Z. Elizabeth Floyd

Immunophenotype of Human Adipose‐Derived Cells: Temporal Changes in Stromal‐Associated and Stem Cell–Associated Markers

Adipose tissue represents an abundant and accessible source of multipotent adult stem cells and is used by many investigators for tissue engineering applications; however, not all laboratories use cells at equivalent stages of isolation and passage. We have compared the immunophenotype of freshly isolated human adipose tissue‐derived stromal vascular fraction (SVF) cells relative to serial‐passaged adipose‐derived stem cells (ASCs). The initial SVF cells contained colony‐forming unit fibroblasts at a frequency of 1:32. Colony‐forming unit adipocytes and osteoblasts were present in the SVF cells at comparable frequencies (1:28 and 1:16, respectively). The immunophenotype of the adipose‐derived cells based on flow cytometry changed progressively with adherence and passage. Stromal cell–associated markers (CD13, CD29, CD44, CD63, CD73, CD90, CD166) were initially low on SVF cells and increased significantly with successive passages. The stem cell–associated marker CD34 was at peak levels in the SVF cells and/or early‐passage ASCs and remained present, although at reduced levels, throughout the culture period. Aldehyde dehydrogenase and the multidrug‐resistance transport protein (ABCG2), both of which have been used to identify and characterize hematopoietic stem cells, are expressed by SVF cells and ASCs at detectable levels. Endothelial cell–associated markers (CD31, CD144 or VE‐cadherin, vascular endothelial growth factor receptor 2, von Willebrand factor) were expressed on SVF cells and did not change significantly with serial passage. Thus, the adherence to plastic and subsequent expansion of human adipose‐derived cells in fetal bovine serum‐supplemented medium selects for a relatively homogeneous cell population, enriching for cells expressing a stromal immunophenotype, compared with the heterogeneity of the crude SVF.

Playing with bone and fat

The relationship between bone and fat formation within the bone marrow microenvironment is complex and remains an area of active investigation. Classical in vitro and in vivo studies strongly support an inverse relationship between the commitment of bone marrow‐derived mesenchymal stem cells or stromal cells to the adipoctye and osteoblast lineage pathways. In this review, we focus on the recent literature exploring the mechanisms underlying these differentiation events and discuss their implications relevant to osteoporosis and regenerative medicine. J. Cell. Biochem. 98: 251–266, 2006.

The Immunogenicity of Human Adipose‐Derived Cells: Temporal Changes In Vitro

Regenerative medical techniques will require an abundant source of human adult stem cells that can be readily available at the point of care. The ability to use unmatched allogeneic stem cells will help achieve this goal. Since adipose tissue represents an untapped reservoir of human cells, we have compared the immunogenic properties of freshly isolated, collagenase‐digested human adipose tissue‐derived stromal vascular fraction cells (SVFs) relative to passaged, plastic‐adherent adipose‐derived stem cells (ASCs). Parallel studies have shown that adherence to plastic and subsequent expansion of human adipose‐derived cells selects for a relatively homogeneous cell population based on immunophenotype. Consistent with these findings, the presence of hematopoietic‐associated markers (CD11a, CD14, CD45, CD86, and histocompatible locus antigen‐DR [HLA‐DR]) detected on the heterogeneous SVF cell population decreased upon subsequent passage of the ASCs. In mixed lymphocyte reactions (MLRs), SVFs, and early passage ASCs stimulated proliferation by allogeneic responder T cells. In contrast, the ASCs beyond passage P1 failed to elicit a response from T cells. Indeed, late passage ASCs actually suppressed the MLR response. Although these results support the feasibility of allogeneic human ASC transplantation, confirmatory in vivo animal studies will be required.

