Pulsatile Hyperglycaemia Induces Vascular Oxidative Stress and GLUT 1 Expression More Potently than Sustained Hyperglycaemia in Rats on High Fat Diet

<div><p>Introduction</p><p>Pulsatile hyperglycaemia resulting in oxidative stress may play an important role in the development of macrovascular complications. We investigated the effects of sustained vs. pulsatile hyperglycaemia in insulin resistant rats on markers of oxidative stress, enzyme expression and glucose metabolism in liver and aorta. We hypothesized that liver’s ability to regulate the glucose homeostasis under varying states of hyperglycaemia may indirectly affect oxidative stress status in aorta despite the amount of glucose challenged with.</p><p>Methods</p><p>Animals were infused with sustained high (SHG), low (SLG), pulsatile (PLG) glucose or saline (VEH) for 96 h. Oxidative stress status and key regulators of glucose metabolism in liver and aorta were investigated.</p><p>Results</p><p>Similar response in plasma lipid oxidation was observed in PLG as in SHG. Likewise, in aorta, PLG and SHG displayed increased expression of glucose transporter 1 (GLUT1), gp-91^PHOX and super oxide dismutase (SOD), while only the PLG group showed increased accumulation of oxidative stress and oxidised low density lipoprotein (oxLDL) in aorta.</p><p>Conclusion</p><p>Pulsatile hyperglycaemia induced relatively higher levels of oxidative stress systemically and in aorta in particular than overt sustained hyperglycaemia thus supporting the clinical observations that pulsatile hyperglycaemia is an independent risk factor for diabetes related macrovascular complications.</p></div

Pulsatile and chronic overt hyperglycaemia increased GLUT1 and gp91^PHOX protein expression in aorta.

<p>Western Blot analyses of GLUT1 (A) and gp91^PHOX (B) in aorta homogenates. Representive immunoblots of GLUT1, gp91^PHOX and β-actin are shown above the bars. Data are means ± SEM, n = 7–8. *p < 0.05 vs. VEH, $p < 0.05 vs. SLG.</p

Chronic sustained hyperglycaemia down regulates key glycolytic enzymes in liver.

<p>After 96 hours of different glucose infusion paradigms liver was analysed for glycogen (A) and triglyceride (B) content and the expression level of GK (protein (C); mRNA (D)); GS (protein (E); mRNA (F)); Screbp1c (protein (G); mRNA (H)). Representive immunoblots of GK, GS, Screbp1c and β-actin are shown above the bars. Data are means ± SEM, n = 7–8. *p < 0.05, **p < 0.001 and ***p<0.0001 vs. VEH. $p < 0.05 vs. SLG. £p < 0.05 vs. SHG.</p

Sustained but not Pulsatile hyperglycaemia increases oxidative stress in liver.

<p>Liver MDA (A), SOD activity (B) and protein expression of gp91^PHOX (C) were determined in liver homogenates. Representive immunoblots of gp91^PHOX and β-actin are shown above the bars. Data are means ± SEM, n = 7–8. *p<0.05, ***p<0.0001 vs. VEH.</p

Pulsatile hyperglycaemia induced increased oxidative stress status and accumulation of oxLDL in aorta.

<p>Aorta homogenates were analysed for MDA content (A), SOD activity (B) and oxLDL (C) accumulation. Data are means ± SEM, n = 7–8. *p < 0.05 vs. VEH, $p < 0.05 vs. SLG.</p

Pulsatile hyperglycaemia increases systemic oxidative stress status independent of glycemic exposure.

<p>Plasma glucose (A) and insulin (B) was followed throughout the complete study period. The PLG group were subjected to nine glucose pulses daily and the glucose levels were determined at selected time points covering the complete circadian rhythm. Plasma MDA (C) and 8-IsoP (D) was monitored daily at the exact same time point. Data are only means for plasma glucose and insulin and means ± SEM for plasma MDA and 8-IsoP, n = 7–8. *p < 0.05 vs. VEH.</p

α-MSH (100 nM) stimulated 2-Deoxy Glucose uptake and TBC1D1 S237, T596 and S700 phosphorylation +/- H89 in dissected soleus explants from WT mice.

<p>A: Soleus muscle was dissected, stimulated and 2-DG was measured as described (n indicated in the individual bars). B: Phosphorylation of TBC1D1 was measured in soleus muscle using WB as described. TBC1D1 S237, T596 and S700 phosphorylation is normalized to total TBC1D1. Findings are shown as a representative immunoblot and pooled data quantified in bar graphs as arbitrary units. 2-way RM ANOVA was used to calculate statistical significance (*p < 0.05, **p < 0.01, ***p < 0.001 vs. vehicle). ^# indicates a significant effect of genotype (^#p < 0.05, ^##p < 0.01, ^###p < 0.001). Data generated in the experiment are only obtained from experiment day 4.</p

α-MSH (100 nM) stimulated TBC1D1 S237 and T596 phosphorylation in WT and AMPK KD mice.

<p>TBC1D1 S237 and T596 phosphorylation sites were measured in soleus muscle using WB as described (n indicated in the individual bars). Phosphorylation of TBC1D1 S237 and T596 is normalized to total TBC1D1. Findings are shown as representative immunoblots and pooled data is quantified in bar graphs as arbitrary units. 2-way RM ANOVA was used to calculate statistical significance (*p < 0.05, **p < 0.01, ***p < 0.001 vs. vehicle). ^# indicates a significant effect of genotype (^#p < 0.05, ^##p < 0.01, ^###p < 0.001).</p

α-MSH stimulated 2-Deoxy Glucose uptake in soleus and extensor digitorum longus (EDL) muscle explants.

<p>3.A: soleus explants from WT and AMPK KD mice were stimulated with α-MSH (100 nM). Data is presented as the mean ± SEM of pooled data from a series of experiments (see individual bars). 3.B: EDL explants from WT and AMPK KD mice were stimulated with α-MSH (100 nM). 3.C: Phosphorylation of Akt was measured in soleus explants after α-MSH-stimulation (100 nM). Data is presented as the mean ± SEM. 2-way RM ANOVA was used to calculate statistical significance (*p < 0.05, **p < 0.01, ***p < 0.001 vs. vehicle). ^# indicates a significant effect of genotype (^#p < 0.05, ^##p < 0.01, ^###p < 0.001).</p

Identification and in Vivo and in Vitro Characterization of Long Acting and Melanocortin 4 Receptor (MC4-R) Selective α-Melanocyte-Stimulating Hormone (α-MSH) Analogues

We report in vitro and in vivo data of new α-melanocyte-stimulating hormone (α-MSH) analogues which are N-terminal modified with a long chain fatty acid derivative. While keeping the pharmacophoric motif (d-Phe-Arg-Trp) fixed, we tried to improve selectivity and physicochemical parameters like solubility and stability of these analogues by replacing amino acids further away from the motif. Receptor specific changes in binding affinity to the melanocortin receptors were observed between the acetyl derivatives and the fatty acid analogues. Furthermore, amino acids at the N-terminal of α-MSH (Ser-Tyr-Ser) not considered to be part of the pharmacophore were found to have an influence on the MC4/MC1 receptor selectivity. While the acetyl analogues have an in vivo effect for around 7 h, the long chain fatty acid analogues have an effect up to 48 h in an acute feeding study in male Sprague–Dawley rats after a single subcutaneous administration