scholarly journals Influence of Acute High Glucose on Protein Abundance Changes in Murine Glomerular Mesangial Cells

2016 ◽  
Vol 2016 ◽  
pp. 1-11 ◽  
Author(s):  
Michelle T. Barati ◽  
James C. Gould ◽  
Sarah A. Salyer ◽  
Susan Isaacs ◽  
Daniel W. Wilkey ◽  
...  
Keyword(s):  
Protein Expression ◽  
Posttranslational Modifications ◽  
High Glucose ◽  
Mesangial Cells ◽  
Expression Patterns ◽  
Growth Conditions ◽  
Glomerular Mesangial Cells ◽  
Two Dimensional Gel Electrophoresis ◽  
Glucose Levels ◽  
Flight Mass Spectrometry

The effects of acute exposure to high glucose levels as experienced by glomerular mesangial cells in postprandial conditions and states such as in prediabetes were investigated using proteomic methods. Two-dimensional gel electrophoresis and matrix assisted laser desorption ionization time of flight mass spectrometry methods were used to identify protein expression patterns in immortalized rat mesangial cells altered by 2 h high glucose (HG) growth conditions as compared to isoosmotic/normal glucose control (NG⁎) conditions. Unique protein expression changes at 2 h HG treatment were measured for 51 protein spots. These proteins could be broadly grouped into two categories: (1) proteins involved in cell survival/cell signaling and (2) proteins involved in stress response. Immunoblot experiments for a protein belonging to both categories, prohibitin (PHB), supported a trend for increased total expression as well as significant increases in an acidic PHB isoform. Additional studies confirmed the regulation of proteasomal subunit alpha-type 2 and the endoplasmic reticulum chaperone and oxidoreductase PDI (protein disulfide isomerase), suggesting altered ER protein folding capacity and proteasomal function in response to acute HG. We conclude that short term high glucose induces subtle changes in protein abundances suggesting posttranslational modifications and regulation of pathways involved in proteostasis.

Downregulation of TRPC6 protein expression by high glucose, a possible mechanism for the impaired Ca2+ signaling in glomerular mesangial cells in diabetes

2007 ◽  
Vol 293 (4) ◽  
pp. F1381-F1390 ◽  
Author(s):  
Sarabeth Graham ◽  
Min Ding ◽  
Sherry Sours-Brothers ◽  
Thomas Yorio ◽  
Jian-Xing Ma ◽  
...  
Keyword(s):  
Patch Clamp ◽  
Protein Expression ◽  
High Glucose ◽  
Mesangial Cells ◽  
Membrane Currents ◽  
Maximum Effect ◽  
Ang Ii ◽  
Glomerular Mesangial Cells ◽  
Fura 2 ◽  
Fluorescence Measurements

The present study was performed to investigate whether transient receptor potential (TRPC)6 participated in Ca2+ signaling of glomerular mesangial cells (MCs) and expression of this protein was altered in diabetes. Western blots and real-time PCR were used to evaluate the expression level of TRPC6 protein and mRNA, respectively. Cell-attached patch-clamp and fura-2 fluorescence measurements were utilized to assess angiotensin II (ANG II)-stimulated membrane currents and Ca2+ responses in MCs. In cultured human MCs, high glucose significantly reduced expression of TRPC6 protein, but there was no effect on either TRPC1 or TRPC3. The high glucose-induced effect on TRPC6 was time and dose dependent with the maximum effect observed on day 7 and at 30 mM glucose, respectively. In glomeruli isolated from streptozotocin-induced diabetic rats, TRPC6, but not TRPC1, was markedly reduced compared with the glomeruli of control rats. Furthermore, TRPC6 mRNA in MCs was also significantly decreased by high glucose as early as 1 day after treatment with maximal reduction on day 4. Patch-clamp experiments showed that ANG II-stimulated membrane currents in MCs were significantly attenuated or enhanced by knockdown or overexpression of TRPC6, respectively. Fura-2 fluorescence measurements revealed that the ANG II-induced Ca2+ influxes were markedly inhibited in MCs with TRPC6 knockdown, reminiscent of the impaired Ca2+ entry in response to ANG II in high glucose-treated MCs. These results suggest that the TRPC6 protein expression in MCs was downregulated by high glucose and the deficiency of TRPC6 protein might contribute to the impaired Ca2+ signaling of MCs seen in diabetes.

