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Obesity is caused by a prolonged positive energy balance1,2. Whether reduced energy expenditure stemming from reduced activity levels contributes is debated3,4. Here we show that in both sexes, total energy expenditure (TEE) adjusted for body composition and age declined since the late 1980s, while adjusted activity energy expenditure increased over time. We use the International Atomic Energy Agency Doubly Labelled Water database on energy expenditure of adults in the United States and Europe (n = 4,799) to explore patterns in total (TEE: n = 4,799), basal (BEE: n = 1,432) and physical activity energy expenditure (n = 1,432) over time. In males, adjusted BEE decreased significantly, but in females this did not reach significance. A larger dataset of basal metabolic rate (equivalent to BEE) measurements of 9,912 adults across 163 studies spanning 100 years replicates the decline in BEE in both sexes. We conclude that increasing obesity in the United States/Europe has probably not been fuelled by reduced physical activity leading to lowered TEE. We identify here a decline in adjusted BEE as a previously unrecognized factor.

Fat distribution is sexually dimorphic and is associated with metabolic disease risk. It is unknown if prepubertal sex-hormone signaling influences adult fat distribution. Here, we show that karyotypically male androgen-insensitive mice exhibit pronounced subcutaneous adiposity compared with wild-type males and females. This subcutaneous adipose bias emerges prior to puberty and is not due to differences in adipocyte size or rates of adipogenesis between visceral and subcutaneous fat. Instead, we find that androgen-insensitive mice lack an adequate progenitor pool for normal visceral-fat expansion during development, thus increasing the subcutaneous-to-visceral-fat ratio. Obesogenic visceral-fat expansion is likewise inhibited in these mice, yet their metabolic health is similar to wild-type animals with comparable total fat mass. Taken together, these data show that adult fat distribution can be determined prior to the onset of puberty by the relative number of progenitors that seed nascent adipose depots.

Establishing metabolic programming begins during fetal and postnatal development, and early-life lipid exposures play a critical role during neonatal adipogenesis. We define how neonatal consumption of a low omega-6 to -3 fatty acid ratio (n6/n3 FA ratio) establishes FA oxidation in adipocyte precursor cells (APCs) before they become adipocytes.

Summary: Adipocyte progenitor cells (APCs) provide the reservoir of regenerative cells to produce new adipocytes, although their identity in humans remains elusive. Using FACS analysis, gene expression profiling, and metabolic and proteomic analyses, we identified three APC subtypes in human white adipose tissues. The APC subtypes are molecularly distinct but possess similar proliferative and adipogenic capacities. Adipocytes derived from APCs with high CD34 expression exhibit exceedingly high rates of lipid flux compared with APCs with low or no CD34 expression, while adipocytes produced from CD34− APCs display beige-like adipocyte properties and a unique endocrine profile. APCs were more abundant in gluteofemoral compared with abdominal subcutaneous and omental adipose tissues, and the distribution of APC subtypes varies between depots and in patients with type 2 diabetes. These findings provide a mechanistic explanation for the heterogeneity of human white adipose tissue and a potential basis for dysregulated adipocyte function in type 2 diabetes. : Raajendiran et al. report the identification of three adipocyte progenitor cell (APC) subtypes that reside in human adipose tissues. These APCs have distinct molecular phenotypes yet retain similar adipogenic potential. The APCs give rise to adipocytes with divergent metabolic and endocrine capacities and their distribution varies in type 2 diabetes patients. Keywords: adipose tissue, lipid metabolism, adipokine, obesity, type 2 diabetes, adipogenesis, adipocyte progenitor cell, beige adipocyte, lipolysis

SUMMARY We found that exposure of adult animals to caloriedense foods rapidly abolished expression of mitofusin 2 (Mfn2), a gene promoting mitochondrial fusion and mitochondrion-endoplasmic reticulum interactions, in white and brown fat. Mfn2 mRN was also robustly lower in obese human subjects compared with lean controls. Adipocyte-specific knockdown of Mfn2 in adult mice led to increased food intake, adiposity, and impaired glucose metabolism on standard chow as well as on a diet with high calorie content. The body weight and adiposity of mature adipocyte-specific Mfn2 knockout mice on a standard diet were similar to those of control mice on a high-fat diet. The transcriptional profile of the adipose tissue in adipocyte-specific Mfn2 knockout mice was consistent with adipocyte proliferation, increased lipogenesis at the tissue level, and decreased glucose utilization at the systemic level. These observations suggest a possible crucial role for mitochondrial dynamics in adipocytes in initiating systemic metabolic dysregulation., Graphical Abstract, In Brief Mancini et al. find that the mitochondrial fusion protein Mfn2 is lower in adipose tissue of mice on a high-fat diet and that of obese humans and that this protein in the fat is important for systemic control of metabolism.

