorption phase, during the first 4 h of the dark (active) period of their diurnal cycle. An initial sample of blood was taken from the tip of the tail, following which the rat was placed in a polyethylene cone restraining device (Decapi-cone; Braintree Scientific, Braintree, MA) for 15 min (21). A second blood sample was taken while the animal was in the cone, after which it was released and returned to its cage. A final blood sample was taken at 60 min from the start of restraint.
Meal tolerance test.
A meal tolerance test was performed at 24 wk of age, following a standardized protocol (47). Animals were previously subjected to a mock procedure 1 wk before the meal tolerance test. The rats were deprived of food over the light (inactive) period, and the test was conducted in the first 3 h of the dark period. Conscious, unrestrained rats were subjected to three blood samplings during each session. Initially, animals were placed on a heated table to ensure vasodilation of the tail, and 0.5 ml of blood was taken from the tip of the tail (time 0). Rats were then replaced in their cages and given a 5-g food pellet (the test meal). Timing began when 50% of the test meal had been consumed, and samples of blood were taken at 30 and 60 min for the analysis of glucose and insulin. All rats ate the full test meal within 15 min of presentation.
Plasma glucose was determined using a glucose oxidase assay procedure (Diagnostic Chemicals, Charlottetown, PEI, Canada). Insulin was assayed by rat ELISA assay (Mercodia, Uppsala, Sweden). Plasma triglyceride (L-type TG H), total cholesterol (Cholesterol E), and low-density lipoprotein (LDL) cholesterol (L-type LDL-C) assays were obtained from Wako Pure Chemicals USA (Richmond, VA). High-density lipoprotein (HDL) cholesterol was assayed using direct HDL assay (Diagnostic Chemicals). Unesterified FA in the serum was measured using an assay kit (NEFAC; Wako Pure Chemicals USA) and a 96-well plate reader (8). Lipids were extracted from samples of liver, aorta, and skeletal muscle (7); the chloroform phase was dried and then dissolved in 100 μl of propan-1-ol. Triglyceride concentrations were then measured using 20 μl of propanol solution and 200 μl of TG assay reagent. Urine albumin measurements were performed on a Beckman Coulter LX20i analyzer using an immunoturbidimetric method.
Rate of lipolysis.
The rate of lipolysis was measured using adipose tissue (∼100 mg) in preference to isolated adipocytes, since the latter are highly fragile when isolated from obese rats (8). Fresh adipose tissue was preincubated at 37°C for 30 min in 900 μl of medium, as described previously (24). A 100-μl sample of medium was collected before 100 μl of new medium that either did or did not contain norepinephrine (final concentration: 11.1 μmol/l) was added. Samples were then incubated for a further 3 h at 37°C, after which the clear medium was collected. Medium collected following the 30-min and 3-h incubation periods was assayed for glycerol content, and the resulting values were used to calculate the amount of glycerol released during the 3-h incubation period.
The rate of FA oxidation in resting intact soleus muscle and in slices of liver was determined using [14C]palmitic acid (18). Briefly, fresh soleus muscle (∼100 mg) was tied lightly to a tungsten wire with suture material so as to maintain its normal resting length. A sample of liver (∼100 mg) was cut into small pieces. The muscle and liver samples were crimp-sealed in 25-ml serum vials with 2 ml of Krebs-Henseleit buffer containing 0.2 mmol/l [1-14C]palmitate (0.083 Ci/mol) adsorbed onto BSA (0.2% wt/vol) and 11 mmol/l glucose. The vials were gassed with 95% O2-5% CO2 via a stainless steel needle inserted through the seal, and effluent gas was collected through a second needle and bubbled through 1.5 ml of hyamine hydroxide (ICN Pharmaceuticals Canada, Montreal, QC, Canada). Vials were incubated for 2 h at 37°C, after which the reaction was stopped by injecting 1 ml of 8% perchloric acid through the seal. After a further 30 min of gassing and collection of the effluent, 10 ml of ACS scintillation cocktail (Amersham Canada, Oakville, ON, Canada) was added to the hyamine solution and radioactivity was determined by scintillation counting.
