Non-stroke admissions to a hyperacute stroke unit.

A significant proportion of patients presenting to hyperacute stroke units (HSUs) are diagnosed with non-stroke (NS). This study aimed to assess the rate and diagnoses of NS patients admitted to a HSU and the implications for clinical service provision. Admissions to the HSU at the Southern General Hospital, Glasgow, were retrospectively assessed (March 2007-September 2007). NS patients were identified by two parallel ascertainment methods and NS diagnosis was confirmed by case-note and discharge letter review. Of 375 presentations, 116 (31%) were due to NS. NS diagnosis was more likely for local referrals than from regional hospitals (41% versus 19%; P = 0.0002). Compared with stroke/transient ischaemic attack patients, NS patients were significantly younger, more likely to have an magnetic resonance imaging (MRI) scan and had a shorter length of hospital stay. Common NS diagnoses were migraine (22%), functional neurological disorder (14%), syncope (12%) and seizure (6%). NS patients who had an MRI scan were more likely to have a length of stay ≥2 days (75% versus 53%; P = 0.03). NS makes up one-third of acute stroke-like presentations with a high frequency of neurological conditions. NS patients tend to be younger and require significant investigation. The increased use of MRI and neurological services has implications for providing a hyperacute stroke service.

Tools for the identification of bioactives impacting the metabolic syndrome: screening of a botanical extract library using subcutaneous and visceral human adipose-derived stem cell-based assays.

Plant extracts continue to represent an untapped source of renewable therapeutic compounds for the treatment and prevention of illnesses including chronic metabolic disorders. With the increase in worldwide obesity and its related morbidities, the need for identifying safe and effective treatments is also rising. As such, use of primary human adipose-derived stem cells represents a physiologically relevant cell system to screen for bioactive agents in the prevention and treatment of obesity and its related complications. By using these cells in a primary screen, the risk and cost of identifying artifacts due to interspecies variation and immortalized cell lines is eliminated. We demonstrate that these cells can be formatted into 384-well high throughput screens to rapidly identify botanical extracts that affect lipogenesis and lipolysis. Additionally, counterscreening with human primary stem cells from distinct adipose depots can be routinely performed to identify tissue specific responses. In our study, over 500 botanical extracts were screened and 16 (2.7%) were found to affect lipogenesis and 4 (0.7%) affected lipolysis.

Metabolic remodeling of human skeletal myocytes by cocultured adipocytes depends on the lipolytic state of the system.

OBJECTIVE: Adipocyte infiltration of the musculoskeletal system is well recognized as a hallmark of aging, obesity, and type 2 diabetes. Intermuscular adipocytes might serve as a benign storage site for surplus lipid or play a role in disrupting energy homeostasis as a result of dysregulated lipolysis or secretion of proinflammatory cytokines. This investigation sought to understand the net impact of local adipocytes on skeletal myocyte metabolism. RESEARCH DESIGN AND METHODS: Interactions between these two tissues were modeled using a coculture system composed of primary human adipocytes and human skeletal myotubes derived from lean or obese donors. Metabolic analysis of myocytes was performed after coculture with lipolytically silent or activated adipocytes and included transcript and metabolite profiling along with assessment of substrate selection and insulin action. RESULTS: Cocultured adipocytes increased myotube mRNA expression of genes involved in oxidative metabolism, regardless of the donor and degree of lipolytic activity. Adipocytes in the basal state sequestered free fatty acids, thereby forcing neighboring myotubes to rely more heavily on glucose fuel. Under this condition, insulin action was enhanced in myotubes from lean but not obese donors. In contrast, when exposed to lipolytically active adipocytes, cocultured myotubes shifted substrate use in favor of fatty acids, which was accompanied by intracellular accumulation of triacylglycerol and even-chain acylcarnitines, decreased glucose oxidation, and modest attenuation of insulin signaling. CONCLUSIONS: The effects of cocultured adipocytes on myocyte substrate selection and insulin action depended on the metabolic state of the system. These findings are relevant to understanding the metabolic consequences of intermuscular adipogenesis.

Use of adipose-derived stem cells in high-throughput screening to identify modulators of lipogenesis.

