Computerized Determination of Adipocyte Size

Objective: Fat cell size is a fundamental parameter in the study of adipose tissue metabolism, because it markedly influences the cellular rates of metabolism. Previous techniques for the sizing of adipocytes are often complicated or time‐consuming. The aim of this study was to develop a new, computerized method for rapid and accurate determination of adipocyte size in a cell suspension obtained by incubating human or rat adipose tissue biopsies with collagenase. Research Methods and Procedures: The cell suspension was placed between a siliconized glass slide and a cover slip. Using the reference method [designated as (R)], the cell diameters were determined manually using a microscope with a calibrated ocular. The new method presented here [designated as (C)] was based on computerized image analysis. Results: After two well‐defined corrective adjustments, measurements with (R) and (C) agreed very well. The small remaining differences seemed, in fact, to depend on inconsistencies in (R). Discussion: We propose that (C) constitutes a valuable tool to study fat cell size, because this method is fast and allows the assessment of a sufficient number of cells to get reliable data on size distribution. Furthermore, images of cell preparations may be stored for future reference.     

Endothelial cells of adipose tissues: A niche of adipogenesis

Comment on: Tran KV, et al. Cell Metab 2012; 15:222-9.     

UCP1 mRNA does not produce heat

Because of the possible role of brown adipose tissue and UCP1 in metabolic regulation, even in adult humans, there is presently considerable interest in quantifying, from in-vitro data, the thermogenic capacities of brown and brite/beige adipose tissues. An important issue is therefore to establish which parameters are the most adequate for this. A particularly important issue is the relevance of UCP1 mRNA levels as estimates of the degree of recruitment and of the thermogenic capacity resulting from differences in physiological conditions and from experimental manipulations. By solely following UCP1 mRNA levels in brown adipose tissue, the conclusion would be made that the tissue's highest activation occurs after only 6 h in the cold and then successively decreases to being only some 50% elevated after 1 month in the cold. However, measurement of total UCP1 protein levels per depot ("mouse") reveals that the maximal thermogenic capacity estimated in this way is reached first after 1 month but represents an approx. 10-fold increase in thermogenic capacity. Since this in-vitro measure correlates quantitatively and temporally with the acquisition of nonshivering thermogenesis, this must be considered the most physiologically relevant parameter. Similarly, observations that cold acclimation barely increases UCP1 mRNA levels in classical brown adipose tissue but leads to a 200-fold increase in UCP1 mRNA levels in brite/beige adipose tissue depots may overemphasise the physiological significance of these depots, as the high fold-increases are due to very low initial levels, and the UCP1 mRNA levels reached are at least an order of magnitude lower than in brown adipose tissue; furthermore, based on total UCP1 protein amounts, the brite/beige depots attain only about 10% of the thermogenic capacity of the classical brown adipose tissue depots. Consequently, inadequate conclusions may be reached if UCP1 mRNA levels are used as a proxy for the metabolic significance of recruited versus non-recruited brown adipose tissue and for estimating the metabolic significance of brown versus brite/beige adipose tissues. This article is part of a Special Issue entitled Brown and White Fat: From Signaling to Disease.     

Lipolysis-derived linoleic acid drives beige fat progenitor cell proliferation

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Brown and white adipose tissue lipid composition in three successive progenies of rats: Effects of ethanol exposure

