Discovery of a Potent 9‐Deazaxanthine‐based Agent for the Treatment of Obesity‐Related Non‐alcoholic Fatty Liver Disease

A series of deazaxanthine‐based derivatives were rationally prepared and evaluated. 8g exhibited the most potent glucose‐lowering effect on HepG2 cell line and modulated adiponectin and leptin expression in 3T3‐L1 adipocytes. Oral administration of 8g at 25 mg/kg/day for 4 weeks manifested therapeutic effects on high‐fat diet‐induced non‐alcoholic fatty liver disease (NAFLD) by decreasing the weights of the body, liver, and fat. 8g also modulated the serum levels of fasting glucose and adiponectin, triglycerides, low‐density lipoprotein‐cholesterol, and alanine aminotransferase, as well as the hepatic concentrations of triglycerides, total cholesterol. Moreover, 8g significantly decreased steatosis and blocked the increase of adipocytes and the size of adipose tissues from NAFLD. In the DIO mice model, 8g ameliorated the obesity‐related symptoms and normalized serum biomarkers.     

Neutrophil gelatinase-associated lipocalin (NGAL) as a new biomarker for non--acute kidney injury (AKI) diseases

Neutrophil gelatinase-associated lipocalin, or NGAL, an acute phase protein, is part of the lipocalin family. NGAL is highly induced in inflammatory conditions and ischemia, and is a critical component of innate immunity to bacterial infection. Recently, NGAL has been proven as an emerging biomarker for predicting acute kidney injury (AKI). Meanwhile, numerous studies have also demonstrated that NGAL may be a potential biomarker for the diagnosis, prediction, prevention, and prognosis of non--AKI diseases such as chronic kidney diseases, vascular disorders, cancer, preeclampsia, and allergies. This article systematically reviews the clinical utilities of NGAL as a new biomarker for non--AKI diseases.     

Microarray profiling of gene expression in human adipocytes in response to anthocyanins

Adipocyte dysfunction is strongly associated with the development of obesity and insulin resistance. It is accepted that the regulation of adipocytokine secretion or the adipocyte specific gene expression is one of the most important targets for the prevention of obesity and amelioration of insulin sensitivity. Recently, we demonstrated that anthocyanins, which are pigments widespread in the plant kingdom, have the potency of anti-obesity in mice and the enhancement adipocytokine secretion and its gene expression in adipocytes. In this study, we have shown the gene expression profile in human adipocytes treated with anthocyanins (cyanidin 3-glucoside; C3G or cyanidin; Cy). The human adipocytes were treated with 100 microM C3G, Cy or vehicle for 24 h. The total RNA from the adipocytes was isolated and carried out GeneChip microarray analysis. Based on the gene expression profile, we demonstrated the significant changes of adipocytokine expression (up-regulation of adiponectin and down-regulation of plasminogen activator inhibitor-1 and interleukin-6). Some of lipid metabolism related genes (uncoupling protein2, acylCoA oxidase1 and perilipin) also significantly induced in both common the C3G or Cy treatment groups. These studies have provided an overview of the gene expression profiles in human adipocytes treated with anthocyanins and demonstrated that anthocyanins can regulate adipocytokine gene expression to ameliorate adipocyte function related with obesity and diabetes that merit further investigation.     

Early detection of renal injury using urinary vanin‐1 in rats with experimental colitis

Renal complications are often detected in patients with inflammatory bowel disease (IBD). Because conventional markers such as serum creatinine and β2‐microglobulin are not sensitive and/or specific, a new renal biomarker is needed. We have recently identified urinary vanin‐1 as an early biomarker for the detection of nephrotoxicant‐induced renal injury. In this study, we compared the usefulness of urinary vanin‐1 with other newly developed biomarkers [urinary monocyte chemoattractant protein‐1 (MCP‐1), kidney injury molecule‐1 (Kim‐1) and N‐acetyl‐beta‐D‐glucosaminidase (NAG)] for the detection of renal complications in rats with experimental colitis. On day 2 after intracolonic injection of 2,4,6‐trinitrobenzene sulfonic acid (TNBS), male Wistar rats developed not only colitis, but histologically evident renal injury. Urinary vanin‐1 started to elevate on day 1, whereas serum creatinine and urinary excretions of glucose, total protein, albumin, Kim‐1, MCP‐1 and NAG significantly increased only on day 2. The mRNA expressions of vanin‐1 and Kim‐1 significantly increased in the kidney, but not in the colon. In addition, vanin‐1 did not appear in the blood. On the other hand, colonic mRNA expression and the serum concentration of MCP‐1 were significantly higher in the TNBS‐treated rats than in the control animals. These results suggest that, in contrast to MCP‐1, urinary vanin‐1 and Kim‐1 mainly originated from the kidney rather than the colon in this model. Compared with Kim‐1 and MCP‐1, vanin‐1 might be an earlier biomarker for the detection of renal injury in rats with experimental colitis. Copyright © 2013 John Wiley & Sons, Ltd.     

