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Ablation of eNOS Does Not Promote Adipose Tissue Inflammation 3 4 5 6 Thomas J. Jurrissen1, Ryan D. Sheldon1,2, Michelle L. Gastecki1, Makenzie L. 7 Woodford1, Terese M. Zidon1, R. Scott Rector1,2,3, Victoria J. Vieira-Potter1, Jaume 8 Padilla1,4,5 9 10 1Nutrition and Exercise Physiology, University of Missouri, Columbia, MO 11 2Research Service-Harry S Truman Memorial Veterans Affairs Medical Center, 12 Columbia, MO 13 3Medicine-Division of Gastroenterology and Hepatology, University of Missouri, 14 Columbia, MO 15 4Dalton Cardiovascular Research Center, University of Missouri, Columbia, MO 16 5Child Health, University of Missouri, Columbia, MO 17 18 19 20 21 22 Running Title: eNOS deficiency and adipose tissue inflammation 23 24 Key Words: Obesity, western diet, adipose tissue, endothelial nitric oxide synthase 25 26 27 28 29 30 31 32 33 Correspondence: 34 Jaume Padilla, Ph.D. 35 Department of Nutrition & Exercise Physiology 36 204 Gwynn Hall 37 University of Missouri 38 Columbia, MO 65211 39 padillaja@missouri.edu 40
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ABSTRACT 42
Adipose tissue (AT) inflammation is a hallmark characteristic of obesity and an 43
important determinant of insulin resistance and cardiovascular disease; therefore, a 44
better understanding of factors regulating AT inflammation is critical. It is well 45
established that reduced vascular endothelial nitric oxide (NO) bioavailability promotes 46
arterial inflammation; however, the role of NO in modulating inflammation in AT remains 47
disputed. In the present study, ten-week old C57BL6 wild-type and eNOS knockout 48
male mice were randomized to either a control diet (10% kcal from fat) or Western diet 49
(44.9% kcal from fat, 17% sucrose, and 1% cholesterol) for 18 weeks (n=7-8/group). In 50
wild-type mice, Western diet-induced obesity led to increased visceral white AT 51
expression of inflammatory genes (e.g., MCP1, TNFα, CCL5 mRNAs) and markers of 52
macrophage infiltration (e.g., CD68, ITGAM, EMR1, CD11C mRNAs and Mac-2 53
protein), as well as reduced markers of mitochondrial content (e.g., OXPHOS complex I 54
and IV protein). Unexpectedly, these effects of Western diet on visceral white AT were 55
not accompanied by decreases in eNOS phosphorylation at Ser1177 or increases in 56
eNOS phosphorylation at Thr495. Also counter to expectations, eNOS knockout mice, 57
independent of the diet, were leaner and did not exhibit greater white or brown AT 58
inflammation compared to wild-type mice. Collectively, these findings do not support the 59
hypothesis that reduced NO production from eNOS contributes to obesity-related AT 60
inflammation. 61
62
63
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INTRODUCTION 64
With obesity, expansion of visceral white adipose tissue (AT) is accompanied by 65
infiltration of immune cells and increased expression of inflammatory cytokines (7). A 66
better understanding of the factors regulating AT inflammation is important given the 67
cumulative evidence implicating AT inflammation as a determinant of obesity-related 68
insulin resistance and cardiovascular disease (3, 14). While it is well-known that 69
reduced vascular endothelial nitric oxide (NO) bioavailability, a hallmark characteristic of 70
obesity and type 2 diabetes, contributes to arterial inflammation (4, 6, 9, 10, 15), 71
conflicting evidence exists regarding the role of NO in modulating inflammation in AT. 72
One study revealed that male eNOS knockout mice exhibit increased expression of 73
inflammatory genes and markers of immune cell infiltration in visceral white (epididymal) 74
AT (5); however, that finding does not appear to be reinforced by data from others (12, 75
13). In a microarray analysis of visceral white AT from female eNOS knockout versus 76
wild-type mice, inflammatory genes were not reported amongst the genes with 77
significantly altered expression (13); however, since data on inflammatory genes were 78
not reported, conclusions cannot be drawn regarding whether or not those eNOS 79
knockout mice had greater or equivalent AT inflammation compared to controls. We 80
recently showed in lean and obese male rats that chronic administration of L-NAME, a 81
