scholarly journals Identification of Insulin-Mimetic Plant Extracts: From an In Vitro High-Content Screen to Blood Glucose Reduction in Live Animals

Molecules ◽  
2021 ◽  
Vol 26 (14) ◽  
pp. 4346
Author(s):  
Verena Stadlbauer ◽  
Cathrina Neuhauser ◽  
Tobias Aumiller ◽  
Alexander Stallinger ◽  
Marcus Iken ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Blood Glucose ◽  
Plant Extracts ◽  
Glucose Transporter ◽  
Glut4 Translocation ◽  
In Ovo ◽  
Insulin Mimetic ◽  
Glucose Levels ◽  
Intracellular Translocation

Type 2 diabetes mellitus (T2DM) is linked to insulin resistance and a loss of insulin sensitivity, leading to millions of deaths worldwide each year. T2DM is caused by reduced uptake of glucose facilitated by glucose transporter 4 (GLUT4) in muscle and adipose tissue due to decreased intracellular translocation of GLUT4-containing vesicles to the plasma membrane. To treat T2DM, novel medications are required. Through a fluorescence microscopy-based high-content screen, we tested more than 600 plant extracts for their potential to induce GLUT4 translocation in the absence of insulin. The primary screen in CHO-K1 cells resulted in 30 positive hits, which were further investigated in HeLa and 3T3-L1 cells. In addition, full plasma membrane insertion was examined by immunostaining of the first extracellular loop of GLUT4. The application of appropriate inhibitors identified PI3 kinase as the most important signal transduction target relevant for GLUT4 translocation. Finally, from the most effective hits in vitro, four extracts effectively reduced blood glucose levels in chicken embryos (in ovo), indicating their applicability as antidiabetic pharmaceuticals or nutraceuticals.

scholarly journals Insulin Mimetic Properties of Extracts Prepared from Bellis perennis

Molecules ◽  
2018 ◽  
Vol 23 (10) ◽  
pp. 2605 ◽  
Author(s):  
Renate Haselgrübler ◽  
Verena Stadlbauer ◽  
Flora Stübl ◽  
Bettina Schwarzinger ◽  
Ieva Rudzionyte ◽  
...  
Keyword(s):  
Blood Glucose ◽  
Glucose Transporter ◽  
Living Organism ◽  
Cell System ◽  
Glut4 Translocation ◽  
In Ovo ◽  
Screening Assay ◽  
Glucose Levels ◽  
Blood Glucose Levels

Diabetes mellitus (DM) and consequential cardiovascular diseases lead to millions of deaths worldwide each year; 90% of all people suffering from DM are classified as Type 2 DM (T2DM) patients. T2DM is linked to insulin resistance and a loss of insulin sensitivity. It leads to a reduced uptake of glucose mediated by glucose transporter 4 (GLUT4) in muscle and adipose tissue, and finally hyperglycemia. Using a fluorescence microscopy-based screening assay we searched for herbal extracts that induce GLUT4 translocation in the absence of insulin, and confirmed their activity in chick embryos. We found that extracts prepared from Bellis perennis (common daisy) are efficient inducers of GLUT4 translocation in the applied in vitro cell system. In addition, these extracts also led to reduced blood glucose levels in chicken embryos (in ovo), confirming their activity in a living organism. Using high-performance liquid chromtaography (HPLC) analysis, we identified and quantified numerous polyphenolic compounds including apigenin glycosides, quercitrin and chlorogenic acid, which potentially contribute to the induction of GLUT4 translocation. In conclusion, Bellis perennis extracts reduce blood glucose levels and are therefore suitable candidates for application in food supplements for the prevention and accompanying therapy of T2DM.

scholarly journals Specialized sorting of GLUT4 and its recruitment to the cell surface are independently regulated by distinct Rabs

2013 ◽  
Vol 24 (16) ◽  
pp. 2544-2557 ◽  
Author(s):  
L. Amanda Sadacca ◽  
Joanne Bruno ◽  
Jennifer Wen ◽  
Wenyong Xiong ◽  
Timothy E. McGraw
Keyword(s):  
Plasma Membrane ◽  
Glucose Uptake ◽  
Glucose Transporter ◽  
Receptor Activation ◽  
Glut4 Translocation ◽  
Insulin Stimulation ◽  
Transport Vesicles ◽  
Glucose Transporter Glut4