scholarly journals Impairment of hepatic nuclear factor-4α binding to the Stim1 promoter contributes to high glucose-induced upregulation of STIM1 expression in glomerular mesangial cells

2015 ◽  
Vol 308 (10) ◽  
pp. F1135-F1145 ◽  
Author(s):  
Yanxia Wang ◽  
Sarika Chaudhari ◽  
Yuezhong Ren ◽  
Rong Ma
Keyword(s):  
Protein Expression ◽  
High Glucose ◽  
Mesangial Cells ◽  
Therapeutic Option ◽  
Binding Activity ◽  
Nuclear Factor ◽  
Glomerular Mesangial Cells ◽  
Expression Levels ◽  
293 Cells ◽  
Interfering Rna

The present study was carried out to investigate if hepatic nuclear factor (HNF)4α contributed to the high glucose-induced increase in stromal interacting molecule (STIM)1 protein abundance in glomerular mesangial cells (MCs). Western blot and immunofluorescence experiments showed HNF4α expression in MCs. Knockdown of HNF4α using a small interfering RNA approach significantly increased mRNA expression levels of both STIM1 and Orai1 and protein expression levels of STIM1 in cultured human MCs. Consistently, overexpression of HNF4α reduced expressed STIM1 protein expression in human embryonic kidney-293 cells. Furthermore, high glucose treatment did not significantly change the abundance of HNF4α protein in MCs but significantly attenuated HNF4α binding activity to the Stim1 promoter. Moreover, knockdown of HNF4α significantly augmented store-operated Ca2+ entry, which is known to be gated by STIM1 and has recently been found to be antifibrotic in MCs. In agreement with those results, knockdown of HNF4α significantly attenuated the fibrotic response of high glucose. These results suggest that HNF4α negatively regulates STIM1 transcription in MCs. High glucose increases STIM1 expression levels by impairing HNF4α binding activity to the Stim1 promoter, which subsequently releases Stim1 transcription from HNF4α repression. Since the STIM1-gated store-operated Ca2+ entry pathway in MCs has an antifibrotic effect, inhibition of HNF4α in MCs might be a potential therapeutic option for diabetic kidney disease.

scholarly journals Effect of Elevated Glucose on Endothelin-Induced Store-Operated and Non-Store-Operated Calcium Influx in Renal Mesangial Cells

2000 ◽  
Vol 11 (7) ◽  
pp. 1225-1235
Author(s):  
LETA K. NUTT ◽  
ROGER G. O'NEIL
Keyword(s):  
Calcium Signaling ◽  
High Glucose ◽  
Mesangial Cells ◽  
Calcium Influx ◽  
Calcium Entry ◽  
Calcium Signal ◽  
Glomerular Mesangial Cells ◽  
Glucose Levels ◽  
Elevated Glucose ◽  
Acute And Chronic Exposure

Abstract. Early diabetic nephropathy exhibits renal glomerular hyperfiltration and an increase in renal plasma flow. The hyperfiltration is a dysfunctional state that may arise from a hyperglycemic-induced hypocontractility of glomerular mesangial cells that may be associated with depressed Ca2+signaling events. The present study was designed to determine the effects of acute (minutes) and chronic (days) elevated glucose levels on endothelin-induced calcium signaling with a particular emphasis on the potential influence on stores and store-operated Ca2+influx (SOCI ; also called capacitative calcium entry) in glomerular mesangial cells. Primary cultures of rat mesangial cells were grown in either high (30 mM) or normal (5 mM) glucose-containing media and tested in the presence of either high (30 mM) or normal (5 mM) glucose levels. Intracellular calcium levels were monitored with the calcium-sensitive fluorophore fura-2 before and after treatment with either endothelin-1 (10 nM), to induce typical Ca2+signals, or the endoplasmic reticulum (ER) Ca-ATPase inhibitor thapsagargin (1 μM), to unload ER Ca2+stores. Both acute and chronic exposure to high glucose levels depressed the endothelin-induced calcium signal. However, neither release of Ca2+from stores nor SOCI were depressed by high glucose levels. In contrast, an endothelin-induced calcium entry pathway (likely receptor-operated calcium influx), separate from SOCI, was markedly depressed in the presence of both acute and chronic high glucose levels. The depressant effect of high glucose was rapidly (minutes) reversible upon returning to normal glucose levels. It is concluded that high glucose levels depress endothelin-induced calcium signaling in rat mesangial cells by inhibiting non-SOCI Ca2+entry pathways, namely the receptor-operated Ca2+influx pathway. The glucose-induced alterations in the receptor-operated calcium influx pathway may, in part, contribute to the depressed contractile state of glomerular cells during periods of hyperglycemia.