Do regional differences in adipocyte biology provide new pathophysiological insights?

Obesity is an increasing health problem in many countries. Striking differences exist in the magnitude of the impact of different obesities on comorbidities. Individuals with peripheral obesity ('pears') possess fat distributed subcutaneously in gluteofemoral areas and the lower part of the abdomen, and are at little risk of metabolic complications. Conversely, individuals with upper-body obesity ('apples') accumulate fat in subcutaneous and visceral deposits and are more prone to metabolic and cardiovascular problems, particularly when visceral fat deposits are abundant. In this article, whether the different risk factors for obesity of 'apples' and 'pears' are largely related to the heterogeneity of function and responsiveness of the adipocytes from visceral and subcutaneous deposits is questioned. Possible pharmacological approaches to the treatment of obesity and related diseases are also considered.     

Direct adipotropic actions of atorvastatin: differentiation state-dependent induction of apoptosis, modulation of endocrine function, and inhibition of glucose uptake

Statins exert anti-inflammatory, anti-atherogenic actions. The mechanisms responsible for these effects remain only partially elucidated. Diabetes and obesity are characterized by low-grade inflammation. Metabolic and endocrine adipocyte dysfunction is known to play a crucial role in the development of these disorders and the related cardiovascular complications. Thus, direct modulation of adipocyte function may represent a mechanism of pleiotropic statin actions. We investigated effects of atorvastatin on apoptosis, differentiation, endocrine, and metabolic functions in murine white and brown adipocyte lines. Direct exposure of differentiating preadipocytes to atorvastatin strongly reduced lipid accumulation and diminished protein expression of the differentiation marker CCAAT/enhancer binding protein-beta (CEBP-beta). In fully differentiated adipocytes, however, lipid accumulation remained unchanged after chronic atorvastatin treatment. Furthermore, cell viability was reduced in response to atorvastatin treatment in proliferating and differentiating preadipocytes, but not in differentiated cells. Moreover, atorvastatin induced apoptosis and inhibited protein kinase B (AKT) phosphorylation in proliferating and differentiating preadipocytes, but not in differentiated adipocytes. On the endocrine level, direct atorvastatin treatment of differentiated white adipocytes enhanced expression of the pro-inflammatory adipokine interleukin-6 (IL-6), and downregulated expression of the insulin-mimetic and anti-inflammatory adipokines visfatin and adiponectin. Finally, these direct adipotropic endocrine effects of atorvastatin were paralleled by the acute inhibition of insulin-induced glucose uptake in differentiated white adipocytes, while protein expression of the thermogenic uncoupling protein-1 (UCP-1) in brown adipocytes remained unchanged. Taken together, our data for the first time demonstrate direct differentiation state-dependent effects of atorvastatin including apoptosis, modulation of pro-inflammatory and glucostatic adipokine expression, and insulin resistance in adipose cells. These differential interactions may explain variable clinical observations.     

