Outcomes of the UAMS summer undergraduate research program to increase diversity in research and health professions.

The University of Arkansas for Medical Sciences (UAMS) Summer Undergraduate Research Program (SURP) aims to increase diversity in research and health-related careers. The SURP provides underrepresented minority (URM) and disadvantaged students with research, mentoring, and networking experiences; real-life surgical observations; and simulated cardiovascular demonstrations. A postprogram survey was developed to assess program outcomes and explore ways of improving the program to stimulate URM and disadvantaged students' interest in research and health-related careers. This is a report of those postprogram survey findings. Using a survey research design, an online survey was emailed to participants (n = 88). Data were collected for 6 weeks beginning March 2020. There were 37 multiple-choice and open-ended questions regarding education, career choices, and program experiences. Responses were downloaded to statistical software for analyses. Quantitative data were analyzed using descriptive statistics. Major themes were identified for qualitative data. Responses were received from 44.3% (n = 39) of former SURP participants. Overall, 59% stated that the SURP influenced their career goals. When asked about mentor-mentee relationships, 69.3% responded that their interactions were excellent or good; 61.5% maintained contact with their mentor after the SURP. Finally, 79% indicated their SURP experience was excellent or good, and 84.6% would recommend the SURP to others. The SURP has been successful at providing URM and disadvantaged students with positive research experiences and long-term mentor-mentee relationships and has influenced educational and/or career goals. Programs that expose URM and disadvantaged students to basic, clinical, and/or translational research are beneficial for stimulating interest in research and health-related careers.NEW & NOTEWORTHY Mentor-mentee relationships were extremely beneficial as many of the former participants maintained contact with their summer mentor after the program ended. This assessment also revealed that exposing underrepresented and minority students to research has a long-lasting effect on career and educational goals.

Summer undergraduate biomedical research program for underrepresented minority students in a rural, low-income state.

Introduction: Diversity can enhance the agenda and quality of biomedical research, but a dearth of underrepresented minorities and women serve as biomedical researchers. The study purpose was to examine the impact of the a summer undergraduate research program on self-efficacy in research, scientific communication, and leadership as well as scientific identity, valuing objectives of the scientific community, and intent to pursue a biomedical research career. Methods: Underrepresented minority and female undergraduate students participated in a mentored research experience in a rural, low-income state. Results: Students' self-efficacy in research, scientific communication, and leadership as well as scientific identity, valuing objectives of the scientific community, and intent to pursue a biomedical research career increased post-program compared to pre-program. Conclusion: This study supports implementation of a biomedical summer undergraduate research program for URM and women in a poor, rural, settings.

Performance measures of racially underrepresented Ph.D. students in biomedical sciences: The UAMS IMSD Program Outcomes.

The purpose of this study was to identify performance measures of racially underrepresented minority (RUM) Ph.D. trainees who needed additional training initiatives to assist with completing the UAMS biomedical science degree. A sample of 37 trainees in the 10-year NIH-NIGMS funded Initiative for Maximizing Student Development (IMSD) program at the University of Arkansas for Medical Sciences (UAMS) were examined. Descriptive statistics and correlations examined process measures (GRE scores, GPAs, etc.) and outcome measures (time-to-degree, publications, post-doctoral fellowship, etc.) While differences were found, there were no statistically significant differences between how these two groups (Historically Black Colleges and Universities (HBCUs) and Predominately White Institutions (PWIs)) of students performed over time as Ph.D. students. Graduates who scored lower on the verbal section of the GRE also had a higher final graduate school grade point average in graduates who received their undergraduate training from HBCUs. Of the graduates who received their undergraduate training from PWIs, graduates who scored lower on the quantitative section of the GRE had higher numbers of publications. These findings stimulate the need to 1) reduce reliance on the use of the GRE in admission committee decisions, 2) identify psychometrically valid indicators that tailored to assess outcome variables that are relevant to the careers of biomedical scientists, and 3) ensure the effective use of the tools in making admission decisions.

Association of scavenger receptors in adipose tissue with insulin resistance in nondiabetic humans.

