STK38 is a PPARγ-interacting protein promoting adipogenesis.

Peroxisome proliferator-activated receptor-γ (PPARγ) is the master regulator of adipogenesis, but knowledge about how PPARγ is regulated at the protein level is very limited. We aimed to identify PPARγ-interacting proteins which modulate PPARγ's protein levels and transactivating activities in human adipocytes. We expressed Flag-tagged PPARγ in human preadipocytes as bait to capture PPARγ-associated proteins, followed by mass spectroscopy and proteomics analysis, which identified serine/threonine kinase 38 (STK38) as a major PPARγ-associated protein. Protein pulldown studies confirmed this protein-protein interaction in transfected cells, and reporter assays demonstrated that STK38 enhanced PPARγ's transactivating activities without requiring STK38's kinase activity. In cell-based assays, STK38 increased PPARγ protein stability, extending PPARγ's half-life from ~1.08 to 1.95 h. Notably, in human preadipocytes, the overexpression of STK38 enhanced adipogenesis, whereas knockdown impaired the process in a PPARγ-dependent manner. Thus, we discovered that STK38 is a novel PPARγ-cofactor promoting adipogenesis, likely through stabilization of PPARγ.

Recombinant Fc-Elabela fusion protein has extended plasma half-life andmitigates post-infarct heart dysfunction in rats.

Activation of the apelin receptor, or APJ, by apelin is considered a therapeutic avenue for cardiovascular disease, including heart failure. Recently, a novel endogenous ligand for APJ named Elabela (ELA) has been discovered and is known to possess anti-heart failure activity in animal models. However, the short in vivo half-life of ELA constrains its clinical potential. To extend its half-life in vivo, we attempted to make IgG-Fc-ELA fusion proteins. We found that Fc-ELA-32 fusion proteins are cleaved during protein production, whereas Fc-ELA-21 fusion proteins are expressed intact, so we focused our studies on the latter. The Fc-ELA-21 fusion protein retained its functionality in vitro and had a half-life of approximately 44 h in circulation in mice after subcutaneous injection. Daily injection of the fusion protein in MI rats for 4 weeks significantly mitigated heart dysfunction with respect to hemodynamics. At the cellular and tissue levels, treatment of Fc-ELA-21 fusion protein significantly increased angiogenesis, promoted cardiomyocyte proliferation and reduced apoptosis and heart fibrosis near the infarct area. In comparison, ELA-21 had a half-life of 13 min and showed no significant cardioprotective activities. These data suggest that Fc-ELA-21 may be a potential therapeutic for heart failure.

Direct conversion of human myoblasts into brown-like adipocytes by engineered super-active PPARγ.

OBJECTIVE: To determine whether super-activation of PPARγ can reprogram human myoblasts into brown-like adipocytes and to establish a new cell model for browning research. METHODS: To enhance the PPARγ signaling, M3, the transactivation domain of MyoD, was fused to PPARγ. PPARγ and M3-PPARγ-lentiviral vectors were used to convert human myoblasts into adipocytes. Brown adipocyte markers of the reprogrammed adipocytes were assessed by qPCR and protein analyses. White adipocytes differentiated from subcutaneous stromal vascular cells and perithyroid brown fat tissues were used as references. RESULTS: In transient transfections, M3-PPARγ had a stronger constitutive activity than PPARγ by reporter assay. Although the transduction of either PPARγ or M3-PPARγ induced adipogenesis in myoblasts, M3-PPARγ drastically induced the brown adipocyte markers of UCP1, CIDEA, and PRDM16 by 1,050, 2.4, and 5.0 fold, respectively and increased mitochondria contents by 4 fold, compared to PPARγ. CONCLUSIONS: Super-activation of PPARγ can effectively convert human myoblasts into brown-like adipocytes and a new approach to derive brown-like adipocytes.

Hepatic ALT isoenzymes are elevated in gluconeogenic conditions including diabetes and suppressed by insulin at the protein level.

