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Understanding how differences in animal husbandry practices affect the reproducibility of research results is critical. We sought to understand how different beddings might influence dietary obesity studies. We compared the effects of paper and corncob bedding on weight gain, metabolism, and gut microbiome (GM) of mice fed a high-fat diet (HFD) or a normal diet (ND) and evaluated effects on fecal and cecal microbiomes collected from these cohorts after euthanasia. Male C57BL/6J mice at 5 wk age were allowed to acclimate to the facility and the assigned bedding for one week before being placed on HFD or remaining on the ND for 12 wk. Fecal pellets and cecal samples were collected and frozen for batched 16S sequencing. Mice had similar body weight, visceral gonadal white adipose tissue (GWAT), subcutaneous inguinal white adipose tissue (IWAT), liver and spleen weights and metabolic changes regardless of the bedding type. Baseline microbiota differences were detected one week after bedding assignment. After 12 wk, the GM showed significant differences depending on both bedding and diet. The effects of the bedding were not significantly different between endpoint fecal and cecal GM, despite the inherent differences in microbiota in fecal and cecal samples. A correlation was detected between diet and the relative abundance of Bacteroidetes and Verrucomicrobia: Akkermansia. In conclusion, this study demonstrates the importance of considering bedding type when performing dietary experiments.

Obesity-induced inflammation is a risk factor for metabolic impairment leading to diabetes and cardiovascular disease. We, and others, have shown that sex differences exist in adipose tissue (AT) and myeloid inflammatory responses to high-fat diet (HFD) and lipolysis. However, the causal cellular mechanisms behind this sexual dimorphism are not well defined. Our objective was to investigate sex differences in lipid storage and metabolism that drive sexually dimorphic inflammatory responses. C57Bl/6J mice were fed a normal diet or 60%kcal%fat (HFD) for 16 weeks. AT macrophages (ATMs) were sorted for RNA sequencing. Animals were gonadectomized (GX) prior to starting HFD and gonadal white AT (GWAT) was collected for lipidomics. To directly model androgen receptor (AR) activation, GX mice were given dihydrotestosterone (DHT) to study AR-specific effects. RNA sequencing identified upregulated genes (Lpl, Ctsb, and Cd36) linked to a metabolically-activated macrophage only in male HFD ATMs. Furthermore, pathways associated with mitochondrial depolarization and fatty acid (FA) metabolism differed significantly by sex. GWAT lipidomics identified shifts in lipid species both by sex and hormone status, and differences were also observed in acylcarnitines and saturation of ceramide species that are implicated in inflammation. In addition, males showed elevated levels of key precursor free FAs (palmitic, linoleic, and arachidonic acid) when compared to female and GX mice. DHT treatment did not completely mimic male pro-inflammatory responses but induced GWAT crown-like structures in both sexes. DHT failed to upregulate inflammatory markers (Il6 and Mcp1) but showed impaired expression of adipogenic genes (Pparg and Pgc1α) in GX mice. Altogether, our data suggest there are sex-specific alterations in GWAT lipid storage and metabolism that may drive sexually dimorphic ATM polarization, which is only partially reversed by AR activation. Disclosure R. Mohan: None. M. Varghese: None. S. Abrishami: None. K. Singer: None. Funding National Institutes of Health - NIDDK (R01DK115583)

Obesity is associated with systemic inflammation and immune cell recruitment to metabolic tissues. Sex differences have been observed where male mice challenged with high fat diet (HFD) exhibit greater adipose tissue inflammation than females demonstrating a role for sex hormones in differential inflammatory responses. Circulating monocytes that respond to dietary lipids and chemokines and produce cytokines are the primary source of recruited adipose tissue macrophages (ATMs). In this study, we investigated sexual dimorphism in biological pathways in HFD-fed ATMs from male and female mice by RNA-seq. We also conducted chemotaxis assays to investigate sex differences in the migration of monocytes isolated from bone marrow from male and female mice toward a dietary saturated lipid — palmitate (PA), and a chemokine — monocyte chemoattractant protein 1 (MCP1), factors known to stimulate myeloid cells in obesity. ATM RNA-Seq demonstrated sex differences of both metabolic and inflammatory activation, including pathways for chemokine signaling and leukocyte trans-endothelial migration. In vivo monocyte transfer studies demonstrated that male monocytes traffic to female adipose tissue to generate ATMs more readily. In chemotaxis assays, lean male monocytes migrated in greater numbers than females toward PA and MCP1. With short-term HFD, male and female monocytes migrated similarly, but in chronic HFD, male monocytes showed greater migration than females upon PA and MCP1 stimulation. Studies with monocytes from toll-like receptor 4 knockout mice (Tlr4-/-) demonstrated that both males and females showed decreased migration than WT in response to PA and MCP1 implying a role for TLR4 in monocyte influx in response to meta-inflammation. Overall, these data demonstrate the role of sexual dimorphism in monocyte recruitment and response to metabolic stimuli that may influence meta-inflammation in obesity.

