Jason DeFuria

Adipocyte Death, Adipose Tissue Remodeling and Obesity Complications

OBJECTIVE—We sought to determine the role of adipocyte death in obesity-induced adipose tissue (AT) inflammation and obesity complications. RESEARCH DESIGN AND METHODS—Male C57BL/6 mice were fed a high-fat diet for 20 weeks to induce obesity. Every 4 weeks, insulin resistance was assessed by intraperitoneal insulin tolerance tests, and epididymal (eAT) and inguinal subcutaneous AT (iAT) and livers were harvested for histological, immunohistochemical, and gene expression analyses. RESULTS—Frequency of adipocyte death in eAT increased from <0.1% at baseline to 16% at week 12, coincident with increases in 1) depot weight; 2) AT macrophages (ATMΦs) expressing F4/80 and CD11c; 3) mRNA for tumor necrosis factor (TNF)-α, monocyte chemotactic protein (MCP)-1, and interleukin (IL)-10; and 4) insulin resistance. ATMΦs in crown-like structures surrounding dead adipocytes expressed TNF-α and IL-6 proteins. Adipocyte number began to decline at week 12. At week 16, adipocyte death reached ∼80%, coincident with maximal expression of CD11c and inflammatory genes, loss (40%) of eAT mass, widespread collagen deposition, and accelerated hepatic macrosteatosis. By week 20, adipocyte number was restored with small adipocytes, coincident with reduced adipocyte death (fourfold), CD11c and MCP-1 gene expression (twofold), and insulin resistance (35%). eAT weight did not increase at week 20 and was inversely correlated with liver weight after week 12 (r = −0. 85, P < 0.001). In iAT, adipocyte death was first detected at week 12 and remained ≤3%. CONCLUSIONS—These results implicate depot-selective adipocyte death and MΦ-mediated AT remodeling in inflammatory and metabolic complications of murine obesity.

B cells promote inflammation in obesity and type 2 diabetes through regulation of T-cell function and an inflammatory cytokine profile

Patients with type 2 diabetes (T2D) have disease-associated changes in B-cell function, but the role these changes play in disease pathogenesis is not well established. Data herein show B cells from obese mice produce a proinflammatory cytokine profile compared with B cells from lean mice. Complementary in vivo studies show that obese B cell–null mice have decreased systemic inflammation, inflammatory B- and T-cell cytokines, adipose tissue inflammation, and insulin resistance (IR) compared with obese WT mice. Reduced inflammation in obese/insulin resistant B cell–null mice associates with an increased percentage of anti-inflammatory regulatory T cells (Tregs). This increase contrasts with the sharply decreased percentage of Tregs in obese compared with lean WT mice and suggests that B cells may be critical regulators of T-cell functions previously shown to play important roles in IR. We demonstrate that B cells from T2D (but not non-T2D) subjects support proinflammatory T-cell function in obesity/T2D through contact-dependent mechanisms. In contrast, human monocytes increase proinflammatory T-cell cytokines in both T2D and non-T2D analyses. These data support the conclusion that B cells are critical regulators of inflammation in T2D due to their direct ability to promote proinflammatory T-cell function and secrete a proinflammatory cytokine profile. Thus, B cells are potential therapeutic targets for T2D.

Dietary Blueberry Attenuates Whole-Body Insulin Resistance in High Fat-Fed Mice by Reducing Adipocyte Death and Its Inflammatory Sequelae

Adipose tissue (AT) inflammation promotes insulin resistance (IR) and other obesity complications. AT inflammation and IR are associated with oxidative stress, adipocyte death, and the scavenging of dead adipocytes by proinflammatory CD11c+ AT macrophages (ATMPhi). We tested the hypothesis that supplementation of an obesitogenic (high-fat) diet with whole blueberry (BB) powder protects against AT inflammation and IR. Male C57Bl/6j mice were maintained for 8 wk on 1 of 3 diets: low-fat (10% of energy) diet (LFD), high-fat (60% of energy) diet (HFD) or the HFD containing 4% (wt:wt) whole BB powder (1:1 Vaccinium ashei and V. corymbosum) (HFD+B). BB supplementation (2.7% of total energy) did not affect HFD-associated alterations in energy intake, metabolic rate, body weight, or adiposity. We observed an emerging pattern of gene expression in AT of HFD mice indicating a shift toward global upregulation of inflammatory genes (tumor necrosis factor-alpha, interleukin-6, monocyte chemoattractant protein 1, inducible nitric oxide synthase), increased M1-polarized ATMPhi (CD11c+), and increased oxidative stress (reduced glutathione peroxidase 3). This shift was attenuated or nonexistent in HFD+B-fed mice. Furthermore, mice fed the HFD+B were protected from IR and hyperglycemia coincident with reductions in adipocyte death. Salutary effects of BB on adipocyte physiology and ATMPhi gene expression may reflect the ability of BB anthocyanins to alter mitogen-activated protein kinase and nuclear factor-kappaB stress signaling pathways, which regulate cell fate and inflammatory genes. These results suggest that cytoprotective and antiinflammatory actions of dietary BB can provide metabolic benefits to combat obesity-associated pathology.

