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Obesity (Silver Spring). Author manuscript; available in PMC 2017 October 01. Published in final edited form as: Obesity (Silver Spring). 2016 October ; 24(10): 2140–2149. doi:10.1002/oby.21620.
Obesity alters immune and metabolic profiles: new insight from obese-resistant mice on high fat diet Shannon K. Boi1,*, Claire M. Buchta2,*, Nicole A. Pearson3, Meghan B. Francis4, David K. Meyerholz5, Justin L. Grobe3, and Lyse A. Norian1,4,6 1Graduate
Biomedical Sciences, University of Alabama at Birmingham, Birmingham AL, USA
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2Department
of Urology The University of Iowa Carver College of Medicine, Iowa City, IA, USA
3Department
of Pharmacology, The Obesity Research and Education Initiative, and the Fraternal Order of Eagles’ Diabetes Research Center, The University of Iowa Carver College of Medicine, Iowa City, IA, USA
4Department
of Nutrition Sciences, University of Alabama at Birmingham, Birmingham AL, USA
5Department
of Pathology, The University of Iowa Carver College of Medicine, Iowa City, IA, USA
6Nutrition
Obesity Research Center and Comprehensive Cancer Center, University of Alabama at Birmingham, Birmingham AL, USA
Abstract Author Manuscript
Objective—Diet-induced obesity has been shown to alter immune function in mice, but distinguishing the effects of obesity from changes in diet composition is complicated. We hypothesized that immunological differences would exist between diet-induced obese (DIO) and obese-resistant (OB-Res) mice fed the same high-fat diet (HFD). Methods—BALB/c mice were fed either standard chow or HFD to generate lean or DIO and OB-Res mice, respectively. Resulting mice were analyzed for serum immunologic and metabolic profiles, and cellular immune parameters.
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Results—BALB/c mice on HFD can be categorized as DIO or OB-Res, based on body weight versus lean controls. DIO mice are physiologically distinct from OB-Res mice, whose serum Insulin, Leptin, GIP, and Eotaxin concentrations remain similar to lean controls. DIO mice have increased macrophage+ crown-like structures in white adipose tissue, although macrophage percentages were unchanged from OB-Res and lean mice. DIO mice also have decreased splenic CD4+ T cells, elevated serum GM-CSF, and increased splenic CD11c+ dendritic cells, but
Corresponding Author: Lyse A. Norian, Ph.D., Department of Nutrition Sciences, Comprehensive Cancer Center, and Nutrition Obesity, Research Center; University of Alabama at Birmingham, WTI 320C, 1824 6th Ave South, Birmingham, AL 35233, Phone: 205-996-0152. *Authors contributed equally to this work. CM Buchta Current Address: Carter Immunology Center, University of Virginia, Charlottesville, VA, USA Disclosure(s): The authors have no conflicts of interest to disclose. Author Contributions: SKB: analyzed data and wrote manuscript; CMB performed experiments, analyzed data, and wrote manuscript; NAP performed experiments and analyzed data; MBF wrote manuscript; DKM performed experiments, analyzed data, and wrote manuscript; JLG performed experiments, analyzed data, and wrote manuscript; LAN performed experiments, analyzed data, and wrote manuscript.
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impaired dendritic cell stimulatory capacity (p < 0.05 versus lean controls). These parameters were unaltered in OB-Res mice versus lean controls. Conclusions—Diet-induced obesity results in alterations in immune and metabolic profiles that are distinct from effects caused by HFD alone. Keywords Diet-induced obesity; obese; obese-resistant; immune function; dendritic cell
Introduction
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Obesity is accompanied by adverse health effects that include cardiovascular disease, diabetes, and increased cancer risk (1, 2). Mounting evidence suggests that obesity is also associated with impaired immune responses to vaccinations, infections, and tumor growth (3–5), although contrary evidence does exist (6). With ~35% of the United States adult population obese (7), a better understanding of the complex immunologic implications of obesity is needed. Models of diet-induced obese animals/diet-induced obesity (DIO), wherein animals are fed a high-fat (HFD) or other obesogenic diet, allow investigators to manipulate caloric intake and diet composition in genetically intact animals. Most HFD models utilize inbred mouse strains, with C57BL/6 being the most common (8, 9). The C57BL/6 DIO mouse model is characterized by insulin resistance, particularly in males, so permits evaluation of obesity in combination with type 2 diabetes (10). In contrast, the BALB/c strain is more resistant to developing both obesity and type 2 diabetes (11, 12). However, use of DIO BALB/c mice may at times be beneficial due to diminished confounding effects of type 2 diabetes.
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Most DIO studies contain one important caveat: control groups are composed of lean mice that are fed different rodent diets. This approach renders it impossible to distinguish between effects of dietary composition on physiological outcomes versus effects of obesity per se. Evaluating these relative contributions is important, as some dietary components can alter biological function and immune responses in the absence of obesity (13, 14).
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Here, we present a refinement of the DIO BALB/c model, based upon identification of a subset of genetically intact mice that are highly resistant to becoming obese. As we and others have reported, obesity has complex effects on immune responses, and dendritic cell function in particular (4, 15). Thus, we used this model to examine effects of obesity on immune and metabolic profiles of DIO and Obese-resistant (OB-Res) mice fed the same HFD. Our data illustrate that obesity alters multiple immune parameters beyond effects from HFD alone. Furthermore, we identify OB-Res BALB/c mice as important controls that lend new insight into the complex interplay of diet and obesity.
