Normal Weight Mice Research Articles

Diet-Induced Obesity Affects Muscle Regeneration After Murine Blunt Muscle Trauma-A Broad Spectrum Analysis.

Injury to skeletal muscle affects millions of people worldwide. The underlying regenerative process however, is a very complex mechanism, time-wise highly coordinated, and subdivided in an initial inflammatory, a regenerative and a remodeling phase. Muscle regeneration can be impaired by several factors, among them diet-induced obesity (DIO). In order to evaluate if obesity negatively affects healing processes after trauma, we utilized a blunt injury approach to damage the extensor iliotibialis anticus muscle on the left hind limb of obese and normal weight C57BL/6J without showing any significant differences in force input between normal weight and obese mice. Magnetic resonance imaging (MRI) of the injury and regeneration process revealed edema formation and hemorrhage exudate in muscle tissue of normal weight and obese mice. In addition, morphological analysis of physiological changes revealed tissue necrosis, immune cell infiltration, extracellular matrix (ECM) remodeling, and fibrosis formation in the damaged muscle tissue. Regeneration was delayed in muscles of obese mice, with a higher incidence of fibrosis formation due to hampered expression levels of genes involved in ECM organization. Furthermore, a detailed molecular fingerprint in different stages of muscle regeneration underlined a delay or even lack of a regenerative response to injury in obese mice. A time-lapse heatmap determined 81 differentially expressed genes (DEG) with at least three hits in our model at all-time points, suggesting key candidates with a high impact on muscle regeneration. Pathway analysis of the DEG revealed five pathways with a high confidence level: myeloid leukocyte migration, regulation of tumor necrosis factor production, CD4-positive, alpha-beta T cell differentiation, ECM organization, and toll-like receptor (TLR) signaling. Moreover, changes in complement-, Wnt-, and satellite cell-related genes were found to be impaired in obese animals after trauma. Furthermore, histological satellite cell evaluation showed lower satellite cell numbers in the obese model upon injury. Ankrd1, C3ar1, Ccl8, Mpeg1, and Myog expression levels were also verified by qPCR. In summary, increased fibrosis formation, the reduction of Pax7+ satellite cells as well as specific changes in gene expression and signaling pathways could explain the delay of tissue regeneration in obese mice post trauma.

Feasibility Evaluation of Myocardial Cannabinoid Type 1 Receptor Imaging in Obesity: A Translational Approach

ObjectivesThe aim of this study was to evaluate the feasibility of targeted imaging of myocardial cannabinoid type 1 receptor (CB1-R) and its potential up-regulation in obese mice with translation to humans using [11C]-OMAR and positron emission tomography (PET)/computed tomography (CT). BackgroundActivation of myocardial CB1-R by endocannabinoids has been implicated in cardiac dysfunction in diabetic mice. Obesity may lead to an up-regulation of myocardial CB1-R, potentially providing a mechanistic link between obesity and the initiation and/or progression of cardiomyopathy. MethodsBinding specificity of [11C]-OMAR to CB1-R was investigated by blocking studies with rimonabant in mice. The heart was harvested from each mouse, and its radioactivity was determined by γ-counter. Furthermore, [11C]-OMAR dynamic micro-PET/CT was carried out in obese and normal-weight mice. Ex vivo validation was performed by droplet digital polymerase chain reaction (absolute quantification) and RNAscope Technology (an in situ ribonucleic acid analysis platform). Subsequently, myocardial CB1-R expression was probed noninvasively with intravenous injection of CB1-R ligand [11C]-OMAR and PET/CT in humans with advanced obesity and normal-weight human control subjects, respectively. ResultsRimonabant significantly blocked OMAR uptake in the heart muscle compared with vehicle, signifying specific binding of OMAR to the CB1-R in the myocardium. The myocardial OMAR retention quantified by micro-PET/CT in mice was significantly higher in obese compared with normal-weight mice. Absolute quantification of CB1-R gene expression with droplet digital polymerase chain reaction and in situ hybridization confirmed CB1-R up-regulation in all major myocardial cell types (e.g., cardiomyocytes, endothelium, vascular smooth muscle cells, and fibroblasts) of obese mice. Obese mice also had elevated myocardial levels of endocannabinoids anandamide and 2-arachidonoylglycerol compared with lean mice. Translation to humans revealed higher myocardial OMAR retention in advanced obesity compared with normal-weight subjects. ConclusionsNoninvasive imaging of cardiac CB1-R expression in obesity is feasible applying [11C]-OMAR and PET/CT. These results may provide a rationale for further clinical testing of CB1-R-targeted molecular imaging in cardiometabolic diseases.

Comparison of Fatty Acid and Gene Profiles in Skeletal Muscle in Normal and Obese C57BL/6J Mice before and after Blunt Muscle Injury.