Drug discovery efforts have an increasing focus on functional cell-based screening to identify compounds that modulate targets presented in a relevant format. Historically, immortalized cell lines have been used in primary and secondary screens due to their ease of manipulation, transformation, and propagation. However, more researchers are using primary cells that present their drug targets in their natural context. Human primary cell isolation and propagation procedures have become efficient enough to provide these cells in the necessary scale for early stage drug discovery. Adult human stem cells provide an opportunity for investigating multiple pathways of differentiation, development, regeneration, and toxicity using a single cell source and type. Adipose-derived stem cells (ASCs) are an attractive adult human primary stem cell for drug discovery due their abundance in adipose tissue, ease of isolation, and propagation in culture. They can be expanded in high numbers and retain their unique properties to differentiate into multiple lineages. In this chapter, we describe a protocol to identify modulators of human ASC lipogenesis following partial differentiation to adipocytes.

Trans-10, cis-12, but not cis-9, trans-11, conjugated linoleic acid attenuates lipogenesis in primary cultures of stromal vascular cells from human adipose tissue.

We have previously shown that both a commercially available mixture of conjugated linoleic acid (CLA) isomers and the trans-10, cis-12 isomer of CLA reduced the triglyceride (TG) content and induced apoptosis in differentiating cultures of murine 3T3-L1 preadipocytes. However, the influence of CLA isomers on differentiating human (pre)adipocytes is unknown. Therefore, we conducted a series of studies using primary cultures of stromal vascular cells isolated from human adipose tissue to determine: 1) the influence of seeding density and thiazolidinedione (TZD) concentration on TG content; 2) the chronic dose response of cis-9, trans-11 CLA vs. trans-10, cis-12 CLA on TG content; 3) whether chronic linoleic acid supplementation could rescue the TG content of CLA-treated cultures; and 4) whether trans-10, cis-12-mediated reduction in cellular TG was due to decreased lipogenesis and/or increased lipolysis. In expt. 1, the TG content [micromol/(L x 10(6) cells)] increased as both seeding density and TZD concentration increased. For example, cultures seeded at 4 x 10(4) cells/cm(2) and supplemented with 10 micromol/L BRL 49653 had 10-fold more TG than similarly seeded cultures without BRL 49653. In expt. 2, TG content decreased as the level of trans-10, cis-12 CLA increased from 1 to 10 micromol/L, whereas the TG content increased with increasing concentrations of either linoleic acid or cis-9, trans-11 CLA. In expt. 3, linoleic acid supplementation restored the TG content of cultures treated with trans-10, cis-12 CLA compared with cultures treated with CLA alone, suggesting that attenuation of TG content by CLA is reversible. In expt. 4, glucose incorporation into total lipid decreased with increasing levels of trans-10, cis-12 CLA, whereas neither CLA isomer acutely affected lipolysis. These data suggest that the reported antiobesity actions of a supplement containing a crude mixture of CLA isomers given to humans may be due to inhibition of lipogenesis by the trans-10, cis-12 isomer.

Thiazolidinediones and glucocorticoids synergistically induce differentiation of human adipose tissue stromal cells: biochemical, cellular, and molecular analysis.

While adipocyte differentiation has been studied extensively in murine cultures, the lack of a readily available preadipocyte model has hindered equivalent studies in man. We describe methods for the isolation and culture of primary human stromal cells from surgical adipose tissue specimens. In vitro, the stromal cells rapidly differentiate in response to a combination of adipogenic agents. Among these, glucocorticoids and thiazolidinediones act together to induce the formation of lipid vacuoles within the cells. These morphologic changes accompany the increased expression of 2 characteristic adipocyte proteins, the cytoplasmic enzyme glycerol phosphate dehydrogenase (GPDH) and the secreted cytokine leptin. Likewise, stromal cell differentiation results in elevated mRNA levels for the fatty acid binding protein aP2 and the adipogenic regulatory transcription factors CCAAT/enhancer binding protein alpha (C/EBPalpha) and peroxisome proliferator-activated receptor gamma (PPARgamma) in addition to leptin. The in vitro differentiated stromal cells exhibit a lipolytic response to beta-adrenergic agonists, comparable to that reported with primary human adipocytes. These studies demonstrate the validity of human adipose tissue-derived stromal cells as a reliable in vitro model for investigations of adipocyte metabolism in humans.

Adipogenic potential of human adipose derived stromal cells from multiple donors is heterogeneous.