Adipocyte glucose uptake in response to insulin is essential for physiological glucose homeostasis: stimulation of adipocytes with insulin results in insertion of the glucose transporter GLUT4 into the plasma membrane and subsequent glucose uptake. Here we establish that RAB10 and RAB14 are key regulators of GLUT4 trafficking that function at independent, sequential steps of GLUT4 translocation. RAB14 functions upstream of RAB10 in the sorting of GLUT4 to the specialized transport vesicles that ferry GLUT4 to the plasma membrane. RAB10 and its GTPase-activating protein (GAP) AS160 comprise the principal signaling module downstream of insulin receptor activation that regulates the accumulation of GLUT4 transport vesicles at the plasma membrane. Although both RAB10 and RAB14 are regulated by the GAP activity of AS160 in vitro, only RAB10 is under the control of AS160 in vivo. Insulin regulation of the pool of RAB10 required for GLUT4 translocation occurs through regulation of AS160, since activation of RAB10 by DENND4C, its GTP exchange factor, does not require insulin stimulation.

scholarly journals Separation of Insulin Signaling into Distinct GLUT4 Translocation and Activation Steps

2004 ◽  
Vol 24 (17) ◽  
pp. 7567-7577 ◽  
Author(s):  
Makoto Funaki ◽  
Paramjeet Randhawa ◽  
Paul A. Janmey
Keyword(s):  
Plasma Membrane ◽  
Glucose Uptake ◽  
Glucose Transporter ◽  
Time Lag ◽  
Inhibitory Effect ◽  
Glut4 Translocation ◽  
Glucose Levels ◽  
Increase Glucose Uptake ◽  
A Cell ◽  
Peptide Treatment

ABSTRACT GLUT4 (glucose transporter 4) plays a pivotal role in insulin-induced glucose uptake to maintain normal blood glucose levels. Here, we report that a cell-permeable phosphoinositide-binding peptide induced GLUT4 translocation to the plasma membrane without inhibiting IRAP (insulin-responsive aminopeptidase) endocytosis. However, unlike insulin treatment, the peptide treatment did not increase glucose uptake in 3T3-L1 adipocytes, indicating that GLUT4 translocation and activation are separate events. GLUT4 activation can occur at the plasma membrane, since insulin was able to increase glucose uptake with a shorter time lag when inactive GLUT4 was first translocated to the plasma membrane by pretreating the cells with this peptide. Inhibition of phosphatidylinositol (PI) 3-kinase activity failed to inhibit GLUT4 translocation by the peptide but did inhibit glucose uptake when insulin was added following peptide treatment. Insulin, but not the peptide, stimulated GLUT1 translocation. Surprisingly, the peptide pretreatment inhibited insulin-induced GLUT1 translocation, suggesting that the peptide treatment has both a stimulatory effect on GLUT4 translocation and an inhibitory effect on insulin-induced GLUT1 translocation. These results suggest that GLUT4 requires translocation to the plasma membrane, as well as activation at the plasma membrane, to initiate glucose uptake, and both of these steps normally require PI 3-kinase activation.

scholarly journals Fluorescence Microscopy-Based Quantitation of GLUT4 Translocation: High Throughput or High Content?

2020 ◽  
Vol 21 (21) ◽  
pp. 7964
Author(s):  
Verena Stadlbauer ◽  
Peter Lanzerstorfer ◽  
Cathrina Neuhauser ◽  
Florian Weber ◽  
Flora Stübl ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Fluorescence Microscopy ◽  
High Throughput ◽  
Glucose Transporter ◽  
Large Cell ◽  
Live Cells ◽  
Glut4 Translocation ◽  
Plasma Membrane Vesicles ◽  
Innovative Strategy ◽  
Insulin Mimetic

Due to the global rise of type 2 diabetes mellitus (T2DM) in combination with insulin resistance, novel compounds to efficiently treat this pandemic disease are needed. Screening for compounds that induce the translocation of glucose transporter 4 (GLUT4) from the intracellular compartments to the plasma membrane in insulin-sensitive tissues is an innovative strategy. Here, we compared the applicability of three fluorescence microscopy-based assays optimized for the quantitation of GLUT4 translocation in simple cell systems. An objective-type scanning total internal reflection fluorescence (TIRF) microscopy approach was shown to have high sensitivity but only moderate throughput. Therefore, we implemented a prism-type TIR reader for the simultaneous analysis of large cell populations grown in adapted microtiter plates. This approach was found to be high throughput and have sufficient sensitivity for the characterization of insulin mimetic compounds in live cells. Finally, we applied confocal microscopy to giant plasma membrane vesicles (GPMVs) formed from GLUT4-expressing cells. While this assay has only limited throughput, it offers the advantage of being less sensitive to insulin mimetic compounds with high autofluorescence. In summary, the combined implementation of different fluorescence microscopy-based approaches enables the quantitation of GLUT4 translocation with high throughput and high content.

