Brown adipose tissue whitening leads to brown adipocyte death and adipose tissue inflammation.

In mammals, white adipose tissue (WAT) stores and releases lipids, whereas brown adipose tissue (BAT) oxidizes lipids to fuel thermogenesis. In obese individuals, WAT undergoes profound changes; it expands, becomes dysfunctional, and develops a low-grade inflammatory state. Importantly, BAT content and activity decline in obese subjects, mainly as a result of the conversion of brown adipocytes to white-like unilocular cells. Here, we show that BAT "whitening" is induced by multiple factors, including high ambient temperature, leptin receptor deficiency, ß-adrenergic signaling impairment, and lipase deficiency, each of which is capable of inducing macrophage infiltration, brown adipocyte death, and crown-like structure (CLS) formation. Brown-to-white conversion and increased CLS formation were most marked in BAT from adipose triglyceride lipase (Atgl)-deficient mice, where, according to transmission electron microscopy, whitened brown adipocytes contained enlarged endoplasmic reticulum, cholesterol crystals, and some degenerating mitochondria, and were surrounded by an increased number of collagen fibrils. Gene expression analysis showed that BAT whitening in Atgl-deficient mice was associated to a strong inflammatory response and NLRP3 inflammasome activation. Altogether, the present findings suggest that converted enlarged brown adipocytes are highly prone to death, which, by promoting inflammation in whitened BAT, may contribute to the typical inflammatory state seen in obesity.

p53 regulates expression of uncoupling protein 1 through binding and repression of PPARγ coactivator-1α.

The tumor suppressor p53 (TRP53 in mice) is known for its involvement in carcinogenesis, but work during recent years has underscored the importance of p53 in the regulation of whole body metabolism. A general notion is that p53 is necessary for efficient oxidative metabolism. The importance of UCP1-dependent uncoupled respiration and increased oxidation of glucose and fatty acids in brown or brown-like adipocytes, termed brite or beige, in relation to energy balance and homeostasis has been highlighted recently. UCP1-dependent uncoupled respiration in classic interscapular brown adipose tissue is central to cold-induced thermogenesis, whereas brite/beige adipocytes are of special importance in relation to diet-induced thermogenesis, where the importance of UCP1 is only clearly manifested in mice kept at thermoneutrality. We challenged wild-type and TRP53-deficient mice by high-fat feeding under thermoneutral conditions. Interestingly, mice lacking TRP53 gained less weight compared with their wild-type counterparts. This was related to an increased expression of Ucp1 and other PPARGC1a and PPARGC1b target genes but not Ppargc1a or Ppargc1b in inguinal white adipose tissue of mice lacking TRP53. We show that TRP53, independently of its ability to bind DNA, inhibits the activity of PPARGC1a and PPARGC1b. Collectively, our data show that TRP53 has the ability to regulate the thermogenic capacity of adipocytes through modulation of PPARGC1 activity.

Adipsin is an adipokine that improves ß cell function in diabetes.

A hallmark of type 2 diabetes mellitus (T2DM) is the development of pancreatic ß cell failure, which results in insulinopenia and hyperglycemia. We show that the adipokine adipsin has a beneficial role in maintaining ß cell function. Animals genetically lacking adipsin have glucose intolerance due to insulinopenia; isolated islets from these mice have reduced glucose-stimulated insulin secretion. Replenishment of adipsin to diabetic mice treated hyperglycemia by boosting insulin secretion. We identify C3a, a peptide generated by adipsin, as a potent insulin secretagogue and show that the C3a receptor is required for these beneficial effects of adipsin. C3a acts on islets by augmenting ATP levels, respiration, and cytosolic free Ca(2+). Finally, we demonstrate that T2DM patients with ß cell failure are deficient in adipsin. These findings indicate that the adipsin/C3a pathway connects adipocyte function to ß cell physiology, and manipulation of this molecular switch may serve as a therapy in T2DM.

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.

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 ß3-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.

Impaired local production of proresolving lipid mediators in obesity and 17-HDHA as a potential treatment for obesity-associated inflammation.