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Methods Animals and diets
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Female BALB/cAnNCr and C57BL/6NCr mice were purchased (Charles River/NCI, Frederick, MD) at 7–8 weeks of age, maintained on standard chow (Teklad 7013, 18% kcal fat, 3.1 kcal/g) for 1 week then randomly assigned to standard chow or HFD (Research Diets #12492, 60% kcal fat, 5.24 kcal/g). Due to accelerated weight gain, C57BL/6NCr mice were maintained on HFD for 10 weeks to induce obesity, whereas BALB/c mice were maintained on diet for 20 weeks as previously reported (5). DUC18 TCR transgenic mice have been described (16, 17). Mice were housed five to a cage in standard static cages under pathogenfree conditions in 12:12 light:dark cycles at 25°C with ad libitum access to food and water at the University of Iowa Animal Care Facility, which is fully AALAC accredited. All animal procedures were approved by the University of Iowa IACUC. After appropriate time on feed, all mice were weighed, and the mean and standard deviation (s.d.) of lean (standard chow) groups were calculated. Blood glucose measurement C57BL/6NCr and BALB/c mice were maintained on standard chow or HFD as described above. Mice were fasted overnight, blood was obtained from the tail vein, and blood glucose measured via glucometer (Roche Diabetes Care). Cytokine and chemokine evaluation by Multiplex Array
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Serum samples were obtained from lean, OB-Res, and DIO mice and frozen at −80°C until use. Serum cytokine and chemokine concentrations were determined via BioPlex Pro Mouse Diabetes Assay and BioPlex Pro Mouse Cytokine Assay and run on a Bio-Rad BioPlex analyzer (Bio-Rad). Surface staining for flow cytometry Spleens and adipose tissue (pooled gonadal/renal fat pads) were harvested and processed as described (5, 6). Cells were stained with antibodies, then results were acquired using a BD LSR II (BD Biosciences) and analyzed with FlowJo software (Tree Star). CD19-BV510 and Vβ8.3-PE (BD Biosciences). I-Ad-A488, CD8α-A700, CD4-APC, CD11c-APC/Cy7, CD3ε-PE, CD11b-PE/Cy7, CD54-FITC, CD40-PE, H2Kd-FITC, I-Ad-PE, CD11c-biotin, SA-APC/Cy7 and TruStain FcX (BioLegend). CD86-APC (eBioscience). T cell proliferation assay
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Splenic DCs were purified from pooled lean, OB-Res, and DIO mice using CD11c microbeads (Miltenyi Biotec). DCs were pulsed with tumor extracellular signal-regulated kinase (tERK) peptide and naïve CD8+ DUC18 T cell proliferation was evaluated as described (17). Immunohistochemistry White adipose tissues (WAT) were stained with anti-IBA-1 (ionized calcium-binding adapter molecule 1), a defined macrophage marker (18). Tissues were examined by a veterinary
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pathologist using the post-examination masking method (19). To assess incidences of crownlike structures (CLS), three random WAT samples (20x magnification) were evaluated for total area and number of CLS. Values were averaged and reported as number of CLS/mm2. The number of IBA+ cells in the WAT interstitium were enumerated (n=3 random samples, 100x magnification), averaged for each tissue, and reported as macrophage number/mm2. During enumeration, solitary macrophages were recorded as a distinct pool from macrophages within CLS. Energy balance by bomb calorimetry
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Energy balance was evaluated using bomb calorimetry as described (20, 21). Briefly, mice were placed into single-mouse sized metabolic cages (Nalgene/Tecniplast) at room temperature with ad libitum access to food and water. Food intake, water intake, urine output, and fecal output were quantified to measure behavioral changes. Fecal samples were desiccated and caloric content was determined using a semi-micro bomb calorimeter (Parr). Digestive efficiency was determined as the ratio of calories absorbed to calories consumed. Energy efficiency was determined as the change in body mass divided by total calories absorbed. Resting Metabolic Rate (RMR) evaluation by respirometry RMR was determined using continuous respirometry as described (22, 23). Briefly, animals were placed into thermally-controlled chambers maintained at 30°C. Oxygen and carbon dioxide composition of effluent air, and chamber air flow rates (corrected to STP) during bouts of rest were used to calculate rates of oxygen consumption and carbon dioxide production. Using the equation derived from Lusk (24), RMR was calculated.
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Statistical analyses Statistical analyses were accomplished utilizing Prism, Version 6.07 (GraphPad). Gaussian distribution was assessed using D’Agostino-Pearson omnibus normality test. Data were analyzed using two-tailed unpaired Student t-tests with (carrots; ^) or without (asterisks; *) Welch’s correction (threshold of 3 s.d.), as appropriate. Non-parametric analyses were performed using two-tailed Mann-Whitney U tests (pound signs; #). Significance indicated as ^ = * = # = p < 0.05; ^^ = ** = ## = p < 0.01; ^^^ = *** = ### = p < 0.001; ^^^^ = **** = #### = p < 0.0001.
Results Distinguishing DIO from OB-Res mice on HFD
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DIO murine models are widely used to study biological and physiological implications of obesity, but comparisons to lean mice on standard chow introduce problems related to distinguishing effects of obesity from effects of diet. Previously, we noted that within BALB/c strains, individual mice showed remarkable heterogeneity to HFD, with some being highly resistant to obesity (5). Here, we leveraged this divergence to examine metabolic and immune profiles in HFD-fed DIO versus OB-Res mice.
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As expected (25, 26), all C57BL/6NCr mice showed significant weight gain after 10 weeks on HFD (average weight gain versus chow controls = 25.73 gm/ mouse) (Fig. 1A). As per standard convention, these mice are referred to as DIO. In contrast, BALB/c female mice showed less dramatic and more variable weight gain on HFD, even after 20 weeks (average weight gain = 6.86 gm/mouse versus chow controls) (Fig. 1B). As previously, we defined mice with DIO as having a final body weight >3 s.d. above the mean weight of the standard chow cohort (dotted line, Fig. 1B) (5). Using this definition, 50% of BALB/c mice were classified as having DIO, versus 100% of C57BL/6NCr mice (Fig. 1A). Male BALB/c mice followed a similar trend, with 66% of mice on HFD classified as obese (data not shown). BALB/c mice that remained within one s.d. of the lean cohort’s mean weight were designated OB-Res. Onset of the DIO versus OB-Res phenotype was detectable in as few as 4–6 weeks on HFD (Fig. 1C). To determine whether these phenotypic weight differences were due to hierarchical cage status, mice showing an OB-Res or DIO phenotype were separated at 10 weeks post-HFD administration and co-housed with like counterparts (ex: OB-Res with OB-Res) for 12 additional weeks. All mice retained their original adiposity phenotypes (Fig. 1D), suggesting that intra-cage social behaviors do not substantially impact DIO status in this model. DIO mice had significantly larger gonadal and renal pads of WAT that accounted for a greater percentage of total body weight (Fig. 1E), consistent with prior findings (27). DIO mice have increased blood glucose, insulin, and leptin concentrations not seen in OBRes or lean mice
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Increased fasting blood glucose and elevated serum insulin and leptin concentrations are characteristic of obesity and type 2 diabetes. As reported (9, 26), DIO C57BL/6NCr mice exhibited a significant increase in fasting blood glucose compared to lean controls (Fig. 2A). Consistent with prior reports, BALB/c mice on HFD were resistant to hyperglycemia (28) (Fig. 2B). Although the absolute concentrations (mean = 62.8 mg/dL for DIO BALB/c mice) were lower than those observed for DIO C57BL/6NCr mice (mean = 115.6 mg/dL), DIO BALB/c mice did show elevated fasting blood glucose concentrations versus lean controls (Fig. 2B). BALB/c OB-Res mice had blood glucose levels that were equivalent to lean counterparts, despite being on HFD for 20 weeks. In addition, BALB/c DIO mice had increased serum insulin (Fig. 2C) and leptin (Fig. 2D) as compared to OB-Res mice and lean controls (Figs. 2C/2D). Thus, only DIO mice on HFD showed key hallmarks of obesity, whereas OB-Res mice on HFD resembled lean chow-fed controls. OB-Res mice are resistant to weight gain due to increased energy expenditure relative to DIO mice
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Leptin is an adipokine that regulates food intake and satiety; therefore decreased serum leptin in BALB/c OB-Res mice suggested that their phenotype might be due to altered food consumption or digestive efficiency. To investigate this, BALB/c DIO and OB-Res mice were singly housed in metabolic cages and given ad libitum access to HFD. Food intake, as measured in both g/day and kcal/day, was equivalent in DIO and OB-Res mice (Table I). Fecal output was also comparable. Once food is consumed, it must be digested and metabolized into useable energy forms. Therefore, we measured calories absorbed from food by DIO versus OB-Res mice using bomb calorimetry, and found these to be equivalent, Obesity (Silver Spring). Author manuscript; available in PMC 2017 October 01.