Subcutaneous obesity is associated with better metabolic health than visceral obesity. Here, we leverage two mouse models with differing quantities of visceral and subcutaneous fat to assess the role of fat distribution in glucose homeostasis. Interestingly, we found genetic ablation of inguinal subcutaneous fat does not exacerbate obesity-associated impairments in glucose metabolism. Consistent with this observation, mutant mice that preferentially accrue subcutaneous fat display a similar metabolic profile to controls with equal fat mass. Importantly, the increased subcutaneous adiposity in these mice occurs downstream of androgen receptor deficiency and is not driven by elevated adiponectin activity. Rather, it is caused by diminished adipocyte precursor seeding in nascent visceral fat and proportionally greater growth of subcutaneous fat. Thus, the pattern of obesogenic fat mass expansion can be determined early in development without impacting glucose metabolism. This suggests that different mechanisms underlying biased fat accumulation exert different effects on glucometabolic health.

Catecholamine-induced lipolysis, the first step in the generation of energy substrates by the hydrolysis of triglycerides, declines with age. The defect in the mobilization of free fatty acids in the elderly is accompanied by increased visceral adiposity, lower exercise capacity, failure to maintain core body temperature during cold stress, and reduced ability to survive starvation. Although catecholamine signalling in adipocytes is normal in the elderly, how lipolysis is impaired in ageing remains unknown. Here we show that adipose tissue macrophages regulate the age-related reduction in adipocyte lipolysis in mice by lowering the bioavailability of noradrenaline. Unexpectedly, unbiased whole-transcriptome analyses of adipose macrophages revealed that ageing upregulates genes that control catecholamine degradation in an NLRP3 inflammasome-dependent manner. Deletion of NLRP3 in ageing restored catecholamine-induced lipolysis by downregulating growth differentiation factor-3 (GDF3) and monoamine oxidase A (MAOA) that is known to degrade noradrenaline. Consistent with this, deletion of GDF3 in inflammasome-activated macrophages improved lipolysis by decreasing levels of MAOA and caspase-1. Furthermore, inhibition of MAOA reversed the age-related reduction in noradrenaline concentration in adipose tissue, and restored lipolysis with increased levels of the key lipolytic enzymes adipose triglyceride lipase (ATGL) and hormone sensitive lipase (HSL). Our study reveals that targeting neuro-immunometabolic signalling between the sympathetic nervous system and macrophages may offer new approaches to mitigate chronic inflammation-induced metabolic impairment and functional decline.

Over the past 2 decades, the incidence of childhood obesity has risen dramatically. This recent rise in childhood obesity is particularly concerning as adults who were obese during childhood develop type II diabetes that is intractable to current forms of treatment compared with individuals who develop obesity in adulthood. While the mechanisms responsible for the exacerbated diabetic phenotype associated with childhood obesity is not clear, it is well known that childhood is an important time period for the establishment of normal white adipose tissue in humans. This association suggests that exposure to obesogenic stimuli during adipose development may have detrimental effects on adipose function and metabolic homeostasis. In this study, we identify the period of development associated with puberty, postnatal days 18–34, as critical for the establishment of normal adipose mass in mice. Exposure of mice to high fat diet only during this time period results in metabolic dysfunction, increased leptin expression, and increased adipocyte size in adulthood in the absence of sustained increased fat mass or body weight. These findings indicate that exposure to obesogenic stimuli during critical developmental periods have prolonged effects on adipose tissue function that may contribute to the exacerbated metabolic dysfunctions associated with childhood obesity.

Adipose tissue is composed of anatomically distinct depots that mediate several important aspects of energy homeostasis. The past two decades have witnessed increased research effort to elucidate the ontogenetic basis of adipose form and function. In this Review, we discuss advances in our understanding of adipose tissue development with particular emphasis on the embryonic patterning of depot-specific adipocyte lineages and adipocyte differentiation in vivo. Micro-environmental cues and other factors that influence cell identity and cell behavior at various junctures in the adipocyte lineage hierarchy are also considered.

The embryonic origin of distinct fat depots and the role for ontogeny in specifying the functional differences among adipocyte lineages between and within depots is unclear. Using a Cre/Lox-based strategy to track the fate of major mesodermal subcompartments in mice we present evidence that fewer than 50% of interscapular brown adipocytes are derived from progenitors of the central dermomyotome. Furthermore, we demonstrate that depot-specific adipocyte lineages spatially diverge as early as gastrulation and that perigonadal adipocytes arise from separate mesodermal subcompartments in males and females. Last, we show adipocyte precursors (APs) of distinct lineages within the same depot exhibit indistinguishable responses to a high fat diet, indicating ontogenetic differences between APs do not necessarily correspond to functional differences in this context. Altogether, these findings shed light on adipose tissue patterning and suggest the behavior of adipocyte lineage cells is not strictly determined by developmental history.

