Malabika Datta

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.

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‐1β, levels of which are significantly elevated in varied diseases especially diabetes where it is believed to significantly contribute to pancreatic β‐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. J. Cell. Physiol. 227: 1791–1795, 2012.

Inhibition of Insulin Receptor Gene Expression and Insulin Signaling by Fatty Acid: Interplay of PKC Isoforms Therein

Fatty acids are known to play a key role in promoting the loss of insulin sensitivity causing insulin resistance and type 2 diabetes. However, underlying mechanism involved here is still unclear. Incubation of rat skeletal muscle cells with palmitate followed by I125- insulin binding to the plasma membrane receptor preparation demonstrated a two-fold decrease in receptor occupation. In searching the cause for this reduction, we found that palmitate inhibition of insulin receptor (IR) gene expression effecting reduced amount of IR protein in skeletal muscle cells. This was followed by the inhibition of insulin-stimulated IRβ tyrosine phosphorylation that consequently resulted inhibition of insulin receptor substrate 1 (IRS 1) and IRS 1 associated phosphatidylinositol-3 kinase (PI3 Kinase), phosphoinositide dependent kinase-1 (PDK 1) phosphorylation. PDK 1 dependent phosphorylation of PKCζ and Akt/PKB were also inhibited by palmitate. Surprisingly, although PKCΕ phosphorylation is PDK1 dependent, palmitate effected its constitutive phosphorylation independent of PDK1. Time kinetics study showed translocation of palmitate induced phosphorylated PKCΕ from cell membrane to nuclear region and its possible association with the inhibition of IR gene transcription. Our study suggests one of the pathways through which fatty acid can induce insulin resistance in skeletal muscle cell.

Systems Biology Approach Reveals Genome to Phenome Correlation in Type 2 Diabetes

Genome-wide association studies (GWASs) have discovered association of several loci with Type 2 diabetes (T2D), a common complex disease characterized by impaired insulin secretion by pancreatic β cells and insulin signaling in target tissues. However, effect of genetic risk variants on continuous glycemic measures in nondiabetic subjects mainly elucidates perturbation of insulin secretion. Also, the disease associated genes do not clearly converge on functional categories consistent with the known aspects of T2D pathophysiology. We used a systems biology approach to unravel genome to phenome correlation in T2D. We first examined enrichment of pathways in genes identified in T2D GWASs at genome-wide or lower levels of significance. Genes at lower significance threshold showed enrichment of insulin secretion related pathway. Notably, physical and genetic interaction network of these genes showed robust enrichment of insulin signaling and other T2D pathophysiology related pathways including insulin secretion. The network also overrepresented genes reported to interact with insulin secretion and insulin action targeting antidiabetic drugs. The drug interacting genes themselves showed overrepresentation of insulin signaling and other T2D relevant pathways. Next, we generated genome-wide expression profiles of multiple insulin responsive tissues from nondiabetic and diabetic patients. Remarkably, the differentially expressed genes showed significant overlap with the network genes, with the intersection showing enrichment of insulin signaling and other pathways consistent with T2D pathophysiology. Literature search led our genomic, interactomic, transcriptomic and toxicogenomic evidence to converge on TGF-beta signaling, a pathway known to play a crucial role in pancreatic islets development and function, and insulin signaling. Cumulatively, we find that GWAS genes relate directly to insulin secretion and indirectly, through collaborating with other genes, to insulin resistance. This seems to support the epidemiological evidence that environmentally triggered insulin resistance interacts with genetically programmed β cell dysfunction to precipitate diabetes.

Gene expression profiling and pathway analysis identify the integrin signaling pathway to be altered by IL-1β in human pancreatic cancer cells: Role of JNK

The pro-inflammatory cytokine, IL-1β, is a critical component of the persistent inflammatory milieu that pancreatic cancer cells frequently encounter. Although several studies report diverse mechanisms responsible for this association, yet a comprehensive global analysis of the effect of IL-1β in these cells is not clearly evident. In this study, we performed whole genome transcriptome analysis of control and IL-1β treated human pancreatic MIA PaCa-2 cells, validated the most targeted pathway and evaluated the role of JNK therein. 225 Genes were up-regulated and 1215 were down-regulated and these were categorized into biological processes and cellular pathways using the PANTHER classification system. The altered genes categorized into significant biological processes that included those of cell cycle, mitosis, transport and intracellular protein trafficking. The integrin signaling pathway emerged as harboring the maximum number of differentially expressed genes. Two important genes of this pathway, namely vinculin and α5-integrin were validated and both depicted significant inhibition by IL-1β that was prevented in the presence of JNK siRNA. In a wound healing assay, IL-1β increased the migratory rate of MIA PaCa-2 and Panc-1 cells that was abrogated by JNK inhibition. Additionally, vinculin and α-integrin siRNAs also increased the migration of these cells along the wound edge. These results suggest that in these pancreatic cancer cells, IL-1β inhibits components of the integrin signaling pathway in a JNK dependent manner that contributes to their increased migratory potential. Therefore, JNK might be potentially targeted to prevent the migration and invasion of pancreatic cancer cells.