Aging, like obesity, is associated with metabolic and inflammatory alterations within adipose tissue in older individuals. Younger females are protected from adipose inflammation, but older post-menopausal females exhibit exaggerated visceral adiposity correlated with increased disease risk. Obesity accelerates the onset and progression of age-associated diseases, but it is unclear if aging and obesity drive adipose tissue dysfunction in a sexually dimorphic fashion. We investigated adipose tissue metabolism and inflammation in a diet-induced obesity model in young and old mice. We identified age related sex differences in adipose tissue macrophages (ATMs), fibrosis and lipid metabolism in male and female visceral fat depot (GWAT). Although aging normalized body weights between the sexes, females remained protected from proinflammatory ATMs and stimulated lipolysis failed to adversely affect the inflammatory state even with obesity. Older obese males had augmented CD11c+ ATMs and higher insulin levels, while females showed increased visceral adiposity and exaggerated Pparγ, and Pgc1α expression. Obesity in aging demonstrated similar expression of GWAT p53, p16, p21, Timp1 and Tgfβ1 in both sexes. Our studies suggest that even with aging, female GWAT shows an attenuated inflammatory response compared to males due to an efficient oxidative metabolism combined with an active tissue remodeling state.

This study investigated the effects of elevated fatty acid (FA) supply from adipose tissue on the ultrastructure of cardiac lipid droplets (LDs) and the expression and organization of LD scaffold proteins perilipin-2 (PLIN2) and perilipin-5 (PLIN5). Stimulation of adipocyte lipolysis by fasting (24 h) or β3-adrenergic receptor activation by CL316, 243 (CL) increased cardiac triacylglycerol (TAG) levels and LD size, whereas CL treatment also increased LD number. LDs were tightly associated with mitochondria, which was maintained during LD expansion. Electron tomography (ET) studies revealed continuity of LD and smooth endoplasmic reticulum (SER), suggesting interconnections among LDs. Under fed ad libitum conditions, the cristae of mitochondria that apposed LD were mostly organized perpendicularly to the tangent of the LD surface. Fasting significantly reduced, whereas CL treatment greatly increased, the perpendicular alignment of mitochondrial cristae. Fasting and CL treatment strongly upregulated PLIN5 protein and PLIN2 to a lesser extent. Immunofluorescence and immuno-electron microscopy demonstrated strong targeting of PLIN5 to the cardiac LD-mitochondrial interface, but not to the mitochondrial matrix. CL treatment augmented PLIN5 targeting to the LD-mitochondrial interface, whereas PLIN2 was not significantly affected. Together, our results support the concept that the interface between LD and cardiac mitochondria represents an organized and dynamic “metabolic synapse” that is highly responsive to FA trafficking.

We describe a novel gold-templating method for fabricating stable, biodegradable, bile salt microparticles with controlled geometry., Bile acids are proposed as therapeutic agents for various diseases, including liver diseases and obesity. However, oral or subcutaneous administration of a solubilized version of these drugs has limited efficacy and imposes unwanted side effects. Here, we describe a gold-templating method for fabricating stable, bile salt—cholate or deoxycholate—microparticles. The gold ions’ reduction at the oil-water interface in a double emulsion solvent evaporation process enables a gold–bile salt interaction and the formation of bile salt particles. We demonstrate that composite microparticles release cholate/deoxycholate into solution via a surface erosion process. We illustrate these particles’ capability to lyse adipocytes, both in vitro and in vivo, with minimal side effects, contrary to the Food and Drug Administration–approved salt solution that leads to severe inflammation and ulceration. Overall, particle-based cholate/deoxycholate opens opportunities for localized delivery of these salts, improving efficacy while minimizing side effects associated with oral and subcutaneous use.

