GRP75 mediates endoplasmic reticulum-mitochondria coupling during palmitate-induced pancreatic ß-cell apoptosis.

The endoplasmic reticulum (ER) and mitochondria are structurally connected with each other at specific sites termed mitochondria-associated membranes (MAMs). These physical links are composed of several tethering proteins and are important during varied cellular processes, such as calcium homeostasis, lipid metabolism and transport, membrane biogenesis, and organelle remodeling. However, the attributes of specific tethering proteins in these cellular functions remain debatable. Here, we present data to show that one such tether protein, glucose regulated protein 75 (GRP75), is essential in increasing ER-mitochondria contact during palmitate-induced apoptosis in pancreatic insulinoma cells. We demonstrate that palmitate increased GRP75 levels in mouse and rat pancreatic insulinoma cells as well as in mouse primary islet cells. This was associated with increased mitochondrial Ca2+ transfer, impaired mitochondrial membrane potential, increased ROS production, and enhanced physical coupling between the ER and mitochondria. Interestingly, GRP75 inhibition prevented these palmitate-induced cellular aberrations. Additionally, GRP75 overexpression alone was sufficient to impair mitochondrial membrane potential, increase mitochondrial Ca2+ levels and ROS generation, augment ER-mitochondria contact, and induce apoptosis in these cells. In vivo injection of palmitate induced hyperglycemia and hypertriglyceridemia, as well as impaired glucose and insulin tolerance in mice. These animals also exhibited elevated GRP75 levels accompanied by enhanced apoptosis within the pancreatic islets. Our findings suggest that GRP75 is critical in mediating palmitate-induced ER-mitochondrial interaction leading to apoptosis in pancreatic islet cells.

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

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.

Circulatory miR-98-5p levels are deregulated during diabetes and it inhibits proliferation and promotes apoptosis by targeting PPP1R15B in keratinocytes.

Although deregulated circulatory miRNA signatures during diabetes have been identified for some years now, the effects of such miRNAs on several target tissues are not yet thoroughly investigated. The skin that is nourished by components present in the circulation exhibits several notable abnormal features during diabetes. We, therefore, hypothesized that such altered circulatory miRNA levels might be critical in the onset and progression of impaired skin health during diabetes. RNA sequencing from blood samples of normal and type 2 diabetic human subjects identified 9 upregulated and 19 downregulated miRNAs. miR-98-5p was significantly downregulated and its overexpression down-regulated PPP1R15B levels in HaCaT cells and this was prevented by the miR-98-5p inhibitor. This was validated in human primary epidermal keratinocytes and further supported by a dual reporter luciferase assay of the PPP1R15B 3'UTR where miR-98-5p significantly decreased the luciferase activity which was prevented in the presence of the miRNA inhibitor and by mutation in the miRNA binding site. By targeting PPP1R15B, miR-98-5p increases levels of p-eIF2α, BiP and CHOP. Consequently, there was induction of apoptosis accompanied with decreased proliferation in the presence of miR-98-5p. Conversely, miR-98-5p inhibition alone inhibited apoptosis and promoted proliferation. Taken together, our data suggest that by targeting PPP1R15B, miR-98-5p induces apoptosis and decreases proliferation. As opposed to this since circulatory miR-98-5p levels are decreased in diabetes, we believe that this decrease in the circulation that feeds the skin layers might be a major contributor of hyperproliferation as seen in the skin during diabetes.Abbreviations: miRNAs: MicroRNAs; PPP1R15B: PPP1R15B: Protein Phosphatase 1 Regulatory Subunit 15B; TGFßR1: Transforming Growth Factor Beta Receptor 1; ER: Endoplasmic Reticulum; Bip: Binding Immunoglobulin Protein; Chop: CCAAT-enhancer-binding protein homologous protein; p-eIF2α: Eukaryotic Translation Initiation Factor 2a; Bax: Bcl2-associated X protein; Bcl-2: B-cell CLL/lymphoma 2; PCNA: Proliferating Cell Nuclear Antigen; K5: Cytokeratin 5; qRT-PCR: Quantitative Real-Time PCR; ESCC: Oesophageal squamous cell carcinoma; HCC: Hepatocellular carcinoma; CTHRC1: Collagen triple helix repeat containing 1; SALL4: Sal-like protein 4; TNFα: Tumour Necrosis Factor alpha; PGC-1ß: Peroxisome Profilerator-activated receptor-γ coactivator-1ß; IGF2BP1: Insulin-like growth factor 2 mRNA binding protein 1.

