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ANIMAL NUTRITION |
,
,,2
* Department of Animal Sciences,
and
Department of Veterinary Clinical Medicine, and
Division of Nutritional Sciences, University of Illinois, Urbana 61801
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Abstract |
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 Top Abstract INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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Key Words: adipose tissue • body composition • cat • gene expression • ovariohysterectomy • physical activity
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INTRODUCTION |
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TopAbstractINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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; Lund et al., 2005). Although gonadectomy is an effective strategy for controlling overpopulation, it is a risk factor for feline obesity. The effects of estrogen on metabolic rate and disease risk are evident but poorly understood. Estrogen not only has receptors in the hypothalamus, but has also been shown to interact with leptin and increase its action, suggesting a role in appetite regulation (Cooke and Naaz, 2004). Estrogen has also been associated with increased voluntary exercise, with ambulatory activity decreasing in ovariectomized rats (Shimomura et al., 1990). Although these interactions have been demonstrated, the mechanisms contributing to increased food intake and BW gain in spayed cats are unknown.
Because altered glucose and lipid metabolism accompany BW gain, identifying differences in skeletal muscle and adipose tissue after spaying is of interest. Skeletal muscle plays an important role in glucose metabolism and accounts for approximately 80% of insulin-mediated glucose uptake (Defronzo et al., 1981), a response that is diminished in obese individuals. In addition to its role in energy storage, adipose tissue is now accepted as an active endocrine organ intricately involved in energy homeostasis and metabolism. The metabolic dysfunction that occurs in muscle after spaying, with BW gain, or both, is not entirely understood, but may be partly attributed to low-grade inflammation. It is now well accepted that much of this inflammation is derived from inflammatory mediators produced by immune cells present in adipose tissue. Because estrogen receptors are expressed in several cell types in adipose tissue including preadipocytes and macrophages (Gulshan et al., 1990; Venkov et al., 1996), studying the effects of spaying on adipose tissue function is justified.
Recent feline genome sequencing initiatives have allowed the measurement of gene transcripts that may aid in our understanding of spaying and its effect on adipose and muscle metabolism. The objectives of this experiment were 2-fold. Because the effects of food restriction to maintain BW following spaying have been poorly studied, we first aimed to evaluate the effects of spaying and 12 wk of food restriction on BW, body composition, activity levels, blood metabolites, and mRNA abundance of genes associated with lipid metabolism and inflammation in subcutaneous adipose and skeletal muscle tissues of adult cats. Second, we aimed to measure these same outcomes in cats fed ad libitum for an additional 12 wk.
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MATERIALS AND METHODS |
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TopAbstractINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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Animals and Diet
Eight adult (mean age = 1.6 ± 0.13 yr old; mean BW = 3.52 ± 0.72 kg) female domestic shorthair cats (Liberty Research Inc., Waverly, NY) were used. Cats were individually housed in stainless steel cages measuring 0.61 x 0.61 x 0.61 m in the animal facility of the Edward R. Madigan Laboratory at the University of Illinois. Room dimensions were 7.32 m wide x 9 m long x 3.96 m high. The room was climate-controlled (22°C) with a 16 h light:8 h dark cycle. Cats were fed a commercially available, nutritionally complete dry cat food (Iams Original Formula Cat Food, Procter and Gamble, Dayton, OH) throughout the experiment and were given ad libitum access to water. The primary ingredients in the diet included chicken, chicken byproduct meal, corn grits, corn meal, chicken fat, fish meal, and dried beet pulp. Analyzed dietary chemical composition is listed in Table 1.
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Experimental design is presented in Figure 1. Four weeks before the experiment, cats were acclimated to the diet and fed at a level to maintain BW. During this time, cats were also acclimated to a daily activity schedule that allowed for group interaction but individual housing and feeding. This period also allowed for an accurate estimate of the caloric intake required to maintain a stable BW before treatment. Cats were housed in pens individually for 7 h/d (0800 to 0900 h; 1000 to 1200 h; 1500 to 1900 h) and group housed for 17 h/d in the room, including the dark period. After the baseline period (wk 0), all activity and body composition measurements were performed and blood and tissue samples were collected. Cats were then spayed and studied for 24 additional wk. At wk 0 (pre-spay), 6, 12, 18, and 24, fasted blood samples were collected for glucose, leptin, ghrelin, triacylglyceride (TG), NEFA, fructosamine, and insulin analysis. Subcutaneous abdominal adipose and LM biopsies also were collected at wk 0, 12, and 24.