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indicating comparable digestive efficiency in OB-Res and DIO mice. However, energy efficiency (the amount of weight gain for any given calorie consumed) was significantly lower in OB-Res mice than in DIO mice (mean 3.65 vs 10.09 mg BW per kcal absorbed, p = 0.006). This means that for each calorie consumed, OB-Res mice used a smaller fraction for weight gain. Thus, the total energy expenditure of OB-Res animals is significantly greater than for DIO mice.
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Extending these data, aerobic resting metabolic rate (RMR) was determined using respirometry at thermoneutrality. DIO mice (n=15, 37.41±0.99 g) and OB-Res mice (n=18, 26.20±0.82 g, P20 weeks, it is unlikely that gut dysbiosis accounts for changes in weight gain. Mice exhibit coprophagia, so co-housed mice typically have similar gut microbiomes. At present, the behavioral, biological and/or metabolic causes for differences in energy expenditure and the subsequent development of DIO versus OB-Res status remain unknown. Future studies to dissect the relative contributions of aerobic versus anaerobic RMR, the gut microbiome, physical activity, and potential stress responses to the OB-Res phenotype are required.
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The effects of obesity on immune function are likely complex and highly dependent upon diet composition. Reports have shown that immune function can be impacted by physical activity (35, 36), and stress hormones (i.e., glucocorticoids) (37); therefore, it is possible that immune alterations of OB-Res versus DIO hosts could also be attributed to increased physical activity and/or stress responses. We previously demonstrated that immune responses to renal tumors are impaired in DIO BALB/c, as fewer effector CD8+ T cells infiltrate tumor-bearing kidneys (5). Others have reported impaired lymphatic drainage and
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DC migration to lymph nodes (38), and heightened/pathogenic CD8+ T cells in the lung after influenza challenge (39) in DIO versus chow-fed lean C57BL/6 mice, and alterations in lung metabolomic signatures in genetically modified obese versus wild type mice on chow diet (40). In contrast, Khan et al. found no changes in memory CD8+ T cell responses in DIO C57BL/6, BALB/c, or outbred Swiss Webster mice following viral challenge with either LCMV or influenza (6). In each of these prior studies, including our own, DIO mice on HFD were compared to lean mice on standard chow. The use of OB-Res mice, as described in our current study, will add an important new tool to future studies on DIO, as inclusion of this group will permit identification of immunological changes caused by dietary composition, versus outright obesity.
Conclusions Author Manuscript
Our current study provides two main advances to our understanding of the biological implications of obesity: 1) we refined the BALB/c model of DIO so as to study OB-Res animals on the same HFD, and 2) we illustrate that obesity alters multiple parameters of immune function even when diet is controlled. Moving forward, highly obese-resistant BALB/c mice could be utilized to further delineate the immunological and broader biological consequences of obesity versus diet.
Acknowledgments Funding: NIH grant # R01CA181088 to LAN, NIH grant #HL084207 and American Diabetes Association grant #1-14-BS-079 to JLB, NIH/NCI CPCTP R25 grant #CA47888 to SKB. The authors would like to thank Drs. Aliasger Salem and David Lubaroff for critical input to this project.
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Abbreviations HFD
high fat diet
DIO
diet-induced obesity or diet-induced obese
OB-res
obese-resistant
DC
dendritic cell
WAT
white adipose tissue
References Author Manuscript
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What is already known about this subject? 1.
Murine models of high fat diet-induced obesity have been used to describe changes in immune function with obesity onset.
2.
Controls for diet-induced obesity models typically include mice on standard chow.
3.
Changes in diet composition in the absence of obesity can alter immune function; thus prior conclusions based upon comparison of high fat diet-induced obese mice to lean mice on standard chow may be erroneously describing effects of diet rather than obesity.
What does this study add?
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1.
We provide a refinement to diet-induced obesity models, via characterization of a subset of obese-resistant mice on high fat diet.
2.
When diet is controlled, obesity causes multiple systemic alterations in metabolic proteins and cytokines.
3.
When diet is controlled, obesity causes multiple changes in cellular immune cell composition and function.
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Figure 1.