OBJECTIVE: Scavenger receptors play crucial roles in the pathogenesis of atherosclerosis, but their role in insulin resistance has not been explored. We hypothesized that scavenger receptors are present in human adipose tissue resident macrophages, and their gene expression is regulated by adiponectin and thaizolidinediones. METHODS AND RESULTS: The gene expression of scavenger receptors including scavenger receptor-A (SRA), CD36, and lectin-like oxidized LDL receptor-1 (LOX-1) were studied in subcutaneous adipose tissue of nondiabetic subjects and in vitro. Adipose tissue SRA expression was independently associated with insulin resistance. Pioglitazone downregulated SRA gene expression in adipose tissue of subjects with impaired glucose tolerance and decreased LOX-1 mRNA in vitro. Macrophage LOX-1 expression was decreased when macrophages were cocultured with adipocytes or when exposed to adipocyte conditioned medium. Adding adiponectin neutralizing antibody resulted in a 2-fold increase in LOX-1 gene expression demonstrating that adiponectin regulates LOX-1 expression. CONCLUSIONS: Adipose tissue scavenger receptors are strongly associated with insulin resistance. Pioglitazone and adiponectin regulate gene expression of SRA and LOX-1, and this may have clinical implications in arresting the untoward sequalae of insulin resistance and diabetes, including accelerated atherosclerosis.

Stearoyl-coenzyme A desaturase 1 gene expression increases after pioglitazone treatment and is associated with peroxisomal proliferator-activated receptor-gamma responsiveness.

CONTEXT AND OBJECTIVE: Stearoyl-coenzyme A desaturase (SCD1) is the rate-limiting enzyme that converts palmitoyl- and stearoyl-coenzyme A to palmitoleoyl- and oleoyl-cownzyme A, respectively. SCD-deficient mice are protected from obesity, and the ob/ob mouse has high levels of SCD. This study was designed to better characterize SCD1 gene and protein expression in humans with varying insulin sensitivity. DESIGN, PARTICIPANTS, AND SETTING: In a university hospital clinical research center setting, SCD1 gene expression was measured in sc adipose and vastus lateralis muscle of 86 nondiabetic subjects; 10 wk of pioglitazone (45 mg daily) and metformin (1000 mg twice daily) treatment were assessed in 36 impaired glucose-tolerant subjects. Adipocytes were treated with pioglitazone, and SCD1 expression was attenuated with small interfering RNA (siRNA) to examine other adipocyte genes. RESULTS: There was no significant relationship between adipose or muscle SCD1 mRNA and either body mass index or insulin sensitivity. After pioglitazone (but not metformin) treatment, there was a 2-fold increase in SCD1 mRNA and protein in adipose tissue. Pioglitazone also increased SCD1 in vitro. There were significant positive correlations between SCD1 and peroxisomal proliferator-activated receptor gamma (PPARgamma) as well as other PPARgamma-responsive genes, including lipin-beta, AGPAT2, RBP4, adiponectin receptors, CD68, and MCP1. When SCD1 expression was inhibited with a siRNA, lipin-beta, AGPAT2, and the adiponectin R2 receptor expression were decreased, and adipocyte MCP-1 was increased. CONCLUSIONS: SCD1 is closely linked to PPARgamma expression in humans, and is increased by PPARgamma agonists. The change in expression of some downstream PPARgamma targets after SCD1 knockdown suggests that PPARgamma up-regulation of SCD1 leads to increased lipogenesis and potentiation of adiponectin signaling.

Expression of p107 and p130 during human adipose-derived stem cell adipogenesis.

Within the first 24h of hormonally stimulated adipocyte differentiation, murine 3T3-L1 preadipocytes undergo a mitotic expansion phase prior to terminal differentiation. During this time, the cell cycle regulatory proteins, p130 and p107 undergo dramatic differential expression and the transient increase in expression of p107 appears to be required for terminal differentiation. Recently, human adipose-derived human stem cells (hASC) of mesenchymal origin have been used as a model of human adipocyte differentiation and we sought to determine if differentiating hASC undergo clonal expansion and if the regulated expression of p130/p107 was similar to that observed during 3T3-L1 adipogenesis. Results indicate that differentiating hASC, unlike 3T3-L1 cells do not undergo clonal expansion and p130 expression gradually diminishes across differentiation. However, p107 expression is transiently increased during hASC differentiation in a manner analogous to 3T3-L1 cells suggesting a similar role for p107 in terminal differentiation in human adipocytes.

