Stephen Scott

Evidence that pioglitazone, metformin and pentoxifylline are inhibitors of glycation

Enhanced formation and accumulation of advanced glycation end products (AGEs) have been proposed to play a major role in the pathogenesis of diabetic complications, and atherosclerosis, leading to the development of a range of diabetic complications including nephropathy, retinopathy and neuropathy. Several potential drug candidates as AGE inhibitors have been reported recently. Aminoguanidine is the first drug extensively studied. However, there are no currently available medications known to block AGE formation. We have previously reported a number of novel and structurally diverse compounds as potent inhibitors of glycation and AGE formation. We have now studied several of the existing drugs, which are in therapeutic practice for lowering blood sugar or the treatment of peripheral vascular disease in diabetic patients, for possible inhibitory effects on glycation. We show that that three compounds; pioglitazone, metformin and pentoxifylline are also inhibitors of glycation.

Angiotensin II Signaling in Vascular Smooth Muscle Cells Under High Glucose Conditions

The mechanisms responsible for the accelerated cardiovascular disease in diabetes, as well as the increased hypertrophic effects of angiotensin II (Ang II) under hyperglycemic conditions, are not very clear. We examined whether the culture of vascular smooth muscle cells (VSMC) under hyperglycemic conditions to simulate the diabetic state can lead to increased activation of key growth- and stress-related kinases, such as the mitogen-activated protein kinases (MAPKs), in the basal state and in response to Ang II. Treatment of porcine VSMC for short time periods (0.5 to 3 hours) with high glucose (HG; 25 mmol/L) markedly increased the activation of the extracellular signal-regulated kinase (ERK1/2) and c-Jun/N-terminal kinase (JNK) relative to cells cultured in normal glucose (NG; 5.5 mmol/L). p38 MAPK also was activated by HG, and this effect remained sustained for several hours. Ang II treatment increased the activity of all 3 families of MAPKs. Ang II-induced ERK activation was potentiated nearly 2-fold in cells treated with HG for 0.5 hour. However, Ang II-induced JNK was not altered. In VSMC cultured for 24 hours with HG, Ang II and HG displayed an additive response on p38 MAPK activity. MAPKs can lead to activation of transcription factors such as activator protein-1 (AP-1). HG alone significantly increased AP-1 DNA-binding activity. Furthermore, Ang II and HG combined had additive effects on AP-1 activity. These results suggest that increased activation of specific MAPKs and downstream transcription factors, such as AP-1, may be key mechanisms for the increased VSMC growth potential of HG alone and of Ang II under HG conditions.

Dietary magnesium prevents fructose-induced insulin insensitivity in rats.

Increased dietary fructose may produce insulin insensitivity and elevate blood pressure in rats. It is possible that the reduced magnesium content of the high-fructose commercial diet used in some studies may play a role in these abnormalities because it is known that magnesium deficiency can produce insulin insensitivity and increased angiotensin II action in humans. To study this, we maintained rats for 9 weeks on either a normal control diet, a standard high-fructose diet, or the same high-fructose diet supplemented with magnesium. Glucose uptake was assessed using a perfused rat hindquarter preparation sequentially with 0, 900, and 120,000 pmol/L of added insulin. Basal serum glucose, plasma insulin, and basal glucose uptake in the absence of insulin were similar among all three groups. However, insulin sensitivity, defined as glucose uptake in the presence of 900 pmol/L insulin minus basal, was depressed in the high-fructose compared with the control group (1.02 +/- 0.38 to 1.77 +/- 0.57 mumol/g per hour, P < .05). In contrast, the high-fructose group supplemented with normal magnesium had similar insulin sensitivity as the control group (2.09 +/- 0.69 mumol/g per hour). Total serum magnesium was reduced in the high-fructose group compared with control or high-fructose plus magnesium-supplemented groups. Blood pressure and fasting insulin levels were also lower in the magnesium-supplemented group. These results suggest that magnesium deficiency and not fructose ingestion per se leads to insulin insensitivity in skeletal muscle and changes in blood pressure.