T Cell Recruitment and Th1 Polarization in Adipose Tissue During Diet-Induced Obesity in C57BL/6 mice

The role of adaptive immunity in obesity‐associated adipose tissue (AT) inflammation and insulin resistance (IR) is controversial. We employed flow cytometry and quantitative PCR to assess T‐cell recruitment and activation in epididymal AT (eAT) of C57BL/6 mice during 4–22 weeks of a high‐fat diet (HFD (60% energy)). By week 6, eAT mass and stromal vascular cell (SVC) number increased threefold in mice fed HFD, coincident with onset of IR. We observed no increase in the proportion of CD3+ SVCs or in gene expression of CD3, interferon‐γ (IFN‐γ), or regulated upon activation, normal T‐cell expressed and secreted (RANTES) during the first 16 weeks of HFD. In contrast, CD11c+ macrophages (MΦ) were enriched sixfold by week 8 (P < 0.01). SVC enrichment for T cells (predominantly CD4+ and CD8+) and elevated IFN‐γ and RANTES gene expression were detected by 20–22 weeks of HFD (P < 0.01), coincident with the resolution of eAT remodeling. HFD‐induced T‐cell priming earlier in the obesity time course is suggested by (i) elevated (fivefold) interleukin‐12 (IL‐12)p40 gene expression in eAT by week 12 (P ≤ 0.01) and (ii) greater IFN‐γ secretion from phorbol myristate acetate (PMA)/ionophore‐stimulated eAT explants at week 6 (onefold, P = 0.08) and week 12 (fivefold, P < 0.001). In conclusion, T‐cell enrichment and IFN‐γ gene induction occur subsequent to AT macrophage (ATMΦ) recruitment, onset of IR and resolution of eAT remodeling. However, enhanced priming for IFN‐γ production suggests the contribution of CD4+ and/or CD8+ effectors to cell‐mediated immune responses promoting HFD‐induced AT inflammation and IR.

Adipose tissue inflammation and reduced insulin sensitivity in ovariectomized mice occurs in the absence of increased adiposity.

Menopause promotes central obesity, adipose tissue (AT) inflammation, and insulin resistance (IR). Both obesity and the loss of estrogen can activate innate and adaptive immune cells (macrophages, T cells). The respective impacts of weight gain and loss of ovarian hormones on AT inflammation and IR are poorly understood. Here we determined the temporal kinetics of fat accretion, AT inflammation, and IR over a 26-wk time course in ovariectomized (OVX) mice, a model of menopause. OVX and sham-operated (SHM) C57BL6 mice were fed a normal chow diet. Weight, body composition (magnetic resonance imaging), total and regional adiposity, activity, food intake, AT crown-like structures, biohumoral measures, and insulin sensitivity (insulin tolerance testing and homeostatic model assessment) were determined at wk 12, 20, and 26. Macrophages and T cells from perigonadal AT were immunophenotyped by fluorescence-associated cell sorting, and perigonadal adipose tissue (PGAT) gene expression was quantified by quantitative PCR. OVX mice (≈ 31 g) became fatter than SHM mice (≈ 26 g) by wk 12, but mice were equally insulin sensitive. PGAT of OVX mice contained more T cells but expressed higher levels of M2-MΦ (arginase-1) and T cell-regulatory (cytotoxic T-lymphocyte antigen 4) genes. At wk 20, both OVX and SHM mice weighed approximately 35 g and were equally insulin sensitive with comparable amounts of PGAT and total body fat. OVX mice became less insulin sensitive than SHM mice by wk 26, coincident with the down-regulation of PGAT arginase-1 (-20-fold) and cytotoxic T-lymphocyte antigen 4 (2-fold) and up-regulation of M1/Th1 genes CD11c (+2-fold), IL12p40 (+2-fold), and interferon-γ (+78-fold). Ovarian hormone loss in mice induces PGAT inflammation and IR by mechanisms that can be uncoupled from OVX-induced obesity.