Wnt10b deficiency promotes coexpression of myogenic and adipogenic programs in myoblasts.

Adult myoblasts retain plasticity in developmental potential and can be induced to undergo myogenic, adipogenic, or osteoblastogenic differentiation in vitro. In this report, we show that the balance between myogenic and adipogenic potential in myoblasts is controlled by Wnt signaling. Furthermore, this balance is altered during aging such that aspects of both differentiation programs are coexpressed in myoblasts due to decreased Wnt10b abundance. Mimicking Wnt signaling in aged myoblasts through inhibition of glycogen synthase kinase or through overexpression of Wnt10b resulted in inhibition of adipogenic gene expression and sustained or enhanced myogenic differentiation. On the other hand, myoblasts isolated from Wnt10b null mice showed increased adipogenic potential, likely contributing to excessive lipid accumulation in actively regenerating myofibers in vivo in Wnt10b-/- mice. Whereas Wnt10b deficiency contributed to increased adipogenic potential in myoblasts, the augmented myogenic differentiation potential observed is likely the result of a compensatory increase in Wnt7b during differentiation of Wnt10b-/- myoblasts. No such compensation was apparent in aged myoblasts and in fact, both Wnt5b and Wnt10b were down-regulated. Thus, alteration in Wnt signaling in myoblasts with age may contribute to impaired muscle regenerative capacity and to increased muscle adiposity, both characteristic of aged muscle.

Retinol binding protein 4 expression in humans: relationship to insulin resistance, inflammation, and response to pioglitazone.

CONTEXT: Retinol binding protein 4 (RBP4) was recently found to be expressed and secreted by adipose tissue, and was strongly associated with insulin resistance. OBJECTIVE: The aim was to determine the relationship between RBP4 and obesity, insulin resistance, and other markers of insulin resistance in humans. DESIGN AND PATIENTS: RBP4 mRNA levels in adipose tissue and muscle of nondiabetic human subjects with either normal or impaired glucose tolerance (IGT) were studied, along with plasma RBP4. RBP4 gene expression was also measured in adipose tissue fractions, and from visceral and sc adipose tissue (SAT) from surgical patients. SETTING: The study was conducted at University Hospital and General Clinical Research Center. INTERVENTION: Insulin sensitivity (S(I)) was measured, and fat and muscle biopsies were performed. In IGT subjects, these procedures were performed before and after treatment with metformin or pioglitazone. MAIN OUTCOME MEASURES: The relationship between RBP4 expression and obesity, S(I), adipose tissue inflammation, and intramyocellular lipid level, and response to insulin sensitizers was measured. RESULTS: RBP4 was expressed predominantly from the adipocyte fraction of SAT. Although SAT RBP4 expression and the plasma RBP4 level demonstrated no significant relationship with body mass index or S(I), there was a strong positive correlation between RBP4 mRNA and adipose inflammation (monocyte chemoattractant protein-1 and CD68), and glucose transporter 4 mRNA. Treatment of IGT subjects with pioglitazone resulted in an increase in S(I) and an increase in RBP4 gene expression in both adipose tissue and muscle, but not in plasma RBP4 level, and the in vitro treatment of cultured adipocytes with pioglitazone yielded a similar increase in RBP4 mRNA. CONCLUSIONS: RBP4 gene expression in humans is associated with inflammatory markers, but not with insulin resistance. The increase in RBP4 mRNA after pioglitazone treatment is unusual, suggesting a complex regulation of this novel adipokine.

Human visfatin expression: relationship to insulin sensitivity, intramyocellular lipids, and inflammation.