The current study was done to assess if heterogeneity existed in the degree of adipogenesis in stromal cells (preadipocytes) from multiple donors. In addition to conventional lipid-based methods, we have employed a novel signal amplification technology, known as branched DNA, to monitor expression of an adipocyte specific gene product aP2. The fatty acid binding protein aP2 increases during adipocyte differentiation and is induced by thiazolidinediones and other peroxisome proliferator activated receptor gamma ligands. The current work examined the adipogenic induction of aP2 mRNA levels in human adipose tissue stromal cells derived from 12 patients (mean age +/- SEM, 38.9 +/- 3.1) with mild to moderate obesity (mean body mass index +/- SEM, 27.8 +/- 2.4). Based on branched DNA technology, a rapid and sensitive measure of specific RNAs, the relative aP2 level in adipocytes increased by 679 +/- 93-fold (mean +/- SEM, n=12) compared to preadipocytes. Normalization of the aP2 mRNA levels to the housekeeping gene, glyceraldehyde phosphate dehydrogenase, did not significantly alter the fold induction in a subset of 4 patients (803.6 +/- 197.5 vs 1118.5 +/- 308.1). Independent adipocyte differentiation markers were compared between adipocytes and preadipocytes in parallel studies. Leptin secretion increased by up to three-orders of magnitude while measurements of neutral lipid accumulation by Oil Red O and Nile Red staining increased by 8.5-fold and 8.3-fold, respectively. These results indicate that preadipocytes isolated from multiple donors displayed varying degrees of differentiation in response to an optimal adipogenic stimulus in vitro. This work also demonstrates that branched DNA measurement of aP2 is a rapid and sensitive measure of adipogenesis in human stromal cells. The linear range of this assay extends up to three-orders of magnitude and correlates directly with independent measures of cellular differentiation.

Prospective study of apolipoprotein E genotype and functional outcome following ischemic stroke.

BACKGROUND: The apolipoprotein E (APOE) epsilon 4 allele is a marker of adverse outcome following head injury and intracerebral hemorrhage. Transgenic animal data in a focal cerebral ischemia model suggest that the epsilon 4 allele increases infarct size and functional impairment. OBJECTIVE: To determine if APOE genotype is associated with functional recovery from ischemic stroke. DESIGN: Prospective study. SETTING: Stroke service at a university teaching hospital. PATIENTS: Patients with clinical and neuroimaging findings (computed tomography or magnetic resonance imaging) compatible with an acute ischemic stroke. MAIN OUTCOME: Functional outcome by Barthel index (BI) and modified Rankin scale (mRS) was compared for epsilon 3/epsilon 3 patients vs epsilon 4 carriers and vs epsilon 2 carriers at 1 and 3 months. Univariate predictors of 3-month outcome were examined in a multivariate analysis. RESULTS: One hundred eighty nine patients were enrolled: 100 women, 89 men (mean +/- SD age, 69.4 +/- 11.0 years). There were 25 epsilon 2 alleles (frequency, 0.07), 292 epsilon 3 alleles (0.77), and 61 epsilon 4 alleles (0.16). Baseline National Institutes of Health Stroke Scale scores and Oxfordshire Community Stroke Project classifications were similar in all groups (epsilon 3/epsilon 3, epsilon 4, and epsilon 2 carriers). One-month (BI, P = .64; mRS, P = .59) and 3-month (BI, P = .87; mRS, P = .73) outcomes were not associated with possession of either epsilon 4 or the epsilon 2 allele. Baseline National Institutes of Health Stroke Scale scores (P < .001) and age (P = .002) were significant predictors of 3-month BI and mRS outcomes in multivariate analyses. CONCLUSIONS: Although there is a robust influence of APOE polymorphism on functional recovery after some types of brain injury in humans, it does not exert a major influence on injury severity or functional recovery following ischemic stroke. Arch Neurol. 2000;57:1480-1484

Dehydroepiandrosterone and related steroids alter 3T3-L1 preadipocyte proliferation and differentiation.

