animal
Animal (2009), 3:9, pp 1287–1298 & The Animal Consortium 2009
doi:10.1017/S1751731109004868
Ovariohysterectomy alters body composition and adipose and
skeletal muscle gene expression in cats fed a high-protein
or moderate-protein diet
B. M. Vester1, S. M. Sutter1, T. L. Keel2, T. K. Graves2 and K. S. Swanson11
Department of Animal Sciences, University of Illinois, Urbana, IL 61801, USA; 2Department of Veterinary Clinical Medicine, University of Illinois, Urbana,
IL 61801, USA
(Received 14 October 2008; Accepted 4 May 2009; First published online 28 May 2009)
The objective of this study was to measure changes in body composition, physical activity and adipose and skeletal muscle
gene expression of cats fed a high-protein (HP) diet or moderate-protein (MP) diet, following ovariohysterectomy. Eight cats
were randomized onto HP or MP diets and were fed those diets for several months prior to baseline. All cats underwent an
ovariohysterectomy at baseline (week 0) and were allowed ad libitum access to dietary treatments for 24 weeks. Food intake
was measured daily, and BW and body condition score were measured weekly. Blood, adipose and skeletal muscle tissue
samples were collected, physical activity was measured, and body composition was determined using DEXA (dual-energy X-ray
absorptiometry) at weeks 0, 12 and 24. Caloric intake increased soon after ovariohysterectomy, resulting in increased ( P , 0.05)
BW at weeks 12 and 24 compared to week 0. Body condition score and body fat percentage increased ( P , 0.05) over time.
Blood glucose increased ( P , 0.05) linearly over time. Non-esterified fatty acids were decreased ( P , 0.05) at weeks 12 and
24 compared to week 0. Blood leptin increased ( P , 0.05) over time. Total physical activity decreased ( P , 0.05) from week
0 to weeks 12 and 24 in all cats. Adipose tissue mRNA abundance of adiponectin, hormone sensitive lipase, toll-like receptor-4,
uncoupling protein-2 (UCP2) and vascular endothelial growth factor decreased ( P , 0.05) linearly over time, regardless of diet.
Skeletal muscle mRNA abundance for glucose transporter-1, hormone sensitive lipase and UCP2 were decreased ( P , 0.05),
regardless of dietary treatment. Our research noted metabolic changes following ovariohysterectomy that are in agreement with
gene expression changes pertaining to lipid metabolism. Feeding cats ad libitum after ovariohysterectomy is inadvisable.
Keywords: cat, protein, ovariohysterectomy, obesity
Implications
Feline obesity is increasing, and one of the risk factors for
obesity in female cats is ovariohysterectomy. High-protein (HP)
diets have been utilized in felines to promote weight loss
while maintaining lean body mass and to control diabetes, and
have been shown in rodents and humans to modify satiety
and food intake. This study evaluated the use of a HP diet to
ameliorate post-ovariohysterectomy weight gain in cats. Our
results indicate that a HP diet did not ameliorate weight gain,
but did result in changes in adipose and skeletal muscle gene
expression that may be of interest in future studies.
Introduction
Obesity is a widespread problem in domestic cat populations of developed countries. It is estimated that 25% to
-
E-mail: [email protected]
34% of cats are overweight or obese (Scarlett et al., 1994;
Donoghue and Scarlett, 1998; Allan et al., 2000; Lund et al.,
2005; Freeman et al., 2006). As in humans, feline obesity
leads to hyperlipidemia, insulin resistance and glucose
intolerance. The main risk factors for feline obesity are
middle age and ovariohysterectomy or neutering (Scarlett
et al., 1994; Donoghue and Scarlett, 1998; Robertson,
1999; Lund et al., 2005). Middle-aged cats (5 to 11 years of
age) appear to have the highest risk of developing obesity
(Lund et al., 2005). Although neutering increases the risk
of obesity, the benefits of controlling pet overpopulation
outweigh this risk.
Authors often report that ovariohysterectomized cats have
decreased metabolic energy requirements, increased food
intake and altered blood hormone concentrations, but without explanation. Research aimed at identifying mechanisms
responsible for increased feline obesity following ovariohysterectomy or neutering, and the influence diet may have
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Vester, Sutter, Keel, Graves and Swanson
on its development, has been limited. Measuring changes in
gene transcripts (mRNA) may identify key mechanisms by
which neutering influences energy homeostasis and targets
for dietary intervention.
The effects of dietary composition on appetite, body
composition and tissue gene expression in rodents and
humans are well documented, but similar data are lacking
in the cat. HP diets increase satiety response, which has
been attributed to increased hormone concentrations,
including glucagon like peptide-1 and cholecystokinin that
are known to curb appetite (Liddle et al., 1986; Backus
et al., 1995; Lejeune et al., 2006). The exact mechanisms
by which satiety is reached and the hormone interactions
that occur are still unknown. HP diets (50% crude protein;
58 g/kg BW0.67) increase heat production (Hoenig et al.,
2007), maintain insulin sensitivity (Hoenig et al., 2007) and
maintain lean body mass during weight loss (Laflamme and
Hannah, 2005) in cats. Thus, a HP diet may be protective of
weight gain following ovariohysterectomy or neutering.
Estrogen plays a key role in adipose tissue deposition and
function (Cooke and Naaz, 2004), and decreases leptin
receptor expression in the hypothalamus and adipose tissue
(Meli et al., 2004). Estrogen plays a role in spontaneous
physical activity, and therefore, its removal can lead to
decreased muscle mass and strength (Brown, 2008). These
findings suggest that estrogen plays a role not only in
appetite regulation but also influences tissues important in
energy balance, lipid metabolism and glucose transport. An
earlier study in our lab reported changes in feline adipose
and skeletal muscle tissue mRNA following ovariohysterectomy (Belsito et al., 2009), but was focused on foodrestriction rather than dietary composition.