CONTEXT: Visfatin (VF) is a recently described adipokine preferentially secreted by visceral adipose tissue (VAT) with insulin mimetic properties. OBJECTIVE: The aim of this study was to examine the association of VF with insulin sensitivity, intramyocellular lipids (IMCL), and inflammation in humans. DESIGN AND PATIENTS: VF mRNA was examined in paired samples of VAT and abdominal sc adipose tissue (SAT) obtained from subjects undergoing surgery. Plasma VF and VF mRNA was also examined in SAT and muscle tissue, obtained by biopsy from well-characterized subjects with normal or impaired glucose tolerance, with a wide range in body mass index (BMI) and insulin sensitivity (S(I)). SETTING: The study was conducted at a University Hospital and General Clinical Research Center. INTERVENTION: S(I) was measured, and fat and muscle biopsies were performed. In impaired glucose tolerance subjects, these procedures were performed before and after treatment with pioglitazone or metformin. MAIN OUTCOME MEASURES: We measured the relationship between VF and obesity, S(I), adipose tissue inflammation, IMCL, and response to insulin sensitizers. RESULTS: No significant difference in VF mRNA was seen between SAT and VAT depots. VAT VF mRNA associated positively with BMI, whereas SAT VF mRNA decreased with BMI. SAT VF correlated positively with S(I), and the association of SAT VF mRNA with S(I) was independent of BMI. IMCL and markers of inflammation (adipose CD68 and plasma TNFalpha) were negatively associated with SAT VF. Impaired glucose tolerance subjects treated with pioglitazone showed no change in SAT VF mRNA despite a significant increase in S(I). Plasma VF and muscle VF mRNA did not correlate with BMI or S(I) or IMCL, and there was no change in muscle VF with either pioglitazone or metformin treatments. CONCLUSION: SAT VF is highly expressed in lean, more insulin-sensitive subjects and is attenuated in subjects with high IMCL, low S(I), and high levels of inflammatory markers. VAT VF and SAT VF are regulated oppositely with BMI.

Lipin expression is attenuated in adipose tissue of insulin-resistant human subjects and increases with peroxisome proliferator-activated receptor gamma activation.

Lipin-alpha and -beta are the alternatively spliced gene products of the Lpin1 gene, whose product lipin is required for adipocyte differentiation. Lipin deficiency causes lipodystrophy, fatty liver, and insulin resistance in mice, whereas adipose tissue lipin overexpression results in increased adiposity but improved insulin sensitivity. To assess lipin expression and its relation to insulin resistance in humans, we examined lipin-alpha and -beta mRNA levels in subjects with normal or impaired glucose tolerance. We found higher expression levels of both lipin isoforms in lean, insulin-sensitive subjects. When compared with normal glucose-tolerant subjects, individuals with impaired glucose tolerance were more insulin resistant, demonstrated higher levels of intramyocellular lipids (IMCLs), and expressed approximately 50% lower levels of lipin-alpha and -beta. In addition, there was a strong inverse correlation between adipose tissue lipin expression and muscle IMCLs but no evidence for an increase in muscle lipid oxidation. After treatment of the impaired glucose-tolerant subjects with insulin sensitizers for 10 weeks, pioglitazone (but not metformin) resulted in a 60% increase in the insulin sensitivity index (Si) and a 32% decrease in IMCLs (both P < 0.01), along with an increase in lipin-beta (but not lipin-alpha) expression by 200% (P < 0.005). Lipin expression in skeletal muscle, however, was not related to obesity or insulin resistance. Hence, high adipose tissue lipin expression is found in insulin-sensitive subjects, and lipin-beta expression increases following treatment with pioglitazone. These results suggest that increased adipogenesis and/or lipogenesis in subcutaneous fat, mediated by the LPIN1 gene, may prevent lipotoxicity in muscle, leading to improved insulin sensitivity.

Expression of CD68 and macrophage chemoattractant protein-1 genes in human adipose and muscle tissues: association with cytokine expression, insulin resistance, and reduction by pioglitazone.