Vascular function studies.
The vascular function of aortic rings, with intact endothelium, was assessed using established methods (41). Briefly, rats were anesthetized using isofluorane in oxygen. The chest cavity was exposed and the thoracic aorta excised, trimmed of adhering fat and connective tissue, and cut into 3-mm-long transverse rings. Aortic rings were mounted on stainless steel hooks under 1.5-g resting tension in 10-ml organ baths and bathed at 37°C in Krebs solution (containing in mmol/l: 116 NaCl, 5.4 KCl, 1.2 CaCl2, 2 MgCl2, 1.2 Na2PO4, 10 glucose, and 19 NaHCO3) and gassed with 95% O2 and 5% CO2. Tension was recorded isometrically with Grass FTO3C transducers (Grass Medical Instruments, Quincy, MA) and displayed on a Digi-Med tissue force analyzer (Model 210; Micro-Med, Louisville, KY) linked to an IBM-compatible computer that acquired data digitally using DMSI 210/4 (Micro-Med) software.
The contractile response of endothelium-intact rings of aortae to phenylephrine (PE) was assessed through concentration-response curves for PE (1 nmol/l to 300 mmol/l). The basal nitric oxide (NO)-mediated relaxation of aortic rings (precontracted with PE to 80% of maximal contraction) was assessed by determining the concentration response to the endothelial NO-releasing agent acetylcholine (ACh) and the NO donor sodium nitroprusside. Direct assessment of NO-mediated effects was also determined through addition of NG-nitro-L-arginine methyl ester, at 10−4 mol/l, to inhibit NO synthase activity (33).
Hearts were cut transversely into four blocks, fixed in formalin, subjected to conventional processing, embedded in a single paraffin block, and sectioned followed by hematoxylin and eosin staining. Heart sections were examined blind by an experienced observer and the number of ischemic lesions identified in each of the sections summed for each heart. The lesions were categorized by four stages, as described previously (34, 36).
Results are expressed as means ± SE and were analyzed with SigmaStat (Jandel Scientific, San Rafael, CA) and plotted using SigmaPlot (Systat Software, San Jose, CA) and Prism (Graphpad, San Diego, CA). Results were compared using one- and two-way analysis of variance followed by multiple comparison tests. Concentration-response curves were analyzed using the ALLFIT program (15), which fits the complete data set to the logistic equation and permits independent testing of differences between individual parameters. A value of P < 0.05 was taken as being statistically significant.
Experiment 1: Metabolic Effects of Pair Feeding in Adolescent cp/cp Rats
Food intake and body weight.
Figure 1 (left) shows food intake and body weight of cp/cp rats that were either freely fed or pair fed over the period from 8 to 12 wk of age. Data from freely fed +/? control rats is also shown for reference. Despite having the same daily food intake over each 24-h period as rats fed ad libitum, the pair fed cp/cp rats showed an 8% lower body weight (P < 0.05) from 9 wk of age and beyond.
Figure 2 depicts normal fed-state insulin and glucose levels (baseline values) and values following 15-min restraint and the 45-min recovery period. The cp/cp rats had higher fed-state plasma insulin levels than +/? rats, as reported previously (43, 47). Pair-fed cp/cp rats showed markedly lower (50%, P < 0.001) insulin levels at baseline compared with the freely fed cp/cp animals. Freely fed cp/cp rats had reduced plasma insulin levels following 15-min restraint (P < 0.05) and a further decrease after the 45-min recovery period (P < 0.01). Similarly, freely fed +/? rats had significantly decreased plasma insulin after the recovery period (P < 0.05) but not immediately after restraint. Pair-fed cp/cp rats, like the +/? rats, had no change in insulin levels in response to the restraint stress, with a small decrease in the recovery period. Pair-fed cp/cp rats also had lower baseline plasma glucose concentrations that were not altered by the restraint stress (Fig. 2).