Glucose‐Stimulated Increment in Oxygen Consumption Rate as a Standardized Test of Human Islet Quality

Standardized assessment of islet quality is imperative for clinical islet transplantation. We have previously shown that the increment in oxygen consumption rate stimulated by glucose (ΔOCRglc) can predict in vivo efficacy of islet transplantation in mice. To further evaluate the approach, we studied three factors: islet specificity, islet composition and agreement between results obtained by different groups. Equivalent perifusion systems were set up at the City of Hope and the University of Washington and the values of ΔOCRglc obtained at both institutions were compared. Islet specificity was determined by comparing ΔOCRglc in islet and nonislet tissue. The ΔOCRglc ranged from 0.01 to 0.19 nmol/min/100 islets (n = 14), a wide range in islet quality, but the values obtained by the two centers were similar. The contribution from nonislet impurities was negligible (ΔOCRglc was 0.12 nmol/min/100 islets vs. 0.007 nmol/min/100 nonislet clusters). The ΔOCRglc was statistically independent of percent beta cells, demonstrating that ΔOCRglc is governed more by islet quality than by islet composition. The ΔOCRglc, but not the absolute level of OCR, was predictive of reversal of hyperglycemia in diabetic mice. These demonstrations lay the foundation for testing ΔOCRglc as a measurement of islet quality for human islet transplantation.

Magnesium deficiency and glucose metabolism in rat adipocytes

We examined the effect of reducing ambient and intracellular free Mg ion ([Mg]i) concentrations on insulin action in epididymal adipocytes from male Sprague-Dawley rats in terms of (1) cellular transport of nonmetabolizable 2-deoxyglucose, (2) [U-14C]glucose oxidation to CO2, and (3) D-[3H]glucose incorporation into triglycerides. There were no significant differences in basal or insulin-stimulated transport of 2-deoxyglucose between adipocytes cultured in physiologic (1.24 mmol) or low (0.16 mmol) Mg for up to 24 hours. In contrast, insulin-stimulated but not basal [U-14C]glucose oxidation to CO2 was significantly reduced in adipocytes cultured in low versus physiologic Mg (P < .05 to .01). Similarly, there were no differences in basal glucose incorporation into triglycerides between cells cultured in low or physiologic Mg media for up to 24 hours. However, long-term (24-hour) but not short-term (2-hour) exposure of cells to low Mg was associated with a significant 30% reduction in insulin-stimulated D-[3H]glucose incorporation into triglycerides. When adipocytes incubated in low Mg were reincubated in high Mg (1.24 or 5 mmol) for 30 minutes, normal insulin-stimulated D-[3H]glucose incorporation into triglycerides was restored. Incubation of adipocytes in low Mg (0.16 mmol) for 24 hours resulted in a significant decrease in [Mg]i (264 +/- 89 v 437 +/- 125 micromol/cell [mean +/- SEM]) as compared with cells incubated in physiologic Mg (1.24 mmol; P < .01). These data support a role for intracellular Mg deficiency in the development of insulin resistance and suggest that the effect occurs at a site(s) distal to glucose entry into the cell. The effect of Mg deficiency on insulin action appears to be reversible.

Evidence That Angiotensin II and Lipoxygenase Products Activate c-Jun NH2-Terminal Kinase