Proteomic Analysis of Primary Cultures of Human Adipose-derived Stem Cells Modulation by Adipogenesis

Adipogenesis plays a critical role in energy metabolism and is a contributing factor to the obesity epidemic. This study examined the proteome of primary cultures of human adipose-derived adult stem (ADAS) cells as an in vitro model of adipogenesis. Protein lysates obtained from four individual donors were compared before and after adipocyte differentiation by two-dimensional gel electrophoresis and tandem mass spectroscopy. Over 170 individual protein features in the undifferentiated adipose-derived adult stem cells were identified. Following adipogenesis, over 40 proteins were up-regulated by ≥2-fold, whereas 13 showed a ≥3-fold reduction. The majority of the modulated proteins belonged to the following functional categories: cytoskeleton, metabolic, redox, protein degradation, and heat shock protein/chaperones. Additional immunoblot analysis documented the induction of four individual heat shock proteins and confirmed the presence of the heat shock protein 27 phosphoserine 82 isoform, as predicted by the proteomic analysis, as well as the crystallin α phosphorylated isoforms. These findings suggest that the heat shock protein family proteome warrants further investigation with respect to the etiology of obesity and type 2 diabetes.

Regulation of Adipogenesis by Natural and Synthetic REV-ERB Ligands

The nuclear hormone receptor, REV-ERB, plays an essential role in adipogenesis. Rev-erbalpha expression is induced in 3T3-L1 cells during adipogenesis, and overexpression of this receptor leads to expression of adipogenic genes. We recently demonstrated that the porphyrin heme functions as a ligand for REV-ERB, and binding of heme is required for the receptors activity. We therefore hypothesized that REV-ERB ligands may play a role in regulation of adipogenesis. We detected an increase intracellular heme levels during 3T3-L1 adipogenesis that correlated with induction of aminolevulinic acid synthase 1 (Alas1) expression, the rate-limiting enzyme in heme biosynthesis. If the increase in Alas1 expression was blocked, adipogenesis was severely attenuated, indicating that induction of expression of Alas1 and the increase in heme synthesis is critical for differentiation. Inhibition of heme synthesis during adipogenesis leads to decreased recruitment of nuclear receptor corepressor to the promoter of a REV-ERB target gene, suggesting alteration of REV-ERB activity. Treatment of 3T3-L1 cells with a synthetic REV-ERB ligand, SR6452, resulted in induction of adipocyte differentiation to a similar extent as treatment with the peroxisomal proliferator-activated receptor-gamma agonist, rosiglitazone. Combination of SR6452 and rosiglitazone had an additive effect on stimulation of adipocyte differentiation. These results suggest that heme, functioning as a REV-ERB ligand, is an important signaling molecule for induction of adipogenesis. Moreover, synthetic small molecule ligands for REV-ERB are effective modulators of adipogenesis and may be useful for treatment of metabolic diseases.

Modulation of peroxisome proliferator-activated receptor γ stability and transcriptional activity in adipocytes by resveratrol

The peroxisome proliferator-activated receptor (PPAR) gamma is essential for the formation and function of adipocytes. It is also involved in regulating insulin sensitivity and is the functional target of the thiazolidinedione class of insulin-sensitizing drugs. Whereas thiazolidinediones activate PPARgamma and decrease PPARgamma protein levels, genetic models indicate that decreased expression of PPARgamma is also associated with increased insulin sensitivity. In this study, we show that resveratrol modulates PPARgamma protein levels in 3T3-L1 adipocytes via inhibition of PPARgamma gene expression coupled with increased ubiquitin-proteasome-dependent degradation of PPARgamma proteins. Resveratrol-mediated decreases in PPARgamma expression are associated with repression of PPARgamma transcriptional activity when assayed using a panel of PPARgamma target genes in adipocytes. Finally, we demonstrate that resveratrol inhibits insulin-dependent changes in glucose uptake and glycogen levels and decreases insulin receptor substrate 1 and glucose transporter 4 protein levels, indicating that resveratrol represses insulin sensitivity in adipocytes. These results indicate that the resveratrol-mediated effects in adipocytes involve regulation of PPARgamma expression and transcriptional activity along with decreased responsiveness to insulin.

Isolation of human adipose-derived stem cells from lipoaspirates.

