Ultrastructural features of cultured mature adipocyte precursors from adipose tissue in multiple symmetric lipomatosis

The pathogenesis of multiple symmetric lipomatosis (MSL) is as yet unknown; however, some studies seem to indicate the neoplastic nature of this lesion. In this study we have examined the ultrastructural features of the adipocyte precursors of patients with MSL. The cells were isolated by collagenase, cultured, and then examined at the moment of their confluence in a monolayer. Their ultrastructural features were compared with those of cells of the abdominal subcutaneous adipose tissue taken from patients with nonsystemic, surgically treated illnesses. When examined immediately after isolation, the cells from normal tissue did not show significant differences from MSL cells. In culture, however, MSL grew more quickly into a monolayer and showed numerous nuclear pockets and cytoplasmic microfilaments, which were not seen in cells from normal tissue. These differences appear compatible with the neoplastic nature of the MSL adipocyte precursors.     

Ultrastructural Features of Cultured Mature Adipocyte Precursors from Adipose Tissue in Multiple Symmetric Lipomatosis

The pathogenesis of multiple symmetric lipomatosis (MSL) is as yet unknown; however, some studies seem to indicate the neoplastic nature of this lesion. In this study we have examined the ultrastructural features of the adipocyte precursors of patients with MSL. The cells were isolated by collagenase, cultured, and then examined at the moment of their confluence in a monolayer. Their ultrastructural features were compared with those of cells of the abdominal subcutaneous adipose tissue taken from patients with non-systemic, surgically treated illnesses.

When examined immediately after isolation, the cells from normal tissue did not show significant differences from MSL cells. In culture, however, MSL grew more quickly into a monolayer and showed numerous nuclear pockets and cytoplasmic microf ila-ments, which were not seen in cells from normal tissue. These differences appear compatible with the neoplastic nature of the MSL adipocyte precursors.     

Hibernoma: a possible model of brown fat histogenesis

Hibernomas are composed of cells remarkably similar to those observed in brown adipose tissue. A surgically resected hibernoma was observed to contain cells at all stages of maturation from immature, relatively lipid free cells to multiloculated and finally uniloculated adipocytes. The ultrastructural features of adipocytes contained in the hibernoma were compared to previously reported features of differentiating preadipocytes and lipid depleted mature adipocytes derived from human white adipose tissue. This comparison confirmed the existence of distinct mitochondrial and cytoplasmic membrane component differences at all developmental stages and suggests that brown and white adipose tissue are two unique histologic types.     

The pre- and postnatal development and ageing of interscapular brown adipose tissue in the rat

Summary Developmental and ageing changes in interscapular brown adipose tissue have been studied in white Wistar rats by light, fluorescence and electron microscopy.Cellular aggregation was noted in the fetal interscapular area on the 15th day of gestation and vascularised primitive lobules of brown adipose tissue became unequivocally identifiable on the 17th day in utero. Brown adipocyte precursors appeared to be derived directly from mesenchymal cells and were uniquely characterised by larger (0.8–1.7 m diameter) mitochondria. Numbers of these precursor cells (pre-adipocytes) were seen in mitosis during intrauterine life. Pericapillary cells similar in appearance to embryonic pre-adipocytes were regularly observed within brown fat lobules throughout later life.Cardinal features noted in mature brown adipose tissue were parenchymal cells with a multilocular lipid distribution and numerous large mitochondria with distinctive inparallel cristae, as well as an extensive vascular network and a dense catecholaminergic vasomotor and parenchymal innervation.Brown adipocytes generally retained a multilocular lipid distribution into old age, and although the catecholaminergic fluorescence of the nerves supplying the tissue was reduced, a widespread distribution of noradrenergic vasomotor and parenchymal nerves and of nexuses between brown adipocytes continued to be demonstrable by electron microscopy in the brown adipose tissue of senile rats.     