scholarly journals Inhibition of insulin-stimulated phosphorylation of the intracellular domain of phospholemman decreases insulin-dependent GLUT4 translocation in streptolysin-O-permeabilized adipocytes

1999 ◽  
Vol 343 (1) ◽  
pp. 151-157 ◽  
Author(s):  
Otto WALAAS ◽  
Robert S. HORN ◽  
S. Ivar WALAAS
Keyword(s):  
Plasma Membrane ◽  
Protein Kinase C ◽  
Protein Kinase ◽  
Glucose Transporter ◽  
Insulin Signalling ◽  
Glut4 Translocation ◽  
Intracellular Domain ◽  
Kinase C ◽  
Streptolysin O

A variety of studies indicate that protein kinase C might be involved in the insulin signalling cascade leading to translocation of the insulin-regulated glucose transporter GLUT4 from intracellular pools to the plasma membrane. Phospholemman is a plasma-membrane protein kinase C substrate whose phosphorylation is increased by insulin in intact muscle [Walaas, Czernik, Olstad, Sletten and Walaas (1994) Biochem. J. 304, 635-640]. The present study examined whether the inhibition of phospholemman phosphorylation modulates the effects of insulin on GLUT4 translocation. For this purpose, a synthetic peptide derived from the intracellular domain of phospholemman with the phosphorylatable serine residues replaced with alanine residues was prepared. This peptide was found to decrease the protein kinase C-catalysed phosphorylation of a synthetic phospholemman peptide in vitro. When introduced into streptolysin-O-permeabilized adipocytes, the peptide decreased the effects of insulin on both the phosphorylation of phospholemman and the recruitment of GLUT4 to the plasma membrane. Similarly, the internalization of phospholemman antibodies, which also decreased the protein kinase C-mediated phosphorylation of the synthetic phospholemman peptide in vitro, decreased the effect of insulin on GLUT4 translocation in the adipocytes. The results suggest that phosphorylation of the intracellular domain of phospholemman might be involved in modulating the insulin-induced translocation of GLUT4 to the plasma membrane.

The synaptic vesicle protein, cysteine-string protein, is associated with the plasma membrane in 3T3-L1 adipocytes and interacts with syntaxin 4

2001 ◽  
Vol 114 (2) ◽  
pp. 445-455
Author(s):  
L.H. Chamberlain ◽  
M.E. Graham ◽  
S. Kane ◽  
J.L. Jackson ◽  
V.H. Maier ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Blood Glucose ◽  
Glucose Transporter ◽  
Cell Types ◽  
Secretory Vesicle ◽  
Insulin Stimulation ◽  
Glucose Levels ◽  
Cysteine String Protein ◽  
Syntaxin 4 ◽  
Vesicle Protein

Adipocytes and muscle cells play a major role in blood glucose homeostasis. This is dependent upon the expression of Glut4, an insulin-responsive facilitative glucose transporter. Glut4 is localised to specialised intracellular vesicles that fuse with the plasma membrane in response to insulin stimulation. The insulin-induced translocation of Glut4 to the cell surface is essential for the maintenance of optimal blood glucose levels, and defects in this system are associated with insulin resistance and type II diabetes. Therefore, a major focus of recent research has been to identify and characterise proteins that regulate Glut4 translocation. Cysteine-string protein (Csp) is a secretory vesicle protein that functions in presynaptic neurotransmission and also in regulated exocytosis from non-neuronal cells. We show that Csp1 is expressed in 3T3-L1 adipocytes and that cellular levels of this protein are increased following cell differentiation. Combined fractionation and immunofluorescence analyses reveal that Csp1 is not a component of intracellular Glut4-storage vesicles (GSVs), but is associated with the adipocyte plasma membrane. This association is stable, and not affected by either insulin stimulation or chemical depalmitoylation of Csp1. We also demonstrate that Csp1 interacts with the t-SNARE syntaxin 4. As syntaxin 4 is an important mediator of insulin-stimulated GSV fusion with the plasma membrane, this suggests that Csp1 may play a regulatory role in this process. Syntaxin 4 interacts specifically with Csp1, but not with Csp2. In contrast, syntaxin 1A binds to both Csp isoforms, and actually exhibits a higher affinity for the Csp2 protein. The results described raise a number of interesting questions concerning the intracellular targeting of Csp in different cell types, and suggest that the composition and synthesis of GSVs may be different from synaptic and other secretory vesicles. In addition, the interaction of Csp1 with syntaxin 4 suggests that this Csp isoform may play a role in insulin-stimulated fusion of GSVs with the plasma membrane.