Adipose tissue is as an abundant and accessible source of stem cells with multipotent properties suitable for tissue engineering and regenerative medical applications. Here, we describe methods from our own laboratory and the literature for the isolation and expansion of adipose-derived stem cells (ASCs). We present a large-scale procedure suitable for processing >100-ml volumes of lipoaspirate tissue specimens by collagenase digestion and a related procedure suitable for processing adipose tissue aspirates without digestion.

Bioactives from bitter melon enhance insulin signaling and modulate acyl carnitine content in skeletal muscle in high-fat diet-fed mice

Bioactive components from bitter melon (BM) have been reported to improve glucose metabolism in vivo, but definitive studies on efficacy and mechanism of action are lacking. We sought to investigate the effects of BM bioactives on body weight, muscle lipid content and insulin signaling in mice fed a high-fat diet and on insulin signaling in L6 myotubes. Male C57BL/6J mice were randomly divided into low-fat diet control (LFD), high-fat diet (HFD) and HFD plus BM (BM) groups. Body weight, body composition, plasma glucose, leptin, insulin and muscle lipid profile were determined over 12 weeks. Insulin signaling was determined in the mouse muscle taken at end of study and in L6 myotubes exposed to the extract. Body weight, plasma glucose, insulin, leptin levels and HOMA-IR values were significantly lower in the BM-fed HFD group when compared to the HFD group. BM supplementation significantly increased IRS-2, IR β, PI 3K and GLUT4 protein abundance in skeletal muscle, as well as phosphorylation of IRS-1, Akt1 and Akt2 when compared with HFD (P<.05 and P<.01). BM also significantly reduced muscle lipid content in the HFD mice. BM extract greatly increased glucose uptake and enhanced insulin signaling in L6 myotubes. This study shows that BM bioactives reduced body weight, improved glucose metabolism and enhanced skeletal muscle insulin signaling. A contributing mechanism to the enhanced insulin signaling may be associated with the reduction in skeletal muscle lipid content. Nutritional supplementation with this extract, if validated for human studies, may offer an adjunctive therapy for diabetes.

Controlling a Master Switch of adipocyte development and insulin sensitivity: Covalent Modifications of PPARγ

Adipocytes are highly specialized cells that play a central role in lipid homeostasis and the maintenance of energy balance. Obesity, an excessive accumulation of adipose tissue, is a major risk factor for the development of Type 2 diabetes mellitus (T2DM), cardiovascular disease, and hypertension. A variety of studies suggest that obesity and T2DM can be linked to a breakdown in the regulatory mechanisms that control the expression and transcriptional activity of PPARγ. PPARγ is a nuclear hormone receptor that functions as a master switch in controlling adipocyte differentiation and development. Also important in controlling glucose homeostasis and insulin sensitivity, PPARγ is a ligand-dependent transcription factor that is the functional receptor for the anti-diabetic thiazolidinediones (TZDs). In the last fifteen years, a variety of covalent modifications of PPARγ activity have been identified and studied. These covalent modifications include phosphorylation, ubiquitylation, O-GlcNAcylation and SUMOylation. Covalent modifications of PPARγ represent key regulatory mechanisms that control both PPARγ protein stability and transcriptional activity. A variety of PPARγ transgenic models, including mice heterozygous for PPARγ, have demonstrated the importance of PPARγ expression in glucose homeostasis and insulin resistance. In the following review, we have highlighted the regulation of PPARγ by covalent modifications, the interplay between these interactions and how these post-translational modifications impact metabolic disease states.

Circadian Rhythms and the Regulation of Metabolic Tissue Function and Energy Homeostasis

Circadian oscillators play an indispensable role in the coordination of physiological processes with the cyclic changes in the physical environment. A significant number of recent clinical and molecular studies suggest that circadian biology may play an important role in the regulation of adipose and other metabolic tissue functions. In this discussion, we present the hypothesis that circadian dysfunction may be involved in the pathogenesis of obesity, type 2 diabetes, and the metabolic syndrome.