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Abnormal fibroblast function underlies poor wound healing in patients with diabetes; however, the mechanisms that impair wound healing are poorly defined. Here, we evaluated fibroblasts from individuals who had type 1 diabetes (T1D) for 50 years or more (Medalists, n = 26) and from age-matched controls (n = 7). Compared with those from controls, Medalist fibroblasts demonstrated a reduced migration response to insulin, lower VEGF expression, and less phosphorylated AKT (p-AKT), but not p-ERK, activation. Medalist fibroblasts were also functionally less effective at wound closure in nude mice. Activation of the δ isoform of protein kinase C (PKCδ) was increased in postmortem fibroblasts from Medalists, fibroblasts from living T1D subjects, biopsies of active wounds of living T1D subjects, and granulation tissues from mice with streptozotocin-induced diabetes. Diabetes-induced PKCD mRNA expression was related to a 2-fold increase in the mRNA half-life. Pharmacologic inhibition and siRNA-mediated knockdown of PKCδ or expression of a dominant-negative isoform restored insulin signaling of p-AKT and VEGF expression in vitro and improved wound healing in vivo. Additionally, increasing PKCδ expression in control fibroblasts produced the same abnormalities as those seen in Medalist fibroblasts. Our results indicate that persistent PKCδ elevation in fibroblasts from diabetic patients inhibits insulin signaling and function to impair wound healing and suggest PKCδ inhibition as a potential therapy to improve wound healing in diabetic patients., INTRODUCTION Chronic foot ulcers are one of the most common and serious complications of diabetes, resulting in more than 80,000 amputations per year as well as high health care costs [...]

miRNAs are important regulators of biological processes in many tissues, including the differentiation and function of brown and white adipocytes. The endoribonuclease dicer is a major component of the miRNA-processing pathway, and in adipose tissue, levels of dicer have been shown to decrease with age, increase with caloric restriction, and influence stress resistance. Here, we demonstrated that mice with a fat-specific KO of dicer develop a form of lipodystrophy that is characterized by loss of intra-abdominal and subcutaneous white fat, severe insulin resistance, and enlargement and 'whitening' of interscapular brown fat. Additionally, KO of dicer in cultured brown preadipocytes promoted a white adipocyte-like phenotype and reduced expression of several miRNAs. Brown preadipocyte whitening was partially reversed by expression of miR-365, a miRNA known to promote brown fat differentiation; however, introduction of other miRNAs, including miR-346 and miR-362, also contributed to reversal of the loss of the dicer phenotype. Interestingly, fat samples from patients with HIV- related lipodystrophy exhibited a substantial downregulation of dicer mRNA expression. Together, these findings indicate the importance of miRNA processing in white and brown adipose tissue determination and provide a potential link between this process and HIV-related lipodystrophy., Introduction Adipose tissue is a heterogeneous tissue. White adipose tissue (WAT) is the major site of energy storage and is mainly located in subcutaneous and intra-abdominal locations, whereas brown adipose [...]

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. Key Words: Aging--Preadipocyte--Fat cell progenitor. doi: 10.1093/gerona/glp213

White adipose tissue is intimately involved in the regulation of immunity and inflammation. We reported that human mesentefic 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-dimethyl-thiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl) -2H-tetra-zolium (MTS), bromodeoxyuridine (BrdU), caspase-3, and TUNEL assays, as well as Western immunoanalysis. SP ([10.sup.-7] M) increased MTS and proliferation (BrdU) and time dependently (15-30 min) induced Akt, EGF receptor, IGF receptor, integrin [alpha]V[beta]3, 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 [micro]M) 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.sup.-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 Crohn's disease. neuropeptide; creeping fat; Crohn's disease; adipocyte; proliferation; protein kinase C-[theta]; Akt