Injury and obesity are two major health burdens affecting millions of people worldwide. Obesity is recognized as a state of chronic inflammation accompanied by various co-morbidities like T2D or cardiovascular diseases. There is increasing evidence that obesity impairs muscle regeneration, which is mainly due to chronic inflammation and to excessive accumulation of lipids in adipose and non-adipose tissue. To compare fatty acid profiles and changes in gene expression at different time points after muscle injury, we used an established drop tower-based model with a defined force input to damage the extensor iliotibialis anticus on the left hind limb of female C57BL/6J mice of normal weight and obese mice. Although most changes in fatty acid content in muscle tissue are diet related, levels of eicosaenoic (normal weight) and DHG-linolenic acid (obese) in the phospholipid and docosahexaenoic acid (normal weight) in the triglyceride fraction are altered after injury. Furthermore, changes in gene transcription were detected in 3829 genes in muscles of normal weight mice, whereas only 287 genes were altered in muscles of obese mice after trauma. Alterations were found within several pathways, among them notch-signaling, insulin-signaling, sonic hedgehog-signaling, apoptosis related pathways, fat metabolism related cholesterol homeostasis, fatty acid biosynthetic process, fatty acid elongation, and acyl-CoA metabolic process. We could show that genes involved in fat metabolism are affected 3 days after trauma induction mostly in normal weight but not in obese mice. The strongest effects were observed in normal weight mice for Alox5ap, the activating protein for leukotriene synthesis, and Apobec1, an enzyme substantial for LDL synthesis. In summary, we show that obesity changes the fat content of skeletal muscle and generally shows a negative impact upon blunt muscle injury on various cellular processes, among them fatty acid related metabolism, notch-, insulin-, sonic hedgehog-signaling, and apoptosis.

Hypothesis: Induction of biomarkers for detection of colonic neoplasms.

The signing of the National Cancer Act of 1971 by President Nixon marked the beginning of our war on cancer. More than 45 years later, the war is still going steady, with the enemy being almost as strong as in 1971. Furthermore, the increasing rates of obesity not only among adults, but among children and adolescents, are the likely cause for the 30-year trend of colon cancer (CC) becoming a disease of the younger population in the U.S. These trends, however, have not spurred the development of novel screening approaches for CC. Considering the need for a sensitive and non-invasive detection of early stage neoplastic lesions in the colon, we propose the development of a test based on a novel concept - the concept of induced biomarkers. The proposal is based upon our findings that the food additives propolis and gamma-cyclodextrin (gCD) (a) decrease the neoplastic burden in normal weight and obese ApcMin mice, a model of early stage intestinal neoplasia, and (b) elicit significant changes in the serum proteome in ApcMin mice. We posit that gCD and propolis induce the release of neoplasm-associated biomarkers in systemic circulation (e.g., metabolites, neoplastic, apoptotic, and immune response proteins), and these markers could be used to detect early stage intestinal neoplasms. Additional dietary bioactives may also elicit a complement of induced markers. The hypothesis could be ascertained by utilizing a mouse model, the Apc+/1638Nmice, as well as through human subject studies that integrate proteomics and metabolomics analyses. The concept of detecting inducible markers of colonic neoplasms is novel, and is substantiated by the significant physiological effects of gCD and propolis on neoplastic colonic cells in culture and on early neoplastic development in ApcMinmice. The long-term objective is to develop a minimally invasive method that detects early stage neoplastic development in the human colon.

TLR4-NF-κB Signal Involved in Depressive-Like Behaviors and Cytokine Expression of Frontal Cortex and Hippocampus in Stressed C57BL/6 and ob/ob Mice.

Studies found that elevated levels of cytokines such as interleukin- (IL-) 1β, IL-6, and tumor necrosis factor-α (TNF-α) are closely associated with the pathogenesis of depression. Obesity providing a low-grade inflammation state was proposed to be implicated in susceptibility to depression in obesity. However, the alterations of cytokines and the TLR4-NF-κB signal in the brain of normal-weight and obese mice under stress have not been fully elucidated. This study used chronic unpredictable mild stress (CUMS) to induce a depressive-like behavior in an animal model and examine depressive-like behaviors, memory changes, and serum corticosterone levels, as well as the expressions of cytokines and NF-κB in the frontal cortex and hippocampus. We aimed to observe the role of neuroinflammation in susceptibility to depression in obesity under CUMS. In addition, we investigated the protective effect of inhibiting the TLR4-NF-κB signal. Our results demonstrated that CUMS induced depressive-like behavior and spatial memory damage, higher level of serum corticosterone, and overexpression of cytokines and NF-κB in the frontal cortex and hippocampus in both C57BL/6 and ob/ob mice. ob/ob mice displayed serious behavioral disorder and higher levels of IL-1β, IL-6, TNF-α, and NF-κB. Our results concluded that a hyperactive TLR4-NF-κB signal and higher level of cytokines are involved in susceptibility to depression in stressed obese mice.