The effect of ethanol exposure on the fatty acid composition of brown and white adipose tissue in three successive rat progenies at the end of an experimental period (24 weeks) was studied. Ethanol‐treated rats received a standard rat chow diet and 5, 10 and 15% ethanol in the ad libitum drinking fluid over 3 successive weeks. Then a concentration of 20% ethanol was maintained for 5 additional weeks up to the end of the experimental period. The males and females in the ethanol treated group were mated to obtain the 1st generation of offspring. Then female and male rats from the 1st generation were mated to obtain the 2nd generation. Finally, males and females from the 2nd generation were mated to obtain the 3rd generation of ethanol treated rats. Another group served as control and received only water and a standard rat chow diet. The control group was handled in the same way as the other experimental groups. In the 1st and 2nd generations the percentage of stearic acid (18:0) decreased and palmitoleic (16:ln7) and oleic acid (18:ln9) increased in both adipose tissues of ethanol‐treated rats with respect to control. Additionally, n‐3 and n‐6 series were reduced both in brown and white adipose tissues. In the 3rd generation the fatty acid composition of the white adipose tissue was similar to that of control rats. Thus, no significant difference in essential fatty acids and oleic acid (18:ln9) were found. However, the fatty acid composition of the brown adipose tissue, in the 3rd generation, was similar to that observed in the 1st and 2nd generation. Thus, a decrease in essential fatty acids and an increase in oleic acid (18:ln9) was found. This suggests adaptation to ethanol consumption during successive progenies in white adipose tissue. However, in brown adipose tissue the values indicate a triglyceride storing during the thermogenesis, which is more important to newborns.     

The Yellow Agouti Mutation Alters Some But Not All Responses to Diet and Exercise

Objective: Effects of ectopic expression of the agouti signaling protein were studied on responses to diet restriction and exercise in C57BL/6J (B6) mice and obese B6 mice congenic for the yellow agouti mutation [B6.Cg‐A^y (A^y)]. Research Methods and Procedures: Adult male A^y mice were either kept sedentary or exercised on a running wheel and fed ad libitum or diet restricted until weight matched to ad libitum‐fed B6 control mice. Body composition, plasma lipids, leptin, and adiponectin were measured. mRNA levels for leptin, adiponectin, lipoprotein lipase, and pyruvate dehydrogenase kinase 4 were measured in a visceral (epididymal) and a subcutaneous (femoral) fat depot by real‐time polymerase chain reaction. Results: Correlations among traits exhibited one of three patterns: similar lines for B6 and A^y mice, different slopes for B6 and A^y mice, and/or different intercepts for B6 and A^y mice. Correlations involving plasma leptin, mesenteric and epididymal adipose weights, or low‐density lipoprotein‐cholesterol were most likely to have different slopes and/or intercepts in B6 and A^y mice. mRNA levels for leptin, Acrp30, pyruvate dehydrogenase kinase 4, and lipoprotein lipase in epididymal adipose tissue were not correlated with corresponding levels in femoral adipose tissue. Discussion: The agouti protein interferes with leptin signaling at melanocortin receptors in the hypothalamus of A^y mice. Our results are consistent with the hypothesis that the melanocortin portion of the leptin‐signaling pathway mediates effects primarily on certain fat depots and on some, but not all, components of cholesterol homeostasis.     

Characterization and comparison of adipose tissue‐derived cells from human subcutaneous and omental adipose tissues

Different fat depots contribute differently to disease and function. These differences may be due to the regional variation in cell types and inherent properties of fat cell progenitors. To address the differences of cell types in the adipose tissue from different depots, the phenotypes of freshly isolated adipose tissue‐derived cells (ATDCs) from subcutaneous (SC) and omental (OM) adipose tissues were compared using flow cytometry. Our results showed that CD31^?CD34⁺CD45^?CD90^‐CD105^?CD146⁺ population, containing vascular smooth muscle cells and pericytes, was specifically defined in the SC adipose tissue while no such population was observed in OM adipose tissue. On the other hand, CD31^?CD34⁺CD45^?CD90^?CD105^?CD146^? population, which is an undefined cell population, were found solely in OM adipose tissue. Overall, the SC adipose tissue contained more ATDCs than OM adipose tissue, while OM adipose tissue contained more blood‐derived cells. Regarding to the inherent properties of fat cell progenitors from the two depots, adipose‐derived stem cells (ADSCs) from SC had higher capacity to differentiate into both adipogenic and osteogenic lineages than those from OM, regardless of that the proliferation rates of ADSCs from both depots were similar. The higher differentiation capacity of ADSCs from SC adipose tissue suggests that SC tissue is more suitable cell source for regenerative medicine than OM adipose tissue. Copyright © 2009 John Wiley & Sons, Ltd.     