Obesity-induced chronic low-grade inflammation originates from adipose tissue and is crucial for obesity-driven metabolic deterioration, including insulin resistance and type 2 diabetes. Chronic inflammation may be a consequence of a failure to actively resolve inflammation and could result from a lack of local specialized proresolving lipid mediators (SPMs), such as resolvins and protectins, which derive from the n-3 polyunsaturated fatty acids eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA). We assessed obesity-induced changes of n-3-derived SPMs in adipose tissue and the effects of dietary EPA/DHA thereon. Moreover, we treated obese mice with SPM precursors and investigated the effects on inflammation and metabolic dysregulation. Obesity significantly decreased DHA-derived 17-hydroxydocosahexaenoic acid (17-HDHA, resolvin D1 precursor) and protectin D1 (PD1) levels in murine adipose tissue. Dietary EPA/DHA treatment restored endogenous biosynthesis of n-3-derived lipid mediators in obesity while attenuating adipose tissue inflammation and improving insulin sensitivity. Notably, 17-HDHA treatment reduced adipose tissue expression of inflammatory cytokines, increased adiponectin expression, and improved glucose tolerance parallel to insulin sensitivity in obese mice. These findings indicate that impaired biosynthesis of certain SPM and SPM precursors, including 17-HDHA and PD1, contributes to adipose tissue inflammation in obesity and suggest 17-HDHA as a novel treatment option for obesity-associated complications.

In vivo physiological transdifferentiation of adult adipose cells.

Grafts of adipose tissue from adult Rosa26 mice from different sites of the body, irrespective of the sex of the donor, share with the mammary fat the property of giving rise to milk-secreting epithelial cells when exposed to the microenvironment of the mammary gland in pregnant and lactating females. To rule out the possibility that the labeled mammary glandular tissue was derived from stem cells associated with the stroma vascular part of the grafts, we injected into the mammary gland a pure suspension of adipocytes obtained by treating a fragment of adipose tissue with collagenase. X-gal-positive cells were inserted into the alveoli of the native gland, and electron microscopy showed that the labeled cells had transformed into milk-secreting glandular cells. At the site of the adipocyte injection, the labeled alveoli contained a mixture of X-gal-positive and X-gal-negative cells, and a single epithelial cell was occasionally stained in an otherwise unlabeled alveolus. This suggests that growing ducts individually recruit adjacent adipocytes that transdifferentiate into secretory epithelial cells as they became part of the glandular alveoli. After dissociation, the isolated adipocytes retained the morphology and protein markers typical of differentiated fat cells but expressed high levels of stem cell genes and the reprogramming transcription factor Klf4. Thus, the well-documented osteogenic, chondrogenic, myogenic, and angiogenic transformation of preadipocytes associated with the stroma vascular component of the adipose tissue may reflect an intrinsic capability of adipocytes to reprogram their gene expression and transform into different cytotypes.

Neuronal protein tyrosine phosphatase 1B deficiency results in inhibition of hypothalamic AMPK and isoform-specific activation of AMPK in peripheral tissues.

PTP1B(-/-) mice are resistant to diet-induced obesity due to leptin hypersensitivity and consequent increased energy expenditure. We aimed to determine the cellular mechanisms underlying this metabolic state. AMPK is an important mediator of leptin's metabolic effects. We find that alpha1 and alpha2 AMPK activity are elevated and acetyl-coenzyme A carboxylase activity is decreased in the muscle and brown adipose tissue (BAT) of PTP1B(-/-) mice. The effects of PTP1B deficiency on alpha2, but not alpha1, AMPK activity in BAT and muscle are neuronally mediated, as they are present in neuron- but not muscle-specific PTP1B(-/-) mice. In addition, AMPK activity is decreased in the hypothalamic nuclei of neuronal and whole-body PTP1B(-/-) mice, accompanied by alterations in neuropeptide expression that are indicative of enhanced leptin sensitivity. Furthermore, AMPK target genes regulating mitochondrial biogenesis, fatty acid oxidation, and energy expenditure are induced with PTP1B inhibition, resulting in increased mitochondrial content in BAT and conversion to a more oxidative muscle fiber type. Thus, neuronal PTP1B inhibition results in decreased hypothalamic AMPK activity, isoform-specific AMPK activation in peripheral tissues, and downstream gene expression changes that promote leanness and increased energy expenditure. Therefore, the mechanism by which PTP1B regulates adiposity and leptin sensitivity likely involves the coordinated regulation of AMPK in hypothalamus and peripheral tissues.