Guanylin and uroguanylin mRNA expression is increased following Roux-en-Y gastric bypass, but guanylins do not play a significant role in body weight regulation and glycemic control

AimTo determine whether intestinal expression of guanylate cyclase activator 2A (GUCA2A) and guanylate cyclase activator 2B (GUCA2B) genes is regulated in obese humans following Roux-en-Y gastric bypass (RYGB), and to evaluate the corresponding guanylin (GN) and uroguanylin (UGN) peptides for potentially contributing to the beneficial metabolic effects of RYGB. MethodsEnteroendocrine cells were harvested peri- and post-RYGB, and GUCA2A/GUCA2B mRNA expression was compared. GN, UGN and their prohormones (proGN, proUGN) were administered subcutaneously in normal-weight mice to evaluate effects on food intake and glucose regulation. The effect of pro-UGN or UGN overexpression, using adeno-associated virus (AAV) vectors, was assessed in diet-induced obese (DIO) mice. Intracerebroventricular administration of GN and UGN was performed in rats for assessment of putative centrally mediated effects on food intake. GN and UGN, as well as their prohormones, were evaluated for effects on glucose-stimulated insulin secretion (GSIS) in rat pancreatic islets and perfused rat pancreas. ResultsGUCA2A and GUCA2B mRNA expression was significantly upregulated in enteroendocrine cells after RYGB. Peripheral administration of guanylins or prohormones did not influence food intake, oral glucose tolerance, and GSIS. Central administration of GN and UGN did not affect food intake in rats. Chronic AVV-mediated overexpression of UGN and proUGN had no effect on body weight or glucose homeostasis in DIO mice. ConclusionGN and UGN, as well as their prohormones, do not seem to play a significant role in body weight regulation and glycemic control, suggesting that guanylin-family peptides do not show promise as targets for the treatment of obesity or diabetes.

Gene expression and epigenetic aberrations in F1-placentas fathered by obese males.

Gene expression and/or epigenetic deregulation may have consequences for sperm and blastocysts, as well as for the placenta, together potentially contributing to problems observed in offspring. We previously demonstrated specific perturbations of fertilization, blastocyst formation, implantation, as well as aberrant glucose metabolism and adiposity in offspring using a mouse model of paternal obesity. The current investigation analyzed gene expression and methylation of specific CpG residues in F1 placentas of pregnancies fathered by obese and normal-weight male mice, using real-time PCR and bisulfite pyrosequencing. Our aim was to determine if paternal obesity deregulated placental gene expression and DNA methylation when compared to normal-weight males. Gene methylation of sperm DNA was analyzed and compared to placentas to address epigenetic transmission. Of the 10 paternally expressed genes (Pegs), 11 genes important for development and transport of nutrients, and the long-terminal repeat Intracisternal A particle (IAP) elements, derived from a member of the class II endogenous retroviral gene family, we observed a significant effect of paternal diet-induced obesity on deregulated expression of Peg3, Peg9, Peg10, and the nutrient transporter gene Slc38a2, and aberrant DNA methylation of the Peg9 promoter in F1 placental tissue. Epigenetic changes in Peg9 were also found in sperm from obese fathers. We therefore propose that paternal obesity renders changes in gene expression and/or methylation throughout the placental genome, which could contribute to the reproductive problems related to fertility and to the metabolic, long-term health impact on offspring.

Diet-Induced Obesity Does Not Alter Tigecycline Treatment Efficacy in Murine Lyme Disease.

Obese individuals more frequently suffer from infections, as a result of increased susceptibility to a number of bacterial pathogens. Furthermore, obesity can alter antibiotic treatment efficacy due to changes in drug pharmacokinetics which can result in under-dosing. However, studies on the treatment of bacterial infections in the context of obesity are scarce. To address this research gap, we assessed efficacy of antibiotic treatment in diet-induced obese mice infected with the Lyme disease pathogen, Borrelia burgdorferi. Diet-induced obese C3H/HeN mice and normal-weight controls were infected with B. burgdorferi, and treated during the acute phase of infection with two doses of tigecycline, adjusted to the weights of diet-induced obese and normal-weight mice. Antibiotic treatment efficacy was assessed 1 month after the treatment by cultivating bacteria from tissues, measuring severity of Lyme carditis, and quantifying bacterial DNA clearance in ten tissues. In addition, B. burgdorferi-specific IgG production was monitored throughout the experiment. Tigecycline treatment was ineffective in reducing B. burgdorferi DNA copies in brain. However, diet-induced obesity did not affect antibiotic-dependent bacterial DNA clearance in any tissues, regardless of the tigecycline dose used for treatment. Production of B. burgdorferi-specific IgGs was delayed and attenuated in mock-treated diet-induced obese mice compared to mock-treated normal-weight animals, but did not differ among experimental groups following antibiotic treatment. No carditis or cultivatable B. burgdorferi were detected in any antibiotic-treated group. In conclusion, obesity was associated with attenuated and delayed humoral immune responses to B. burgdorferi, but did not affect efficacy of antibiotic treatment.