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). Body Composition
Lean mass, fat mass, and bone mineral mass were determined using dual-energy x-ray absorptiometry, which has been previously validated in cats (Speakman et al., 2001). Determinations were conducted after ovariohysterectomy and tissue biopsy, while still under sedation. Cats were placed in ventral recumbency and body composition was analyzed using a Hologic model QDR-4500 Fan Beam x-ray Bone Densitometer and software (Hologic Inc., Waltham, MA).
Activity Level Assessment
Voluntary activity levels was evaluated using Actical activity collars (Mini Mitter, Bend, OR), which were worn around the neck for 9 consecutive days before wk 0, 12, and 24. The collars contained omnidirectional sensors capable of accurately incorporating both intensity and duration of movements. Once the collars were removed, the Actical software analyzed the data compiled by the collar and converted it into arbitrary numbers referred to as activity counts. In the current data set, average activity was presented in activity counts per epoch (epoch length = 0.25 min). Actical software allowed for the determination of average activity counts per epoch during the light and dark periods without human interference.
Ovariohysterectomy
Food was withheld for at least 8 h before ovariohysterectomy. Cats were given free access to water. Cats were premedicated with ketamine and equal parts valium (11 mg/kg intramuscular), and anesthesia was induced and maintained with isoflurane in 100% oxygen. Briefly, a ventral medial incision was made and ovariohysterectomy was performed by T. K. Graves and T. Keel using standard techniques. Postsurgery, butorphanol (0.22 mg/kg) was administered to alleviate discomfort. All cats were completely recovered within 2 wk postsurgery, returning to their normal food intake and activity levels.
Sample Collection and Analysis
Representative diet samples were collected and ground using a Wiley mill (model 4, Thomas Scientific, Swedesboro, NJ) through a 2-mm screen and dry ice in preparation for chemical analyses. Diet samples were analyzed for DM and OM according to AOAC (1984). Crude protein was determined according to AOAC (1995) using a Leco Nitrogen/Protein Determinator (model FP-2000, Leco Corporation, St. Joseph, MI). Fat concentrations were measured by acid hydrolysis (AACC, 1983) followed by ether extraction (Budde, 1952). Gross energy was measured by use of a bomb calorimeter (Model 1261, Parr Instrument Co., Moline, IL). Total dietary fiber concentration was determined according to Prosky et al. (1992).
Fasting (8 h) blood samples (3 and 8 mL) were collected at wk 0, 6, 12, 18, and 24 via jugular venipuncture. One sample (3 mL) was collected into BD Vacutainer tubes (#367835, 5.4 mg of K2 EDTA, Becton, Dickinson, and Company, Franklin Lakes, NJ) for plasma. The second sample (8 mL) was first used for glucose measurement (1 drop needed) using the glucose oxidase method by utilizing a Precision G Blood Glucose Testing System (Medisense, Bedford, MA). The remainder of the second blood sample was transferred to the appropriate serum separator tubes (#367985, Becton, Dickinson, and Company). All tubes were centrifuged (13,000 x g for 15 min at 4°C) within 1 h of collection. Following centrifugation, supernatant was collected, separated into respective cryovials, and stored at –80°C until further analysis.
Serum fructosamine, TG, and NEFA were analyzed by the University of Illinois Clinical Diagnostic Laboratory (Urbana). Plasma leptin concentrations were determined using a commercially available RIA kit (Multispecies Leptin RIA Kit, Linco Research, St. Louis, MO) previously validated in cats (Backus et al., 2000). Serum insulin concentrations were determined using a commercial RIA kit (Double Antibody RIA, Diagnostic Systems Laboratories Inc., Webster, TX) validated in the cat by the manufacturer. Serum total ghrelin concentrations were determined using a commercially available enzyme immunoassay kit (Ghrelin Rat EIA Kit, Phoenix Pharmaceuticals Inc., Belmont, CA) following a 10-fold dilution of serum. Insulin, leptin, and ghrelin data were analyzed using GraphPad Software (Prism Software, San Diego, CA) before statistical analyses were performed.
At wk 0, 12, and 24, approximately 1 g of subcutaneous abdominal adipose tissue and 1 g of LM was collected following an overnight fast. Cats were sedated using atipamazole (0.02 mg/kg), atropine (0.04 mg/kg), and medetomadine (0.02 mg/kg). Samples were immediately flash frozen in liquid nitrogen and stored at –80°C until further analyses.