Fat depot sizes peak in middle age but decrease by advanced old age. This phenomenon is associated with ectopic fat deposition, decreased adipocyte size, impaired differentiation of preadipocytes into fat cells, decreased adipogenic transcription factor expression, and increased fat tissue inflammatory cytokine generation. To define the mechanisms contributing to impaired adipogenesis with aging, we examined the release of TNF[alpha], which inhibits adipogeneszs, and the expression of CCAAT/enhancer-binding protein (C/EBP) homologous protein (CHOP), which blocks activity of adipogenic C/EBP family members, in preadipocytes cultured from young, middle-aged, and old rats. Medium conditioned by fat tissue, as well as preadipocytes, from old rats impeded lipid accumulation by preadipocytes from young animals. More TNF[alpha] was released by preadipocytes from old than young rats. Differences in TNF[alpha]-converting enzyme, TNF[alpha] degradation, or the presence of macrophages in cultures were not responsible. TNF[alpha] induced rat preadipocyte CHOP expression. CHOP was higher in undifferentiated preadipocytes from old than younger animals. Over-expression of CHOP in young rat preadipocytes inhibited lipid accumulation. TNF[alpha] short interference RNA reduced CHOP and partially restored lipid accumulation in old rat preadipocytes. CHOP normally increases during late differentiation, potentially modulating the process. This late increase in CHOP was not affected substantially by aging: CHOP was similar in differentiating preadipocytes and fat tissue from old and young animals. Hypoglycemia, which normally causes an adaptive increase in CHOP, was less effective in inducing CHOP in preadipocytes from old than younger animals. Thus increased TNF[alpha] release by undifferentiated preadipocytes with elevated basal CHOP contributes to impaired adipogenesis with aging. adipocytes; CCAAT/enhancer-binding protein-[zeta]; growth arrest and DNA damage 153; differentiation; stress response; hypoglycemia

Aging is associated with metabolic syndrome, tissue damage by cytotoxic lipids, and altered fatty acid handling. Fat tissue dysfunction may contribute to these processes. This could result, in part, from age-related changes in preadipocytes, since they give rise to new fat cells throughout life. To test this hypothesis, preadipocytes cultured from rats of different ages were exposed to oleic acid, the most abundant fatty acyl moiety in fat tissue and the diet. At fatty acid concentrations at which preadipocytes from young animals remained viable, cells from old animals accumulated lipid in multiple small lipid droplets and died, with increased apoptotic index, caspase activity, BAX, and p53. Rather than inducing apoptosis, oleic acid promoted adipogenesis in preadipocytes from young animals, with appearance of large lipid droplets. CCAAT/enhancer-binding protein-[alpha] (C/EBP[alpha]) and peroxisome proliferator-activated receptor-[gamma] (PPAR[gamma]) increased to a greater extent in cells from young than old animals after oleate exposure. Oleic acid, but not glucose, oxidation was impaired in preadipocytes and fat cells from old animals. Expression of carnitine palmitoyltransferase (CPT)-1, which catalyzes the rate-limiting step in fatty acid [beta]-oxidation, was not reduced in preadipocytes from old animals. At lower fatty acid levels, constitutively active CPT I expression enhanced [beta]-oxidation. At higher levels, CPT I was not as effective in enhancing [beta]-oxidation in preadipocytes from old as young animals, suggesting that mitochondrial dysfunction may contribute. Consistent with this, medium-chain acyl-CoA dehydrogenase expression was reduced in preadipocytes from old animals. Thus preadipocyte fatty acid handling changes with aging, with increased susceptibly to lipotoxicity and impaired fatty acid-induced adipogenesis and [beta]-oxidation. fatty acid metabolism; oleic acid; peroxisome proliferator-activated receptor-[gamma]; CCAAT/enhancer-binding protein-[alpha]; [beta]-oxidation

Anatomically separate fat depots differ in size, function, and contribution to pathological states, such as the metabolic syndrome. We isolated preadipocytes from different human fat depots to determine whether the basis for this variation is partly attributable to differences in inherent properties of fat cell progenitors. We found that genome-wide expression profiles of primary preadipocytes cultured in parallel from abdominal subcutaneous, mesenteric, and omental fat depots were distinct. Interestingly, visceral fat was not homogeneous. Preadipocytes from one of the two main visceral depots, mesenteric fat, had an expression profile closer to that of subcutaneous than omental preadipocytes, the other main visceral depot. Expression of genes that regulate early development, including homeotic genes, differed extensively among undifferentiated preadipocytes isolated from different fat depots. These profiles were confirmed by real-time PCR analysis of preadipocytes from additional lean and obese male and female subjects. We made preadipocyte strains from single abdominal subcutaneous and omental preadipocytes by expressing telomerase. Depot-specific developmental gene expression profiles persisted for 40 population doublings in these strains. Thus, human fat cell progenitors from different regions are effectively distinct, consistent with different fat depots being separate mini-organs. visceral fat; bomeotic genes; telomerase; metabolic syndrome

Author(s): Thomas Thomou; Marcelo A. Mori; Jonathan M. Dreyfuss; Masahiro Konishi; Masaji Sakaguchi; Christian Wolfrum; Tata Nageswara Rao; Jonathon N. Winnay; Ruben Garcia-Martin; Steven K. Grinspoon; Phillip Gorden; C. Ronald [...]

