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.

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.

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.

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.

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.

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.

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.

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.

Long non-coding RNA H19 inhibition promotes hyperglycemia in mice by upregulating hepatic FoxO1 levels and promoting gluconeogenesis.

In a previous report from our laboratory, it was reported that hepatic levels of the long non-coding RNA (lncRNA), H19 are decreased in diabetic mice which elevates hepatic gluconeogenesis and glucose output. But, the mechanisms of H19 inhibition in elevating gluconeogenic genes' transcription and promoting hepatic glucose output were not known. In this study, we aimed to decipher this regulatory role of H19 on glucose metabolism and on FoxO1, an important transcriptional regulator of gluconeogenesis. While H19 inhibition in HepG2 cells increased the levels of FoxO1, its overexpression led to significant inhibition in FoxO1 levels, thereby identifying H19 as an important regulator of FoxO1. Our data also demonstrates that in the absence of H19, there is increased occupancy of p53 on the FoxO1 promoter that possibly is responsible for increased FoxO1 transcription. In vivo silencing of H19 in normal mice caused hyperglycemia, hyperinsulinemia and impaired glucose, insulin, and pyruvate tolerance. Serum triglyceride and cholesterol levels, however, did not show any change. H19 inhibition significantly elevated the hepatic levels of FoxO1 and all the gluconeogenic genes. While fasting increased gluconeogenic genes' transcription, the levels of H19 were decreased and these patterns reversed upon refeeding the mice. Thus, gluconeogenic genes and H19 levels show inverse patterns of expression, and these results indicate towards an important regulatory role of the lncRNA, H19. It acts as an upstream regulator of gluconeogenesis by regulating the transcription of FoxO1, an important transcription factor of gluconeogenic genes, and hence, regulates hepatic glucose metabolism. KEY MESSAGES: H19 regulates FoxO1 transcript and protein levels. H19 inhibition increases p53 occupancy on the FoxO1 promoter that promotes FoxO1 transcription. H19 inhibition in vivo induces hyperglycemia and impairs glucose, insulin, and pyruvate tolerance. In vivo H19 inhibition increases the hepatic transcript levels of gluconeogenic genes and FoxO1. Transcript levels of H19 and gluconeogenic genes are inversely regulated during fed and fasted states.

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.

RNA sequencing of db/db mice liver identifies lncRNA H19 as a key regulator of gluconeogenesis and hepatic glucose output.

Liver plays a key role in maintaining glucose homeostasis and impaired hepatic glucose metabolism is associated with type 2 diabetes. In the present study, we used RNA sequencing to profile the transcriptome of the livers of diabetic db/db mice as compared to the normal db/+ mice and identified 218 differentially expressed genes. Amongst these, there were 3 lncRNAs that were significantly downregulated and H19 was the most altered lncRNA in the livers of db/db mice. H19 expression significantly correlated with the expression of genes of the glycolysis and gluconeogenesis pathways, which suggest that altered hepatic H19 levels can directly or indirectly modulate their expression. Inhibition of H19 using specific siRNA in HepG2 cells and primary mouse hepatocytes significantly increased the levels of gluconeogenic genes. This was subsequently accompanied by increased hepatic glucose output. Further,H19 depletion in HepG2 cells impaired insulin signaling and increased nuclear localization of FoxO1, an important transcriptional regulator of gluconeogenic gene expression. Our results reveal a novel link between decreased H19 levels and impaired gluconeogenesis via regulation of FoxO1 nuclear levels. These put forth interesting observations on the regulatory role of H19 in altering hepatic physiology during diabetes.

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.

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

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 db/db 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 Cdc20b gene, while being downregulated in the skeletal muscle of diabetic mice, was the most highly upregulated during HDAC inhibition. The host gene, Cdc20b, was also significantly upregulated during HDAC inhibition. Bioinformatics analyses identified a common promoter for both Cdc20b 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 db/db mice skeletal muscle. Further, in SAHA-treated C2C12 cells, we observed augmented occupancy of acetylated histones on the Cdc20b/miR-449a promoter, which possibly promotes their upregulation. In vivo injection of SAHA to db/db 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.

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.

Elevated Hepatic miR-22-3p Expression Impairs Gluconeogenesis by Silencing the Wnt-Responsive Transcription Factor Tcf7.

Levels of miR-22-3p, a highly abundant hepatic microRNA, are abnormally increased in mouse models of insulin resistance and type 2 diabetes, yet its contribution to deregulated hepatic metabolism under diseased states is not well understood. Here, we unravel a novel link between elevated hepatic miR-22-3p expression and impaired gluconeogenesis in diabetic db/db mice via the regulation of Tcf7 (transcription factor 7). Our data demonstrate that miR-22-3p binds to the 3' untranslated region of TCF7 and downregulates it, and this microRNA-mediated regulation of TCF7 increases the expression of enzymes of the gluconeogenic pathway in HepG2 cells. Small interfering RNA-mediated knockdown of TCF7 in HepG2 cells also causes similar upregulation of gluconeogenic genes. Furthermore, in vivo silencing of miR-22-3p by antagomiR administration lowered random as well as fasting glucose levels in diabetic mice. miR-22-3p antagonism improved glucose tolerance and insulin sensitivity. Importantly, the hepatic Tcf7 levels were restored along with reduced hepatic glucose output, which was also reflected by the decreased expression of gluconeogenic genes. Our results support a critical role for miR-22-3p and its target, Tcf7, in the pathogenesis of diabetes by upregulating gluconeogenesis. Moreover, targeting the miR-22/Tcf7/Wnt axis might hold therapeutic potential for the treatment of altered hepatic physiology during insulin resistance and type 2 diabetes.

Transcriptome profiling identifies p53 as a key player during calreticulin deficiency: Implications in lipid accumulation.

Calreticulin (CRT) is an endoplasmic reticulum (ER) resident calcium binding protein that is involved in several cellular activities. Transcriptome analyses in CRT knockdown HepG2 cells revealed 253 altered unique genes and subsequent in silico protein-protein interaction network and MCODE clustering identified 34 significant clusters, of which p53 occupied the central hub node in the highest node-rich cluster. Toward validation, we show that CRT knockdown leads to inhibition of p53 protein levels. Both, CRT and p53 siRNA promote hepatic lipid accumulation and this was accompanied by elevated SREBP-1c and FAS levels. p53 was identified to bind at -219 bp on the SREBP-1c promoter and in the presence of CRT siRNA, there was decreased occupancy of p53 on this binding element. This was associated with increased SREBP-1c promoter activity and both, mutation in this binding site or p53 over-expression antagonised the effects of CRT knockdown. We, therefore, identify a negatively regulating p53 binding site on the SREBP-1c promoter that is critical during hepatic lipid accumulation. These results were validated in mouse primary hepatocytes and toward a physiological relevance, we report that while the levels of CRT and p53 are reduced in the fatty livers of diabetic db/db mice, SREBP-1c levels are significantly elevated. Our results suggest that decreased CRT levels might be involved in the development of a fatty liver by preventing p53 occupancy on the SREBP-1c promoter and thereby facilitating SREBP-1c up-regulation and consequently, lipid accumulation.

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.