miR-449a regulates insulin signalling by targeting the Notch ligand, Jag1 in skeletal muscle cells.

BACKGROUND: miR-449a, an intronic miRNA, is highly down-regulated in the skeletal muscle during diabetes. Its levels are epigenetically regulated by altered acetylation/deacetylation on the promoter that it shares with its host gene, Cdc20b. However, the cellular role of this epigenetically regulated miRNA in the muscle during diabetes is not well understood. Here, we sought to unravel the crosstalk between altered miR-449a expression and impaired skeletal muscle metabolism. METHODS: Predicted targets of miR-449a were extracted using online available target prediction tools. Differentiated C2C12 cells were transfected with the miR-449a mimic and/or its inhibitor and the levels of the target mRNA and protein was evaluated by qRT-PCR and Western Blot analysis. This was validated by luciferase wild type and mutated constructs of the target 3'UTR. Inhibition of Notch signalling was assessed by evaluating the transcript levels of Notch target genes, Hes1 and Hey1 and the status of NICD (Notch Intracellular domain) by immunofluoresence microscopy. Effect of miR-449a on insulin signalling was evaluated by monitoring insulin induced PI3K and AKT phosphorylation and glucose uptake. RESULTS: Our data demonstrate that in C2C12 skeletal muscle cells, miR-449a binds to the 3'UTR of Jag1, an important Notch ligand, and down-regulates, both its transcript and protein levels. This was, however, prevented in the presence of the miR-449a inhibitor that suggests the specificity of the miRNA effect. This was validated in human primary skeletal muscle cells where miR-449a decreased Jag1 protein levels and this was prevented in the presence of the miR-449a inhibitor. This miR-449a-Jag1 interaction subsequently affects the Notch signalling pathway as was evident by the fact that miR-449a decreased the levels of NICD and consequently, the levels of Notch target genes, Hes1 and Hey1 were significantly inhibited. miR-449a and Notch pathway inhibition using DAPT, significantly increased insulin stimulated PI3K and AKT phosphorylation and these were prevented in the presence of the miR-449a inhibitor. CONCLUSION: Our results indicate towards a critical role for miR-449a and its target, Jag1 in regulating Notch signalling and insulin signalling in the skeletal muscle and imply that targeting this axis might hold therapeutic potential for impaired skeletal muscle metabolism during diabetes.

Lnc-ing non-coding RNAs with metabolism and diabetes: roles of lncRNAs.

Type 2 diabetes is a complex metabolic disorder characterized by insulin resistance and pancreatic ß-cell dysfunction. Deregulated glucose and lipid metabolism are the primary underlying manifestations associated with this disease and its complications. Long non-coding RNAs (lncRNAs) are a novel class of functional RNAs that regulate a variety of biological processes by a diverse interplay of mechanisms including recruitment of epigenetic modifiers, transcriptional and post-transcriptional regulation, control of mRNA decay, and sequestration of transcription factors. Although the underlying causes that define the diabetic phenotype are extremely intricate, most of the studies in the last decades were mostly centered on protein-coding genes. However, current opinion in the recent past has authenticated the contributions of diverse lncRNAs as critical regulatory players during the manifestation of diabetes. The current review will highlight the importance of lncRNAs in regulating cellular processes that govern metabolic homeostasis in key metabolic tissues. A more in-depth understanding of lncRNAs may enable their exploitation as biomarkers or for therapeutic applications during diabetes and its associated complications.