scholarly journals Trigonelline regulates Wnt/β-catenin signaling in glomerular mesangial cells under high glucose conditions

2021 ◽  
Author(s):  
Chen Chen ◽  
Yan Shi ◽  
Zhen Chen ◽  
Xiangjun Li ◽  
Bo Sun ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Flow Cytometry ◽  
High Glucose ◽  
Cell Cycle Progression ◽  
Cell Injury ◽  
Mesangial Cells ◽  
Animal Experiments ◽  
Glomerular Mesangial Cells ◽  
Glucose Levels ◽  
Elevated Glucose

Abstract Background: Trigonelline have hypoglycemic effects. In previous animal experiments, we observed that trigonelline (TRL) treat-ment attenuated metabolic abnormalities associated with hyperglycemic conditions in the experimental DN model. In streptozotocin (STZ)-induced rats, TRL treatment reduced albuminuria, lowered blood sugar, improved renal function and alleviated the pathological alterations within the glomerulus. Methods: We stimulated human mesangial cells (HMC) with high glucose (30 mmol / L) medium. HMCs were transfected with β-catenin plasmid or siRNA to investigate the effect of trigonelline on high glucose-induced excessive proliferation and apoptosis of HMCs, and to understand its mechanism of action. Cell viability was measured by MTT assay. Flow cytometry was used to detect the cell cycle. Cell apoptosis was evaluated by flow cytometry and terminal dUTP transferase nick end labeling (TUNEL) assay. Protein and mRNA expression of β-catenin, Wnt5a, TCF4, Cyclin D1, and CDK4 were detected by western blotting and RT-PCR, respectively. Results: Trigonelline inhibited cell proliferation by blocking cell-cycle progression at the G1 phase and decreased apoptosis via the Wnt/β-catenin pathway. Elevated glucose levels enhanced the expression of β-catenin, an important modulator of diabetic nephropathy, while trigonelline restored up-regulation. Conclusions: High glucose and high expression of β-catenin could lead to cell injury; however, this effect was mitigated by trigonelline via managing the canonical Wnt/β-catenin signaling pathway.

The Mechanism of miR-192 in Regulating High Glucose-Induced MCP-1 Expression in Rat Glomerular Mesangial Cells

2019 ◽  
Vol 19 (7) ◽  
pp. 1055-1063
Author(s):  
Fenqin Chen ◽  
Guozhu Wei ◽  
Yang Zhou ◽  
Xiaoyu Ma ◽  
Qiuyue Wang
Keyword(s):  
Protein Expression ◽  
Mrna Expression ◽  
Cell Lines ◽  
Western Blotting ◽  
High Glucose ◽  
Messenger Rna ◽  
Mesangial Cells ◽  
Inflammatory Factor ◽  
Glomerular Mesangial Cells ◽  
Regulatory Loop