Susceptibility of Overweight Mice to Liver Injury as a Result of the ZnO Nanoparticle-Enhanced Liver Deposition of Pb2.

The prevalence of the applications of nanomaterials in consumer products and water treatment facilities increases the chance that humans will be exposed to both nanoparticles and environmental pollutants such as heavy metals. Co-exposure to nanoparticles and heavy metals may adversely affect human health, especially in susceptible populations such as overweight subjects. To evaluate the impact of such co-exposures, we orally administered zinc oxide nanoparticles (ZNPs; 14 or 58 nm) and/or Pb(Ac)2 at tolerable doses to both healthy overweight and healthy normal weight mice. The ZNPs enhanced the deposition of Pb in all major organs in the overweight mice compared with that in the normal mice. As a result, higher levels of hepatic reactive oxygen species, pro-inflammatory cytokines, and liver injury were observed in the overweight mice but not in the normal weight mice. Our findings underscore a potentially enhanced risk of nanoparticle/heavy metal co-exposure in the susceptible overweight population.

Hypoxic Exercise Training Promotes apelin/APJ Expression in Skeletal Muscles of High Fat Diet-Induced Obese Mice.

Apelin, an endogenous ligand of the G-protein-coupled receptor APJ, is a novel myokine and may play a key role in regulating energy metabolism. The purpose of the present study was to investigate the effects of hypoxic exposure, exercise, and hypoxic exercise training on the expression of apelin and APJ in skeletal muscle of obese mice. Sixty two-months old C57BL/6J mice were randomly divided into two groups: Ten in normal diet group (N) and 50 in the high fat diet (HFD) groups. After two months of feeding, the HFD mice, whose body weight was 20% higher than the average weight of the N group, were selected as obese mice and further allocated into four groups: Control (C), Exercise (E), Hypoxia (H), and Exercise plus Hypoxia (E+H), at 8-9 mice/group. Besides body weight, measured variables in skeletal muscle were protein/mRNA levels of apelin/APJ, AMPKα-Thr172 phosphorylation, hypoxia inducible factor-1α (HIF-1α), mRNA levels of peroxisome proliferator-activated receptor α (PPARα), estrogen-related receptor (ERRα), and nuclear respiratory factor 1 (NRF1). Obese mice had significantly lower mRNA and protein expressions of apelin/ APJ in skeletal muscles than the normal body weight mice. After four weeks of interventions, hypoxic exercise training decreased body weight and increased mRNA and protein expressions of apelin and APJ, mRNA expression of ERRα, and protein expression of HIF-1α. These results indicate that changes of body weight may be associated with the levels of apelin/APJ expressions in skeletal muscle.

Abstract 4090: Metabolic and microenvironment changes in the mammary of calorie restricted, normal weight, and obese mice throughout the lifespan

Abstract Background: Breast tissue remodeling occurs with age and is marked by a progressive increase in stromal tissue. Advancing age is also associated with increased senescence among stromal cells that promote a protumorigenic microenvironment by adopting a senescence-associated secretory phenotype (SASP). In previous studies, calorie restriction has been effective in cancer prevention through inhibiting cellular senescence and SASP. However, obesity has shown to be cancer promoting with inconsistent findings on its role in cellular senescence. Purpose: We tested the hypothesis that calorie restriction is protective to the protumorigenic, age-related changes that occur within the mammary microenvironment while diet-induced obesity accelerates these changes. Methods: Six week old female mice were randomized to receive either a low-fat control regimen (10 kcal% fat), a 30% calorie restricted (CR) regimen relative to control, or a high-fat diet induced obesity (DIO, 60 kcal% fat) regimen, resulting in control, CR, and DIO mice respectively. A subset of mice was sacrificed at 1, 3, 5, 12 and 20 months following diet initiation with mammary fat pads (MFP) harvested and serum stored for further analysis. H&E staining was analyzed using a digital algorithm to quantify the composition of adipose, epithelia and stoma in the MFP tissue. RNA was extracted from MFP sections and rt-PCR was preformed to analyze gene expression of CDKN2a, the gene encoding p16(INK4a) a well known marker for senescence. Results: CR significantly decreased body weight and decreased serum IGF-1, insulin, leptin and estradiol and significantly increased adiponectin relative to control and DIO mice at all time points. MFP composition remained relatively stable from 1 and 12-month time points. However, there was an apparent non-significant trend from 12 and 20-month time points, with increasing stromal tissue and a decrease in adipose tissue, which was most drastic in CR mice. Gene expression analysis showed an increase in CDKN2a expression in control mice relative to CR mice at 5 and 20-month time points. Additionally, control and CR mice demonstrated an increase in CDKN2a expression with age from 5 months to 20-months. Conclusions: Stromal composition of murine MFP displayed an age-related increase that is consistent with findings in human breast tissue. Despite this uniform increase, we see a decrease in the age-related acquisition of a senescence phenotype in CR mice relative to control supporting the notion that CR is protective to this protumorigenic change that occurs with advancing age. Ongoing analysis of gene expression in DIO mice will determine the role of obesity in MFP cellular senescence and confirmation of senescent phenotypes via β-galactosidase staining and analysis of SASP associated gene expression. Citation Format: Laura A. Smith, Emily L. Rossi, Laura W. Bowers, Emma H. Allot, Sarah Dunlap, Liza Makowski, Bentley Midkiff, Melissa Troester, Stephen D. Hursting. Metabolic and microenvironment changes in the mammary of calorie restricted, normal weight, and obese mice throughout the lifespan. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4090.

