Thomas Thomou

Mechanisms and Metabolic Implications of Regional Differences among Fat Depots

Fat distribution is closely linked to metabolic disease risk. Distribution varies with sex, genetic background, disease state, certain drugs and hormones, development, and aging. Preadipocyte replication and differentiation, developmental gene expression, susceptibility to apoptosis and cellular senescence, vascularity, inflammatory cell infiltration, and adipokine secretion vary among depots, as do fatty-acid handling and mechanisms of enlargement with positive-energy and loss with negative-energy balance. How interdepot differences in these molecular, cellular, and pathophysiological properties are related is incompletely understood. Whether fat redistribution causes metabolic disease or whether it is a marker of underlying processes that are primarily responsible is an open question.

Fat Depot–Specific Characteristics Are Retained in Strains Derived From Single Human Preadipocytes

Fat depots vary in size, function, and potential contribution to disease. Since fat tissue turns over throughout life, preadipocyte characteristics could contribute to this regional variation. To address whether preadipocytes from different depots are distinct, we produced preadipocyte strains from single abdominal subcutaneous, mesenteric, and omental human preadipocytes by stably expressing human telomere reverse transcriptase (hTERT). These strains could be subcultured repeatedly and retained capacity for differentiation, while primary preadipocyte adipogenesis and replication declined with subculturing. Primary omental preadipocytes, in which telomeres were longest, replicated more slowly than mesenteric or abdominal subcutaneous preadipocytes. Even after 40 population doublings, replication, abundance of the rapidly replicating preadipocyte subtype, and resistance to tumor necrosis factor α–induced apoptosis were highest in subcutaneous, intermediate in mesenteric, and lowest in omental hTERT-expressing strains, as in primary preadipocytes. Subcutaneous hTERT-expressing strains accumulated more lipid and expressed more adipocyte fatty acid–binding protein (aP2), peroxisome proliferator–activated receptor γ2, and CCAAT/enhancer-binding protein α than omental cells, as in primary preadipocytes, while hTERT abundance was similar. Thus, despite dividing 40 population doublings, hTERT strains derived from single preadipocytes retained fat depot–specific cell dynamic characteristics, consistent with heritable processes contributing to regional variation in fat tissue function.

Adipose-derived circulating miRNAs regulate gene expression in other tissues

Adipose tissue is a major site of energy storage and has a role in the regulation of metabolism through the release of adipokines. Here we show that mice with an adipose-tissue-specific knockout of the microRNA (miRNA)-processing enzyme Dicer (ADicerKO), as well as humans with lipodystrophy, exhibit a substantial decrease in levels of circulating exosomal miRNAs. Transplantation of both white and brown adipose tissue—brown especially—into ADicerKO mice restores the level of numerous circulating miRNAs that are associated with an improvement in glucose tolerance and a reduction in hepatic Fgf21 mRNA and circulating FGF21. This gene regulation can be mimicked by the administration of normal, but not ADicerKO, serum exosomes. Expression of a human-specific miRNA in the brown adipose tissue of one mouse in vivo can also regulate its 3′ UTR reporter in the liver of another mouse through serum exosomal transfer. Thus, adipose tissue constitutes an important source of circulating exosomal miRNAs, which can regulate gene expression in distant tissues and thereby serve as a previously undescribed form of adipokine.

Aging and Regional Differences in Fat Cell Progenitors – A Mini-Review

Fat mass and fat tissue distribution change dramatically throughout life. In old age, fat becomes dysfunctional and is redistributed from subcutaneous to intra-abdominal visceral depots as well as other ectopic sites, including bone marrow, muscle and the liver. These changes are associated with increased risk of metabolic syndrome. Fat tissue is a nutrient storage, endocrine and immune organ that undergoes renewal throughout the lifespan. Preadipocytes, which account for 15–50% of cells in fat tissue, give rise to new fat cells. With aging, declines in preadipocyte proliferation and differentiation likely contribute to increased systemic exposure to lipotoxic free fatty acids. Age-related fat tissue inflammation is related to changes that occur in preadipocytes and macrophages in a fat depot-dependent manner. Fat tissue inflammation frequently leads to further reduction in adipogenesis with aging, more lipotoxicity and activation of cellular stress pathways that, in turn, exacerbate inflammatory responses of preadipocytes and immune cells, establishing self-perpetuating cycles that lead to systemic dysfunction. In this review, we will consider how inherent, age-related, depot-dependent alterations in preadipocyte function contribute to age-related fat tissue redistribution and metabolic dysfunction.

