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.

Calcium is involved in formation of high molecular weight adiponectin.

BACKGROUND: Adiponectin, an adipocyte-specific secretory protein, is known to circulate as different isoforms in the blood stream. METHODS: Using sucrose gradients and Western blotting on nondenaturing gels, adiponectin isoforms were examined in human serum, plasma, adipose tissue, and cells. The medium from human adipose tissue and human and mouse adipocytes were also examined for changes in isoform formation upon treatment with EGTA. RESULTS: Comparison of adiponectin complexes revealed distinct differences in distribution of high molecular weight (HMW) forms between human serum and plasma, with an apparent difference in molecular weight. Variation in molecular weight suggested a probable dissociation of the HMW isoforms in the presence of EDTA in the plasma. Examination of human serum samples treated with EDTA or EGTA showed a partial dissociation of the HMW isoform, while the addition of excess calcium, but not magnesium, to human plasma resulted in partial restoration of HMW adiponectin. When human adipose tissue-secreted adiponectin was treated with EGTA, there was a decrease in the HMW isoform by 61% (+/- 1.89%) and a corresponding increase in low molecular weight (LMW) and middle molecular weight (MMW) isoforms, compared to untreated samples. Analysis of mouse and human adipocytes also showed a reduction in HMW isoforms with a corresponding increase in MMW and LMW isoforms upon treatment with EGTA. The Simpson-Golabi-Behmel syndrome (SGBS) human adipocyte cell line, which primarily synthesizes LMW isoforms, produced increasing amounts of HMW adiponectin upon treatment with calcium in a dose-dependent manner. CONCLUSION: These data indicate that calcium promotes the formation of HMW adiponectin, and calcium sequestration decreases HMW adiponectin. Because of the importance of HMW adiponectin in insulin sensitivity, these data demonstrate the importance of assay conditions and sample preparation in the measurement of adiponectin isoforms.

Thrombospondin-1 is an adipokine associated with obesity, adipose inflammation, and insulin resistance.

OBJECTIVE: We examined the relationship between the expression of thrombospondin (TSP)1, an antiangiogenic factor and regulator of transforming growth factor-beta activity, obesity, adipose inflammation, and insulin resistance. RESEARCH DESIGN AND METHODS: TSP1 gene expression was quantified in subcutaneous adipose tissue (SAT) of 86 nondiabetic subjects covering a wide range of BMI and insulin sensitivity, from visceral adipose (VAT) and SAT from 14 surgical patients and from 38 subjects with impaired glucose tolerance randomized to receive either pioglitazone or metformin for 10 weeks. An adipocyte culture system was also used to assess the effects of pioglitazone and coculture with macrophages on TSP1 gene expression. RESULTS: TSP1 mRNA was significantly associated with obesity (BMI) and insulin resistance (low insulin sensitivity index). Relatively strong positive associations were seen with markers of inflammation, including CD68, macrophage chemoattractant protein-1, and plasminogen activator inhibitor (PAI)-1 mRNA (r >/= 0.46, P = 0.001 for each), that remained significant after controlling for BMI and S(i). However, TSP1 mRNA was preferentially expressed in adipocyte fraction, whereas inflammatory markers predominated in stromal vascular fraction. Coculture of adipocytes and macrophages augmented TSP1 gene expression and secretion from both cell types. Pioglitazone (not metformin) treatment resulted in a 54% decrease (P < 0.04) in adipose TSP gene expression, as did in vitro pioglitazone treatment of adipocytes. CONCLUSIONS: TSP1 is a true adipokine that is highly expressed in obese, insulin-resistant subjects; is highly correlated with adipose inflammation; and is decreased by pioglitazone. TSP1 is an important link between adipocytes and macrophage-driven adipose tissue inflammation and may mediate the elevation of PAI-1 that promotes a prothrombotic state.

Amyloid precursor protein modulates beta-catenin degradation.

BACKGROUND: The amyloid precursor protein (APP) is genetically associated with Alzheimer's disease (AD). Elucidating the function of APP should help understand AD pathogenesis and provide insights into therapeutic designs against this devastating neurodegenerative disease. RESULTS: We demonstrate that APP expression in primary neurons induces beta-catenin phosphorylation at Ser33, Ser37, and Thr41 (S33/37/T41) residues, which is a prerequisite for beta-catenin ubiquitinylation and proteasomal degradation. APP-induced phosphorylation of beta-catenin resulted in the reduction of total beta-catenin levels, suggesting that APP expression promotes beta-catenin degradation. In contrast, treatment of neurons with APP siRNAs increased total beta-catenin levels and decreased beta-catenin phosphorylation at residues S33/37/T41. Further, beta-catenin was dramatically increased in hippocampal CA1 pyramidal cells from APP knockout animals. Acute expression of wild type APP or of familial AD APP mutants in primary neurons downregulated beta-catenin in membrane and cytosolic fractions, and did not appear to affect nuclear beta-catenin or beta-catenin-dependent transcription. Conversely, in APP knockout CA1 pyramidal cells, accumulation of beta-catenin was associated with the upregulation of cyclin D1, a downstream target of beta-catenin signaling. Together, these data establish that APP downregulates beta-catenin and suggest a role for APP in sustaining neuronal function by preventing cell cycle reactivation and maintaining synaptic integrity. CONCLUSION: We have provided strong evidence that APP modulates beta-catenin degradation in vitro and in vivo. Future studies may investigate whether APP processing is necessary for beta-catenin downregulation, and determine if excessive APP expression contributes to AD pathogenesis through abnormal beta-catenin downregulation.