Src family kinases suppress differentiation of brown adipocytes and browning of white adipocytes

Brown adipocytes and beige adipocytes can expend energy, generate heat, and increase whole‐body energy expenditure. The detailed mechanisms of adipogenesis and thermogenesis of these cells are still obscure. Here, we show that Src family kinases (SFKs) regulate both brown adipogenesis and browning of white adipocytes. To identify factors involved in brown adipogenesis, we first examined the effect of several chemical inhibitors on the differentiation of brown preadipocytes isolated from mouse brown adipose tissue (BAT) and found that treatment with PP2, the specific inhibitor of SFKs, promoted the differentiation. Another inhibitor of SFKs, PP1, also promoted the brown adipogenesis, whereas an inactive analogue of PP2, PP3, did not. Moreover, over‐expression of C‐terminal Src kinase (CSK), the negative regulator of SFKs, also promoted brown adipogenesis. Next, we examined the effect of inhibition of SFKs on the differentiation of white preadipocytes isolated from white adipose tissue (WAT). Our results showed that either PP2 treatment or CSK‐over‐expression generated Ucp1‐positive beige adipocytes, thus inducing browning of white adipocytes. Finally, our analysis showed that the expression levels and activity of SFKs in WAT were much higher than in BAT. These results taken together suggest that SFKs regulate differentiation and browning of fat cells in vivo.     

4E-BP1 regulates the differentiation of white adipose tissue

4E Binding protein 1 (4E-BP1) suppresses translation initiation. The absence of 4E-BP1 drastically reduces the amount of adipose tissue in mice. To address the role of 4E-BP1 in adipocyte differentiation, we characterized 4E-BP1^−/− mice in this study. The lack of 4E-BP1 decreased the amount of white adipose tissue and increased the amount of brown adipose tissue. In 4E-BP1^−/− MEF cells, PPARγ coactivator 1 alpha (PGC-1α) expression increased and exogenous 4E-BP1 expression suppressed PGC-1α expression. The level of 4E-BP1 expression was higher in white adipocytes than in brown adipocytes and showed significantly greater up-regulation in white adipocytes than in brown adipocytes during preadipocyte differentiation into mature adipocytes. The amount of PGC-1α was consistently higher in HB cells (a brown preadipocyte cell line) than in HW cells (a white preadipocyte cell line) during differentiation. Moreover, the ectopic over-expression of 4E-BP1 suppressed PGC-1α expression in white adipocytes, but not in brown adipocytes. Thus, the results of our study indicate that 4E-BP1 may suppress brown adipocyte differentiation and PGC-1α expression in white adipose tissues.     

Adipogenesis of murine embryonic stem cells in a three-dimensional culture system using electrospun polymer scaffolds

A mechanistic understanding of adipose tissue differentiation is critical for the treatment and prevention of obesity and type 2 diabetes. Conventional in vitro models of adipogenesis are preadipocytes or freshly isolated adipocytes grown in two-dimensional (2D) cultures. Optimal results using in vitro tissue culture models can be expected only when adipocyte models closely resemble adipose tissue in vivo. Thus the design of an in vitro three-dimensional (3D) model which faithfully mimics the in vivo environment is needed to effectively study adipogenesis. Pluripotent embryonic stem (ES) cells are a self-renewing cell type that can readily be differentiated into adipocytes. In this study, a 3D culture system was developed to mimic the geometry of adipose tissue in vivo. Murine ES cells were seeded into electrospun polycaprolactone scaffolds and differentiated into adipocytes in situ by hormone induction as demonstrated using a battery of gene and protein expression markers along with the accumulation of neutral lipid droplets. Insulin-responsive Akt phosphorylation, and beta-adrenergic stimulation of cyclic AMP synthesis were demonstrated in ES cell-derived adipocytes. Morphologically, ES cell-derived adipocytes resembled native fat cells by scanning electron and phase contrast microscopy. This tissue engineered ES cell-matrix model has potential uses in drug screening and other therapeutic developments.     

