Andreas Leiherer

Phytochemicals and their impact on adipose tissue inflammation and diabetes.

Type 2 diabetes mellitus is an inflammatory disease and the mechanisms that underlie this disease, although still incompletely understood, take place in the adipose tissue of obese subjects. Concurrently, the prevalence of obesity caused by Western diets excessive energy intake and the lack of exercise escalates, and is believed to be causative for the chronic inflammatory state in adipose tissue. Overnutrition itself as an overload of energy may induce the adipocytes to secrete chemokines activating and attracting immune cells to adipose tissue. But also inflammation-mediating food ingredients like saturated fatty acids are believed to directly initiate the inflammatory cascade. In addition, hypoxia in adipose tissue as a direct consequence of obesity, and its effect on gene expression in adipocytes and surrounding cells in fat tissue of obese subjects appears to play a central role in this inflammatory response too. In contrast, revisiting diet all over the world, there are also some natural food products and beverages which are associated with curative effects on human health. Several natural compounds known as spices such as curcumin, capsaicin, and gingerol, or secondary plant metabolites catechin, resveratrol, genistein, and quercetin have been reported to provide an improved health status to their consumers, especially with regard to diabetes, and therefore have been investigated for their anti-inflammatory effect. In this review, we will give an overview about these phytochemicals and their role to interfere with inflammatory cascades in adipose tissue and their potential for fighting against inflammatory diseases like diabetes as investigated in vivo.

Identification of Hypoxia-Induced Genes in Human SGBS Adipocytes by Microarray Analysis

Hypoxia in adipose tissue is suggested to be involved in the development of a chronic mild inflammation, which in obesity can further lead to insulin resistance. The effect of hypoxia on gene expression in adipocytes appears to play a central role in this inflammatory response observed in obesity. However, the global impact of hypoxia on transcriptional changes in human adipocytes is unclear. Therefore, we compared gene expression profiles of human Simpson-Golabi-Behmel syndrome (SGBS) adipocytes under normoxic or hypoxic conditions to detect hypoxia-responsive genes in adipocytes by using whole human genome microarrays. Microarray analysis showed more than 500 significantly differentially regulated mRNAs after incubation of the cells under low oxygen levels. To gain further insight into the biological processes, hypoxia-regulated genes after 16 hours of hypoxia were classified according to their function. We identified an enrichment of genes involved in important biological processes such as glycolysis, response to hypoxia, regulation of cellular component movement, response to nutrient levels, regulation of cell migration, and transcription regulator activity. Real-time PCR confirmed eight genes to be consistently upregulated in response to 3, 6 and 16 hours of hypoxia. For adipocytes the hypoxia-induced regulation of these genes is shown here for the first time. Moreover in six of these eight genes we identified HIF response elements in the proximal promoters, specific for the HIF transcription factor family members HIF1A and HIF2A. In the present study, we demonstrated that hypoxia has an extensive effect on gene expression of SGBS adipocytes. In addition, the identified hypoxia-regulated genes are likely involved in the regulation of obesity, the incidence of type 2 diabetes, and the metabolic syndrome.

Importance of protease cleavage sites within and flanking human immunodeficiency virus type 1 transframe protein p6* for spatiotemporal regulation of protease activation.

ABSTRACT The human immunodeficiency virus type 1 (HIV-1) protease (PR) has recently been shown to be inhibited by its propeptide p6* in vitro. As p6* itself is a PR substrate, the primary goal of this study was to determine the importance of p6* cleavage for HIV-1 maturation and infectivity. For that purpose, short peptide variants mimicking proposed cleavage sites within and flanking p6* were designed and analyzed for qualitative and quantitative hydrolysis in vitro. Proviral clones comprising the selected cleavage site mutations were established and analyzed for Gag and Pol processing, virus maturation, and infectivity in cultured cells. Amino-terminal cleavage site mutation caused aberrant processing of nucleocapsid proteins and delayed replication kinetics. Blocking the internal cleavage site resulted in the utilization of a flanking site at a significantly decreased hydrolysis rate in vitro, which however did not affect Gag-Pol processing and viral replication. Although mutations blocking cleavage at the p6* carboxyl terminus yielded noninfectious virions exhibiting severe Gag processing defects, mutations retarding hydrolysis of this cleavage site neither seemed to impact viral infectivity and propagation in cultured cells nor seemed to interfere with overall maturation of released viruses. Interestingly, these mutants were shown to be clearly disadvantaged when challenged with wild-type virus in a dual competition assay. In sum, we conclude that p6* cleavage is absolutely essential to allow complete activation of the PR and subsequent processing of the viral precursors.