BACKGROUND: Alanine transaminase (ALT) plays an important role in gluconeogenesis by converting alanine into pyruvate for glucose production. Early studies have shown that ALT activities are upregulated in gluconeogenic conditions and may be implicated in the development of diabetes. ALT consists of two isoforms, ALT1 and ALT2, with distinctive subcellular and tissue distributions. Whether and how they are regulated are largely unknown. METHODS: By using Western blotting analysis, we measured hepatic ALT isoforms at the protein level in obese and diabetic animals and in Fao hepatoma cells treated with dexamethasone and insulin. In addition, we measured glucose output in Fao cells over-expressing ALT1 and ALT2. RESULTS: Both ALT isoforms in the liver were increased in diabetic Goto-Kakizaki rats and during fasting. However, in ob/ob mice, only ALT2, but not ALT1, protein levels were elevated, and the increase of ALT2 was correlated with that of ALT activity. We further demonstrated that, in vitro, both ALT1 and ALT2 were induced by glucocorticoid dexamethasone, but suppressed by insulin in Fao cells. Finally, we showed that the over-expression of ALT1 and ALT2 in Fao cells directly increased glucose output. CONCLUSIONS: We have shown the similarity and difference in the regulation of ALT isoforms in gluconeogenic conditions at the protein level, supporting that ALT isoenzymes play an important role in glucose metabolism and may be implicated the development of insulin resistance and diabetes.

Elabela-apelin receptor signaling pathway is functional in mammalian systems.

Elabela (ELA) or Toddler is a recently discovered hormone which is required for normal development of heart and vasculature through activation of apelin receptor (APJ), a G protein-coupled receptor (GPCR), in zebrafish. The present study explores whether the ELA-APJ signaling pathway is functional in the mammalian system. Using reverse-transcription PCR, we found that ELA is restrictedly expressed in human pluripotent stem cells and adult kidney whereas APJ is more widely expressed. We next studied ELA-APJ signaling pathway in reconstituted mammalian cell systems. Addition of ELA to HEK293 cells over-expressing GFP-AJP fusion protein resulted in rapid internalization of the fusion receptor. In Chinese hamster ovarian (CHO) cells over-expressing human APJ, ELA suppresses cAMP production with EC50 of 11.1 nM, stimulates ERK1/2 phosphorylation with EC50 of 14.3 nM and weakly induces intracellular calcium mobilization. Finally, we tested ELA biological function in human umbilical vascular endothelial cells and showed that ELA induces angiogenesis and relaxes mouse aortic blood vessel in a dose-dependent manner through a mechanism different from apelin. Collectively, we demonstrate that the ELA-AJP signaling pathways are functional in mammalian systems, indicating that ELA likely serves as a hormone regulating the circulation system in adulthood as well as in embryonic development.

Niemann-Pick Disease Type C: Induced Pluripotent Stem Cell-Derived Neuronal Cells for Modeling Neural Disease and Evaluating Drug Efficacy.

Niemann-Pick disease type C (NPC) is a rare neurodegenerative disorder caused by recessive mutations in the NPC1 or NPC2 gene that result in lysosomal accumulation of unesterified cholesterol in patient cells. Patient fibroblasts have been used for evaluation of compound efficacy, although neuronal degeneration is the hallmark of NPC disease. Here, we report the application of human NPC1 neural stem cells as a cell-based disease model to evaluate nine compounds that have been reported to be efficacious in the NPC1 fibroblasts and mouse models. These cells are differentiated from NPC1 induced pluripotent stem cells and exhibit a phenotype of lysosomal cholesterol accumulation. Treatment of these cells with hydroxypropyl-ß-cyclodextrin, methyl-ß-cyclodextrin, and δ-tocopherol significantly ameliorated the lysosomal cholesterol accumulation. Combined treatment with cyclodextrin and δ-tocopherol shows an additive or synergistic effect that otherwise requires 10-fold higher concentration of cyclodextrin alone. In addition, we found that hydroxypropyl-ß-cyclodextrin is much more potent and efficacious in the NPC1 neural stem cells compared to the NPC1 fibroblasts. Miglustat, suberoylanilide hydroxamic acid, curcumin, lovastatin, pravastatin, and rapamycin did not, however, have significant effects in these cells. The results demonstrate that patient-derived NPC1 neural stem cells can be used as a model system for evaluation of drug efficacy and study of disease pathogenesis.

Correlation of circulating MMP-9 with white blood cell count in humans: effect of smoking.

