OMEGA-3 FATTY ACIDS, OBESITY-RELATED DISEASES, AND ADIPOKINES IN DOGS AND ...

341 Pages·2011·3.36 MB·English

OMEGA-3 FATTY ACIDS, OBESITY-RELATED DISEASES, AND ADIPOKINES IN DOGS
AND CATS

By

Michal Mazaki-Tovi

A DISSERTATION

Submitted to
Michigan State University
in partial fulfillment of the requirements
for the degree of

DOCTOR OF PHILOSOPHY

Pathobiology

2011
ABSTRACT

OMEGA-3 FATTY ACIDS, OBESITY-RELATED DISEASES, AND ADIPOKINES IN DOGS
AND CATS

By
Michal Mazaki-Tovi

The incidence of obesity in dogs and cats is increasing in recent years. In humans, obesity
has been associated with the metabolic syndrome, which consists of a cluster of disorders
including, insulin resistance, type II diabetes mellitus, hyperlipidemia, hepatic steatosis,
hypertension, and atherosclerosis. Although naturally-occurring metabolic syndrome has not
been described in dogs or cats, some components of this syndrome occur in diseases related to
obesity, either as a predisposing factor (i.e. feline hepatic lipidosis and feline diabetes mellitus), a
complicating factor (i.e. canine diabetes mellitus), or a consequence (i.e. canine
hypothyroidism). Decreased adiponectin concentration has been implicated in the pathogenesis
of the metabolic syndrome in humans. Findings regarding leptin are less consistent and either
leptin deficiency or resistance was suggested. Consumption of n3-polyunsaturated fatty acids
(PUFAs) has beneficial effects on various metabolic alterations of the syndrome. The present
studies aimed to determine alterations in adipokines concentrations in obesity-related diseases in
dogs and cats and to determine the effect of n3PUFAs on adipokines in health and disease.
In healthy dogs, circulating concentrations of docosapentaenoic acid were positively
associated with concentrations of adiponectin and leptin; dietary fish oil supplementation for 30
days yielded an increase in adiponectin. In healthy cats, associations of n3PUFAs with
adipokines were differential with body condition. Concentrations of eicosapentaenoic acid and
docosahexaenoic acid were positively associated with adiponectin concentrations in obese cats;
while in non-obese cats, concentrations of eicosapentaenoic acid were negatively associated with
concentrations of adiponectin and positively associated with concentrations of leptin.
In canine adipose tissue culture, eicosapentaenoic acid stimulated adiponectin secretion
by mature adipocytes and inhibited inteleukin-6 secretion by stromovascular cells. In contrast,
palmitic acid inhibited adiponectin secretion by mature adipocytes and stimulated inteleukin-6
secretion by stromovascular cells. In feline adipose tissue culture, arachidonic acid stimulated
inteleukin-6 secretion by stromovascular cells. Therefore, the stimulatory effect of n3PUFAs on
adiponectin may be direct or mediated through inhibition of IL6. Moreover, these effects may be
conveyed by n3PUFAs directly or by substitution of n6PUFAs or a saturated fatty acid.
Feline hepatic lipidosis was associated with hyperadiponectinemia, which is likely related
to liver injury, and with hyperleptinemia, which is suggested to be specific to lipidosis. Canine
hypothyroidism was associated with hyperleptinemia, which was associated thyroid hormone
deficiency, and with hyperadiponectinemia, suggestive of adiponectin resistance. Diabetes
mellitus was associated with hyperleptinemia in cats and hypoleptinemia in dogs, consistent with
the opposite alterations in insulin secretion in these diseases.
In healthy animals, docosapentaenoic acid (dogs) or eicosapentaenoic acid (obese cats)
was negatively associated with concentrations of triglyceride, supporting a hypolipidemic effect
n3PUFAs. A negative association between n3PUFA and triglyceride was present in cats with
hepatic lipidosis, but not the other diseases studied, suggesting preservation of a hypolipidemic
effect of n3PUFA in overt feline hepatic lipidosis. Eicosapentaenoic was negatively associated
with insulin concentrations in obese cats, supporting an insulin-sensitizing effect.
Copyright by
MICHAL MAZAKI-TOVI
2011
ACKNOWLEDGMENTS

This is a great opportunity to express my respect to my guidance committee members,
Dr. Patricia Schenck (Head of committee), Dr. Sarah Abood, Dr. James Wagner, and Dr. P.S.
Mohankumar, for their valued and constructive direction of the work presented here. I would
like to thank Dr. Steve Bolin, who has provided me with indispensable assistance in
developing the methods for primary adipose tissue culture in dogs and cats. I would also like to
thank all who invested time and effort in my education and instruction as well as in helping
with the enrollment of dogs and cats into the different studies. These persons include Mr. Justin
Zyskowsky for instruction of methods of lipid extraction and fatty acids analysis using gas
chromatography, Ms. Susan Beyerlein for instruction of performing radioimmunoassays, Ms.
Enass Bassiouny for instruction of performing ELISAs, Dr. Mary Dee Sist and Mr. Alex
Schram for enrolling dogs into the fish oil supplementation study, and residents in the
Departments of Internal Medicine at Michigan State University and Hebrew University of
Jerusalem for enrolling cases of feline hepatic lipidosis, canine hypothyroidism, and feline and
canine diabetes mellitus.
The studies were supported by a grant from the American Academy of Veterinary
Nutrition / Waltham and a grant from the Margery Seeger Companion Animal Fund, College
of Veterinary medicine, Michigan State University.