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Individual examination of age-matched BALB/c mice on high fat diet identifies unique populations of DIO and OB-Res mice. (A) Body weight (g) of C57BL/6NCr mice was calculated at the end of 10 weeks for mice fed either standard chow or HFD. Dots represent individual mice. (B) Body weights of BALB/c mice at the end of 20 weeks for mice fed either standard chow or HFD. DIO mice were defined as having body weights > lean cohort mean + 3 standard deviations. OB-Res mice were defined as having body weights within 1 standard deviation of the lean cohort mean. (C) Stratification of individual BALB/c mouse body weights over the course of 20 weeks on standard or high fat diet. (D) Body weight (g) of BALB/c mice at 10 weeks of HFD administration (pre-separation) and at 22 weeks of HFD administration (post-separation). After 10 weeks on diet, mice were removed from original cages and co-housed with similar adiposity phenotypes (ex: OB-Res with OB-Res) for 12 additional weeks. Statistical significance was determined using a two-tailed MannWhitney U test (#p < 0.05, ##p< 0.01). (E) Gonadal and renal fat pads were removed from lean, OB-Res, and DIO mice, weighed, and the WAT weight was calculated as a percentage of total body weight. Statistical significance was determined using a two-tailed unpaired student t test (****p < 0.0001).
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DIO mice have hallmarks of obesity not seen in OB-Res mice on HFD. (A) C57BL/6NCr mice were maintained on feed for 10 weeks. Mice were then fasted overnight and serum blood glucose concentration (mg/dL) was calculated. Statistical significance was determined using a two-tailed unpaired student t test (****p < 0.0001). (B) BALB/c mice were maintained on feed for 20 weeks. Mice were then fasted overnight and serum blood glucose concentration (mg/dL) was calculated. Statistical significance was determined using a twotailed Mann-Whitney U test (##p < 0.01). (C) BALB/c serum was analyzed to obtain concentrations of insulin. Statistical significance was determined using a two-tailed unpaired student t test with Welch’s correction (^^p < 0.01). (D) BALB/c serum was analyzed to obtain concentrations of leptin. Statistical significance was determined using a two-tailed unpaired student t test with Welch’s correction (^^^p < 0.001).
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Figure 3.
Serum analyses reveal unique obesogenic and proinflammatory differences between DIO, OB-Res, and lean BALB/c mice. Serum was collected from BALB/c mice maintained on standard chow or HFD for 20 weeks and analyzed via Multiplex array where n= 8 lean mice, 13 OB-Res, and 10 DIO mice. Statistical significance was determined using either a twotailed unpaired student t test (*p < 0.05), or a two-tailed Mann-Whitney U test (#p < 0.05, ##p < 0.01, ###p < 0.001), as appropriate.
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Figure 4.
No differences in leukocytic infiltration into WAT in DIO, OB-Res or lean mice. BALB/c mice (n= 9–10 mice per group) were maintained on standard chow or high fat diet for 20 weeks and gonadal and renal fat pads were collected. The stromal vascular fraction was then isolated and percentages of CD4+ and CD8+ T cells, CD19+ B cells, CD11c+ DC (CD11c+/I-Ad+/ CD11bint-low), and CD11b+ (CD11b+/I-Ad+/CD11clow) macrophages were determined by flow cytometry.
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Macrophage localization within CLS in WAT distinguishes DIO mice from OB-Res and lean counterparts. (A) BALB/c mice were maintained on standard chow or high fat diet for 20 weeks and gonadal and renal fat pads were collected. Immunohistochemical staining of IBA1+ macrophages in WAT was performed. Black arrows indicate single IBA1+ macrophages, red arrow indicates IBA1+ macrophages within a representative crown-like structure (CLS). (B) Enumeration of total IBA1+ macrophages and CLS in WAT from n= 5 each lean, OB-Res and DIO mice. Statistical significance was determined using a two-tailed Mann-Whitney U test (#p < 0.05, ##p < 0.01).
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Figure 6.
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DIO mice have immune alterations not present in OB-Res or lean mice. (A) BALB/c mice (n= 10 per group) were maintained on standard chow or HFD for 20 weeks prior to spleen harvest. Percentages of CD4+ and CD8+ T cells, CD19+ B cells, CD11b+ (CD11b+/I-Ad+/ CD11clow) macrophages, and CD11chigh/I-Ad+ dendritic cells were determined by flow cytometric analysis. Statistical significance was determined using a two-tailed unpaired student t test (*p < 0.05, **p < 0.01) or a two-tailed Mann-Whitney U test (##p < 0.01), as appropriate. (B) Serum was collected from indicated mice (n= 8 lean mice, 13 OB-Res, and 10 DIO mice) and analyzed via Multiplex for concentrations of granulocyte macrophage colony-stimulating factor (GM-CSF) (pg/mL). Statistical significance was determined using a two-tailed Mann Whitney U test (##p < 0.01).
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Obesity, but not HFD administration, is associated with impaired DC stimulatory capacity. (A) Expression of surface markers CD86, CD40, CD54, MHC Class I (H-2Kd) and MHC Class II (I-Ad) on gated splenic CD11chigh DC were analyzed by flow cytometry. Statistical significance was determined using a two-tailed Mann-Whitney U test (#p < 0.05). (B) CD11chigh / MHC II+/ Gr-1neg splenic DC were sort-purified from indicated mice, pulsed with exogenous peptide, then used to stimulate naive antigen-specific CD8+ T cells. T cell proliferation was measured by incorporation of 3H-thymidine (counts per minute) at 72 hours. Data are pooled from 2–5 individual experiments, with 2–3 mice pooled per
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experiment to generate sufficient numbers of DC. Statistical significance was determined by using a two-tailed Mann-Whitney U test (#p < 0.05).
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Table I
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Energy balance evaluation of DIO and OB-Res mice reveals differences in energy efficiency. BALB/c mice were maintained on high fat diet for 20 weeks. Mice (n= 13 DIO and 12 OB-Res) were then singly housed in metabolic cages for 3–5 days and given ad libitum access to high fat diet chow. Caloric densities of food and fecal samples were determined by bomb calorimetry. DIO
OB-Res
t-test p=
38.62
25.35
0.001
2.04
1.81
2.44
2.45
0.09
0.39
14.15
14.18
0.5
2.27
0.42
0.42
0.03
0.06
1.80
1.85
0.17
0.23
12.34
12.25
0.57
2.06
87.30
85.90
1.43
2.09
10.09
3.65
1.01
1.4
Body weight (g)
sem Food intake (g/day)
sem
0.990
Food intake (kcal/day)
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sem
0.990
Fecal output (g/day)
sem
0.930
Fecal output (kcal/day)
sem
0.873
Calories absorbed (kcal/day)
sem
0.967
Digestive Efficiency (% consumed)
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sem
0.595
Energy efficiency (D mg BW/kcal absorbed)
sem
0.006
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Obesity (Silver Spring). Author manuscript; available in PMC 2017 October 01. Published in final edited form as: Obesity (Silver Spring). 2016 October ; 24(10): 2140–2149. doi:10.1002/oby.21620.