The presence of adipocytes in mammalian bone marrow (BM) has been recognized histologically for decades, yet, until recently, these cells have received little attention from the research community. Advancements in mouse transgenics and imaging methods, particularly in the last 10 years, have permitted more detailed examinations of marrow adipocytes than ever before and yielded data that show these cells are critical regulators of the BM microenvironment and whole-body metabolism. Indeed, marrow adipocytes are anatomically and functionally separate from brown, beige, and classic white adipocytes. Thus, areas of BM space populated by adipocytes can be considered distinct fat depots and are collectively referred to as marrow adipose tissue (MAT) in this review. In the proceeding text, we focus on the developmental origin and physiologic functions of MAT. We also discuss the signals that cause the accumulation and loss of marrow adipocytes and the ability of these cells to regulate other cell lineages in the BM. Last, we consider roles for MAT in human physiology and disease.

The production of new adipocytes requires the differentiation of adipocyte precursor (AP) cells residing within the adipose tissue stromal-vascular compartment. The objective was to obtain an immortalized primary adipogenic cell line derived from FACS isolated committed APs using the conditional expression of SV40 T antigen. Adipocyte precursors were isolated from white adipose tissue (WAT) using FACS to remove non-adipogenic cell populations from mice expressing a conditionally regulated SV40 T antigen. APs were maintained by continuous culture and induced to undergo adipogenic differentiation. Adipogenesis, determined by Oil Red O staining, was assessed with each passage and compared to wildtype controls. Adipogenic capability was rapidly lost with increased passage number in committed APs with concurrent reduction in cell proliferation and expression of essential late adipogenic genes, including Pparγ and C/ebpα. Thus, FACS purified committed APs have limited capability to undergo expansion and subsequent adipogenic differentiation in vitro even if they are immortalized with the SV40 T antigen.

Adipocytes were identified in human bone marrow more than a century ago, yet until recently little has been known about their origin, development, function or interactions with other cells in the bone marrow. Little functional significance has been attributed to these cells, a paradigm that still persists today. However, we now know that marrow adipose tissue increases with age and in response to a variety of physiologic induction signals. Bone marrow adipocytes have recently been shown to influence other cell populations within the marrow and can affect whole body metabolism by the secretion of a defined set of adipokines. Recent research shows that marrow adipocytes are distinct from white, brown and beige adipocytes, indicating that the bone marrow is a distinct adipose depot. This review will highlight recent data regarding these areas and the interactions of marrow adipose tissue (MAT) with cells within and outside of the bone marrow.

SUMMARY While therapeutic modulation of miRNAs provides a promising approach for numerous diseases, the promiscuous nature of miRNAs raises concern over detrimental off-target effects. miR-33 has emerged as a likely target for treatment of cardiovascular diseases. However, the deleterious effects of long-term anti-miR-33 therapies and predisposition of miR-33−/− mice to obesity and metabolic dysfunction exemplify the possible pitfalls of miRNA-based therapies. Our work provides an in-depth characterization of miR-33−/− mice and explores the mechanisms by which loss of miR-33 promotes insulin resistance in key metabolic tissues. Contrary to previous reports, our data do not support a direct role for SREBP-1-mediated lipid synthesis in promoting these effects. Alternatively, in adipose tissue of miR-33−/− mice, we observe increased pre-adipocyte proliferation, enhanced lipid uptake, and impaired lipolysis. Moreover, we demonstrate that the driving force behind these abnormalities is increased food intake, which can be prevented by pair feeding with wild-type animals., In Brief While anti-miR-33 therapies offer promise for treating cardiovascular disease, deletion of miR-33 causes obesity and metabolic dysfunction. Price et al. elucidate how miR-33 deficiency affects metabolic functions in different tissues. Rather than finding that dysregulation of SREBP-1-mediated lipid synthesis primarily drives these effects, they demonstrate that these changes depend on increased food consumption.

Excess ECM and fibrosis of white adipose tissue (WAT) is associated with tissue dysfunction and type 2 diabetes. In this issue of Cell Metabolism, Marcelin et al. (2017) elucidate a key mechanism behind WAT fibrosis in which the activation of PDGFRα on adipocyte precursors drives this population toward a fibrotic phenotype.