Interplay between the miRNome and the epigenetic machinery: Implications in health and disease.

Epigenetics refers to functionally relevant genomic changes that do not involve changes in the basic nucleotide sequence. Majorly, these are of two types: DNA methylation and histone modifications. Small RNA molecules called miRNAs are often thought to mediate post-transcriptional epigenetic changes by mRNA degradation or translational attenuation. While DNA methylation and histone modifications have their own independent effects on various cellular events, several reports are suggestive of an obvious interplay between these phenomena and the miRNA regulatory program within the cell. Several miRNAs like miR-375, members of miR-29 family, miR-34, miR-200, and others are regulated by DNA methylation and histone modifications in various types of cancers and metabolic diseases. On the other hand, miRNAs like miR-449a, miR-148, miR-101, miR-214, and miR-128 target members of the epigenetic machinery and their dysregulation leads to diverse cellular aberrations. In spite of being independent cellular events, emergence of such reports that suggest a connection between DNA methylation, histone modification, and miRNA function in several diseases indicate that this connecting axis offers a valuable target with great therapeutic potential that might be exploited for disease management. We review the current status of crosstalk between the major epigenetic modifications and the miRNA machinery and discuss this in the context of health and disease.

Downregulation of miRNAs during Delayed Wound Healing in Diabetes: Role of Dicer.

Delayed wound healing is a major complication associated with diabetes and is a result of a complex interplay among diverse deregulated cellular parameters. Although several genes and pathways have been identified to be mediating impaired wound closure, the role of microRNAs (miRNAs) in these events is not very well understood. Here, we identify an altered miRNA signature in the prolonged inflammatory phase in a wound during diabetes, with increased infiltration of inflammatory cells in the basal layer of the epidermis. Nineteen miRNAs were downregulated in diabetic rat wounds (as compared with normal rat wound, d 7 postwounding) together with inhibited levels of the central miRNA biosynthesis enzyme, Dicer, suggesting that in wounds of diabetic rats, the decreased levels of Dicer are presumably responsible for miRNA downregulation. Compared with unwounded skin, Dicer levels were significantly upregulated 12 d postwounding in normal rats, and this result was notably absent in diabetic rats that showed impaired wound closure. In a wound-healing specific quantitative reverse transcriptase-polymerase chain reaction (RT-PCR) array, 10 genes were significantly altered in the diabetic rat wound and included growth factors and collagens. Network analyses demonstrated significant interactions and correlations between the miRNA predicted targets (regulators) and the 10 wound-healing specific genes, suggesting altered miRNAs might fine-tune the levels of these genes that determine wound closure. Dicer inhibition prevented HaCaT cell migration and affected wound closure. Altered levels of Dicer and miRNAs are critical during delayed wound closure and offer promising targets to address the issue of impaired wound healing.

Role of calmodulin-calmodulin kinase II, cAMP/protein kinase A and ERK 1/2 on Aeromonas hydrophila-induced apoptosis of head kidney macrophages.

The role of calcium (Ca2+) and its dependent protease calpain in Aeromonas hydrophila-induced head kidney macrophage (HKM) apoptosis has been reported. Here, we report the pro-apoptotic involvement of calmodulin (CaM) and calmodulin kinase II gamma (CaMKIIg) in the process. We observed significant increase in CaM levels in A. hydrophila-infected HKM and the inhibitory role of BAPTA/AM, EGTA, nifedipine and verapamil suggested CaM elevation to be Ca2+-dependent. Our studies with CaM-specific siRNA and the CaM inhibitor calmidazolium chloride demonstrated CaM to be pro-apoptotic that initiated the downstream expression of CaMKIIg. Using the CaMKIIg-targeted siRNA, specific inhibitor KN-93 and its inactive structural analogue KN-92 we report CaM-CaMKIIg signalling to be critical for apoptosis of A. hydrophila-infected HKM. Inhibitor studies further suggested the role of calpain-2 in CaMKIIg expression. CaMK Kinase (CaMKK), the other CaM dependent kinase exhibited no role in A. hydrophila-induced HKM apoptosis. We report increased production of intracellular cAMP in infected HKM and our results with KN-93 or KN-92 implicate the role of CaMKIIg in cAMP production. Using siRNA to PKACA, the catalytic subunit of PKA, anti-PKACA antibody and H-89, the specific inhibitor for PKA we prove the pro-apoptotic involvement of cAMP/PKA pathway in the pathogenicity of A. hydrophila. Our inhibitor studies coupled with siRNA approach further implicated the role of cAMP/PKA in activation of extracellular signal-regulated kinase 1 and 2 (ERK 1/2). We conclude that the alteration in intracellular Ca2+ levels initiated by A. hydrophila activates CaM and calpain-2; both pathways converge on CaMKIIg which in turn induces cAMP/PKA mediated ERK 1/2 phosphorylation leading to caspase-3 mediated apoptosis of infected HKM.

