Scaffold proteins are known to regulate important cellular processes by interacting with multiple proteins to modulate molecular responses. RACK1 (Receptor for Activated C Kinase 1) is a WD-40 type scaffold protein, conserved in eukaryotes, from Chlamydymonas to plants and humans, expresses ubiquitously and plays regulatory roles in diverse signal transduction and stress response pathways. Here we present the use of Arabidopsis RACK1A, the predominant isoform of a 3-member family, as a bait to screen a split-ubiquitin based cDNA library. In total 97 proteins from dehydration, salt stress, ribosomal and photosynthesis pathways are found to potentially interact with RACK1A. False positive interactions were eliminated following extensive selection based growth potentials. Confirmation of a sub-set of selected interactions is demonstrated through the co-transformation with individual plasmid containing cDNA and the respective bait. Interaction of diverse proteins points to a regulatory role of RACK1A in the cross-talk between signaling pathways. Promoter analysis of the stress and photosynthetic pathway genes revealed conserved transcription factor binding sites. RACK1A is known to be a multifunctional protein and the current identification of potential interacting proteins and future in vivo elucidations of the physiological basis of such interactions will shed light on the possible molecular mechanisms that RACK1A uses to regulate diverse signaling pathways.
AimsType 2 diabetes is associated with endothelial dysfunction leading to cardiovascular disease. CD34+ endothelial Progenitor Cells (EPCs) are responsible for endothelial repair and neo-angiogenesis and can be used as a cardiovascular disease risk biomarker. This study investigated whether the addition of saxagliptin, a DPP-IV inhibitor, to metformin, may reduce cardiovascular disease risk in addition to improving glycemic control in Type 2 diabetes patients.MethodsIn 12 week, double-blind, randomized placebo-controlled trial, 42 subjects already taking metformin 1–2 grams/day were randomized to placebo or saxagliptin 5 mg. Subjects aged 40–70 years with diabetes for < 10 years, with no known cardiovascular disease, BMI 25–39.9, HbA1C 6–9% were included. We evaluated EPCs number, function, surface markers and gene expression, in addition to arterial stiffness, blood biochemistries, resting energy expenditure, and body composition parameters. A mixed model regression to examine saxagliptin vs placebo, accounting for within-subject autocorrelation, was done with SAS (p < 0.05).ResultsAlthough there was no significant increase in CD34+ cell number, CD31+ cells percentage increased. Saxagliptin increased migration (in response to SDF1α) with a trend of higher colony formation count. MNCs cytometry showed higher percentage of CXCR4 double positivity for both CD34 and CD31 positive cells, indicating a functional improvement. Gene expression analysis showed an upregulation in CD34+ cells for antioxidant SOD1 (p < 0.05) and a downregulation in CD34− cells for IL-6 (p < 0.01). For arterial stiffness, both augmentation index and systolic blood pressure measures went down in saxagliptin subjects (p < 0.05).ConclusionSaxagliptin, in combination with metformin, can help improve endothelial dysfunction in early diabetes before macrovascular complications appear.Trial registration Trial is registered under clinicaltrials.gov, NCT02024477Electronic supplementary materialThe online version of this article (10.1186/s12933-018-0709-9) contains supplementary material, which is available to authorized users.
Background The incidence of obesity and diabetes is increasing rapidly. Optimal management is still elusive. Obesity associated with type 2 diabetes is known to cause adipose tissue inflammation, increase oxidative stress, and cause white fat hyperplasia and mitochondrial dysfunction. In this study, we investigated whether mitochondrial and cytosolic antioxidant-upregulated mesenchymal stem cell (MSC) delivery reduces oxidative stress and subsequently improves glucose tolerance, reduce systemic inflammation, and improves fatty liver disease in diet-induced obese (DIO) mouse models. Methods Antioxidant genes Sod2 (mitochondrial) and catalase (cytosolic) or null (control) were upregulated in human adipose tissue-derived MSCs using adenoviral constructs. Modified MSCs were then delivered intraperitoneally into mice that were fed a 45% or 60% high-fat diet (HFD), and animals were followed for 4 weeks. Results Over 4 weeks, body weight remained stable; however, we noted a significant reduction in liver fat content by histological analysis and liver triglyceride assay. Triglyceride assay ( p < 0.01) confirmed reduced liver fat accumulation in animals that received either Sod2- or Cat-MSCs. There was a lower plasma level of inflammatory marker TNFα, measured in mice that were fed either 45% or 60% HFD and received Sod2- or Cat-MSCs, indicating reduced systemic inflammation. Ucp1 mRNA was upregulated approximately 100–1000-fold for omental fat and 10–100-fold for pericardial fat compared to the Null-MSC-receiving group. Pcgc1a and Prdm16 mRNA upregulation was also noted particularly for pericardial fat. Glucose tolerance showed a positive improvement trend with a lower area under the curve (AUC) values for both Sod2- and Cat-MSCs groups in comparison to control. For mice fed with 60% HFD and that received Sod2-MSCs, glucose levels were significantly lower than control (* p < 0.05) at a time point of 60 min in the glycemic curve during glucose tolerance test. Conclusion Reduction of oxidative stress post-antioxidant-upregulated MSC delivery, intraperitoneally, reduces systemic inflammation and fat accumulation in the liver. There is evidence of an increase in browning of white adipose tissue depots with concomitant improvement of glucose tolerance in a weight-independent fashion. Antioxidant-upregulated MSC delivery may be a safe yet effective therapy for obesity and prediabetes and improves related complication such as non-alcoholic fatty liver disease. Electronic supplementary material The online version of this article (10.1186/s13287-019-1393-8) contains supplementary material, which is available to authorized users.