Sex- and Depot-Dependent Differences in Adipogenesis in Normal-Weight Humans

To elucidate cellular mechanisms of sex‐related differences in fat distribution, we determined body fat distribution (dual‐energy X‐ray absorptiometry and single‐slice abdominal computed tomography (CT)), adipocyte size, adipocyte number, and proportion of early‐differentiated adipocytes (aP2+CD68−) in the stromovascular fraction (SVF) in the upper and lower body of normal‐weight healthy men (n = 12) and premenopausal women (n = 20) (age: 18–49 years, BMI: 18–26 kg/m2). Women had more subcutaneous and less visceral fat than men. The proportion of early differentiated adipocytes in the subcutaneous adipose tissue SVF of women was greater than in men (P = 0.01), especially in the femoral depot, although in vitro adipogenesis, as assessed by peroxisome proliferator activated receptor‐γ (PPARγ) expression, was not increased in femoral preadipocytes cultured from women compared with men. In women, differentiation of femoral preadipocytes was less than that of abdominal subcutaneous preadipocytes (P = 0.04), and femoral subcutaneous preadipocytes tended to be more resistant to tumor necrosis factor‐α (TNFα)–induced apoptosis (P = 0.06). Thus, turnover and utilization of the preadipocyte pool may be reduced in lower vs. the upper‐body fat in women. Collectively, these data indicate that the microenvironment, rather than differences in inherent properties of preadipocytes between genders, may explain the gynoid obesity phenotype and higher percent body fat in women compared to men.

ASC-1, PAT2, and P2RX5 are cell surface markers for white, beige, and brown adipocytes

The cell surface markers ASC-1, PAT2, and P2RX5 can be used to mark and identify brown, beige, and white adipocytes in both rodents and humans. Fat Cells Gain New Identities There’s “good fat” and there’s “bad fat.” Good fat is considered to be brown adipose tissue (BAT), which burns calories. Bad fat can be white adipose tissue (WAT), which stores lipids as energy and, in excess, contributes to obesity. When brown fat cells, or adipocytes, develop within white fat, they are called “beige.” Sorting out these different adipocyte subtypes within the human body has been challenging but will be important in uncovering the underlying mechanisms for obesity and its comorbidities, such as type 2 diabetes. To this end, Ussar and colleagues have now identified three new surface markers of white, beige, and brown fat cells. These markers—ASC-1, PAT2, and P2RX5—were first selected in silico, then confirmed in mouse WAT and BAT, and lastly verified in human adipose tissue biopsies. ASC-1, PAT2, and P2RX5 are located in the plasma membrane of adipocytes, thus making them prime targets for imaging fat locations within the body and for directing therapeutics toward particular fat depots. White, beige, and brown adipocytes are developmentally and functionally distinct but often occur mixed together within individual depots. To target white, beige, and brown adipocytes for diagnostic or therapeutic purposes, a better understanding of the cell surface properties of these cell types is essential. Using a combination of in silico, in vitro, and in vivo methods, we have identified three new cell surface markers of adipose cell types. The amino acid transporter ASC-1 is a white adipocyte–specific cell surface protein, with little or no expression in brown adipocytes, whereas the amino acid transporter PAT2 and the purinergic receptor P2RX5 are cell surface markers expressed in classical brown and beige adipocytes in mice. These markers also selectively mark brown/beige and white adipocytes in human tissue. Thus, ASC-1, PAT2, and P2RX5 are membrane surface proteins that may serve as tools to identify and target white and brown/beige adipocytes for therapeutic purposes.

Concerted Action of Aldehyde Dehydrogenases Influences Depot-Specific Fat Formation

Vitamin A metabolite retinoic acid (RA) regulates life-sustaining differentiation processes and metabolic homeostasis. The aldehyde dehydrogenase-1 (Aldh1) family of enzymes (Aldh1a1, a2, and a3) catalyzes RA production from retinaldehyde and thereby controls concentrations of this transcriptionally active metabolite. The hierarchy of Aldh1 functions in adipose tissue has not been elucidated. We hypothesized that Aldh1 enzymes produce endogenous RA and regulate adipogenesis and fat formation in a fat depot-specific manner. We demonstrate that adipogenesis in vitro is accompanied by RA production generated primarily by Aldh1a1. In Aldh1a1-deficient adipocytes, adipogenesis is impaired compared with wild-type adipocytes due to markedly reduced expression of PPARγ regulated through zinc-finger protein 423 (ZFP423)-dependent mechanisms. These effects were recovered to some extent either by RA stimulation or overexpression of any of the Aldh1 enzymes in Aldh1a1(-/-) cells arguing that Aldh1a1 plays a dominant role in autocrine RA production. In vivo studies in C57/BL6 and Aldh1a1(-/-) mice on a regular diet revealed that multiple Aldh1 enzymes regulate differences in the formation of sc and visceral fat. In Aldh1a1(-/-) mice, visceral fat essentially lacked all Aldh1 expression. This loss of RA-producing enzymes was accompanied by 70% decreased expression of ZFP423, PPARγ, and Fabp4 in visceral fat of Aldh1a1(-/-) vs. wild-type mice and by the predominant loss of visceral fat. Subcutaneous fat of Aldh1a1(-/-) mice expressed Aldh1a3 for RA production that was sufficient to maintain expression of ZFP423 and PPARγ and sc fat mass. Our data suggest a paradigm for regulation of fat depots through the concerted action of Aldh1 enzymes that establish RA-dependent tandem regulation of transcription factors ZFP423 and PPARγ in a depot-specific manner.