miR-107 orchestrates ER stress induction and lipid accumulation by post-transcriptional regulation of fatty acid synthase in hepatocytes.

MicroRNAs, a class of small non-coding RNAs, are believed to regulate several biological pathways and processes and are implicated in several diseases. They mostly regulate the levels of their target genes at the post transcriptional stage by primarily binding to the 3' UTR. Elevated hepatic levels of miR-107 are a consistent feature associated with several obese and diabetic models. Here, we show that miR-107 post-transcriptionally regulates fatty acid synthase (FASN) by binding to its 3' UTR and reduces its protein levels and the 3'UTR luciferase reporter activity, which are blunted by the miR-107 inhibitor and mutation in the miR-107 binding site in the 3' UTR. Knock-down of endogenous miR-107 levels increased FASN levels in a dose-dependent manner. Overexpression of miR-107 led to significant accumulation of malonyl CoA, accompanied by ER stress induction. All these events were prevented in the presence of the miR-107 inhibitor. While overexpression of FASN could attenuate miR-107 mediated ER stress markers' induction; the ER stress inhibitor, 4-phenyl-butyric acid did not rescue miR-107 induced FASN inhibition. This was followed by increased triglyceride formation and lipid accumulation in the presence of miR-107. These indicate that miR-107 inhibits FASN levels by binding to its 3' UTR and this interaction promotes ER stress induction and malonyl CoA and lipid accumulation in HepG2 cells and primary hepatocytes. Our results suggest that increased levels of miR-107 are critical in promoting lipid accumulation in hepatocytes and this might form the basis of diverse etiologies encountered in a fatty liver.

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

Although 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.

LncRNA H19 inhibition impairs endoplasmic reticulum-mitochondria contact in hepatic cells and augments gluconeogenesis by increasing VDAC1 levels.

Inspite of exerting independent cellular functions, the endoplasmic-reticulum (ER) and the mitochondria also physically connect at specific sites termed mitochondria-associated ER membranes (MAMs) and these sites consist of several tethering proteins that play varied roles in diverse cellular processes. However, the regulation of these tethering proteins within the cell is relatively less studied. Here, we show that several MAM proteins are significantly altered in the liver during diabetes and among these, the lncRNA, H19 regulates the levels of VDAC1. Inhibition of H19 expression using H19 specific siRNA altered VDAC1, mitochondrial Ca2+ and oxygen consumption rate, ATP and ROS levels and enhanced ER and mitochondria coupling in Hepa 1-6 cells. While H19 inhibition did not impact lipid accumulation, levels of gluconeogenic genes were significantly increased. JNK-phosphorylation and IRS1-Ser307-phosphorylation were increased by H19 inhibition and this was associated with abrogation of insulin-stimulated AKT (Ser-473) phosphorylation and glucose uptake in Hepa 1-6 cells. While inhibition of VDAC1 expression using siRNAs and with metformin significantly rescued the effects of H19 inhibition, VDAC1 overexpression alone exerted effects similar to H19 inhibition, suggesting that VDAC1 increase mediates the adverse effects of H19. In-vivo H19 inhibition using specific siRNAs increased hepatic VDAC1, pJNK and pIRS1 (Ser307) levels and decreased AKT (Ser-473) phosphorylation in mice. These suggest an important role of the H19-VDAC1 axis in ER-mitochondria coupling and regulation of gluconeogenesis in the liver during diabetes.

mir-98-5p regulates gluconeogenesis and lipogenesis by targeting PPP1R15B in hepatocytes.

