The Influence of Maternal High Fat Diet During Lactation on Offspring Hematopoietic Priming.

Obesity and metabolic diseases are rising among women of reproductive age, increasing offspring metabolic risk. Maternal nutritional interventions during lactation present an opportunity to modify offspring outcomes. We previously demonstrated in mice that adult male offspring have metabolic impairments and increased adipose tissue macrophages (ATM) when dams are fed high fat diet (HFD) during the postnatal lactation window (HFD PN). We sought to understand the effect of HFD during lactation on early-life inflammation. HFD PN offspring were evaluated at postnatal day 16 to 19 for tissue weight and gene expression. Profiling of adipose tissue and bone marrow immune cells was conducted through lipidomics, in vitro myeloid colony forming unit assays, and flow cytometry. HFD PN mice had more visceral gonadal white adipose tissue (GWAT) and subcutaneous fat. Adipose tissue RNA sequencing demonstrated enrichment of inflammation, chemotaxis, and fatty acid metabolism and concordant changes in GWAT lipidomics. Bone marrow (BM) of both HFD PN male and female offspring had increased monocytes (CD45+Ly6G-CD11b+CD115+) and B cells (CD45+Ly6G-CD11b-CD19+). Similarly, serum from HFD PN offspring enhanced in vitro BM myeloid colonies in a toll-like receptor 4-dependent manner. We identified that male HFD PN offspring had increased GWAT pro-inflammatory CD11c+ ATMs (CD45+CD64+). Maternal exposure to HFD alters milk lipids enhancing adiposity and myeloid inflammation even in early life. Future studies are needed to understand the mechanisms driving this pro-inflammatory state of both BM and ATMs, the causes of the sexually dimorphic phenotypes, and the feasibility of intervening in this window to improve metabolic health.

Constitutive bone marrow adipocytes suppress local bone formation.

BM adipocytes (BMAd) are a unique cell population derived from BM mesenchymal progenitors and marrow adipogenic lineage precursors. Although they have long been considered to be a space filler within bone cavities, recent studies have revealed important physiological roles in hematopoiesis and bone metabolism. To date, the approaches used to study BMAd function have been confounded by contributions by nonmarrow adipocytes or by BM stromal cells. To address this gap in the field, we have developed a BMAd-specific Cre mouse model to deplete BMAds by expression of diphtheria toxin A (DTA) or by deletion of peroxisome proliferator-activated receptor gamma (Pparg). We found that DTA-induced loss of BMAds results in decreased hematopoietic stem and progenitor cell numbers and increased bone mass in BMAd-enriched locations, including the distal tibiae and caudal vertebrae. Elevated bone mass appears to be secondary to enhanced endosteal bone formation, suggesting a local effect caused by depletion of BMAd. Augmented bone formation with BMAd depletion protects mice from bone loss induced by caloric restriction or ovariectomy, and it facilitates the bone-healing process after fracture. Finally, ablation of Pparg also reduces BMAd numbers and largely recapitulates high-bone mass phenotypes observed with DTA-induced BMAd depletion.

Lipolysis of bone marrow adipocytes is required to fuel bone and the marrow niche during energy deficits.

To investigate roles for bone marrow adipocyte (BMAd) lipolysis in bone homeostasis, we created a BMAd-specific Cre mouse model in which we knocked out adipose triglyceride lipase (ATGL, Pnpla2 gene). BMAd-Pnpla2-/- mice have impaired BMAd lipolysis, and increased size and number of BMAds at baseline. Although energy from BMAd lipid stores is largely dispensable when mice are fed ad libitum, BMAd lipolysis is necessary to maintain myelopoiesis and bone mass under caloric restriction. BMAd-specific Pnpla2 deficiency compounds the effects of caloric restriction on loss of trabecular bone in male mice, likely due to impaired osteoblast expression of collagen genes and reduced osteoid synthesis. RNA sequencing analysis of bone marrow adipose tissue reveals that caloric restriction induces dramatic elevations in extracellular matrix organization and skeletal development genes, and energy from BMAd is required for these adaptations. BMAd-derived energy supply is also required for bone regeneration upon injury, and maintenance of bone mass with cold exposure.

Monocyte Trafficking and Polarization Contribute to Sex Differences in Meta-Inflammation.

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.

