Circulating long noncoding RNA GAS5 levels are correlated to prevalence of type 2 diabetes mellitus

AbstractBackgroundDiabetes mellitus (DM), a metabolic disease, is characterized by impaired fasting glucose levels. Type 2 DM is adult onset diabetes. Long non-coding RNAs (lncRNAs) regulate gene expression and multiple studies have linked lncRNAs to human diseases.MethodsSerum samples obtained from 96 participating veterans at JAH VA were deposited in the Research Biospecimen Repository. We used a two-stage strategy to identify an lncRNA whose levels correlated with T2DM. Initially we screened five serum samples from diabetic and non-diabetic individuals using lncRNA arrays. Next, GAS5 lncRNA levels were analyzed in 96 serum samples using quantitative PCR. Receiver operating characteristic (ROC) analysis was performed to determine the optimal cutoff GAS5 for diagnosis of DM.ResultsOur results demonstrate that decreased GAS5 levels in serum were associated with diabetes in a cohort of US military veterans. The ROC analysis revealed an optimal cutoff GAS5 value of less than or equal to 10. qPCR results indicated that individuals with absolute GAS5<10ng/μl have almost twelve times higher odds of having diabetes (Exact Odds Ratio [OR]=11.79 (95% CI: 3.97, 37.26), p<0.001). Analysis indicated area under curve (AUC) of ROC of 0.81 with 85.1% sensitivity and 67.3% specificity in distinguishing non-diabetic from diabetic subjects. The positive predictive value is 71.4%.ConclusionlncRNA GAS5 levels are correlated to prevalence of T2DM.General SignificanceAssessment of GAS5 in serum along with other parameters offers greater accuracy in identifying individuals at-risk for diabetes

Methods and compositions for diagnosis and management of diabetes and metabolic syndrome

Described herein are assays, methods, and devices for diagnosing/prognosing diabetes, metabolic syndrome, pre-diabetic state and/or the early-onset of diabetes in a subject. The assays, methods, and devices described herein can be configured to detect one or more long-coding RNAs in a sample from a subject

Identification of Sortilin Alternatively Spliced Variants in Mouse 3T3L1 Adipocytes

Type 2 diabetes mellitus is a metabolic disorder defined by systemic insulin resistance. Insulin resistance in adipocytes, an important regulator of glucose metabolism, results in impaired glucose uptake. The trafficking protein, sortilin, regulates major glucose transporter 4 (Glut4) movement, thereby promoting glucose uptake in adipocytes. Here, we demonstrate the presence of an alternatively spliced sortilin variant (Sort17b), whose levels increase with insulin resistance in mouse 3T3L1 adipocytes. Using a splicing minigene, we show that inclusion of alternative exon 17b results in the expression of Sort17b splice variant. Bioinformatic analysis indicated a novel intrinsic disorder region (IDR) encoded by exon 17b of Sort17b. Root mean square deviation (RMSD) and root mean square fluctuation (RMSF) measurements using molecular dynamics demonstrated increased flexibility of the protein backbone within the IDR. Using protein–protein docking and co-immunoprecipitation assays, we show robust binding of Glut4 to Sort17b. Further, results demonstrate that over-expression of Sort17b correlates with reduced Glut4 translocation and decreased glucose uptake in adipocytes. The study demonstrates that insulin resistance in 3T3L1 adipocytes promotes expression of a novel sortilin splice variant with thus far unknown implications in glucose metabolism. This knowledge may be used to develop therapeutics targeting sortilin variants in the management of type 2 diabetes and metabolic syndrome