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TABLE OF CONTENTS

LIST OF TABLES……………………………………………………………………………….. viii

LIST OF FIGURES……………………………………………………………………………… xi

LIST OF ABBREVIATIONS……………………………………………………………………. xiv

CHAPTER 1 – ADIPOKINES AND N3PUFAS IN HEALTHY DOGS AND CATS..………... 1
1.1. Introduction…………………………………………………………………………….. 2
1.1.1. Obesity, adipose tissue, and adipokines……………………………….…….. 3
1.1.2. Dietary FAs and beneficial effects of n3PUFAs………………………......... 20
1.1.3. Hypotheses………………………….……………………………………….. 29
1.2. Studies: study aims, methods, and results…...…………………………………………. 33
1.2.1. Associations among circulating concentrations of adipokines and n3PUFAs,
body condition, age, and gender in healthy dogs...…………………………. 34
1.2.2. Effect of fish oil supplementation on circulating concentration of
adipokines in healthy dogs………………………………………………….. 46
1.2.3. Associations among circulating concentrations of adipokines and n3PUFAs,
body condition, age, and gender in healthy cats..…………………………... 54
1.2.4. Associations between FAs or troglitazone treatment, body condition, or
anatomic location and adipokines secretion in primary canine adipose
tissue culture……………………................................................................... 65
1.2.5. Associations between FAs or troglitazone treatment, body condition, or
anatomic location and adipokines secretion in primary feline adipose tissue
culture.………………………………………………………………………. 79
1.3. Discussion………...……………………………………………………………………. 88
1.3.1. Introductory comments……………………………………………….……... 89
1.3.2. Effect of body condition on adipokines........................................................... 92
1.3.3. Effect of gender on adipokines...……………................................................. 98
1.3.4. Effect of age on adipokines………………………………………………......100
1.3.5. Effect of adipose tissue anatomic location on adipokines…………………... 102
1.3.6. Effect of troglitazone on adipokines………………………………………… 105
1.3.7. Effect of n3PUFAs on adipokines…............................................................... 108
1.3.8. Effect of n3PUFAs on insulin and triglyceride……………………………… 124
1.3.9. Summary ..................................................................................................…... 128

CHAPTER 2 - ADIPOKINES AND N3PUFAS IN OBESITY-RELATED DISEASES IN
DOGS AND CATS………….……………………………………….…………. 131
2.1. Introduction……….………………………………………………………………......... 132
2.1.1. Obesity-related diseases, adipokines, and n3PUFAs...……………………… 133
2.1.2. Feline hepatic lipidosis……………………………………………………… 135
2.1.3. Canine hypothyroidism……………………………………………………… 137
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2.1.4. Canine and feline diabetes mellitus…………………………………………. 139
2.1.5. Hypotheses………………………………………………………………….. 141
2.2. Studies: study aims, methods, and results……………………………………………… 142
2.2.1. Circulating concentrations of adipokines and n3PUFAs in feline hepatic
lipidosis……………………………………………………………….…...... 143
2.2.2. Circulating concentrations of adipokines and n3PUFAs in canine
hypothyroidism……………………………………………………………… 162
2.2.3. Circulating concentrations of adipokines and n3PUFAs in feline diabetes
mellitus ……………………………………………………………………... 178
2.2.4. Circulating concentrations of adipokines and n3PUFAs in canine diabetes
mellitus……………………………………………………………………… 194
2.3. Discussion……………………………………………………………………………… 210
2.3.1. Feline Hepatic lipidosis………………………………………………………211
2.3.2. Canine hypothyroidism……………………………………………………… 219
2.3.4. Canine and feline diabetes mellitus…………………………………………. 228
2.3.5. Summary …………………………..………………………………………... 236

CHAPTER 3 - PRESENT LIMITATIONS AND FUTURE DIRECTIONS……………………. 238

APPENDICES…………………………………………………………………………………… 245
A. Assay interference testing……………………………………………………………….. 246
B. Stability of long-chain FAs in fish oil gel capsules…………………………..………….. 251
C. Procedures protocols…………………………………………………………………….. 253
1. ELISAs………………………………………………………………………….. 253
2. RIAs……………………………………………………………………………... 272
3. Total lipid determination and FA analysis…………………………...………….. 278
4. Primary adipose tissue culture…………………………………………………... 281
5. Preparation of serum samples for interference testing study……………………. 291

REFERENCES…………………………………………………………………...……………… 292

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LIST OF TABLES

Table 1. Correlation coefficients among measures of body condition, age, and circulating
concentrations of adipokines, insulin, glucose, and triglyceride in 62 healthy dogs……. 38

Table 2. Correlation coefficients among measures of body condition, age, and circulating
concentrations of adipokines, insulin, glucose, and triglyceride in 24 obese and 38 non-
obese healthy dogs……………………………………………………………………….. 39