NOS inhibitor, did not increase markers of inflammation in AT (12) but did in the liver 82
(17); nevertheless, these data should be interpreted with the understanding that L-83
NAME inhibits production of NO from all three NOS enzymes (eNOS, iNOS, nNOS). 84
Based on these contrasting findings, we set out the present study to more 85
comprehensively interrogate the role of NO in modulating AT inflammation. We 86
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assessed several markers of inflammation in visceral white AT of eNOS knockout and 87
wild-type male mice fed a control versus Western diet. Given that brown AT is more 88
vascularized than white AT and that eNOS is predominantly expressed in endothelial 89
cells, the impact of eNOS deficiency on inflammatory markers was also examined in the 90
interscapular brown AT depot. If, indeed, obesity-induced AT inflammation is partially 91
mediated by reduced NO signaling, then we would expect the AT of lean eNOS 92
deficient mice to phenotypically resemble that of obese mice. That is, we would 93
hypothesize that the pro-inflammatory effect of eNOS deletion would normalize 94
differences in AT inflammation between lean and obese mice. Further, we hypothesized 95
that eNOS ablation would have greater inflammatory effects in brown AT compared to 96
white AT. 97
METHODS 98
Experimental Design 99
Male C57BL6 wild-type and eNOS knockout mice (B6.129P2-Nos3tm1Unc/J) on 100
a C57BL6 background were purchased from Jackson Laboratory (Bar Harbor, ME) at 101
10 weeks of age. Mice were randomized to either normal chow (i.e., control diet) or 102
Western diet (n=7-8/group) ad libitum for 18 weeks. Control diet (3.85 kcal/g of food) 103
contained 10% kcal fat, 70% kcal carbohydrate, and 20% kcal protein, with 3.5% kcal 104
sucrose (D12110704, custom formulated; Research Diets Inc., New Brunswick, NJ). 105
Western diet (4.68 kcal/g of food) contained 44.9% kcal fat, 35.1% kcal carbohydrate, 106
and 20% kcal protein, with 1% cholesterol and 17% kcal sucrose (D09071604, custom 107
formulated; Research Diets Inc.). All mice were pair-housed under standard 108
temperature conditions (~22°C) and humidity with a light cycle from 0700 to 1900 and a 109
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dark cycle from 1900 to 0700. At 28 weeks of age, mice were euthanized following a 5-110
hour fast and tissues were harvested for analysis. All procedures were approved in 111
advance by the University of Missouri Institutional Animal Care and Use Committee. 112
Body composition 113
The percent body fat was measured by a nuclear magnetic resonance imaging 114
whole-body composition analyzer (EchoMRI 4in1/1100; Echo Medical Systems, 115
Houston, TX). This noninvasive measure was performed on conscious mice. 116
Fasting Blood Parameters 117
Plasma glucose, cholesterol, triglycerides, alanine aminotransferase, aspartate 118
aminotransferase, and non-esterified fatty acids (NEFA) assays were performed by a 119
commercial laboratory (Comparative Clinical Pathology Services, Columbia, MO) on an 120
Olympus AU680 automated chemistry analyzer (Beckman-Coulter, Brea, CA) using 121
assays according to manufacturer’s guidelines. Plasma insulin concentrations were 122
determined using a commercially available, mouse-specific ELISA (Alpco Diagnostics, 123
Salem, NH). Plasma nitrate + nitrite (NOx) levels were measured using the 124
Nitrate/Nitrite Fluorometric Assay Kit (Cayman Chemical, #780051, Ann Arbor, MI) 125
according to manufacturer’s instructions. 126
Histological Assessments 127
Formalin-fixed epididymal white AT samples were processed through paraffin 128
embedment, sectioned at 5 µm, stained with Mac-2 antibody (CL8942AP, Cedarlane), a 129
macrophage marker. Sections were evaluated via an Olympus BX34 photomicroscope 130
(Olympus, Melville, NY) and images were taken at 10x magnification via an Olympus 131
SC30 Optical Microscope Accessory CMOS color camera. Objective quantification of 132
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macrophage infiltration was done by determining the positive Mac-2 stained area per 133
10x field of view using Image J software (NIH public domain; National Institutes of 134