Obesity alters immune and metabolic profiles: new insight from obese-resistant mice on high fat diet Shannon K. Boi1,*, Claire M. Buchta2,*, Nicole A. Pearson3, Meghan B. Francis4, David K. Meyerholz5, Justin L. Grobe3, and Lyse A. Norian1,4,6 1Graduate
Biomedical Sciences, University of Alabama at Birmingham, Birmingham AL, USA
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2Department
of Urology The University of Iowa Carver College of Medicine, Iowa City, IA, USA
3Department
of Pharmacology, The Obesity Research and Education Initiative, and the Fraternal Order of Eagles’ Diabetes Research Center, The University of Iowa Carver College of Medicine, Iowa City, IA, USA
4Department
of Nutrition Sciences, University of Alabama at Birmingham, Birmingham AL, USA
5Department
of Pathology, The University of Iowa Carver College of Medicine, Iowa City, IA, USA
6Nutrition
Obesity Research Center and Comprehensive Cancer Center, University of Alabama at Birmingham, Birmingham AL, USA
Abstract Author Manuscript
Objective—Diet-induced obesity has been shown to alter immune function in mice, but distinguishing the effects of obesity from changes in diet composition is complicated. We hypothesized that immunological differences would exist between diet-induced obese (DIO) and obese-resistant (OB-Res) mice fed the same high-fat diet (HFD). Methods—BALB/c mice were fed either standard chow or HFD to generate lean or DIO and OB-Res mice, respectively. Resulting mice were analyzed for serum immunologic and metabolic profiles, and cellular immune parameters.
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Results—BALB/c mice on HFD can be categorized as DIO or OB-Res, based on body weight versus lean controls. DIO mice are physiologically distinct from OB-Res mice, whose serum Insulin, Leptin, GIP, and Eotaxin concentrations remain similar to lean controls. DIO mice have increased macrophage+ crown-like structures in white adipose tissue, although macrophage percentages were unchanged from OB-Res and lean mice. DIO mice also have decreased splenic CD4+ T cells, elevated serum GM-CSF, and increased splenic CD11c+ dendritic cells, but
Corresponding Author: Lyse A. Norian, Ph.D., Department of Nutrition Sciences, Comprehensive Cancer Center, and Nutrition Obesity, Research Center; University of Alabama at Birmingham, WTI 320C, 1824 6th Ave South, Birmingham, AL 35233, Phone: 205-996-0152. *Authors contributed equally to this work. CM Buchta Current Address: Carter Immunology Center, University of Virginia, Charlottesville, VA, USA Disclosure(s): The authors have no conflicts of interest to disclose. Author Contributions: SKB: analyzed data and wrote manuscript; CMB performed experiments, analyzed data, and wrote manuscript; NAP performed experiments and analyzed data; MBF wrote manuscript; DKM performed experiments, analyzed data, and wrote manuscript; JLG performed experiments, analyzed data, and wrote manuscript; LAN performed experiments, analyzed data, and wrote manuscript.
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impaired dendritic cell stimulatory capacity (p < 0.05 versus lean controls). These parameters were unaltered in OB-Res mice versus lean controls. Conclusions—Diet-induced obesity results in alterations in immune and metabolic profiles that are distinct from effects caused by HFD alone. Keywords Diet-induced obesity; obese; obese-resistant; immune function; dendritic cell
Introduction
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Obesity is accompanied by adverse health effects that include cardiovascular disease, diabetes, and increased cancer risk (1, 2). Mounting evidence suggests that obesity is also associated with impaired immune responses to vaccinations, infections, and tumor growth (3–5), although contrary evidence does exist (6). With ~35% of the United States adult population obese (7), a better understanding of the complex immunologic implications of obesity is needed. Models of diet-induced obese animals/diet-induced obesity (DIO), wherein animals are fed a high-fat (HFD) or other obesogenic diet, allow investigators to manipulate caloric intake and diet composition in genetically intact animals. Most HFD models utilize inbred mouse strains, with C57BL/6 being the most common (8, 9). The C57BL/6 DIO mouse model is characterized by insulin resistance, particularly in males, so permits evaluation of obesity in combination with type 2 diabetes (10). In contrast, the BALB/c strain is more resistant to developing both obesity and type 2 diabetes (11, 12). However, use of DIO BALB/c mice may at times be beneficial due to diminished confounding effects of type 2 diabetes.
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Most DIO studies contain one important caveat: control groups are composed of lean mice that are fed different rodent diets. This approach renders it impossible to distinguish between effects of dietary composition on physiological outcomes versus effects of obesity per se. Evaluating these relative contributions is important, as some dietary components can alter biological function and immune responses in the absence of obesity (13, 14).
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Here, we present a refinement of the DIO BALB/c model, based upon identification of a subset of genetically intact mice that are highly resistant to becoming obese. As we and others have reported, obesity has complex effects on immune responses, and dendritic cell function in particular (4, 15). Thus, we used this model to examine effects of obesity on immune and metabolic profiles of DIO and Obese-resistant (OB-Res) mice fed the same HFD. Our data illustrate that obesity alters multiple immune parameters beyond effects from HFD alone. Furthermore, we identify OB-Res BALB/c mice as important controls that lend new insight into the complex interplay of diet and obesity.