Obesity-induced chronic inflammation is associated with metabolic disease. Results from mouse models utilizing a high-fat diet (HFD) have indicated that an increase in activated macrophages, including CD11c+ adipose tissue macrophages (ATMs), contributes to insulin resistance. Obesity primes myeloid cell production from hematopoietic stem cells (HSCs) and Toll-like receptor 4 (TLR4), and the downstream TIR domain-containing adapter protein-inducing interferon-β (TRIF)- and MyD88-mediated pathways regulate production of similar myeloid cells after lipopolysaccharide stimulation. However, the role of these pathways in HFD-induced myelopoiesis is unknown. We hypothesized that saturated fatty acids and HFD alter myelopoiesis by activating TLR4 pathways in HSCs, differentially producing pro-inflammatory CD11c+ myeloid cells that contribute to obesity-induced metabolic disease. Results from reciprocal bone marrow transplants (BMTs) with Tlr4-/- and WT mice indicated that TLR4 is required for HFD-induced myelopoiesis and production of CD11c+ ATMs. Experiments with homozygous knockouts of Irakm (encoding a suppressor of MyD88 inactivation) and Trif in competitive BMTs revealed that MyD88 is required for HFD expansion of granulocyte macrophage progenitors and that Trif is required for pregranulocyte macrophage progenitor expansion. A comparison of WT, Tlr4-/-, Myd88-/-, and Trif-/- mice on HFD demonstrated that TLR4 plays a role in the production of CD11c+ ATMs, and both Myd88-/- and Trif-/- mice produced fewer ATMs than WT mice. Moreover, HFD-induced TLR4 activation inhibited macrophage proliferation, leading to greater accumulation of recruited CD11c+ ATMs. Our results indicate that HFD potentiates TLR4 and both its MyD88- and TRIF-mediated downstream pathways within progenitors and adipose tissue and leads to macrophage polarization.

Men have a statistically higher risk of metabolic and cardiovascular disease than premenopausal women, but the mechanisms mediating these differences are elusive. Chronic inflammation during obesity contributes to disease risk and is significantly more robust in males. Prior work demonstrated that compared with obese males, obese females have reduced proinflammatory adipose tissue macrophages (ATMs). Given the paucity of data on how sex hormones contribute to macrophage responses in obesity, we sought to understand the role of sex hormones in promoting obesity-induced myeloid inflammation. We used gonadectomy, estrogen receptor–deficient alpha chimeras, and androgen-insensitive mice to model sex hormone deficiency. These models were evaluated in diet-induced obesity conditions (high-fat diet [HFD]) and in vitro myeloid assays. We found that ovariectomy increased weight gain and adiposity. Ovariectomized females had increased ATMs and bone marrow myeloid colonies compared with sham-gonadectomized females. In addition, castrated males exposed to HFD had improved glucose tolerance, insulin sensitivity, and adiposity with fewer Ly6chi monocytes and bone marrow myeloid colonies compared with sham-gonadectomized males, although local adipose inflammation was enhanced. Similar findings were observed in androgen-insensitive mice; however, these mice had fewer CD11c+ ATMs, implying a developmental role for androgens in myelopoiesis and adipose inflammation. We concluded that gonadectomy results in convergence of metabolic and inflammatory responses to HFD between the sexes, and that myeloid estrogen receptor alpha contributes minimally to diet-induced inflammatory responses, whereas loss of androgen-receptor signaling improves metabolic and inflammatory outcomes. These studies demonstrate that sex hormones play a critical role in sex differences in obesity, metabolic dysfunction, and myeloid inflammation.

Age associated chronic inflammation is a major contributor to diseases with advancing age. Adipose tissue function is at the nexus of processes contributing to age-related metabolic disease and mediating longevity. Hormonal fluctuations in aging potentially regulate age-associated visceral adiposity and metabolic dysfunction. Visceral adiposity in aging is linked to aberrant adipogenesis, insulin resistance, lipotoxicity and altered adipokine secretion. Age-related inflammatory phenomena depict sex differences in macrophage polarization, changes in T and B cell numbers, and types of dendritic cells. Sex differences are also observed in adipose tissue remodeling and cellular senescence suggesting a role for sex steroid hormones in the regulation of the adipose tissue microenvironment. It is crucial to investigate sex differences in aging clinical outcomes to identify and better understand physiology in at-risk individuals. Early interventions aimed at targets involved in adipose tissue adipogenesis, remodeling and inflammation in aging could facilitate a profound impact on health span and overcome age-related functional decline.