To examine the role of adipose-resident macrophages in insulin resistance, we examined the gene expression of CD68, a macrophage marker, along with macrophage chemoattractant protein-1 (MCP-1) in human subcutaneous adipose tissue using real-time RT-PCR. Both CD68 and MCP-1 mRNAs were expressed in human adipose tissue, primarily in the stromal vascular fraction. When measured in the adipose tissue from subjects with normal glucose tolerance, covering a wide range of BMI (21-51 kg/m2) and insulin sensitivity (S(I)) (0.6-8.0 x 10(-4)min(-1).microU(-1).ml(-1)), CD68 mRNA abundance, which correlated with the number of CD68-positive cells by immunohistochemistry, tended to increase with BMI but was not statistically significant. However, there was a significant inverse relation between CD68 mRNA and S(I) (r=-0.55, P=0.02). In addition, there was a strong positive relationship among adipose tissue CD68 mRNA, tumor necrosis factor-alpha (TNF-alpha) secretion in vitro (r=0.79, P<0.005), and plasma interleukin-6 (r=0.67, P < 0.005). To determine whether improving S(I) in subjects with impaired glucose tolerance (IGT) was associated with decreased CD68 expression, IGT subjects were treated for 10 weeks with pioglitazone or metformin. Pioglitazone increased S(I) by 60% and in the same subjects reduced both CD68 and MCP-1 mRNAs by >50%. Furthermore, pioglitazone resulted in a reduction in the number of CD68-positive cells in adipose tissue and reduced plasma TNF-alpha. Metformin had no effect on any of these measures. Thus, treatment with pioglitazone reduces expression of CD68 and MCP-1 in adipose tissue, apparently by reducing macrophage numbers, resulting in reduced inflammatory cytokine production and improvement in S(I).

Comparative expression profiling in primary and immortalized endothelial cells: changes in gene expression in response to hydroxy methylglutaryl-coenzyme A reductase inhibition.

Immortalized cell lines offer significant logistical advantages over primary cells when used for in-vitro studies. Immortalized cells may, however, exhibit important differences relative to their primary cell counterparts. In this study, microarrays were used to make a genome-wide comparison between primary human umbilical vein endothelial cells (HUVECs) and EA.hy926, an immortalized HUVEC cell line, in their baseline properties and in their response to inhibition of the mevalonate pathway with an inhibitor of hydroxy methylglutaryl-coenzyme A reductase (statin). HUVECs and EA.hy926 were incubated with control medium, atorvastatin, mevalonate, or a combination of atorvastatin and mevalonate for 24 h. Gene expression profiles were obtained in duplicates using Affymetrix Human Genome U133A 2.0 arrays (Santa Clara, California, USA). Probe-sets were selected according to the following criteria: a twofold or greater increase/decrease in atorvastatin-treated cells compared with untreated cells; a twofold or greater reversal of the effect of atorvastatin by combined treatment with atorvastatin and mevalonate; no significant change in gene expression in cells treated with mevalonate alone compared with untreated cells. Most genes that were expressed by untreated HUVECs, were also expressed by untreated EA.hy926 cells. EA.hy926 cells, however, constitutively expressed a large number of additional genes, many of which were related to cell cycle control and apoptosis. Atorvastatin induced differential expression (> or = twofold) of 103 genes in HUVECs (10 up, 93 down) and 466 genes in EA.hy926 cells (198 up, 268 down). Applying the above selection criteria, thrombomodulin and tissue plasminogen activator were up-regulated in both cell types, whereas, connective tissue growth factor, thrombospondin-1, and cysteine-rich angiogenic inducer 61 were down-regulated. In conclusion, EA.hy926 cells retain most of the characteristics of endothelial cells under baseline conditions as well as after treatment with atorvastatin. It is necessary, however, to carefully select and validate changes in genes that are the focus of studies when using EA.hy926 cells. While this cell line is highly useful in studies on some genes, including genes encoding molecules involved in regulating thrombohemorrhagic homeostasis, they appear to be less suited for studies focused on other genes, particularly those involved in the regulation of cell proliferation and apoptosis.

Microarray analysis of differentiation-specific gene expression during 3T3-L1 adipogenesis.

