Regional differences of insulin action in adipose tissue: insights from in vivo and in vitro studies

Adipose tissue is now recognized to have a multitude of functions that are of importance in the regulation of energy balance and substrate metabolism. Different hormones, in particular insulin and catecholamines, govern the storage and utilization of energy in the triglyceride depots. In addition, adipocytes produce several different substances with endocrine or paracrine functions, which regulate the overall energetic homeostasis. With excess energy storage, obesity develops, leading to increased risk for type 2 diabetes and cardiovascular disease. The distribution of body fat appears to be even more important than the total amount of fat. Abdominal and, in particular, visceral adiposity is strongly linked to insulin resistance, type 2 diabetes, hypertension and dyslipidaemia, leading to increased risk of cardiovascular disease. The adverse metabolic impact of visceral fat has been attributed to distinct biological properties of adipocytes in this depot compared with other adipose tissue depots. Indeed, regional variations in the metabolic activity of fat cells have been observed. Furthermore, expression studies aiming at defining the unique biological properties of adipose tissues from distinct anatomical sites have identified depot-related differences in the protein content of fat-produced molecules. In this review we wish to summarize important results from the literature and also some recent data from our own work. The main scope is to describe the biological functions of adipose tissue, and to focus on metabolic, hormonal, and signalling differences between fat depots.     

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.     

Multiple symmetric lipomatosis. Ultrastructural investigation of the tissue and preadipocytes in primary culture

Multiple symmetric lipomatosis (MSL) is characterized by enlarging, painless fat deposits in the neck and upper trunk. The pathogenesis of MSL is unknown. Owing to localization of MSL fat deposits in the neck and interscapular region, it has been suggested that they could originate from brown fat. However, the histological appearance of MSL adipose tissue is that of white fat, with prevailing monovacuolar adipocytes. Nevertheless, MSL adipocytes are smaller than adipocytes of the common white adipose tissue and show peculiar metabolic features. The ultrastructure of MSL lesions has been not described. The present work investigated the ultrastructural morphology of MSL adipose tissue and lipomatous adipocyte precursors maintained in long-term culture. Samples of lipomatous tissue were obtained from patients affected with MSL undergoing surgical lipectomy. Portion of the tissue was processed for electron microscopy; the rest was digested with collagenase, and isolated preadipocytes from the stromal-vascular fraction were cultured up to 15 days. Cultured cells were prepared for electron microscopy in situ and their morphology compared with human white adipose tissue preadipocytes and rat brown preadipocytes cultured in parallel. Results show the following. 1) Adipocytes of MSL are not monovacuolar and resemble the largest adipocytes that can be found in rat and human brown fat. 2) Some morphological features of MSL adipocyte precursors resemble brown adipocyte more than white: cultured MSL preadipocytes transiently develop large mitochondria with parallel cristae resembling those of the brown fat cell and maintain a multivacuolar lipid deposit in culture, i.e. a typical feature of brown preadipocytes. 3) Some morphological features suggest a neoplastic nature of MSL adipocytes. Taken together, these findings suggest that MSL is a neoplastic disease which could originate in brown fat.     

Host rather than graft origin of Matrigel-induced adipose tissue in the murine tissue-engineering chamber

We have recently shown that Matrigel-filled chambers containing fibroblast growth factor-2 (FGF2) and placed around an epigastric pedicle in the mouse were highly adipogenic. Contact of this construct with pre-existing tissue or a free adipose graft was required. To further investigate the mechanisms underpinning formation of new adipose tissue, we seeded these chambers with human adipose biopsies and human adipose-derived cell populations in severe combined immunodeficient mice and assessed the origin of the resultant adipose tissue after 6 weeks using species-specific probes. The tissues were negative for human-specific vimentin labeling, suggesting that the fat originates from the murine host rather than the human graft. This was supported by the strong presence of mouse-specific Cot-1 deoxyribonucleic acid labeling, and the absence of human Cot-1 labeling in the new fat. Even chambers seeded with FGF2/Matrigel containing cultured human stromal-vascular fraction (SVF) labeled strongly only for human vimentin in cells that did not have a mature adipocyte phenotype; the newly formed fat tissue was negative for human vimentin. These findings indicate that grafts placed in the chamber have an inductive function for neo-adipogenesis, rather than supplying adipocyte-precursor cells to generate the new fat tissue, and preliminary observations implicate the SVF in producing inductive factors. This surprising finding opens the door for refinement of current adipose tissue-engineering approaches.     

