Dead adipocytes, detected as crown-like structures, are prevalent in visceral fat depots of genetically obese mice

Accumulation of visceral fat is a key phenomenon in the onset of obesity-associated metabolic disorders. Macrophage infiltration induces chronic mild inflammation widely considered as a causative factor for insulin resistance and eventually diabetes. We previously showed that >90% of macrophages infiltrating the adipose tissue of obese animals and humans are arranged around dead adipocytes, forming characteristic crown-like structures (CLS). In this study we quantified CLS in visceral and subcutaneous depots from two strains of genetically obese mice, db/db and ob/ob. In both strains, CLS were prevalent in visceral compared with subcutaneous fat. Adipocyte size and CLS density exhibited a positive correlation both in visceral and in subcutaneous depots; however, the finding that adipocyte size was smallest and CLS density highest in visceral fat suggests a different susceptibility of visceral and subcutaneous adipocytes to death. Visceral fat CLS density was 3.4-fold greater in db/db than in ob/ob animals, which at the age at which our experimental strain was used are more prone to glucose metabolic disorders.     

Revascularization determines volume retention and gene expression by fat grafts in mice

Autologous fat transplantation is a popular and useful technique in plastic and reconstructive surgery. The efficiency and survival of such grafts is predictable in many cases, but there are still issues to be resolved, such as how to improve graft volume retention. To address the issue of volume retention, we studied the effect of revascularization from the recipient on the size and function of adipocytes in fat grafts. Treatment of mice with TNP-470, an angiogenesis inhibitor, reduced blood flow from the recipient into the graft after subcutaneous transplantation of epididymal fat. The weight of transplanted tissues and the size of adipocytes in the grafts were significantly lower in mice treated with TNP-470 (TNP mice) than in control mice. Expression of genes for enzymes related to lipid accumulation was decreased in the grafts of TNP mice compared with control mice. Moreover, the expression of adipocyte-derived angiogenic peptides, VEGF and leptin, was significantly lower in the grafts of TNP mice than in grafts from control animals. The expression of VEGF and leptin by cultured human adipocytes was increased in the presence of conditioned medium from cultured vascular endothelial cells. These results show that the inhibition of the revascularization of fat grafts after transplantation reduces graft volume retention and cellular function. Early and adequate revascularization may be important for both the supply of nutrients and vasoactive interactions between vascular endothelial cells and adipocytes in graft.     

Beneficial effects of subcutaneous fat transplantation on metabolism

Subcutaneous (SC) and visceral (VIS) obesity are associated with different risks of diabetes and the metabolic syndrome. To elucidate whether these differences are due to anatomic location or intrinsic differences in adipose depots, we characterized mice after transplantation of SC or VIS fat from donor mice into either SC or VIS regions of recipient mice. The group with SC fat transplanted into the VIS cavity exhibited decreased body weight, total fat mass, and glucose and insulin levels. These mice also exhibited improved insulin sensitivity during hyperinsulinemic-euglycemic clamps with increased whole-body glucose uptake, glucose uptake into endogenous fat, and insulin suppression of hepatic glucose production. These effects were observed to a lesser extent with SC fat transplanted to the SC area, whereas VIS fat transplanted to the VIS area was without effect. These data suggest that SC fat is intrinsically different from VIS fat and produces substances that can act systemically to improve glucose metabolism.     

Free Fatty Acids,Lipopolysaccharide and IL-1α Induce Adipocyte Manganese Superoxide Dismutase Which Is Increased in Visceral Adipose Tissues of Obese Rodents

Excess fat storage in adipocytes is associated with increased generation of reactive oxygen species (ROS) and impaired activity of antioxidant mechanisms. Manganese superoxide dismutase (MnSOD) is a mitochondrial enzyme involved in detoxification of ROS, and objective of the current study is to analyze expression and regulation of MnSOD in obesity. MnSOD is increased in visceral but not subcutaneous fat depots of rodents kept on high fat diets (HFD) and ob/ob mice. MnSOD is elevated in visceral adipocytes of fat fed mice and exposure of differentiating 3T3-L1 cells to lipopolysaccharide, IL-1α, saturated, monounsaturated and polyunsaturated free fatty acids (FFA) upregulates its level. FFA do not alter cytochrome oxidase 4 arguing against overall induction of mitochondrial enzymes. Upregulation of MnSOD in fat loaded cells is not mediated by IL-6, TNF or sterol regulatory element binding protein 2 which are induced in these cells. MnSOD is similarly abundant in perirenal fat of Zucker diabetic rats and non-diabetic animals with similar body weight and glucose has no effect on MnSOD in 3T3-L1 cells. To evaluate whether MnSOD affects adipocyte fat storage, MnSOD was knocked-down in adipocytes for the last three days of differentiation and in mature adipocytes. Knock-down of MnSOD does neither alter lipid storage nor viability of these cells. Heme oxygenase-1 which is induced upon oxidative stress is not altered while antioxidative capacity of the cells is modestly reduced. Current data show that inflammation and excess triglyceride storage raise adipocyte MnSOD which is induced in epididymal adipocytes in obesity.     