Background: Although the pathogenetic mechanism of Diabetic Kidney Disease (DKD) has not been elucidated, an inflammatory mechanism may be a potential contributor. Monocyte chemotactic protein-1 (MCP-1) is suggested to be implicated in the development of DKD by playing a role in the infiltration of monocyte/macrophage. The aim of this study was to investigate the expression of MCP-1 under high glucose conditions, as well as the effects of microRNA-192 (miR-192) under these conditions, and to study the regulatory mechanism of MCP-1 in DKD. <p></p> Methods: Rat glomerular mesangial cells were cultured in high glucose or isotonic mannitol. The messenger RNA(mRNA) expression of miR-192, miR-200b, miR-200c, E-box-binding homeobox 1 (Zeb1), and MCP-1 was then detected by real-time PCR, and the protein expression of Zeb1 and MCP- 1 was assessed by western blotting. The rat mesangial cells were transfected with an miR-192 inhibitor, NC inhibitor , and transfected with siRNA Zeb1, siNC. The cells were then cultured in high glucose to detect the mRNA expression of miR-192, miR-200b, miR-200c, Zeb1, and MCP-1 using realtime PCR, and Zeb1 and MCP-1 protein expression were determined by western blotting. <p></p> Results: MiR-192, miR-200b, miR-200c, and MCP-1 were overexpressed, whereas Zeb1 was downregulated when cultured in high glucose (P < 0.05). After transfection with an miR-192 inhibitor, the expression of miR-192, miR-200b, miR-200c, and MCP-1 was downregulated, whereas Zeb1 was increased, and these differences were statistically significant (P < 0.05). The observed changes in the expression in the NC inhibitor transfection group were similar to that of non-transfected cell lines. Silencing the expression of Zeb1 resulted in a significant increase in the expression of miR-192, miR- 200b, miR-200c, and MCP-1 (P < 0.05). The observed changes in the SiNC transfection group were similar to those of non-transfected cell lines. <p></p> Conclusions: MiR-192 expression was upregulated to increase the expression of inflammatory factor MCP-1 by inhibiting the expression of Zeb1, which was mediated by breaking the regulatory loop of Zeb1 and miR-200b/c in rat mesangial cells cultured in high glucose.

Modulation of intestinal cholesterol absorption by high glucose levels: impact on cholesterol transporters, regulatory enzymes, and transcription factors

2008 ◽  
Vol 295 (5) ◽  
pp. G873-G885 ◽  
Author(s):  
Z. Ravid ◽  
M. Bendayan ◽  
E. Delvin ◽  
A. T. Sane ◽  
M. Elchebly ◽  
...  
Keyword(s):  
Transcription Factors ◽  
Protein Expression ◽  
High Glucose ◽  
Cholesterol Absorption ◽  
Cholesterol Homeostasis ◽  
Cholesterol Transport ◽  
Intestinal Cholesterol Absorption ◽  
Cholesterol Uptake ◽  
Glucose Levels

Growing evidence suggests that the small intestine may contribute to excessive postprandial lipemia, which is highly prevalent in insulin-resistant/Type 2 diabetic individuals and substantially increases the risk of cardiovascular disease. The aim of the present study was to determine the role of high glucose levels on intestinal cholesterol absorption, cholesterol transporter expression, enzymes controlling cholesterol homeostasis, and the status of transcription factors. To this end, we employed highly differentiated and polarized cells (20 days of culture), plated on permeable polycarbonate filters. In the presence of [14C]cholesterol, glucose at 25 mM stimulated cholesterol uptake compared with Caco-2/15 cells supplemented with 5 mM glucose ( P < 0.04). Because combination of 5 mM glucose with 20 mM of the structurally related mannitol or sorbitol did not change cholesterol uptake, we conclude that extracellular glucose concentration is uniquely involved in the regulation of intestinal cholesterol transport. The high concentration of glucose enhanced the protein expression of the critical cholesterol transporter NPC1L1 and that of CD36 ( P < 0.02) and concomitantly decreased SR-BI protein mass ( P < 0.02). No significant changes were observed in the protein expression of ABCA1 and ABCG8, which act as efflux pumps favoring cholesterol export out of absorptive cells. At the same time, 3-hydroxy-3-methylglutaryl-coenzyme A reductase activity was decreased ( P < 0.007), whereas ACAT activity remained unchanged. Finally, increases were noted in the transcription factors LXR-α, LXR-β, PPAR-β, and PPAR-γ along with a drop in the protein expression of SREBP-2. Collectively, our data indicate that glucose at high concentrations may regulate intestinal cholesterol transport and metabolism in Caco-2/15 cells, thus suggesting a potential influence on the cholesterol absorption process in Type 2 diabetes.

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