Haploinsufficiency of the retinoblastoma protein gene reduces diet-induced obesity, insulin resistance, and hepatosteatosis in mice.

Brown adipose tissue activity dissipates energy as heat, and there is evidence that lack of the retinoblastoma protein (pRb) may favor the development of the brown adipocyte phenotype in adipose cells. In this work we assessed the impact of germ line haploinsufficiency of the pRb gene (Rb) on the response to high-fat diet feeding in mice. Rb(+/-) mice had body weight and adiposity indistinguishable from that of wild-type (Rb(+/+)) littermates when maintained on a standard diet, yet they gained less body weight and body fat after long-term high-fat diet feeding coupled with reduced feed efficiency and increased rectal temperature. Rb haploinsufficiency ameliorated insulin resistance and hepatosteatosis after high-fat diet in male mice, in which these disturbances were more marked than in females. Compared with wild-type littermates, Rb(+/-) mice fed a high-fat diet displayed higher expression of peroxisome proliferator-activated receptor (PPAR)gamma as well as of genes involved in mitochondrial function, cAMP sensitivity, brown adipocyte determination, and tissue vascularization in white adipose tissue depots. Furthermore, Rb(+/-) mice exhibited signs of enhanced activation of brown adipose tissue and higher expression levels of PPARalpha in liver and of PPARdelta in skeletal muscle, suggestive of an increased capability for fatty acid oxidation in these tissues. These findings support a role for pRb in modulating whole body energy metabolism and the plasticity of the adipose tissues in vivo and constitute first evidence that partial deficiency in the Rb gene protects against the development of obesity and associated metabolic disturbances.

Partial lipodystrophy and insulin resistant diabetes in a patient with a homozygous nonsense mutation in CIDEC.

Lipodystrophic syndromes are characterized by adipose tissue deficiency. Although rare, they are of considerable interest as they, like obesity, typically lead to ectopic lipid accumulation, dyslipidaemia and insulin resistant diabetes. In this paper we describe a female patient with partial lipodystrophy (affecting limb, femorogluteal and subcutaneous abdominal fat), white adipocytes with multiloculated lipid droplets and insulin-resistant diabetes, who was found to be homozygous for a premature truncation mutation in the lipid droplet protein cell death-inducing Dffa-like effector C (CIDEC) (E186X). The truncation disrupts the highly conserved CIDE-C domain and the mutant protein is mistargeted and fails to increase the lipid droplet size in transfected cells. In mice, Cidec deficiency also reduces fat mass and induces the formation of white adipocytes with multilocular lipid droplets, but in contrast to our patient, Cidec null mice are protected against diet-induced obesity and insulin resistance. In addition to describing a novel autosomal recessive form of familial partial lipodystrophy, these observations also suggest that CIDEC is required for unilocular lipid droplet formation and optimal energy storage in human fat.

Complement abnormalities in acquired lipodystrophy revisited.

CONTEXT: Lipodystrophy is a heterogeneous condition characterized by an inherited or acquired deficiency in the number of adipocytes required for the storage of energy as triglycerides. Acquired lipodystrophy is frequently associated with other autoimmune disorders. One well-studied form is characterized by the selective loss of upper body fat in association with activation of the alternative complement pathway by C3 nephritic factor, low complement factor C3, and mesangiocapillary glomerulonephritis. OBJECTIVE: We now describe an immunologically distinct form of acquired generalized lipodystrophy, with evidence of activation of the classical complement pathway (low C4) and autoimmune hepatitis. Patients and Research Design: Three unrelated patients with acquired lipodystrophy and low complement C4 levels are described. In vitro analysis of the complement pathway was undertaken to determine the reason for the low C4 complement levels. Biopsies were obtained from liver, bone marrow, and adipose tissue for histological analysis. RESULTS: All three patients manifested near-total lipodystrophy, chronic hepatitis with autoimmune features, and low C4 complement levels. Additional autoimmune diseases, including severe hemolytic anemia, autoimmune thyroid disease, and polyneuropathy, were variably present. Detailed studies of complement pathways suggested constitutive classical pathway activation. CONCLUSIONS: Although the previously described syndrome, which typically results in a cephalad pattern of partial lipodystrophy, results from activation of the alternative complement pathway, this form, in which lipodystrophy is generalized, is associated with activation of the classical pathway. Future therapeutic approaches to these disorders may benefit from being tailored to their distinct immunopathogenesis.