Effect of aging and cellularity on lipolysis in isolated mouse fat cells

The effects of age and cellularity on lipolysis have been investigated in isolated epididymal fat cells from both Swiss albino mice and Sprague-Dawley rats. No significant lipolytic response to glucagon could be demonstrated with adipocytes from either young or old mice, while glycerol output was increased by this hormone with fat cells from young rats. Larger adipocytes from older mice showed significantly greater isoproterenol-stimulated lipolysis than those from younger animals if the glycerol output was expressed on a per cell basis. However, the lipolytic response per cell appeared to be equivalent in young and old rat adipocytes with either isoproterenol or ACTH-(1-24). In a complete aging study, relationships between body weight, epididymal fat pad weight and cellularity were examined covering the life span of the mouse. ACTH-(1-24)- and dibutyryl cyclic AMP-stimulated lipolysis increased with age and cell size but fell at senescence when adipocyte size diminished. Although an effect of aging per se cannot be ruled out with the experimental techniques used in the present study, a dominant influence of adipocyte size on the lipolytic process was demonstrated.     

Effect of cell size, age and anatomical location on the lipolytic response of adipocytes

Studies were conducted on the norepinephrine (NE) stimulated lipolytic sensitivity of adipocytes from epididymal (Epi), perirenal (PR), subcutaneous (SC) and mesentric (M) depots from young (7–8 wk.) and adult (14–16 wk.) male rats. In the young rats dose response curves to NE were similar for Epi, PR and M depots whereas adipocytes from the SC depot showed a diminished effect over the mid-portion of the curve. This difference could not be ascribed to differences in cell size. In the adult rats glycerol release in the Epi depot in response to NE was identical to the younger rats which was in marked contrast to the other depots in which glycerol release was decreased in comparison to the younger animals. This decreased responsiveness was probably largely a result of age and not changes vn adipocyte size within a given depot. In these older rats, glycerol release was greatest in the Epi cells, least in the SC and M depots, and intermediate in PR. When young rats were subjected to a 72-hour fast, loss of triglyceride per cell was the same in all depots as predicted by the $\text{in vitro}$ data whereas in old rats (610 g), triglyceride loss was proportional to cell size with Epi ≥ PR > SC ≥ M. This was also essentially in agreement with the $\text{in vitro}$ lipolytic data from adult rats. These data demonstrate lipolytic differences between depots that are minimal in young rats and which are accentuated with age.     

Large size cells in the visceral adipose depot predict insulin resistance in the canine model

Adipocyte size plays a key role in the development of insulin resistance. We examined longitudinal changes in adipocyte size and distribution in visceral (VIS) and subcutaneous (SQ) fat during obesity‐induced insulin resistance and after treatment with CB‐1 receptor antagonist, rimonabant (RIM) in canines. We also examined whether adipocyte size and/or distribution is predictive of insulin resistance. Adipocyte morphology was assessed by direct microscopy and analysis of digital images in previously studied animals 6 weeks after high‐fat diet (HFD) and 16 weeks of HFD + placebo (PL; n = 8) or HFD + RIM (1.25 mg/kg/day; n = 11). At 6 weeks, mean adipocyte diameter increased in both depots with a bimodal pattern only in VIS. Sixteen weeks of HFD+PL resulted in four normally distributed cell populations in VIS and a bimodal pattern in SQ. Multilevel mixed‐effects linear regression with random‐effects model of repeated measures showed that size combined with share of adipocytes >75 µm in VIS only was related to hepatic insulin resistance. VIS adipocytes >75 µm were predictive of whole body and hepatic insulin resistance. In contrast, there was no predictive power of SQ adipocytes >75 µm regarding insulin resistance. RIM prevented the formation of large cells, normalizing to pre‐fat status in both depots. The appearance of hypertrophic adipocytes in VIS is a critical predictor of insulin resistance, supporting the deleterious effects of increased VIS adiposity in the pathogenesis of insulin resistance.     