Total RNA Extraction and Quantitative Reverse Transcriptase-PCR
Total cellular RNA was isolated from adipose tissue and skeletal muscle using Trizol (Invitrogen, Carlsbad, CA). The concentration and purity of RNA was determined using an ND-1000 spectrophotometer (Nanodrop Technologies, Wilmington, DE). Conversion of RNA to complementary (c)DNA was carried out using methods described by the ABI cDNA Archive kit (Applied Bio-systems, Foster City, CA). Isolated cDNA was amplified using quantitative reverse transcriptase-PCR on an ABI Prism 7900HT Sequence Detection System (Applied Biosystems). Gene-specific primers for adiponectin, IL-6, IL-1, uncoupling protein 2, lipoprotein lipase (LPL), vascular endothelial growth factor, chemokine receptor 5, estrogen receptor 1, hormone-sensitive lipase (HSL), leptin, toll-like receptor 4, tumor necrosis factor 
(TNF-), and glyceraldehyde-3-phosphate dehydrogenase, were designed using Primer Express 2.0 Software (Perkin Elmer, Boston, MA; Table 2). The mRNA abundance for all of the genes was tested in adipose tissue, whereas only estrogen receptor 1, uncoupling protein 2, LPL, and HSL were measured in skeletal muscle.
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Statistical Analyses
The experimental design consisted of a single factor (week) experiment with repeated measures. Data were analyzed using the MIXED procedure (SAS Inst. Inc., Cary, NC). Cat and week were tested as fixed effects. Least squares means for food intake and BW data were compared with wk 0, and least squares means for all other data were compared between week by using the least significant difference method when the main effects were significant. Results are presented as means ± SEM. The correlation between leptin and body fat mass was analyzed using the Pearson correlation in SAS. Relative calculated levels of mRNA in fat and skeletal muscle tissues were analyzed using MIXED procedure of SAS. Again, cat and week were tested as fixed effects. A probability of P < 0.05 was considered significant and P < 0.10 was considered to be a trend.
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RESULTS |
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TopAbstractINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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To maintain ideal BW during the first 12 wk after spaying, food intake was decreased, with decreases (P < 0.05) occurring by wk 7 (30.6% by wk 12; Figure 2). After 12 wk, cats were fed ad libitum. As expected, a large increase in food intake resulted from wk 12 to 13, followed by a gradual decline that was still greater (P < 0.05) at wk 23 than at baseline (40.5% increase from wk 12 to 24). Food intake at wk 24 was not different from baseline.
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Total fat mass and bone mineral content were greater (P < 0.05) at wk 24 compared with wk 0 and 12 (Table 3). Lean mass also was greater at wk 24 compared with wk 0 and tended (P < 0.10) to be greater than at wk 12. Body fat percentage did not differ from wk 0 to 12, but was greater (P < 0.001) at wk 24 compared with wk 0 and 12. Although lean mass (g) increased from wk 0 to 24, the dramatic change in fat mass resulted in decreased (P < 0.05) lean mass percentage at wk 24 compared with wk 0 and 12. Bone mineral content percentage increased (P < 0.05) from wk 0 to 24, which was likely the result of an increased need for skeletal support of a greater BW.
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From wk 0 to 24, total daily activity decreased by 52%, decreasing (P < 0.001) from 30.0 to 14.3 ± 1.82 mean activity counts per epoch. In the first 12 wk, activity during light hours did not change, but activity during the dark period decreased (P < 0.001) dramatically (Figure 3). From wk 12 to 24, total activity decreased (P < 0.05) further, primarily because of the decreased (P < 0.05) activity during light hours.
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By wk 24, cats had increased (P < 0.001) serum glucose and TG concentrations compared with wk 0, 6, 12, and 18 (Table 4). Circulating leptin also tended to be greater (P < 0.10) at wk 18 and 24 compared with wk 0.
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Adipose LPL mRNA abundance was decreased (P < 0.05) at wk 12 and 24 compared with wk 0 (Table 5). Similarly, HSL mRNA abundance was decreased (P < 0.05) at wk 24 compared with wk 0 and 12. In this study, TNF- mRNA tended to decrease (P < 0.10) by wk 12 compared with wk 0, but was not different at wk 24 (Table 5). Adipose IL-6 mRNA expression was increased (P < 0.05) at wk 12 and 24 compared with wk 0 (Table 5). Adipose leptin mRNA expression levels decreased (P < 0.05) at wk 12, but were not different at wk 24 compared with wk 0 (Table 5). Adipose adiponectin mRNA was not different between wk 0 and 12, but was decreased (P < 0.05) at wk 24 (Table 5). Skeletal muscle mRNA abundance was not changed (P > 0.05) throughout the experiment (data not shown).