The purpose of the present study was to determine if the anti-adipogenic effects of dehydroepiandrosterone (DHEA) are mediated solely by DHEA or by one or more of its downstream metabolites. In Experiment 1, preconfluent proliferating cultures of 3T3-L1 preadipocytes were incubated for either 24 or 72 h with 0, 1, 5 or 25 microM DHEA, DHEA sulfate (DHEAS), testosterone, estrone and 17beta-estradiol. Pregnenolone, a precursor of DHEA(S), was also tested at these concentrations. After 24 h of incubation, DHEAS, 17beta-estradiol and estrone at the 1 microM level stimulated preadipocyte proliferation. In contrast, DHEA and 17beta-estradiol at the 25 microM level attenuated proliferation to a greater extent than all other steroids. After 72 h of incubation, DHEA and 17beta-estradiol at the 25 microM level attenuated proliferation to a greater extent than all other steroids. In Experiment 2, post-confluent cultures of differentiating 3T3-L1 preadipocytes were incubated for 6 days with 0, 5, 30, or 60 microM levels of these steroids. Preadipocyte differentiation, as assessed by lipid content and glycerol-3-phosphate dehydrogenase activity, decreased markedly when treated with 30 and 60 microM DHEA, 17beta-estradiol, estrone and pregnenolone. In contrast, DHEAS had no impact on preadipocyte proliferation or differentiation. These results suggest that the anti-adipogenic actions of DHEA in adipose tissue may be mediated, in part, by one or more of its distal metabolites, including 17beta-estradiol.

Dehydroepiandrosterone alters the growth of stromal vascular cells from human adipose tissue.

OBJECTIVE: The purpose of this study was to determine if the antiobesity actions of dehydroepiandrosterone (DHEA) observed in vivo are due to an influence on the proliferation and differentiation of primary cultures of stromal-vascular (SV) cells isolated from human adipose tissue. DESIGN: SV cells were isolated from subcutaneous adipose tissue obtained from a young adult female undergoing elective liposuction. For the proliferation assay (Experiment 1), cultures were fed proliferation media containing 0, 5, 25 or 100 microM DHEA for 3d. At the end of this treatment period, cultures were either prepared for counting or for determining their metabolic activity using the Alamar Blue staining procedure. For the differentiation assays (Experiment 2), cultures were fed differentiation media containing 0, 25 or 50 microM DHEA for 20 d. At the end of this treatment period, cultures were either prepared for lipid staining using Oil Red O or for marker enzyme analysis (glycerol-3-phosphate dehydrogenase activity; GPDH). To determine if the stimulatory effects of DHEA on SV cell differentiation were dependent on the presence of thiazolidinediones (Experiment 3), cultures of differentiating SV cells were incubated in the presence and absence of BRL 49653 and either 0, 25 or 50 microM DHEA. RESULTS: In Experiment 1, cultures treated with 25 and 100 microM DHEA had fewer cells than cultures treated with either 0 or 5 microM DHEA. Alamar Blue staining decreased as the level of DHEA in the cultures increased. In Experiment 2, cultures treated with DHEA had more lipid and GPDH activity than control cultures. In Experiment 3, cultures treated with BRL 49653 had more triglyceride than cultures treated without BRL 49653. Likewise, cultures treated with DHEA had more triglyceride than their non-DHEA controls. Regardless of the BRL status, cultures supplemented with DHEA had more triglyceride than control cultures. CONCLUSION: These data suggest that in cultures of SV cells from human adipose tissue, DHEA supplementation attenuates proliferation and enhances differentiation. These data support the hypothesis that DHEA directly attenuates preadipocyte proliferation in humans as we previously demonstrated in primary cultures of pig and rat SV cells and in cultures of 3T3-L1 preadipocytes. In contrast, DHEA stimulated the differentiation of human preadipocytes, which is contrary to its actions in differentiating cultures of preadipocytes from animals.

Dehydroepiandrosterone reduces proliferation and differentiation of 3T3-L1 preadipocytes.

