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 Ͻ0.1% at baseline to 16% at week 12, coincident with increases in 1) depot weight; 2) AT macrophages (ATM⌽s) expressing F4/80 and CD11c; 3) mRNA for tumor necrosis factor (TNF)-␣, monocyte chemotactic protein (MCP)-1, and interleukin (IL)-10; and 4) insulin resistance. ATM⌽s in crown-like structures surrounding dead adipocytes expressed TNF-␣ and IL-6 proteins. Adipocyte number began to decline at week 12. At week 16, adipocyte death reached ϳ80%, 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 ϭ Ϫ0. 85, P Ͻ 0.001). In iAT, adipocyte death was first detected at week 12 and remained Յ3%.CONCLUSIONS-These results implicate depot-selective adipocyte death and M⌽-mediated AT remodeling in inflammatory and metabolic complications of murine obesity. Diabetes

Structural disruption of gut microbiota and associated inflammation are considered important etiological factors in high fat diet (HFD)-induced metabolic syndrome (MS). Three candidate probiotic strains, Lactobacillus paracasei CNCM I-4270 (LC), L. rhamnosus I-3690 (LR) and Bifidobacterium animalis subsp. lactis I-2494 (BA), were individually administered to HFD-fed mice (108 cells day−1) for 12 weeks. Each strain attenuated weight gain and macrophage infiltration into epididymal adipose tissue and markedly improved glucose–insulin homeostasis and hepatic steatosis. Weighted UniFrac principal coordinate analysis based on 454 pyrosequencing of fecal bacterial 16S rRNA genes showed that the probiotic strains shifted the overall structure of the HFD-disrupted gut microbiota toward that of lean mice fed a normal (chow) diet. Redundancy analysis revealed that abundances of 83 operational taxonomic units (OTUs) were altered by probiotics. Forty-nine altered OTUs were significantly correlated with one or more host MS parameters and were designated ‘functionally relevant phylotypes'. Thirteen of the 15 functionally relevant OTUs that were negatively correlated with MS phenotypes were promoted, and 26 of the 34 functionally relevant OTUs that were positively correlated with MS were reduced by at least one of the probiotics, but each strain changed a distinct set of functionally relevant OTUs. LC and LR increased cecal acetate but did not affect circulating lipopolysaccharide-binding protein; in contrast, BA did not increase acetate but significantly decreased adipose and hepatic tumor necrosis factor-α gene expression. These results suggest that Lactobacillus and Bifidobacterium differentially attenuate obesity comorbidities in part through strain-specific impacts on MS-associated phylotypes of gut microbiota in mice.

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.immunometabolism | lymphocytes M ultiple studies support the concept that inflammation strongly associates with insulin resistance (IR), which, in addition to loss of islet function, defines type 2 diabetes (T2D) (1). Work implicating B cells in IR/T2D is limited. We showed B cells from T2D subjects secrete a proinflammatory cytokine profile, including an extraordinary inability to secrete the potent anti-inflammatory cytokine IL-10 and an elevated production of proinflammatory IL-8 compared with B cells from non-T2D subjects (2). Given the importance of B-cell IL-10 in preventing numerous inflammatory diseases (3, 4) and the links between IL-8 and T2D (5, 6), these data suggest that altered B-cell cytokine production plays an important role in initiating or promoting IR/T2D. Published analyses further support a role for B cells in IR and include studies of B cell-null New Zealand Obese (NZO) mice, which, in contrast to B cell-sufficient NZOs, fail to develop IR in response to obesity (7). These findings have been recently reproduced in studies showing obese B cell-null or B cell-depleted mice have less inflammation and IR than obese WT mice (8). Interestingly, T-cell cytokine production is decreased in obese B cell-null mouse adipose tissue (AT) (8), which raises the possibility that, in addition to production of a proinflammatory cytokine profile, B cells may function in IR by regulating the T cell-mediated inflammation known to drive disease pathogenesis (9, 10). We identified a proinflammatory T-cell ratio [defined by increased Th17 cells plus decreased regulatory T cells (Tregs)] in T2D patients that mirror...

We report a new cellular mechanism of rod photoreceptor adaptation in vivo, which is triggered by daylight levels of illumination. The mechanism involves a massive light-dependent translocation of the photoreceptor-specific G protein, transducin, between the functional compartments of rods. To characterize the mechanism, we developed a novel technique that combines serial tangential cryodissection of the rat retina with Western blot analysis of protein distribution in the sections. Up to 90% of transducin translocates from rod outer segments to other cellular compartments on the time scale of tens of minutes. The reduction in the transducin content of the rod outer segments is accompanied by a corresponding reduction in the amplification of the rod photoresponse, allowing rods to operate in illumination up to 10-fold higher than would otherwise be possible.