During cellular differentiation and development, it is recognized that many complex molecular mechanisms as well as precise patterns of differentially expressed genes occur in directing precursor cells toward a given lineage. Using microarray-based technology, we examined gene expression across the course of 3T3-L1 adipocyte differentiation. Total cellular RNA was isolated at times 0, 2, 8, 16, 24, 48, and 96 h following treatment with either standard hormonal inducers of differentiation; insulin, dexamethasone, isobutylmethylxanthine (IDX), or IDX plus trichostatin A (TsA), a histone deacetylase inhibitor and potent adipogenic inhibitor. cRNA was synthesized from cellular RNA and hybridized to high density Affymetrix MG_U74Av2 microarray gene chips containing 12,488 cDNA/Expressed Sequence Tags (ESTs) probe sets. From the IDX-only treated cells, all probe sets that were either unchanged or differentially expressed less than 2-fold throughout differentiation with respect to time 0 preadipocytes were excluded from further analyses. This selection resulted in a net of 1686 transcripts, 859 were increased in expression, and 827 were decreased in expression at least 2-fold across differentiation. To focus in on genes that were more specific to differentiation, the same analysis was performed on IDX plus TsA-treated non-differentiating cells and all probe sets from the IDX-only group that exhibited similar expression profiles in the non-differentiating TsA-treated group were excluded leaving a total of 1016 transcripts that were regulated only under differentiating conditions. Six hundred and thirty-six of these transcripts were elevated at least 2-fold and 380 exhibited a decrease in expression relative to time 0 preadipocytes. This group of genes was further analyzed using hierarchical clustering and self-organizing maps and resulted in the identification of numerous genes not previously known to be regulated during adipocyte differentiation. Many of these genes may well represent novel adipogenic mediators and markers of adipogenesis.

Microarray analysis of gene expression during early adipocyte differentiation.

The molecular mechanisms that regulate cellular differentiation during development and throughout life are complex. It is now recognized that precise patterns of differentially expressed genes ultimately direct a particular cell toward a given lineage and many of these are regulated during the earliest stages of differentiation. Using a microarray-based expression analysis, we have examined gene expression profiles during the first 24 h of 3T3-L1 adipocyte differentiation. RNA was isolated at times 0, 2, 8, 16, and 24 h following stimulation of differentiation and hybridized in duplicate to high density Affymetrix microarray gene chips containing a series of 13,179 cDNA/expressed sequence tag (EST) probe sets. Two hundred and eighty-five cDNA/ESTs were shown to have at least a fivefold change in expression levels during this time course and both hierarchical and self-organizing map clustering analysis was performed to categorize them by expression profiles. Several genes known to be regulated during this time period were confirmed and Western blot analysis of the proteins encoded by some of the identified genes revealed expression profiles similar to their mRNA counterparts. As expected, many of the genes identified have not been examined in such a critical time period during adipogenesis and may well represent novel adipogenic mediators.

Activation of an adipogenic program in adult myoblasts with age.

Myoblasts isolated from mouse hindlimb skeletal muscle demonstrated increased adipogenic potential as a function of age. Whereas myoblasts from 8-month-old adult mice did not significantly accumulate terminal markers of adipogenesis regardless of culture conditions, myoblasts from 23-month-old mice accumulated fat and expressed genes characteristic of differentiated adipocytes, such as the fatty acid binding protein aP2. This change in differentiation potential was associated with a change in the abundance of the mRNA encoding the transcription factor C/EBPalpha, and in the relative abundance of PPARgamma2 to PPARgamma1 mRNAs. Furthermore, PPARgamma activity appeared to be regulated at the level of phosphorylation, being more highly phosphorylated in myoblasts isolated from younger animals. Although adipogenic gene expression in myoblasts from aged animals was activated, presumably in response to PPARgamma and C/EBPalpha, unexpectedly, myogenic gene expression was not effectively repressed. The Wnt signaling pathway may also alter differentiation potential in muscle with age. Wnt-10b mRNA was more abundantly expressed in muscle tissue and cultured myoblasts from adult compared with aged mice, resulting in stabilization of cytosolic beta-catenin, that may potentially contribute to inhibition of adipogenic gene expression in adult myoblasts. The changes reported here, together with those reported in bone marrow stroma with age, suggest that a default program may be activated in mesenchymal cells with increasing age resulting in a more adipogenic-like phenotype. Whether this change in differentiation potential contributes to the increased adiposity in muscle with age remains to be determined.