Effect of Addition of WZB117 as an Inhibitor of Glucose Transporter 1 for Venous Blood Glucose Determination

2020 ◽  
Author(s):  
Lei Zhang ◽  
Yaqiong Ran ◽  
Yan Zhu ◽  
Qianna Zhen
Keyword(s):  
Blood Glucose ◽  
Glucose Level ◽  
Glucose Transporter ◽  
Venous Blood ◽  
Sugar Transport ◽  
Glucose Transporter 1 ◽  
Glucose Determination ◽  
Glucose Levels ◽  
Significant Difference ◽  
Blood Glucose Determination

Abstract Objective Sodium fluoride (NaF) has been applied to inhibit glycolysis in venous specimens for decades. However, it has had little effect on the rate of glycolysis in the first 1 to 2 hours, resulting in a decrease of glucose, so a more efficient method is needed. Recently, we discovered that WZB117, a specific Glut1 inhibitor, restricts glycolysis by inhibiting the passive sugar transport of human red blood cells and cancer cells. The purpose of this study was to evaluate the results of intravenous blood glucose determination after the addition of WZB117. Methods Venous specimens from 40 pairs of healthy volunteers were collected for several days and placed in tubes containing NaF plus EDTA-disodium (Na2) without WZB117 (the A group); citric acid, trisodium citrate, and EDTA-Na2 without WZB117 (B group); and NaF plus EDTA-Na2 with WZB117 (C group). The glucose concentration was measured after venipuncture and compared with test tubes treated for 1 hour, 2 hours, and 3 hours before centrifugation. Glucose level was determined by the hexokinase method. The paired t-test was used to examine differences in glucose values at baseline and at different time points. The number of misdiagnoses and the misdiagnosis rate were calculated at 2 diagnostic stages: high risk of diabetes (glucose level of 6.1 mmol/L) and diagnosis of diabetes (glucose level of 7.0 mmol/L). Results Glucose levels decreased by 1.0% at 1 hour and by 2.1% at 3 hours in the C group tubes and simultaneously decreased by 1.7% at 1 hour and by 2.5% at 3 hours in the B group tubes. In contrast, glucose levels decreased by 4.1% at 1 hour and by 6.3% at 3 hours in the A group tubes. There was a statistically significant difference in glucose levels measured in the A group tubes and B group tubes at 1 hour, 2 hours, and 3 hours. The misdiagnosis rate of clinical diagnosis in diabetes was highest in the A group tubes (7.0‰ at 1 hour, 0.1‰ at 3 hours at 7.0 mmol/L point; 14.6‰ at 1 hour, 0.4‰ at 3 hours at 6.1 mmol/L point) and lowest in the C group tubes (2.95‰ at 1 hour, 0‰ at 3 hours at 7.0 mmol/L point; 4.8‰ at 1 hour, 0.1‰ at 3 hours at 6.1 mmol/L point). Conclusion The tube addition of WZB117 is more suitable for minimizing glycolysis and has no effect on glucose levels even if specimens are left uncentrifuged for up to 3 hours.

scholarly journals Oral DhHP-6 for the Treatment of Type 2 Diabetes Mellitus

2019 ◽  
Vol 20 (6) ◽  
pp. 1517 ◽  
Author(s):  
Kai Wang ◽  
Yu Su ◽  
Yuting Liang ◽  
Yanhui Song ◽  
Liping Wang
Keyword(s):  
Diabetes Mellitus ◽  
Type 2 Diabetes ◽  
Type 2 Diabetes Mellitus ◽  
Blood Glucose ◽  
Enzymatic Activity ◽  
Glucose Levels ◽  
Blood Glucose Levels

Type 2 diabetes mellitus (T2DM) is associated with pancreatic β-cell dysfunction which can be induced by oxidative stress. Deuterohemin-βAla-His-Thr-Val-Glu-Lys (DhHP-6) is a microperoxidase mimetic that can scavenge reactive oxygen species (ROS) in vivo. In our previous studies, we demonstrated an increased stability of linear peptides upon their covalent attachment to porphyrins. In this study, we assessed the utility of DhHP-6 as an oral anti-diabetic drug in vitro and in vivo. DhHP-6 showed high resistance to proteolytic degradation in vitro and in vivo. The degraded DhHP-6 product in gastrointestinal (GI) fluid retained the enzymatic activity of DhHP-6, but displayed a higher permeability coefficient. DhHP-6 protected against the cell damage induced by H2O2 and promoted insulin secretion in INS-1 cells. In the T2DM model, DhHP-6 reduced blood glucose levels and facilitated the recovery of blood lipid disorders. DhHP-6 also mitigated both insulin resistance and glucose tolerance. Most importantly, DhHP-6 promoted the recovery of damaged pancreas islets. These findings suggest that DhHP-6 in physiological environments has high stability against enzymatic degradation and maintains enzymatic activity. As DhHP-6 lowered the fasting blood glucose levels of T2DM mice, it thus represents a promising candidate for oral administration and clinical therapy.