Effects of propolis and gamma-cyclodextrin on intestinal neoplasia in normal weight and obese mice.

Obesity is associated with colorectal cancer (CRC). This effect might be attributed to adipokine‐supported signaling. We have established that propolis suppresses survival signaling in CRC cells in vitro; therefore, we ascertained the ability of a propolis supplement to modulate intestinal neoplastic development in C57BL/6J‐ApcMin/+/J mice in the lean and obese state. To induce obesity, mice were fed with a Western diet containing 40% fat. Since the propolis supplement includes gamma‐cyclodextrin, the interventions included diets supplemented with or without gamma‐cyclodextrin. The animals were administered the following diets: (1) control diet, (2) control diet/gamma‐cyclodextrin, (3) control diet/propolis, (4) Western diet, (5) Western diet/gamma‐cyclodextrin, and (6) Western diet/propolis. Western diet, resulting in obesity, accelerated neoplastic progression, as evidenced by the larger size and higher grade dysplasia of the neoplasms. In the context of normal weight, gamma‐cyclodextrin and propolis affected neoplastic progression, as determined by the size of the lesions and their grade of dysplasia. A statistically significant decrease in the number of adenomas was detected in mice fed a control diet with the propolis supplement (61.8 ± 10.6 vs. 35.3 ± 7.6, P = 0.008). Although there was no significant difference in the polyp numbers between the six groups, the mice with the lowest number and size of adenomas were those fed a Western diet with gamma‐cyclodextrin. This unexpected outcome might be explained by the increased levels of apoptosis detected in the intestinal tissues of these obese mice. We posit that butyrate derived from the metabolism of gamma‐cyclodextrin may contribute to the decreased neoplastic burden in the context of obesity; however, future studies are required to address this possibility.

A novel thermoregulatory role for PDE10A in mouse and human adipocytes.

Phosphodiesterase type 10A (PDE10A) is highly enriched in striatum and is under evaluation as a drug target for several psychiatric/neurodegenerative diseases. Preclinical studies implicate PDE10A in the regulation of energy homeostasis, but the mechanisms remain unclear. By utilizing small‐animal PET/MRI and the novel radioligand [18F]‐AQ28A, we found marked levels of PDE10A in interscapular brown adipose tissue (BAT) of mice. Pharmacological inactivation of PDE10A with the highly selective inhibitor MP‐10 recruited BAT and potentiated thermogenesis in vivo. In diet‐induced obese mice, chronic administration of MP‐10 caused weight loss associated with increased energy expenditure, browning of white adipose tissue, and improved insulin sensitivity. Analysis of human PET data further revealed marked levels of PDE10A in the supraclavicular region where brown/beige adipocytes are clustered in adults. Finally, the inhibition of PDE10A with MP‐10 stimulated thermogenic gene expression in human brown adipocytes and induced browning of human white adipocytes. Collectively, our findings highlight a novel thermoregulatory role for PDE10A in mouse and human adipocytes and promote PDE10A inhibitors as promising candidates for the treatment of obesity and diabetes.