Therefore, the objective of this study was to determine
the effects of ovariohysterectomy on changes in body
composition, blood metabolite concentrations, physical
activity levels and adipose and skeletal muscle tissue mRNA
abundance in cats fed a HP or moderate-protein (MP) diet.
We hypothesized that HP-fed cats would regulate food
intake, resulting in less weight gain and fewer metabolic
changes following ovariohysterectomy, as compared with
MP-fed cats.
Material and methods
Animals and diet
Eight adult (mean age 5 2.0 6 0.02 years of age; mean
BW 5 4.19 6 0.37 kg) female domestic shorthair cats (Liberty
Research, Inc., Waverly, NY, USA) were used. Cats were
individually housed in stainless steel cages (0.61 m 3 0.61
m 3 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 3 9 m long 3 3.96 m high. The room was
climate controlled (228C) with a 16 h light : 8 h dark cycle.
Cats were randomized to HP or MP diets (Table 1) and were
fed these diets for several months prior to baseline. Cats were
allowed ad libitum access to food and water throughout the
study. All procedures were approved by the University of
Table 1 Ingredient and chemical composition of the high-protein (HP)
and moderate-protein (MP) diets fed to cats
Item
As fed (g/kg)
Chicken meal
Potato product
Chicken fat
Eggs
Herring meal
Beet pulp
Natural flavors
Herring oil
Premium cat vitamin premix1,2
Salt
Premium cat mineral premix1,3
Potassium chloride
Dried chicory root
Natural antioxidant, dry
DL methionine
Chemical composition
Dry matter
g/kg dry matter
Organic matter
Crude protein
Acid hydrolyzed fat
Total dietary fiber
Gross energy (kcal/g)
Metabolizable energy4 (kcal/g)
MP
HP
308.4
403.8
127.9
50.0
50.0
30.0
10.0
3.4
6.5
2.5
1.5
3.9
1.0
0.3
0.7
647.9
106.9
85.7
50.0
50.0
30.0
10.0
6.6
6.5
2.5
1.5
1.0
1.0
0.5
0.0
942.2
948.3
912.8
343.4
192.3
68.8
5.2
4.4
891.9
528.8
235.0
20.1
5.5
4.8
1
Trouw Nutrition (Highland, IL, USA).
Composition of mineral premix (g/kg): calcium carbonate, 360.2; zinc
sulfate, 208.3; Optimin zinc, 166.7; iron sulfate, 77.4; Optimin iron, 53.3;
copper sulfate, 35.7; Optimin copper, 3.0; manganese sulfate, 23.4; Optimin
manganese, 16.7; selenium, 12.0; carrier (soybean oil), 10.0; Optimin cobalt,
3.8; iodine, 1.8; cobalt carbonate, 0.6.
3
Composition of vitamin premix (g/kg): carrier (pea fiber), 728.4; calcium
carbonate, 170.9; vitamin E 50% adsorbate, 40.0; betaine (source of
choline), 26.0; carrier (soybean oil), 10.0; nicotinic acid, 9.6; vitamin A, 4.0;
D-calcium pantothenate, 2.7; vitamin B1 (thiamine mononitrate), 2.7; vitamin
B2 (riboflavin), 1.25; beta carotene, 1.0; vitamin B12, 1.0; vitamin D3, 0.8;
biotin, 0.7; vitamin B6 (pyridoxine), 0.7; folic acid, 0.2.
4
Calculated according to NRC (2006), predictive equations for metabolizable
energy in cat foods for prepared cat foods using total dietary fiber.
2
Illinois Institutional Animal Care and Use Committee prior to
experimentation.
Experimental design
Cats were evaluated using a completely randomized design
with repeated measures over time. Cats were acclimated to
a physical activity schedule according to Belsito et al.
(2009). Cats were housed in pens individually for 7 h/day
and group housed for 17 h/day in the room, including
the dark period. Food was available only during times cats
were individually housed in cages. Cats underwent ovariohysterectomy at week 0 and were evaluated for an additional 24 weeks. Baseline (week 0) body composition and
physical activity were assessed and 12 h food-restricted
blood samples, subcutaneous adipose tissue and skeletal
muscle (longissimus dorsi) tissue were collected prior to
ovariohysterectomy. All measurements were repeated at
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High-protein diet following ovariohysterectomy
12 and 24 weeks after ovariohysterectomy. Cats were weighed
(GSE 350; Mettler Toledo, Inc., Columbus, OH, USA) and body
condition score (Laflamme, 1997) determined weekly.
Body composition
Lean mass, fat mass and bone mineral content (BMC) were
determined using DEXA (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, USA).
Physical activity level assessment
Voluntary physical activity levels were evaluated using
Actical activity collars (Mini Mitter, Bend, OR, USA), which
were worn around the neck for seven consecutive days prior
to weeks 0, 12 and 24. Collars contain omnidirectional
sensors capable of accurately incorporating both intensity
and duration of movements. Once the collars were
removed, Actical software analyzed data compiled by the
collar and converted it into arbitrary numbers referred to as
‘activity counts’. In the current dataset, average activity was
presented as activity counts per epoch (epoch length 5
0.25 min). Actical software allowed for the determination
of average activity counts per epoch during light and dark
periods without human interference.
Sample collection
Food was withheld for at least 12 h prior to ovariohysterectomy and tissue biopsies. Tissue biopsies were collected
during ovariohystectomy at week 0. Cats were allowed free
access to water. For all tissue biopsies, cats were sedated
with butorphanol (0.02 mg/kg), atropine (0.04 mg/kg) and
medetomadine (0.02 mg/kg; intramuscular). Anesthesia was
induced and maintained with isoflurane in 100% oxygen
during ovariohysterectomy. Food-restricted blood samples
(via jugular venipuncture, prior to isoflurane exposure) and
approximately 1 g of subcutaneous abdominal adipose
tissue and 1 g of longissimus dorsi muscle were collected
following an overnight fast. Tissue samples were immediately flash frozen in liquid nitrogen and stored at 2808C
until further analyses.
