detection-small-gtpase-prenylation-gtp-binding-using-membrane

Detection of Small GTPase Prenylation and GTP Binding Using Membrane Fractionation and  GTPase-linked Immunosorbent Assay

The Rho GTPase family belongs to the Ras superfamily and includes approximately 20 members in humans. Rho GTPases are important in the regulation of ...

University of Manitoba

The prevalence of type 2 diabetes (T2D) has increased dramatically over the past two decades, not only among adults but also among adolescents. T2D is a systemic disorder affecting every organ system and is especially damaging to the cardiovascular system, predisposing individuals to severe cardiac and vascular complications. The precise mechanisms that cause T2D are an area of active research. Most current theories suggest that the process begins with peripheral insulin resistance that precedes failure of the pancreatic β-cells to secrete sufficient insulin to maintain normoglycemia. A growing body of literature has highlighted multiple aspects of mitochondrial function, including oxidative phosphorylation, lipid homeostasis, and mitochondrial quality control in the regulation of peripheral insulin sensitivity. Whether the cellular mechanisms of insulin resistance in adults are comparable to that in adolescents remains unclear. This review will summarize both clinical and basic studies that shed light on how alterations in skeletal muscle mitochondrial function contribute to whole body insulin resistance and will discuss the evidence supporting high-intensity exercise training as a therapy to circumvent skeletal muscle mitochondrial dysfunction to restore insulin sensitivity in both adults and adolescents.

Targeting skeletal muscle mitochondria to prevent type 2 diabetes in youth.

The aim of the present study was to determine the in vitro effect(s) of a bovine-based human breast milk fortifier (HMF) on human intestinal cells. HMF increases the expression of BCL2/adenovirus E1B 19 kDa protein-interacting protein (Bnip3) and cell death; the prostaglandin analogue misoprostol will rescue this effect.

Human Milk Fortification Increases Bnip3 Expression Associated With Intestinal Cell Death In Vitro.

The mevalonate (MEV) cascade is responsible for cholesterol biosynthesis and the formation of the intermediate metabolites geranylgeranylpyrophosphate (GGPP) and farnesylpyrophosphate (FPP) used in the prenylation of proteins. Here we show that the MEV cascade inhibitor simvastatin induced significant cell death in a wide range of human tumor cell lines, including glioblastoma, astrocytoma, neuroblastoma, lung adenocarcinoma, and breast cancer. Simvastatin induced apoptotic cell death via the intrinsic apoptotic pathway. In all cancer cell types tested, simvastatin-induced cell death was not rescued by cholesterol, but was dependent on GGPP- and FPP-depletion. We confirmed that simvastatin caused the translocation of the small Rho GTPases RhoA, Cdc42, and Rac1/2/3 from cell membranes to the cytosol in U251 (glioblastoma), A549 (lung adenocarcinoma) and MDA-MB-231(breast cancer). Simvastatin-induced Rho-GTP loading significantly increased in U251 cells which were reversed with MEV, FPP, GGPP. In contrast, simvastatin did not change Rho-GTP loading in A549 and MDA-MB-231. Inhibition of geranylgeranyltransferase I by GGTi-298, but not farnesyltransferase by FTi-277, induced significant cell death in U251, A549, and MDA-MB-231. These results indicate that MEV cascade inhibition by simvastatin induced the intrinsic apoptosis pathway via inhibition of Rho family prenylation and depletion of GGPP, in a variety of different human cancer cell lines.

Mevalonate Cascade Inhibition by Simvastatin Induces the Intrinsic Apoptosis Pathway via Depletion of Isoprenoids in Tumor Cells.

Myocardin is a cardiac- and smooth muscle-enriched transcriptional co-activator that was originally described as an interacting partner of the serum response factor. Shortly after myocardin's discovery, a wealth of published literature described the role of myocardin as a regulator of smooth muscle differentiation and phenotype modulation, while gene-targeting studies confirmed the essential role of myocardin in vascular development. More recently, myocardin has been implicated as an important regulator of cardiac myocyte differentiation in studies demonstrating direct programming of fibroblasts towards the cardiac lineage. This function of myocardin has been attributed to its physical interaction with cardiac-enriched transcription factors such as MEF2C, GATA4, and TBX5. Moreover, conditional knockout models have revealed a critical role for myocardin during cardiac chamber maturation, and a surprising function for myocardin in the regulation of cardiomyocyte proliferation, cell death, and possibly mitochondrial function. This review summarizes the literature surrounding the cardiac-specific roles of myocardin during development and post-natal cardiac remodeling. In addition, we take a bioinformatics and computational approach to discuss known and predicted interactions and biological functions of myocardin, which suggests areas for future research.

Regulation of cardiac myocyte cell death and differentiation by myocardin.