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.

APP-BP1 inhibits Abeta42 levels by interacting with Presenilin-1.

BACKGROUND: The beta-amyloid precursor protein (APP) is sequentially cleaved by the beta- and then gamma-secretase to generate the amyloid beta-peptides Abeta40 and Abeta42. Increased Abeta42/Abeta40 ratios trigger amyloid plaque formations in Alzheimer's disease (AD). APP binds to APP-BP1, but the biological consequence is not well understood. RESULTS: We report that when the endogenous APP-BP1 was suppressed by small interfering RNAs (siRNAs), cell-associated Abeta42 was dramatically increased in APP695 expressing primary neurons. The accumulation of Abeta42 was accompanied by significant increases in APP and APP-CTF in APP-BP1 siRNA expressing neurons. In contrast, APP-BP1 overexpression in primary neurons significantly decreased the levels of Abeta and endogenous APP but not APLPs. We also investigated the potential mechanism of APP-BP1-mediated APP processing. APP-BP1 co-precipitated with Presenilin-1 (PS1) in native rat brain extracts, co-migrated with the gamma-secretase components in brain membrane extracts in glycerol gradient centrifugation, and colocalized in primary neurons. Further, the endogenous PS1-CTF was significantly downregulated by APP-BP1 expression. CONCLUSION: Our data suggest that APP-BP1 may inhibit Abeta42 production by interacting with PS1 under physiological conditions.

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.

Pioglitazone increases secretion of high-molecular-weight adiponectin from adipocytes.

Adiponectin is an adipocyte-derived serum protein that plays important roles in energy homeostasis, obesity, and insulin sensitivity. Using sucrose gradients and Western blotting of nondenaturing gels, we examined the adiponectin isoforms secreted from human adipose tissue, human and mouse adipocytes, and cell lines in response to pioglitazone added in vitro. The predominant form secreted from adipose tissue in vitro was the high-molecular-weight (HMW) isoform, with small amounts of low-molecular-weight (LMW) forms present. The addition of pioglitazone (1-3 micromM) in vitro increased the secretion of the HMW isoform, with no significant effect on the other isoforms. Human adipose tissue was also examined for changes in adiponectin mRNA levels upon pioglitazone treatment. No difference was detected, suggesting that the effect of pioglitazone is not at the transcriptional level but, rather, at a posttranscriptional phase of the secretory pathway. Additional experiments were conducted to determine whether adiponectin expression was mechanistically similar in other adipose cells. Examination of primary human adipocytes revealed an increase in intracellular HMW isoform with a decline in LMW forms following pioglitazone treatment, with a corresponding increase in the secreted HMW form. Similar results were observed with primary mouse adipocytes, 3T3-F422A cells, and SGBS human adipocyte cells, although differences in the distribution of HMW and LMW isoforms were apparent between cell types. Although there are differences in isoforms between species, in all cases pioglitazone served to increase the secretion of the HMW form of adiponectin.

Pioglitazone induces apoptosis of macrophages in human adipose tissue.

Metabolic syndrome and type 2 diabetes mellitus are associated with an increased number of macrophage cells that infiltrate white adipose tissue (WAT). Previously, we demonstrated that the treatment of subjects with impaired glucose tolerance (IGT) with the peroxisome proliferator-activated receptor gamma (PPARgamma) agonist pioglitazone resulted in a decrease in macrophage number in adipose tissue. Here, adipose tissue samples from IGT subjects treated with pioglitazone were examined for apoptosis with terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling (TUNEL) staining. TUNEL-positive cells were identified, and there was a significant 42% increase in TUNEL-positive cells following pioglitazone treatment. Overlay experiments with anti-CD68 antibody demonstrated that most of the TUNEL-positive cells were macrophages. To determine whether macrophage apoptosis was a direct or indirect effect of pioglitazone treatment, human THP1 cells were treated with pioglitazone in vitro, demonstrating increased TUNEL staining in a dose- and time-dependent manner. Furthermore, the appearance of the active proteolytic subunits of caspase-3 and caspase-9 were detected in cell lysate from THP1 cells and also increased in a dose- and time-dependent manner following pioglitazone treatment. Pretreatment with a PPARgamma inhibitor, GW9662, prevented pioglitazone induction of the apoptotic pathway in THP1 cells. Differentiated human adipocytes did not show any significant increase in apoptosis after treatment in vitro with piolgitazone. These findings indicate that PPARgamma has distinct functions in different cell types in WAT, such that pioglitazone reduces macrophage infiltration by inducing apoptotic cell death specifically in macrophages through PPARgamma activation.

Secreted beta-amyloid precursor protein activates microglia via JNK and p38-MAPK.