The release of FA into the circulation is a major component of the response to stress. Compared with animals in the fed state (baseline values), fasted rats +/? and cp/cp at 12 wk of age showed significantly elevated plasma FA levels (P < 0.001; Fig. 3), with the cp/cp rats having higher levels than the +/? animals (P < 0.0005). Although baseline (fed state) FA concentrations were not different between freely fed +/? and cp/cp rats, pair-fed cp/cp rats had ∼75% higher levels than freely fed cp/cp rats (P < 0.01). FA levels of freely fed cp/cp rats increased nonsignificantly during the period of restraint, with a further increase during recovery (P < 0.01). The significantly higher FA levels of pair-fed cp/cp rats at time 0 were further exacerbated both during and following restraint (P < 0.001).
Fat pads and lipolysis.
Retroperitoneal fat pads of the cp/cp rats are larger than those of the +/? rats, reflecting their obese status (Table 1). Basal lipolytic activity, expressed per gram of tissue, did not differ with genotype. However, when stimulated with norepinephrine, adipose tissue from the +/? rats showed significantly higher rates of lipolysis, on a per gram basis, than did that from the cp/cp rats. When expressed on a per fat pad basis, the basal and norepinephrine-stimulated release of glycerol from adipose tissue of the cp/cp rats remained about fourfold higher than that from the +/? rats due to higher fat pad weights. Rate of lipolysis by adipose tissue of cp/cp rats was not significantly affected by pair feeding under the experimental conditions.
The rate of FA oxidation by soleus muscle showed no significant difference between freely fed +/? and cp/cp rats (Fig. 4) but was significantly reduced in the pair-fed cp/cp animals (P < 0.05). Livers of 12-wk-old cp/cp rats had a much lower rate of FA oxidation than did those of +/? rats, with pair feeding having no significant effect.
Tissue triglyceride content.
Soleus muscle TG concentrations were significantly higher in cp/cp than in +/? rats and unaffected by pair feeding (Fig. 4). TG content of the liver was also significantly greater in the cp/cp than in the +/? rats, and this was reduced by pair feeding (P < 0.05).
Experiment 2: Metabolic and Physiological Effects of Pair Feeding in Adult cp/cp Rats
Food intake and body weight.
Figure 1 (right) shows food intake and body weights over the period of 12 to 24 wk of age. In these mature rats, there was no difference in body weights between the freely fed and pair-fed animals.
Meal tolerance test.
Meal tolerance tests performed on rats at 24 wk of age (Fig. 5) showed much higher fasting and postprandial insulin levels in freely fed control cp/cp rats than in +/? rats. Also, reflecting the insulin-resistant state of these animals, plasma glucose concentrations, fasting and following the test meal, were elevated in the cp/cp rats. Fasting plasma insulin levels of the pair-fed cp/cp rats were not significantly elevated above those of the freely fed cp/cp rats but were significantly elevated 30 min after the meal challenge (P < 0.05). Plasma glucose concentrations of the pair-fed rats were consistently lower than those of the freely fed animals, with this difference being highly significant 60 min after the meal challenge (P < 0.001).
Plasma lipid concentrations.
Pair feeding caused a significant decrease in plasma TG concentrations of the cp/cp rat (P < 0.01; Fig. 6). There was no change in the plasma concentrations of total, LDL, or HDL cholesterol in the pair-fed animals (data not shown).
As shown in Fig. 7, aortic vascular function is significantly impaired in the cp/cp rat, compared with the +/? rat, with elevated contractile response to the noradrenergic agonist PE and impaired ACh-mediated relaxation. Figure 7 also shows a reduced contractile response to PE by the aortic rings from pair-fed cp/cp rats. The calculated parameters in Table 2 show that the reduction in the hypercontractile response to PE in the pair-fed rats was significant (P < 0.001), but there was no significant improvement in the ACh-mediated relaxation.
Glomerular dysfunction, and resultant albuminuria, is an element of the microvascular disease associated with the metabolic syndrome. Figure 8 shows that pair feeding causes a significant reduction in the elevated urinary albumin loss of the cp/cp rat.
Ischemic myocardial lesions.