Effects of 6-month daily supplementation with oral beta-carotene in combination or not with benzo[a]pyrene on cell-cycle markers in the lung of ferrets.

Epidemiological studies have demonstrated that people who eat more fruits and vegetables (rich in carotenoids) and people who have higher serum beta-carotene (BC) levels have a lower risk of cancer, particularly lung cancer. However, the two main human intervention studies of BC supplementation (the ATBC and the CARET trials) revealed an increased risk of lung cancer among smokers and asbestos workers. Previous studies carried out in the ferret have reported that BC effects are related to dose. Here, we treated ferrets with two concentrations of oral BC (0.8 and 3.2 mg/kg body weight per day) for 6 months, using BC in a formulation also containing dl-alpha-tocopherol and ascorbyl palmitate. The effect of the smoke-derived carcinogenic agent benzo[a]pyrene (BP), with or without low-dose BC, was also analysed. We determined the protein levels and mRNA expression levels of activator protein 1 (c-Jun and c-Fos), c-Myc, cyclin D1, proliferating cellular nuclear antigen and retinoic acid receptor beta. We did not find higher levels of cell proliferation markers in the lung of ferrets treated with BC or signals of squamous metaplasia lesions either. On the other hand, although no evident signals of pulmonary carcinogenesis were observed in animals exposed to BP, BC supplementation in these animals may prevent against excess cell proliferation, since this reestablishes Jun protein and cyclin D1 mRNA levels in the lung of BP-exposed animals. In summary, these results show that the combination of BC with alpha-tocopherol and ascorbyl palmitate does not induce pro-oxidant effects in the lung of ferrets.

Remodeling of white adipose tissue after retinoic acid administration in mice.

A reduced brown adipose phenotype in white adipose tissue (WAT) may contribute to obesity and type 2 diabetes in humans. Retinoic acid, the carboxylic form of vitamin A, triggers in rodents a reduction of body weight and adiposity and an increased expression of uncoupling proteins in brown adipose tissue and skeletal muscle. In this study, we investigated possible remodeling effects of all-trans retinoic acid (ATRA) in WAT depots. Changes in the expression of genes related to thermogenesis and fatty acid oxidation and levels of phosphorylated retinoblastoma protein were analyzed in WAT depots of adult NMRI male mice acutely injected with ATRA or vehicle, together with biometric and blood parameters. Body fat loss after ATRA treatment was unaccompanied by any increase in circulating nonesterified fatty acids or ketone bodies and accompanied by increased rectal temperature. The treatment triggered an up-regulation of the mRNA levels of uncoupling proteins 1 and 2, peroxisome proliferator-activated receptor gamma coactivator-1alpha, peroxisome proliferator-activated receptor alpha, muscle- and liver-type carnitine palmitoyltransferase 1, and subunit II of cytochrome oxidase in different WAT depots. Levels of phosphorylated retinoblastoma protein in WAT depots were increased after ATRA treatment. Adipocyte size was reduced, and the number of multilocular adipocytes was increased in inguinal WAT of ATRA-treated mice. The results indicate that ATRA favors the acquisition of brown adipose tissue-like properties in WAT. Understanding the mechanisms and effectors involved in the remodeling of WAT can contribute to new avenues of prevention and treatment of obesity and type 2 diabetes.

Adipocyte death defines macrophage localization and function in adipose tissue of obese mice and humans.