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Methods Animals and diets
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Female BALB/cAnNCr and C57BL/6NCr mice were purchased (Charles River/NCI, Frederick, MD) at 7–8 weeks of age, maintained on standard chow (Teklad 7013, 18% kcal fat, 3.1 kcal/g) for 1 week then randomly assigned to standard chow or HFD (Research Diets #12492, 60% kcal fat, 5.24 kcal/g). Due to accelerated weight gain, C57BL/6NCr mice were maintained on HFD for 10 weeks to induce obesity, whereas BALB/c mice were maintained on diet for 20 weeks as previously reported (5). DUC18 TCR transgenic mice have been described (16, 17). Mice were housed five to a cage in standard static cages under pathogenfree conditions in 12:12 light:dark cycles at 25°C with ad libitum access to food and water at the University of Iowa Animal Care Facility, which is fully AALAC accredited. All animal procedures were approved by the University of Iowa IACUC. After appropriate time on feed, all mice were weighed, and the mean and standard deviation (s.d.) of lean (standard chow) groups were calculated. Blood glucose measurement C57BL/6NCr and BALB/c mice were maintained on standard chow or HFD as described above. Mice were fasted overnight, blood was obtained from the tail vein, and blood glucose measured via glucometer (Roche Diabetes Care). Cytokine and chemokine evaluation by Multiplex Array
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Serum samples were obtained from lean, OB-Res, and DIO mice and frozen at −80°C until use. Serum cytokine and chemokine concentrations were determined via BioPlex Pro Mouse Diabetes Assay and BioPlex Pro Mouse Cytokine Assay and run on a Bio-Rad BioPlex analyzer (Bio-Rad). Surface staining for flow cytometry Spleens and adipose tissue (pooled gonadal/renal fat pads) were harvested and processed as described (5, 6). Cells were stained with antibodies, then results were acquired using a BD LSR II (BD Biosciences) and analyzed with FlowJo software (Tree Star). CD19-BV510 and Vβ8.3-PE (BD Biosciences). I-Ad-A488, CD8α-A700, CD4-APC, CD11c-APC/Cy7, CD3ε-PE, CD11b-PE/Cy7, CD54-FITC, CD40-PE, H2Kd-FITC, I-Ad-PE, CD11c-biotin, SA-APC/Cy7 and TruStain FcX (BioLegend). CD86-APC (eBioscience). T cell proliferation assay
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Splenic DCs were purified from pooled lean, OB-Res, and DIO mice using CD11c microbeads (Miltenyi Biotec). DCs were pulsed with tumor extracellular signal-regulated kinase (tERK) peptide and naïve CD8+ DUC18 T cell proliferation was evaluated as described (17). Immunohistochemistry White adipose tissues (WAT) were stained with anti-IBA-1 (ionized calcium-binding adapter molecule 1), a defined macrophage marker (18). Tissues were examined by a veterinary
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pathologist using the post-examination masking method (19). To assess incidences of crownlike structures (CLS), three random WAT samples (20x magnification) were evaluated for total area and number of CLS. Values were averaged and reported as number of CLS/mm2. The number of IBA+ cells in the WAT interstitium were enumerated (n=3 random samples, 100x magnification), averaged for each tissue, and reported as macrophage number/mm2. During enumeration, solitary macrophages were recorded as a distinct pool from macrophages within CLS. Energy balance by bomb calorimetry
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Energy balance was evaluated using bomb calorimetry as described (20, 21). Briefly, mice were placed into single-mouse sized metabolic cages (Nalgene/Tecniplast) at room temperature with ad libitum access to food and water. Food intake, water intake, urine output, and fecal output were quantified to measure behavioral changes. Fecal samples were desiccated and caloric content was determined using a semi-micro bomb calorimeter (Parr). Digestive efficiency was determined as the ratio of calories absorbed to calories consumed. Energy efficiency was determined as the change in body mass divided by total calories absorbed. Resting Metabolic Rate (RMR) evaluation by respirometry RMR was determined using continuous respirometry as described (22, 23). Briefly, animals were placed into thermally-controlled chambers maintained at 30°C. Oxygen and carbon dioxide composition of effluent air, and chamber air flow rates (corrected to STP) during bouts of rest were used to calculate rates of oxygen consumption and carbon dioxide production. Using the equation derived from Lusk (24), RMR was calculated.
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Statistical analyses Statistical analyses were accomplished utilizing Prism, Version 6.07 (GraphPad). Gaussian distribution was assessed using D’Agostino-Pearson omnibus normality test. Data were analyzed using two-tailed unpaired Student t-tests with (carrots; ^) or without (asterisks; *) Welch’s correction (threshold of 3 s.d.), as appropriate. Non-parametric analyses were performed using two-tailed Mann-Whitney U tests (pound signs; #). Significance indicated as ^ = * = # = p < 0.05; ^^ = ** = ## = p < 0.01; ^^^ = *** = ### = p < 0.001; ^^^^ = **** = #### = p < 0.0001.
Results Distinguishing DIO from OB-Res mice on HFD
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DIO murine models are widely used to study biological and physiological implications of obesity, but comparisons to lean mice on standard chow introduce problems related to distinguishing effects of obesity from effects of diet. Previously, we noted that within BALB/c strains, individual mice showed remarkable heterogeneity to HFD, with some being highly resistant to obesity (5). Here, we leveraged this divergence to examine metabolic and immune profiles in HFD-fed DIO versus OB-Res mice.
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As expected (25, 26), all C57BL/6NCr mice showed significant weight gain after 10 weeks on HFD (average weight gain versus chow controls = 25.73 gm/ mouse) (Fig. 1A). As per standard convention, these mice are referred to as DIO. In contrast, BALB/c female mice showed less dramatic and more variable weight gain on HFD, even after 20 weeks (average weight gain = 6.86 gm/mouse versus chow controls) (Fig. 1B). As previously, we defined mice with DIO as having a final body weight >3 s.d. above the mean weight of the standard chow cohort (dotted line, Fig. 1B) (5). Using this definition, 50% of BALB/c mice were classified as having DIO, versus 100% of C57BL/6NCr mice (Fig. 1A). Male BALB/c mice followed a similar trend, with 66% of mice on HFD classified as obese (data not shown). BALB/c mice that remained within one s.d. of the lean cohort’s mean weight were designated OB-Res. Onset of the DIO versus OB-Res phenotype was detectable in as few as 4–6 weeks on HFD (Fig. 1C). To determine whether these phenotypic weight differences were due to hierarchical cage status, mice showing an OB-Res or DIO phenotype were separated at 10 weeks post-HFD administration and co-housed with like counterparts (ex: OB-Res with OB-Res) for 12 additional weeks. All mice retained their original adiposity phenotypes (Fig. 1D), suggesting that intra-cage social behaviors do not substantially impact DIO status in this model. DIO mice had significantly larger gonadal and renal pads of WAT that accounted for a greater percentage of total body weight (Fig. 1E), consistent with prior findings (27). DIO mice have increased blood glucose, insulin, and leptin concentrations not seen in OBRes or lean mice
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Increased fasting blood glucose and elevated serum insulin and leptin concentrations are characteristic of obesity and type 2 diabetes. As reported (9, 26), DIO C57BL/6NCr mice exhibited a significant increase in fasting blood glucose compared to lean controls (Fig. 2A). Consistent with prior reports, BALB/c mice on HFD were resistant to hyperglycemia (28) (Fig. 2B). Although the absolute concentrations (mean = 62.8 mg/dL for DIO BALB/c mice) were lower than those observed for DIO C57BL/6NCr mice (mean = 115.6 mg/dL), DIO BALB/c mice did show elevated fasting blood glucose concentrations versus lean controls (Fig. 2B). BALB/c OB-Res mice had blood glucose levels that were equivalent to lean counterparts, despite being on HFD for 20 weeks. In addition, BALB/c DIO mice had increased serum insulin (Fig. 2C) and leptin (Fig. 2D) as compared to OB-Res mice and lean controls (Figs. 2C/2D). Thus, only DIO mice on HFD showed key hallmarks of obesity, whereas OB-Res mice on HFD resembled lean chow-fed controls. OB-Res mice are resistant to weight gain due to increased energy expenditure relative to DIO mice
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Leptin is an adipokine that regulates food intake and satiety; therefore decreased serum leptin in BALB/c OB-Res mice suggested that their phenotype might be due to altered food consumption or digestive efficiency. To investigate this, BALB/c DIO and OB-Res mice were singly housed in metabolic cages and given ad libitum access to HFD. Food intake, as measured in both g/day and kcal/day, was equivalent in DIO and OB-Res mice (Table I). Fecal output was also comparable. Once food is consumed, it must be digested and metabolized into useable energy forms. Therefore, we measured calories absorbed from food by DIO versus OB-Res mice using bomb calorimetry, and found these to be equivalent, Obesity (Silver Spring). Author manuscript; available in PMC 2017 October 01.