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DISCUSSION |
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TopAbstractINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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Similar to what has been reported by other research groups, our data suggest that caloric restriction of approximately 30% is required to avoid BW gain following ovariohysterectomy in cats. Flynn et al. (1996) and Hoenig et al. (2002) reported a reduced caloric intake of 37 and 13%, respectively, to maintain baseline BW in spayed cats. Because cats were fed to maintain BW for the first 12 wk, body composition and blood metabolite concentrations were not altered during this period. This allowed for the measurement of activity and gene expression changes due to spaying and food restriction without the bias of BW gain.
Effects of Spaying and Food Restriction on Physical Activity
Reduced physical activity has been suggested as a contributing factor to increased BW gain following ovariohysterectomy but has not been quantitatively measured in cats. Flynn et al. (1996) subjectively measured activity in cats following ovariohysterectomy, reporting no significant differences from pre-spay. However, in that study, cats were only observed for 5 to 10 min/d over the course of a few weeks. The use of activity collars in this experiment allowed for continuous monitoring over several days during dark and light periods and without human interference. Our results demonstrated a dramatic decrease in physical activity after gonadectomy and food restriction, especially during the dark period. To our knowledge, these data are the first of its kind to be reported in spayed cats. Given this interesting outcome, further research is needed to distinguish cause-and-effect relationships between physical activity and the physiological and gene expression changes observed in this experiment.
Effects of Spaying and Food Restriction on Adipose mRNA Abundance
Lipid metabolism is commonly altered following ovariohysterectomy, BW gain, or both (Hoenig et al., 2006), but is rarely tested in spayed cats that are food-restricted. Lipoprotein lipase and HSL are key regulators of lipid storage and affect blood lipid concentrations. The effects of estrogen on LPL activity remain controversial. For example, Palin et al. (2003) reported decreased LPL protein expression at their greatest dose (E2 10–7 mol/L) of estrogen, but increased expression at lesser doses (E2 10–12 mol/L). Other research conducted in mice has shown that estrogen results in decreased LPL activity (Venkov et al., 1996). In the current experiment, wk 12 adipose mRNA abundance of HSL was unchanged, but that of LPL was decreased. However, blood lipid concentrations at 12 wk were not changed. Thus, because mRNA abundance and enzyme activities may not always agree with one another, further research is required to determine whether the decreased LPL mRNA is physiologically relevant.
One mechanism by which LPL is thought to be affected is through inhibition by proinflammatory cytokines such as TNF- and IL-6 that are known to increase with obesity (Greenberg et al., 1992; Kern et al., 1995). Cytokine response to spaying with food restriction has not been well studied. To our knowledge, IL-6 mRNA expression has not been reported in feline adipose tissue. Elevated proinflammatory cytokine concentrations following reduced estrogen production have been reported in some human studies (Cheleuitte et al., 1998), but not others (Rogers and Eastell, 1998). Increased IL-6 expression at wk 12 in the current study suggests that estrogen withdrawal may increase proinflammatory cytokine production and is independent of BW gain. Increased IL-6 also correlates with the decreased LPL expression, supporting its inhibitory role. Tumor necrosis factor- mRNA, however, tended to be decreased in adipose tissue at wk 12. More research is needed to determine mechanisms by which estrogen directly or indirectly affects cytokine mRNA and protein abundance.
Gonadal steroids modulate central nervous system effectors of energy homeostasis that are targets of leptin action. Whereas androgens are anabolic in nature, estrogens are catabolic and decrease food intake and BW (reviewed by Mystkowski and Schwartz, 2000). Mechanisms by which these effects occur, however, are poorly defined. Because estrogen receptors are present in adipose tissue (Pedersen et al., 1996), estrogen is thought to have a direct effect on leptin biosynthesis. Studies using rat adipocytes in culture have supported this hypothesis, demonstrating increased leptin secretion with the administration of estrogenic compounds (Machinal et al., 1999). Although BW and fat mass did not change during food restriction and spaying in the current experiment, leptin mRNA abundance was greatly decreased (approximately 23% of baseline expression) in adipose tissue. These data suggest that estrogen withdrawal and food restriction to maintain BW result in decreased leptin secretion. Because circulating leptin was not different, further study is warranted.