Buprenorphine‐Induced Respiratory Depression Varies as a Function of Leptin‐Related Genotype in Normal Weight and Obese Mice

Most obesity is polygenic with comorbidities that include increased reports of pain (J Pain Res 8: 399, 2015) and opiate‐induced respiratory depression (Anesthesiology 122: 659, 2015). Adipocytes produce leptin and obese, leptin‐deficient mice exhibit state‐dependent respiratory depression (Resp Physiol 119: 173, 2000) as well as altered nociception (Neuroscience 275: 531, 2014). Here we report pre‐clinical studies testing the hypothesis that buprenorphine‐induced respiratory depression is greater in mice lacking leptin or leptin receptors than in normal mice. These studies have quantified buprenorphine‐induced changes in breathing in C57BL/6J (B6) mice that have normal levels of leptin; B6.BKS(D)‐Leprdb/J (db/db) mice that lack leptin receptors, are obese, and diabetic; and B6.Cg‐Lepob/J (ob/ob) mice that lack leptin and are obese. Mice (10 males and 10 females of each genotype) were purchased from The Jackson Laboratory and studied in accordance with the FASEB Statement of Principles for animal research. Mice received intraperitoneal injections (0.3 mL) of saline (control) or buprenorphine at concentrations of 0.1, 0.3, 1.0, 3.0, or 10 mg/kg. After each injection, breathing was measured for one hour using whole body plethysmography (DSI, St. Paul, MN, USA). At all doses, minute ventilation (mL/min/g body weight) of B6 mice was about three‐fold greater than in obese db/db and ob/ob mice. Two‐way ANOVA revealed that minute ventilation varied significantly by dose for male (F=3.47; df=5,135; p=0.0056) and female (F=4.18; df=5,135; p=0.0015) mice, by genotype for males (F=159; df=2,27; p<0.0001) and females (F=332; df=2,27; p<0.0001), and the dose by genotype interaction was statistically significant for both male and female mice. Respiratory duty cycle (duration of inspiration/total respiratory cycle time), the proportion of the respiratory cycle spent in the active inspiratory phase, at all doses was lower in B6 than in ob/ob and db/db mice. Duty cycle also varied by dose in males (F=6.93; df‐5,135; p<0.001) and females (F=23.75; df=5,135; p<0.0001), by genotype in males (F=48; df=2,27; p<0.0001) and females (F=19.76; df=2,27; p<0.0001), and the interaction term was statistically significant. Inspiratory flow (tidal volume/duration of inspiration) in mL/s, an index of ventilatory drive, was lower at every buprenorphine dose in both sexes of ob/ob and db/db mice than in the B6 mice. Inspiratory flow varied by dose for males (F=19.72;df‐5,135; p<0.0001) and females (F=29.9;df=5,135;p<0.0001), by genotype for males (F=29.97;df=2,27; p<0.0001) and females (F=8.79;df=2,27; p=0.001), and the dose by genotype interaction was statistically significant. Taken together, these data support the interpretation that the desirable ceiling effect of buprenorphine for respiratory depression is diminished in obese mice with dysfunctional leptin. Ongoing studies include mice with phenotypes of diet‐induced obesity and normal leptin.Support or Funding InformationNIH Grant HL065272 and Departments of Anesthesiology and Psychology

What Do Obesity, Memory Problems, Stress, and Depression Have in Common?

Epigenetic links have been posited for the apparent relationships among obesity, cognitive decline, stress, and depression. Two studies in mice examine these relationships. In mice with high-fat diet-induced obesity (DIO) and normal-weight mice, Heyward and colleagues elucidated the mechanism of the well-established association of obesity and memory impairment. On memory tests that required little exertion (because DIO mice are less active), DIO mice had preserved object recognition, but hippocampus-dependent spatial memory was impaired and long-term potentiation was reduced. Global …

Abstract P2-05-22: Pre-clinical findings on obesity reversal and breast cancer progression: Targeting persistent inflammation

