Gene Expression Profiling and Network Analysis Reveals Lipid and Steroid Metabolism to Be the Most Favored by TNFα in HepG2 Cells

BACKGROUND: The proinflammatory cytokine, TNFalpha, is a crucial mediator of the pathogenesis of several diseases, more so in cases involving the liver wherein it is critical in maintaining liver homeostasis since it is a major determiner of hepatocyte life and death. Gene expression profiling serves as an appropriate strategy to unravel the underlying signatures to envisage such varied responses and considering this, gene transcription profiling was examined in control and TNFalpha treated HepG2 cells. METHODS AND FINDINGS: Microarray experiments between control and TNFalpha treated HepG2 cells indicated that TNFalpha could significantly alter the expression profiling of 140 genes; among those up-regulated, several GO (Gene Ontology) terms related to lipid and fat metabolism were significantly (p<0.01) overrepresented indicating a global preference of fat metabolism within the hepatocyte and those within the down-regulated dataset included genes involved in several aspects of the immune response like immunoglobulin receptor activity and IgE binding thereby indicating a compromise in the immune defense mechanism(s). Conserved transcription factor binding sites were identified in identically clustered genes within a common GO term and SREBP-1 and FOXJ2 depicted increased occupation of their respective binding elements in the presence of TNFalpha. The interacting network of "lipid metabolism, small molecule biochemistry" was derived to be significantly overrepresented that correlated well with the top canonical pathway of "biosynthesis of steroids". CONCLUSIONS: TNFalpha alters the transcriptome profiling within HepG2 cells with an interesting catalog of genes being affected and those involved in lipid and steroid metabolism to be the most favored. This study represents a composite analysis of the effects of TNFalpha in HepG2 cells that encompasses the altered transcriptome profiling, the functional analysis of the up- and down- regulated genes and the identification of conserved transcription factor binding sites. These could possibly determine TNFalpha mediated alterations mainly the phenotypes of hepatic steatosis and fatty liver associated with several hepatic pathological states

miR-135a targets IRS2 and regulates insulin signaling and glucose uptake in the diabetic gastrocnemius skeletal muscle

AbstractAlthough aberrant miRNA signatures are associated with diabetes, yet, the status and role of altered miRNAs in the diabetic skeletal muscle is currently poorly understood. Here, we report that 41 miRNAs are altered in the diabetic gastrocnemius skeletal muscle and of these, miR-135a that is identified as a critical regulator of myogenesis, is significantly up-regulated. IRS2 is predicted as its potential putative target and its levels are down-regulated in the diabetic gastrocnemius skeletal muscle. In C2C12 cells, while miR-135a levels decreased during differentiation, IRS2 levels were up-regulated. miR-135a significantly reduced IRS2 protein levels and its 3′UTR luciferase reporter activity and these were blunted by the miR-135a inhibitor and mutation in the miR-135a binding site. Knock-down of endogenous miR-135a levels increased IRS2 at the mRNA and protein levels. miR-135a also attenuated insulin stimulated phosphorylation and activation of PI3Kp85α and Akt and glucose uptake. miR-135a levels were also found to be elevated in the human diabetic skeletal muscle. In-vivo silencing of miR-135a alleviated hyperglycemia, improved glucose tolerance and significantly restored the levels of IRS2 and p-Akt in the gastrocnemius skeletal muscle of db/db mice without any effect on their hepatic levels. These suggest that miR-135a targets IRS2 levels by binding to its 3′UTR and this interaction regulates skeletal muscle insulin signaling

H19 inhibition increases HDAC6 and regulates IRS1 levels and insulin signaling in the skeletal muscle during diabetes

Abstract Background Histone deacetylases (HDACs) that catalyze removal of acetyl groups from histone proteins, are strongly associated with several diseases including diabetes, yet the precise regulatory events that control the levels and activity of the HDACs are not yet well elucidated. Methods Levels of H19 and HDACs were evaluated in skeletal muscles of normal and diabetic db/db mice by Western Blot analysis. C2C12 cells were differentiated and transfected with either the scramble or H19 siRNA and the levels of HDACs and Prkab2, Pfkfb3, Srebf1, Socs2, Irs1 and Ppp2r5b were assessed by Western Blot analysis and qRT-PCR, respectively. Levels of H9, HDAC6 and IRS1 were evaluated in skeletal muscles of scramble/ H19 siRNA injected mice and chow/HFD-fed mice. Results Our data show that the lncRNA H19 and HDAC6 exhibit inverse patterns of expression in the skeletal muscle of diabetic db/db mice and in C2C12 cells, H19 inhibition led to significant increase in HDAC activity and in the levels of HDAC6, both at the transcript and protein levels. This was associated with downregulation of IRS1 levels that were prevented in the presence of the HDAC inhibitor, SAHA, and HDAC6 siRNA suggesting the lncRNA H19-HDAC6 axis possibly regulates cellular IRS1 levels. Such patterns of H19, HDAC6 and IRS1 expression were also validated and confirmed in high fat diet-fed mice where as compared to normal chow-fed mice, H19 levels were significantly inhibited in the skeletal muscle of these mice and this was accompanied with elevated HDAC6 levels and decreased IRS1 levels. In-vivo inhibition of H19 led to significant increase in HDAC6 levels and this was associated with a decrease in IRS1 levels in the skeletal muscle. Conclusions Our results suggest a critical role for the lncRNA H19-HDAC6 axis in regulating IRS1 levels in the skeletal muscle during diabetes and therefore restoring normal H19 levels might hold a therapeutic potential for the management of aberrant skeletal muscle physiology during insulin resistance and type 2 diabetes