Sex hormones regulate metainflammation in diet-induced obesity in mice.

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.

Weight Gain, Glucose Tolerance, and the Gut Microbiome of Male C57BL/6J Mice Housed on Corncob or Paper Bedding and Fed Normal or High-Fat Diet.

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.

Enhanced Myeloid Leukocytes in Obese Children and Adolescents at Risk for Metabolic Impairment.

Objective: We aimed to examine if myeloid leukocyte profiles are associated with metabolic impairment in children and adolescents with obesity, and if sex, age, or race influence this relationship. Methods: 282 children ages 8-17 were evaluated. Predictor measures were absolute neutrophil counts (ANC), absolute monocyte count, monocyte subtypes and C reactive protein (CRP). Outcome variables were waist circumference, fasting glucose and insulin, HOMA-IR, HbA1c (%) and lipid profiles. Pearson correlation coefficients were used to determine associations between predictor and outcome variables. Wilcoxon two-sample tests were used to evaluate differences by sex. Results: CRP (p < 0.0001), ANC (p < 0.0018), and classical monocytes (p = 0.05) were significantly higher in children with obesity. CRP, ANC and classical monocytes showed positive correlations with waist circumference, insulin, HOMA-IR and triglycerides. CRP was positively associated with ANC overall (p = 0.05). ANC demonstrated positive correlation with monocytes (p < 0.001). The associations between predictor and outcome variables were influenced by sex, race, and age. Conclusions: CRP and myeloid leukocyte populations, specifically classical monocytes and neutrophils associate with both body composition and metabolic parameters in children with obesity suggesting that these cells may play a critical role in metabolic impairment. Race, gender and age interactions between monocytes and metabolic parameters were significant.

Female adipose tissue has improved adaptability and metabolic health compared to males in aged 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 Tgfß1 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.

Lactational High-Fat Diet Exposure Programs Metabolic Inflammation and Bone Marrow Adiposity in Male Offspring.

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.

Inflammatory responses to dietary and surgical weight loss in male and female mice.

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.

Sex Differences in Inflammatory Responses to Adipose Tissue Lipolysis in Diet-Induced Obesity.

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 ß-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.

TLR4, TRIF, and MyD88 are essential for myelopoiesis and CD11c+ adipose tissue macrophage production in obese mice.

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-ß (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.

Changes in Skeletal Integrity and Marrow Adiposity during High-Fat Diet and after Weight Loss.

The prevalence of obesity has continued to rise over the past three decades leading to significant increases in obesity-related medical care costs from metabolic and non-metabolic sequelae. It is now clear that expansion of body fat leads to an increase in inflammation with systemic effects on metabolism. In mouse models of diet-induced obesity, there is also an expansion of bone marrow adipocytes. However, the persistence of these changes after weight loss has not been well described. The objective of this study was to investigate the impact of high-fat diet (HFD) and subsequent weight loss on skeletal parameters in C57Bl6/J mice. Male mice were given a normal chow diet (ND) or 60% HFD at 6 weeks of age for 12, 16, or 20 weeks. A third group of mice was put on HFD for 12 weeks and then on ND for 8 weeks to mimic weight loss. After these dietary challenges, the tibia and femur were removed and analyzed by micro computed-tomography for bone morphology. Decalcification followed by osmium staining was used to assess bone marrow adiposity, and mechanical testing was performed to assess bone strength. After 12, 16, or 20 weeks of HFD, mice had significant weight gain relative to controls. Body mass returned to normal after weight loss. Marrow adipose tissue (MAT) volume in the tibia increased after 16 weeks of HFD and persisted in the 20-week HFD group. Weight loss prevented HFD-induced MAT expansion. Trabecular bone volume fraction, mineral content, and number were decreased after 12, 16, or 20 weeks of HFD, relative to ND controls, with only partial recovery after weight loss. Mechanical testing demonstrated decreased fracture resistance after 20 weeks of HFD. Loss of mechanical integrity did not recover after weight loss. Our study demonstrates that HFD causes long-term, persistent changes in bone quality, despite prevention of marrow adipose tissue accumulation, as demonstrated through changes in bone morphology and mechanical strength in a mouse model of diet-induced obesity and weight loss.

Cell therapy with a tissue-engineered kidney reduces the multiple-organ consequences of septic shock.