Sample analyses
Representative diet samples were collected and ground
using a Wiley mill (model 4; Thomas Scientific, Swedesboro,
NJ, USA) through a 2 mm screen and dry ice in preparation
for chemical analyses. Diet samples were analyzed for dry
matter (DM) and organic matter according to the Association of Official Analytical Chemists (1984). Crude protein
was determined according to the Association of Official
Analytical Chemists (1995) using a Leco Nitrogen/Protein
Determinator (model FP-2000; Leco Corporation, St. Joseph,
MI, USA). Fat concentrations were measured by acid
hydrolysis (American Association of Cereal Chemists, 1983)
followed by ether extraction (Budde, 1952). Gross energy
was measured using a bomb calorimeter (Model 1261; Parr
Instrument Co., Moline, IL, USA). Total dietary fiber concentration was determined according to Prosky et al. (1992).
Food-restricted blood samples were immediately analyzed
for glucose using the glucose oxidase method (Precision G
Blood Glucose Testing System; Medisense, Bedford, MA, USA).
Blood was then transferred to the appropriate collection tubes,
and all tubes were centrifuged (30 min at 1300 3 g; 48C)
within 1 h of collection. Following centrifugation, supernatant
was collected, separated into respective cryovials and stored at
2808C until further analyses.
Serum fructosamine, triglyceride (TG), nonesterified fatty
acid (NEFA) and thyroxine concentrations were analyzed by
the University of Illinois Clinical Diagnostic Laboratory.
Plasma leptin concentrations were determined using a
commercially available radioimmunoassay kit (Multispecies
Leptin RIA Kit; Linco Research, St. Louis, MO, USA; interassay variation 7.9%; intra-assay variation 4.8%). Serum
insulin concentrations were determined using a commercial
RIA kit (Double Antibody RIA; Diagnostic Systems Laboratories Inc., Webster, TX, USA), validated in cats 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, USA; inter-assay variation 12.5%; intra-assay
variation 8.7%), following a ten-fold dilution of serum. All
blood analysis kits were previously validated for use in our
laboratory (Belsito et al., 2009). Insulin, leptin and ghrelin
data were analyzed using GraphPad Software (Prism Software, San Diego, CA, USA) before statistical analyses were
performed.
Total RNA extraction and quantitative reverse
transcriptase-PCR
Total cellular RNA was isolated from adipose tissue and
skeletal muscle using Trizol (Invitrogen, Carlsbad, CA, USA).
The concentration and purity of RNA was determined using
a ND-1000 spectrophotometer (Nanodrop Technologies,
Wilmington, DE, USA). Conversion of RNA to cDNA was
done using a cDNA Archive kit (Applied Biosystems, Foster
City, CA, USA) according to manufacturer’s instructions.
Isolated cDNA was amplified using real time reverse transcriptase-PCR on an ABI PRISM 7900HT Sequence Detection
System (Applied BioSystems). Gene-specific primers (Supplementary Table 1) were designed using Primer Express 2.0
Software (Perkin Elmer, Boston, MA, USA) or from published literature (Zini et al., 2009). Genes with available
mRNA sequence and having roles in lipid metabolism and
transport (peroxisome proliferator-activating receptor gamma
isoform-1 (PPARg1); peroxisome proliferator-activating receptor gamma isoform-2 (PPARg); hormone sensitive lipase (HSL);
lipoprotein lipase (LPL)), glucose metabolism and transport
(insulin receptor (IR); glucose transporter 1 (GLUT1); glucose
transporter 4 (GLUT4)), inflammation (interleukin 1 (IL-1);
interleukin 6 (IL-6); tumor necrosis factor alpha (TNF-a);
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Vester, Sutter, Keel, Graves and Swanson
Results
monocyte chemotactic protein 1 (MCP-1); chemokine receptor
5 (CCR5); toll-like receptor 4 (TLR4)) and genes having various
roles in adipose tissue (uncoupling protein 2 (UCP2); leptin;
adiponectin; vascular endothelial growth factor (VEGF);
estrogen receptor 1 (ESR1); plasminogen activator inhibitor 1
(PAI1)) were evaluated in adipose tissue samples. Skeletal
muscle tissue was analyzed for UCP2, LPL, HSL, GLUT1 and
GLUT4 mRNA abundance.
The PCR mixture contained 5 and 50 ng/ml of cDNA from
adipose and skeletal muscle tissue, respectively, 15 pmol of
each primer, and 5 ml SYBR Green. The final volume was
adjusted to 10 ml using sterile deionized water. The 18S
rRNA gene was used as an internal standard. Data analyses
were conducted with sequence detection system software
(Applied BioSystems). All target genes were normalized to
18S prior to statistical analyses.
Dry matter intake increased (P , 0.001) following ovariohysterectomy and was greater (P , 0.001) at weeks 12 and 24
compared to week 0 (Figure 1). Intake was lower (P , 0.001)
at week 24 compared to week 12, but remained higher than
baseline. Intake was highest at week 8 (81.6 g DM/day).
Caloric intake over the entire course of the study (determined
from calculated metabolizable energy (ME)) tended to be
greater (P 5 0.08) in HP-fed cats. BW and body condition
scores (BCS) increased (P , 0.001) from week 0 (mean
BW 5 4.18 kg; mean BCS 5 6.0) to weeks 12 (mean BW 5
5.08 kg; mean BCS 5 7.0) and 24 (mean BW 5 5.21 kg; mean
BCS 5 7.0), regardless of dietary treatment (Figure 2). Cats
consumed less (P , 0.05) food from week 12 to 24; however,
BW did not differ from week 12 to 24.
Body composition did not change due to diet; however,
body fat mass (g) increased (P , 0.05) over time (Table 2).