Embedding Permanent Watermarks in Synthetic Genes

As synthetic biology advances, labeling of genes or organisms, like other high-value products, will become important not only to pinpoint their identity, origin, or spread, but also for intellectual property, classification, bio-security or legal reasons. Ideally information should be inseparably interlaced into expressed genes. We describe a method for embedding messages within open reading frames of synthetic genes by adapting steganographic algorithms typically used for watermarking digital media files. Text messages are first translated into a binary string, and then represented in the reading frame by synonymous codon choice. To aim for good expression of the labeled gene in its host as well as retain a high degree of codon assignment flexibility for gene optimization, codon usage tables of the target organism are taken into account. Preferably amino acids with 4 or 6 synonymous codons are used to comprise binary digits. Several different messages were embedded into open reading frames of T7 RNA polymerase, GFP, human EMG1 and HIV gag, variously optimized for bacterial, yeast, mammalian or plant expression, without affecting their protein expression or function. We also introduced Vigenère polyalphabetic substitution to cipher text messages, and developed an identifier as a key to deciphering codon usage ranking stored for a specific organism within a sequence of 35 nucleotides.

Uncoupling Human Immunodeficiency Virus Type 1 gag and pol Reading Frames: Role of the Transframe Protein p6* in Viral Replication

ABSTRACT Apart from its regulatory role in protease (PR) activation, little is known about the function of the human immunodeficiency virus type 1 transframe protein p6* in the virus life cycle. p6* is located between the nucleocapsid and PR domains in the Gag-Pol polyprotein precursor and is cleaved by PR during viral maturation. We have recently reported that the central region of p6* can be extensively mutated without abolishing viral infectivity and replication in vitro. However, mutagenesis of the entire p6*-coding sequence in the proviral context is not feasible without affecting the superimposed frameshift signal or the overlapping p1-p6gag sequences. To overcome these limitations, we created a novel NL4-3-derived provirus by displacing the original frameshift signal to the 3′ end of the gag gene, thereby uncoupling the p6* gene sequence from the p1-p6gag reading frame. The resulting virus (AL) proved to be replication competent in different cell cultures and thus represents an elegant tool for detailed analysis of p6* function. Hence, extensive deletions or substitutions were introduced into the p6* gene sequence of the AL provirus, and effects on particle release, protein processing, and viral infectivity were evaluated. Interestingly, neither the deletion of 63% of all p6* residues nor the partial substitution by a heterologous sequence affected virus growth and infectivity, suggesting that p6* is widely dispensable for viral in vitro replication. However, the insertion of a larger reporter sequence interfered with virus production and maturation, implying that the length or conformation of this spacer region might be critical for p6* function.

Angiopoietin-like protein 4 significantly predicts future cardiovascular events in coronary patients