CHANGES IN microRNA (miR) PROFILE AND EFFECTS OF miR-320 IN INSULIN-RESISTANT 3T3-L1 ADIPOCYTES

• 1 MicroRNAs (miRNAs) play essential roles in many biological processes. It is known that aberrant miRNA expression contributes to some pathological conditions. However, it is not known whether miRNAs play any role in the development of insulin resistance in adipocytes, a key pathophysiological link between obesity and diabetes.
• 2 To investigate the function of miRNAs in the development of insulin resistance, using miRNA microarray analysis we compared miRNA expression profiles between normal insulin‐sensitive 3T3‐L1 adipocytes and 3T3‐L1 adipocytes rendered insulin resistant following treatment with high glucose (25 mmol/L) and high insulin (1 µmol/L). Furthermore, adipocytes were transfected with specific antisense oligonucleotides against miRNA‐320 (anti‐miR‐320 oligo) and the effects on the development of insulin resistance were evaluated.
• 3 We identified 50 upregulated and 29 downregulated miRNAs in insulin‐resistant (IR) adipocytes, including a 50‐fold increase in miRNA‐320 (miR‐320) expression. Using bioinformatic techniques, the p85 subunit of phosphatidylinositol 3‐kinase (PI3‐K) was found to be a potential target of miR‐320. In experiments with anti‐miR‐320 oligo, insulin sensitivity was increased in IR adipocytes, as evidenced by increases in p85 expression, phosphorylation of Akt and the protein expression of the glucose transporter GLUT‐4, as well as insulin‐stimulated glucose uptake. These beneficial effects of anti‐miR‐320 oligo were observed only in IR adipocytes and not in normal adipocytes.
• 4 In conclusion, the miRNA profile changes in IR adipocytes compared with normal 3T3‐L1 adipocytes. Anti‐miR‐320 oligo was found to regulate insulin resistance in adipocytes by improving insulin–PI3‐K signalling pathways. The findings provide information regarding a potentially new therapeutic strategy to control insulin resistance.

     

New insights into adipose cell biology

During the last past Years, obesity had become a major public health problem, and new aspects of fat cells biology have been unraveled. First, since the discovery of leptin, adipocytes have been recognized as true endocrine cells secreting a variety of factors in a regulated manner. The role of these factors on the development of obesity-associated metabolic complications is becoming increasingly clear. Also, the process of fat cell differentiation has been uncovered, leading to the possibility of efficient targeting protein expression in adipose tIssue. Finally, lines of transgenic mice have been created, some of which are totally resistant to obesity. These models led to the identification of new potential adipose targets for the treatment of obesity.     

<Emphasis Type="Italic">Salacia reticulata</Emphasis> has therapeutic effects on obesity

Salacia reticulata Wight (S. reticulata) is a herbal medicine used for treatment of early diabetes in Ayurvedic medicine. In previous reports, the extract of S. reticulata showed preventive effects on obesity and various metabolic disorders and a suppressive effect on differentiation in premature adipocytes. The aim of this research was to elucidate the therapeutic efficacy of the extract of S. reticulata on obesity and various metabolic disorders in 12-week-old TSOD mice with obesity and metabolic disorders and in mature 3T3-L1 adipocytes. In TSOD mice, S. reticulata therapy produced a reduction in body weight and mesenteric fat accumulation, an improvement in abnormal glucose metabolism, and an increase in adiponectin level in plasma. In addition, the mRNA expressions of hormone-sensitive lipase (HSL) and adiponectin were increased in mesenteric fat. In in vitro experiments, S. reticulata therapy produced suppression of intracellular triacylglycerol accumulation and enhancement of glycerol release into the medium in mature 3T3-L1 cells. The mRNA expressions of lipogenesis factor (peroxisome proliferator-activated receptor γ, lipoprotein lipase, CD36, and fatty acid binding protein 4) were down-regulated, while the expressions of lipolysis factor (adipose tissue triacylglycerol lipase and HSL) and adiponectin were up-regulated. Moreover, the extract of S. reticulata enhanced the expression of total AMP-activated protein kinase α (AMPKα) and phosphorylated AMPKα in mature adipocytes. These findings demonstrate that the extract of S. reticulata has therapeutic effects on obesity and metabolic disorders by enhancing lipogenesis genes and suppressing lipolysis genes through the activation of AMPKα in adipocytes.     