Macrophage infiltration of white adipose tissue (WAT) is implicated in the metabolic complications of obesity. The precipitating event(s) and function(s) of macrophage infiltration into WAT are unknown. We demonstrate that >90% of all macrophages in WAT of obese mice and humans are localized to dead adipocytes, where they fuse to form syncytia that sequester and scavenge the residual "free" adipocyte lipid droplet and ultimately form multinucleate giant cells, a hallmark of chronic inflammation. Adipocyte death increases in obese (db/db) mice (30-fold) and humans and exhibits ultrastructural features of necrosis (but not apoptosis). These observations identify necrotic-like adipocyte death as a pathologic hallmark of obesity and suggest that scavenging of adipocyte debris is an important function of WAT macrophages in obese individuals. The frequency of adipocyte death is positively correlated with increased adipocyte size in obese mice and humans and in hormone-sensitive lipase-deficient (HSL-/-) mice, a model of adipocyte hypertrophy without increased adipose mass. WAT of HSL-/- mice exhibited a 15-fold increase in necrotic-like adipocyte death and formation of macrophage syncytia, coincident with increased tumor necrosis factor-alpha gene expression. These results provide a novel framework for understanding macrophage recruitment, function, and persistence in WAT of obese individuals.

Regional-dependent increase of sympathetic innervation in rat white adipose tissue during prolonged fasting.

White adipose tissue (WAT) is innervated by the sympathetic nervous system. A role for WAT sympathetic noradrenergic nerves in lipid mobilization has been suggested. To gain insight into the involvement of nerve activity in the delipidation process, WAT nerves were investigated in rat retroperitoneal and epididymal depots after prolonged fasting. A significant increase in tyrosine hydroxylase (TH) content was found in epididymal and, especially, retroperitoneal WAT by Western blotting. Accordingly, an increased immunoreactivity for TH was detected by immunohistochemistry in epididymal and, especially, retroperitoneal vascular and parenchymal noradrenergic nerves. Neuropeptide Y (NPY)-containing nerves were found around arteries and in the parenchyma. Double-staining experiments and confocal microscopy showed that most perivascular and some parenchymal noradrenergic nerves also contained NPY. Detection of protein gene product (PGP) 9.5, a general marker of peripheral nerves, by Western blotting and PGP 9.5-TH by double-staining experiments showed significantly increased noradrenergic nerve density in fasted retroperitoneal, but not epididymal depots, suggesting that formation of new nerves takes place in retroperitoneal WAT in fasting conditions. On the whole, these data confirm the important role of sympathetic noradrenergic nerves in WAT lipid mobilization during fasting but also raise questions about the physiological role of regional-dependent nerve adjustments and their functional significance in relation to white adipocyte secretory products.

Morphology of ferret subcutaneous adipose tissue after 6-month daily supplementation with oral beta-carotene.

Adipose tissue is an important retinoid depot and retinoids are known to influence white and brown adipocyte metabolism. Identifying nutrients that can affect the biological activity of the adipose organ would be of great medical interest in the light of the current obesity epidemic and related disorders in developed countries. The vast majority of mammal studies of chronic administration of oral beta-carotene have used murine models, while few have employed mammals exhibiting uptake and processing of intestinal beta-carotene similar to those of humans. While rodents transform practically all ingested beta-carotene into retinol, in ferrets, as in humans, part of the beta-carotene is absorbed and released into the circulation intact. We studied the effects of 6-month daily administration of two doses of oral beta-carotene (0.8 or 3.2 mg/kg/day) on ferret body weight, size of body fat depots, and, using morphological and morphometric methods, on subcutaneous (inguinal) white adipose tissue (WAT). Because of the oral mode of administration, liver, stomach, and small and large intestine were also studied. Control animals received the vehicle. Data show that at the end of treatment the higher dose induced significantly higher body weight compared with controls and significantly higher inguinal fat depot compared with animals treated with the lower dose. In addition, chronic treatment with beta-carotene induced a dose-dependent hypertrophy of white adipocytes and increased neoangiogenesis in subcutaneous WAT in all treated ferrets. Vasculogenesis was independent of adipocyte hypertrophy. We also found focally evident liver steatosis in the ferrets treated with the higher dose of beta-carotene. The other gastrointestinal tract organs studied were not significantly different from those of control animals.