Reactive microglia are thought to play a role in the pathogenesis of Alzheimer's disease (AD) and are localized to the senile plaques that are associated with cognitive decline. The beta-amyloid precursor protein (betaAPP) is over-expressed in the dystrophic neurites near such plaques, and secreted forms of betaAPP (sAPPalpha) activate inflammatory responses in microglia. To characterize the mechanisms by which sAPPalpha activates microglia, we assayed its effects on MAP kinases, including c-Jun N-terminal kinases (JNK), extracellular signal-regulated protein kinases (ERK), and p38-MAPK. sAPPalpha was found to rapidly activate JNKs, ERKs and p38-MAPK in a dose-dependent manner. The JNK inhibitor SP600125 and the p38 inhibitor SB203580 independently reduced both nitrite accumulation and induction of inflammatory nitric oxide synthase (iNOS). By contrast, inhibition of the ERK pathway with U0126 did not appreciably affect either outcome measure. These findings suggest that sAPP activates the ERK, JNK and p38 classes of MAP kinases but that only JNK and p38-MAPK are critical for activation of microglia by sAPPalpha, a process that compromises neuronal function and survival.

Cytokines and the aging brain - what we don't know might help us.

Cognitive aspects of aging represent a grave challenge for our societal circumstances as members of the baby-boom generation spiral toward a collective 'senior moment'. In addition, age-related changes in the CNS can contribute to motor deficits and other somatic aberrations. Inflammation and its regulation by cytokines have been connected to many aspects of aging, and mechanisms addressed here provide a rationale for this. Nevertheless, a role for cytokines in normal aging of the human brain has not been confirmed, and it seems to be possible to ameliorate both cognitive decline and cytokine elevation via lifestyle choices. So ignorance of the brain should not prohibit development of successful strategies for delaying or avoiding neurological deficits.

Induction of serine racemase expression and D-serine release from microglia by amyloid beta-peptide.

BACKGROUND: Roles for excitotoxicity and inflammation in Alzheimer's disease have been hypothesized. Proinflammatory stimuli, including amyloid beta-peptide (Abeta), elicit a release of glutamate from microglia. We tested the possibility that a coagonist at the NMDA class of glutamate receptors, D-serine, could respond similarly. METHODS: Cultured microglial cells were exposed to Abeta. The culture medium was assayed for levels of D-serine by HPLC and for effects on calcium and survival on primary cultures of rat hippocampal neurons. Microglial cell lysates were examined for the levels of mRNA and protein for serine racemase, the enzyme that forms D-serine from L-serine. The racemase mRNA was also assayed in Alzheimer hippocampus and age-matched controls. A microglial cell line was transfected with a luciferase reporter construct driven by the putative regulatory region of human serine racemase. RESULTS: Conditioned medium from Abeta-treated microglia contained elevated levels of D-serine. Bioassays of hippocampal neurons with the microglia-conditioned medium indicated that Abeta elevated a NMDA receptor agonist that was sensitive to an antagonist of the D-serine/glycine site (5,7-dicholorokynurenic acid; DCKA) and to enzymatic degradation of D-amino acids by D-amino acid oxidase (DAAOx). In the microglia, Abeta elevated steady-state levels of dimeric serine racemase, the apparent active form of the enzyme. Promoter-reporter and mRNA analyses suggest that serine racemase is transcriptionally induced by Abeta. Finally, the levels of serine racemase mRNA were elevated in Alzheimer's disease hippocampus, relative to age-matched controls. CONCLUSIONS: These data suggest that Abeta could contribute to neurodegeneration through stimulating microglia to release cooperative excitatory amino acids, including D-serine.

Alpha-synuclein aggregation.

Alpha-synuclein is a major component of Lewy bodies in Parkinson's disease and is found associated with several other forms of dementia. As with other neurodegenerative diseases, the ability of alpha-synuclein to aggregate and form fibrillar deposits seems central to its pathology. We have defined a sequence within the NAC region of alpha-synuclein that is necessary for aggregation. Exploitation of chemically modified analogues of this peptide may produce inhibitors of aggregation.

Inhibition of fibril formation and toxicity of a fragment of alpha-synuclein by an N-methylated peptide analogue.

Alpha-synuclein has been linked to amyloidogenesis in Parkinson's disease and other neurodegenerative disorders. We have previously shown that a peptide comprising residues 68-78 of alpha-synuclein is the minimum fragment that, like alpha-synuclein itself, forms amyloid fibrils and exhibits toxicity towards cells in culture. Hughes et al. [J. Biol. Chem. 275 (2000) 25109] showed that an N-methylated derivative of Abeta(25-35) inhibited the formation of fibrils by Abeta(25-35) and reduced its toxicity. We have now extended this concept to an amyloidogenic alpha-synuclein-based peptide. Alpha-synuclein(68-78), N-methylated at G1y73, was compared to non-methylated peptide. Whereas alpha-synuclein(68-78) formed fibrils and was toxic to cells, the N-methylated analogue had neither of these properties. Moreover, an equimolar mixture of the non-methylated and methylated peptides formed very few fibrils and toxicity was markedly reduced.