Abstract Background Obesity is an established risk and progression factor for several intrinsic subtypes of breast cancer, including basal-like breast cancer (BLBC). Increased local and systemic levels of pro-inflammatory mediators, which typically accompany obesity, can independently influence tumor growth. Currently, it is unclear whether weight loss can reverse the enhancing effects of obesity on breast tumorigenesis. We hypothesize that chronic obesity results in epigenetic reprogramming, which may mediate residual inflammation, and lead to persistent mammary tumor growth despite moderate weight loss. Methods Female C57BL/6 mice (n=51) were administered a control (10% kcal from fat) or diet-induced obesity (DIO; 60% kcal from fat) regimen. After 17 weeks, DIO mice either continued on DIO diet or were switched to control diet to stimulate gradual weight loss, subsequently designated as Formerly Obese (FOb). Mice were orthotopically injected with Wnt-1 mammary tumor cells (a model of BLBC) at week 25, and monitored for an additional 9 weeks, then killed and their tumors excised, measured and stored for subsequent analysis. In an ongoing tumor study, we will randomize mice to the control or DIO regimen, switching half the DIO mice to the control diet after 15 weeks, resulting in normal weight, obese, and FOb mice. Within each of these groups, the mice will be further randomized to +/- supplementation with Sulindac, a non-steroidal anti-inflammatory drug (NSAID), starting at the time of the diet switch. After 10 weeks of +/- Sulindac supplementation, mice will receive orthotopic injections of a mesenchymal derivative of an MMTV-Wnt-1 transgenic tumor, the M-Wnt cell line, and will continue on diet and treatment until euthanization. Results In our initial study, body weight, adiposity, and serum levels of leptin and insulin were similar in FOb and control mice, but serum levels of IL-6 were similar in FOb and DIO mice, and significantly higher than controls. Moreover, tumor burden, the mammary gland expression of key inflammatory genes, and the prevalence of hypermethylated inflammation-related genes in DNA from mammary tissue were comparable in DIO and FOb mice and significantly higher than in control mice, and there was high concordance with DNA methylation profiles in breast DNA from obese versus normoweight women participating in the UNC Normal Breast Study. Conclusions Our preclinical findings suggest that modest weight loss may not be sufficient to reverse the effects of chronic obesity on epigenetic reprogramming and inflammatory signals that are associated with obesity-related mammary tumor progression. Moreover, we have identified several genes with concordant obesity-related hypermethylation in humans and mice; which were unchanged in FOb mice. Thus combining weight loss regimens with epigenetic or inflammatory modulators may be an effective strategy for breaking the obesity-breast cancer link. We are currently assessing whether combining moderate weight loss with anti-inflammatory interventions (Sulindac or omega-3 fatty acids), or reprograming metabolism with a bariatric surgical intervention, is more effective than moderate weight loss alone at offsetting the persistent enhancing effects of chronic obesity on BLBC. Citation Format: Rossi EL, Bowers LW, Khatib SA, Doerstling SS, Hursting SD. Pre-clinical findings on obesity reversal and breast cancer progression: Targeting persistent inflammation. [abstract]. In: Proceedings of the Thirty-Eighth Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2015 Dec 8-12; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(4 Suppl):Abstract nr P2-05-22.

Reversing the Effects of Maternal Obesity

Obese women tend to give birth to overweight offspring who have a predisposition to metabolic disease and obesity. This effect has now been reversed in mice by infusing pregnant mothers with adiponectin, a hormone that helps manage metabolism [1]. The effects of maternal obesity on the fetus seem to be mediated mainly through the placenta, and include increased transport of nutrients across the placenta as a result of elevated insulin signaling, leading to excess fetal growth. There have been hints that adiponectin, which is produced by fat cells, might counteract some of these effects—for instance, circulating levels of adiponectin are low in mouse and human mothers who are obese or have gestational diabetes. Irving Aye et al. now report that restoring normal levels of adiponectin reverses the effects of maternal obesity in mice. Consistent with previous studies, the researchers found that overweight pregnant mice had a host of problems: elevated fasting serum insulin and leptin, elevated placental insulin signaling, activation of regulators such as mTORC1 that respond to insulin, increased expression of glucose and amino acid transporters in the placenta, and increased flux of these nutrients across the placenta. Their offspring were about 29 percent heavier than offspring from normal-weight mice and also had high blood sugar. Treatment with adiponectin reversed all of these effects, and resulted in offspring of normal weight with normal glucose levels. The researchers propose that adiponectin operates by restoring normal placental insulin signaling and fetal nutrient supply, leading to normal-weight offspring. These findings have the potential to be translated to humans, but one caveat is that that adiponectin treatment in mice can result in underweight offspring if given to normal-weight mothers. An intervention is urgently needed, as about two-thirds of mothers in the United States enter pregnancy being overweight or obese—contributing to a spiraling population-wide obesity epidemic. Future studies testing the value of this potential therapy may use an oral form of adiponectin, which recently has been developed.

Triglyceride sensing in the reward circuitry: A new insight in feeding behaviour regulation.

In both developed and emerging countries, sedentary life style and over exposition to high energy dense foods has led to a thermodynamic imbalance and consequently obesity. Obesity often involves a behavioural component in which, similar to drugs abuse, compulsive consumption of palatable food rich in lipids and sugar drives energy intake far beyond metabolic demands. The hypothalamus is one of the primary integration sites of circulating energy-related signals like leptin or ghrelin and is therefore considered as one of the main central regulators of energy balance. However, food intake is also modulated by sensory inputs, such as tastes and odours, as well as by affective or emotional states. The mesolimbic pathway is well established as a key actor of the rewarding aspect of feeding. Particularly, the hedonic and motivational aspects of food are closely tied to the release of the neurotransmitter dopamine (DA) in striatal structure such as the Nucleus Accumbens (Nacc). In both rodent and humans several studies shows an attenuated activity of dopaminergic signal associated with obesity and there is evidence that consumption of palatable food per se leads to DA signalling alterations. Furthermore impaired cognition in obese mice is improved by selectively lowering triglycerides (TG) and intracerebroventricular administration of TG induces by itself acquisition impairment in several cognitive paradigms in normal body weight mice. Together, these observations raise the possibility that nutritional lipids, particularly TG, directly affect cognitive and reward processes by modulating the mesolimbic pathway and might contribute to the downward spiral of compulsive consumption of palatable food and obesity. This review is an attempt to capture recent evolution in the field that might point toward a direct action of nutritional lipid in the reward circuitry.