Two alternative procedures for isolating adipofibroblasts from sheep skeletal muscle

Methods to isolate cells used in the study of adipocyte differentiation are lengthy, potentially damaging to the cells collected, and usually result in a mixed group of cells which are difficult to define clearly. Additionally, much work done on the differentiation of fibroblasts to preadipocytes or preadipocytes to adipocytes has relied on the use of populations of cells which are either embryonic in origin or from genetically unique animals. We therefore report two simple and rapid protocols for obtaining relatively pure populations of preadipocytes from perimuscular fat and from intramuscular fat depots of normal sheep skeletal muscle. In the first procedure, a finely minced preparation of perimuscular adipose tissue is placed directly into flasks for ceiling culture. During the second procedure, free-floating adipocytes, resulting from the enzymatic preparation of satellite cells from skeletal muscle, are placed into ceiling culture. Cells from both isolation procedures attach, grow, and are later harvested for use. These cells demonstrate proliferation and differentiation abilities of normal preadipocytes. Cell populations may be expanded for use in cloning pure populations of adipocytes or used directly in studies to identify mechanisms of adipocyte development and intercellular communication with myogenic cells.     

Insulin resistance and diabetes mellitus in transgenic mice expressing nuclear SREBP-1c in adipose tissue: model for congenital generalized lipodystrophy

Overexpression of the nuclear form of sterol regulatory element-binding protein-1c (nSREBP-1c/ADD1) in cultured 3T3-L1 preadipocytes was shown previously to promote adipocyte differentiation. Here, we produced transgenic mice that overexpress nSREBP-1c in adipose tissue under the control of the adipocyte-specific aP2 enhancer/promoter. A syndrome with the following features was observed: (1) Disordered differentiation of adipose tissue. White fat failed to differentiate fully, and the size of white fat depots was markedly decreased. Brown fat was hypertrophic and contained fat-laden cells resembling immature white fat. Levels of mRNA encoding adipocyte differentiation markers (C/EBPα, PPARγ, adipsin, leptin, UCP1) were reduced, but levels of Pref-1 and TNFα were increased. (2) Marked insulin resistance with 60-fold elevation in plasma insulin. (3) Diabetes mellitus with elevated blood glucose (>300 mg/dl) that failed to decline when insulin was injected. (4) Fatty liver from birth and elevated plasma triglyceride levels later in life. These mice exhibit many of the features of congenital generalized lipodystrophy (CGL), an autosomal recessive disorder in humans.     

Ageing is associated with brown adipose tissue remodelling and loss of white fat browning in female C57BL/6 mice

Fat storage changes throughout life and affects body metabolism. Ageing impact on brown (BAT) and white adipose tissue (WAT) deserves attention, especially in females, because they are less prone to age‐induced weight gain. While in male mice the impact of ageing on adipose tissue remodelling is well characterized, the effects in female mice remain largely unclear. Thus, we investigated BAT and WAT remodelling during ageing in female C57BL/6 mice. At 3 months, body weight was 24 ± 0.3 g (mean±SD), and it increased from 6 to 9 months of age (+20%, P < 0.0001). Oral glucose tolerance test showed no disturbance of glucose metabolism. All WAT depots became heavier, and white adipocytes hypertrophied. The subcutaneous and visceral WAT had clusters of beige cells in younger mice, but they were progressively lost by ageing, indicating loss of WAT browning. Older mice had hypertrophied classic brown adipocytes that had larger cytoplasmic lipid droplets than younger mice. Pearson's correlation showed that WAT mass has a weak correlate with BAT mass, although white adipocyte diameter has a strong correlation with classic brown adipocyte size. In conclusion, our results indicate that female C57BL/6 mice have a progressive age‐dependent loss of subcutaneous and visceral WAT browning, and this process runs in parallel with BAT morphological changes towards a fat storer phenotype, independent of cycling or disturbances in glucose metabolism.     