The study of adipose tissue in vivo has been significantly advanced through the use of genetic mouse models. While the aP2-Cre(BI) and aP2-Cre(Salk) lines have been widely used to target adipose tissue, the specificity of these lines for adipocytes has recently been questioned. Here we characterize Cre recombinase activity in multiple cell populations of the major adipose tissue depots of these and other Cre lines using the membrane-Tomato/membrane-GFP (mT/mG) dual fluorescent reporter. We find that the aP2-Cre(BI) and aP2-Cre(Salk) lines lack specificity for adipocytes within adipose tissue, and that the aP2-Cre(BI) line does not efficiently target adipocytes in white adipose depots. Alternatively, the Adiponectin-CreERT line shows high efficiency and specificity for adipocytes, while the PdgfRα-CreERUCL and PdgfRα-CreERJHU lines do not efficiently target adipocyte precursor cells in the major adipose depots. Instead, we show that the PdgfRα-Cre line is preferable for studies targeting adipocyte precursor cells in vivo.

Pigment epithelium-derived factor (PEDF), the protein product of the SERPINF1 gene, has been linked to distinct diseases involving adipose or bone tissue, the metabolic syndrome, and osteogenesis imperfecta (OI) type VI. Since mesenchymal stem cell (MSC) differentiation into adipocytes vs. osteoblasts can be regulated by specific factors, PEDF-directed dependency of murine and human MSCs was assessed. PEDF inhibited adipogenesis and promoted osteoblast differentiation of murine MSCs, osteoblast precursors, and human MSCs. Blockade of adipogenesis by PEDF suppressed peroxisome proliferator-activated receptor-γ (PPARγ), adiponectin, and other adipocyte markers by nearly 90% compared with control-treated cells (P50% compared with controls, illustrating its systemic role as a negative regulator of adipogenesis. Bones from KO mice demonstrated a reduction in mineral content recapitulating the OI type VI phenotype. These results demonstrate that the human diseases associated with PEDF reflect its ability to modulate MSC differentiation.—Gattu, A. K., Swenson, E. S., Iwakiri, Y., Samuel, V. T., Troiano, N., Berry, R., Church, C. D., Rodeheffer, M. S., Carpenter, T. O., Chung, C. Determination of mesenchymal stem cell fate by pigment epithelium-derived factor (PEDF) results in increased adiposity and reduced bone mineral content.

There is growing interest in the relationship between bone mineral density, bone strength, and fat depots. Marrow adipose tissue, a well-established component of the marrow environment, is metabolically distinct from peripheral fat depots, but its functional significance is unknown.In this review, we discuss animal and human data linking the marrow adipose tissue depot to parameters of bone density and integrity as well as the potential significance of marrow adipose tissue in metabolic diseases associated with bone loss, including type 1 diabetes mellitus and anorexia nervosa. Potential hormonal determinants of marrow adipose tissue are also discussed.We conclude that whereas most animal and human data demonstrate an inverse association between marrow adipose tissue and measures of bone density and strength, understanding the functional significance of marrow adipose tissue and its hormonal determinants will be critical to better understanding its role in skeletal integrity and the role of marrow adipose tissue in the pathophysiology of bone loss.

Mature adipocytes are generated through the proliferation and differentiation of precursor cells. Our previous studies identified adipocyte progenitors in white adipose tissue (WAT) as Lin(-):CD29(+):CD34(+):Sca-1(+):CD24(+) (CD24(+)) cells that are capable of generating functional WAT (ref. ). Here, we employ several Cre recombinase mouse models to identify the adipocyte cellular lineage in vivo. Although it has been proposed that white adipocytes are derived from endothelial and haematopoietic lineages, we find that neither of these lineages label white adipocytes. However, platelet-derived growth factor receptor α (PdgfRα)-Cre trace labels all white adipocytes. Analysis of WAT from PdgfRα-Cre reporter mice identifies CD24(+) and Lin(-):CD29(+):CD34(+):Sca-1(+): CD24(-) (CD24(-)) cells as adipocyte precursors. We show that CD24(+) cells generate the CD24(-) population in vivo and the CD24(-) cells express late markers of adipogenesis. From these data we propose a model where the CD24(+) adipocyte progenitors become further committed to the adipocyte lineage as CD24 expression is lost, generating CD24(-) preadipocytes. This characterization of the adipocyte cellular lineage will facilitate the study of the mechanisms that regulate WAT formation in vivo and WAT mass expansion in obesity.