Validation of an Elliptical Anthropometric Model to Estimate Visceral Compartment Area

Objective: The visceral compartment is a surrogate for visceral adipose tissue. Cross‐sectional visceral compartment area (VCA) has been approximated from waist circumference using a circular model. However, the two‐dimensional shape of the abdomen is rarely circular. This study validated an elliptical model of cross‐sectional total abdominal area (TAA), subcutaneous adipose tissue (SAT) area, and VCA at the L₄–L₅ level. Research Methods and Procedures: We analyzed magnetic resonance images (MRIs) at the level of the L₄–L₅ intervertebral space from 35 subjects with a wide range of abdominal adiposity. Waist circumference, abdominal thickness (midline sagittal diameter), abdominal width (coronal diameter at one‐half of abdominal thickness), and abdominal SAT thickness at four sites (front, back, right, and left) were measured from MRI images using an image analysis software. The same anatomical regions were also estimated from anthropometrics purely by geometric formulae of circular and elliptical models. A simple linear regression model was used to interpret the association strength between anthropometric estimates and MRI measures. Results: Estimated TAA by either model was strongly related to MRI TAA (r² = 0.98, p < 0.0001). The SAT and VCA by MRI analysis showed a stronger association with calculation from an elliptical model (r² = 0.95 and 0.88, respectively; p < 0.001) than a circular model (r² = 0.69 and 0.25, respectively; p < 0.001). The absolute prediction residuals and variances were significantly smaller with an elliptical model than a circular model (p < 0.0001). Discussion: An elliptical anthropometric model might be superior to a circular model to estimate abdominal SAT and VCA.     

京尼平对小鼠棕色和白色脂肪组织UCP1表达的影响

目的:棕色脂肪组织活化和白色脂肪组织棕化是改善减肥的良好策略。本研究利用冷刺激作为阳性对照,观察京尼平对小鼠脂肪组织活化与棕化的作用。方法:8周龄雄性C57BL/6J小鼠30只,随机分为正常对照组、京尼平组、冷刺激组, 每组10只。京尼平组小鼠腹腔注射给予京尼平处理(15 mg/(kg·d),连续9 d),对照组用生理盐水处理,冷刺激组小鼠在室温(22℃±2℃)下处理4 d后,置于4℃环境中进行冷刺激处理5 d(24 h/d)。检测各组小鼠每天摄食量、体重和体温变化,取肩胛下区、腹股沟区及附睾周围部分脂肪组织观察形态学的变化,测定棕色脂肪组织、皮下白色脂肪组织以及内脏白色脂肪组织解偶联蛋白1(UCP1)的表达。结果:与正常对照组相比,京尼平组小鼠白色脂肪湿重下降16%,冷刺激组下降28%,均有明显差异(P＜0.05);京尼平组和冷刺激组白色脂肪组织颜色变深,HE染色显示脂肪细胞内的脂滴变小,数量增加;京尼平组小鼠的皮下、内脏白色脂肪组织和棕色3种脂肪组织中的UCP1表达量均明显增加(P＜0.05)。结论:京尼平通过上调UCP1的表达促进棕色脂肪组织活化和白色脂肪组织棕化,此效应是京尼平降脂减轻体重的作用机制之一。     

妊娠期糖尿病血清和脂肪组织内脂素的表达

目的:探讨妊娠期糖尿病孕妇内脂素水平与糖代谢的关系.方法:检测血清中内脂素、FIN、FIG水平,计算HOMA-IR,用RT-PCR法检测脂肪组织中内脂素mRNA的表达.结果:(1)妊娠期糖尿病组孕妇血清内脂素、FPG、HOMA-IR、FIN明显高于对照组.(2)妊娠期糖尿病组孕妇内脏脂肪组织中的内脂素的表达明显高于对照组,且妊娠期糖尿病组孕妇内脏组织中内脂素的表达明显高于表皮脂肪组织.(3)血清中内脂素的水平与内脏脂肪组织及HOMA-IR呈正相关.结论:妊娠期糖尿病孕妇脂肪组织中内脂素表达上调,导致血液循环中内脂素水平升高,参与GDM孕妇血糖调节.     