Brown adipocyte precursor cells: a morphological study

The origin of brown adipocyte precursor cells is to date unknown. Some authors believe they arise from vascular cells, others from interstitial cells. The purpose of the present ultrastructural study was to find markers in rat fetal and perinatal adipose tissue that can be used to identify brown adipose precursor cells. The study was carried out on the interscapular brown adipose tissue of fetal (fetuses of 19 and 21 days) and perinatal rats (pups of 4 and 12 hours and of 1, 3, 5, 7, 9, 11, 13, and 15 days). The analysis focused on stem cells and showed the characteristic presence of typical mitochondria which make their identification as brown adipocyte precursor cells inequivocal. These cells were frequently observed in a pericytic position. Also some endothelial cells were characterised by typical mitochondria and abundant glycogen. These data seem to support the hypothesis that brown adipocytes originate from vascular cells.     

Morphology of adipose cells in lambs at birth and during subsequent transition of brown to white adipose tissue in cold and in warm conditions

Light and electron microscopy were used to study the morphology of adipose cells in newborn Merino lambs (Ovis aries). Postnatal changes in morphology were also examined in naturally fed Merino lambs held at 26°C or at 3°C for up to 32 days from the time of birth, and in lambs fasted for up to three days at 26°C or at 3°C since birth. All adipose cells examined in the newborn lambs showed the morphological characteristics of brown adipose cells; no white adipose cells could be found. The brown adipose tissue in normally fed lambs was replaced progressively by white adipose tissue during the first two or three weeks of life, and this replacement was retarded in the lambs held at 3°C. During replacement a continuous spectrum of cells with morphological characteristics between those of brown and white adipose cells were seen. No degenerating brown adipose cells were observed; and, apart from brown adipose cells, no cells were identified that could have been precursors of white adipose cells. This evidence suggests that in various body regions of lambs white adipose cells are derived from brown adipose cells. Lambs fasted at 3°C or under conditions that approached thermoneutrality (26°C) showed rapid depletion of lipid from brown adipose cells; hence brown adipose tissue of the lamb differs from that in the newborn rabbit in which this tissue is not readily depleted of fat during starvation under thermoneutral conditions.     

Oxygen consumption in undifferentiated versus differentiated adipogenic mesenchymal precursor cells

To date, no adequate implant material for the correction of soft tissue defects such as after extensive deep burns, tumor resections or in congenital defects is available. A biohybrid composed of viable adipose precursor cells and an optimised matrix could help towards a solution. Morphologically, preadipocytes resemble fibroblasts and have not yet built a large cytoplasmic lipid droplet as found in differentiated adipocytes. Additionally, preadipocytes are smaller than mature adipocytes allowing a quicker revascularization after transplantation. Furthermore, transplanted preadipocytes can form adipose tissue in vivo whereas the transplantation of mature adipocytes often gives poor results, i.e. oil cysts or shrinkage of the transplant. Since these observations point to differences in metabolic activity between preadipocytes and adipocytes, we investigated the oxygen consumption of preadipocytes stimulated to undergo differentiation, and fibroblasts, by measuring the respiration with a Clark-type oxygen electrode. Preadipocytes had a significantly lower oxygen consumption than mature adipocytes. This advantage in respiration and the better revascularization of undifferentiated adipose tissue cells allow the development of innovative transplants and point to preadipocytes as promising tool to improve transplantations in adipose tissue reconstruction.     