Comprehensive miRNome and in silico analyses identify the Wnt signaling pathway to be altered in the diabetic liver

Aberrant microRNA expression patterns underlie the pathogenesis of diverse diseases, however in a disease as complex as diabetes where the liver exhibits deregulations of normal metabolic processes, the status and role of microRNAs are not yet completely understood. In a step towards unraveling this correlation, we assessed the global microRNA expression profiles in the control and diabetic (db/db) mice liver. These db/db mice were on a C57BLKS/J background and they exhibit diabetic phenotypes that are remarkably similar to those in humans. microRNA microarray profiling revealed 11 miRNAs to be up-regulated and 2 to be down-regulated in the db/db mice liver. Predicted targets of these differentially expressed microRNAs were retrieved from miRanda and TargetScan and the maximum number of commonly predicted targets mapped onto the Wnt signaling pathway that is otherwise conventionally associated with organogenesis and development. Towards validation of this prediction, we found that major components of the Wnt signaling pathway are inhibited in the db/db mice liver. A significant number of these down-regulated genes of the Wnt signaling pathway are predicted targets to the up-regulated miRNAs and specifically our results show that miR-34a and miR-22 decreased the protein levels of their targets. Overexpression of miR-34a and miR-22 and also inhibition of Wnt signaling using specific inhibitors led to increased lipid accumulation in HepG2 cells. Our data suggest that the Wnt signaling pathway could contribute towards the deregulated hepatic behavior in these animals and an altered hepatic miRNA signature could be playing a regulatory role herein.

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.

Tumour necrosis factor-α attenuates insulin action on phosphoenolpyruvate carboxykinase gene expression and gluconeogenesis by altering the cellular localization of Foxa2 in HepG2 cells

Circulating tumour necrosis factor‐α (TNFα) levels, which are elevated in obesity‐associated insulin resistance and diabetes, inhibit insulin signalling at several points in the signalling cascade. The liver is critical in maintaining circulating glucose levels and, in a preliminary investigation using the human hepatoma (HepG2) cell line in this study, we demonstrated the role of TNFα in the regulation of this phenomenon and determined the underlying molecular mechanisms. As the transcription factor Foxa2 has been implicated, in part, in the regulation of gluconeogenic genes, we studied the effects of TNFα and/or insulin on its cellular status in hepatocytes, followed by an assessment of its occupancy on the phosphoenolpyruvate carboxykinase (PEPCK) promoter. Preincubation of cells with TNFα, followed by insulin, significantly prevented insulin‐mediated nuclear exclusion of Foxa2 and substantially increased its nuclear concentration. Foxa2 was subsequently found to occupy its binding element on the PEPCK promoter. TNFα alone, however, did not alter the status of cellular Foxa2 or its occupancy on the PEPCK promoter. TNFα preincubation also significantly attenuated insulin‐induced inhibition of the expression of gluconeogenic enzymes and hepatic glucose production. Insulin inhibition of PEPCK expression and the preventive effect of TNFα could be partially but significantly restored in the presence of Foxa2 siRNA. Several other well‐known mediators of insulin action in the liver in general and of gluconeogenic genes in particular include Foxo1, PGC‐1 and SREBP‐1c. Our results indicate that another transcription factor, Foxa2, is at least partly responsible for the attenuating effect of TNFα on insulin action on PEPCK expression and glucose production in HepG2 cells.

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.

Aeromonas hydrophila induced head kidney macrophage apoptosis in Clarias batrachus involves the activation of calpain and is caspase-3 mediated

The mechanism of macrophage cytotoxicity induced by Aeromonas hydrophila is yet unresolved. We observed A. hydrophila induces Head Kidney Macrophage (HKM) apoptosis in Clarias batrachus, as evident from Hoechst 33342 and AnnexinV-Propidium Iodide staining and presence of oligonucleosomal DNA ladder. Initiation of apoptosis required the bacteria to be alive, be actively phagocytosed into HKM and was dependent on host proteins. Elevated cytosolic calcium and consequent calpain activity that declined following pre-incubation with EGTA, verapamil and nifedipine implicates the role of calcium influx through voltage gated calcium channels and calpain in A. hydrophila-induced HKM apoptosis. Though, calpain-1 and -2 were involved, calpain-2 appeared to be more important in the process. EGTA, verapamil, nifedipine and calpain-2 inhibitor reduced caspase-3 activity and apoptosis. We conclude that A. hydrophila alters cytosolic calcium homeostasis initiating the activation of calpains, more specifically calpain-2, which leads to caspase-3 mediated HKM apoptosis in C. batrachus.