Metabolic Syndrome Is Associated with Increased Oxo-Nitrative Stress and Asthma-Like Changes in Lungs.

Epidemiological studies have shown an increased obesity-related risk of asthma. In support, obese mice develop airway hyperresponsiveness (AHR). However, it remains unclear whether the increased risk is a consequence of obesity, adipogenic diet, or the metabolic syndrome (MetS). Altered L-arginine and nitric oxide (NO) metabolism is a common feature between asthma and metabolic syndrome that appears independent of body mass. Increased asthma risk resulting from such metabolic changes would have important consequences in global health. Since high-sugar diets can induce MetS, without necessarily causing obesity, studies of their effect on arginine/NO metabolism and airway function could clarify this aspect. We investigated whether normal-weight mice with MetS, due to high-fructose diet, had dysfunctional arginine/NO metabolism and features of asthma. Mice were fed chow-diet, high-fat-diet, or high-fructose-diet for 18 weeks. Only the high-fat-diet group developed obesity or adiposity. Hyperinsulinemia, hyperglycaemia, and hyperlipidaemia were common to both high-fat-diet and high-fructose-diet groups and the high-fructose-diet group additionally developed hypertension. At 18 weeks, airway hyperresponsiveness (AHR) could be seen in obese high-fat-diet mice as well as non-obese high-fructose-diet mice, when compared to standard chow-diet mice. No inflammatory cell infiltrate or goblet cell metaplasia was seen in either high-fat-diet or high-fructose-diet mice. Exhaled NO was reduced in both these groups. This reduction in exhaled NO correlated with reduced arginine bioavailability in lungs. In summary, mice with normal weight but metabolic obesity show reduced arginine bioavailability, reduced NO production, and asthma-like features. Reduced NO related bronchodilation and increased oxo-nitrosative stress may contribute to the pathogenesis.

PP.33.29

Objective: Obesity is associated with development of cardiovascular risk factors such as arterial hypertension, dyslipidemia, atherosclerosis and metabolic syndrome. RhoA associated kinase has been identified to play an important role in modulating vascular effects. In arterial hypertension abnormalities in RhoA/Rho-kinase signalling lead to augmented contractile response on vasoconstrictors. The pleiotrophic effect of statins is linked to this by inhibition of RhoA isoprenylation. Our aim was to explore the vascular changes of diet induced obesity leading to hypertension and the effects of Pravastatin incubation on vascular contractility. Design and method: Aortas from mice, fed with high-fat-diet (HFD, 51% of energy from fat) for two weeks, were incubated either with Pravastatin (10 μM) or without for 1 or 24 hours. A second group was treated with HFD and Pravastatin 50 mg/kg/d in drinking water. To examine vasoconstrictor response, dose-response-curves to norepinephrine, high potassium and acetylcholine were performed on a wired myograph. Blood pressure was measured by determining the tail blood volume. Results: Mice fed with HFD developed an increased blood pressure compared to normal weight mice. A two weeks treatment of Pravastatin showed a significant decrease in blood pressure compared to mice treated only with HFD (systolic blood pressure control: 138.5 ± 3.3 mmHg vs. 2wks Pravastatin: 125.3 ± 2.3mmHg). In vitro treatment with Pravastatin for 24 h attenuated the contractile response to norepinephrine and high potassium whereas incubation for one hour had no significant effect on vascular contractility (obese control: logEC50 -7.650 vs. Pravastatin 24 h: logEC50 -7.315). In vivo, a two week treatment with Pravastatin showed a significant reduction of contractile response to vasoconstrictors (obese control: logEC50 -6.880 vs. Pravastatin 2wks: logEC50 -6.593). Dose-response curves showed a shift to the right depending on length of treatment. Sensitivity against vasoconstrictors was significantly decreased. Acetylcholine dependent dilatation was not affected by treatment. A two weeks HFD treatment did not lead to endothelial dysfunction. Conclusions: Our study shows that a Pravastatin treatment of obese mice for just two weeks has beneficial effects on the cardiovascular system. The treatment prevented an increase in blood pressure and vascular smooth muscle cell contractility in obese mice.