BACKGROUND Angiopoietin-like protein 4 (ANGPTL4) has been associated with cardiometabolic disorders including dyslipidemia and atherosclerosis in animal studies; in humans, however, its impact on metabolic traits and cardiovascular risk remains unclear. METHODS We examined the association of plasma ANGPTL4 levels with the metabolic syndrome (harmonized consensus definition), with angiographically determined coronary artery disease (CAD), and with the risk of future cardiovascular events in a cohort of 490 patients undergoing coronary angiography for the evaluation of stable CAD. In addition, we investigated the influence of the tagging single nucleotide polymorphisms (SNPs) rs4076317, rs2278236, rs1044250, and rs11672433 as well as variant rs116843064 (E40K) of the ANGPTL4 gene on cardiovascular risk in a larger sample of 983 angiographied coronary patients including the above mentioned 490 subjects. RESULTS Plasma ANGPTL4 was significantly higher in patients with the metabolic syndrome than in subjects without the metabolic syndrome (26.0 ± 19.4 ng/ml vs. 22.2 ± 19.7 ng/ml; p = 0.008). No significant association was found between ANGPTL4 and angiographically characterized coronary atherosclerosis. Prospectively, however, plasma ANGPTL4 significantly predicted future cardiovascular events both univariately (HR1.45 [1.16-1.82], p = 0.001) and after adjustment for standard cardiovascular risk factors (1.26 [1.01-1.58]; p = 0.045). Concordantly, rs4076317, rs2278236, and rs1044250 significantly affected the risk of future cardiovascular events (adjusted HRs 0.70 [0.54-0.90]; p = 0.005, 0.76 [0.61-0.94]; p = 0.012, and 1.30 [1.03-1.62]; p = 0.025, respectively). CONCLUSIONS We conclude that plasma ANGPTL4 levels as well as ANGPTL4 variants significantly predict cardiovascular events independently of conventional cardiovascular risk factors.

High plasma omentin predicts cardiovascular events independently from the presence and extent of angiographically determined atherosclerosis.

BACKGROUND AND AIMS No prospective data on the power of the adipocytokine omentin to predict cardiovascular events are available. We aimed at investigating i) the association of plasma omentin with cardiometabolic risk markers, ii) its association with angiographically determined coronary atherosclerosis, and iii) its power to predict cardiovascular events. METHODS We measured plasma omentin in 295 patients undergoing coronary angiography for the evaluation of established or suspected stable coronary artery disease (CAD), of whom 161 had significant CAD with coronary artery stenoses ≥50% and 134 did not have significant CAD. RESULTS Over 3.5 years, 17.6% of our patients suffered cardiovascular events, corresponding to an annual event rate of 5.0%. At baseline, plasma omentin was not significantly associated with metabolic syndrome stigmata and did not differ significantly between patients with and subjects without significant CAD (17.2 ± 13.6 ng/ml vs. 17.5 ± 15.1 ng/ml; p = 0.783). Prospectively, however, cardiovascular event risk significantly increased over tertiles of omentin (12.1%, 13.8%, and 29.5%, for tertiles 1 through 3; ptrend = 0.003), and omentin as a continuous variable significantly predicted cardiovascular events after adjustment for age, gender, BMI, diabetes, hypertension, LDL cholesterol, HDL cholesterol, and smoking (standardized adjusted hazard ratio (HR) 1.41 [95% CI 1.16-1.72]; p < 0.001), as well as after additional adjustment for the presence and extent of significant CAD at baseline (HR 1.59 [95% CI 1.29-1.97, p < 0.001). CONCLUSION From this first prospective evaluation of the cardiovascular risk associated with omentin we conclude that elevated plasma omentin significantly predicts cardiovascular events independently from the presence and extent of angiographically determined baseline CAD.

High plasma chemerin is associated with renal dysfunction and predictive for cardiovascular events - Insights from phenotype and genotype characterization.