BACKGROUND: Matrix metalloproteinase-9 (MMP-9) is an emerging biomarker for several disease conditions, where white blood cell (WBC) count is also elevated. In this study, we examined the relationship between MMP-9 and WBC levels in apparently healthy smoking and non-smoking human subjects. METHODS: We conducted a cross-sectional study to assess the relationship of serum MMP-9 with WBC in 383 men and 356 women. Next, we divided the male population (women do not smoke in this population) into three groups: never (n = 243), current (n = 76) and former (n = 64) smokers and compared the group differences in MMP-9 and WBC levels and their correlations within each group. RESULTS: Circulating MMP-9 and WBC count are significantly correlated in men (R(2) = 0.13, p<0.001) and women (R(2) = 0.19, p<0.001). After stratification by smoking status, MMP-9 level was significantly higher in current smokers (mean ± SE; 663.3±43.4 ng/ml), compared to never (529.7±20.6) and former smokers (568±39.3). WBC count was changed in a similar pattern. Meanwhile, the relationship became stronger in current smokers with increased correlation coefficient of r = 0.45 or R(2) = 0.21 (p<0.001) and steeper slope of ß = 1.16±0.30 (p<0.001) in current smokers, compared to r = 0.26 or R(2) = 0.07 (p<0.001) and ß = 0.34±0.10 (p<0.001) in never smokers. CONCLUSIONS: WBC count accounts for 13% and 19% of MMP-9 variance in men and women, respectively. In non-smoking men, WBC count accounts for 7% of MMP-9 variance, but in smoking subjects, it accounts for up to 21% of MMP-9 variance. Thus, we have discovered a previously unrecognized correlation between the circulating MMP-9 and WBC levels in humans.

Transmembrane emp24 protein transport domain 6 is selectively expressed in pancreatic islets and implicated in insulin secretion and diabetes.

OBJECTIVES: The objective of the study was to identify pancreatic islet-selective gene(s) that may play a functional role in islet biology and diabetes development. METHODS: Through bioinformatics, we identified and cloned a pancreas-enriched complementary DNA encoding transmembrane emp24 protein transport domain 6 (TMED6) and examined its mRNA and protein expression in tissues and islet cell lines by Northern analysis and immunofluorescence histochemistry. We also studied the role of TMED6 in insulin secretion using a knockdown approach and its gene expression changes during the development of diabetes in Goto-Kakizaki rats. RESULTS: TMED6 is selectively expressed in pancreatic islets and belongs to the EMP24_GP25L superfamily, which is known to be involved in protein trafficking and secretion. Northern analysis revealed that TMED6 mRNA is highly and selectively expressed in pancreas. Immunofluorescence histochemistry of mouse pancreas showed that TMED6 expression is restricted to pancreatic islets with higher levels in α cells than ß cells. Knockdown of TMED6 gene expression in Min6 ß cells decreased insulin secretion. Moreover, TMED6 gene expression was significantly lower in diabetic Goto-Kakizaki rats. CONCLUSIONS: TMED6 may play a functional role in islet biology, particularly in hormone production or secretion, and its dysregulation may be implicated in the development of diabetes.

Mapping of loci from Solanum lycopersicoides conferring resistance or susceptibility to Botrytis cinerea in tomato.

Cultivated tomato (Solanum lycopersicum, syn. Lycopersicon esculentum) is susceptible to the necrotrophic ascomycete and causal agent of gray mold, Botrytis cinerea. Resistance to this fungal pathogen is elevated in wild relatives of tomato, including Solanum lycopersicoides. An introgression line population (IL) containing chromosomal segments of S. lycopersicoides within the background of tomato cv. VF36 was used to screen the genome for foliar resistance and susceptibility to B. cinerea. Based on this screen, putative quantitative trait loci (QTL) were identified, five for resistance and two for susceptibility. Four resistance QTL decreased infection frequency while the fifth reduced lesion diameter. One susceptibility QTL increased infection frequency whereas the other increased lesion diameter. Overlapping chromosomal segments provided strong evidence for partial resistance on chromosomes 1 and 9 and for elevated susceptibility on chromosome 11. Segregation analysis confirmed the major resistance QTL on the long arm of chromosome 1 and susceptibility on chromosome 11. Linkage of partial resistance to chromosome 9 could not be confirmed. The usefulness of these data for resistance breeding and for map-based cloning of foliar resistance to B. cinerea is discussed.

Expression, purification, and initial characterization of human alanine aminotransferase (ALT) isoenzyme 1 and 2 in High-five insect cells.