Table 3. Associations between DPA and adiponectin, leptin, insulin, glucose, and triglyceride
in 62 healthy dogs…………………….………………………………………………….. 41

Table 4. Associations between total lipid and adiponectin, leptin, insulin, glucose, and
triglyceride in 62 healthy dogs…………………………………………………………… 42

Table 5. Associations between %BF and adiponectin, leptin, insulin, glucose, and triglyceride
in 62 healthy dogs……………………………………………..…………...……………. 43

Table 6. Associations between age and adiponectin, leptin, insulin, glucose, and triglyceride in
62 healthy dogs………………………...…………...……………………………………. 44

Table 7. Correlation coefficients among measures of body condition, age, and circulating
concentrations of adipokines, insulin, glucose, and triglyceride in 55 healthy cats…....... 59

Table 8. Correlation coefficients among measures of body condition, age, and circulating
concentrations of adipokines, insulin, glucose, and triglyceride in 21 obese and 34 non-
obese healthy cats ….……………………………………………………………...….…. 60

Table 9. Associations between gender and adiponectin, leptin, insulin, glucose, triglyceride,
and cholesterol in 55 healthy neutered cats .……………………………………….…… 61

Table 10. Associations between age and adiponectin, leptin, insulin, glucose, triglyceride, and
cholesterol in 55 healthy cats…………………………………………………………….. 61

Table 11. Associations between EPA and adiponectin, leptin, insulin, glucose, triglyceride, and
cholesterol in 34 non-obese cats…………………………………………………………. 63

Table 12. Associations between EPA and adiponectin, leptin, insulin, glucose, triglyceride, and
cholesterol in 21 obese cats……………………………………………………………... 64

Table 13. A summary of associations between body condition and adipokines concentrations in
serum and medium……………………………………………………………………... 97
viii
Table 14. A summary of associations between gender and adipokines concentrations in
serum…………………………………………………………………………………….. 99

Table 15. A summary of associations between age and adipokines concentrations in
serum…………………………………………………………………………………….. 101

Table 16. A summary of associations between adipose tissue anatomic location and adipokines
concentrations in medium………………………………………………………………... 104

Table 17. A summary of associations between troglitazone treatment and adipokines
concentrations in medium………………………………………………………………... 107

Table 18. A summary of associations between n3PUFAs and adipokines concentrations in
serum, effect of dietary fish oil supplementation on serum concentrations, and effect of FAs
treatment on medium concentration ……………………………………………………...122

Table 19. A summary of associations between n3PUFAs and concentrations of insulin and
triglyceride and effect of dietary fish oil supplementation………………………………. 127

Table 20. Biochemical changes in cats with liver diseases……………………….…….…… 149

Table 21. Serum concentrations of hormones, glucose, and lipids in cats with liver diseases and
healthy cats………………………………………………………………………………..151

Table 22. Associations between total n3PUFAs and adiponectin, leptin, insulin, glucose,
triglyceride, and cholesterol in 10 obese cats with HL…………………………...……… 159

Table 23. Biochemical changes in dogs with hypothyroidism………………………………. 167

Table 24. Serum concentrations of hormones, glucose, and lipids in dogs with hypothyroidism
and healthy dogs……………………………………………………………………….… 169

Table 25. Biochemical changes in cats with DM……………………………….……...……. 183

Table 26. Serum concentrations of hormones, glucose, and lipids in cats with DM and healthy
cats……………………………………………………………………….………………. 185

Table 27. Biochemical changes in dogs with DM……………..…………….………………. 199

Table 28. Serum concentrations of hormones, glucose, and lipids in dogs with DM and healthy
dogs……………………………………………………………………….…………........ 201

Table 29. Associations between total n3PUFAs and adiponectin, leptin, insulin, glucose,
triglyceride, and cholesterol in dogs with DM…………………………….…………….. 209

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Table 30. A summary of associations between serum concentrations of adiponectin, leptin,
insulin, glucose, triglyceride, and cholesterol in cats with liver disease……...…………. 218

Table 31. A summary of associations between serum concentrations of adiponectin, leptin,
insulin, glucose, triglyceride, and cholesterol in dogs with hypothyroidism……………. 227

Table 32. A summary of associations between serum concentrations of adiponectin, leptin,
insulin, glucose, triglyceride, and cholesterol in cats and dogs with DM……………….. 235

Table 33. Effect of hemoglobin, lipid, and bilirubin on measured concentrations of leptin,
adiponectin, insulin, IL6 and TNFα in serum pools from dogs and cats………………... 248

Table 34. Hanks’ Balanced Salt Solution………………………………….………………... 285

Table 35. Dulbecco’s Modified Eagle’s Medium /Ham’s Nutrient Mixture F-12..…..……... 285

Table 36. Supplements added to medium……………………………………....…………… 287

Table 37. Preparation of growth factors stock solutions…………………………………….. 287

Table 38. Preparation of medium with growth factors…………………….………………... 288

Table 39. Preparation of FAs stock solution………………………….……………………... 288

x