Background Endothelial progenitor cells (EPCs) has been shown to be dysfunctional in both type 2 diabetes mellitus (T2DM) and chronic kidney disease (CKD) leading to poor regeneration of endothelium and renal perfusion. EPCs have been shown to be a robust cardiovascular disease (CVD) risk indicator. Effect of sodium glucose channel inhibitors (SGLT2i) such as Canagliflozin (CG) on a cellular biomarker such as CD34+ve progenitor cells, which may help predict CVD risk, in patients with T2DM with established CKD has not been explored. Methods This is a pilot study where 29 subjects taking metformin and/or Insulin were enrolled in a 16 week, double blind, randomized placebo matched trial, with a low dose 100 mg CG as the intervention group compared to matched placebo. Type 2 diabetes subjects (30–70 years old), with hemoglobin A1c (HbA1c) of 7–10%, were enrolled. CD34+ve cell number, migratory function, gene expression along with vascular parameters such as arterial stiffness, serum biochemistry pertaining to cardio-metabolic health, resting energy expenditure and body composition were measured. Data were collected at week 0, 8 and 16. A mixed model regression analysis was done and p value less than 0.05 was considered statistically significant. Results A significant expression of CXCR4 receptor with a concomittant increase in migratory function of CD34+ve cells was observed in CG treated group as compared to placebo group. Gene expression analysis of CD34+ve cells showed an increase in expression of antioxidants (superoxide dismutase 2 or SOD2, Catalase and Glutathione Peroxidase or GPX) and notable endothelial markers (PECAM1, VEGF-A, and NOS3). A significant reduction in glucose and HbA1c levels were observed along with improved systolic and diastolic blood pressure in the CG group. A significant increase in adiponectin (p = 0.006) was also noted in treatment group. Urinary exosomal protein leak in urine, examining podocyte health (podocalyxin, Wilm’s tumor and nephrin) showed reduction with CG Conclusion Low dose Canagliflozin has a beneficial effect on CD34+ cell function, serum biochemistry and urinary podocyte specific exosomes in type 2 diabetes.
Background: Endothelial Progenitor cells (EPCs) has been shown to be dysfunctional in both type 2 diabetes mellitus (T2DM) and chronic kidney disease (CKD) leading to poor regeneration of endothelium and renal perfusion. EPCs have been shown to be a robust cardiovascular disease (CVD) risk indicator. Cellular mechanisms of DPP4 inhibitors such as linagliptin (LG) on CVD risk, in patients with T2DM with established CKD has not been established. Linagliptin, a DPP4 inhibitor when added to insulin, metformin or both may improve endothelial dysfunction in a diabetic kidney disease (DKD) population. Methods: 31 subjects taking metformin and/or Insulin were enrolled in this 12 weeks, double blind, randomized placebo matched trial, with 5 mg LG compared to placebo. Type 2 diabetes subjects (30-70 years old), HbA1c of 6.5-10%, CKD Stage 1-3 were included. CD34+ cell number, migratory function, gene expression along with vascular parameters such as arterial stiffness, biochemistry, resting energy expenditure and body composition were measured. Data were collected at week 0, 6 and 12. A mixed model regression analysis was done with p value < 0.05 considered significant. Results: A double positive CD34/CD184 cell count had a statistically significant increase (p < 0.02) as determined by flow cytometry in LG group where CD184 is SDF1a cell surface receptor. Though mRNA differences in CD34+ve was more pronounced CD34-cell mRNA analysis showed increase in antioxidants (superoxide dismutase 2 or SOD2, Catalase and Glutathione Peroxidase or GPX) and prominent endothelial markers (PECAM1, VEGF-A, vWF and NOS3). Arterial stiffness measures such as augmentation Index (AI) (p < 0.04) and pulse wave analysis (PWV) were improved (reduced in stiffness) in LG group. A reduction in LDL: HDL ratio was noted in treatment group (p < 0.04). Urinary exosome protein examining podocyte health (podocalyxin, Wilms tumor and nephrin) showed reduction or improvement.
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