In vivo differentiation of brown adipocytes in adult mice: An electron microscopic study

The differentiation of brown adipocyte precursor cells was studied in interscapular brown adipose tissue of adult mice by electron microscopy. Different stages of cell differentiation were characterized in situ. Previous autoradiographic studies suggested that interstitial cells represent the precursor cells of fully differentiated brown adipocytes. The present observations provide morphological evidence for a progressive differentiation of interstitial stem cells into mature brown adipocytes. Four typical stages of development were identified: (1) interstitial cells, (2) protoadipocytes, (3) preadipocytes, and (4) mature brown adipocytes. Interstitial stem cells were small spindle shaped cells, situated between brown adipocytes and characterized by a high nuclear-cytoplasmic ratio, the scarcity of organelles, and the absence of lipid inclusions. Protoadipocytes resembled interstitial cells except that they contained a few tiny lipid droplets in their cytoplasm. Preadipocytes had a larger cytoplasm enclosing many mitochondria and lipid droplets; the smooth endoplasmic reticulum was well developed surrounding the lipid droplets, and was closely associated with the mitochondria. Preadipocytes had the typical structure of growing cells, developing long cytoplasmic processes between and around blood capillaries. Mature brown adipocytes represented the final stage of differentiation. Almost all their cellular volume was occupied by lipid droplets and numerous mitochondria with very dense cristae. Brown adipocytes were also characterized by a tight association with blood capillaries, as expected from metabolically active cells requiring oxygen and substrates. These observations provide direct ultrastructural evidence for a progressive differentiation of interstitial cells into brown adipocytes with a continuum of intermediate cellular types.     

Ultrastructural features of highly active antiretroviral therapy-associated partial lipodystrophy

Chronic treatment with highly active antiretroviral therapy (HAART) results in a novel variety of partial lipodystrophy, combining lipoatrophic and hypertrophic areas. We have previously reported the histopathological features of this disease and have also shown that adipocyte apoptosis is involved in its origin. With the aim of further elucidating the mechanisms underlying this peculiar disorder, we performed an ultrastructural study of the adipocytes of ten HIV-1-infected patients treated with HAART for 20-42 months. In all ten cases, two main sets of ultrastructural changes were identified. Some adipocytes showed disruption of cell membranes, fragmented cytoplasmic rims, irregular cell outlines, and eventually fat droplets laying free in the connective tissue, with a histiocytic reaction around them. In addition, many adipocytes showed variable compartmentalization of fat droplets with decrease in cell size and abundant, mitochondria-rich cytoplasm. Often, a dual "white and brown" fat appearance was observed with a large unilocular vacuole surrounded by a rim of multilocular cytoplasm containing smaller isometric fat droplets and numerous mitochondria. These findings suggest that HAART-associated partial lipodystrophy is probably the result of a remodeling process of fat cells involving variable combinations of apoptosis, defective lipogenesis, and increased metabolic activity in different adipose areas of the body.     

Quantitative image analysis in adipose tissue using an automated image analysis system: differential effects of peroxisome proliferator-activated receptor-alpha and -gamma agonist on white and brown adipose tissue morphology in AKR obese and db/db diabetic mice

Morphometric analysis of adipocytes is widely used to demonstrate the effects of antiobesity drugs or anti-diabetic drugs on adipose tissues. However, adipocyte morphometry has been quantitatively performed by manual object extraction using conventional image analysis systems. The authors have developed an automated quantitative image analysis method for adipose tissues using an innovative object-based quantitative image analysis system (eCognition). Using this system, it has been shown quantitatively that morphological features of adipose tissues of mice treated with peroxisome proliferator-activated receptor (PPAR) agonists differ dramatically depending on the type of PPAR agonist. Marked alteration of morphological characteristics of brown adipose tissue (BAT) treated with GI259578A, a PPAR-alpha agonist, was observed in AKR/J (AKR) obese mice. Furthermore, there was a 22.8% decrease in the mean size of adipocytes in white adipose tissue (WAT) compared with vehicle. In diabetic db/db mice, the PPAR-gamma agonist GW347845X decreased the mean size of adipocytes in WAT by 15.4% compared with vehicle. In contrast to changes in WAT, GW347845X increased the mean size of adipocytes in BAT greatly by 96.1% compared with vehicle. These findings suggest that GI259578A may activate fatty acid oxidation in BAT and that GW347845X may cause adipocyte differentiation in WAT and enhancement of lipid storage in BAT.     