Honokiol enhances adipocyte differentiation by potentiating insulin signaling in 3T3-L1 preadipocytes

Adipose tissue plays an essential role in energy homeostasis as a metabolic and endocrine organ. Accordingly, adipocytes are emerging as a major drug target for obesity and obesity-mediated metabolic syndrome. Dysfunction of enlarged adipocytes in obesity is involved in obesity-mediated metabolic syndrome. Adipocytokines, such as adiponectin released from small adipocytes, are able to prevent these disorders. In this study, we found that honokiol, an ingredient of Magnolia officinalis used in traditional Chinese and Japanese medicines, enhanced adipocyte differentiation in 3T3-L1 preadipocytes. Oil Red O staining showed that treatment with honokiol in the presence of insulin dose-dependently increased lipid accumulation in 3T3-L1 preadipoyctes although its activity was weak compared with rosiglitazone. During adipocyte differentiation, the expression of peroxisome proliferator-activated receptor γ2 (PPARγ2) mRNA and PPARγ target genes such as adipocyte protein 2 (aP2), adiponectin, and GLUT4 was induced by treatment with 10?μM honokiol. However, honokiol failed to show direct binding to the PPARγ ligand-binding domain in vitro. In preadipocytes, treatment with honokiol in the presence of insulin increased the phosphorylation of extracellular signal-regulated kinase (ERK) 1/2 protein and Akt protein, early insulin signaling pathways related to adipocyte differentiation, compared with insulin-only treatment. Taken together, our results suggest that honokiol promotes adipocyte differentiation through increased expression of PPARγ2 mRNA and potentiation of insulin signaling pathways such as the Ras/ERK1/2 and phosphoinositide-3-kinase (PI3K)/Akt signaling pathways.     

Tumour necrosis factor‐α inhibition with lenalidomide alleviates tissue oxidative injury and apoptosis in ob/ob obese mice

Lenalidomide (Revlimid; Selleck Chemicals, Houston, TX, USA), an analogue of thalidomide, possesses potent cytokine modulatory capacity through inhibition of cytokines such as tumour necrosis factor (TNF)‐α, a cytokine pivotal for the onset and development of complications in obesity and diabetes mellitus. The present study was designed to evaluate the effect of lenalidomide on oxidative stress, protein and DNA damage in multiple organs in an ob/ob murine model of obesity. To this end, C57BL/6 lean and ob/ob obese mice were administered lenalidomide (50 mg/kg per day, p.o.) for 5 days. Oxidative stress, protein and DNA damage were assessed using the conversion of reduced glutathione (GSH) to oxidized glutathione (GSSG), carbonyl formation and Comet assay, respectively. Apoptosis was evaluated using caspase 3 activity, and levels of Bax, Bcl‐2, Bip, caspase 8, caspase 9 and TNF‐α were assessed using western blot analysis. Lenalidomide treatment did not affect glucose clearance in lean or ob/ob mice. Obese mice exhibited a reduced GSH/GSSG ratio in the liver, gastrocnemius skeletal muscle and small intestine, as well as enhanced protein carbonyl formation, DNA damage and caspase 3 activity in the liver, kidney, skeletal muscle and intestine; these effects were alleviated by lenalidomide, with the exception of obesity‐associated DNA damage in the liver and kidney. Western blot analysis revealed elevated TNF‐α, Bax, Bcl‐2, Bip, caspase 8 and caspase 9 in ob/ob mice with various degrees of reversal by lenalidomide treatment. Together, these data indicate that lenalidomide protects against obesity‐induced tissue injury and protein damage, possibly in association with antagonism of cytokine production and cytokine‐induced apoptosis and oxidative stress.     

Quantitative comparison of adipocytokine gene expression during adipocyte maturation in non-obese and obese rats

Adipocytokines secreted from adipocytes have been extensively analyzed due to their role as key factors in various complications of obesity, including arterial sclerosis, liver steatosis, insulin resistance, and diabetes. Several in vivo and in vitro studies have suggested that adipocyte maturation is related to fluctuations in adipocytokine secretion. However, the relationship between adipocyte maturation and adipocytokine levels has not been fully elucidated. Therefore, we sought to clarify the link between adipocytokine gene expression and adipocyte maturation through systematic analysis. We quantified mRNA for six adipocytokine genes: adiponectin, resistin, leptin, plasminogen activator inhibitor 1 (PAI-1), heparin-binding epidermal growth factor (EGF)-like growth factor (HB-EGF), and visfatin, in adipose tissue, in primary cultured adipocytes obtained from an obese Zucker rat, and in the preadipocyte cell line 3T3-L1. Moreover, to elucidate the role of adipocytokines in adipocyte maturation, adipocytokine expression levels were analyzed during maturation. Although fluctuations in adipocytokine gene expression were heterogeneous, gene expression was highly similar during maturation of primary cultured adipocytes from obese and non-obese rats, suggesting that the maturation process is independent from processes that lead to obesity. Moreover, the expression patterns of adiponectin, resistin and leptin mRNA in 3T3-L1 cells were highly similar to those in primary cultured adipocytes, indicating that these adipocytokines could be common maturation markers for primary cultured adipocytes obtained from obese and non-obese rats, and for preadipocyte cell lines.     