Several reports suggest that circulatory miRNAs are deregulated in diverse diseases and used as markers for disease diagnosis and prognosis. Here we show that miR-98-5p, that is down-regulated in the circulation during diabetes, regulates hepatic gluconeogenesis and lipogenesis by targeting PPP1R15B. miR-98-5p overexpression significantly decreased the transcript and protein levels of PPP1R15B in hepatic HepG2 cells and increased p-eIF2α expression and these were prevented in the presence of its inhibitor. Two major hepatic hallmarks during diabetes i.e. hepatic lipid accumulation and glucose output were explored towards physiological relevance. As compared to scramble, overexpression of miR-98-5p decreased the transcript levels of both gluconeogenic and lipogenic genes together with a significant reduction in hepatic glucose production and fat accumulation in HepG2 cells. Using PASTAA to detect common transcription factors regulating these altered genes, CREB emerged as the most significantly enriched transcription factor. While miR-98-5p overexpression did not change the transcript levels of CREB, there was a significant change in its protein levels. While similar effects on gluconeogenic and lipogenic gene expression were detected using the PPP1R15B siRNA, the opposite was observed in the presence of miR-98-5p inhibitor alone. All these suggest that by targeting PPP1R15B, miR-98-5p regulates hepatic steatosis and glucose output; deregulation of which are characteristic hepatic features during diabetes. Therapeutic intervention of the miR-98/PPP1R15B axis might offer a potential strategy to target aberrant hepatic metabolism during diabetes.

The critical role of JNK in the ER-mitochondrial crosstalk during apoptotic cell death.

Apoptosis or programmed cell death is an extremely coordinated phenomenon that involves the participation of a complex interacting crosstalk between the endoplasmic reticulum and mitochondria. This involves a series of signaling molecules like stress kinases, caspases, Bcl-2 family of proteins, etc. that coordinately induce apoptosis by releasing apoptotic proteins from the mitochondria and mediate DNA damage of the cell. Among the stress kinases, JNK, a member of the MAPK family has been believed to be critically mediating these apoptotic phenomena. The involvement of JNK has been clouded by controversies because of its role both as a pro-apoptotic and an anti-apoptotic mediator. A very significant initiator of JNK activation is the pro-inflammatory cytokine, IL-1ß, levels of which are significantly elevated in varied diseases especially diabetes where it is believed to significantly contribute to pancreatic ß-cell death. During apoptotic cell death, the endoplasmic reticulum and the mitochondrion participate in a relay of cellular events that determine the onset of the classical apoptotic pathways. Here we discuss the details of this ER-mitochondrial crosstalk and the role of JNK herein that ultimately culminates into apoptotic cell death that is evident in various pathophysiological conditions.

miR-539-5p regulates Srebf1 transcription in the skeletal muscle of diabetic mice by targeting DNA methyltransferase 3b.