In obesity, high levels of saturated fatty acids (SFAs) contribute to adipose tissue inflammation and dysfunction. Obesity-induced macrophage infiltration leads to insulin resistance, but the adipocyte itself may play a role in generating the inflammatory milieu. Given our recent findings of the role of TLR4 in myeloid biasing in obesity, we next investigated the role of TLR4 in adipocyte generated inflammatory responses to SFAs and lipopolysaccharides. We used WT and Tlr4−/- ear mesenchymal stem cell derived adipocytes (EMSC Ad) and bone marrow dendritic cells (BMDCs) to evaluate cell specific responses. Our work demonstrates a role for TLR4 in adipocyte- immune cell crosstalk and that SFA derived metabolites from adipocytes may induce proinflammatory stimulation of immune cells in a TLR4 independent manner.

With the increasing prevalence of obesity in women of reproductive age there is a need to understand the ramifications of this on offspring. The purpose of this study is to investigate the programming effects of maternal obesity during preconception and the preconception/gestational period on adiposity and adipose tissue inflammation in offspring using an animal model. Adult female C57Bl/6J mice were assigned either normal diet, high fat diet (HFD) prior to pregnancy, or HFD prior to and through pregnancy. Some offspring were maintained on normal diet while others started HFD later in life. Offspring were assessed for body composition and metabolic responses. Lipid storing tissues were evaluated for expansion and inflammation. Male offspring from the preconception group had the greatest weight gain, most subcutaneous adipose tissue, and largest liver mass when introduced to postnatal HFD. Male offspring of the preconception/gestation group had worsened glucose tolerance and an increase in resident (CD11c−) adipose tissue macrophages (ATMs) when exposed to postnatal HFD. Female offspring had no significant difference in any parameter between the diet treatment groups. In conclusion, this study demonstrates that prenatal and pregnancy windows have independent programming effects on offspring. Preconception exposure affects body composition and adiposity while gestation exposure affects metabolism and tissue immune cell phenotypes.

Background Weight loss by surgery or lifestyle changes is strongly recommended for obese individuals to improve metabolic health, but the underlying impairments that persist from a history of obesity remain unclear. Recent investigations demonstrate a persistent inflammatory state with weight loss and bariatric surgery, but the mechanism and impact are not fully understood. Additionally, these studies have not been performed in females although women are the majority of individuals undergoing weight loss interventions. Methods The goal of this study was to determine the sex differences in metabolically induced inflammation after dietary weight loss (WL) or bariatric surgery. Following a 60% high-fat diet (HFD) for 12 weeks, C57Bl/6j mice underwent either a dietary switch to normal chow for WL or vertical sleeve gastrectomy (VSG) and were evaluated 8 weeks after intervention. WL effects on myelopoiesis were further evaluated with bone marrow chimeras. Results Both sexes had a decrease in adiposity and total weight following WL or VSG intervention. With HFD, females had very little inflammation and no further increase with WL, but males had persistent inflammation even after WL despite metabolic improvement. Interestingly, after VSG, myeloid inflammation was increased in the livers of males and to a lesser extent in females. Conclusions These studies demonstrate that regardless of sex, it is critical to assess an individuals’ history of obesity rather than just rely on current weight status in medical decision-making. There are long-lasting effects on tissue inflammation in both sexes especially with surgical weight loss. Dietary change is overall most effective to improve meta-inflammation in obese males on its own or in combination with surgical weight loss.