Health, Bethesda, MD). The average of six 10x fields of view was used per animal. 135
Adipocyte size was calculated based on 100 adipocytes/animal obtained from the same 136
six 10x fields as performed previously (19). Briefly, cross-sectional areas of the 137
adipocytes were obtained from perimeter tracings using Image J software. All 138
procedures were performed by an investigator who was blinded to the groups. 139
RNA Extraction and Quantitative Real-time RT-PCR 140
Epididymal white and interscapular brown AT as well as aorta samples were 141
homogenized in TRIzol solution using a tissue homogenizer (TissueLyser LT, Qiagen, 142
Valencia, CA). Total RNA was isolated according to the Qiagen’s RNeasy lipid tissue 143
protocol and assayed using a Nanodrop spectrophotometer (Thermo Scientific, 144
Wilmington, DE) to assess purity and concentration. The MU DNA Core confirmed our 145
RNA extraction produced optimal RNA integrity (all RQN’s>8.0). First-strand cDNA was 146
synthesized from total RNA using the High Capacity cDNA Reverse Transcription kit 147
(Applied Biosystems, Carlsbad, CA). Quantitative real-time PCR was performed as 148
previously described using the ABI StepOne Plus sequence detection system (Applied 149
Biosystems). Primer sequences were designed using the NCBI Primer Design tool and 150
have been previously published (19). All primers were purchased from IDT (Coralville, 151
IA). A 20 μL reaction mixture containing 10 μL iTaq UniverSYBR Green SMX (BioRad, 152
Hercules, CA) and the appropriate concentrations of gene-specific primers plus 4 μL of 153
cDNA template were loaded in a single well of a 96-well plate. All PCR reactions were 154
performed in duplicate under thermal conditions as follows: 95ºC for 10 min, followed by 155
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40 cycles of 95ºC for 15 s and 60ºC for 45 s. A dissociation melt curve analysis was 156
performed to verify the specificity of the PCR products. GAPDH was used as house-157
keeping control gene. GAPDH cycle threshold (CT) was not different among the groups 158
of animals. mRNA expression values are presented as 2ΔCT whereby ΔCT = GAPDH CT 159
- gene of interest CT. mRNA levels were normalized to the wild-type control diet group. 160
Western blotting 161
Triton X-100 tissue lysates were used to produce Western blot-ready Laemmli 162
samples. Protein samples (10 µg/lane) were separated by SDS-PAGE, transferred to 163
polyvinylidene difluoride membranes, and probed with primary antibodies. eNOS 164
(#610296, 1:500) and phospho-specific eNOS at Ser1177 (#61239, 1:250) and Thr495 165
(#612706, 1:500) antibodies were purchased from BD Biosciences. Akt (#4691, 1:500), 166
phospho-specific Akt at Ser473 (#4060, 1:250), Mac-2 (#127335, 1:1000), iNOS 167
(#13120, 1:500) and GAPDH (#5174, 1:1000) antibodies were purchased from Cell 168
Signaling. OXPHOS (#ab110413, 1:2000) cocktail antibody was purchased from 169
Abcam. Nitrotyrosine (#5411; 1:500) antibody was purchased from Millipore. Intensity of 170
individual protein bands were quantified using FluoroChem HD2 (AlphaView, version 171
3.4.0.0), and expressed as ratio to control band GAPDH. 172
Statistical Analysis 173
A 2x2 (genotype x diet) analysis of variance (ANOVA) was used to evaluate the 174
effects of eNOS deficiency and Western diet for all dependent variables. When a 175
significant genotype by diet interaction existed, LSD post-hoc test was used for pair-176
wise comparisons. All data are presented as mean ± standard error (SE). For all 177
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statistical tests, the alpha level was set at 0.05. All statistical analyses were performed 178
with SPSS V23.0. 179
RESULTS 180
As shown in Fig 1, independent of genotype, mice fed a Western diet had larger 181
body weights, greater adiposity and consumed more kilocalories per day compared to 182
control diet-fed mice (p<0.05). eNOS knockout mice weighted less, had reduced 183
adiposity, and consumed fewer kilocalories per week compared to wild-type mice 184
(p<0.05). Table 1 summarizes the fasting blood characteristics. Independent of 185
genotype, compared to control-fed mice, Western diet-fed mice had greater plasma total 186