Direct and indirect effects of leptin on adipocyte metabolism

Leptin is hypothesized to function as a negative feedback signal in the regulation of energy balance. It is produced primarily by adipose tissue and circulating concentrations correlate with the size of body fat stores. Administration of exogenous leptin to normal weight, leptin responsive animals inhibits food intake and reduces the size of body fat stores whereas mice that are deficient in either leptin or functional leptin receptors are hyperphagic and obese, consistent with a role for leptin in the control of body weight. This review discusses the effect of leptin on adipocyte metabolism. Because adipocytes express leptin receptors there is the potential for leptin to influence adipocyte metabolism directly. Adipocytes also are insulin responsive and receive sympathetic innervation, therefore leptin can also modify adipocyte metabolism indirectly. Studies published to date suggest that direct activation of adipocyte leptin receptors has little effect on cell metabolism in vivo, but that leptin modifies adipocyte sensitivity to insulin to inhibit lipid accumulation. In vivo administration of leptin leads to a suppression of lipogenesis, an increase in triglyceride hydrolysis and an increase in fatty acid and glucose oxidation. Activation of central leptin receptors also contributes to the development of a catabolic state in adipocytes, but this may vary between different fat depots. Leptin reduces the size of white fat depots by inhibiting cell proliferation both through induction of inhibitory circulating factors and by contributing to sympathetic tone which suppresses adipocyte proliferation. This article is part of a Special Issue entitled: Modulation of Adipose Tissue in Health and Disease.     

Combined leptin actions on adipose tissue and hypothalamus are required to deplete adipocyte fat in lean rats: implications for obesity treatment

Intense hyperleptinemia completely depletes adipocyte fat of normal rats within 14 days. To determine the mechanism, epididymal fat pads from normal wild-type (+/+) and obese (fa/fa) Zucker Diabetic Fatty (ZDF) donor rats were transplanted into normal +/+ and fa/fa ZDF recipients. Hyperleptinemia induced by adenovirus-leptin administration depleted all fat from native fat pads and from fat transplants from +/+ donors but not from transplants from ZDF(fa/fa) donors with defective leptin receptors. In both native and transplanted +/+ fat pads, large numbers of mitochondria were apparent, and genes involved in fatty acid oxidation were up-regulated. However, +/+ fat pads transplanted into fa/fa recipients did not respond to hyperleptinemia, suggesting lack of an essential leptin-stimulated cohormone(s). In +/+ but not in fa/fa rats, plasma catecholamine levels rose, and both P-STAT3 and P-CREB increased in adipose tissue, suggesting that both direct and indirect (hypothalamic) leptin receptor-mediated actions of hyperleptinemia are involved in depletion of adipocyte fat.     

Description of obesity in the PBB/Ld mouse.

A new strain of obese mouse, the PBB/Ld, has been studied in terms of fat pad cellularity, serum insulin and blood glucose levels, and response to gold thioglucose injections. Age-matched C57B1/6J mice were used as controls. Adipocyte size and number in the major fat depots were determined at various ages from weanling to maturity in the PBB/Ld and C57B1/6J strains. Results indicated that obesity in the PBB/Ld was due to hypertrophy of adipocytes in retroperitoneal and subcutaneous fat depots and to hypertrophy and hyperplasia in the epididymal fat pad. PBB/Ld mice also developed hyperinsulinemia and hyperglycemia and these findings have been discussed in terms of the developmental changes in fat pad cellularity. The injection of gold thioglucose led to increased food intake in both PBB/Ld and C57B1/6J mice. Hyperphagia was also present in the PBB/LD control group, but increased efficiency of converting calories to body weight was not observed in this group when compared to control C57B1/6J mice. The characteristics of obesity seen in the PBB/Ld mouse are discussed and comparisons are made to similar studies in other rodent models of obesity.     

Effects of obesity on the relationship of leptin mRNA expression and adipocyte size in anatomically distinct fat depots in mice

In support of leptin's physiological role as humoral signal of fat mass, we have shown that adipocyte volume is a predominant determinant of leptin mRNA levels in anatomically distinct fat depots in lean young mice in the postabsorptive state. In this report, we investigated how obesity may affect the relationship between leptin mRNA levels and adipocyte volume in anatomically distinct fat depots in mice with genetic (Lep(ob)/Lep(ob) and A(y)/+), diet-induced, and aging-related obesity. In all of the obese mice examined, tissue leptin mRNA levels relative to the average adipocyte volume were lower in the perigonadal and/or retroperitoneal than in the inguinal fat depots and were lower than those of the lean young mice in the perigonadal fat depot. A close, positive correlation between leptin mRNA level and adipocyte volume was present from small to hypertrophic adipocytes within each perigonadal and inguinal fat pad in the obese mice, but the slopes of the regression lines relating leptin mRNA level to adipocyte volume were significantly lower in the perigonadal than in the inguinal fat pads of the same mice. These results suggest that obesity per se is associated with a decreased leptin gene expression per unit of fat mass in mice and that the positive correlation between leptin mRNA level and adipocyte volume is an intrinsic property of adipocytes that is not disrupted by adipocyte hypertrophy in obese mice.     