There were relatively few stage 1 myocardial lesions (areas of early ischemia or necrosis) in the hearts (Fig. 9). The frequency of stage 1 lesions was lower, but not significantly, in the hearts of pair-fed cp/cp rats. Similarly, there were relatively few stage 2 lesions (foci of chronic inflammatory cells without cell dropout) seen, and there were no significant differences between groups. Stage 3 lesions (areas with strong inflammatory cell infiltration and cell lysis) were infrequent in hearts of the +/? animals but present at a significant frequency in the hearts of freely fed cp/cp rats. Hearts of pair-fed cp/cp rats had significantly fewer stage 3 lesions (>50%, P < 0.05) than the freely fed cp/cp animals. Stage 4 lesions were infrequent in these 24-wk-old hearts, reflecting the cumulative nature of these old scarred lesions and the relatively young age of the rats, with no significant differences between groups.
Although a hormonally driven hyperphagia (55) with resultant obesity and insulin resistance is a prominent and critical element of the metabolic syndrome, abnormal stress responses are also clearly important (21, 22, 35). The response to stress may be related to the vascular dysfunction, which leads to end-stage cardiovascular disease (27, 32) but may also involve other metabolic elements (19, 35). This is consistent with our earlier work showing exaggerated metabolic response by the cp/cp rats to restraint and handling stress (21, 26). Overall, our results would indicate an integrated response to the ObR defect-mediated hyperphagia, obesity, and insulin resistance at central nervous system, metabolic, and cardiovascular levels.
Pair feeding of rats, with the resultant enforced intermittent food intake, would appear to constitute a relatively minor environmental change. In contrast to overt food restriction employed in other studies (5, 15, 28, 50), there was no reduction in total caloric intake and therefore only a modest, albeit important, effect on body weight of adolescent animals, with no effect in mature rats (Fig. 1). However, pair feeding 12 wk old cp/cp rats in experiment 1 caused a modest, but significant, decrease in weight gain and a significant decrease in insulin resistance, indicated by reduced concentrations of both insulin and glucose in the fed state. Unlike the freely fed rats, pair-fed cp/cp rats responded to short restraint stress with no change in insulin levels and a decrease during the recovery period, resembling the +/? freely fed rats. The fall in insulin concentration with a constant plasma glucose concentration implies an increased response to insulin during the stress and/or recovery periods and represents a significant improvement in the metabolic status of the animals. These effects are similar to those reported by others in Sprague-Dawley and Zucker fatty rats (11, 23) but are more extreme due to the metabolic dysfunction of the cp/cp rat and were enhanced by the stress of pair feeding. Neither +/? nor cp/cp rats showed the rise in plasma glucose seen in both fa/fa Zucker rats (10) and in cp/cp rats of our somewhat different earlier study (26).
Mature, 24-wk-old, cp/cp rats that were pair fed (experiment 2) showed no significant increase in fasting insulin levels compared with freely fed controls but exhibited a significant increase in insulin immediately postmeal challenge with no change in glucose concentration (30 min; Fig. 5). Thus, there is no evidence of an increase in insulin sensitivity and a possible small increase in insulin resistance, in contrast to the improvement in insulin sensitivity shown by young adult rats in experiment 1. These differences in response to pair feeding reflect metabolic changes as the obese cp/cp rats age and mature.
Plasma FA concentrations are sensitive to both stress and insulin/glucose metabolism, illustrated by the significantly greater FA levels in fasted rats and the greater increase in response to stress of the pair-fed cp/cp rats (Fig. 3). The FA response to stress in the present results was similar to, but lower than, that seen in earlier studies (26). Lower FA concentrations in this study may reflect differences in experimental protocol from our previous study (26) in which the animals were implanted with venous cannulae for blood sampling. The increase in FA seen during the recovery phase in the present study resembled that reported for handled rats but not for nonhandled rats, with handling constituting a significant form of stress to the animal (26). Animals in the present study were also handled in association with the tail-bleeding procedure but were familiarized with this less aversive procedure in advance. Interestingly, there is a contrast between the rise in FA seen in fa/fa (lean) Zucker rats studied under a more extreme restraint stress exposure (11) and the absence of response by the +/? rats under the brief cone restraint of this study. As well, cp/cp rats showed significant rises in FA during and following restraint, whereas no such increases were seen in fa/fa (obese) rats by Chaouloff et al. (11). The contrasting results are consistent with the different nature of the fa mutation (an amino acid substitution that leads to reduced affinity of the ObR receptor for leptin) and the cp mutation (a stop codon that leads to the absence of all isoforms of the ObR) (12). In this study, pair-fed cp/cp rats had prestress FA levels that were almost double those of the freely fed rats, with an exaggerated response to the restraint stress that was comparable with that induced by the fasted state. These levels were comparable with those seen in our earlier study with rats that were handled (26) and indicate a major exacerbation of the stress response in pair-fed rats.