cholesterol, low density lipoprotein, high density lipoprotein, alanine aminotransferase, 187
insulin, and HOMA-IR, and reduced plasma nitrite + nitrate (p<0.05). Despite being 188
leaner, eNOS knockout mice had greater plasma NEFAs compared to wild-type mice 189
(p<0.05). 190
Figure 2A illustrates representative histological images of epididymal white AT 191
stained with Mac-2, a macrophage marker, and quantification is presented in panel B. 192
Western diet-fed mice had greater Mac-2 staining in white AT than mice fed a control 193
diet (p<0.05), independent of genotype. Similarly, adipocyte size was greater in Western 194
diet-fed mice compared to control diet-fed mice (p<0.05) and smaller in eNOS knockout 195
mice compared to wild-type mice (Fig 2C, p<0.05). 196
Gene expression data in epididymal white AT and interscapular brown AT are 197
summarized in Figure 3A and B, respectively. In white AT, mRNA levels related to 198
inflammation [e.g., MCP-1, TNF-α, CCL5], immune cell infiltration [e.g., CD68, ITGAM, 199
EMR1 and CD11c], oxidative stress [e.g., p22phox and p47phox] were all elevated with 200
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Western diet (p<0.05) and not affected by eNOS ablation (p>0.05). Adiopokine leptin 201
mRNA was increased and adiponectin mRNA was decreased with Western diet 202
(p<0.05) but not affected by loss of eNOS (p>0.05). In brown AT, mRNA levels related 203
to inflammation [MCP1, TNF-α, CCR2, and IL-6], immune cell infiltration [e.g., CD68, 204
ITGAM, and CD11c], oxidative stress [e.g., p22phox and p47phox], endoplasmic 205
reticulum stress [e.g., DDIT3], anti-clotting protein PAI-1, and the adipokine leptin were 206
all elevated with Western diet (p<0.05) and, again, not affected by eNOS abrogation 207
(p>0.05). 208
eNOS protein content in epididymal white AT was undetectable in eNOS 209
knockout mice (Fig 4, p<0.05). No differences in total eNOS, p-eNOS at Ser1177, or p-210
eNOS at Thr495 were observed with Western diet in wild-type mice (p>0.05). iNOS 211
protein content was also unaltered with Western diet or eNOS ablation (p>0.05). In 212
addition, there were no effects of diet or genotype on Akt and p-Akt at Ser473 (p>0.05). 213
Similar to findings obtained by immunohistochemistry (Fig 2) and gene expression (Fig 214
3), Mac-2 protein content was greater in Western diet-fed mice (Fig 4, p<0.05), 215
independent of genotype. Furthermore, consistent with findings in gene expression (Fig 216
3), we found a Western diet-induced increase in nitrotyrosine, a marker of oxidative 217
stress (p<0.05). Western diet reduced mitochondrial oxidative phosphorylation complex 218
I and IV protein content in both genotypes (p<0.05) and complex II protein content in 219
eNOS knockout (p<0.05). No effects of diet or genotype were observed in complexes III 220
or V (p>0.05). 221
In stark contrast with AT data (Fig 3), aortic gene expression markers of immune 222
cell infiltration [CD68, ITGAM, EMR1, and CD11c], oxidative stress [p22phox and 223
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p47phox], endoplasmic reticulum stress [DDIT3, HSPA1A, HSP5A (p=0.072)], and anti-224
clotting protein PAI-1 were significantly upregulated in eNOS knockout compared to 225
wild-type mice (Fig 5, p<0.05, unless stated otherwise). Also, in contrast to the results 226
observed in AT, no diet effects were found in the aorta (p>0.05). 227
DISCUSSION 228
We found that increased white AT inflammation with Western diet-induced 229
obesity was not accompanied by changes in eNOS phosphorylation at Ser1177 and 230
Thr495, the two phosphorylation sites that mainly regulate eNOS activity. Furthermore, 231
ablation of eNOS did not increase AT inflammation in either white or brown AT. 232
Collectively, these findings do not support the hypothesis that reduced NO production 233
from eNOS contributes to obesity-related AT inflammation. 234
Our findings are in discordance with results from Handa et al. (5), a study also 235
using the eNOS knockout mouse model. In that previous study, the authors reported 236
that eNOS deficiency, but surprisingly not 4 weeks of high-fat diet feeding, promoted 237
inflammation in epididymal AT (5). Our results indicate that markers of inflammation in 238