Tissue growth and maintenance requires stem cell populations that self-renew, proliferate, and differentiate. Maintenance of white adipose tissue (WAT) requires the proliferation and differentiation of adipocyte stem cells (ASCs) to form postmitotic, lipid-filled mature adipocytes. Here we use the dynamic adipogenic program that occurs during hair growth to uncover an unrecognized regulator of ASC self-renewal and proliferation, PDGFA, which activates AKT signaling to drive and maintain the adipogenic program in the skin. Pdgfa expression is reduced in aged ASCs and is required for ASC proliferation and maintenance in the dermis, but not in other WATs. Our molecular and genetic studies uncover PI3K/AKT2 as a direct PDGFA target that is activated in ASCs during WAT hyperplasia and is functionally required for dermal ASC proliferation. Our data therefore reveal active mechanisms that regulate ASC self-renewal in the skin and show that distinct regulatory mechanisms operate in different WAT depots.

The sexually dimorphic distribution of adipose tissue influences the development of obesity-associated pathologies. The accumulation of visceral white adipose tissue (VWAT) that occurs in males is detrimental to metabolic health, while accumulation of subcutaneous adipose tissue (SWAT) seen in females may be protective. Here, we show that adipocyte hyperplasia contributes directly to the differential fat distribution between the sexes. In male mice, high-fat diet (HFD) induces adipogenesis specifically in VWAT, while in females HFD induces adipogenesis in both VWAT and SWAT in a sex hormone-dependent manner. We also show that the activation of adipocyte precursors (APs), which drives adipocyte hyperplasia in obesity, is regulated by the adipose depot microenvironment and not by cell-intrinsic mechanisms. These findings indicate that APs are plastic cells, which respond to both local and systemic signals that influence their differentiation potential independent of depot origin. Therefore, depot-specific AP microenvironment niches coordinate adipose tissue growth and distribution.

White adipose tissue (WAT) is essential for maintaining metabolic function, especially during obesity. The intronic microRNAs miR-33a and miR-33b, located within the genes encoding sterol regulatory element-binding protein 2 (SREBP-2) and SREBP-1, respectively, are transcribed in concert with their host genes and function alongside them to regulate cholesterol, fatty acid, and glucose metabolism. SREBP-1 is highly expressed in mature WAT and plays a critical role in promoting in vitro adipocyte differentiation. It is unknown whether miR-33b is induced during or involved in adipogenesis. This is in part due to loss of miR-33b in rodents, precluding in vivo assessment of the impact of miR-33b using standard mouse models. This work demonstrates that miR-33b is highly induced upon differentiation of human preadipocytes, along with SREBP-1. We further report that miR-33b is an important regulator of adipogenesis, as inhibition of miR-33b enhanced lipid droplet accumulation. Conversely, overexpression of miR-33b impaired preadipocyte proliferation and reduced lipid droplet formation and the induction of peroxisome proliferator-activated receptor γ (PPARγ) target genes during differentiation. These effects may be mediated by targeting of HMGA2, cyclin-dependent kinase 6 (CDK6), and other predicted miR-33b targets. Together, these findings demonstrate a novel role of miR-33b in the regulation of adipocyte differentiation, with important implications for the development of obesity and metabolic disease.

In vertebrates, adipose tissue is the main storage site for lipids within specialized lipid-laden mature adipocytes. While many species have evolved cells capable of lipid storage, the adipocyte represents a unique specialized cell involved in fuel storage, endocrine, nervous and immune function. However, the adipocytes are not the only cell type in mammals that can accumulate lipid droplets. The ectopic accumulation of lipid in non-adipose tissues including the liver, skeletal muscle, bone, pancreas, and heart in combination with its excessive accumulation in adipose tissue contributes to metabolic disease. Determining the lipid processing components that are necessary and sufficiently for lipid accumulation in adipose and non-adipose tissues, in addition to endocrine function, will lead to a clearer definition of an adipocyte.

Obesity is characterized by an expansion of white adipose tissue mass that results from an increase in the size and the number of adipocytes. However, the mechanisms responsible for the formation of adipocytes during development and the molecular mechanisms regulating their increase and maintenance in adulthood are poorly understood. Here, we report the use of leptin-luciferase BAC transgenic mice to track white adipose tissue (WAT) development and guide the isolation and molecular characterization of adipocytes during development using DNA microarrays. These data reveal distinct transcriptional programs that are regulated during murine WAT development in vivo. By using a de novo cis-regulatory motif discovery tool (FIRE), we identify two early gene clusters whose promoters show significant enrichment for NRF2/ETS transcription factor binding sites. We further demonstrate that Ets transcription factors, but not Nrf2, are regulated during early adipogenesis and that Ets2 is essential for the normal progression of the adipocyte differentiation program in vitro. These data identify ETS2 as a functionally important transcription factor in adipogenesis and its possible role in regulating adipose tissue mass in adults can now be tested. Our approach also provides the basis for elucidating the function of other gene networks during WAT development in vivo. Finally these data confirm that although gene expression during adipogenesis in vitro recapitulates many of the patterns of gene expression in vivo, there are additional developmental transitions in pre and post-natal adipose tissue that are not evident in cell culture systems.