Hormone-sensitive lipase (HSL) is the predominant lipase effector of catecholamine-stimulated lipolysis in adipocytes. HSL-dependent lipolysis in response to catecholamines is mediated by protein kinase A (PKA)-dependent phosphorylation of perilipin A (Peri A), an essential lipid droplet (LD)-associated protein.It is believed that perilipin phosphorylation is essential for the translocation of HSL from the cytosol to the LD, a key event in stimulated lipolysis. Using adipocytes retrovirally engineered from murine embryonic fibroblasts of perilipin null mice (Peri؊/؊ MEF), we demonstrate by cell fractionation and confocal microscopy that up to 50% of cellular HSL is LD-associated in the basal state and that PKA-stimulated HSL translocation is fully supported by adenoviral expression of a mutant perilipin lacking all six PKA sites (Peri A⌬1-6). PKA-stimulated HSL translocation was confirmed in differentiated brown adipocytes from perilipin null mice expressing an adipose-specific Peri A⌬1-6 transgene. Thus, PKA-induced HSL translocation was independent of perilipin phosphorylation. However, Peri A⌬1-6 failed to enhance PKA-stimulated lipolysis in either MEF adipocytes or differentiated brown adipocytes. Thus, the lipolytic action(s) of HSL at the LD surface requires PKA-dependent perilipin phosphorylation. In Peri؊/؊ MEF adipocytes, PKA activation significantly enhanced the amount of HSL that could be cross-linked to and co-immunoprecipitated with ectopic Peri A. Notably, this enhanced cross-linking was blunted in Peri؊/؊ MEF adipocytes expressing Peri A⌬1-6. This suggests that PKA-dependent perilipin phosphorylation facilitates (either direct or indirect) perilipin interaction with LD-associated HSL. These results redefine and expand our understanding of how perilipin regulates HSL-mediated lipolysis in adipocytes.The enzymatic hydrolysis of stored neutral lipid in adipocytes is an exquisitely regulated process that maintains whole body energy homeostasis in response to physiological demands. In both white and brown adipose tissue (BAT) 4 basal (constitutive) rates of adipocyte lipolysis are rapidly and dramatically up-regulated by lipolytic hormones such as catecholamines (1, 2). In white adipose tissue, catecholamine-induced lipolysis provides fatty acids as fuel to peripheral tissues during times of energy need, such as fasting and exercise (3-5). In BAT of human newborns and rodents, catecholamine-stimulated lipolysis provides fatty acids for heat production (via ␤-oxidation and mitochondrial uncoupling) in response to hypothermia (i.e. adaptive thermogenesis) (2, 6). Fatty acids that are released during adipocyte lipolysis also function as modulators of glucose and insulin action and insulin production (7,8). Moreover, the dysregulated release of fatty acids from adipocytes that occurs in obesity is implicated in the etiology of obesity-related complications, including type 2 diabetes (8 -10). Thus, in addition to its role in whole body energy homeostasis, the regulation of adipocyte lipolysis is vital to me...

OBJECTIVETo identify, localize, and determine M1/M2 polarization of epidydimal adipose tissue (eAT) macrophages (Φs) during high-fat diet (HFD)-induced obesity.RESEARCH DESIGN AND METHODSMale C57BL/6 mice were fed an HFD (60% fat kcal) or low-fat diet (LFD) (10% fat kcal) for 8 or 12 weeks. eATMΦs (F4/80+ cells) were characterized by in vivo fluorescent labeling, immunohistochemistry, fluorescence-activated cell sorting, and quantitative PCR.RESULTSRecruited interstitial macrophage galactose-type C-type lectin (MGL)1+/CD11c− and crown-like structure–associated MGL1−/CD11c+ and MGL1med/CD11c+ eATMΦs were identified after 8 weeks of HFD. MGL1med/CD11c+ cells comprised ∼65% of CD11c+ eATMΦs. CD11c+ eATMΦs expressed a mixed M1/M2 profile, with some M1 transcripts upregulated (IL-12p40 and IL-1β), others downregulated (iNOS, caspase-1, MCP-1, and CD86), and multiple M2 and matrix remodeling transcripts upregulated (arginase-1, IL-1Ra, MMP-12, ADAM8, VEGF, and Clec-7a). At HFD week 12, each eATMΦ subtype displayed an enhanced M2 phenotype as compared with HFD week 8. CD11c+ subtypes downregulated IL-1β and genes mediating antigen presentation (I-a, CD80) and upregulated the M2 hallmark Ym-1 and genes promoting oxidative metabolism (PGC-1α) and adipogenesis (MMP-2). MGL1med/CD11c+ eATMΦs upregulated additional M2 genes (IL-13, SPHK1, CD163, LYVE-1, and PPAR-α). MGL1med/CD11c+ ATMΦs expressing elevated PGC-1α, PPAR-α, and Ym-1 transcripts were selectively enriched in eAT of obese mice fed pioglitazone for 6 days, confirming the M2 features of the MGL1med/CD11c+ eATMΦ transcriptional profile and implicating PPAR activation in its elicitation.CONCLUSIONSThese results 1) redefine the phenotypic potential of CD11c+ eATMΦs and 2) suggest previously unappreciated phenotypic and functional commonality between murine and human ATMΦs in the development of obesity and its complications.

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.

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 (60% energy) fat diet (HFD). By week 6, eAT mass and stromal vascular cell (SVC) number increased 3-fold 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, IFNγ, 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 6-fold by week 8 (p < 0.01). SVC enrichment for T cells (predominantly CD4+ and CD8+) and elevated IFNγ 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 (1) elevated (5-fold) IL-12p40 gene expression in eAT by week 12 (p ≤ 0.01) and (2) greater IFNγ secretion from PMA/ionophorestimulated eAT explants at week 6 (1 fold, p = 0.08) and week 12 (5 fold, p < 0.001). In summary, T cell enrichment and IFNγ gene induction occur subsequent to ATMφ recruitment, onset of IR and resolution of eAT remodeling. However, enhanced priming for IFNγ production suggests the contribution of CD4+ and/or CD8+ effectors to cell-mediated immune responses promoting HFDinduced AT inflammation and IR.