Omental and subcutaneous adipose tissue steroid levels in obese men

We examined plasma and fat tissue sex steroid levels in a sample of 28 men aged 24.8-62.2 years (average BMI value of 46.3 +/- 12.7 kg/m(2)). Abdominal adipose tissue biopsies were obtained during general or obesity surgery. Omental and subcutaneous adipose tissue steroid levels were measured by gas chromatography and chemical ionization mass spectrometry after appropriate extraction procedures. BMI and waist circumference were negatively correlated with plasma testosterone (r = -0.49 and -0.50, respectively, p < 0.01) and dihydrotestosterone (r = -0.58 and -0.56, respectively, p < 0.01), and positively associated with estrone levels (r = 0.64 and 0.62, respectively, p < 0.001). Regional differences in adipose tissue steroid levels were observed for dihydrotestosterone (p < 0.005), androstenedione (p < 0.0001) and dehydroepiandrosterone levels (p < 0.05), which were all significantly more concentrated in omental versus subcutaneous fat. Positive significant associations were found between circulating level of a steroid and its concentration in omental and subcutaneous adipose tissue, for estrone (r = 0.72 and 0.57, respectively, p < 0.01), testosterone (r = 0.66 and 0.58, respectively, p < 0.01) and dihydrotestosterone (r = 0.58 and 0.45, respectively, p < 0.05). Positive correlations were observed between plasma dehydroepiandrosterone-sulfate and omental (r = 0.56, p < 0.01) as well as subcutaneous adipose tissue dehydroepiandrosterone level (r = 0.38, p = 0.05). Positive significant associations were found between omental adipocyte responsiveness to positive lipolytic stimuli (isoproterenol, dibutyryl cyclic AMP and forskolin) and plasma or omental fat tissue androgen levels. In conclusion, although plasma androgen or estrogen levels are strong correlates of adipose tissue steroid content both in the omental and subcutaneous fat depots, regional differences may be observed. Androgen concentration differences in omental versus subcutaneous adipose tissue suggest a depot-specific impact of these hormones on adipocyte function and metabolism.     

New scanning electron microscopic method for determination of adipocyte size in humans and mice

Objective: To evaluate a new scanning electronic microscopic (EM) method for assessing fat cell sizes and compare fat cell size distribution in human adipose tissue from different fat depots before and after weight loss. Research Methods and Procedures: Identical human fat tissue biopsies were separated into two fractions: one used to prepare a fat cell suspension by collagenase digestion followed by photomicrography (collagenase method) and the other fixed in formalin for EM analysis. The EM method was evaluated further by determining fat cell sizes from lean and ob/ob mice. Finally, the EM method was used to assess fat cell sizes in biopsies of different human depots from before and after weight loss. Results: Fat cell size distributions measured by the two methods were not identical, but differences were generally small. The EM method reproduced the well‐documented fat cell size difference between lean and ob/ob mice. Large variation was detected in fat cell distributions among three depots in humans. Weight loss reduced fat cell sizes in subjects with large baseline fat cells but had no effect in subjects with small baseline fat cell sizes. Discussion: Our results suggest that the EM method may be a useful alternative for fat cell size analysis of clinical samples.     