SummaryIn mammalian skin, multiple types of resident cells are required to create a functional tissue and support tissue homeostasis and regeneration. The cells that compose the epithelial stem cell niche for skin homeostasis and regeneration are not well defined. Here, we identify adipose precursor cells within the skin and demonstrate that their dynamic regeneration parallels the activation of skin stem cells. Functional analysis of adipocyte lineage cells in mice with defects in adipogenesis and in transplantation experiments revealed that intradermal adipocyte lineage cells are necessary and sufficient to drive follicular stem cell activation. Furthermore, we implicate PDGF expression by immature adipocyte cells in the regulation of follicular stem cell activity. These data highlight adipogenic cells as skin niche cells that positively regulate skin stem cell activity, and suggest that adipocyte lineage cells may alter epithelial stem cell function clinically.

The increased white adipose tissue (WAT) mass associated with obesity is the result of both hyperplasia and hypertrophy of adipocytes. However, the mechanisms controlling adipocyte number are unknown in part because the identity of the physiological adipocyte progenitor cells has not been defined in vivo. In this report, we employ a variety of approaches, including a noninvasive assay for following fat mass reconstitution in vivo, to identify a subpopulation of early adipocyte progenitor cells (Lin(-):CD29(+):CD34(+):Sca-1(+):CD24(+)) resident in adult WAT. When injected into the residual fat pads of A-Zip lipodystrophic mice, these cells reconstitute a normal WAT depot and rescue the diabetic phenotype that develops in these animals. This report provides the identification of an undifferentiated adipocyte precursor subpopulation resident within the adipose tissue stroma that is capable of proliferating and differentiating into an adipose depot in vivo.

To better define the molecular mechanisms underlying leptin release from adipocytes, we developed a novel protocol that maximizes leptin production from 3T3-L1 adipocytes. The addition of a PPARgamma agonist to the Isobutylmethylxanthine/Dexamethasone/Insulin differentiation cocktail increased leptin mRNA levels by 5-fold, maintained insulin sensitivity, and yielded mature phenotype in cultured adipocytes. Under these conditions, acute insulin stimulation for 2 h induced a two-fold increase in leptin secretion, which was independent of new protein synthesis, and was not due to alterations in glucose metabolism. Stimulation with insulin for 15 min induced the same level of leptin release and was blocked by Brefeldin A. Inhibiting PI 3-kinase with wortmannin had no effect on insulin stimulation of leptin secretion. These studies show that insulin can stimulate leptin release via a PI3K independent mechanism and provide a cellular system for studying the effect of insulin and potentially other mediators on leptin secretion.

Adipocytes were identified in the bone marrow (BM) more than 100 years ago, yet until recently little has been known about their formation, function or interactions with other BM cells. We know now that marrow adipose tissue (MAT) increases with age, and in response to a variety of stressors. BM adipocytes have also been shown to have regulatory roles through their interactions with multiple cell populations within the marrow. Furthermore, recent and ongoing research is uncovering the cellular origin of BM adipocytes, implicating marrow adipose tissue as a distinct adipose depot. This chapter will highlight recent data regarding these areas, and the interaction of MAT with cells in the skeletal, hematopoietic and immune systems.

The formation of brown, white and beige adipocytes have been a subject of intense scientific interest in recent years due to the growing obesity epidemic in the United States and around the world. This interest has led to the identification and characterization of specific tissue resident progenitor cells that give rise to each adipocyte population in vivo. However, much still remains to be discovered about each progenitor population in terms of their "niche" within each tissue and how they are regulated at the cellular and molecular level during healthy and diseased states. While our knowledge of brown, white and beige adipose tissue is rapidly increasing, little is still known about marrow adipose tissue and its progenitor despite recent studies demonstrating possible roles for marrow adipose tissue in regulating the hematopoietic space and systemic metabolism at large. This chapter focuses on our current knowledge of brown, white, beige and marrow adipose tissue with a specific focus on the formation of each tissue from tissue resident progenitor cells.

Mitochondrial dysfunction causes numerous human diseases and is widely believed to be involved in aging. However, mechanisms through which compromised mitochondrial gene expression elicits the reported variety of cellular defects remain unclear. The amino-terminal domain (ATD) of yeast mitochondrial RNA polymerase is required to couple transcription to translation during expression of mitochondrial DNA-encoded oxidative phosphorylation subunits. Here we report that several ATD mutants exhibit reduced chronological life span. The most severe of these (harboring the rpo41-R129D mutation) displays imbalanced mitochondrial translation, conditional inactivation of respiration, elevated production of reactive oxygen species (ROS), and increased oxidative stress. Reduction of ROS, via overexpression of superoxide dismutase (SOD1 or SOD2 product), not only greatly extends the life span of this mutant but also increases its ability to respire. Another ATD mutant with similarly reduced respiration (rpo41-D152A/D154A) accumulates only intermediate levels of ROS and has a less severe life span defect that is not rescued by SOD. Altogether, our results provide compelling evidence for the “vicious cycle” of mitochondrial ROS production and lead us to propose that the amount of ROS generated depends on the precise nature of the mitochondrial gene expression defect and initiates a downward spiral of oxidative stress only if a critical threshold is crossed.