Gene Expression Profiling of the Hepatic Transcriptome in Presence of TNF-alpha

Diabetes mellitus, often simply termed Diabetes, is a syndrome characterized by disordered metabolism and high blood sugar. It is caused due to low levels of insulin hormone or from abnormal resistance to insulin in its target tissues. World Health Organization estimates that India will alone have 79.4 million diabetic patients in 2030. One of its major form Type 2 diabetes, is often associated with obesity, hypertension, elevated cholesterol and metabolic syndrome. Changes in life style, such as consumption of high-calorie diet and lack of exercise, have increased the global prevalence not only of diabetes but also of obesity. Type 2 diabetes is characterized by insulin resistance in target tissue, occurs due to several reasons and one of them being the proinflammatory cytokine, TNF-&#x3b1;. It is also known as the link between diabetes and obesity. High levels of TNF-&#x3b1; interfere with insulin signaling to cause the effect and to further investigate into the situation, gene transcription profiling was examined in control and TNF-&#x3b1; treated HepG2 cells. Results indicated that TNF-&#x3b1; could significantly alter the expression of a significant number of genes that were identified to be related to lipid and fat metabolism on one hand and to immunoglobulin receptor activity and IgE binding thereby on the other thereby indicating global dysregulation of fat metabolism and compromise in immune defense mechanism(s) within the hepatocyte by TNF-&#x3b1;. Pathway analysis revealed &#x201c;biosynthesis of steroids&#x201d; to be most effected. All these indicate TNF-&#x3b1; to be significantly altering the transcriptome profiling within HepG2 cells with genes involved in lipid and steroid metabolism being the most favoured and this could explain one of the underlying mechanisms of TNF-&#x3b1; action in the liver. &#xd;&#xa;&#xd;&#xa

Differential regulation of leydig cell 3&#946;-hydroxysteroid dehydrogenase/&#916;<SUP>5</SUP>-&#916;<SUP>4</SUP>-isomerase activity by gonadotropin and thyroid hormone in a freshwater perch, Anabas testudineus (Bloch)

Leydig cells were isolated from the perch testes belonging to the pre-spawning stage by collagenase treatment and mechanical separation followed by percoll gradient. They were incubated in vitro either for 5 h or at different times in the absence (control) or presence of piscine gonadotropin (GTH, 2 &#956;g (1&#215;106 cells)-1) or 3,5,3'-triiodothyronine (T3, 50 ng (1&#215;106 cells)-1) or T3-induced protein (TIP, 2 &#956;g (1&#215;106 cells)-1). 3&#946;-hydroxysteroid dehydrogenase/&#916;5-&#916;4-isomerase (3&#946;HSD) activity was determined by the conversion of [3H]&#916;5-dehydroepiandrosterone (DHEA) to [3H]&#916;4-androstenedione or [3H]&#916;5-pregnenolone to [3H]&#916;4-progesterone (P4) or by spectrophotometric estimation of NADH formation from NAD. T3 significantly increased (P&lt;0.01) both &#916;5-DHEA to &#916;4-androstenedione and &#916;5-pregnenolone to &#916;4-P4 conversion in Leydig cells indicating stimulation of 3&#946;-HSD activity. T3 stimulation of 3&#946;-HSD activity could be inhibited by cycloheximide (50 &#956;g ml-1) suggesting the involvement of T3-induced protein (TIP) which was isolated and purified earlier in this laboratory from goat Leydig cells [15]. Addition of TIP or GTH significantly stimulated Leydig cell 3&#946;-HSD activity (P&lt;0.01). However, there was a difference between TIP and GTH stimulation in time kinetic study where TIP enhanced 3&#946;-HSD activity at 1 h (P&lt;0.05), reached its peak at 3 h (P&lt;0.01) and then plateaued till 8 h. GTH, on the other hand, did not show any stimulation of 3&#946;-HSD activity for 2 h, stimulation was marked only at 3 h (P&lt;0.05), reached a peak at 6 h (P&lt;0.01) and then leveled off. Determination of Km and Vmax of the enzyme showed an increase in the velocity of reaction by GTH with unaltered Km, TIP increased both velocity and affinity of the enzyme. GTH significantly increased the synthesis of 3&#946;-HSD protein at 3 h (P&lt;0.01) reaching maximal stimulation at 6 h which clearly coincided with the enzyme activity. In contrast, TIP had no effect on 3&#946;-HSD protein synthesis, but its direct addition to 3&#946;-HSD enzyme preparation in vitro caused significant augmentation of the enzyme activity (P&lt;0.01) suggesting thereby its modulatory effect on the enzyme. Results, therefore, show that although both T3 and GTH stimulated perch testicular Leydig cell 3&#946;-HSD activity, T3 effect was not direct but mediated via TIP and there is a clear distinction between GTH and TIP stimulation. GTH increased the enzyme activity by stimulating 3&#946;-HSD protein synthesis while TIP acts directly on the enzyme modulating it from less active to more active state