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indicating comparable digestive efficiency in OB-Res and DIO mice. However, energy efficiency (the amount of weight gain for any given calorie consumed) was significantly lower in OB-Res mice than in DIO mice (mean 3.65 vs 10.09 mg BW per kcal absorbed, p = 0.006). This means that for each calorie consumed, OB-Res mice used a smaller fraction for weight gain. Thus, the total energy expenditure of OB-Res animals is significantly greater than for DIO mice.
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Extending these data, aerobic resting metabolic rate (RMR) was determined using respirometry at thermoneutrality. DIO mice (n=15, 37.41±0.99 g) and OB-Res mice (n=18, 26.20±0.82 g, P20 weeks, it is unlikely that gut dysbiosis accounts for changes in weight gain. Mice exhibit coprophagia, so co-housed mice typically have similar gut microbiomes. At present, the behavioral, biological and/or metabolic causes for differences in energy expenditure and the subsequent development of DIO versus OB-Res status remain unknown. Future studies to dissect the relative contributions of aerobic versus anaerobic RMR, the gut microbiome, physical activity, and potential stress responses to the OB-Res phenotype are required.
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The effects of obesity on immune function are likely complex and highly dependent upon diet composition. Reports have shown that immune function can be impacted by physical activity (35, 36), and stress hormones (i.e., glucocorticoids) (37); therefore, it is possible that immune alterations of OB-Res versus DIO hosts could also be attributed to increased physical activity and/or stress responses. We previously demonstrated that immune responses to renal tumors are impaired in DIO BALB/c, as fewer effector CD8+ T cells infiltrate tumor-bearing kidneys (5). Others have reported impaired lymphatic drainage and
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DC migration to lymph nodes (38), and heightened/pathogenic CD8+ T cells in the lung after influenza challenge (39) in DIO versus chow-fed lean C57BL/6 mice, and alterations in lung metabolomic signatures in genetically modified obese versus wild type mice on chow diet (40). In contrast, Khan et al. found no changes in memory CD8+ T cell responses in DIO C57BL/6, BALB/c, or outbred Swiss Webster mice following viral challenge with either LCMV or influenza (6). In each of these prior studies, including our own, DIO mice on HFD were compared to lean mice on standard chow. The use of OB-Res mice, as described in our current study, will add an important new tool to future studies on DIO, as inclusion of this group will permit identification of immunological changes caused by dietary composition, versus outright obesity.
Conclusions Author Manuscript
Our current study provides two main advances to our understanding of the biological implications of obesity: 1) we refined the BALB/c model of DIO so as to study OB-Res animals on the same HFD, and 2) we illustrate that obesity alters multiple parameters of immune function even when diet is controlled. Moving forward, highly obese-resistant BALB/c mice could be utilized to further delineate the immunological and broader biological consequences of obesity versus diet.
Acknowledgments Funding: NIH grant # R01CA181088 to LAN, NIH grant #HL084207 and American Diabetes Association grant #1-14-BS-079 to JLB, NIH/NCI CPCTP R25 grant #CA47888 to SKB. The authors would like to thank Drs. Aliasger Salem and David Lubaroff for critical input to this project.
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Abbreviations HFD
high fat diet
DIO
diet-induced obesity or diet-induced obese
OB-res
obese-resistant
DC
dendritic cell
WAT
white adipose tissue
References Author Manuscript
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What is already known about this subject? 1.
Murine models of high fat diet-induced obesity have been used to describe changes in immune function with obesity onset.
2.
Controls for diet-induced obesity models typically include mice on standard chow.
3.
Changes in diet composition in the absence of obesity can alter immune function; thus prior conclusions based upon comparison of high fat diet-induced obese mice to lean mice on standard chow may be erroneously describing effects of diet rather than obesity.
What does this study add?
Author Manuscript
1.
We provide a refinement to diet-induced obesity models, via characterization of a subset of obese-resistant mice on high fat diet.
2.
When diet is controlled, obesity causes multiple systemic alterations in metabolic proteins and cytokines.
3.
When diet is controlled, obesity causes multiple changes in cellular immune cell composition and function.
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Figure 1.
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Individual examination of age-matched BALB/c mice on high fat diet identifies unique populations of DIO and OB-Res mice. (A) Body weight (g) of C57BL/6NCr mice was calculated at the end of 10 weeks for mice fed either standard chow or HFD. Dots represent individual mice. (B) Body weights of BALB/c mice at the end of 20 weeks for mice fed either standard chow or HFD. DIO mice were defined as having body weights > lean cohort mean + 3 standard deviations. OB-Res mice were defined as having body weights within 1 standard deviation of the lean cohort mean. (C) Stratification of individual BALB/c mouse body weights over the course of 20 weeks on standard or high fat diet. (D) Body weight (g) of BALB/c mice at 10 weeks of HFD administration (pre-separation) and at 22 weeks of HFD administration (post-separation). After 10 weeks on diet, mice were removed from original cages and co-housed with similar adiposity phenotypes (ex: OB-Res with OB-Res) for 12 additional weeks. Statistical significance was determined using a two-tailed MannWhitney U test (#p < 0.05, ##p< 0.01). (E) Gonadal and renal fat pads were removed from lean, OB-Res, and DIO mice, weighed, and the WAT weight was calculated as a percentage of total body weight. Statistical significance was determined using a two-tailed unpaired student t test (****p < 0.0001).