Under stimulation by norepinephrine, the rate of lipolysis, per gram of retroperitoneal fat, was higher in the +/? rats, as expected from the smaller size and greater number of adipocytes per gram tissue in these animals. However, when expressed on a per fat pad basis, +/? rats had lower rates of lipolysis, reflecting a smaller mass of adipose tissue (26). Thus, higher steady-state circulating FA concentrations in the fasted state seen in the cp/cp rats compared with the +/? rats were due to the greater adiposity of the former and, therefore, potentially greater release of FA. The origin of the increased plasma FA response to the immobilization stress in the pair-fed rats (Fig. 3) does not appear to lie in enhanced catecholamine sensitivity of peripheral fat tissues, as Table 1 shows no effect of pair feeding on the rate of lipolysis. We speculate that the underlying mechanism is driven by either the central nervous system and/or a systemic hormonal response to the pair feeding through the sympathoadrenal axis (11, 13).
The rates of FA oxidation found in both the muscle and liver of JCR:LA-cp rats were qualitatively similar to those reported by Iso et al. (19) in the ZDF rat. Results in Fig. 4 are uniformly about 50% of those found in the ZDF rat (19), probably as a consequence of the differences between the fa and cp mutations and the different background genotypes of the two strains (12, 56). The greater FA increase in the plasma of the pair-fed rats may be related to the lower rate of oxidation in muscle tissue secondary to chronic stress (Fig. 4). The TG content of skeletal (soleus) muscle of the cp/cp rat is fourfold higher than that of the +/? rat at 12 wk of age, and we have shown previously that this is directly related to the peripheral insulin resistance (42). The liver of the cp/cp rat has a 10-fold higher TG content than that of the +/? rat, probably because of the greatly enhanced lipid synthesis and metabolism (46). In comparison, pair-fed cp/cp rats had a threefold reduction in hepatic TG content that parallels the elevated plasma FA levels in the fed state, both basal and in response to immobilization stress (Fig. 3). The reduction in TG content does not involve enhanced FA oxidation in either liver or skeletal muscle, as Fig. 4 shows reduced FA oxidation in the pair-fed rats. Reduced hepatic FA oxidation is consistent with oversupply of glucose (46), which was not altered by pair feeding (food intake being unaltered). However, the reduced TG content of liver (but not soleus muscle) of pair-fed cp/cp rats suggests diversion of FA to some other, at this point unidentified, tissue and presumably oxidation. Similarly, the reduced plasma TG concentration seen in pair-fed cp/cp rats (Fig. 6) is consistent with diversion of FA to oxidation and away from TG synthesis and secretion as VLDL (37, 40, 51). These changes seen with pair feeding are consistent with the increase in insulin sensitivity and reduction in plasma insulin levels, with consequent increase in peripheral glucose uptake and oxidation and reduction in the bias to FA metabolism characteristic of the prediabetic insulin-resistant state.
Vascular Function and Myocardial/Renal Disease
Macrovascular dysfunction, evident in increased contractile response of aortic rings to PE and impaired endothelium-dependent relaxation, as seen in Fig. 7, is a major complication of the hyperinsulinemic prediabetic state (17). It is associated, in the cp/cp rat, with ischemic lesions in the heart (36) and is reduced by interventions that are cardioprotective in this model (38, 39, 42). The reduction in PE-mediated vascular contraction in the pair-fed 24-wk-old cp/cp rats was associated with a reduction in plasma TG concentrations but not with lower cholesterol, indicating that noncholesterol lipid elements were involved in the vasculopathy. The reduced vascular dysfunction is reflected in the lower incidence of stage 3 ischemic lesions in the heart (Fig. 9).
