Adipocyte là gì

Katherine J. Strissel, Zlatina Stancheva, Hideaki Miyoshi, James W. Perfield II, Jason DeFuria, Zoe Jiông chồng, Andrew S. Greenberg and Martin S. ObinAddress correspondence & reprint requests to Martin S. Obin or Andrew S. Greenberg, USDA, HNRCA at Tufts University, Boston, MA 02111. E-mail: or



OBJECTIVE—We sought to determine the role of adipocyte death in obesity-induced adipose tissue (AT) inflammation and obesity complications.

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RESEARCH DESIGN AND METHODS—Male C57BL/6 mice were fed a high-fat diet for đôi mươi weeks to lớn induce obesity. Every 4 weeks, insulin resistance was assessed by intraperitoneal insulin tolerance tests, và epididymal (eAT) and inguinal subcutaneous AT (iAT) & livers were harvested for histological, immunohistochemical, and ren expression analyses.

RESULTS—Frequency of adipocyte death in eAT increased from

Adipose tissue (AT) macrophages (ATMΦs) promote obesity-associated AT inflammation and insulin resistance (1–5). Infiltrating ATMΦs secrete proinflammatory mediators that are elevated in AT of obese mice and humans & are implicated in the development of insulin resistance, including tumor necrosis factor (TNF)-α, interleukin (IL)-6, and monocyte chemotactic protein (MCP)-1 (5–9). In obese mice & humans, MΦs infiltrate AT as circulating monocytes in response to AT secretion of MCP-1, which recruits monocytes expressing the C-C chemokine receptor (CCR)2 (1,2,10–12,13). CCR2+ MΦs expressing the MΦ differentiation marker F4/80 & CD11c, a dendritic cell (DC) marker (13) upregulated in MΦ foam cells (14), were recently reported to lớn infiltrate AT of obese mice và were distinguished from noninflammatory resident ATMΦs (F4/80+/CCR2−/CD11c−) isolated from nonobese mice (12,15). However, the relationship between this obesity-associated ATMΦ “phenotypic switch” (12) & the development and progression of obesity complications is unclear (16,17).

In addition to lớn their roles in innate immunity, MΦs perkhung vital functions in developmental and homeostatic tissue remodeling (18,19). In AT, MΦs promote angiogenesis và vascular remodeling required for postnatal growth of mouse epididymal AT (eAT) (20). ATMΦ production of matrix-degrading proteinases is implicated in the matrix remodeling associated with adipocyte enlargement & AT expansion (21). We previously hypothesized that infiltrating ATMΦs play an important role(s) in obesity-associated AT remodeling, based on our observation that ATMΦs in obese mice and humans localize to lớn dead adipocytes, which increase in frequency in obesity (22). At sites of adipocyte death, ATMΦs aggregate to size a crown-lượt thích structure (CLS) that envelopes & ingests the moribund adipocyte and its potentially cytotoxic remnant lipid droplet (22). As a consequence of lipid scavenging, ATMΦs within CLS become lipid-laden “foam cells” (22). MΦ fusion within CLS is common, with multinucleate giant cells (MGCs) frequently observed (22). We propose that clearance of dead adipocytes by ATMΦs is an initial remodeling sự kiện required for AT repair và differentiation of new adipocytes at sites of adipocyte loss. MΦ-mediated cell killing is a feature of various forms of tissue remodeling (18), rendering it plausible that ATMΦs actively participate in adipocyte execution (22).