Alpha-syntrophin deficient mice are protected from adipocyte hypertrophy and ectopic triglyceride deposition in obesity

Alpha-syntrophin (SNTA) is a molecular adapter protein which is expressed in adipocytes. Knock-down of SNTA in 3T3-L1 preadipocytes increases cell proliferation, and differentiated adipocytes display small lipid droplets. These effects are both characteristics of healthy adipose tissue growth which is associated with metabolic improvements in obesity. To evaluate a role of SNTA in adipose tissue morphology and obesity associated metabolic dysfunction, SNTA deficient mice were fed a standard chow or a high fat diet. Mice deficient of SNTA had less fat mass and smaller adipocytes in obesity when compared to control animals. Accordingly, these animals did not develop liver steatosis and did not store excess triglycerides in skeletal muscle upon high fat diet feeding. SNTA?/? animals were protected from hyperinsulinemia and hepatic insulin resistance. Of note, body-weight, food uptake, and serum lipids were normal in the SNTA null mice. SNTA was induced in adipose tissues but not in the liver of diet induced obese and ob/ob mice. In human subcutaneous and visceral fat of seven patients SNTA was similarly expressed and was not associated with body mass index. Current data demonstrate beneficial effects of SNTA deficiency in obesity which is partly attributed to smaller adipocytes and reduced white adipose tissue mass. Higher SNTA protein in fat depots of obese mice may contribute to adipose tissue hypertrophy and ectopic lipid deposition which has to be confirmed in humans.     

Immunological contributions to adipose tissue homeostasis

Adipose tissue is composed of many functionally and developmentally distinct cell types, the metabolic core of which is the adipocyte. The classification of “adipocyte” encompasses three primary types – white, brown, and beige – with distinct origins, anatomic distributions, and homeostatic functions. The ability of adipocytes to store and release lipids, respond to insulin, and perform their endocrine functions (via secretion of adipokines) is heavily influenced by the immune system. Various cell populations of the innate and adaptive arms of the immune system can resist or exacerbate the development of the chronic, low-grade inflammation associated with obesity and metabolic dysfunction. Here, we discuss these interactions, with a focus on their consequences for adipocyte and adipose tissue function in the setting of chronic overnutrition. In addition, we will review the effects of diet composition on adipose tissue inflammation and recent evidence suggesting that diet-driven disruption of the gut microbiota can trigger pathologic inflammation of adipose tissue.     

Multiple symmetric lipomatosis - a reflection of new concepts about obesity

Multiple symmetric lipomatosis (MSL) is characterized by subcutaneous accumulation of nonencapsulated adipose tissue. In type 2 MSL accumulation occurs on proximal limbs, upper back and hips. This sometimes unrecognized disease is similar to an exaggerated female fat distribution and can be confused with simple obesity. Obesity is a heterogeneous disorder and we suppose that type 2 MSL might have a place on the edge of the obesity spectrum. Several contemporary concepts about adipose tissue could be recognized in the model of MSL. Changes in fat distribution among different depots of adipose tissue in obesity have emerged as origin of its metabolic complications. Decreased insulin resistance and raised adiponectin have been found in MSL just as in some other conditions with accumulation of the subcutaneous adipose tissue (SAT). In that context, MSL may present as a model for possible favourable metabolic impact of SAT depots. Adipogenesis in MSL is not a consequence of energy excess but it is an active hyperplastic proliferation of SAT. This kind of behaviour of some adipocytes in several subcutaneous areas in MSL suggests that the energy unrelated adipogenesis could contribute to the expansion of at least a part of SAT depot in obesity in general. Contrary to current concept that the signals for adipogenesis are dependent only on the energy equation, allowing this additional mechanism would imply a new approach to issues of obesity, foremost to differentiate its particular types for which these concepts may be relevant.