The purpose of these studies was to determine whether the antiobesity actions of dehydroepiandrosterone (DHEA) and DHEA-sulfate (DHEAS) observed in vivo are due to an influence on proliferation and/or differentiation in monolayer cultures of 3T3-L1 preadipocytes. For the proliferation study (Exp. 1), cells were grown in plating medium containing DHEA at 0, 5, 25, 50, or 100 microM for 1-4 d. DHEAS was added at the 100 microM level only. For the differentiation study (Exp. 2), cultures were grown in plating medium containing DHEA at 0, 5, 30, 60, 120, or 240 microM for 2-6 d. DHEAS was added at the 240 microM level only. In Exp. 3, the effect of DHEA on mature adipocytes was determined by exposing adipocytes grown in plating medium to DHEA at 0, 75, 125, and 250 microM for 1-4 d. In Exp. 1, preadipocyte proliferation decreased as the level of DHEA increased in cultures of 3T3-L1 cells. DHEAS had no effect on preadipocyte proliferation. The antiproliferative effect of DHEA was partially reversed by the addition of 1 microM mevalonic acid to proliferating cultures containing 25 microM DHEA. In Exp. 2, preadipocyte differentiation decreased as the level of DHEA in the cultures increased. In contrast, neither DHEAS nor mevalonic acid treatment influenced preadipocyte differentiation decreased as the level and duration of DHEA treatment increased in cultures of mature adipocytes. These data support the hypothesis that DHEA, but not DHEAS, is the active form of the steroid that attenuates obesity via altering preadipocyte proliferation and differentiation. The addition of 1 microM mevalonic acid to cultures of 3T3-L1 preadipocytes partially reversed DHEA's antiproliferative effects.

Dehydroepiandrosterone-sulfate (DHEAS) reduces adipocyte hyperplasia associated with feeding rats a high-fat diet.

OBJECTIVE: To determine if chronic administration of a low level of dehydroepiandrosterone-sulfate (DHEAS) (10 micrograms/ml drinking water) attenuates adiposity in male Osborne-Mendel rats fed low-fat (11% of kcals) vs high fat (46% of kcals) diets. DESIGN: Rats were randomly assigned to one of four treatment groups for 6 wk in this 2 x 2 factorial study. The main effects tested were diet (low vs high fat) and DHEAS (- or +). SUBJECTS: Male Osborne-Mendel rats (initial body wt approximately 265 g). MEASUREMENTS: Adipocyte mass, size and number from two major fat depots (retroperitoneal, epididymal); mass of one subcutaneous adipose depot (inguinal); serum levels of triglycerides, insulin, glucose and DHEAS; brown adipose tissue (BAT) mass; body weight gain, food and water consumption, and residual carcass composition. RESULTS: DHEAS treatment had no effect on weight gain, food consumption or water intake. DHEAS-treated rats fed the high-fat diet had smaller fat pads containing fewer adipocytes and less carcass lipid than the non DHEAS-treated rats fed the high-fat diet. In contrast, DHEAS-treated rats fed the low-fat diet had similar levels of adipose tissue mass and cellularity compared to control animals fed the low-fat diet. CONCLUSION: Administration of a low dose of DHEAS (10 micrograms/ml or 0.8 mg/kg body wt/d) in the drinking water of young male Osborne-Mendel rats fed a high-fat diet for 6 wk reduced carcass lipid, fat depot mass and retroperitoneal and epididymal adipocyte number compared to their high-fat-fed cohorts. In this study, the antiobesity effects of DHEAS were specific to the level of dietary fat used.

Effects of acute administration of dehydroepiandrosterone-sulfate on adipose tissue mass and cellularity in male rats.

OBJECTIVE: To determine if short term (2 week) treatment of growing male rats with low levels of dehydroepiandrosterone-sulfate (DHEAS) can reduce adiposity and serum triglycerides. DESIGN: Rats were administered either normal drinking water or drinking water supplemented with 10 (D10) or 100 (D100) micrograms/ml DHEAS for 14 d. SUBJECTS: Twenty-one male Sprague-Dawley rats (initial body weight 280 g). MEASUREMENTS: Adipocyte mass, size and number from three major fat depots (retroperitoneal, epididymal, inguinal); serum levels of triglycerides, insulin, IGF-1 and DHEAS; brown adipose tissue (BAT) mass, uncoupling protein content and enzyme activity; body weight gain, food and water consumption; carcass composition. RESULTS: DHEAS treatment had no effect on weight gain, food consumption or water intake. In contrast, rats treated with both levels of DHEAS had lighter fat pads, fewer epididymal and retroperitoneal adipocytes, less carcass lipid, lower levels of serum triglycerides and greater BAT mass and UCP content than control rats. Moreover, rats administered 100 micrograms/ml DHEAS had smaller and fewer epididymal adipocytes and fewer inguinal adipocytes than the D10 and the control rats. CONCLUSION: Acute treatment of growing male rats with low levels (10 micrograms/ml drinking water or 0.7 mg/kg body wt/d) of DHEAS reduces carcass lipid, adipose tissue mass and cellularity as well as serum triglycerides without altering food intake and body weight gain or causing hepatomegaly.