Aging, Depot Origin, and Preadipocyte Gene Expression

Fat distribution changes with aging. Inherent changes in fat cell progenitors may contribute because fat cells turn over throughout life. To define mechanisms, gene expression was profiled in preadipocytes cultured from epididymal and perirenal depots of young and old rats. 8.4% of probe sets differed significantly between depots, particularly developmental genes. Only 0.02% differed with aging, despite using less stringent criteria than for comparing depots. Twenty-five genes selected based on fold change with aging were analyzed in preadipocytes from additional young, middle-aged, and old animals by polymerase chain reaction. Thirteen changed significantly with aging, 13 among depots, and 9 with both. Genes involved in inflammation, stress, and differentiation changed with aging, as occurs in fat tissue. Age-related changes were greater in perirenal than epididymal preadipocytes, consistent with larger declines in replication and adipogenesis in perirenal preadipocytes. Thus, age-related changes in preadipocyte gene expression differ among depots, potentially contributing to fat redistribution and dysfunction.

Increased CUG triplet repeat-binding protein-1 predisposes to impaired adipogenesis with aging

Preadipocyte differentiation capacity declines between middle and old age. Expression of the adipogenic transcription factors, CCAAT/enhancer-binding protein (C/EBP) α and peroxisome proliferator-activated receptor γ (PPARγ), is lower in differentiating preadipocytes from old than young animals, although no age-related changes occur in C/EBPβ mRNA, which is upstream of C/EBPα and PPARγ. C/EBPβ-liver-enriched inhibitory protein (C/EBPβ-LIP), a truncated C/EBPβ isoform that is a dominant inhibitor of differentiation, increases with aging in rat fat tissue and preadipocytes. CUG triplet repeat-binding protein-1 (CUGBP1) binds to C/EBPβ mRNA, increasing C/EBPβ-LIP translation. Abundance and nucleotide binding activity of CUGBP1 increased with aging in preadipocytes. CUGBP1 overexpression in preadipocytes from young animals increased C/EBPβ-LIP and impaired adipogenesis. Decreasing CUGBP1 in preadipocytes from old rats by RNA interference reduced C/EBPβ-LIP abundance and promoted adipogenesis. Tumor necrosis factor-α, levels of which are elevated in fat tissue with aging, increased CUGBP1 protein, CUGBP1 binding activity, and C/EBPβ-LIP in preadipocytes from young rats. Thus, CUGBP1 contributes to regulation of adipogenesis in primary preadipocytes and is responsive to tumor necrosis factor-α. With aging, preadipocyte CUGBP1 abundance and activity increases, resulting in enhanced translation of the C/EBPβ-LIP isoform, thereby blocking effects of adipogenic transcription factors, predisposing preadipocytes from old animals to resist adipogenesis. Altered translational processing, possibly related to changes in cytokine milieu and activation of stress responses, may contribute to changes in progenitor differentiation and tissue function with aging.

Substance P promotes expansion of human mesenteric preadipocytes through proliferative and antiapoptotic pathways

White adipose tissue is intimately involved in the regulation of immunity and inflammation. We reported that human mesenteric preadipocytes express the substance P (SP)-mediated neurokinin-1 receptor (NK-1R), which signals proinflammatory responses. Here we tested the hypothesis that SP promotes proliferation and survival of human mesenteric preadipocytes and investigated responsible mechanism(s). Preadipocytes were isolated from mesenteric fat biopsies during gastric bypass surgery. Proliferative and antiapoptotic responses were delineated in 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS), bromodeoxyuridine (BrdU), caspase-3, and TUNEL assays, as well as Western immunoanalysis. SP (10(-7) M) increased MTS and proliferation (BrdU) and time dependently (15-30 min) induced Akt, EGF receptor, IGF receptor, integrin alphaVbeta3, phosphatidylinositol 3-kinase, and PKC-theta phosphorylation. Furthermore, pharmacological antagonism of Akt and PKC-theta activation significantly attenuated SP-induced preadipocyte proliferation. Exposure of preadipocytes to the proapoptotic Fas ligand (FasL, 100 microM) resulted in nuclear DNA fragmentation (TUNEL assay), as well as increased cleaved poly (ADP-ribose) polymerase, cleaved caspase-7, and caspase-3 expression. Cotreatment with SP almost completely abolished these responses in a NK-1R-dependent fashion. SP (10(-7) M) also time dependently stimulated expression 4E binding protein 1 and phosphorylation of p70 S6 kinase, which increased protein translation efficiency. SP increases preadipocyte viability, reduces apoptosis, and stimulates proliferation, possibly via cell cycle upregulation and increased protein translation efficiency. SP-induced proliferative and antiapoptotic pathways in fat depots may contribute to development of the creeping fat and inflammation characteristic of Crohns disease.