Alanine aminotransferase (ALT) is a key enzyme for gluconeogenesis as well as a widely used serum marker for liver injury. We have identified two ALT isoenzymes, ALT1 and ALT2, which are encoded by separate genes. In this study, we described the expression, purification and initial characterization of human ALT1 and ALT2 proteins in High-five insect cells. Human ALT1 and ALT2 were expressed as His-tagged fusion proteins by recombinant baculovirus in insect cells and purified into homogeneity in one step by using immobilized Ni2+-affinity chromatography. Tag-free ALT1 and ALT2 were obtained by cleavage of enterokinase digestion and used for initial characterization of the enzymes. The specific ALT activity of purified fusion or His-tag-removed ALT1 was about 15-fold higher than that of ALT2 and their enzymatic activities decreased quickly at 37 degrees C and -20 degrees C, but were well preserved at -80 degrees C. Nevertheless, the ALT1 and ALT2 activities remained stable in a buffer containing 25% glycerol. The pH profile was similar between hALT1 and hALT2 in that both enzymes remained fully active between pH 6.5 and 8.0. The purified ALT recombinant proteins can not only be used as a reference protein standard for the ALT assay in clinical chemistry, but also will be useful for understanding the biochemical and biological significance of the isoenzymes and for developing ALT isoform-specific assays for clinical or preclinical diagnostic use.

Consequences of lipid droplet coat protein downregulation in liver cells: abnormal lipid droplet metabolism and induction of insulin resistance.

OBJECTIVE: Accumulation of intracellular lipid droplets (LDs) in non-adipose tissues is recognized as a strong prognostic factor for the development of insulin resistance in obesity. LDs are coated with perilipin, adipose differentiation-related protein, tail interacting protein of 47 kd (PAT) proteins that are thought to regulate LD turnover by modulating lipolysis. Our hypothesis is that PAT proteins modulate LD metabolism and therefore insulin resistance. RESEARCH DESIGN AND METHODS: We used a cell culture model (murine AML12 loaded with oleic acid) and small interfering RNA to directly assess the impact of PAT proteins on LD accumulation, lipid metabolism, and insulin action. PAT proteins associated with excess fat deposited in livers of diet-induced obese (DIO) mice were also measured. RESULTS: Cells lacking PAT proteins exhibited a dramatic increase in LD size and a decrease in LD number. Further, the lipolytic rate increased by approximately 2- to 2.5-fold in association with increased adipose triglyceride lipase (ATGL) at the LD surface. Downregulation of PAT proteins also produced insulin resistance, as indicated by decreased insulin stimulation of Akt phosphorylation (P < 0.001). Phosphoinositide-dependent kinase-1 and phosphoinositide 3-kinase decreased, and insulin receptor substrate-1 307 phosphorylation increased. Increased lipids in DIO mice livers were accompanied by changes in PAT composition but also increased ATGL, suggesting a relative PAT deficiency. CONCLUSIONS: These data establish an important role for PAT proteins as surfactant at the LD surface, packaging lipids in smaller units and restricting access of lipases and thus preventing insulin resistance. We suggest that a deficiency of PAT proteins relative to the quantity of ectopic fat could contribute to cellular dysfunction in obesity and type 2 diabetes.

Omentin plasma levels and gene expression are decreased in obesity.

Central obesity and the accumulation of visceral fat are risk factors for the development of type 2 diabetes and cardiovascular disease. Omentin is a protein expressed and secreted from visceral but not subcutaneous adipose tissue that increases insulin sensitivity in human adipocytes. To determine the impact of obesity-dependent insulin resistance on the regulation of two omentin isoforms, gene expression and plasma levels were measured in lean, overweight, and obese subjects. Omentin 1 was shown to be the major circulating isoform in human plasma. Lean subjects had significantly higher plasma omentin 1 levels than obese and overweight subjects. In addition, higher plasma omentin 1 levels were detected in women compared with men. Plasma omentin 1 levels were inversely correlated with BMI, waist circumference, leptin levels, and insulin resistance as measured by homeostasis model assessment and positively correlated with adiponectin and HDL levels. Both omentin 1 and omentin 2 gene expression were decreased with obesity and were highly correlated with each other in visceral adipose tissue. In summary, decreased omentin levels are associated with increasing obesity and insulin resistance. Therefore, omentin levels may be predictive of the metabolic consequences or co-morbidities associated with obesity.