Obesity is a global health epidemic, closely associated with cardiovascular diseases (CVD), hypertension, type 2 diabetes, and atherosclerosis. Obese adipose tissue is a site and source for inflammation that is strongly associated with these metabolic diseases. In obese mice models, males exhibit profound adipose tissue macrophage (ATM) accumulation in gonadal white adipose tissue (GWAT) linked to insulin resistance, while females are protected even with enhanced adiposity. This dampened inflammation may directly explain reduced metabolic and cardiovascular disease in young women. However, it is not clear what metabolic and inflammatory responses occur in post-menopausal models. We hypothesized an increased inflammatory response would occur in obese older females. We challenged male and female C57Bl6/j mice to 16-24 weeks of 60% high fat diet (HFD) starting at 6 weeks of age (young) or 10 month of age (old). Animal body weights and metabolic depot (adipose and liver) weights were measured at the completion of diet challenge. Flow cytometry was used to determine ATM populations in visceral (GWAT) compared to subcutaneous inguinal WAT (IWAT). Lipolysis was stimulated with a beta3 adrenergic receptor (ADRB3) agonist CL-316, 243 and free fatty acids (FFA), glycerol, and triglyceride (TG) levels assessed in serum and liver. Obesity in young mice led to fat accumulation in GWAT and IWAT of both males and females. In old male animals, fat pads did not expand, and liver weights were as a result larger than in females. Both young and old females had reduced adipose inflammation compared to males. ADRB3 activation had no effect on adipose tissue weights but significantly increased liver weights in obese young females. Obese CL treated young and old female mice showed elevated FFA compared to levels induced in males. Lipolysis promoted appearance of crown like structures (CLS) in both obese young and old males and females, due to CD11c- ATM accumulation. Obesity during aging continues to lead to enhanced inflammation in males along with steatosis but females continue to be protected with appropriate storage of fatty acids with very little induced inflammation. Sex differences in inflammation that persist in aging demonstrate that differences in meta-inflammation may explain sex differences in metabolic and cardiovascular diseases regardless of age and need to be further explored for targeted treatment strategies.

Males are known to have profound adipose tissue macrophage (ATM) accumulation in gonadal white adipose tissue (GWAT) during obesity, whereas females are protected from such an inflammatory response even with increased adiposity. The inflammatory tone in males is linked to insulin resistance and might be the underlying cause for sex differences in metabolic disease. Factors regulating the meta-inflammatory response remain unclear but enhanced lipid storage in females may explain the reduced inflammatory response to high-fat diets. In this study, we evaluated lean and obese females with stimulated lipolysis to understand whether a stress release of free fatty acids (FFAs) could induce female ATMs. We demonstrate that in both lean and obese females, GWAT CD11c(−) resident ATMs accumulate with β-3 adrenergic receptor–stimulated lipolysis. Lipolysis elevated serum FFA, triglyceride, and IL-6 levels in females that corresponded to significant phosphorylated hormone-sensitive lipase and adipose triglyceride lipase protein expression in obese female GWAT compared with males. Increased lipolytic response in obese females was associated with crown-like structures and induced Il6, Mcp1, Arg1, and Mgl1 expression in obese female GWAT, suggesting an environment of lipid clearance and adipose remodeling. With this finding we next investigated whether lipid storage and lipolytic mediators differed by sex. Diacylglycerol, ceramides, phospholipids, and certain fatty acid species associated with inflammation were elevated in male GWAT compared with obese female GWAT. Overall, our data demonstrate a role for GWAT lipid storage and lipolytic metabolites to induce inflammation in males and induce remodeling in females that might explain sex differences in overall metabolic health.

PURPOSE OF REVIEW: As the ongoing epidemic of adult and childhood obesity grows, it puts a greater burden on individuals and the healthcare system due to increased prevalence of obesity-associated diseases. An important area that has gained much attention recently is the sex and gender difference related to obesity and associated complications. Basic science and clinical studies have now improved our understanding of obesity and have discovered adipose tissue biology to be key in metabolism. RECENT FINDINGS: There is evidence related to the sex dichotomy in obesity in a variety of areas including adipocyte function, sex hormone effects, genetics, and metabolic inflammation leading to critical differences in adipose tissue biology. SUMMARY: The sex and gender difference in adipose tissue is a factor that should be considered when studying an individuals’ risk for obesity and metabolic dysfunction. This understanding is important for strategizing treatment and prevention measures.