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. Copyright © 2010 S. Karger AG, Basel [ABSTRACT FROM AUTHOR]

Aging is associated with metabolic syndrome, tissue damage by cytotoxic lipids, and altered fatty acid handling. Fat tissue dysfunction may contribute to these processes. This could result, in part, from age-related changes in preadipocytes, since they give rise to new fat cells throughout life. To test this hypothesis, preadipocytes cultured from rats of different ages were exposed to oleic acid, the most abundant fatty acyl moiety in fat tissue and the diet. At fatty acid concentrations at which preadipocytes from young animals remained viable, cells from old animals accumulated lipid in multiple small lipid droplets and died, with increased apoptotic index, caspase activity, BAX, and p53. Rather than inducing apoptosis, oleic acid promoted adipogenesis in preadipocytes from young animals, with appearance of large lipid droplets. CCAAT/enhancer-binding protein-α (C/EBPα) and peroxisome proliferator-activated receptor-γ (PPAR-γ) increased to a greater extent in cells from young than old animals after oleate exposure. Oleic acid, but not glucose, oxidation was impaired in preadipocytes and fat cells from old animals. Expression of carnitine palmitoyltransferase (CPT)-1, which catalyzes the rate-limiting step in fatty acid p-oxidation, was not reduced in preadipocytes from old animals. At lower fatty acid levels, constitutively active CPT1 expression enhanced p-oxidation. At higher levels, CPT1 was not as effective in enhancing n-oxidation in preadipocytes from old as young animals, suggesting that mitochondrial dysfunction may contribute. Consistent with this, medium-chain acyl-CoA dehydrogenase expression was reduced in preadipocytes from old animals. Thus preadipocyte fatty acid handling changes with aging, with increased susceptibly to lipotoxicity and impaired fatty acid-induced adipogenesis and 3-oxidation. [ABSTRACT FROM AUTHOR]

Anatomically separate fat depots differ in size, function, and contribution to pathological states, such as the metabolic syndrome. We isolated preadipocytes from different human fat depots to determine whether the basis for this variation is partly attributable to differences in inherent properties of fat cell progenitors. We found that genome-wide expression profiles of primary preadipocytes cultured in parallel from abdominal subcutaneous, mesenteric, and omental fat depots were distinct. Interestingly, visceral fat was not homogeneous. Preadipocytes from one of the two main visceral depots, mesenteric fat, had an expression profile closer to that of subcutaneous than omental preadipocytes, the other main visceral depot. Expression of genes that regulate early development, including homeotic genes, differed extensively among undifferentiated preadipocytes isolated from different fat depots. These profiles were confirmed by real-time PCR analysis of preadipocytes from additional lean and obese male and female subjects. We made preadipocyte strains from single abdominal subcutaneous and omental preadipocytes by expressing telomerase. Depot-specific developmental gene expression profiles persisted for 40 population doublings in these strains. Thus, human fat cell progenitors from different regions are effectively distinct, consistent with different fat depots being separate mini-organs. [ABSTRACT FROM AUTHOR]

Aging increases the risk of type 2 diabetes, and this can be prevented by dietary restriction (DR). We have previously shown that DR inhibits the downregulation of miRNAs and their processing enzymes - mainly Dicer - that occurs with aging in mouse white adipose tissue (WAT). Here we used fat-specific Dicer knockout mice (AdicerKO) to understand the contributions of adipose tissue Dicer to the metabolic effects of aging and DR. Metabolomic data uncovered a clear distinction between the serum metabolite profiles of Lox control and AdicerKO mice, with a notable elevation of branched-chain amino acids (BCAA) in AdicerKO. These profiles were associated with reduced oxidative metabolism and increased lactate in WAT of AdicerKO mice and were accompanied by structural and functional changes in mitochondria, particularly under DR. AdicerKO mice displayed increased mTORC1 activation in WAT and skeletal muscle, where Dicer expression is not affected. This was accompanied by accelerated age-associated insulin resistance and premature mortality. Moreover, DR-induced insulin sensitivity was abrogated in AdicerKO mice. This was reverted by rapamycin injection, demonstrating that insulin resistance in AdicerKO mice is caused by mTORC1 hyperactivation. Our study evidences a DR-modulated role for WAT Dicer in controlling metabolism and insulin resistance.