white AT were upregulated by 18 weeks of Western diet-induced obesity, but not by 239
eNOS deficiency. We do not have a clear explanation for the discrepancies in results 240
between these two studies. One possibility could be the use of different strains of 241
eNOS knockout mice; however, the strain used in that previous publication (5) was not 242
reported. Another explanation could be related to the use of different diets. Indeed, in 243
Handa et al. (5) obesity was induced with a diet containing 60% kcal from fat, 20% kcal 244
from carbohydrate, and 20% kcal from protein (5.24 kcal/g of food); whereas in our 245
study the Western diet contained 44.9% kcal from fat, 35.1% kcal carbohydrate, and 246
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20% kcal protein, with 1% cholesterol (4.68 kcal/g of food). The control diets were 247
comparable between studies in macro-nutrient composition (10% kcal from fat, 70% 248
kcal from carbohydrate, and 20% kcal from protein; 3.85 kcal/g of food) but levels of 249
sucrose were markedly different (34.5% kcal in Handa et al. vs. 3.5% kcal in our study). 250
Another important difference between study designs to highlight is that our mice were 251
given the respective diets for 18 weeks, whereas in Handa et al. study the diet 252
intervention was 4 weeks. To determine if the lack of effect of eNOS deficiency was 253
limited to white AT, we examined the same genes in interscapular brown AT, a fat depot 254
that is more vascularized. Similar to white AT, we found no evidence of increased 255
inflammation in brown AT from eNOS knockout mice. Interestingly, however, the lack of 256
inflammation could be specific to AT, as we have preliminary data suggesting that there 257
are robust differences in inflammatory markers in the liver of eNOS knockout mice 258
compared to wild-type mice (RSR, unpublished observations). As will be discussed 259
later, inflammation was also detected in vascular tissue of eNOS knockout mice. 260
The present findings are congruent with findings from a genome-wide microarray 261
analysis of visceral white AT from eNOS knockout versus wild-type female mice (13). In 262
that study, no significant differences between eNOS knockout and wild-type mice were 263
reported in regards to inflammatory genes. In support of the notion that reduced 264
production of NO does not contribute to AT inflammation, we recently found that 265
expression of inflammatory genes and markers of immune cell infiltration in white and 266
brown AT were, by and large, unchanged with chronic administration of L-NAME in 267
drinking water in both lean and obese rats (12). However, because L-NAME inhibits all 268
NOS isoforms, we were unable to exclusively test the role of eNOS as source of NO. 269
12
In the present study, the lack of effect of eNOS deficiency on white and brown AT 270
inflammation was in marked contrast with findings in the aorta where loss of eNOS 271
produced increased expression of inflammatory genes and markers of immune cell 272
infiltration. Our data expand on previous work demonstrating the atheroprotective role of 273
vascular NO. Indeed, previous research demonstrates that inhibition of NOS with L-274
NAME upregulates inflammatory and pro-oxidant genes, increases endothelial cell 275
adhesion, and promotes atherosclerotic lesions (1, 4, 6, 12). Further, there is evidence 276
that eNOS deficient mice display aberrant vascular remodeling in response to external 277
carotid artery ligation (15), and mice with eNOS/apoE double knockout reveal 278
accelerated atherosclerosis, aortic aneurysm formation, and ischemic heart disease (8, 279
9). 280
A novel finding of the present study was the observation in the aorta that eNOS 281
deficiency was distinctly associated with increased expression of endoplasmic reticulum 282
(ER) stress markers, including DDIT3, HSP5A, and HSPA1A mRNA (genes encoding 283
for CHOP, GRP78, and HSP72, respectively). ER stress has been implicated in the 284
etiology and progression of atherosclerosis (2, 11, 16, 18) and these data suggest that 285
reduced eNOS-derived NO production promotes ER stress; however, more research is 286
needed to elucidate the intracellular signaling mechanisms underlying the link between 287