The clearance of dead adipocytes is likely to lớn promote proinflammatory ATMΦ activation, reflecting both the necrotic-like morphology of adipocyte death and ATMΦ fusion (22). MΦ fusion, which synergistically increases MΦ absorptive sầu capađô thị, requires TNF-α autocrine/paracrine signaling (23), suggesting that CLS and MGCs may be chronic sources of TNF-α. Moreover, because each dead adipocyte “recruits” dozens of ATMΦs, a low frequency of adipocyte death may be sufficient to cause AT inflammation & promote insulin resistance. However, the proinflammatory milieu at sites of tissue remodeling is typically transient, giving way to a “repair” program that promotes resolution of inflammation and tissue restoration (24). Tissue repair is characterized by MΦ upregulation of anti-inflammatory mediators such as IL-10, IL-4, và TGF-β (24). Thus, the character, magnitude, and physiological impact of obesity-associated AT inflammation will in part reflect the net balance of proinflammatory and anti-inflammatory inputs during AT remodeling at sites of adipocyte death.

Here, using a 20-week course of high-fat (HF) feeding in C57BL/6 mice, we demonstrate that adipocyte death is an early, progressive, & depot-dependent event in diet-induced obesity (DIO) that is significantly correlated with AT expansion, the ATMΦ phenotypic switch, AT inflammation, và development of whole-body toàn thân insulin resistance. Adipocyte death is prevalent in eAT, but not in inguinal AT (iAT), and is associated with matrix deposition & differentiation of new adipocytes. These observations suggest that obesity-associated remodeling in intra-abdominal AT contributes to inflammatory và metabolic complications of obesity (25,26).


Animals & diets.

Experiments were conducted in a viral pathogen–free facility at the Jean Mayer-U.S. Department of Agriculture Human Nutrition Research Center on Aging at Tufts University in accordance with institutional animal care và use committee guidelines. At 5 weeks of age, individually caged male C57BL/6 mice (The Jackson Laboratories) were established in weight-matched groups fed either a low-fat (LF) diet (10% calories from fat; Retìm kiếm Diets no. D12450B) or a HF diet (60% calories from fat; Retìm kiếm Diets no. D12492) for 1, 4, 8, 12, 16, or đôi mươi weeks.

Histology và immunohistochemistry.

Mice were killed by CO2 narcosis/cervical dislocation. Fat & liver were dissected, fixed, embedded in paraffin, & sectioned (22). Sections were stained with hematoxylin & eosin or with Gomori trichrome. Digital images were acquired with an Olympus DX51 light microscope. For each mouse, morphometric data were obtained from ≥500 adipocytes from three or more sections cut at least 50 μm apart. Immunohistochemistry was performed using VectaStain kits (Vector Labs). Antibodies were rat anti-mouse F4/80 (Serotec), Mac-2 (Cedarlane Labs), goat anti-mouse TNF-α, goat anti-mouse IL-6 (Santa Cruz), & rabbit anti-mouse perilipin (27). Negative controls were nonimmune IgG & peptide-neutralized primary antibody.

Adipocyte death.

Dead/dying adipocytes were identified by light microscopy as perilipin-negative lipid droplets surrounded by MΦ crowns (22). The frequency of adipocyte death was calculated from micrographs as (number dead adipocytes/number total adipocytes) × 100.

Adipocyte volume & adipocyte number.

Adipocyte volume was calculated from cross-sectional area obtained from perimeter tracings using Image J software (28) (Sun Microsystems). Adipocyte number was calculated from fat pad weight & adipocyte volume (29) và corrected for percentage of dead adipocytes.

Quantitative sầu PCR.

Adipose tissues (lymph nodes removed) were dissected, frozen in liquid nitrogen, và stored at −70°C. Total RNA was extracted, quantified, and analyzed by SYBR Green real-time PCR on an Applied Biosystems 7300 Real Time PCR system (30). Fold difference in gen expression was calculated as 2−ΔΔCt using cyclophilin B as the endogenous control gene with mice fed the LF diet for 1 week as the “comparer” (31). Genes & primer sequences are listed in Table 1.

Measures of insulin resistance.

Fasted (overnight) serum insulin was measured by enzyme-linked immunosorbent assay using mouse insulin as a standard (Crystal Chem). Intraperitoneal insulin tolerance tests (ITTs) were performed on nonanesthetized mice fasted for 4–6 h in the morning. Glucose measures were obtained from whole–tail vein blood using an automated glucometer at baseline và 15, 30, 45, 60, and 90 min following intraperitoneal injection of human insulin (0.75 mU/kg). Glucose area under the curve (AUC0–90) was determined for HF-fed & LF-fed cohorts and the difference (ΔAUC) reported.