The adipose organ of obesity-prone C57BL/6J mice is composed of mixed white and brown adipocytes

White and brown adipocytes are believed to occupy different sites in the body. We studied the anatomical features and quantitative histology of the fat depots in obesity and type 2 diabetes-prone C57BL/6J mice acclimated to warm or cold temperatures. Most of the fat tissue was contained in depots with discrete anatomical features, and most depots contained both white and brown adipocytes. Quantitative analysis showed that cold acclimation induced an increase in brown adipocytes and an almost equal reduction in white adipocytes; however, there were no significant differences in total adipocyte count or any signs of apoptosis or mitosis, in line with the hypothesis of the direct transformation of white into brown adipocytes. The brown adipocyte increase was accompanied by enhanced density of noradrenergic parenchymal nerve fibers, with a significant correlation between the density of these fibers and the number of brown adipocytes. Comparison with data from obesity-resistant Sv129 mice disclosed a significantly different brown adipocyte content in C57BL/6J mice, suggesting that this feature could underpin the propensity of the latter strain to develop obesity. However, the greater C57BL/6J browning capacity can hopefully be harnessed to curb obesity and type 2 diabetes in patients with constitutively low amounts of brown adipose tissue.     

Influence of dietary macronutrient composition on adiposity and cellularity of different fat depots in Wistar rats

The aim of this study was to investigate the role of dietary macronutrient content on adiposity parameters and adipocyte hypertrophy/hyperplasia in subcutaneous and visceral fat depots from Wistar rats using combined histological and computational approaches. For this purpose, male Wistar rats were distributed into 4 groups and were assigned to different nutritional interventions: Control group (chow diet); high-fat group, HF (60% E from fat); high-fat-sucrose group, HFS (45% E from fat and 17% from sucrose); and high-sucrose group, HS (42% E from sucrose). At day 35, rats were sacrificed, blood was collected, tissues were weighed and fragments of different fat depots were kept for histological analyses with the new softwareAdiposoft. Rats fed with HF, HFS and HS diets increased significantly body weight and total body fat against Control rats, being metabolic impairments more pronounced on HS rats than in the other groups. Cellularity analyses usingAdiposoft revealed that retroperitoneal adipose tissue is histologically different than mesenteric and subcutaneous ones, in relation to bigger adipocytes. The subcutaneous fat pad was the most sensitive to the diet, presenting adipocyte hypertrophy induced by HF diet and adipocyte hyperplasia induced by HS diet. The mesenteric fat pad had a similar but attenuated response in comparison to the subcutaneous adipose tissue, while retroperitoneal fat pad only presented adipocyte hyperplasia induced by the HS diet intake after 35 days of intervention. These findings provide new insights into the role of macronutrients in the development of hyperplastic obesity, which is characterized by the severity of the clinical features. Finally, a new tool for analyzing histological adipose samples is presented.     

5 alpha-reductase activity in rat adipose tissue

We measured the 5 alpha-reductase activity in isolated cell preparations of rat adipose tissue using the formation of [3H]dihydrotestosterone from [3H]testosterone as an endpoint. Stromal cells were prepared from the epididymal fat pad, perinephric fat, and subcutaneous fat of male rats and from perinephric fat of female rats. Adipocytes were prepared from the epididymal fat pad and perinephric fat of male rats. Stromal cells from the epididymal fat pad and perinephric fat contained greater 5 alpha-reductase activity than did the adipocytes from these depots. Stromal cells from the epididymal fat pad contained greater activity than those from perinephric and subcutaneous depots. Perinephric stromal cells from female rats were slightly more active than those from male rats. Estradiol (10(-8) M), when added to the medium, caused a 90% decrease in 5 alpha-reductase activity. Aromatase activity was minimal, several orders of magnitude less than 5 alpha-reductase activity in each tissue studied.     