Myocardin is a transcriptional co-activator required for cardiovascular development, but also promotes cardiomyocyte survival through an unclear molecular mechanism. Mitochondrial permeability transition is implicated in necrosis, while pore closure is required for mitochondrial maturation during cardiac development. We show that loss of myocardin function leads to subendocardial necrosis at E9.5, concurrent with elevated expression of the death gene Nix. Mechanistically, we demonstrate that myocardin knockdown reduces microRNA-133a levels to allow Nix accumulation, leading to mitochondrial permeability transition, reduced mitochondrial respiration, and necrosis. Myocardin knockdown elicits calcium release from the endo/sarcoplasmic reticulum with mitochondrial calcium accumulation, while restoration of microRNA-133a function, or knockdown of Nix rescues calcium perturbations. We observed reduced myocardin and elevated Nix expression within the infarct border-zone following coronary ligation. These findings identify a myocardin-regulated pathway that maintains calcium homeostasis and mitochondrial function during development, and is attenuated during ischemic heart disease. Given the diverse role of Nix and microRNA-133a, these findings may have broader implications to metabolic disease and cancer.

Myocardin regulates mitochondrial calcium homeostasis and prevents permeability transition.

The cellular response to hypoxia involves the activation of a conserved pathway for gene expression regulated by the transcription factor complex called hypoxia-inducible factor (HIF). This pathway has been implicated in both the adaptive response to hypoxia and in several hypoxic-ischemic-related pathologies. Perinatal hypoxic injury, often associated with prematurity, leads to multi-organ dysfunction resulting in significant morbidity and mortality. Using a rodent model of neonatal hypoxia and several representative cell lines, we observed HIF1α activation and down-stream induction of the cell death gene Bnip3 in brain, large intestine, and heart which was mitigated by administration of the prostaglandin E1 analog misoprostol. Mechanistically, we determined that misoprostol inhibits full-length Bnip3 (Bnip3-FL) expression through PKA-mediated NF-κB (P65) nuclear retention, and the induction of pro-survival splice variants. We observed that the dominant small pro-survival variant of Bnip3 in mouse cells lacks the third exon (Bnip3ΔExon3), whereas human cells produce a pro-survival BNIP3 variant lacking exon 2 (BNIP3ΔExon2). In addition, these small Bnip3 splice variants prevent mitochondrial dysfunction, permeability transition, and necrosis triggered by Bnip3-FL by blocking calcium transfer from the sarco/endoplasmic reticulum to the mitochondria. Furthermore, misoprostol and Bnip3ΔExon3 promote nuclear calcium accumulation, resulting in HDAC5 nuclear export, NFAT activation, and adaptive changes in cell morphology and gene expression. Collectively, our data suggests that misoprostol can mitigate the potential damaging effects of hypoxia on multiple cell types by activating adaptive cell survival pathways through Bnip3 repression and alternative splicing.

Misoprostol regulates Bnip3 repression and alternative splicing to control cellular calcium homeostasis during hypoxic stress.

Adding vigorous-intensity intervals (VII) to moderate-intensity exercise prevents immediate declines in blood glucose in type 1 diabetes (T1D) however the intensity required to minimize post-exercise hypoglycemia is unknown. To examine this question, ten sedentary T1D individuals completed four treadmill exercise sessions: a control session of 45 minutes of walking at 45-55% of heart rate reserve (HRR) and three sessions consisting of 60 seconds (VII) at 70%, 80%, or 90% of HRR every 4 minutes during exercise at 45-55% of HRR. We used continuous glucose monitoring (CGM) to measure time ≤3.9 mmol/L, glucose variability, hypoglycemia frequency and area under the curve (AUC) for hypoglycemia and hyperglycemia for 12 hours post-exercise. We also examined growth hormone and cortisol responses during and following exercise. In the 12 hours post-exercise, the percentage of time ≤3.9 mmol/L, glucose variability, and AUC for hypoglycemia and hyperglycemia were similar across conditions. The frequency of hypoglycemic events was highest after the 90% intervals compared to the control arm (12 vs 3 events, p = 0.03). There was a trend towards elevated growth hormone with increasing exercise intensity but cortisol levels were similar across conditions. Adding VII to moderate intensity exercise may increase hypoglycemia risk at higher intensities.

Article	Year
Targeting skeletal muscle mitochondria to prevent type 2 diabetes in youth. Biochemistry and cell biology = Biochimie et biologie cellulaire\| PubMed ID: 26151290	2015
Human Milk Fortification Increases Bnip3 Expression Associated With Intestinal Cell Death In Vitro. Journal of pediatric gastroenterology and nutrition\| PubMed ID: 26505960	2015
Mevalonate Cascade Inhibition by Simvastatin Induces the Intrinsic Apoptosis Pathway via Depletion of Isoprenoids in Tumor Cells. Scientific reports\| PubMed ID: 28344327	2017
Regulation of cardiac myocyte cell death and differentiation by myocardin. Molecular and cellular biochemistry\| PubMed ID: 28631251	2018
Myocardin regulates mitochondrial calcium homeostasis and prevents permeability transition. Cell death and differentiation\| PubMed ID: 29511336	2018
Misoprostol regulates Bnip3 repression and alternative splicing to control cellular calcium homeostasis during hypoxic stress. Cell death discovery\| PubMed ID: 30275982	2018
Vigorous Intervals and Hypoglycemia in Type 1 Diabetes: A Randomized Cross Over Trial. Scientific reports\| PubMed ID: 30367116	2018

Joseph W. Gordon

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