Aberrant DNA methylation is associated with diabetes, but the precise regulatory events that control the levels and activity of DNA methyltransferases (DNMTs) is not well understood. Here we show that miR-539-5p targets Dnmt3b and regulates its cellular levels. miR-539-5p and Dnmt3b show inverse patterns of expression in skeletal muscle of diabetic mice. By binding to the 3' UTR of Dnmt3b, miR-539-5p downregulates its levels in C2C12 cells and in human primary skeletal muscle cells. miR-539-5p-Dnmt3b interaction regulates Srebf1 transcription by altering methylation at CpG islands within Srebf1 in C2C12 cells. Dnmt3b inhibition alone was sufficient to upregulate Srebf1 transcription. In vivo antagonism of miR-539-5p in normal mice induced hyperglycemia and hyperinsulinemia and impaired oral glucose tolerance. These mice had elevated Dnmt3b and decreased Srebf1 levels in skeletal muscle. db/db mice injected with miR-539-5p mimics showed improved circulatory glucose and cholesterol levels. Oral glucose tolerance improved together with normalization of Dnmt3b and Srebf1 levels in skeletal muscle. Our results support a critical role of miR-539-5p and Dnmt3b in aberrant skeletal muscle metabolism during diabetes by regulating Srebf1 transcription; modulating the miR-539-5p-Dnmt3b axis might have therapeutic potential for addressing altered skeletal muscle physiology during insulin resistance and type 2 diabetes.

Identification of perturbed pathways rendering susceptibility to tuberculosis in type 2 diabetes mellitus patients using BioNSi simulation of integrated networks of implicated human genes.

In type 2 diabetes mellitus (T2DM) patients, chronic hyperglycemia and inflammation underlie susceptibility to tuberculosis (TB) and result in poor TB control. Here, an integrative pathway-based approach is used to investigate perturbed pathways in T2DM patients that render susceptibility to TB. We obtained 36 genes implicated in type 2 diabetes-associated tuberculosis (T2DMTB) from the literature. Gene expression analysis on T2DM patient data (GSE26168) showed that DEFA1 is differentially expressed at Padj <0.05. The human host TB susceptibility genes TNFRSF10A, MSRA, GPR148, SLC37A3, PXK, PROK2, REV3L, PGM1, HIST3H2A, PLAC4, LETM2, and EMP2 and hsa-miR-146a microRNA were also differentially expressed at Padj <0.05. We included all these genes and added the remaining 28 genes from the T2DMTB set and the remaining differentially expressed genes at Padj <0.05 in STRING and obtained a well-connected network with high confidence score (≥0.7). Further, we extracted the KEGG pathways at FDR <0.05 and retained only the diabetes and TB pathways. The network was simulated with BioNSi using gene expression data. It is evident from BioNSi analysis that the NF-kappa B and Toll-like receptor pathways are commonly perturbed with high ranking in multiple gene expression datasets of type 2 diabetes versus healthy controls. The other pathways, necroptosis pathway and FoxO signalling pathway, appear perturbed with high ranking in different gene expression datasets. These pathways likely underlie susceptibility to TB in T2DM patients.

LncRNAs in cancer: Regulatory and therapeutic implications.

Long noncoding RNAs (lncRNAs) comprise a class of RNAs that do not code for proteins but are critical in regulating diverse cellular processes and maintaining cell function. In doing so, they have, in recent years, added a potentially new and significant layer of biological regulation. These are more than 200 nucleotides in length and are implicated in a range of diseases and therefore have emerged as potential tools for possible therapeutic intervention. For a disease as complex as cancer, emerging technologies suggest the presence of mutations on genomic loci that do not encode proteins, but give rise to lncRNAs. Aberrant signatures of lncRNAs are now a consistent feature of almost all types of cancers and their associated complications. Analysis and characterisation of functional pathways that lncRNAs are involved with suggest that lncRNAs interact with the chromatin, the protein or with the RNA to demonstrate their cellular effects to modulate proliferation, migration, differentiation, apoptosis and cell death. This review summarizes the current knowledge of lncRNAs, their implications in diverse types of cancer and their possible therapeutic utility.

RNA sequencing reveals potential interacting networks between the altered transcriptome and ncRNome in the skeletal muscle of diabetic mice.