Biohumoral measures.

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Blood was obtained from fasted (overnight) mice by cardiac puncture. Serum leptin, resistin, adiponectin, and MCP-1 were measured by enzyme-linked immunosorbent assay (Lincoplex mouse adipokine; Millipore Bioscience), & nonesterified fatty acids (NEFAs) were measured using the NEFA-C kit (Wako Chemicals).


ANOVA or general linear mã sản phẩm procedures were used in conjunction with Tukey’s honestly significant difference demo (SYSTAT v10). Frequency data were transformed as arcsin√x before statistical analysis. Significance was mix at P Fig. 1A). Dead adipocytes were identified by combined F4/80 và perilipin immunohistochemistry (22) (Fig. 1B). The frequency of adipocyte death during the development of DIO was determined in histological sections of eAT & iAT obtained at weeks 1, 4, 8, 12, 16, & 20 (Fig. 1C). The frequency of adipocyte death in eAT increased progressively from Fig. 1C and D). During this time, adipocyte death was robustly associated with adipocyte size (r = 0.94, P Fig. 2B). Despite a relatively high rate of adipocyte death at week 12, no areas không tính tiền of adipocytes were observed (Fig. 1C), và eAT mass continued khổng lồ increase in the majority of mice examined (Fig. 2D). These observations demonstrate that the frequency of adipocyte death increases progressively during DIO and suggest that sites of adipocyte death are successfully remodeled lớn maintain AT integrity and expansion.

By week 16, however, the rate of adipocyte death in eAT was ∼80% (Fig. 1C & D). At this time, many F4/80− stromal cells were observed in the interstitium between F4/80+ CLS (online appendix Fig. 1 ). Gomori trichrome staining revealed collagene deposition proximal lớn both viable & dead adipocytes, consistent with active sầu matrix remodeling at these sites (Fig. 1E). Both adipocyte number (Fig. 2A) & mean adipocyte size (Fig. 2B) decreased, & depot weight was reduced by 40% (Fig. 2D). Thus, at week 16, the rate of adipocyte death exceeded the rate of tissue repair resulting in net adipocyte and eAT loss.

By week đôi mươi, the frequency of adipocyte death had diminished fivefold lớn levels measured at week 12 (Fig. 1D), & adipocyte-miễn phí areas (Fig. 1C) và collagene staining of the interstitium between adipocytes (Fig. 1E) were rare. These observations indicate the resolution of remodeling with no apparent fibrosis. Notably, at week đôi mươi, adipocyte number of remodeled eAT was greater than fourfold the number observed at week 16 và was comparable lớn the adipocyte number at week 8 (Fig. 2A). A striking feature of remodeled eAT at week trăng tròn was the prevalence of small adipocytes (Fig. 2C). As a consequence, eAT mass remained reduced as compared with week 12, despite restoration of adipocyte number (Fig. 2A and D). In control mice receiving the LF diet, the frequency of adipocyte death never exceeded 1% in eAT (data not shown).

In contrast to lớn eAT, adipocyte death in iAT was not detectable until week 12 & never exceeded 3% (P > 0.05) (Fig. 1C and D). iAT mass increased continuously (>10-fold) throughout DIO, reflecting a 2-fold increase in adipocyte number (P Fig. 2B). The frequency of adipocyte death in iAT through week trăng tròn was positively correlated with adipocyte form size (r = 0.86, P 3 μm3>) was less than half that measured in eAT (Fig. 2B). In control mice fed the LF diet, the frequency of adipocyte death in iAT did not exceed 0.5% (data not shown).