Extended lifespan and reduced adiposity in mice lacking the FAT10 gene.

Significance For the first time we describe a physiological role for HLA-F adjacent transcript 10 (FAT10) in metabolism, obesity, and aging in mammals. We show that FAT10 knockout prevents the development of age-associated obesity in mice while extending lifespan and vigor without the appearance of deleterious developmental effects. In addition, we did not observe an increase in cancer incidence. If the role of FAT10 in humans is similar to mice, then targeting of FAT10 may hold promising therapeutic impact for the treatment of various diseases including obesity and obesity-related diseases and aging associated diseases. The HLA-F adjacent transcript 10 (FAT10) is a member of the ubiquitin-like gene family that alters protein function/stability through covalent ligation. Although FAT10 is induced by inflammatory mediators and implicated in immunity, the physiological functions of FAT10 are poorly defined. We report the discovery that FAT10 regulates lifespan through pleiotropic actions on metabolism and inflammation. Median and overall lifespan are increased 20% in FAT10ko mice, coincident with elevated metabolic rate, preferential use of fat as fuel, and dramatically reduced adiposity. This phenotype is associated with metabolic reprogramming of skeletal muscle (i.e., increased AMP kinase activity, β-oxidation and -uncoupling, and decreased triglyceride content). Moreover, knockout mice have reduced circulating glucose and insulin levels and enhanced insulin sensitivity in metabolic tissues, consistent with elevated IL-10 in skeletal muscle and serum. These observations suggest novel roles of FAT10 in immune metabolic regulation that impact aging and chronic disease.

B cells promote obesity-associated periodontitis and oral pathogen-associated inflammation

Individuals with T2D and PD suffer significantly from the ability of one disease to intensify the other. Disease‐associated inflammation is one mechanism thought to fuel this pathogenic feed‐forward loop. Several lines of evidence indicate that proinflammatory B cells promote T2D and PD; thus, B cells are top candidates for a cell type that predisposes PD in T2D. To test directly the role of B cells in T2D‐associated PD, we compared outcomes from oral Porphyromonas gingivalis challenge of lean WT or B cell‐null mice with outcomes from mice that were obese and insulin‐resistant before challenge. Obese WT mice responded to oral P. gingivalis challenge with significant periodontal bone loss, whereas obese B cell‐null mice were protected completely from PD. By contrast, lean WT and B cell‐null mice suffer similar periodontal bone loss in response to oral pathogen. B cells from obese/insulin‐resistant hosts also support oral osteoclastogenesis and both oral and systemic production of inflammatory cytokines, including pro‐osteoclastogenic TNF‐α and MIP‐2, an ortholog of human IL‐8. B cells furthermore impact AT inflammation in obese, P. gingivalis‐infected hosts. Taken together, these data show that fundamentally different mechanisms regulate PD in lean and obese hosts, with B cells able to promote PD only if the hosts are “primed” by obesity. These results justify more intense analysis of obesity‐associated changes in B cells that predispose PD in human T2D.

Neurogenin 3-Specific Dipeptidyl Peptidase-2 Deficiency Causes Impaired Glucose Tolerance, Insulin Resistance, and Visceral Obesity

The control of glucose metabolism is a complex process, and dysregulation at any level can cause impaired glucose tolerance and insulin resistance. These two defects are well-known characteristics associated with obesity and onset of type 2 diabetes. Here we introduce the N-terminal dipeptidase, DPP2, as a novel regulator of the glucose metabolism. We generated mice with a neurogenin 3 (NGN3)-specific DPP2 knockdown (kd) to explore a possible role of DPP2 in maintaining metabolic homeostasis. These mice spontaneously developed hyperinsulinemia, glucose intolerance, and insulin resistance by 4 months of age. In addition, we observed an increase in food intake in DPP2 kd mice, which was associated with a significant increase in adipose tissue mass and enhanced liver steatosis but no difference in body weight. In accordance with these findings, the mutant mice had a higher rate of respiratory exchange than the control littermates. This phenotype was exacerbated with age and when challenged with a high-fat diet. We report, for the first time, that DPP2 enzyme activity is essential for preventing hyperinsulinemia and maintaining glucose homeostasis. Interestingly, the phenotype of NGN3-DPP2 kd mice is opposite that of DPP4 knockout mice with regard to glucose metabolism, namely the former have normal glucagon-like peptide 1 levels but present with glucose intolerance, whereas the latter have increased glucagon-like peptide 1, which is accompanied by augmented glucose tolerance.