Microvascular dysfunction is the other major facet of the end-stage complications of the prediabetic state and type 2 diabetes. Microvascular damage is evident in microalbuminuria, as shown in Fig. 8, with progression to glomerular sclerosis and eventually renal failure (24, 30). Pair-fed cp/cp rats had significantly lower urinary albumin loss, consistent with improved microvascular function and with the improved macrovascular function. In these relatively young adult rats, there was no improvement in the severity of glomerular sclerosis (results not shown), although other interventions that cause significant reduction in albuminuria have been shown to be associated with reduced glomerular damage (30, 49).
Under the experimental conditions, rats eat the daily allocation of food rapidly (over ∼12 h) and are food deprived for the ≥10-h balance of each diurnal cycle, inducing an intermittent metabolic deprivation. Although the length of time the rats are metabolically deprived is short in human terms, the passage of time and physiological processes are effectively faster in a short-lived species such as the rat (20). With a heart rate about six times that of humans, time and physiological processes effectively run equivalently rapidly. Thus, metabolic deprivation of a rat that normally eats numerous small meals throughout the diurnal cycle for 14 h each day is equivalent to recurring 3-day fasting by a human. The metabolic changes seen in the rats may be related to the stress experienced by animals that perceive that they have an intermittent or unreliable food supply. However, we have recent data (49) that show similar effects in cp/cp rats treated with 2-DG, a synthetic glucose analog that inhibits glucose oxidation and thus energy availability. Importantly, the effects of 2-DG are evident primarily with intermittent (alternating days), and not with continuous, treatment. We suggest that intermittent food deprivation, or metabolic inhibition, has similar effects. In support of this concept, Wan et al. (54) also used an intermittent-treatment 2-DG schedule as an analog to intermittent fasting, which they have shown to have beneficial effects on the cardiovascular system (25).
In previous studies, we have shown that reduction of insulin resistance and the resultant hyperinsulinemia, through a variety of interventions, leads to improved vascular function and sharply reduced myocardial lesion severity and glomerular sclerosis (8, 27, 31, 33, 35–38, 40–43). Intermittent caloric restriction, induced by the pair-feeding protocol, appears to have effects that are very similar to other insulin-sensitizing treatments. The mechanisms underlying the beneficial pathophysiological effects are probably related to reduction of insulin concentrations and thus of the cytokine activity of insulin at high levels (1, 2) and reduction of circulating triglyceride as VLDL and chylomicrons, both of which are atherogenic (31, 52).
The cp/cp rat exhibits exaggerated metabolic responses to changes in physical environment, including intermittent caloric restriction. The responses include lower insulin levels following restraint stress, consistent with an increase in insulin sensitivity, and a greater increase in circulating FA. Metabolism of the pair-fed cp/cp rat appears to be altered to favor FA release, and presumably metabolism, rather than glucose oxidation. Lower insulin levels are potentially beneficial in terms of the cardiovascular disease that accompanies obesity and insulin resistance in these animals and in humans (2, 17). On the other hand, the markedly higher stress-induced FA concentrations in young pair-fed rats fall within a range that could have adverse effects (9), leading to vascular damage and increased cardiovascular disease (2, 27). However, the older pair-fed cp/cp rats had significantly reduced plasma triglyceride levels, improved micro- and macrovascular function, and reduced severity of ischemic myocardial lesions. These constitute significant amelioration of the end-stage disease processes that accompany the metabolic syndrome and are analogous to the previously reported effects of food restriction on degenerative diseases (25, 54). It is possible that similar interactions exist in susceptible human beings.
This work was supported financially by Sanofi-Aventis, Rueil-Malmaison, France; Institut de Recherches Internationales Servier, Courbevoie, France; the NSERC Discovery Program; and the Heart and Stroke Foundation of Alberta and the Northwest Territories. D. N. Brindley is a Medical Scientist of the Alberta Heritage Foundation for Medical Research.
The technical assistance of K. MacNaughton, S. Sokolik, and X. Li is gratefully acknowledged.
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