reduced NO and ER stress. 288
This study’s findings should be considered in light of its limitations. First, our 289
study did not establish whether the reported changes in adipose tissue mRNA and 290
protein levels are attributable to alterations in the phenotype of adipocytes and/or 291
resident immune cells within AT. It should be noted that expression of some 292
13
inflammatory genes is markedly different when assessed in the stromal vascular 293
fractions versus whole tissue (20). As such, future research using FACS analysis is 294
needed to examine the differential inflammatory effects of obesity and eNOS ablation on 295
adipose tissue immune cells (i.e., macrophages, T-cells) versus adipocytes. Second, we 296
have attempted to measure nitrate + nitrite levels in AT homogenates as a surrogate 297
measure of NO; however, the levels were below the detection limits of the assay. Thus, 298
it remains unknown the extent to which AT NO bioavailability is affected by Western diet 299
and eNOS ablation. Our finding that markers of oxidative stress were increased with 300
Western diet suggests that NO bioavailabilty was likely reduced due to NO scavenging 301
by superoxide radicals. Third, despite eNOS deficient mice being smaller, leaner, and 302
revealing a decrease in adipocyte size, they displayed an equivalent degree of AT 303
inflammation. One interpretation of this finding is that, relative to the degree of adiposity, 304
the eNOS knockout mice are more inflamed (i.e., more inflammation per unit of adipose 305
tissue). To examine the effect of eNOS ablation independent of adiposity differences, 306
we performed a secondary analysis which involved the comparison of the 4 leanest 307
mice from the wild-type Western diet group (weight = 41.0±2.0 g; 37.9±4.4 %fat) versus 308
the 4 heaviest mice from the eNOS knockout Western diet group (weight=40.2±1.7 g; 309
38.5±1.2 %fat). We found no significant differences in any of the markers of 310
inflammation between these two subgroups (p values ranged from 0.233 to 0.934), thus 311
suggesting that the lack of genotype effect on AT inflammation may be unrelated to 312
differences in body weight or adiposity. However, to more precisely evaluate the 313
influence of adiposity, future studies should experimentally match body weight and 314
adiposity between wild-type and eNOS knockout mice using a pair-feeding paradigm. 315
14
These effects of eNOS deficiency on body weight and composition may be due to 316
central effects resulting in reduced food and caloric intake (Fig 1F). In our previous 317
chronic L-NAME treatment study in rats it was also noted that NOS inhibition diminished 318
food intake and adiposity (12). 319
In conclusion, we found that increased white AT inflammation with Western diet-320
induced obesity was not accompanied by changes in eNOS phosphorylation at Ser1177 321
and Thr495, two sites of phosphorylation that largely control eNOS activation. In 322
addition, we found no evidence of increased AT inflammation in mice with genetic 323
deletion of eNOS. Therefore, obesity and insulin resistance-associated AT inflammation 324
appears to be unrelated to eNOS-derived NO production. 325
326
ACKNOWLEDGMENTS 327
The authors thank Grace Meers, Rebecca Scroggins, Nick Fleming, Youngmin 328
Park, and T’Keaya Gaines for excellent technical assistance. 329
330
GRANTS 331
This work was supported by a University of Missouri Life Sciences Fellowship (R. 332
D. Sheldon), Veterans Health Administration grant CDA2 1BX001299 (R. S. Rector), 333
and National Institute of Health grants K01 HL-125503 and R21 DK-105368 (J. Padilla). 334
This work was completed with resources and the use of facilities at the Harry S Truman 335
Memorial Veterans Hospital. 336
337
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REFERENCES 339