Effect of Ad Libitum Feeding on Body Composition and Blood Metabolites
As expected, ad libitum feeding resulted in dramatic increases in BW, body composition, and blood metabolite concentrations. Over the 12-wk period, total fat mass increased by approximately 120% compared with about 13% of lean mass. Harper et al. (2001) reported similar data (40% increase in fat mass and 10% in lean mass), suggesting that most BW gained following gonadectomy is in the form of fat. Concentrations of blood glucose, TG, and leptin were increased with BW gain. Because cats may display stress-induced hyperglycemia (Rand et al., 2002), an indicator of blood glucose concentrations over a 2- to 3-wk period (fructosamine) was measured. Although fructosamine concentrations increased with BW gain (data not shown), all values were within the normal range for cats (Bennett, 2002). Hypertriglyceridemia, which is often present in obese and type-2 diabetic cats (Hoenig et al., 2003), was also noted by wk 24. Finally, leptin concentrations tended to increase with BW gain during ad libitum feeding. Despite the lack of large changes in blood leptin concentrations, its relationship with body fat mass was highly correlated (r = 0.80). Therefore, ad libitum feeding resulted in the desired physiologic outcomes for comparison with mRNA abundance and physical activity.
Effect of Ad Libitum Feeding and BW Gain on Adipose mRNA Abundance
Altered adipose and skeletal muscle gene expression levels were expected to accompany the dramatic changes in body composition from wk 12 to 24. Decreased adipose tissue LPL mRNA abundance agrees with the results of Hoenig et al. (2006), who reported decreased LPL mRNA and enzyme activity levels in obese versus lean cats. Those researchers proposed that decreased adipose tissue LPL mRNA expression is compensated for by increased LPL expression in skeletal muscle, potentially increasing lipid deposition in skeletal muscle and contributing to insulin resistance. Adipose HSL mRNA was decreased with BW gain, which contradicts obese feline data reported by Hoenig et al. (2006), but is in agreement with Large et al. (2000) who noted decreased HSL mRNA, protein levels, and activity in obese humans. Decreased adipose HSL and LPL mRNA expression suggest a reduced activity of adipocytes with BW gain, yet the signals for such changes remain unknown.
Adiponectin mRNA abundance was decreased with BW gain, a result reported in other species, including humans (Weyer et al., 2001). Circulating adiponectin concentrations have been positively correlated with insulin sensitivity in humans and cats (Weyer et al., 2001; Hoenig et al., 2007). Although circulating adiponectin was not measured in this experiment, tissue mRNA levels suggest the potential for insulin resistance with sustained obesity. Although blood leptin tended to increase with BW gain, as reported in previous feline studies (Appleton et al., 2000), the lack of change in leptin mRNA was surprising. This discrepancy indicates that adipose tissue mRNA expression may not be indicative of plasma leptin concentrations.
Does Spaying Alter "Set Point"?
Many of the observed changes in this experiment suggest an altered "set-point" BW and metabolic activity within tissues with spaying. A "sliding set point" has been reported in seasonal animals that is dependent on photoperiod (reviewed by Rousseau et al., 2003), but has not been suggested with estrogen withdrawal. Several outcomes of this experiment, however, suggest the possibility of such a response. Briefly, adipose leptin mRNA was dramatically decreased after spaying and food restriction. Even though circulating leptin concentration was not different after 12 wk, this tissue-specific response justifies further study. Adiponectin and HSL mRNA concentrations were numerically decreased as well. Decreased adipose LPL mRNA and increased IL-6 mRNA without change in BW suggest a regulatory capacity of estrogen in these systems as well. Further support comes from the lack of expected responses to extreme BW gain during ad libitum feeding. Despite doubling fat mass from wk 12 to 24, leptin and TNF- mRNA levels were not different from baseline as has been demonstrated in previous feline studies (Hoenig et al., 2006), and mRNA of LPL and HSL continued to be significantly decreased. Given the number of unexpected outcomes and the inability to measure cause-and-effect with such an experiment, further research is necessary to clearly identify the role of estrogen in these biological pathways and determine whether dietary intervention (e.g., those containing estrogenic compounds) may successfully minimize these changes and avoid BW gain following ovariohysterectomy.
We have presented several novel findings in food-restricted cats to maintain BW following ovariohysterectomy. During this period, voluntary physical activity was dramatically reduced, especially during the dark period. Moreover, our data suggest that estrogen withdrawal and food restriction affect the expression of several genes associated with lipid metabolism and inflammation, namely LPL, leptin, and IL-6, that may contribute to changes in appetite, blood lipid concentrations, and insulin sensitivity. Although further research is required for validation, these changes suggest that the set point of a cat may be adjusted by gonadectomy, leading to increased food intake and the physiologic outcomes that follow. By identifying changes in gene expression and physical activity of estrogen withdrawal and food restriction, potential targets for nutritional intervention or lifestyle management have been identified.
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Footnotes |
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2 Corresponding author: ksswanso{at}uiuc.edu
Received for publication January 21, 2008. Accepted for publication November 4, 2008.
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LITERATURE CITED |
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TopAbstractINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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in lean and obese cats. Domest. Anim. Endocrinol. 30:333–344.[Medline]
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