Excessive accumulation of white adipose tissue (WAT) is the defining characteristic of obesity. WAT mass is composed primarily of mature adipocytes, which are generated through the proliferation and differentiation of adipocyte precursors (APs). Although the production of new adipocytes contributes to WAT growth in obesity, little is known about the cellular and molecular mechanisms underlying adipogenesis in vivo. Here, we show that high-fat diet feeding in mice rapidly and transiently induces proliferation of APs within WAT to produce new adipocytes. Importantly, the activation of adipogenesis is specific to the perigonadal visceral depot in male mice, consistent with the patterns of obesogenic WAT growth observed in humans. Furthermore, we find that in multiple models of obesity, the activation of APs is dependent on the phosphoinositide 3-kinase (PI3K)-AKT2 pathway; however, the development of WAT does not require AKT2. These data indicate that developmental and obesogenic adipogenesis are regulated through distinct molecular mechanisms.

Alignment of three fungal mtRNA polymerases revealed conserved amino acid sequences in an amino-terminal region of the Saccharomyces cerevisiae enzyme implicated previously as harboring an important functional domain. Phenotypic analysis of deletion and point mutations, in conjunction with a yeast two-hybrid assay, revealed that Nam1p, a protein involved in RNA processing and translation in mitochondria, binds specifically to this domain. The significance of this interaction in vivo was demonstrated by the fact that the temperature-sensitive phenotype of a deletion mutation (rpo41Delta2), which impinges on this amino-terminal domain, is suppressed by overproducing Nam1p. In addition, mutations in the amino-terminal domain result specifically in decreased steady-state levels of mature mitochondrial CYTB and COXI transcripts, which is a primary defect observed in NAM1 null mutant yeast strains. Finally, one point mutation (R129D) did not abolish Nam1p binding, yet displayed an obvious COX1/CYTB transcript defect. This mutation exhibited the most severe mitochondrial phenotype, suggesting that mutations in the amino-terminal domain can perturb other critical interactions, in addition to Nam1p binding, that contribute to the observed phenotypes. These results implicate the amino-terminal domain of mtRNA polymerases in coupling additional factors and activities involved in mitochondrial gene expression directly to the transcription machinery.

White adipose tissue (WAT) is a heterogeneous tissue composed of lipid-filled adipocytes and several nonadipocyte cell populations, including endothelial, blood, uncharacterized stromal, and adipocyte precursor cells. Although lipid-filled adipocytes account for the majority of WAT volume and mass, nonadipocyte cell populations have critical roles in WAT maintenance, growth, and function. As mature adipocytes are terminally differentiated postmitotic cells, differentiation of adipocyte precursors is required for hyperplastic WAT growth during development and in obesity. In this chapter, we present methods to separate adipocyte precursor cells from other nonadipocyte cell populations within WAT for analysis by flow cytometry or purification by fluorescence-activated cell sorting. Additionally, we provide methods to study the adipogenic capacity of purified adipocyte precursor cells ex vivo.

Adipocytes reside in discrete, well-defined depots throughout the body. In addition to mature adipocytes, white adipose tissue depots are composed of many cell types, including macrophages, endothelial cells, fibroblasts, and stromal cells, which together are referred to as the stromal vascular fraction (SVF). The SVF also contains adipocyte progenitors that give rise to mature adipocytes in those depots. Marrow adipose tissue (MAT) or marrow fat has long been known to be present in bone marrow (BM) but its origin, development, and function remain largely unknown. Clinically, increased MAT is associated with age, metabolic diseases, drug treatment, and marrow recovery in children receiving radiation and chemotherapy. In contrast to the other depots, MAT is unevenly distributed in the BM of long bones. Conventional quantitation relies on sectioning of the bone to overcome issues with distribution but is time-consuming, resource intensive, inconsistent between laboratories and may be unreliable as it may miss changes in MAT volume. Thus, the inability to quantitate MAT in a rapid, systematic, and reproducible manner has hampered a full understanding of its development and function. In this chapter, we describe a new technique that couples histochemical staining of lipid using osmium tetroxide with microcomputerized tomography to visualize and quantitate MAT within the medullary canal in three dimensions. Imaging of osmium staining provides a high-resolution map of existing and developing MAT in the BM. Because this method is simple, reproducible, and quantitative, we expect it will become a useful tool for the precise characterization of MAT.