Thoughts on the source of tissue on subsequent cell culture success

This paper describes attempts to initiate equine adipocyte cultures from necropsy cases with varying intervals from time of death to isolation and culture. Equine adipocytes were isolated from 21 necropsy cases, regardless of the interval from time after death to establishment in primary ceiling cultures. However, while all cultures produced adipocytes, only 2 attempts to produce long-term equine adipocyte cultures from the subcutaneous rump fat depots were successful and not contaminated. Findings from these experiments indicate that it is possible to collect and culture equine adipocytes from necropsy cases with varying intervals of time of death to culturing provided that the issue of contamination is addressed. Viable cells were produced from tissue with an interval of 38.5 hours as well as 45 minutes. This result encourages the continuation of research using equine necropsy cases as a source of adipose tissue.     

The role of adipose tissue dysfunction in the pathogenesis of obesity-related insulin resistance

Research of the past decade has increased our understanding of the role adipose tissue plays in health and disease. Adipose tissue is now recognized as a highly active metabolic and endocrine organ. Adipocytes are of importance in buffering the daily influx of dietary fat and exert autocrine, paracrine and/or endocrine effects by secreting a variety of adipokines. The normal function of adipose tissue is disturbed in obesity, and there is accumulating evidence to suggest that adipose tissue dysfunction plays a prominent role in the development and/or progression of insulin resistance. Obese individuals often have enlarged adipocytes with a reduced buffering capacity for lipid storage, thereby exposing other tissues to an excessive influx of lipids, leading to ectopic fat deposition and insulin resistance in situations where energy intake exceeds energy expenditure. In addition, adipose tissue blood flow is decreased in obesity. This impairment may affect lipid handling in adipose tissue and, thereby, further contribute to excessive fat storage in non-adipose tissues. On the other hand, adipose tissue hypoperfusion may induce hypoxia in this tissue. Adipose tissue hypoxia may result in disturbances in adipokine secretion and increased macrophage infiltration in adipose tissue, events that are frequently observed in obesity. In this review, it is discussed how enlarged adipocytes, an impaired blood flow through adipose tissue, adipose tissue hypoxia, adipose tissue inflammation and macrophage infiltration are interrelated and may induce insulin resistance.     

Possible role of nitric oxide on adipocyte lipolysis in exercise-trained rats

A possible role of nitric oxide (NO) on adipocyte lipolysis was studied in exercise-trained (9 weeks of running) rats. Lipolysis in adipose tissue tended to be greater in trained rats than in control rats. A treatment of adipose tissue with 5 mM N(G)-nitro-L-arginine methyl ester (L-NAME) showed that basal and isoproterenol-stimulated lipolysis were both significantly greater in trained rats than in control rats. In contrast, in isolated adipocytes L-NAME had no effect on lipolysis in either group of rats, though the lipolysis of isolated adipocytes was significantly greater in trained rats than in control rats. Training significantly reduced nitrite/nitrate production in adipocytes, but not in tissue. On the other hand, training increased the protein expression of endothelial nitric oxide synthase (eNOS), but not that of inducible NOS (iNOS) in the extracts of tissue homogenates. In tissue homogenates, eNOS activity but not iNOS activity was significantly greater in trained rats than in control rats. In cellular extracts, training significantly reduced the activities of both NOS's, but the mRNA expressions of both NOS's were not different between groups. The NO donors, S-nitroso-N-acetyl-penicillamine (SNAP) and 1-propamine, 3-(2-hydroxy-2-nitroso-1-propyl-hydrazine) (PAPA-NONOate), significantly inhibited adipocyte lipolysis in response to isoproterenol in both groups. This inhibitory effect of SNAP, but not that of PAPA-NONOate, was greater in the adipocytes of trained rats than in those of the control rats. Thus it is possible that NO is involved in the regulation of lipolysis and that exercise training enhances the responsiveness of adipocytes to extracellular NO with the reduced production of nitrite/nitrate in adipocytes because of decreased activities of NOS's. On the other hand, it is also possible that exercise increases either the activity or the protein expression of eNOS in adipose tissue.     