In Vitro Functional Assessment of 22 Newly Identified CYP2D6 Allelic Variants in the Chinese Population

Cytochrome P450 2D6 (CYP2D6) is one of the most widely investigated CYPs related to genetic polymorphisms and is responsible for one‐quarter of the currently used clinical drugs. We previously detected 22 novel, non‐synonymous, mutated sites in the Chinese population, but nothing is known about the functional effects of these mutations in terms of specific CYP2D6 substrates. In this study, wild‐type CYP2D6, two common allelic variants and 22 newly reported CYP2D6 isoforms were transiently expressed in 293FT cells, and the enzymatic activities of these variants were systematically assessed using dextromethorphan and bufuralol as the probing substrates. Consequently, 19 and 21 allelic variants were found to exhibit significantly decreased enzymatic activities for dextromethorphan and bufuralol, respectively. Of 22 novel CYP2D6 variants, six allelic isoforms (CYP2D6.89, CYP2D6.92, CYP2D6.93, CYP2D6.96, E215K and R440C) exhibited absent or extremely reduced metabolic activities compared with those observed for the wild‐type enzyme. Our in vitro functional data can be useful for CYP2D6 phenotype prediction and provide valuable information for the study of clinical impact of these newly found CYP2D6 variants in China.     

The conformational and biological analysis of a cyclic anti‐obesity peptide from the C‐terminal domain of human growth hormone

Abstract: The three‐dimensional solution structure of anti‐obesity drug (AOD), a 15‐residue, disulfide‐bonded, cyclic peptide, cyclo(6,13)‐H₂N‐Leu‐Arg‐Ile‐Val‐Gln‐Cys‐Arg‐Ser‐Val‐Glu‐Gly‐Ser‐Cys‐Gly‐Phe‐OH, derived from the C‐terminal domain of the human growth hormone (hGH) (residues 177–191) was determined using two‐dimensional ¹H NMR spectroscopy. AOD stimulates lipolysis and inhibits lipogenesis, in vitro, in rodent, porcine and human adipose tissues. These biological effects suggest that AOD is a potential therapeutic candidate for the treatment of obesity. Conformational studies of AOD were conducted in aqueous solution and in water/dimethylsulfoxide mixtures. In general, spectral quality was superior in the water/dimethylsulfoxide mixtures. The cyclic region of AOD in water/dimethylsulfoxide adopts type I β‐turns at residues Ser⁸‐Val⁹‐Glu¹⁰‐Gly¹¹ and Ser¹²‐Cys¹³‐Gly¹⁴‐Phe¹⁵, each preceded by loop‐like structures. Comparison of the conformation of this peptide with residues 177–191 in the native hGH protein X‐ray crystal structure indicates that the synthetic peptide retains some structural similarity to the intact protein. This study provides evidence that the C‐terminal region of hGH is a specific functional domain of the multifunctional hGH protein.     

Disulfide pairing and secondary structure of ASP1, an olfactory‐binding protein from honeybee (Apis mellifera L)

Abstract: In insects, the transport of airborne, hydrophobic odorants and pheromones through the sensillum lymph is accomplished by olfactory‐binding proteins (OBPs). We report the structural characterization of a honeybee OBP called ASP1 found in workers and drones, previously observed to bind queen pheromone components. A novel method based on ion‐spray mass spectrometry analysis of cyanylation‐induced cleavage products of partially reduced protein with Tris(2‐carboxyethyl)phosphine was needed to determine the recombinant ASP1 disulfide bond pairing. It was observed to be Cys(I)‐Cys(III), Cys(II)‐Cys(V), Cys(IV)‐Cys(VI), similar to those already described for other OBPs from honeybee and Bombyx mori suggesting that this pattern occurs commonly throughout the diverse family of insect OBPs. Circular dichroism revealed that ASP1 is an all‐alpha protein in accordance with NMR preliminary data, but unlike lipocalin‐like vertebrate OBPs.