Body fat percentage tended to increase (P 5 0.06) linearly
over time. Lean tissue mass (g) increased (P , 0.05) curvilinear over time, increasing rapidly from week 0 to 12 and
then was stable from week 12 to 24. Lean body mass
percentage decreased (P , 0.05) linearly over time. Bone
mineral mass (g) increased (P , 0.05) linearly over time. At
week 12, bone mineral mass was greater (P , 0.05) in cats
fed HP compared to those fed MP, but this difference was
not present at week 24. Body fat mass was positively
correlated (r 5 0.89; P , 0.001) with BCS.
Food-restricted blood glucose concentrations increased
(P , 0.05) over time, while serum NEFA concentrations
decreased (P , 0.05) from week 0 to weeks 12 and 24,
regardless of dietary treatment (Table 3). Glucose increased
over time in cats, regardless of dietary treatment; however,
HP-fed cats had a greater increase from week 0 to week 12
Statistical analyses
Data were analyzed as repeated measures using the Mixed
Models procedure of SAS (SAS Inst. Inc., Cary, NC, USA)
with the best-fit covariance structure applied. The main
effects of diet and week were tested and the interaction
term was investigated if significant. Initial BW was used as
a covariate when analyzing BW over the course of the
study. Orthogonal contrasts were tested if the effect of
week was significant. If the interaction term was significant, contrasts were used to compare the effect of diet
within each week. Results are presented as means 6
standard error of the mean. The correlation between leptin
and body fat mass was analyzed using Pearson’s Correlation in SAS. A probability of P , 0.05 was considered significant. Due to the small number of animals per treatment,
P , 0.10 was considered a trend.
700
600
*
* * * *
*
*
ME intake, kcal/d
500
400
300
200
100
0
-3 -2 -1 0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24
-100
Week
HP
MP
Figure 1 Food intake of cats fed a high- or moderate-protein diet prior to and following ovariohysterectomy. Line represents the average initial
metabolizable energy requirement calculated based on NRC (2006). Food intake was greater (P , 0.05) in cats fed high-protein diet at weeks 4 to 11 after
ovariohysterectomy. Each point represents the mean of four cats 6 s.d. (* Difference (P , 0.05) due to diet).
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High-protein diet following ovariohysterectomy
7
6.5
Body weight, kg
6
5.5
5
4.5
4
3.5
3
-3 -2 -1 0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24
Week
HP
MP
Figure 2 BW of cats fed a high- or moderate-protein diet prior to and following ovariohysterectomy. BW was greater (P , 0.05) in cats fed high-protein
diet at weeks 9 to 24 after ovariohysterectomy. Each point represents the mean of four cats 6 s.d. (*Difference (P , 0.05) due to diet).
Table 2 Body composition of cats fed a high-protein (HP) or moderate-protein (MP) diet prior to and following ovariohysterectomy
P-value
Item
Fat (g)
Week 0
Week 12
Week 24
Lean (g)
Week 0
Week 12
Week 24
BMC (g)
Week 0
Week 12
Week 24
Fat (%)
Week 0
Week 12
Week 24
Lean (%)
Week 0
Week 12
Week 24
BMC (%)
Week 0
Week 12
Week 24
HP
MP
1202.4 6 373.3
2051.9 6 355.5
2162.1 6 344.7
1076.5 6 480.0
1280.0 6 709.5
1424.0 6 805.6
2836.7 6 278.2
3414.9 6 432.5
3362.0 6 411.5
2705.7 6 192.7
3098.8 6 56.9
3033.6 6 34.7
61.9 6 14.9
84.4 6 23.2
86.6 6 22.5
55.9 6 8.4
57.3 6 8.5
69.6 6 12.7
28.1 6 7.5
35.7 6 1.3
37.4 6 1.4
26.4 6 10.0
26.6 6 11.0
29.0 6 12.2
70.4 6 7.2
62.7 6 1.5
61.1 6 1.5
72.1 6 9.9
72.1 6 10.9
69.4 6 12.1
1.6 6 0.4
2.2 6 0.6
2.2 6 0.6
1.4 6 0.2
1.5 6 0.2
1.8 6 0.3
Diet
Week
Diet*week
0.16
0.001*
0.08
0.22
0.001-
0.13
0.16
0.002*
0.008
0.29
0.06
0.33
0.28
0.05*
0.26
0.16
0.002*
0.008
BMC 5 bone mineral content.
Data reported as means 6 s.d.
*Linear effect of week (P , 0.05).
Curvilinear effect of week (P , 0.05).
compared to MP-fed cats. A significant diet-by-week
interaction was present for blood TG concentrations. While
cats fed HP had increasing blood TG over time, TG in MPfed cats peaked at week 12 and decreased back to baseline
concentrations by week 24. Blood leptin concentrations
increased (P , 0.05) from week 0 to 12, and were greater
(P , 0.05) in MP cats. Leptin concentrations decreased
(P , 0.05) from week 12 to 24, but were still greater than
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Vester, Sutter, Keel, Graves and Swanson
Table 3 Blood concentrations of cats fed a high-protein (HP) or moderate-protein (MP) diet prior to and following ovariohysterectomy
P-value
Item
Glucose (mmol/l)
Week 0
Week 12
Week 24
NEFA (mEq/l)
Week 0
Week 12
Week 24
Triglycerides (mmol/l)
Week 0
Week 12
Week 24
Leptin (mg/l)
Week 0
Week 12
Week 24
HP
MP
5.2 6 5.1
8.7 6 2.4
12.0 6 1.9
5.9 6 2.1
6.8 6 2.4
9.0 6 5.0
0.5 6 0.1
0.4 6 0.1
0.5 6 0.04
0.8 6 0.2
0.3 6 0.1
0.3 6 0.1
0.4 6 0.04
0.5 6 0.09
0.6 6 0.1
0.4 6 0.08
0.6 6 0.3
0.4 6 0.09
2.7 6 1.5
7.0 6 1.3
5.7 6 0.5
Diet
Week
Diet*week
0.28
,0.001*
0.02
0.60
0.01-
0.007
0.13
0.23
0.05
0.02
0.04-
0.41
4.9 6 1.4
7.6 6 5.8
6.5 6 4.6
NEFA 5 non-esterified fatty acids.