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.     

Feast and famine: Adipose tissue adaptations for healthy aging

Proper adipose tissue function controls energy balance with favourable effects on metabolic health and longevity. The molecular and metabolic asset of adipose tissue quickly and dynamically readapts in response to nutrient fluctuations. Once delivered into cells, nutrients are managed by mitochondria that represent a key bioenergetics node. A persistent nutrient overload generates mitochondrial exhaustion and uncontrolled reactive oxygen species (^mtROS) production. In adipocytes, metabolic/molecular reorganization is triggered culminating in the acquirement of a hypertrophic and hypersecretory phenotype that accelerates aging. Conversely, dietary regimens such as caloric restriction or time-controlled fasting endorse mitochondrial functionality and ^mtROS-mediated signalling, thus promoting geroprotection. In this perspective view, we argued some important molecular and metabolic aspects related to adipocyte response to nutrient stress. Finally we delineated hypothetical routes by which molecularly and metabolically readapted adipose tissue promotes healthy aging.     

Molecular determinants of brown adipocyte formation and function

Humans contain essentially two types of adipose tissue: brown adipose tissue (BAT) and white adipose tissue (WAT). The function of WAT is to store fat while that of BAT is to burn fat for heat production. A potential strategy to combat obesity and its related disorders is to induce the conversion of WAT into BAT. In this issue of Genes & Development, Kajimura and colleagues (pp. 1397-1409) have identified a mechanism by which PRDM16, the principal regulator of brown adipocyte formation and function, can simultaneously induce BAT gene expression, while suppressing WAT gene expression. The studies suggest that PRDM16 and its associated coregulators PPARgamma coactivator-1alpha (PGC-1alpha) and C-terminal-binding protein 1/2 (CtBP1/2), which control the switch from WAT to BAT, are potential targets for development of obesity-related therapeutics.     

Structural and biochemical characteristics of various white adipose tissue depots

It is now widely accepted that white adipose tissue (WAT) is not merely a fuel storage organ, but also a key component of metabolic homoeostatic mechanisms. Apart from its major role in lipid and glucose metabolism, adipose tissue is also involved in a wide array of other biological processes. The hormones and adipokines, as well as other biologically active agents released from fat cells, affect many physiological and pathological processes. WAT is neither uniform nor inflexible because it undergoes constant remodelling, adapting the size and number of adipocytes to changes in nutrients' availability and hormonal milieu. Fat depots from different areas of the body display distinct structural and functional properties and have disparate roles in pathology. The two major types of WAT are visceral fat, localized within the abdominal cavity and mediastinum, and subcutaneous fat in the hypodermis. Visceral obesity correlates with increased risk of insulin resistance and cardiovascular diseases, while increase of subcutaneous fat is associated with favourable plasma lipid profiles. Visceral adipocytes show higher lipogenic and lipolytic activities and produce more pro-inflammatory cytokines, while subcutaneous adipocytes are the main source of leptin and adiponectin. Moreover, adipose tissue associated with skeletal muscles (intramyocellular and intermuscular fat) and with the epicardium is believed to provide fuels for skeletal and cardiac muscle contraction. However, increased mass of either epicardial or intermuscular adipose tissue correlates with cardiovascular risk, while the presence of the intramyocellular fat is a risk factor for the development of insulin resistance. This review summarizes results of mainly human studies related to the differential characteristics of various WAT depots.     

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.     

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.     

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.     