Men have a higher risk of metabolic and cardiovascular disease than pre-menopausal women, but the mechanisms of these differences are elusive. Chronic myeloid inflammation during obesity contributes to metabolic disease risk and is significantly more robust in males. Given the paucity of data on male sex hormones contributing to macrophage responses in obesity, our objective was to understand the role of androgens in promoting obesity-induced myeloid inflammation. Male and female C57Bl/6J mice were fed a 60% high fat diet for 24 weeks. Glucose tolerance and insulin sensitivity were measured. Monocyte transfer experiments assessed sex differences in bone marrow myeloid responses to obesity independent of host sex. Gonadectomy, mice deficient of androgen receptor signaling (ARtfm) and monocyte specific androgen receptor (AR) knockout mice (LysMAR-/y) were used to model androgen deficiency. Compared to males, females had dampened inflammatory responses with reduced CD11c+ ATMs and cytokines, even with increased adiposity. RNASeq demonstrated sex differences in gene pathways of both metabolic and inflammatory activation in ATMs. Male monocytes transferred into females remained primed for pro-inflammatory response and induced an increase in adiposity. Castrated males exposed to high fat diet had impaired glucose tolerance and increased insulin sensitivity with increased adiposity and lower numbers of CD11c+ ATMs. These same findings were seen in ARtfm mice. Testosterone enhanced palmitate-stimulated myeloid colony production, and this response was dampened in castrated, ARtfm, and LysMAR-/y mice. These studies demonstrate that androgens play a critical role in enhanced metabolic dysfunction and enhanced myeloid inflammation in response to obesogenic cues. Androgen driven myeloid inflammation leads to metabolic dysfunction in a cell-autonomous manner driving sex-differences in obesity induced meta-inflammation. Disclosure C.A. Griffin: None. S. Abrishami: None. M. Varghese: None. K. Singer: None.

Men and post-menopausal women are at greater risk for cardiovascular diseases and diabetes than younger women. In obese rodent models, males exhibit profound adipose tissue macrophage (ATM) accumulation in gonadal white adipose tissue (GWAT) linked to insulin resistance, while females are protected even with enhanced adiposity. We hypothesize that these inflammatory differences may be a result of altered lipolysis responses and that females would have enhanced ATM accumulation when lipolysis is induced. Lipolysis was stimulated with a beta3 adrenergic receptor (ADRB3) agonist CL-316, 243 in C57Bl6/j male and female mice fed 16 weeks of 60% high fat diet (HFD). GWAT leukocytes were isolated after 18 h and evaluated by flow cytometry. Lipogenic, lipolytic, oxidative and inflammatory gene expression along with immunofluorescence were performed. ADRB3 activation did not alter adipose tissue weights, but significantly reduced liver weights in obese females. Obese CL treated female mice showed elevated serum free fatty acid (FFA) and triglyceride (TG) levels compared to levels induced in males. Lipid overload impaired TG storage and utilization genes- Lpl, Fasn, Acsl1, Hsl, and Glut4 expression in obese females. Lipolysis promoted appearance of crown like structures (CLS) in males and females but only induced Il6 proinflammatory cytokine and Mcp1 chemokine expression in obese female GWAT. Activated lipolysis triggered CD11c- ATM accumulation in lean females but in both obese male and female GWAT. Obese males showed a marked reduction in GWAT dendritic cells (DC) but a significant reduction in both GWAT and inguinal WAT DCs of obese females. While obese females are resistant to inflammation, this study shows that induced lipolysis elevates FFA levels, alters lipid metabolism and increased CD11c- ATMs and CLSs in obese females. Elucidating the link between lipolysis and inflammation can better explain sex differences in overall metabolic health leading to targeted treatment strategies. Disclosure M. Varghese: None. S. Abrishami: None. K. Singer: None.

Obesity-induced chronic inflammation is associated with metabolic disease. Results from mouse models utilizing a high-fat diet (HFD) have indicated that an increase in activated macrophages, including CD11c(+) adipose tissue macrophages (ATMs), contributes to insulin resistance. Obesity primes myeloid cell production from hematopoietic stem cells (HSCs) and Toll-like receptor 4 (TLR4), and the downstream TIR domain–containing adapter protein–inducing interferon-β (TRIF)- and MyD88-mediated pathways regulate production of similar myeloid cells after lipopolysaccharide stimulation. However, the role of these pathways in HFD-induced myelopoiesis is unknown. We hypothesized that saturated fatty acids and HFD alter myelopoiesis by activating TLR4 pathways in HSCs, differentially producing pro-inflammatory CD11c(+) myeloid cells that contribute to obesity-induced metabolic disease. Results from reciprocal bone marrow transplants (BMTs) with Tlr4(−/−) and WT mice indicated that TLR4 is required for HFD-induced myelopoiesis and production of CD11c(+) ATMs. Experiments with homozygous knockouts of Irakm (encoding a suppressor of MyD88 inactivation) and Trif in competitive BMTs revealed that MyD88 is required for HFD expansion of granulocyte macrophage progenitors and that Trif is required for pregranulocyte macrophage progenitor expansion. A comparison of WT, Tlr4(−/−), Myd88(−/−), and Trif(−/−) mice on HFD demonstrated that TLR4 plays a role in the production of CD11c(+) ATMs, and both Myd88(−/−) and Trif(−/−) mice produced fewer ATMs than WT mice. Moreover, HFD-induced TLR4 activation inhibited macrophage proliferation, leading to greater accumulation of recruited CD11c(+) ATMs. Our results indicate that HFD potentiates TLR4 and both its MyD88- and TRIF-mediated downstream pathways within progenitors and adipose tissue and leads to macrophage polarization.