Data reported as means 6 s.d.
*Linear effect of week (P , 0.05).
Curvilinear effect of week (P , 0.05).
Table 4 Physical activity of cats fed a high-protein (HP) or moderate-protein (MP) diet prior to and following ovariohysterectomy1
P-value
Item
Light
Week
Week
Week
Dark
Week
Week
Week
Average
Week
Week
Week
Ratio
Week
Week
Week
0
12
24
0
12
24
HP
MP
24.9 6 3.6
19.5 6 11.5
13.1 6 3.2
31.1 6 12.3
21.4 6 8.7
16.1 6 8.7
30.7 6 8.0
10.7 6 4.9
8.8 6 5.0
Diet
Week
Diet*week
0.45
0.01*
0.81
0.09
,0.001-
0.57
0.26
,0.001-
0.70
0.22
0.02*
0.58
41.1 6 4.1
16.5 6 8.7
14.9 6 6.3
0
12
24
26.9 6 5.0
15.1 6 7.9
11.7 6 1.8
34.4 6 9.0
19.0 6 8.7
15.8 6 7.8
0
12
24
0.8 6 0.1
1.9 6 0.7
2.1 6 1.7
0.8 6 0.3
1.4 6 0.3
1.1 6 0.2
Data reported as means 6 s.d.
1
Activity levels defined as arbitrary units referred to as ‘activity counts.’ Data compiled by activity collars by Actical software. Activity is presented in activity
counts per epoch (epoch length 5 0.25 min).
*Linear effect of week (P , 0.05).
Curvilinear effect of week (P , 0.05).
week 0. Blood leptin concentrations were positively correlated (P , 0.001) with body fat mass (g; r 5 0.80) and BW
(r 5 0.71). Blood insulin, ghrelin, fructosamine and thyroxine concentrations were not changed over time.
Physical activity decreased (P , 0.01) during the light
and dark periods over time following ovariohysterectomy
(Table 4). Activity during the light period was unaffected by
diet, but activity in the dark period tended to be higher
(P , 0.10) in MP-fed cats. The light to dark ratio of activity
increased (P , 0.02) linearly over time, increasing from
week 0 to week 12 and 24.
Adipose tissue mRNA abundance of adiponectin, HSL,
TLR4, UCP2 and VEGF decreased (P , 0.05) linearly over
time, regardless of diet (Table 5). IR mRNA decreased
(P , 0.05) curvilinear over time, while GLUT4 had a curvilinear increase (P , 0.05) over time. Adipose IL-6 mRNA
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High-protein diet following ovariohysterectomy
Table 5 Adipose tissue mRNA abundance (arbitrary units) of cats fed a high-protein (HP) or moderate-protein (MP)
diet prior to and following ovariohysterectomy
P-value
Item
Adiponectin
Week 0
Week 12
Week 24
Glucose transporter 4
Week 0
Week 12
Week 24
Hormone sensitive lipase
Week 0
Week 12
Week 24
Insulin receptor
Week 0
Week 12
Week 24
Interleukin-6
Week 0
Week 12
Week 24
Leptin
Week 0
Week 12
Week 24
Toll-like receptor 4
Week 0
Week 12
Week 24
Uncoupling protein 2
Week 0
Week 12
Week 24
Vascular endothelial growth factor
Week 0
Week 12
Week 24
HP
MP
407.2 6 217.9
129.8 6 338.9
205.1 6 149.9
285.4 6 349.7
104.2 6 779.0
76.0 6 406.7
6.6 6 1.7
23.5 6 5.4
19.8 6 12.2
6.6 6 2.7
23.6 6 6.0
30.3 6 6.7
198.2 6 58.6
120.9 6 63.1
149.1 6 61.9
185.2 6 121.6
88.3 6 25.4
110.5 6 51.6
50.8 6 18.7
30.8 6 5.6
31.0 6 5.9
51.4 6 13.4
30.2 6 4.8
31.5 6 51.6
15.5 6 1.9
24.7 6 10.1
22.8 6 17.3
15.1 6 5.7
62.6 6 6.2
47.3 6 27.4
192.1 6 133.4
266.0 6 157.3
375.2 6 188.1
134.6 6 72.8
183.9 6 104.0
139.8 6 84.0
142.0 6 34.5
77.3 6 32.6
110.8 6 48.6
140.4 6 56.3
113.0 6 24.2
81.9 6 34.7
204.7 6 76.6
79.8 6 28.4
82.8 6 39.0
198.5 6 165.2
63.7 6 2.6
53.8 6 6.7
130.2 6 37.9
33.1 6 21.5
51.2 6 9.0
59.0 6 107.5
65.8 6 11.6
42.5 6 23.9
Diet
Week
Diet*week
0.39
0.02*
0.74
0.35
0.001-
0.20
0.47
0.03
0.91
0.96
0.003-
0.98
0.05
0.24
0.49
0.09
0.37
0.34
0.94
0.03*
0.20
0.62
0.01*
0.96
0.52
0.09
0.10
Data reported as means 6 s.d.
*Linear effect of week (P , 0.05).
Curvilinear effect of week (P , 0.05).
was higher (P , 0.05) in MP-fed cats, while leptin mRNA
tended to be greater (P , 0.10) in HP-fed cats. IL-6 or leptin
mRNA were not changed over time. Adipose adiponectin
mRNA expression was negatively correlated (P , 0.05) with
BW (r 5 20.39). Adipose UCP2 mRNA abundance was
positively correlated (P , 0.04) with physical activity during
the dark period (r 5 0.44).