Compositions and methods for detecting intracellular glucose and analogs thereof

The subject invention pertains to nucleic acid constructs for post-transcriptional control of expression of a polynucleotide encoding a protein in a cell, wherein the constructs include a metabolite responsive instability element such as the glucose-regulated mRNA instability element. The subject invention further pertains to host cells and vectors comprising the nucleic acid constructs of the invention, as well as probes, methods, and kits for detecting metabolite responsive instability elements or mutations thereof. The present invention further concerns a reporter vector useful for detecting intracellular glucose and glucose-analogs, host cells genetically modified with the reporter vector, and methods for detecting intracellular glucose. The present invention utilizes an element that regulates messenger RNA (mRNA) stability in response to a metabolite such as glucose or a glucose analog. This glucose-regulated mRNA instability element has been mapped to the protein kinase C βII (PKCβII) mRNA that was found to decrease in the presence of elevated glucose levels. When cloned into a reporter vector, the region of PKCβII containing the mRNA instability element imparts glucose-sensitive instability to the mRNA that is transcribed, thereby down-regulating the expression of the reporter gene when glucose is elevated. The reporter vector of the present invention may be introduced into host cells, allowing detection of intracellular glucose and glucose analogs within intact, living cells in real-time and, optionally, in a high-throughput format

Gloucose-regulated mRNA instability element

The subject invention pertains to nucleic acid constructs for post-transcriptional control of expression of a ploynucleotide encoding a protein in a eukaryotic cell, wherein the constructs include a metabolite responsive instability element such as the glucose-regulated mRNA instability element. The subject invention further pertains to host cells and vectors comprising the nucleic acid constructs of the invention, as well as probes, methods, and kits for detecting metabolite responsive instability elements or mutations thereof

Exosomes from human adipose-derived stem cells for the treatment of brain injury

Brain injury can be caused by trauma or may occur in stroke or neurodegenerative diseases. The disclosure relates to compositions that can include exosomes isolated from human adipose-derived stem cells (hASC) and methods where exosomes from hASC may be used alone or in combination with insulin for the treatment of brain injury

Compositions and methods for adipocyte modulation

Modified stilbenoids and formulations thereof are provided. The modified stilbenoids can include one or more amino or amino-alkyl substituents. The formulation can be for the treatment or prevention of obesity and the modified stilbenoid present in an effective amount to alleviate or prevent the onset of one or more symptoms of obesity. Methods of making the modified stilbenoids and formulations thereof are provided. Methods are provided for treating a subject with a modified stilbenoid in an effective amount to alleviate or prevent one or more symptoms associated with diabetes, cardiovascular disease, high blood pressure, deep vein thrombosis, osteoarthritis, obstructive sleep apnea, cancer, and non-alcoholic fatty liver disease. The administration of the modified stilbenoids can result in decreased levels of PKC.delta.II, PKC.delta.VIII, substantially unaltered expression of PKC.delta.I; a decreased number of adipocytes; increased weight and/or fat loss; or a combination thereof

Regulation of Human Sortilin Alternative Splicing by Glucagon-like Peptide-1 (GLP1) in Adipocytes

Type 2 diabetes mellitus is a chronic metabolic disease with no cure. Adipose tissue is a major site of systemic insulin resistance. Sortilin is a central component of the glucose transporter -Glut4 storage vesicles (GSV) which translocate to the plasma membrane to uptake glucose from circulation. Here, using human adipocytes we demonstrate the presence of the alternatively spliced, truncated sortilin variant (Sort_T) whose expression is significantly increased in diabetic adipose tissue. Artificial-intelligence-based modeling, molecular dynamics, intrinsically disordered region analysis, and co-immunoprecipitation demonstrated association of Sort_T with Glut4 and decreased glucose uptake in adipocytes. The results show that glucagon-like peptide-1 (GLP1) hormone decreases Sort_T. We deciphered the molecular mechanism underlying GLP1 regulation of alternative splicing of human sortilin. Using splicing minigenes and RNA-immunoprecipitation assays, the results show that GLP1 regulates Sort_T alternative splicing via the splice factor, TRA2B. We demonstrate that targeted antisense oligonucleotide morpholinos reduces Sort_T levels and improves glucose uptake in diabetic adipocytes. Thus, we demonstrate that GLP1 regulates alternative splicing of sortilin in human diabetic adipocytes