scholarly journals Baking Effect on Resistant Starch Digestion from Composite Bread Produced with Partial Wheat Flour Substitution

2020 ◽  
Vol 2020 ◽  
pp. 1-13
Author(s):  
Rafael Grassi de Alcântara ◽  
Heidge Fukumasu ◽  
Paulo Cesar Fabricio Raspantini ◽  
Leonila Ester Reinert Raspantini ◽  
Caroline Joy Steel ◽  
...  
Keyword(s):  
Blood Glucose ◽  
Wheat Flour ◽  
Resistant Starch ◽  
Composite Flour ◽  
Corn Flour ◽  
Glucose Levels ◽  
Baked Goods ◽  
Blood Glucose Levels ◽  
Green Banana

The consumption of composite flour, such as green banana and corn flour, is related to maintain stable blood glucose levels, due to high resistant starch levels. However, most of these studies have conducted analyses of unprocessed food such as flour. Therefore, this study aimed to evaluate the effect of baking on resistant starch concentration and digestion from bread produced with partial wheat flour substitution. Response surface methodology was used to evaluate bread physical-chemical characteristics, and then, sensorial and nutritional qualities of the bread were evaluated. The feasibility of incorporating 40% of corn flour was demonstrated, while incorporation of 20% produced bread with similar characteristics to the control; for green banana flour, these levels were 20 and 10%, respectively. Resistant starch levels of composite breads were also enhanced by in vitro analyses. On the other hand, in vivo blood glucose levels evidenced that the ingestion of breads produced with partial wheat flour substitution by green banana or corn flour promoted a more important peak in blood glucose levels in comparison with control bread, which was never previously presented in the literature. Bread ingestion rapidly increased the blood glucose levels of rats; once during the baking process, starch granules become gelatinized and therefore easily digestible. Furthermore, this study also highlighted the lack and need for future investigation of wheat flour-substituted baked goods, in order to better understand mechanical properties formation and also product digestibility.

scholarly journals Glycemic Variability in Diabetes Increases the Severity of Influenza

mBio ◽  
2020 ◽  
Vol 11 (2) ◽  
Author(s):  
Rebecca J. Marshall ◽  
Pornthida Armart ◽  
Katina D. Hulme ◽  
Keng Yih Chew ◽  
Alexandra C. Brown ◽  
...  
Keyword(s):  
Blood Glucose ◽  
Glycemic Variability ◽  
Endothelial Barrier ◽  
Glucose Levels ◽  
Blood Glucose Levels ◽  
Severe Influenza ◽  
Increased Risk ◽  
History Of

ABSTRACT People with diabetes are two times more likely to die from influenza than people with no underlying medical condition. The mechanisms underlying this susceptibility are poorly understood. In healthy individuals, small and short-lived postprandial peaks in blood glucose levels occur. In diabetes mellitus, these fluctuations become greater and more frequent. This glycemic variability is associated with oxidative stress and hyperinflammation. However, the contribution of glycemic variability to the pathogenesis of influenza A virus (IAV) has not been explored. Here, we used an in vitro model of the pulmonary epithelial-endothelial barrier and novel murine models to investigate the role of glycemic variability in influenza severity. In vitro, a history of glycemic variability significantly increased influenza-driven cell death and destruction of the epithelial-endothelial barrier. In vivo, influenza virus-infected mice with a history of glycemic variability lost significantly more body weight than mice with constant blood glucose levels. This increased disease severity was associated with markers of oxidative stress and hyperinflammation both in vitro and in vivo. Together, these results provide the first indication that glycemic variability may help drive the increased risk of severe influenza in people with diabetes mellitus. IMPORTANCE Every winter, people with diabetes are at increased risk of severe influenza. At present, the mechanisms that cause this increased susceptibility are unclear. Here, we show that the fluctuations in blood glucose levels common in people with diabetes are associated with severe influenza. These data suggest that glycemic stability could become a greater clinical priority for patients with diabetes during outbreaks of influenza.

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