Early expression of p107 is associated with 3T3-L1 adipocyte differentiation.

In response to hormonal stimulation quiescent 3T3-L1 preadipocyte cells reenter the cell cycle and undergo a mitotic expansion phase prior to terminal differentiation. The cell cycle regulatory proteins p130 and p107 undergo dramatic changes in protein levels within 24 h of differentiation. The role of these proteins in regulating adipocyte mitotic clonal expansion and/or differentiation are unclear. It has recently been demonstrated that adipocyte proliferation can be uncoupled from adipocyte differentiation through the use of the pharmacological MEK inhibitor PD98059 or the tyrosine phosphatase inhibitor, sodium vanadate. We examined the expression of p130 and p107 in stimulated 3T3-L1 cells in the presence of either PD98059, U0126 or sodium vanadate. While inhibition of MEK blocked proliferation, the cells underwent differentiation normally. In contrast, vanadate blocked differentiation without affecting proliferation. Inhibition of MEK did not affect the increase in p107 expression in stimulated cells indicating that induction of p107 is independent of MAP kinase signaling. Vanadate treatment caused a significant delay in p107 expression in the first 24 h following stimulation. Under these conditions, p130 expression was relatively unchanged. Our results indicate that a rapid increase in p107 expression correlates with a commitment to undergo adipocyte differentiation. The data further suggest that the rapid induction of p107 is not required for cellular proliferation during the mitotic clonal expansion phase. Taken together, these findings provide correlative data that implicate p107 in the terminal differentiation, but not proliferation, of quiescent preadipocytes following hormonal stimulation.

The effects of temperature and seasons on subcutaneous white adipose tissue in humans: evidence for thermogenic gene induction.

CONTEXT: Although brown adipose tissue (BAT) activity is increased by a cold environment, little is known of the response of human white adipose tissue (WAT) to the cold. DESIGN: We examined both abdominal and thigh subcutaneous (SC) WAT from 71 subjects who were biopsied in the summer or winter, and adipose expression was assessed after an acute cold stimulus applied to the thigh of physically active young subjects. RESULTS: In winter, UCP1 and PGC1α mRNA were increased 4 to 10-fold (p < 0.05) and 1.5 to 2-fold, respectively, along with beige adipose markers, and UCP1 protein was 3-fold higher in the winter. The seasonal increase in abdominal SC WAT UCP1 mRNA was considerably diminished in subjects with a BMI > 30 kg/m(2), suggesting that dysfunctional WAT in obesity inhibits adipose thermogenesis. After applying an acute cold stimulus to the thigh of subjects for 30 min, PGC1α and UCP1 mRNA was stimulated 2.7-fold (p < 0.05) and 1.9-fold (p = 0.07), respectively. Acute cold also induced a 2 to 3-fold increase in PGC1α and UCP1 mRNA in human adipocytes in vitro, which was inhibited by macrophage-conditioned medium and by the addition of TNFα. CONCLUSION: Human SC WAT increases thermogenic genes seasonally and acutely in response to a cold stimulus and this response is inhibited by obesity and inflammation.

Regulation of thrombospondin-1 expression in alternatively activated macrophages and adipocytes: role of cellular cross talk and omega-3 fatty acids.

Thrombospondin-1 (TSP-1) expression in human adipose positively correlates with body mass index and may contribute to adipose dysfunction by activating transforming growth factor-ß and/or inhibiting angiogenesis. Our objective was to determine how TSP-1 is regulated in adipocytes and polarized macrophages using a coculture system and to determine whether fatty acids, including the ω-3 fatty acid docosahexaenoic acid (DHA), regulate TSP-1 expression. Coculture of M1, M2a or M2c macrophages with adipocytes induced TSP-1 gene expression in adipocytes (from 2.4- to 4.2-fold, P<.05), and adipocyte coculture induced TSP-1 gene expression in M1 and M2c macrophages (M1: 8.6-fold, M2c: 26-fold; P<.05). TSP-1 protein levels in the shared media of adipocytes and M2c cells were also strongly induced by coculture (>10-fold, P<.05). DHA treatment during the coculture of adipocytes and M2c macrophages potently inhibited the M2c macrophage TSP-1 mRNA level (97% inhibition, P<.05). Adipocyte coculture induced interleukin (IL)-10 expression in M2c macrophages (10.1-fold, P<.05), and this increase in IL-10 mRNA expression was almost completely blocked with DHA treatment (96% inhibition, P<.05); thus, IL-10 expression closely paralleled TSP-1 expression. Since IL-10 has been shown to regulate TSP-1 in other cell types, we reduced IL-10 expression with siRNA in the M2c cells and found that this caused TSP-1 to be reduced in response to adipocyte coculture by 60% (P<.05), suggesting that IL-10 regulates TSP-1 expression in M2c macrophages. These results suggest that supplementation with dietary ω-3 fatty acids could potentially be beneficial to adipose tissue in obesity by reducing TSP-1 and fibrosis.