Adipocytes and scar tissue form during skeletal muscle degeneration. Two new studies reveal that adipocytes and fibroblasts in skeletal muscle derive from a population of bipotent progenitors that reside within muscle, but are not derived from the muscle lineage. These progenitor cells also have a surprising role in stimulating the restoration of muscle mass during regeneration.

How mitochondrial DNA (mtDNA) copy number is determined and modulated according to cellular demands is largely unknown. Our previous investigations of the related DNA helicases Pif1p and Rrm3p uncovered a role for these factors and the conserved Mec1/Rad53 nuclear checkpoint pathway in mtDNA mutagenesis and stability in Saccharomyces cerevisiae. Here, we demonstrate another novel function of this pathway in the regulation of mtDNA copy number. Deletion of RRM3 or SML1, or overexpression of RNR1, which recapitulates Mec1/Rad53 pathway activation, resulted in an approximately twofold increase in mtDNA content relative to the corresponding wild-type yeast strains. In addition, deletion of RRM3 or SML1 fully rescued the ∼50% depletion of mtDNA observed in a pif1 null strain. Furthermore, deletion of SML1 was shown to be epistatic to both a rad53 and an rrm3 null mutation, placing these three genes in the same genetic pathway of mtDNA copy number regulation. Finally, increased mtDNA copy number via the Mec1/Rad53 pathway could occur independently of Abf2p, an mtDNA-binding protein that, like its metazoan homologues, is implicated in mtDNA copy number control. Together, these results indicate that signaling through the Mec1/Rad53 pathway increases mtDNA copy number by altering deoxyribonucleoside triphosphate pools through the activity of ribonucleotide reductase. This comprises the first linkage of a conserved signaling pathway to the regulation of mitochondrial genome copy number and suggests that homologous pathways in humans may likewise regulate mtDNA content under physiological conditions.

The amino-terminal domain (ATD) of Saccharomyces cerevisiae mitochondrial RNA polymerase has been shown to provide a functional link between transcription and post-transcriptional events during mitochondrial gene expression. This connection is mediated in large part by its interactions with the matrix protein Nam1p and, based on genetic phenotypes, the mitochondrial membrane protein Sls1p. These observations led us to propose previously that mtRNA polymerase, Nam1p, and Sls1p work together to coordinate transcription and translation of mtDNA-encoded gene products. Here we demonstrate by specific labeling of mitochondrial gene products in vivo that Nam1p and Sls1p indeed work together in a pathway that is required globally for efficient mitochondrial translation. Likewise, mutations in the ATD result in similar global reductions in mitochondrial translation efficiency and sensitivity to the mitochondrial translation inhibitor erythromycin. These data, coupled with the observation that the ATD is required to co-purify Sls1p in association with mtDNA nucleoids, suggest that efficient expression of mtDNA-encoded genes in yeast involves a complex series of interactions that localize active transcription complexes to the inner membrane in order to coordinate translation with transcription.

1Mitochondrial translation is largely membrane-associated in S. cerevisiae. Recently, we discovered that the matrix protein Nam1p binds the amino-terminal domain of yeast mtRNA polymerase to couple translation and/or RNA-processing events to transcription. To gain additional insight into these transcription-coupled processes, we performed a genetic screen for genes that suppress the petite phenotype of a point mutation in mtRNA polymerase (rpo41-R129D) when overexpressed. One suppressor identified in this screen was SLS1, which encodes a mitochondrial membrane protein required for assembly of respiratory-chain enzyme complexes III and IV. The mtRNA-processing defects associated with the rpo41-R129D mutation were corrected in the suppressed strain, linking Sls1p to a pathway that includes mtRNA polymerase and Nam1p. This was supported by the observation that SLS1 overexpression rescued the petite phenotype of a NAM1 null mutation. In contrast, overexpression of Nam1p did not rescue the petite phenotype of a SLS1 null mutation, indicating that Nam1p and Sls1p are not functionally redundant but rather exist in an ordered pathway. On the basis of these data, a model in which Nam1p coordinates the delivery of newly synthesized transcripts to the membrane, where Sls1p directs or regulates their subsequent handling by membrane-bound factors involved in translation, is proposed.

Nature Cell Biol. 12, 102–104 (2010); published online 17 January 2010; corrected after print, 21 January 2010 In the version of this News and Views initially published, cells are incorrectly referred to as “α7-integrin+”. They should actually be “α7-integrin−”. This error has been corrected in the HTML and PDF version of the article.