OBJECTIVE: Gram-negative septic shock has a clinical mortality rate approaching 50%. The cause of death is secondary to a systemic inflammatory response syndrome with resulting cardiovascular collapse, ischemic damage to vital organs, and multiple-organ systems failure. Renal tubule cell injury occurs early in septic shock but is not clinically appreciated. Since renal tubule cells appear to play a critical role in the immunoregulation of stress states, renal cell therapy during septic shock may alter the detrimental multiple-organ consequences of systemic Gram-negative infection. The development of a tissue-engineered bioartificial kidney consisting of a conventional hemofiltration cartridge in series with a renal tubule assist device (RAD) containing 109 renal proximal tubule cells may be a new therapeutic approach to this clinical disorder. DESIGN: Laboratory study. SETTING: University medical school. SUBJECTS: Pigs weighing 30-35 kg. INTERVENTIONS: To assess the effect of the bioartificial kidney and the RAD in septic shock, pigs were administered 30 x 10(10) bacteria/kg body weight of Escherichia coli into the peritoneal cavity and within 1 hr were immediately placed in a continuous venovenous hemofiltration extracorporeal circuit with either a sham RAD without cells or a RAD with cells. MEASUREMENTS AND MAIN RESULTS: In this animal model, septic shock resulted within hours in acute tubule necrosis in the kidneys of all animals. Renal cell therapy resulted in significantly higher cardiac outputs and renal blood flow rates in treated animals compared with sham controls. RAD treatment also was associated with significantly lower plasma circulating concentrations of interleukin-6 and interferon-gamma compared with sham-treated animals. IL-6 release rates from peripheral blood mononuclear cells isolated from RAD-treated animals were significantly higher after endotoxin stimulation than those isolated from control animals. These physiologic and molecular alterations were associated with nearly a doubling of the average survival time in the RAD-treated group compared with the sham control group. CONCLUSION: These results demonstrate that renal cell therapy ameliorates cardiac and vascular dysfunction, alters systemic cytokine abnormalities, and improves survival time in a large animal model of Gram-negative septic shock. A cell therapeutic approach with a tissue-engineered bioartificial kidney may be a new treatment modality for this current unmet medical need.

Bioartificial kidney ameliorates gram-negative bacteria-induced septic shock in uremic animals.

The bioartificial kidney (BAK) consists of a conventional hemofiltration cartridge in series with a renal tubule assist device (RAD) containing 10(9) porcine renal proximal tubule cells. BAK replaces filtration, transport, and metabolic and endocrinologic activities of a kidney. Previous work in an acutely uremic dog model demonstrated that BAK ameliorated endotoxin (lipopolysaccharide [LPS])-induced hypotension and altered plasma cytokine levels. To further assess the role of BAK in sepsis in acute renal failure, dogs were nephrectomized and 48 h later administered intraperitoneally with 30 x 10(10) bacteria/kg of E. coli. One hour after bacterial administration, animals were placed in a continuous venovenous hemofiltration circuit with either a sham RAD without cells (n = 6) or a RAD with cells (n = 6). BP, cardiac output, heart rate, pulmonary capillary wedge pressure, and systemic vascular resistance were measured throughout the study. All animals tested were in renal failure, with blood urea nitrogen and serum creatinine concentrations greater than 60 and 6 mg/dl, respectively. RAD treatment maintained significantly better cardiovascular performance, as determined by arterial BP (P < 0.05) and cardiac output (P < 0.02), for longer periods than sham RAD therapy. Consistently, all sham RAD-treated animals, except one, expired within 2 to 9 h after bacterial administration, whereas all RAD-treated animals survived more than 10 h. Plasma levels of TNF-alpha, IL-10, and C-reactive protein (CRP) were measured during cell RAD and sham RAD treatment. IL-10 levels were significantly higher (P < 0.01) during the entire treatment interval in the RAD animals compared with sham controls. These data demonstrated in a pilot large animal experiment that the BAK with RAD altered plasma cytokine levels in acutely uremic animals with septic shock. This change was associated with improved cardiovascular performance and increased survival time. These results demonstrate that the addition of cell therapy to hemofiltration in an acutely uremic animal model with septic shock ameliorates cardiovascular dysfunction, alters systemic cytokine balance, and improves survival time.