The effect of angiotensin II (Ang II) to activate c-Jun amino-terminal kinase (JNK) was studied in a Chinese hamster ovary fibroblast cell line overexpressing the rat vascular type-1a Ang II receptor (CHO-AT1a). Ang II treatment induced a time-dependent activation of JNK. Ang II (10(-7) mol/L) activated JNK activity, with a peak at 30 minutes (9.39 +/- 2.52-fold, n = 7, P < .02 versus control), which was maintained until 3 hours (2.7 +/- 0.65-fold, n = 3, P < .02 versus control). Ang II-induced JNK activation at 30 minutes was inhibited by a specific lipoxygenase (LO) pathway inhibitor, cinnamyl-3,4-dihydroxy-alpha-cyanocinnamate (1 mumol/L) by 87.5% (n = 4, P < .01 versus Ang II-induced JNK activity). The direct addition of 12-HETE also induced a time-dependent JNK activation. 12-HETE (10(-7) mol/L) activated JNK activity, with a peak at 10 minutes (3.43 +/- 0.87-fold, n = 6, P < .02 versus control), which remained elevated until 1 hour. These results suggest that the LO pathway is a mediator of Ang II-induced JNK activation. 15-HETE can also activate JNK at 5 minutes, but this activity was reduced at 30 minutes and could not be seen at 1 hour, indicating that the time course was different from that seen with 12-HETE. N-Acetylcysteine (NAC), an antioxidant, was used to perturb intracellular reactive oxygen intermediate (ROI) levels to assess the role of endogenous ROIs in regulating JNK activity. Pretreatment of cells with 500 mumol/L NAC for 1 hour attenuated approximately 50% of Aug II-induced JNK activation, suggesting that ROIs, at least partially, mediate Ang II-induced JNK activation. Furthermore, 12-HETE-induced JNK activation was reduced by approximately 90% by NAC. Finally, pertussis toxin completely blocked 12-HETE-induced JNK activation, suggesting that Gi-protein signaling participates in 12-HETE-induced effects. These results suggest that LO activation plays a role in mediating Ang II-induced JNK activation in part by altering the redox tone and Gi-protein signaling of cells.

A new advanced glycation inhibitor, LR-90, prevents experimental diabetic retinopathy in rats

Background: Diabetic retinopathy is associated with accumulation of advanced glycation end products in the retinal microvasculature. LR-90 is an effective multistage inhibitor of advanced glycation with renoprotective and anti-inflammatory properties. Aim: To explore the role of LR-90 in the progression of experimental diabetic retinopathy. Methods: Streptozotocin-induced diabetic Sprague–Dawley rats were treated with LR-90 (50 mg/l in drinking water) for up to 32 weeks. At the end of the study, eyes were enucleated and subjected to trypsin digestion and staining with light green/haematoxylin. Acellular capillaries and pericytes were quantified in random fields using light microscopy. Results: In the LR-90-treated diabetic animals, acellular capillary numbers were reduced to 1.63 (0.20) from 2.58 (0.49) (p<0.05) in diabetic controls. LR-90 treatment also restored the pericyte deficit from 18.12 (0.98) in diabetic rats to 24.19 (0.76) (p<0.001). Conclusion: These findings show that LR-90 can effectively inhibit important lesions of diabetic retinopathy. This agent has potential for preventing retinopathy in patients with diabetes.

64Cu labeled sarcophagine exendin-4 for microPET imaging of glucagon like peptide-1 receptor expression

The Glucagon-like peptide 1 receptor (GLP-1R) has become an important target for imaging due to its elevated expression profile in pancreatic islets, insulinoma, and the cardiovascular system. Because native GLP-1 is degraded rapidly by dipeptidyl peptidase-IV (DPP-IV), several studies have conjugated different chelators to a more stable analog of GLP-1 (such as exendin-4) as PET or SPECT imaging agents with various advantages and disadvantages. Based on the recently developed Sarcophagin chelator, here, we describe the construction of GLP-1R targeted PET probes containing monomeric and dimeric exendin-4 subunit. The in vitro binding affinity of BarMalSar-exendin-4 and Mal2Sar-(exendin-4)2 was evaluated in INS-1 cells, which over-express GLP-1R. Mal2Sar-(exendin-4)2 demonstrated around 3 times higher binding affinity compared with BaMalSar-exendin-4. After 64Cu labeling, microPET imaging of 64Cu-BaMalSar-exendin-4 and 64Cu-Mal2Sar-(exendin-4)2 were performed on subcutaneous INS-1 tumors, which were clearly visualized with both probes. The tumor uptake of 64Cu-Mal2Sar-(exendin-4)2 was significantly higher than that of 64Cu-BaMaSarl-exendin-4, which could be caused by polyvalency effect. The receptor specificity of these probes was confirmed by effective blocking of the uptake in both tumor and normal positive organs with 20-fold excess of unlabeled exendin-4. In conclusion, sarcophagine cage conjugated exendin-4 demonstrated persistent and specific uptake in INS-1 insulinoma model. Dimerization of exendin-4 could successfully lead to increased tumor uptake in vivo. Both 64Cu-BaMalSar-exendin-4 and 64Cu-Mal2Sar-(exendin-4)2 hold a great potential for GLP-1R targeted imaging.