Ceramide synthesis regulates biogenesis and packaging of exosomal MALAT1 from adipose derived stem cells, increases dermal fibroblast migration and mitochondrial function

Abstract Background The function of exosomes, small extracellular vesicles (sEV) secreted from human adipose-derived stem cells (ADSC), is becoming increasingly recognized as a means of transferring the regenerative power of stem cells to injured cells in wound healing. Exosomes are rich in ceramides and long noncoding RNA (lncRNA) like metastasis-associated lung adenocarcinoma transcript 1 (MALAT1). We identified putative ceramide responsive cis-elements (CRCE) in MALAT1. We hypothesized that CRCE respond to cellular ceramide levels to regulate sEV MALAT1 packaging. MALAT1 levels by many cells exceed those of protein coding genes and it’s expression is equally high in exosomes. Ceramide also regulates exosome synthesis, however, the contents of exosome cargo via sphingomyelinase and ceramide synthase pathways has not been demonstrated. Methods ADSC were treated with an inhibitor of sphingomyelinase, GW4869, and stimulators of ceramide synthesis, C2- and C6-short chain ceramides, prior to collection of conditioned media (CM). sEV were isolated from CM, and then used to treat human dermal fibroblast (HDF) cultures in cell migration scratch assays, and mitochondrial stress tests to evaluate oxygen consumption rates (OCR). Results Inhibition of sphingomyelinase by treatment of ADSC with GW4869 lowered levels of MALAT1 in small EVs. Stimulation of ceramide synthesis using C2- and C6- ceramides increased cellular, EVs levels of MALAT1. The functional role of sEV MALAT1 was evaluated in HDF by applying EVs to HDF. Control sEV increased migration of HDF, and significantly increased ATP production, basal and maximal respiration OCR. sEV from GW4869-treated ADSC inhibited cell migration and maximal respiration. However, sEV from C2- and C6-treated cells, respectively, increased both functions but not significantly above control EV except for maximal respiration. sEV were exosomes except when ADSC were treated with GW4869 and C6-ceramide, then they were larger and considered microvesicles. Conclusions Ceramide synthesis regulates MALAT1 EV content. Sphingomyelinase inhibition blocked MALAT1 from being secreted from ADSC EVs. Our report is consistent with those of MALAT1 increasing cell migration and mitochondrial MALAT1 altering maximal respiration in cells. Since MALAT1 is important for exosome function, it stands that increased exosomal MALAT1 should be beneficial for wound healing as shown with these assays. Video Abstrac

Dysregulated Alternative Splicing Pattern of PKC during Differentiation of Human Preadipocytes Represents Distinct Differences between Lean and Obese Adipocytes

Obesity and its comorbidities affect millions of people. Here, we demonstrate that human preadipocytes are susceptible to programmed cell death (apoptosis) while mature adipocytes are resistant to apoptosis. The molecular mechanisms underlying the phenotype of apoptosis-resistant adipocytes are lesser known. To study the role of apoptosis and define molecular differences in the developmental process of adipogenesis, human preadipocytes were differentiated in vitro to mature adipocytes. Many genes in the apoptosis pathway are alternatively spliced. Our data demonstrates that during differentiation PKCδ, Bclx, and Caspase9 switch to their prosurvival splice variants along with an increase in Bcl2 expression when the cells terminally differentiate into mature adipocytes. Next we determined the expression pattern of these genes in obesity. Our data indicated high expression of PKCδVIII in adipose tissue of obese patient in different depots. We demonstrate a shift in the in vitro expression of these splice variants in differentiating preadipocytes derived from obese patients along with a decrease in adipogenesis markers. Hence, the programmed splicing of antiapoptotic proteins is a pivotal switch in differentiation that commits adipocytes to a prosurvival pathway. The expression pattern of these genes is dysregulated in obesity and may contribute to adipose tissue dysfunction