Development of the Mouse Dermal Adipose Layer Occurs Independently of Subcutaneous Adipose Tissue and Is Marked by Restricted Early Expression of FABP4

The laboratory mouse is a key animal model for studies of adipose biology, metabolism and disease, yet the developmental changes that occur in tissues and cells that become the adipose layer in mouse skin have received little attention. Moreover, the terminology around this adipose body is often confusing, as frequently no distinction is made between adipose tissue within the skin, and so called subcutaneous fat. Here adipocyte development in mouse dorsal skin was investigated from before birth to the end of the first hair follicle growth cycle. Using Oil Red O staining, immunohistochemistry, quantitative RT-PCR and TUNEL staining we confirmed previous observations of a close spatio-temporal link between hair follicle development and the process of adipogenesis. However, unlike previous studies, we observed that the skin adipose layer was created from cells within the lower dermis. By day 16 of embryonic development (e16) the lower dermis was demarcated from the upper dermal layer, and commitment to adipogenesis in the lower dermis was signalled by expression of FABP4, a marker of adipocyte differentiation. In mature mice the skin adipose layer is separated from underlying subcutaneous adipose tissue by the panniculus carnosus. We observed that the skin adipose tissue did not combine or intermix with subcutaneous adipose tissue at any developmental time point. By transplanting skin isolated from e14.5 mice (prior to the start of adipogenesis), under the kidney capsule of adult mice, we showed that skin adipose tissue develops independently and without influence from subcutaneous depots. This study has reinforced the developmental link between hair follicles and skin adipocyte biology. We argue that because skin adipocytes develop from cells within the dermis and independently from subcutaneous adipose tissue, that it is accurately termed dermal adipose tissue and that, in laboratory mice at least, it represents a separate adipose depot.     

Growth Hormone Treatment of Hypophysectomized Rats Increases Catecholamine-induced Lipolysis and the Number of β-Adrenergic Receptors in Adipocytes: No Differences in the Effects of Growth Hormone on Different Fat Depots

Growth hormone (GH) has a lipolytic effect in adipose tissue but this effect may differ in adipose tissue from various fat depots. This latter possibility was investigated in the present study, in which the effects of GH in vivo on catecholamine-induced lipolysis and the number of β-adrenergic receptors in isolated adipocytes from different fat depots of hypophysectomized rats were investigated. Female and male Sprague-Dawley rats were hypophysectomized or sham-operated at 45 days of age. One week after the operation, hormonal replacement therapy with L-thyroxine and hydrocortisone acetate was given. In addition, groups of rats were treated with GH (1.33 mg/kg per day, given as two daily subcutaneous injections). After 1 week of hormonal treatment, adipocytes were isolated from the parametrial, epididymal and inguinal fat pads, and glycerol release after catecholamine-stimulation and ¹²⁵I-cyanopindolol binding were measured. Hypophysectomy resulted in a marked decrease in the lipolytic response to catecholamines. GH treatment significantly increased catecholamine-induced lipolysis with similar effects in adipocytes from parametrial or epididymal and inguinal fat depots in both female and male rats. There were no differences between norepinephrine compared with isoproterenol-induced responses. ¹²⁵I-cyanopindolol binding was reduced after hypophysectomy and normalized by GH treatment, without differences between parametrial and inguinal adipose tissue regions. We conclude that the lipolytic effects of GH in the rat may partly be mediated by a stimulatory effect on β-adrenergic receptors in adipocytes. In addition, GH exerted similar effect on catecholamine-induced lipolysis and β-adrenergic receptors in adipocytes from parametrial, epididymal and inguinal fat depots.     

Obese adipocytes show ultrastructural features of stressed cells and die of pyroptosis

We previously suggested that, in obese animals and humans, white adipose tissue inflammation results from the death of hypertrophic adipocytes; these are then cleared by macrophages, giving rise to distinctive structures we denominated crown-like structures. Here we present evidence that subcutaneous and visceral hypertrophic adipocytes of leptin-deficient (ob/ob and db/db) obese mice exhibit ultrastructural abnormalities (including calcium accumulation and cholesterol crystals), many of which are more common in hyperglycemic db/db versus normoglycemic ob/ob mice and in visceral versus subcutaneous depots. Degenerating adipocytes whose intracellular content disperses in the extracellular space were also noted in obese mice; in addition, increased anti-reactive oxygen species enzyme expression in obese fat pads, documented by RT-PCR and immunohistochemistry, suggests that ultrastructural changes are accompanied by oxidative stress. RT-PCR showed NLRP3 inflammasome activation in the fat pads of both leptin-deficient and high-fat diet obese mice, in which formation of active caspase-1 was documented by immunohistochemistry in the cytoplasm of several hypertrophic adipocytes. Notably, caspase-1 was not detected in FAT-ATTAC transgenic mice, where adipocytes die of apoptosis. Thus, white adipocyte overexpansion induces a stress state that ultimately leads to death. NLRP3-dependent caspase-1 activation in hypertrophic adipocytes likely induces obese adipocyte death by pyroptosis, a proinflammatory programmed cell death.