1. Cayatte A, Palacino J, Horten K, and Cohen R. Chronic inhibition of nitric 340 oxide production accelerates neointima formation and impairs endothelial function in 341 hypercholesterolemic rabbits. Arterioscler Thromb 14: 1994. 342 2. Chistiakov DA, Sobenin IA, Orekhov AN, and Bobryshev YV. Role of 343 endoplasmic reticulum stress in atherosclerosis and diabetic macrovascular 344 complications. BioMed research international 2014: 610140, 2014. 345 3. Ginsberg HN, and MacCallum PR. The obesity, metabolic syndrome, and type 346 2 diabetes mellitus pandemic: Part I. Increased cardiovascular disease risk and the 347 importance of atherogenic dyslipidemia in persons with the metabolic syndrome and 348 type 2 diabetes mellitus. Journal of the cardiometabolic syndrome 4: 113-119, 2009. 349 4. Gomez-Guzman M, Jimenez R, Sanchez M, Romero M, O'Valle F, Lopez-350 Sepulveda R, Quintela A, Galindo P, Zarzuelo M, Bailon E, Delpon E, Perez-351 Vizcaino F, and Duarte J. Chronic (-)-epicatechin improves vascular oxidative and 352 inflammatory status but not hypertension in chronic nitirc oxide-deficient rats. Br J Nutr 353 106: 1337-1348, 2011. 354 5. Handa P, Tateya S, Rizzo N, Cheng A, Morgan-Stevenson V, Han C, Clowes 355 A, Daum G, O'Brien K, Schwartz M, Chait A, and Kim F. Reduced vascular nitric 356 oxide-cGMP signaling contributes to adipose tissue inflammation during high-fat 357 feeding. Arterioscler Thromb Vasc Biol 31: 2827-2835, 2011. 358 6. Hossain M, Qadri S, and Liu L. Inhibition of nitric oxide synthesis enhances 359 leukocyte rolling and adhesion in human microvasculature. J Inflamm 9: 28, 2012. 360 7. Jacobi D, Stanya KJ, and Lee CH. Adipose tissue signaling by nuclear 361 receptors in metabolic complications of obesity. Adipocyte 1: 4-12, 2012. 362 8. Kuhlencordt P, Gyurko R, Han F, Scherrer-Crosbie M, Aretz T, Hajjar R, 363 Picard M, and Huang P. Accelerated atherosclerosis, aortic aneurysm formation, and 364 ischemic heart disease in apolipoprotein E/endothelial nitric oxide synthase double-365 knockout mice. Circulation 104: 2001. 366 9. Kuhlencordt P, Padmapriya P, Rutzel S, Schodel J, Hu K, Schafer A, Huang 367 P, Ertl G, and Bauersachs J. Ezetimibe potently reduces vascular inflammation and 368 arteriosclerosis in eNOS-deficient ApoE ko mice. Atherosclerosis 202: 48-57, 2009. 369 10. Laroux F, Pavlick K, Hines I, Kawachi S, Harada H, Bharwani S, Hoffman J, 370 and Grisham M. Role of nitric oxide in inflammation. Acta Physiol Scand 173: 113-118, 371 2001. 372 11. McAlpine CS, and Werstuck GH. The development and progression of 373 atherosclerosis: evidence supporting a role for endoplasmic reticulum (ER) stress 374 signaling. Cardiovascular & hematological disorders drug targets 13: 158-164, 2013. 375 12. Padilla J, Jenkins NT, Thorne PK, Lansford KA, Fleming NJ, Bayless DS, 376 Sheldon RD, Rector RS, and Laughlin MH. Differential regulation of adipose tissue 377 and vascular inflammatory gene expression by chronic systemic inhibition of NOS in 378 lean and obese rats. Physiological reports 2: e00225, 2014. 379 13. Razny U, Kiec-Wilk B, Polus A, Wator L, Dyduch G, Partyka L, Bodzioch M, 380 Tomaszewska R, and Wybranska I. The adipose tissue gene expression in mice with 381 different nitric oxide availability. J Physiol Pharmacol 61: 607-618, 2010. 382
16
14. Reaven GM. Insulin resistance: the link between obesity and cardiovascular 383 disease. The Medical clinics of North America 95: 875-892, 2011. 384 15. Rudic R, Shesely E, Maeda N, Smithies O, Segal S, and Sessa W. Direct 385 evidence for the importance of endothelium-derived nitric oxide in vascular remodeling. 386 J Clin Invest 101: 731-736, 1998. 387 16. Scull CM, and Tabas I. Mechanisms of ER stress-induced apoptosis in 388 atherosclerosis. Arterioscler Thromb Vasc Biol 31: 2792-2797, 2011. 389 17. Sheldon RD, Padilla J, Jenkins NT, Laughlin MH, and Rector RS. Chronic 390 NOS inhibition accelerates NAFLD progression in an obese rat model. American journal 391 of physiology Gastrointestinal and liver physiology 308: G540-549, 2015. 392 18. Tabas I. The Role of Endoplasmic Reticulum Stress in the Progression of 393 Atherosclerosis. Circulation research 107: 839-850, 2010. 394 19. Wainright KS, Fleming NJ, Rowles JL, Welly RJ, Zidon TM, Park YM, Gaines 395 TL, Scroggins RJ, Anderson-Baucum EK, Hasty AH, Vieira-Potter VJ, and Padilla 396 J. Retention of sedentary obese visceral white adipose tissue phenotype with 397 intermittent physical activity despite reduced adiposity. American journal of physiology 398 Regulatory, integrative and comparative physiology 309: R594-602, 2015. 399 20. Xu H, Barnes GT, Yang Q, Tan G, Yang D, Chou CJ, Sole J, Nichols A, Ross 400 JS, Tartaglia LA, and Chen H. Chronic inflammation in fat plays a crucial role in the 401 development of obesity-related insulin resistance. J Clin Invest 112: 1821-1830, 2003. 402