Serum levels of leptin và resistin (but not adiponectin) were elevated after one week of HF feeding và thereafter (P Fig. 3). Serum levels of adipokines were not significantly associated with the frequency of adipocyte death during the DIO time course. However, the twofold increase in serum leptin at week 20 (P Fig. 2A) and with continued expansion of iAT (online appendix Fig. 2A and C). Fasting serum NEFAs (online appendix Fig. 4) increased by week 8 (P Fig. 2B & D & online appendix Fig. 2B & C); after week 12, fasting serum NEFAs eventually fell to lớn levels measured at week 4, coincident with loss of eAT mass.

F4/80 and CD11c ren expression tracks the time course of adipocyte death in eAT.

We next used real-time PCR analysis of AT khổng lồ investigate associations between the onset and progression of adipocyte death và changes in ATMΦ marker gene expression. After 4 weeks of DIO, the ATMΦ phenotype was characterized by low expression of F4/80, CD11c, CD68, và CD11b (Fig. 3). These observations suggest that most ATMΦs present in eAT after 4 weeks of DIO are resident (CD11c−) ATMΦs. By DIO week 8, the expression of F4/80, CD11c, & CD11b began lớn rise (Fig. 3). These data are consistent with monocyte recruitment khổng lồ eAT during the initial onset of adipocyte death (Fig. 1D) & with the subsequent differentiation of a submix of these monocytes into lớn F4/80+/CD11c+ ATMΦs. F4/80, CD11c, CD11b, và CD68 ren expression (Fig. 3) tracked the increased frequency of adipocyte death between weeks 8 and 12 (Fig. 1D). As previously shown (22), foamy lipid-laden MΦs within CLS became frequent during this time (data not shown), potentially contributing to elevated CD11c expression by ATMΦs (14). Between weeks 12 và trăng tròn, the temporal pattern of ATMΦ marker ren expression became more dynamic và complex (Fig. 3). Whereas F4/80 expression remained elevated, CD11c expression diminished after week 16. CD11b expression fell progressively between weeks 12 và đôi mươi. In contrast, CD68 ren expression rose progressively through week trăng tròn (Fig. 3) (see discussion).

In iAT, ATMΦ marker gene expression remained low throughout the DIO time course. We noted a modest (approximate twofold) upward trkết thúc in F4/80, CD68, and CD11c expression in iAT between weeks 12 và đôi mươi (online appendix Fig. 2D), coincident with the onset of adipocyte death beginning at week 12 (Fig. 1D). Overall, however, the low frequency of adipocyte death in iAT (Fig. 1C & D) was associated with low ATMΦ gen expression.

Adipocyte death induces ATMΦ expression of proinflammatory hallmarks of obesity.

If adipocyte death is an underlying cause of AT inflammation, CLS surrounding dead adipocytes should express inflammatory mediators that are upregulated in ATMΦs of obese mice. Moreover, the expression levels of these mediators should be positively associated with the frequency of obesity-induced adipocyte death in eAT. Immunohistochemistry (Fig. 4A) demonstrates expression of both TNF-α and IL-6 protein by ATMΦs arranged in CLS around remnant lipid droplets of dead adipocytes. cảnh báo that all ATMΦs within each CLS stain positive for the target cytokine. MGCs also expressed proinflammatory cytokines (Fig. 4B). Thus, scavenging of dead adipocytes is associated with ATMΦ expression of cytokines implicated in the development of insulin resistance.

Moreover, real-time PCR analysis of eAT revealed significant correlations between the frequency of adipocyte death & the expression of inflammatory mediators throughout the DIO time course (Fig. 5). Inflammatory mediators included TNF-α (r = 0.78, P 2-fold (P Fig. 5). lưu ý that gene expression of each of these mediators was maximal at week 16 (coincident with maximal frequency of adipocyte death D>) and then fell at week 20 to levels comparable with or approaching ren expression measured at week 12 (when the frequency of adipocyte death was comparable with that at week 20 D>). Also note that IL-10 expression tracked the temporal pattern of TNF-α và MCP-1 expression (Fig. 5), indicating that anti-inflammatory mechanisms are upregulated in eAT, coincident with the progression of obesity-associated adipocyte death, ATMΦ recruitment, and proinflammatory gene expression.