Skeletal muscle mRNA abundance for GLUT1, HSL and
UCP2 decreased (P , 0.05) over time, while IR mRNA
decreased (P , 0.05) curvilinearly over time, regardless of
dietary treatment (Table 6). 18S rRNA, used as an endogenous control, did not differ due to diet (P . 0.05), week
(P . 0.05), or diet*week (P . 0.05).
Discussion
Given the increasing numbers of obese cats, identifying
methods to prevent obesity following ovariohysterectomy
and neutering are of great importance. Understanding the
mechanisms responsible for the increased weight gain may
enable researchers to develop better feeding strategies.
Diets that increase feelings of satiety, thereby decreasing
energy intake, may be developed. Therefore, the objective
of this study was to evaluate metabolic changes in cats fed
a HP or MP diet after ovariohysterectomy and to identify
relationships between metabolic changes and mRNA
abundance in adipose and skeletal muscle tissue.
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Vester, Sutter, Keel, Graves and Swanson
Table 6 Skeletal muscle tissue mRNA abundance (arbitrary units) of cats fed a high-protein (HP) or moderate-protein
(MP) diet prior to and following ovariohysterectomy
P-value
Item
Glucose transporter 1
Week 0
Week 12
Week 24
Hormone sensitive lipase
Week 0
Week 12
Week 24
Insulin receptor
Week 0
Week 12
Week 24
Uncoupling protein 2
Week 0
Week 12
Week 24
HP
MP
112.1 6 56.8
40.9 6 32.5
27.7 6 16.0
91.8 6 45.5
50.4 6 18.0
24.5 6 16.4
22.5 6 4.3
11.4 6 2.8
7.7 6 5.5
22.6 6 6.9
10.8 6 4.3
17.1 6 22.4
155.7 6 32.6
64.9 6 16.3
57.9 6 15.8
190.1 6 76.0
94.9 6 0.0
76.9 6 27.5
107.0 6 70.8
43.6 6 10.9
26.2 6 16.9
76.1 6 48.2
37.2 6 6.7
22.9 6 19.7
Diet
Week
Diet*week
0.74
0.007
0.74
0.57
0.05
0.49
0.46
0.004-
0.87
0.38
0.01*
0.74
Data reported as means 6 s.d.
*Linear effect of week (P , 0.05).
Curvilinear effect of week (P , 0.05).
While protein concentration was not the only difference
between the HP and MP diets, it constituted the largest
difference between the diets. These diets were formulated
to simulate typical commercial-type diets available on the
market today, using common ingredients and levels of
protein, fat and fiber. Despite our primary interest in dietary
protein concentration, we acknowledge slight differences in
other dietary constituents (dietary fat and fiber concentration and/or source) that may have also influenced the
outcome variables measured in this study.
Food intake and BW increased in all cats following ovariohysterectomy, as has been noted in other studies (Fettman
et al., 1997; Harper et al., 2001; Kanchuk et al., 2003; Nguyen
et al., 2004; Backus et al., 2007; Cave et al., 2007; Belsito
et al., 2009). Earlier research has indicated that an estimated
20% to 30% reduction in food intake is needed to maintain
BW of neutered cats (Flynn et al., 1996; Root et al., 1996;
Belsito et al., 2009). Therefore, BW gain following ovariohysterectomy is not only due to increased food intake, but
may also be due to a lower metabolic rate and decreased
physical activity. In the current study, consumption of a HP
diet did not ameliorate increases in food intake or BW gain. In
fact, there was a significant (P 5 0.05) diet by time interaction, as HP-fed cats consumed more food than MP-fed cats
during week 4 through week 9. At week 12, there was a
large decrease in food intake by HP-fed cats. While it was
expected that cats would decrease their intake at 12 weeks
due to surgery, this drastic change following week 12 was
primarily the result of one cat decreasing food intake after this
time point. Because of the small number of animals per
treatment, one cat drastically changing her intake led to a
large shift in average intakes of the HP-fed cats.
Before undergoing ovariohysterectomy (week 23
through 0), cats consumed 100.2 6 34.55% of their ME
requirement and maintained BW. Within 3 weeks (HP-fed
cats) or 8 weeks (MP-fed cats) after ovariohysterectomy,
cats consumed more ME than at baseline and did so for
several weeks. When cats began to consume at or below
their initial ME requirement (at week 13 for MP-fed cats
and at week 20 for HP-fed cats), they continued to maintain
or gain weight. By week 24, lean body mass was increased
by 15% from week 0. Therefore, it would be expected that
ME requirement would increase above the initial calculated
value. By week 13, however, cats were consuming near or
below their initial ME requirement, but continued to maintain
or even gain BW. Interestingly, cats consumed only 89% of
their initial calculated ME requirement (100 3 BW0.67;
National Research Council, 2006) at week 24.
All cats gained weight following ovariohysterectomy,
with the majority comprising adipose tissue rather than lean
body mass or BMC. Similar to BW gain, HP-fed cats gained
body fat mass faster than MP-fed cats and had increased
BMC earlier in the study, which was likely to support
increased weight gain. Changes in fat and lean mass percentages were similar to values reported by Harper et al.
(2001) in cats , 4 years of age (23.6% fat pre-neutering;
34.4% fat 12 months post-neutering; P , 0.002).
Physical activity has an inverse relationship with BW. In a
review of physical activity in animal models, Tou and Wade
(2002) suggested that decreases in physical activity were in
response to, rather than the cause of, increased BW.
Researchers have reported that BW gains following ovariohysterectomy were due to increased food intake and not
decreased energy expenditure (Flynn et al., 1996; Kanchuk
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High-protein diet following ovariohysterectomy
et al., 2003). The methods used in these studies, however,
did not allow for objective measurements of spontaneous
physical activity without human interference. The availability of activity collars now allows researchers to monitor
activity without human interference and provides objective
data that may be quantified and statistically analyzed.