The Choice of Enzyme for Human Pancreas Digestion Is a Critical Factor for Increasing the Success of Islet Isolation

Background We evaluated 3 commercially available enzymes for pancreatic digestion by comparing key parameters during the islet isolation process, as well as islet quality after isolation. Methods Retrospectively compared and analyzed islet isolations from pancreata using 3 different enzyme groups: liberase HI (n = 63), collagenase NB1/neutral protease (NP) (n = 43), and liberase mammalian tissue-free collagenase/thermolysin (MTF C/T) (n = 115). A standardized islet isolation and purification method was used. Islet quality assessment was carried out using islet count, viability, in vitro glucose-stimulated insulin secretion (GSIS), glucose-stimulated oxygen consumption rate, and in vivo transplantation model in mice. Results Donor characteristics were not significantly different among the 3 enzyme groups used in terms of age, sex, hospital stay duration, cause of death, body mass index, hemoglobin A1c, cold ischemia time, and pancreas weight. Digestion efficacy (percentage of digested tissue by weight) was significantly higher in the liberase MTF C/T group (73.5 ± 1.5 %) when compared to the liberase HI group (63.6 ± 2.3 %) (P < 0.001) and the collagenase NB1/NP group (61.7 ± 2.9%) (P < 0.001). The stimulation index for GSIS was significantly higher in the liberase MTF C/T group (5.3 ± 0.5) as compared to the liberase HI (2.9 ± 0.2) (P < 0.0001) and the collagenase NB1/NP (3.6 ± 2.9) (P = 0.012) groups. Furthermore, the liberase MTF C/T enzymes showed the highest success rate of transplantation in diabetic non-obese diabetic severe combined immunodeficiency mice (65%), which was significantly higher than the liberase HI (42%, P = 0.001) and the collagenase NB1/NP enzymes (41%, P < 0.001). Conclusions Liberase MTF C/T is superior to liberase HI and collagenase NB1/NP in terms of digestion efficacy and GSIS in vitro. Moreover, liberase MTF C/T had a significantly higher success rate of transplantation in diabetic NOD Scid mice compared to liberase HI and collagenase NB1/NP enzymes.

Human Pancreatic Islets Isolated From Donors With Elevated HbA1c Levels: Islet Yield and Graft Efficacy.

The aim of this study was to investigate the effects of elevated donor HbA1c levels (type 2 diabetes, T2D) on the islet yield and functionality postisolation. In this retrospective analysis, donors for islet isolations were classified into two groups: T2D group (HbA1c ≥ 6.5%, n = 18) and normal group (HbA1c < 6.5%, n = 308). Optimum pancreas digestion time (switch time) was significantly higher in the T2D group compared to the normal group (13.7 ± 1.2 vs. 11.7 ± 0.1 min, respectively, p = 0.005). Islet yields were significantly lower in the T2D group compared to the control (T2D vs. control): islet equivalent (IEQ)/g (prepurification 2,318 ± 195 vs. 3,713 ± 114, p = 0.003; postpurification 1,735 ± 175 vs. 2,663 ± 89, p = 0.013) and islet particle number (IPN)/g (prepurification, 2,519 ± 336 vs. 4,433 ± 143, p = 0.001; postpurification, 1,760 ± 229 vs. 2,715 ± 85, p = 0.007). Islets from T2D pancreata had significantly lower viability (T2D vs. control: 91.9 ± 1.6 vs. 94.4 ± 0.3%, p = 0.004) and decreased oxygen consumption rate (DOCR) (T2D vs. control: 0.09 ± 0.01 and 0.21 ± 0.03 nmol O2 100 islets−1 min−1, p = 0.049). The islets isolated from T2D donor pancreata reversed diabetes in NOD-SCID mice in 9% (2/22) compared to islets from control donor pancreata, which reversed diabetes in 67% (175/260, p < 0.001). In conclusion, this study demonstrates that elevated HbA1c (≥6.5%) is associated with impairment of islet function and lower islet yield; however, these islets could not be suitable for clinical applications.