Analysis of in vitro secretion profiles from adipose-derived cell populations

ABSTRACT: BACKGROUND: Adipose tissue is an attractive source of cells for therapeutic purposes because of the ease of harvest and the high frequency of mesenchymal stem cells (MSCs). Whilst it is clear that MSCs have significant therapeutic potential via their ability to secrete immuno-modulatory and trophic cytokines, the therapeutic use of mixed cell populations from the adipose stromal vascular fraction (SVF) is becoming increasingly common. METHODS: In this study we have measured a panel of 27 cytokines and growth factors secreted by various combinations of human adipose-derived cell populations. These were 1. co-culture of freshly isolated SVF with adipocytes, 2. freshly isolated SVF cultured alone, 3. freshly isolated adipocytes alone and 4. adherent adipose-derived mesenchymal stem cells (ADSCs) at passage 2. In addition, we produced an 'in silico' dataset by combining the individual secretion profiles obtained from culturing the SVF with that of the adipocytes. This was compared to the secretion profile of co-cultured SVF and adipocytes. Two-tailed t-tests were performed on the secretion profiles obtained from the SVF, adipocytes, ADSCs and the 'in silico' dataset and compared to the secretion profiles obtained from the co-culture of the SVF with adipocytes. A p-value of < 0.05 was considered statistically different. To assess the overall changes that may occur as a result of co-culture we compared the proteomes of SVF and SVF co-cultured with adipocytes using iTRAQ quantitative mass spectrometry. RESULTS: A co-culture of SVF and adipocytes results in a distinct secretion profile when compared to all other adipose-derived cell populations studied. This illustrates that cellular crosstalk during co-culture of the SVF with adipocytes modulates the production of cytokines by one or more cell types. No biologically relevant differences were detected in the proteomes of SVF cultured alone or co-cultured with adipocytes. CONCLUSIONS: The use of mixed adipose cell populations does not appear to induce cellular stress and results in enhanced secretion profiles. Given the importance of secreted cytokines in cell therapy, the use of a mixed cell population such as the SVF with adipocytes may be considered as an alternative to MSCs or fresh SVF alone.     

The role of protease inhibitors in the pathogenesis of HIV-associated insulin resistance: Cellular mechanisms and clinical implications

HIV-associated insulin resistance frequently presents as relative lack of peripheral adipose tissue storage associated with dyslipidemia. This review discusses explanations for the links between acute and subacute abnormalities in glucose metabolism and chronic changes in adipose tissue distribution. Specifically, the molecular mechanisms by which the HIV protease inhibitor class of drugs may affect the normal stimulatory effect of insulin on glucose and fat storage are reviewed. It is proposed that both chronic inflammation from HIV infection and treatment with some drugs in this class trigger cellular homeostatic stress responses with adverse effects on glucose metabolism. The physiologic outcome is such that total energy storage in the adipocytes is decreased, and the remaining adipocytes resist further energy storage. The excess circulating and dietary lipid metabolites, normally "absorbed" by adipose tissue, are deposited ectopically in lean (muscle and liver) tissue where they impair insulin action. This leads to a pathologic cycle of insulin resistance, lipotoxicity and lipoatrophy and a clinical phenotype of body fat distribution with elevated waist-to-hip ratio similar to the metabolic syndrome.     

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.     

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.     

Sema3a is produced by brown adipocytes and its secretion is reduced following cold acclimation

Heat production in brown adipose tissue (BAT) and brown adipocyte recruitment depend heavily on BAT vascular and parenchymal sympathetic and sensory innervation. The expression and distribution of Sema3a, a recently discovered chemorepellent neuronal factor active on both sympathetic and sensory peripheral nerves, were studied in interscapular rat BAT. In rats maintained in thermoneutral conditions, brown adipocytes produced both active isoforms of Sema3a and showed a distinct peripheral polarized immunostaining pattern. This suggests a role for Sema3a secreted by brown adipocytes in the guidance of axons toward their correct targets. In cold-acclimated rats, where parenchymal nerve density is higher, both the expression and the immunostaining of the two active isoforms were slightly but significantly reduced and the distinct staining pattern was not observed. These data suggest that the secretion of Sema3a is inhibited in the brown adipocytes of cold-acclimated rats. Thus, Sema3a could play a role in the plastic adjustment of BAT innervation observed in different conditions of functional demand.