Metabolic and non-metabolic complications due to obesity are becoming more prevalent, yet our understanding of the mechanisms driving these is not. This is due to individual risk factor variability making it difficult to predict disease outcomes such as diabetes and insulin resistance. Gender is a critical factor in obesity outcomes with women having more adiposity but reduced metabolic complications compared to men. The role of immune system activation during obesity is an emerging field that links adiposity to metabolic syndrome. Furthermore, evidence from animal models suggests that sex differences exist in immune responses and, therefore, could be a possible mechanism leading to sex differences in metabolic disease. While there is still much to learn in the area of sex-differences research, this chapter will review the current knowledge and literature detailing the role of sex and sex hormones on adiposity and metabolically induced inflammation in obesity.

Overnutrition during critical windows of development plays a significant role in life-long metabolic disease risk. Early exposure to excessive nutrition may result in altered programming leading to increased susceptibility to obesity, inflammation, and metabolic complications. This study investigated the programming effects of high-fat diet (HFD) exposure during the lactation period on offspring adiposity and inflammation. Female C57Bl/6J dams were fed a normal diet or a 60% HFD during lactation. Offspring were weaned onto a normal diet until 12 weeks of age when half were re-challenged with HFD for 12 weeks. Metabolic testing was performed throughout adulthood. At 24 weeks, adipose depots were isolated and evaluated for macrophage profiling and inflammatory gene expression. Males exposed to HFD during lactation had insulin resistance and glucose intolerance as adults. After re-introduction to HFD, males had increased weight gain and worsened insulin resistance and hyperglycemia. There was increased infiltration of pro-inflammatory CD11c+ adipose tissue macrophages, and bone marrow was primed to produce granulocytes and macrophages. Bone density was lower due to enhanced marrow adiposity. This study demonstrates that maternal HFD exposure during the lactational window programs offspring adiposity, inflammation, and impaired glucose homeostasis.

Metabolic and non-metabolic complications due to obesity are becoming more prevalent, yet our understanding of the mechanisms driving these is not. This is due to individual risk factor variability making it difficult to predict disease outcomes such as diabetes and insulin resistance. Gender is a critical factor in obesity outcomes with women having more adiposity but reduced metabolic complications compared to men. The role of immune system activation during obesity is an emerging field that links adiposity to metabolic syndrome. Furthermore, evidence from animal models suggests that sex differences exist in immune responses and, therefore, could be a possible mechanism leading to sex differences in metabolic disease. While there is still much to learn in the area of sex-differences research, this chapter will review the current knowledge and literature detailing the role of sex and sex hormones on adiposity and metabolically induced inflammation in obesity.

As all microscopists are taught, resolution can be measured by knowing the closest distance between two points that can be discriminated in the image. There are some occasions when we want to measure something in a digital image, and the nominal resolution isn't sufficient for the task. One of these, extracting a contour from the image, was summarized in a previous research report. The principles would be valid for any filled area, such as a particle or structure from an X-ray dot map. The ground rule was set that the outermost pixel would be selected at every point on the contour. It was clear from the geometry shown in Fig. 1 that only values of 0°, 45°, 90°, 135° and 180° could be measured. Because of the stair step defect, tracing the sequence of pixel locations only gave offsets to 8 pixels, one directly below, two on the corners below, three pixels in the same location above, and one pixel on either side of the subject pixel. Therefore it was impossible to measure the true curvature of the contour.