Published literature provides clear evidence that estrogen
influences physical activity (Wade and Gray, 1979; Mystkowski
and Schwartz, 2000; Ainslie et al., 2001; Garey et al., 2001).
Withdrawal of estrogen in rats, through ovariectomy,
decreased spontaneous physical activity within 7 weeks
of surgery compared to sham-operated controls (Ainslie
et al., 2001). Injection of estrogen in ovariectomized mice
(Swiss-Webster mice; 8 to 10 weeks of age at start of study)
led to increased time spent on the running wheel by these
animals (Garey et al., 2001).
Belsito et al. (2009) reported decreased physical activity
(approximately 64%) in ovariohysterectomized cats that were
limit-fed to maintain BW. In that study, however, limit-feeding may have influenced energy expenditure. The largest
portion of decreased energy expenditure occurred during the
dark period (Belsito et al., 2009). Our data confirm these
findings, as week 24 activity during the light period
decreased by approximately 91%, while activity during the
dark period decreased by approximately 500%, as compared to baseline. Due to the design of the current study, it
was impossible to determine a causal relationship between
BW and physical activity.
Other researchers have noted that ovariohysterectomized
or neutered cats have increased insulin and leptin concentrations, with no change in glucose or TG concentrations
(compared to intact cats) (Fettman et al., 1997; Kanchuk
et al., 2003). In the current study, food-restricted glucose
concentrations increased from week 0 to 24, suggesting
decreased insulin sensitivity with weight gain; however,
food-restricted blood insulin concentrations did not change
over time. Szabo et al. (2000) reported increased blood
glucose concentrations in cats following ovariohysterectomy
(and weight gain) with no increases in insulin concentration. Glucose concentrations reported by Szabo et al. (2000)
were similar to those measured in the current study. Kanchuk
et al. (2003) reported increased insulin concentrations of
neutered male v. intact male cats; however, changes
over time within the neutered males were not statistically
analyzed. In the current study, food-restricted TG concentrations increased in HP cats by week 24 following
ovariohysterectomy. TG concentrations at week 0 in the
current study were similar to those reported by Fettman
et al. (1997) after neutering. The increased blood TG concentrations were likely due to an adaptation to the gluconeogenic state of HP-fed cats, which enabled them to
mobilize fat stores more quickly than the MP-fed ones. This
change may have also been in response to the increased
weight gain of the HP-fed cats.
Leptin concentrations are positively correlated with adipose tissue mass (Appleton et al., 2000; Backus et al.,
2000; Belsito et al., 2009). Backus et al. (2000) used linear
regression and estimated that for every 1 g increase in BW, a
1.2 ng/ml increase in leptin occurred. In the current study,
circulating blood leptin concentrations not only increased over
time in cats fed both diets, but were also greater in MP-fed
cats. While MP-fed cats had increased blood leptin concentrations, HP-fed cats tended to have increased adipose
leptin mRNA abundance. A positive correlation (r 5 0.388;
P , 0.0001) between adipose leptin gene expression and
serum concentrations has been noted in humans studies
(Knerr et al., 2006). In the current study, adipose leptin mRNA
concentrations tended to be correlated with circulating leptin
values (r 5 0.39; P 5 0.07). Cats, similar to other species,
have increased circulating leptin concentrations following
weight gain. Increased blood leptin concentrations, without
decreases in food intake suggests decreased leptin sensitivity
(Appleton et al., 2000; Backus et al., 2000; Martin et al.,
2001).
High-protein (low-carbohydrate) diets may improve leptin
sensitivity in humans. Hayes et al. (2007) noted decreased
blood leptin and increased blood ghrelin concentrations in
individuals fed a low-carbohydrate diet, even though
energy intake was consistent among groups. Therefore, the
authors hypothesized that the low-carbohydrate diet led to
improved leptin signaling. In contrast to the current study in
which cats increased food intake and BW, those individuals
reduced energy intake to lose BW (93.5 6 3.6 kg at baseline; 88.3 6 3.4 kg at end of trial).
Measuring mRNA abundance in adipose and skeletal
muscle tissues may identify tissue-specific metabolic changes that are undetectable in blood. Identifying the changes
that occur following the removal of estrogen may determine
the mechanisms by which increased adiposity occurs,
potentially revealing targets for dietary intervention.
Diet had little influence on gene expression in the current
study, which may have been masked by large BW increases.
Changes in mRNA abundance of target genes were similar to
reported human and rodent data. In contrast to leptin, blood
adiponectin concentrations decrease with increasing adiposity
and are thought to contribute to decreased insulin sensitivity.
In cats, adiponectin has been reported to decrease with
obesity and increase upon losing BW (Hoenig et al., 2007).
Adipose adiponectin mRNA abundance decreased over time
in the current study. As BW increased, cats had increased
food-restricted blood glucose concentrations. Although insulin
sensitivity was not measured in the current study, measurement of adiponectin and glucose concentrations and insulin
sensitivity would be useful in future studies.
Other notable changes in gene expression included
increased adipose GLUT4 expression and decreased adipose
and skeletal muscle IR expression. We hypothesized that
GLUT4 mRNA abundance would decrease with obesity, as
was noted by Brennan et al. (2004) in obese v. lean cats. In
that study, female cats (n 5 17) were studied before and
after 6 months of ad libitum feeding that resulted in obesity.
It is possible that increased GLUT4 mRNA abundance noted
in the current study was a compensatory response to
decreased translation of GLUT4 protein or translocation of
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Vester, Sutter, Keel, Graves and Swanson
GLUT4 protein to the cell membrane. Because GLUT4 protein expression or translocation were not measured in the
current study, this is only speculation. Brennan et al. (2004)
also reported decreased GLUT4 protein expression in
skeletal muscle of obese v. lean cats but no changes in
GLUT1. In the current study, GLUT4 mRNA expression did
not change in skeletal muscle of cats fed either diet.