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FIGURE LEGENDS 406
Figure 1. Body weight (A), percent body fat (B), epididymal AT weight (C), 407 retroperitoneal AT weight (D), interscapular brown AT weight (E), and kilocalories 408 consumed per week (F) in wild-type and eNOS knockout mice fed a control diet 409 versus Western diet. Reported body weight, percent fat, and fat pad weights are at 410 time of sacrifice. Kcal consumed/week values are calculated as an average over the 18-411 week feeding period. Data are expressed as means ± SE. D, main effect of diet; G, 412 main effect of genotype; DxG, diet by genotype interaction. Significant p values (<0.05) 413 are highlighted in bold. N=7 to 8/group. 414
Figure 2. Representative histology images (10X) of visceral white (i.e., epididymal) 415 AT stained for Mac-2 (A), fold differences in Mac-2 positive stained area (B), and 416 average adipocyte size (C) in wild-type and eNOS knockout mice fed a control 417 diet versus Western diet. Data are expressed as means ± SE. D, main effect of diet; 418 G, main effect of genotype; DxG, diet by genotype interaction. Significant p values 419 (<0.05) are highlighted in bold. N=5/group. 420
Figure 3. Visceral white (i.e., epididymal) (A) and brown (i.e. interscapular) (B) AT 421 gene expression in wild-type and eNOS knockout mice fed a control diet versus 422 Western diet. Data are expressed as means ± SE. D Denotes main effect of diet 423 (p<0.05). No significant main effects of genotype or interactions were found (all p>0.05). 424 N=7 to 8/group. 425
Figure 4. Visceral white (i.e., epididymal) AT protein content in wild-type and 426 eNOS knockout mice fed a control diet versus Western diet. Data are expressed as 427 means ± SE. D, main effect of diet; G, main effect of genotype; DxG, diet by genotype 428 interaction. Significant p values (<0.05) are highlighted in bold. N=7 to 8/group. For the 429 nitrotyrosine blot, an average of all bands (between 75kda and 25kda) was calculated. 430
Figure 5. Aorta gene expression in wild-type and eNOS knockout mice fed a 431 control diet versus Western diet. Data are expressed as means ± SE. G, denotes 432 main effect of genotype (p<0.05). No significant main effects of diet or interactions were 433 found (all p>0.05). N=4 to 8/group. 434
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Table 1. Fasting blood characteristics. 442
Variable Wild-type eNOS knockout Control diet Western
diet Control diet Western diet Two-Way
ANOVA Total cholesterol, mg/dl
145.4±6.9 215.6±25.7 134.6±4.9 247.1±25.6 D: p<0.001 G: p=0.590 DxG: p=0.276
LDL cholesterol, mg/dl
7.8±0.6 16.9±2.4 6.0±0.4 20.4±5.3 D: p=0.001 G: p=0.776 DxG: p=0.400
HDL cholesterol, mg/dl
64.3±2.0 96.0±10.5 65.3±3.9 105.9±4.2 D: p<0.001 G: p=0.354 DxG: 0.452
Triglycerides, mg/dl
51.1±4.8 42.0±3.3 59.9±4.1 52.1±9.7 D: p=0.194 G: p=0.148 DxG: p=0.913
NEFA, mmol/l 0.73±0.05 0.69±0.07 0.80±0.05 0.92±0.10 D: p=0.585 G: p=0.042 DxG: p=0.285
ALT, U/l 38.3±4.8 210.6±50.3 42.3±5.3 250.8±72.3 D: p<0.001 G: p=0.631 DxG: p=0.694
AST, U/l 226.9±19.6 288.7±52.9 273.0±50.0 321.9±38.4 D: p=0.189 G: p=0.342 DxG: p=0.876
Insulin, ng/ml 0.8±0.1 2.4±0.5 1.2±0.2 3.2±1.2 D: p=0.017 G: p=0.415 DxG: p=0.786
Glucose, mg/dl 290.5±25.5 309.0±30.4 316.1±14.6 315.5±46.4 D: p=0.786 G: p=0.625 DxG: p=0.770
HOMA-IR 17.5±2.5 57.3±12.4 27.9±5.8 74.9±30.6 D: p=0.021 G: p=0.436 DxG: p=0.840
Nitrate + Nitrite, pmoles/µl
20.6±3.1 11.6±1.4 16.4±2.6 11.2±2.4 D: p=0.011 G: p=0.382 DxG: p=0.455
Abbreviations: LDL, low density lipoprotein; HDL, high density lipoprotein; NEFA; non-443 esterified fatty acids; ALT, alanine aminotransferase; AST, aspartate aminotransferase; 444 HOMA-IR, homeostatic model assessment insulin resistance index. Data are expressed 445 as means ± SE.D, main effect of diet; G, main effect of genotype; DxG, diet by genotype 446 interaction. Significant p values (<0.05) are highlighted in bold. N=7 to 8/group. 447
448
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