For a global epidemic like Type 2 diabetes mellitus (T2DM), while impaired gene regulation is identified as a primary cause of aberrant cellular physiology; in the past few years, non-coding RNAs (ncRNAs) have emerged as important regulators of cellular metabolism. However, there are no reports of comprehensive in-depth cross-talk between these regulatory elements and the potential consequences in the skeletal muscle during diabetes. Here, using RNA sequencing, we identified 465 mRNAs and 12 long non-coding RNAs (lncRNAs), to be differentially regulated in the skeletal muscle of diabetic mice and pathway enrichment analysis of these altered transcripts revealed pathways of insulin, FOXO and AMP-activated protein kinase (AMPK) signaling to be majorly over-represented. Construction of networks showed that these pathways significantly interact with each other that might underlie aberrant skeletal muscle metabolism during diabetes. Gene-gene interaction network depicted strong interactions among several differentially expressed genes (DEGs) namely, Prkab2, Irs1, Pfkfb3, Socs2 etc. Seven altered lncRNAs depicted multiple interactions with the altered transcripts, suggesting possible regulatory roles of these lncRNAs. Inverse patterns of expression were observed between several of the deregulated microRNAs (miRNAs) and the differentially expressed transcripts in the tissues. Towards validation, overexpression of miR-381-3p and miR-539-5p in skeletal muscle C2C12 cells significantly decreased the transcript levels of their targets, Nfkbia, Pik3r1 and Pi3kr1, Cdkn2d, respectively. Collectively, the findings provide a comprehensive understanding of the interactions and cross-talk between the ncRNome and transcriptome in the skeletal muscle during diabetes and put forth potential therapeutic options for improving insulin sensitivity.

IL-1beta induces ER stress in a JNK dependent manner that determines cell death in human pancreatic epithelial MIA PaCa-2 cells.

The proinflammatory cytokine, IL-1beta (Interleukin-1beta) is a significant determinant of pancreatic apoptosis and cell death that are common characteristics during diabetes. Using human derived pancreatic MIA PaCa-2 cells, we describe one of the underlying molecular mechanisms behind this observation. Incubation of these cells with IL-1beta at doses from 0.5 to 3.0 ng/ml caused significant cell death at 36 h. This was accompanied with marked increases in JNK and p38 phosphorylation together with increased levels of the endoplasmic reticulum (ER) stress markers, namely BiP, CHOP, GADD34, ATF4 and sXBP1. IL-1beta also led to increased phosphorylation of eIF2alpha and all these events could be prevented by pretreatment with the JNK inhibitor, SP600125. A time course study indicated that while IL-1beta mediated JNK phosphorylation was induced as early as 2 h of IL-1beta treatment, induction of the ER stress markers was evident at later time points. IL-1beta stimulated JNK phosphorylation was observed even in the presence of the ER stress inhibitor, 4-phenyl butyrate and the decrease in cell viability was significantly prevented in the presence of the JNK inhibitor. All these suggest that JNK activation is a pre-requisite for ER stress induction and cell death. Reports till date have consistently demonstrated JNK activation as a consequence of ER stress induction by IL-1beta in the pancreas. We show here for the first time that the activation of JNK by IL-1beta is a prelude to the subsequent induction of ER stress and cell death. These therefore suggest that the JNK-ER stress axis is critical in deciding the decreased survival status by IL-1beta in MIA PaCa-2 cells.

MicroRNAs in diabetes: tiny players in big disease.

MicroRNAs (miRNAs) are a novel group of universally present small non-coding RNAs that have been implicated in wide ranging physiological processes and thereby are critical in the manifestation of diverse diseases. Since their discovery as developmental regulators in C.elegans, they have come a long way and are currently associated with normal and diverse pathophysiological states including Parkinson's syndrome, cardiac hypertrophy, viral infection, diabetes and several types of cancer. Of special significance is their involvement in diabetes, an area in which several emerging reports point to the fact that these small RNA species could be special and critical in this complex disease and they or their specific inhibitors may be exploited as targets for therapeutic intervention. The stable nature of these miRNAs over mRNAs is an added advantage of them being projected for the same. This review focuses on and discusses the current diabetic epidemic and the potential role(s) of these miRNAs in various physiological processes that lead to the diabetic phenotype with an objective of better understanding the emerging mechanisms of these small molecules in the development and progression of diabetes and its complications.