Biotechnology Input in Fish Breeding

29-38Pisciculture, to increase the fish production, has a crucial limitation. Fish cultured in land-locked water bodies usually do not breed without the hormonal induction. Technology of fish breeding suffered for a long time due to the lack of suitable commercial product to induce the spawning of economically important fish. Hypophysation technique, where pituitary extract was used to induce the breeding, faced more failures than success besides the limitation of the source. After the discovery of a brain peptide, gonadotropin releasing hormone (GnRH), which binds to the membrane receptor of pituitary gonadotroph cell and releases gonadotropic hormone (GTH), the situation has changed dramatically. Final maturation and release of germ cells, spermatozoa and oocyte, to the water for fertilization, depends on an acute surge of GTH which is about 10 times greater than the normal circulatory level of GTH. Injection of GnRH causes this acute GTH surge. GnRH is a decapeptide but its gene encodes 92 amino acid containing large molecule. Extensive post-translational processing is necessary to secrete GnRH from the neural cells. For this reason recombinant DNA technology could not be employed for the production of GnRH. Instead, chemical synthesis of this decapeptide is easier and cheaper. Numbers of fish GnRH structure have been elucidated and depending on salmon GnRH peptide sequence, a chemical analogue has now been marketed under the the name of "Ovaprim". There is a strong research background to understand GnRH mechanism of action and signal transduction pathway involved in GnRH mediated GTH function on germ cell maturation and release. These scientific investigations have contributed significantly in designing the superactive GnRH analogues. The only GnRH available in India is from the brain of a freshwater murrel, Channa punctatus. Combination of two murrel GnRH variants, GnRH I and GnRH II, produces far more superior effects than "Ovaprim". Biotechnology input in fish breeding is no doubt highly appreciable as it provides the cultivators almost a riskless method. However, research in different laboratories is still in progress to have a more potent molecule with the possible addition of some metabolic hormones. Pisciculture is now a booming industry all over the world, which imposes a larger demand for "Ova prim" like products. Murrel GnRH, therefore is expected to be a highly competitive product in the global market

Crystallization and preliminary X-ray analysis of cyclophilin from Leishmania donovani

Cyclophilin from the parasite Leishmania donovani is a protein with peptidylprolyl cis±trans isomerase activity, in addition to being a receptor for the drug cyclosporin. Crystals of the enzyme have been obtained in space group P43212, with unit-cell parameters a = b = 48.73, c = 140.93 A Ê , and diffract to 3.5 A Ê resolution. One molecule per asymmetric unit gives a solvent content and Matthews coef®cient of 46% and 2.3 A Ê 3 Da ÿ1, respectively. Molecular-replacement calculations with human cyclophilin A as the search model give an unambiguous solution in rotation and translation functions

Histone deacetylase inhibition regulates miR-449a levels in skeletal muscle cells

<p>microRNAs (miRNAs) are small non-coding RNAs that regulate cellular processes by fine-tuning the levels of their target mRNAs. However, the regulatory elements determining cellular miRNA levels are not well studied. Previously, we had described an altered miRNA signature in the skeletal muscle of <i>db/db</i> mice. Here, we sought to explore the role of epigenetic mechanisms in altering these miRNAs. We show that histone deacetylase (HDAC) protein levels and activity are upregulated in the skeletal muscle of diabetic mice. In C2C12 cells, HDAC inhibition using suberoylanilide hydroxamic acid (SAHA) altered the levels of 24 miRNAs: 15 were downregulated and 9 were upregulated. miR-449a, an intronic miRNA localized within the <i>Cdc20b</i> gene, while being downregulated in the skeletal muscle of diabetic mice, was the most highly upregulated during HDAC inhibition. The host gene, <i>Cdc20b</i>, was also significantly upregulated during HDAC inhibition. Bioinformatics analyses identified a common promoter for both <i>Cdc20b</i> and miR-449a that harbors significant histone acetylation marks, suggesting the possibility of regulation by histone acetylation-deacetylation. These observations suggest an inverse correlation between miR-449a levels and HDAC activity, in both SAHA-treated skeletal muscle cells and <i>db/db</i> mice skeletal muscle. Further, in SAHA-treated C2C12 cells, we observed augmented occupancy of acetylated histones on the <i>Cdc20b</i>/miR-449a promoter, which possibly promotes their upregulation. <i>In vivo</i> injection of SAHA to <i>db/db</i> mice significantly restored skeletal muscle miR-449a levels. Our results provide insights into the potential regulatory role of epigenetic histone acetylation of the miR-449a promoter that may regulate its expression in the diabetic skeletal muscle.</p