In vivo phenotyping of the ob/ob mouse by magnetic resonance imaging and 1H-magnetic resonance spectroscopy

Objective: We studied ob/ob and wild‐type (WT) mice to characterize the adipose tissues depots and other visceral organs and to establish an experimental paradigm for in vivo phenotyping. Research Methods and Procedures: An in vivo evaluation was conducted using magnetic resonance imaging and ¹H‐magnetic resonance spectroscopy (¹H‐MRS). We used T1‐weighted images and three‐dimensional spin echo T1‐weighted images for the morphological analysis and ¹H‐MRS spectra on all body mass, as well as ¹H‐MRS spectra focalized on specific lipid depots [triglyceride (TG) depots] for a molecular analysis. Results: In ob/ob mice, three‐dimensional evaluation of the trunk revealed that ～64% of the volume consists of white adipose tissue, which is 72% subcutaneous and 28% visceral. In vivo ¹H‐MRS showed that 20.00 ± 6.92% in the WT group and 58.67 ± 6.65% in the ob/ob group of the total proton content is composed of TG protons. In in vivo‐localized spectra of ob/ob mice, we found a polyunsaturation degree of 0.5247 in subcutaneous depots. In the liver, we observed that 48.7% of the proton signal is due to water, whereas in the WT group, the water signal amounted to 82.8% of the total proton signal. With the sequences used, the TG amount was not detectable in the brain or kidneys. Discussion: The present study shows that several parameters can be obtained by in vivo examination of ob/ob mice by magnetic resonance imaging and ¹H‐MRS and that the accumulated white adipose tissue displays low polyunsaturation degree and low hydrolipidic ratio. Relevant anatomical alterations observed in urinary and digestive apparatuses should be considered when ob/ob mice are used in experimental paradigms.     

Incorporation of n-3 fatty acids into phospholipids of rat liver and white and brown adipose tissues: A time-course study during fish-oil feeding

The aim of this study was to determine the time-course incorporation of dietary n-3 polyunsaturated fatty acids into phospholipids of tissues highly involved in lipid and energy metabolism: the liver and the white (WAT) and brown (BAT) adipose tissues. Rats were fed a diet supplemented with 19% fish oil for up to 4 weeks. Minor changes in the relative proportions of tissue phospholipids were observed in the three tissues. Fish-oil feeding induced rapid and large replacements of n-6 fatty acids by n-3 fatty acids. In liver, the 22:6n-3 level increased progressively and reached a plateau after 3 (phosphatidylethanolamine and phosphatidylserine) or 7 days (phosphatidylcholine and phosphatidylinositol). In contrast, the 20:5n-3 level transiently peaked in all liver phospholipids at days 1–3 before reaching a plateau after day 7. In WAT as in BAT the level of n-3 fatty acids increased progressively and reached in all phospholipids a plateau after day 7. As a general trend, in each phospholipid class the 22:6n-3/20:5n-3 ratio was higher in liver than in the two adipose tissues. This study shows that each dietary n-3 fatty acid is incorporated very rapidly into liver, WAT, and BAT phospholipids but according to time courses and at levels that depend simultaneously on the tissue and phospholipid class considered.     

Rapamycin Inhibits Human Adipocyte Differentiation in Primary Culture

Objective: The immunosuppressant drug rapamycin, has been reported to inhibit 3T3‐L1 adipocyte differentiation by interfering with critical postconfluent mitoses that are required early on for successful differentiation of this cell line (clonal expansion phase). In contrast to the murine 3T3‐L1 preadipocyte cell line, human preadipocytes in primary culture do not undergo clonal expansion during differentiation. We investigated whether rapamycin could inhibit human adipocyte differentiation. Research Methods and Procedures: The effect of rapamycin on the induction of differentiation of human preadipocytes in primary culture into adipocytes was measured using Oil Red O staining and glycerol phosphate dehydrogenase activity. Results: We have observed that rapamycin severely curtails human adipocyte differentiation of both omental and abdominal subcutaneous preadipocytes (to 14% and 19% of standard differentiation, respectively). The rapamycin‐mediated inhibition of human adipocyte differentiation could be reversed in the presence of excess amounts of FK‐506, which displaces rapamycin from its intracellular receptor, FKPB12. Measurement of cytosolic protein and [³H]thymidine incorporation into DNA confirmed the absence of proliferation during differentiation of human preadipocytes in primary culture. Discussion: Our data indicate that rapamycin exerts important negative regulatory effects on adipogenesis in human preadipocytes, through a mechanism that does not depend on interruption of clonal expansion.     