The novel adipokine chemerin, encoded by the RARRES2 gene, has been suggested to be linked to insulin resistance and to the metabolic syndrome (MetS). However, no well-defined cardiovascular profile has been reported and the association with coronary artery disease (CAD) is a matter of debate. Because there is a relation between renal dysfunction and CAD, we analyzed plasma chemerin levels and the estimated glomerular filtration rate (eGFR) in 495 patients undergoing coronary angiography for the evaluation of established or suspected stable CAD. Chemerin levels were higher in patients with Type 2 diabetes mellitus (T2DM, n=111) and the metabolic syndrome (MetS, n=147) than in subjects without T2DM (191.5±72.9 vs. 169.7±64.7ng/ml, p=0.001) or the MetS (201.2±71.0 vs. 163,1ng/ml, p<0.001), but did not differ significantly between patients with significant CAD (n=247) and those without significant CAD (177.1±67.0 vs. 171.7±67.2ng/ml, p=0.193). Analysis of covariance using age, sex, and BMI as covariates showed that chemerin was significantly and independently associated with eGFR (F=49.6, p<0.001). After an 8-year follow-up period, patients with high chemerin levels were more often affected by cardiovascular events (HR=1.72 [95% CI 1.19-2.47], p=0.004), even after appropriate adjustment for age, gender, BMI, as well as eGFR (adjusted HR 1.51 [95% CI 1.03-2.23], p=0.037). Given the cardiometabolic role of chemerin, we also applied a Cardio-Metabo Chip analysis and revealed a genome-wide significant association with SNPs (rs55709438, rs2444030, and rs3098423) located at chromosomal region 15q15-23, which were associated with metabolic traits and eGFR. This study for the first time demonstrates that high chemerin concentrations are significantly associated with renal impairment and predictive of cardiovascular events and that 15q15-23 might have an impact on chemerin levels beyond common genetic variations in RARRES2.

Occurrence of the JAK2 V617F mutation in patients with peripheral arterial disease

The acquired JAK2 V617F mutation is common in patients with myeloproliferative neoplasms. We previously showed that JAK2 V617F is also found in coronary patients, most of them affected by coronary atherosclerosis. Peripheral arterial disease (PAD) is another important manifestation of atherosclerosis. However, prevalence of the JAK2 V617F mutation and its effect on clinical or hematologic characteristics is unknown in PAD patients. In the present study we determined the prevalence of JAK2 V617F in a cohort of 287 patients with sonographically proven PAD and compared mutation frequency with mutational status of 997 healthy people from the KORA F4 study. JAK2 V617F screening and quantification of allele burden in both cohorts was performed with same allele‐specific quantitative real‐time PCR method. From a total of 287 PAD patients, 9 individuals were tested positive for the JAK2 V617F mutation. One patient showed elevated hemoglobin values, indicating polycythemia vera. Observed JAK2 V617F frequency (3.1%) in PAD patients showed a 5‐fold, highly significant increase compared with healthy people (P < 0.001). Furthermore, occurrence of the mutation in PAD patients was significantly decreased in patients using aspirin (P = 0.003). We conclude that the prevalence of JAK2 V617F mutation is significantly increased in PAD patients compared to the general population. Future studies are warranted to confirm our observations and to define the underlying mechanisms behind our findings. Am. J. Hematol. 90:E17–E21, 2015.

Evaluation of the prevalence and prospective clinical impact of the JAK2 V617F mutation in coronary patients

The JAK2 V617F mutation is not only found in the majority of patients with myeloproliferative neoplasms (MPN), including essential thrombocythemia (ET), but also has been reported in individuals without overt MPN. A close relation of the JAK2 V617F mutation to atherothrombotic events has been described, at least in patients with MPN. The prevalence of the JAK2 V617F mutation and its clinical impact in coronary patients is unknown. To address this issue, DNA samples from 1,589 subjects undergoing coronary angiography with up to 11 years of follow up were genotyped using allele‐specific real‐time PCR assays. Prevalence of the JAK2 V617F mutation was 1.32% (n = 21) in coronary patients. Two JAK2 V617F positive patients showed baseline platelet counts indicative for ET and a third patient developed ET during follow up, finally resulting in a percentage of 0.188% of ET cases. This corresponds to an up to fivefold accumulation of ET cases in coronary patients compared with the general population. Our study showed no impact of the JAK2 V617F mutation on future atherothrombotic events or overall survival (HR = 1.04 [0.33–3.27]; P = 0.949 and HR = 0.35 [0.05–2.46]; P = 0.288, respectively). Therefore, our data suggest that JAK2 V617F positive coronary patients are not at increased risk for future atherothrombotic complications. Routine mutation screening in coronary patients is, therefore, not warranted. However, number of ET cases appears to be accumulated in coronary patients. For this reason, we recommend JAK2 V617F testing only in coronary patients showing abnormal blood cell counts for further clarification. Am. J. Hematol. 89:295–301, 2014.