A computerized morphometric classification technique based on latent factors reveals major protrusion classes: factors 4, 5, and 7. Previous work showed that factor 4 represented filopodia, 5 the distribution of lamellar cytoplasm, and 7 a blunt protrusion. We explore the relationship of focal contact (FC) characteristics and their integrated actin cables to factors values. The results show that FC maturation/cytoskeletal integration affects factor 5, because FC elongation/integration was correlated with its values. On the contrary, 7 values decreased with maturation, so cable or FC size or their integration must be restricted to form these protrusions. Where integration did occur, the cables showed distinctive size and orientation, as indicated by correlation of 7 values with FC shape. Results obtained with myosin inhibitors support the interpretation that a central, isometric, contractile network puts constraints on both factor 5 and 7 protrusions. We conclude that cells establish functional domains by rearranging the cytoskeleton.

Although growth factor-initiated cascades in cells are networked with mechanisms such as “inside-out signaling”, it is not known how these pathways are integrated. Earlier studies reported that ruffling was enhanced and filopodia reduced in transformed cells. Since dissecting relationships among features was impossible if subjective recognition was relied upon, features in two epithelial cell lines were recognized by latent factor analysis. Factor-based classification revealed four protrusion classes, but none of them corresponded to ruffles. Loss of filopodia, defined by factor 4 (F4) values, accounted for the greatest change in features of oncogenically transformed cells. Factor 5 (F5, lamella) was unchanged during transformation of an airway epithelium cell line. The tumor promoter, phorbol 12-myristate 13-acetate (PMA), increased ruffling but decreased filopodia. F4 retained this relationship to ruffling in untreated cells and at multiple times after treatment. F5 values decreased but were positively correlated with measures of ruffling. Because factors are created as mutually orthogonal variables, this suggested that ruffles were not flagged in factor analysis because they originate from other features. Actin filament capping with sub-micromolar cytochalasin D (Cyto D) suppressed ruffling without affecting F4 or F5. Cyto D increased factor 7 (F7) values, thus showing specificity for this feature. However, cytochalasin treatment of PMA-treated cells that had developed stress fibers increased F4 and decreased F5. The results suggest that PMA changes the state of the cytoskeleton, causing protrusions to show novel responses to Cyto D compared to untreated cells. Results suggest that the factors identify physiologically distinct features.

The shape of cells is largely determined by the arrangement of complexes integrating cytoskeletal structures with focal adhesions (FAs) and contacts (FCs). The transformed phenotype of the cells could be identified mathematically by a computerized morphometric assay. The assay is implemented by analyzing the shape of cells’ interference contours. Previous work established a database of shape variable value which was applied to classify cell features. By deconstructing cell shape, we discovered that filopodia, i.e. thin, flat, tapering projections from the cell edge (indexed by factor #4), and knob-shaped, triangular, or strap-shaped features (indexed by factor #7) were decreased and increased, respectively, in transformed cells. Factor #4 represents a feature that accounts for approximately 15% of the phenotype specifying a cancer cell, and here we explore the hypothesis that filopodia may be subtracted by elaborating the larger protrusions. The objective of this work was to model the determinants of the latter protrusions and test different models to gain insights into this feature. Although ruffling is the main mechanism for motility in cells, there was no correlation between #7 values and ruffle number or ruffling frequency. One model that stable contacts were stationed at the cell edge and the actin protrusion force pushed the membrane out parallel to the line of contacts, is supported by some data. First, data implicated PAK (p21-activated kinase)-PIX (PAK-interacting exchange factor) complex in the dynamics of FCs formation and turnover. Consistent with a role for PAK, studies done by expressing GFP-paxillin in the cells, inducing the formation of neurite-like protrusions, and then mapping the location of the bright spots and streaks (paxillin-containing scaffolds), showed that the range of angles formed between FCs and cell edge was lower in protrusions than in other portions of the edge. However, it was unlikely that actin-myosin interactions exerted outward force on the FCs, as ATPase inhibitor blebbistatin and myosin light chain kinase inhibitor SPC 16524 had no effect on the protrusions. A second model held that the protrusions were anchored by actin integration into FCs in the interior of the cell, e.g. at the boundary between the lamellum and lamellipodium. The size and shape of each such site, as well as its orientation and the x, y coordinates of its centroid were read out in MetaMorph software from digital images. A statistical analysis with these variables, testing for correlations with the factor #7 feature values (representing the neurite-like protrusions), showed that the smaller, more compact FCs were positively correlated with factor # 7. This indicated that widely-distributed, smaller contacts might be important for formation of neurite-like features, which could be consistent with either model. These two models were not mutually exclusive and so the necessity for both mechanisms is being explored. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 5112.