White-to-brown transdifferentiation of omental adipocytes in patients affected by pheochromocytoma

In all mammals, white adipose tissue (WAT) and brown adipose tissue (BAT) are found together in several fat depots, forming a multi-depot organ. Adrenergic stimulation induces an increase in BAT usually referred to as “browning”. This phenomenon is important because of its potential use in curbing obesity and related disorders; thus, understanding its cellular mechanisms in humans may be useful for the development of new therapeutic strategies. Data in rodents have supported the direct transformation of white into brown adipocytes. Biopsies of pure white omental fat were collected from 12 patients affected by the catecholamine-secreting tumor pheochromocytoma (pheo-patients) and compared with biopsies from controls. Half of the omental fat samples from pheo-patients contained uncoupling protein 1 (UCP1)-immunoreactive-(ir) multilocular cells that were often arranged in a BAT-like pattern endowed with noradrenergic fibers and dense capillary network. Many UCP1-ir adipocytes showed the characteristic morphology of paucilocular cells, which we have been described as cytological marker of transdifferentiation. Electron microscopy showed increased mitochondrial density in multi- and paucilocular cells and disclosed the presence of perivascular brown adipocyte precursors. Brown fat genes, such as UCP1, PR domain containing 16 (PRDM16) and β3-adrenoreceptor, were highly expressed in the omentum of pheo-patients and in those cases without visible morphologic re-arrangement. Of note, the brown determinant PRDM16 was detected by immunohistochemistry only in nuclei of multi- and paucilocular adipocytes. Quantitative electron microscopy and immunohistochemistry for Ki67 suggest an unlikely contribution of proliferative events to the phenomenon. The data support the idea that, in adult humans, white adipocytes of pure white fat that are subjected to adrenergic stimulation are able to undergo a process of direct transformation into brown adipocytes. This article is part of a Special Issue entitled Brown and White Fat: From Signaling to Disease.     

Effect of Lifestyle Modification on Adipokine Levels in Obese Subjects with Insulin Resistance

Objective: To study the effect of weight loss in response to a lifestyle modification program on the circulating levels of adipose tissue derived cytokines (adipokines) in obese individuals with insulin resistance. Research Methods and Procedures: Twenty‐four insulin‐resistant obese subjects with varying degrees of glucose tolerance completed a 6‐month program consisting of combined hypocaloric diet and moderate physical activity. Adipokines [leptin, adiponectin, resistin, tumor necrosis factor‐α (TNF‐α), interleukin‐6 (IL‐6)] and highly sensitive C‐reactive protein were measured before and after the intervention. Insulin sensitivity index was evaluated by the frequently sampled intravenous glucose tolerance test. Results: Participants had a 6.9 ± 0.1 kg average weight loss, with a significant improvement in sensitivity index and reduction in plasma leptin (27.8 ± 3 vs. 23.6 ± 3 ng/mL, p = 0.01) and IL‐6 (2.75 ± 1.51 vs. 2.3 ± 0.91 pg/mL, p = 0.012). TNF‐α levels tended to decrease (2.3 ± 0.2 vs. 1.9 ± 0.1 pg/mL, p = 0.059). Adiponectin increased significantly only among diabetic subjects. The reductions in leptin were correlated with the decreases in BMI (r = 0.464, p < 0.05) and with changes in highly sensitive C‐reactive protein (r = 0.466, p < 0.05). Discussion: Weight reduction in obese individuals with insulin resistance was associated with a significant decrease in leptin and IL‐6 and a tendency toward a decrease in circulating TNF‐α, whereas adiponectin was increased only in diabetic subjects. Further studies are needed to elucidate the relationship between changes of adipokines and the health benefits of weight loss.