In the current study, GLUT1 gene expression decreased
over time in skeletal muscle, regardless of dietary treatment. GLUT1 is located in many tissues throughout the
body. It is insulin insensitive and plays a role in basal glucose uptake. Obese and obese diabetic rats had decreased
skeletal muscle GLUT1 mRNA abundance compared to lean
rats of the same genetic line (Kahn and Pedersen, 1993).
Those authors stated, however, that this transporter plays a
small role in overall insulin-resistant glucose uptake, and
therefore may not play a large role in the development of
insulin resistance. The usefulness of GLUT1 expression as
an early indicator of insulin insensitivity, however, is
unknown in cats.
Hormone sensitive lipase and UCP2 both play a role in
fatty acid catabolism. Hormone sensitive lipase releases
fatty acids from the cell while UCP2 plays a role in energy
expenditure. Hormone sensitive lipase enzyme activity,
protein expression and mRNA abundance were lower in
subcutaneous adipose tissue of obese humans as compared
to non-obese controls (Large et al., 1999). Decreased adipose and skeletal muscle HSL expression observed in the
current study may have been due to the removal of estrogen or increased weight gain, which is consistent with
rodent data. Decreased HSL response was consistent with
decreased blood NEFA concentrations. Obese humans also
have impaired fatty acid release from skeletal muscle
compared to lean individuals (Blaak et al., 1994a).
Contrary to our data, Hoenig et al. (2006) reported
increased HSL gene expression in adipose and skeletal
muscle in obese v. lean cats. Those researchers also
reported decreased LPL mRNA abundance in adipose tissue,
but greater mRNA abundance in skeletal muscle of obese v.
lean cats (Hoenig et al., 2006). Given the change in LPL :
HSL, the authors hypothesized that HSL in adipose tissue
was shuttling fat to skeletal muscle. Increased lipid
deposition in skeletal muscle tissue leads to decreased
insulin sensitivity. In the current study, adipose and skeletal
muscle tissue LPL expression levels were unchanged.
Decreases noted in adipose UCP2 expression were consistent with decreased HSL expression. A blunted fat
oxidation response to b-androgenic stimulation may be a
primary factor for increased fat stores and maintaining
adipose tissue deposits following weight loss (Blaak et al.,
1994b). UCP2 activity increases with increased blood lipid
circulation. Therefore, with less fat released from adipose
tissue, UCP2 may also be expected to decrease. Furthermore, decreased UCP2 expression may indicate a decreased
metabolic rate of ovariohysterectomized cats.
Inflammation occurs in adipose tissue with prolonged
obesity. Obese cats have been noted to have increased
inflammatory cytokine mRNA and protein concentrations
(Hoenig et al., 2006; Tanner et al., 2007). Gene expression
of inflammatory markers (TNF-a, IL-1, IL-6, MCP-1 and
PAI1) did not change over time in the current study.
Expression of TLR4 decreased over time in adipose tissue.
Although all cats gained weight, the lack of change in this
regard (BCS only 7/9 at week 24) may be an indication that
extreme obesity (BCS 5 8 or 9/9) is required to elicit an
inflammatory response.
Adipose tissue IL-6 mRNA expression increased in MP-fed
cats, which may indicate increased inflammation. Bastard
et al. (2002) reported that IL-6 might impair insulin action
locally in adipose tissue. The authors noted a strong inverse
correlation between adipose tissue IL-6 concentrations and
insulin-stimulated glucose disposal, and a positive correlation between plasma glucose and obese individuals with
varying levels of insulin resistance. The effect of IL-6 on
insulin resistance may be due to its effect on gene transcription. Chronic exposure of IL-6 to 3T3-L1 adipocytes
inhibited gene transcription of IR substrate-1, GLUT4, and
PPAR-g (Rotter et al., 2003). Given that increases in IL-6 did
not occur in MP-fed cats until after weight gain, it may be
of interest in future studies testing the effect of diet on
insulin resistance and diabetes in cats.
Evaluation of cats fed a HP diet did not appear to
ameliorate any metabolic or gene expression changes
associated with increased weight gain. Feeding a HP diet to
maintain BW following ovariohysterectomy may lead to
different changes that have yet to be evaluated at present.
Further evaluation of HP diets fed to cats with known
insulin resistance may illicit changes in gene expression that
aid in our understanding of feline obesity and comorbidities.
In summary, we report several metabolic and gene
expression changes following ovariohysterectomy in cats.
Notable increases in food intake, BW and body fat were
expected and have been well reviewed in the literature.
Decreased physical activity, especially during the dark period,
indicated that physical activity greatly decreased in ovariohysterectomized cats, contributing to further weight gain.
Our experimental design did not allow for the testing of a
causal relationship between physical activity and weight gain.
We hypothesize, however, that increased food intake leads to
weight gain and consequent decreased activity. The novel
approach of measuring physical activity without human
interference may aid in testing this hypothesis in the future.
Gene expression changes may be a good indicator of
metabolic changes in the body. It was clear that fatty acid
metabolism was altered after ovariohysterectomy and
weight gain, leading to increased storage of fat, coupled
with decreased metabolic rate (i.e. decreased UCP2). These
alterations in fatty acid metabolism may be due to obesity
itself, or as a primary factor contributing to the onset of
obesity. Further research testing the role of estrogens on
adipose gene expression in cats is warranted. Not all blood
metabolite concentrations aligned with changes in mRNA
abundance. The relationship of leptin mRNA expression and
circulating blood concentrations require more study.
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High-protein diet following ovariohysterectomy
Due to metabolic changes that occur in adipose and
skeletal muscle tissue, it is inadvisable to feed cats ad libitum
following ovariohysterectomy, regardless of diet fed. Further
feline research focused on controlling metabolic changes or
influencing satiety factors is needed, as it is possible that
limit-feeding a HP diet to maintain BW will yield different
results. While feeding a HP diet did not control food intake or
ameliorate increasing BW herein, more research is needed to
fully elucidate its role in managing feline obesity.
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