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Author Manuscript Author Manuscript Figure 2.
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DIO mice have hallmarks of obesity not seen in OB-Res mice on HFD. (A) C57BL/6NCr mice were maintained on feed for 10 weeks. Mice were then fasted overnight and serum blood glucose concentration (mg/dL) was calculated. Statistical significance was determined using a two-tailed unpaired student t test (****p < 0.0001). (B) BALB/c mice were maintained on feed for 20 weeks. Mice were then fasted overnight and serum blood glucose concentration (mg/dL) was calculated. Statistical significance was determined using a twotailed Mann-Whitney U test (##p < 0.01). (C) BALB/c serum was analyzed to obtain concentrations of insulin. Statistical significance was determined using a two-tailed unpaired student t test with Welch’s correction (^^p < 0.01). (D) BALB/c serum was analyzed to obtain concentrations of leptin. Statistical significance was determined using a two-tailed unpaired student t test with Welch’s correction (^^^p < 0.001).
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Figure 3.
Serum analyses reveal unique obesogenic and proinflammatory differences between DIO, OB-Res, and lean BALB/c mice. Serum was collected from BALB/c mice maintained on standard chow or HFD for 20 weeks and analyzed via Multiplex array where n= 8 lean mice, 13 OB-Res, and 10 DIO mice. Statistical significance was determined using either a twotailed unpaired student t test (*p < 0.05), or a two-tailed Mann-Whitney U test (#p < 0.05, ##p < 0.01, ###p < 0.001), as appropriate.
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Figure 4.
No differences in leukocytic infiltration into WAT in DIO, OB-Res or lean mice. BALB/c mice (n= 9–10 mice per group) were maintained on standard chow or high fat diet for 20 weeks and gonadal and renal fat pads were collected. The stromal vascular fraction was then isolated and percentages of CD4+ and CD8+ T cells, CD19+ B cells, CD11c+ DC (CD11c+/I-Ad+/ CD11bint-low), and CD11b+ (CD11b+/I-Ad+/CD11clow) macrophages were determined by flow cytometry.
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Author Manuscript Author Manuscript Figure 5.
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Macrophage localization within CLS in WAT distinguishes DIO mice from OB-Res and lean counterparts. (A) BALB/c mice were maintained on standard chow or high fat diet for 20 weeks and gonadal and renal fat pads were collected. Immunohistochemical staining of IBA1+ macrophages in WAT was performed. Black arrows indicate single IBA1+ macrophages, red arrow indicates IBA1+ macrophages within a representative crown-like structure (CLS). (B) Enumeration of total IBA1+ macrophages and CLS in WAT from n= 5 each lean, OB-Res and DIO mice. Statistical significance was determined using a two-tailed Mann-Whitney U test (#p < 0.05, ##p < 0.01).
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Figure 6.
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DIO mice have immune alterations not present in OB-Res or lean mice. (A) BALB/c mice (n= 10 per group) were maintained on standard chow or HFD for 20 weeks prior to spleen harvest. Percentages of CD4+ and CD8+ T cells, CD19+ B cells, CD11b+ (CD11b+/I-Ad+/ CD11clow) macrophages, and CD11chigh/I-Ad+ dendritic cells were determined by flow cytometric analysis. Statistical significance was determined using a two-tailed unpaired student t test (*p < 0.05, **p < 0.01) or a two-tailed Mann-Whitney U test (##p < 0.01), as appropriate. (B) Serum was collected from indicated mice (n= 8 lean mice, 13 OB-Res, and 10 DIO mice) and analyzed via Multiplex for concentrations of granulocyte macrophage colony-stimulating factor (GM-CSF) (pg/mL). Statistical significance was determined using a two-tailed Mann Whitney U test (##p < 0.01).
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Author Manuscript Author Manuscript Author Manuscript Figure 7.
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Obesity, but not HFD administration, is associated with impaired DC stimulatory capacity. (A) Expression of surface markers CD86, CD40, CD54, MHC Class I (H-2Kd) and MHC Class II (I-Ad) on gated splenic CD11chigh DC were analyzed by flow cytometry. Statistical significance was determined using a two-tailed Mann-Whitney U test (#p < 0.05). (B) CD11chigh / MHC II+/ Gr-1neg splenic DC were sort-purified from indicated mice, pulsed with exogenous peptide, then used to stimulate naive antigen-specific CD8+ T cells. T cell proliferation was measured by incorporation of 3H-thymidine (counts per minute) at 72 hours. Data are pooled from 2–5 individual experiments, with 2–3 mice pooled per
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experiment to generate sufficient numbers of DC. Statistical significance was determined by using a two-tailed Mann-Whitney U test (#p < 0.05).
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Table I
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Energy balance evaluation of DIO and OB-Res mice reveals differences in energy efficiency. BALB/c mice were maintained on high fat diet for 20 weeks. Mice (n= 13 DIO and 12 OB-Res) were then singly housed in metabolic cages for 3–5 days and given ad libitum access to high fat diet chow. Caloric densities of food and fecal samples were determined by bomb calorimetry. DIO
OB-Res
t-test p=
38.62
25.35
0.001
2.04
1.81
2.44
2.45
0.09
0.39
14.15
14.18
0.5
2.27
0.42
0.42
0.03
0.06
1.80
1.85
0.17
0.23
12.34
12.25
0.57
2.06
87.30
85.90
1.43
2.09
10.09
3.65
1.01
1.4
Body weight (g)
sem Food intake (g/day)
sem
0.990
Food intake (kcal/day)
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sem
0.990
Fecal output (g/day)
sem
0.930
Fecal output (kcal/day)
sem
0.873
Calories absorbed (kcal/day)
sem
0.967
Digestive Efficiency (% consumed)
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sem
0.595
Energy efficiency (D mg BW/kcal absorbed)
sem
0.006
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