Omega-3 fatty acids reduce adipose tissue macrophages in human subjects with insulin resistance.

Fish oils (FOs) have anti-inflammatory effects and lower serum triglycerides. This study examined adipose and muscle inflammatory markers after treatment of humans with FOs and measured the effects of ω-3 fatty acids on adipocytes and macrophages in vitro. Insulin-resistant, nondiabetic subjects were treated with Omega-3-Acid Ethyl Esters (4 g/day) or placebo for 12 weeks. Plasma macrophage chemoattractant protein 1 (MCP-1) levels were reduced by FO, but the levels of other cytokines were unchanged. The adipose (but not muscle) of FO-treated subjects demonstrated a decrease in macrophages, a decrease in MCP-1, and an increase in capillaries, and subjects with the most macrophages demonstrated the greatest response to treatment. Adipose and muscle ω-3 fatty acid content increased after treatment; however, there was no change in insulin sensitivity or adiponectin. In vitro, M1-polarized macrophages expressed high levels of MCP-1. The addition of ω-3 fatty acids reduced MCP-1 expression with no effect on TNF-α. In addition, ω-3 fatty acids suppressed the upregulation of adipocyte MCP-1 that occurred when adipocytes were cocultured with macrophages. Thus, FO reduced adipose macrophages, increased capillaries, and reduced MCP-1 expression in insulin-resistant humans and in macrophages and adipocytes in vitro; however, there was no measureable effect on insulin sensitivity.

Effect of endoplasmic reticulum stress on inflammation and adiponectin regulation in human adipocytes.

The endoplasmic reticulum (ER) of adipocytes plays a major role in the assembly and secretion of adipokines. The levels of serum adiponectin, secreted by adipocytes, are decreased in insulin resistance, diabetes, and obesity. The role of ER stress in downregulating adiponectin levels has been demonstrated in mouse models of obesity. Studies examining human adipose tissue have indicated that there is an increase in the ER stress transcript HSPA5 with increased body mass index (BMI). However, it is not established whether ER stress results in changes in adiponectin levels or multimerization in human adipocytes. We examined whether the induction of ER stress using tunicamycin, thapsigargin, or palmitate alters the messenger RNA (mRNA) and protein expression of adiponectin and the mRNA expression of chaperones ERP44 and ERO1 in adult-derived human adipocyte stem (ADHAS) cells. ER stress was measured using key indicators of ER stress-HSPA5, ERN1, CHOP, and GADD34, as well as changes in eIF2α phosphorylation. Because ER stress is suggested to be the proximal cause of inflammation in adipocytes, we further examined the change in inflammatory status by quantitating the change in Iκß-α protein following the induction of ER stress. Our studies indicate that: (1) ER stress markers were increased to a higher degree using tunicamycin or thapsigargin compared to palmitate; (2) ER stress significantly decreased adiponectin mRNA in response to tunicamycin and thapsigargin, but palmitate did not decrease adiponectin mRNA levels. In all three instances, the induction of ER stress was accompanied by a decrease in adiponectin protein as well as adiponectin multimerization. All three inducers of ER stress increased tumor necrosis factor-α (TNF-α) mRNA and decreased Iκß-α protein in adipocytes. The data suggest that ER stress modifies adiponectin secretion and induces inflammation in ADHAS cells.