In iAT, DIO-associated TNF-α gen expression was not detected until week 12 (data not shown), and its magnitude remained modest through week đôi mươi (2.5 ± 0.2-fold, P Fig. 1CD). Similarly, IL-10 gene expression did not increase in iAT during the DIO time course & was comparable with levels measured in LF-fed mice (data not shown).

Adipocyte death is associated with insulin resistance.

The initial increase in adipocyte death in eAT between weeks 4 & 8 was coincident with the onphối and progression of hyperinsulinemia & whole-body toàn thân insulin resistance. At week 4, we detected nonsignificant trends for increased fasting serum insulin (Table 2) & for glucose AUC between HF-fed and LF-fed mice (ΔAUC) (P > 0.05) (Table 2). By DIO week 8, fasting insulin levels (Table 2) và homeostasis Mã Sản Phẩm assessment of insulin resistance values (not shown) were significantly elevated (P Table 2). The development of whole-toàn thân insulin resistance by week 8 was coincident with upregulated expression of ATMΦ marker genes CD11b & CD11c (Fig. 3) và TNF-α (Fig. 5). Note that no increases in adipocyte death or ATMΦ infiltration were observed in iAT during the development of insulin resistance (Fig. 1CD). Furthermore, insulin resistance was manifest at week 8 before the observed net loss of adipocytes detected in eAT beginning at week 12 (Fig. 2B). This observation argues against lipoatrophy as a contributing factor in the onset of systemic insulin resistance in this study. Fasting serum insulin levels remained essentially unchanged after week 8 (Table 2). However, insulin resistance continued lớn rise through week 12 (P Table 2), coincident with attainment of maximal adipocyte form size (Fig. 2B), increases in adipocyte death (Fig. 1D), increased expression of F4/80 (Fig. 3) and TNF-α (Fig. 5), and a threefold reduction in adiponectin gen expression (data not shown).

Surprisingly, there was no significant increase in insulin resistance between weeks 12 và 16 (Table 2), although expression of ATMΦ marker genes in eAT remained elevated (Fig. 3), TNF-α gen expression increased (Fig. 5), and adiponectin gene expression was downregulated ∼threefold (data not shown). However, fasting levels of serum NEFAs were significantly reduced at this time (online appendix Fig. 4). By week 20, insulin resistance was improved relative sầu to week 16, with glucose ΔAUC obtained in the ITT of 20-week mice comparable with those obtained at week 8 (Table 2). This attenuated insulin resistance coincided with (partial) resolution of eAT remodeling, characterized by reduced frequency of adipocyte death (Fig. 1D), attenuated expression of CD11b và CD11c (Fig. 3), reduced adipocyte size & depot mass (Fig. 2A, C, và D), và further reductions in serum NEFAs (online appendix Fig. 4). However, F4/80 và TNF-α gen expression in eAT remained elevated at week 20 (Figs. 3 and 5), and adiponectin gen expression remained reduced relative sầu to lớn lean controls (data not shown).

eAT remodeling and hepatic steatosis.

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Clinical loss of adipose mass (lipoatrophy) is associated with hepatomegaly due khổng lồ steatosis (32,33), as is chronic obesity. Beginning at week 12, DIO resulted in a progressive sầu & significant loss of eAT mass (Fig. 2C). Moreover, this loss of mass was coincident with an accelerated increase in liver weight (Fig. 6A). Liver weight was robustly inversely associated with eAT weight beginning at week 12 (r = −0. 85, P Fig. 6B). Consistent with these observations, macrosteatosis was rare in livers of HF-fed mice up lớn week 12 but was widespread thereafter (Fig. 6C). These results suggest that loss of eAT mass due to lớn remodeling may contribute to lớn hepatic steatosis induced by HF-feeding.

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