Deletion of TNF-like weak inducer of apoptosis (TWEAK) protects mice from adipose and systemic impacts of severe obesity.

To investigate the role of TNF‐like weak inducer of apoptosis (TWEAK) in pathological adipose tissue (AT) remodeling and complications of obesity.

B lymphocytes regulate a pro-inflammatory T cell balance in human Type 2 diabetes (P3101)

Wun-Ju Shieh graduated from Taipei Medical University in 1979. He completed an internal medicine residency and infectious disease subspecialty training in 1986. He received a Master of Public Health from Harvard University in 1987, followed by a Ph.D. in Microbiology & Immunology from Vanderbilt University in 1992. Afterwards, he completed a combined anatomical and clinical pathology residency training at Vanderbilt University Medical Center and an infectious disease pathology fellowship at CDC. He has been working as a medical officer and pathologist at CDC since 1995. He has participated many outbreak investigations, and has published more than 120 papers in peer-review journals. The role of pathology in diagnosis of emerging viral zoonosesChi-Chao Chan earned her M.D. from Johns Hopkins University and ophthalmology residency from Stanford University School of Medicine. She has completed two post-doctoral fellowships: ophthalmic pathology at Wilmer Institute, Johns Hopkins and clinical ocular immunology at National Eye Institute, National Institutes of Health. She is the Chief of Immunopathology Section, Laboratory of Immunology and Head of Histopathology Core, National Eye Institute, the federal government medical research institute in the US. She has published 582 papers in peer-reviewed journals, 51 book chapters, and one textbook. She also serves as an editorial board member for 16 medical journals. Diagnosis of primary vitreoretinal lymphoma, a subtype of primary CNS lymphomaEnterohemorrhagic Escherichia coli (EHEC) produce one or more types of Shiga toxins and are foodborne causes of bloody diarrhea. The prototype EHEC strain, Escherichia coli O157:H7, is responsible for both sporadic cases and serious outbreaks worldwide. Infection with E. coli that produce Shiga toxins may lead to diarrhea, hemorrhagic colitis, or (less frequently) hemolytic uremic syndrome, which can cause acute kidney failure. The exact mechanism by which EHEC evokes intestinal and renal disease has not yet been determined. The development of a readily reproducible animal oral-infection model with which to evaluate the full pathogenic potential of E. coli O157:H7 and assess the efficacy of therapeutics and vaccines remains a research priority. Dutch belted (DB) rabbits are reported to be susceptible to both natural and experimental EHEC-induced disease, and New Zealand white (NZW) rabbits are a model for the intestinal manifestations of EHEC infection. In the current study, we compared the pathology caused by E. coli O157:H7 infection in DB and NZW rabbits. Both breeds of rabbits developed clinical signs of disease and intestinal lesions after experimental infection. In addition, one of the infected DB rabbits developed renal lesions. Our findings provide evidence that both breeds are susceptible to E. coli O157:H7 infection and that both may be useful models for investigating EHEC infections of humans.Lymphocytes play key roles in the chronic inflammation critical for T2D pathogenesis. We have shown T2D patients have an elevated ratio of pro- to anti-inflammatory T cells, and B cells that produce a pro-inflammatory cytokine profile. Thus lymphocytes promote T2D-associated inflammation. Numerous studies implicate the pro-inflammatory CD4+ T cell balance in T2D pathogenesis, but mechanisms that underlie elevated CD4+ T cell inflammation are poorly understood. We explored the possibility that the T2D-associated changes we identified in B cell function regulate T cell inflammation. We show that B cells control the T2D-associated increase in Th17-mediated inflammation in T2D patients and in obese/insulin resistant mice. Surprisingly, the disease-associated ability of B cells to regulate T cell function is contact-dependent, despite evidence that B cell cytokines hyper-secreted in T2D patients activate T cells. In contrast, elevated activation of Th1 cytokines is B cell-independent. We conclude that both T cell-intrinsic and T cell-extrinsic (B cell-dependent) changes regulate T cell inflammation in T2D. These data indicate that B cell depletion may partially curb T2D-associated T cell inflammation, and thus disease pathogenesis; however, combinatorial treatments aimed at multiple inflammatory axes may be required for favorable clinical outcomes.