Novel hormonal delivery method using the ink-jet technology: application to pulmonary insulin therapies.

BACKGROUND: A device developed based on ink-jet printer technology can precisely control the size and volume of droplets ejected. Here, we evaluated the application of this technology to the pulmonary administration of insulin mist as a therapeutic measure for diabetes. METHODS: Insulin ejected from the ink-jet device was initially characterized by high-performance liquid chromatography (HPLC) and mass spectrometry. Its effects on D-glucose uptake rate by L6 cells were then investigated. Next, different insulin solutions (with or without additives or ink-jet processing) were subcutaneously administered, and their pharmacodynamic features were evaluated. Finally, decreases in plasma glucose level in rats were examined after ventilator-assisted pulmonary administration of insulin mist. RESULTS: Neither the HPLC nor the mass spectrometry profile of insulin was altered by the ink-jet process. The D-glucose uptake rate by L6 cells that received the recovered aerosolized insulin solution was similar to that of cells treated with control insulin, at 107%. Neither the addition of additives nor the ink-jet process used for insulin aerosolization impaired the plasma glucose-lowering action of subcutaneously injected insulin. Similarly, the efficacy of pulmonary insulin administration was not affected by the additives or the ink-jet process. Plasma glucose levels showed a trend towards decreasing after ventilator-assisted pulmonary administration of insulin mist. Plasma insulin level increased 30 min after the inhalation. CONCLUSIONS: The ink-jet process did not affect the quality or biological activity of insulin, suggesting the potential use of the ink-jet device for insulin inhalation therapy for diabetes.

Genetic association of glutathione peroxidase-1 with coronary artery calcification in type 2 diabetes: a case control study with multi-slice computed tomography.

BACKGROUND: Although oxidative stress by accumulation of reactive oxygen species (ROS) in diabetes has become evident, it remains unclear what genes, involved in redox balance, would determine susceptibility for development of atherosclerosis in diabetes. This study evaluated the effect of genetic polymorphism of enzymes producing or responsible for reducing ROS on coronary artery calcification in type 2 diabetes (T2D). METHODS: An index for coronary-arteriosclerosis, coronary artery calcium score (CACS) was evaluated in 91 T2D patients using a multi-slice computed tomography. Patients were genotyped for ROS-scavenging enzymes, Glutathione peroxidase-1 (GPx-1), Catalase, Mn-SOD, Cu/Zn-SOD, as well as SNPs of NADPH oxidase as ROS-promoting elements, genes related to onset of T2D (CAPN10, ADRB3, PPAR gamma, FATP4). Age, blood pressure, BMI, HbA1c, lipid and duration of diabetes were evaluated for a multivariate regression analysis. RESULTS: CACS with Pro/Leu genotype of the GPx-1 gene was significantly higher than in those with Pro/Pro (744 +/- 1,291 vs. 245 +/- 399, respectively, p = 0.006). In addition, genotype frequency of Pro/Leu in those with CACS >or= 1000 was significantly higher than in those with CACS < 1000 (45.5% vs. 18.8%; OR = 3.61, CI = 0.97-13.42; p = 0.045) when tested for deviation from Hardy-Weinberg's equilibrium. Multivariate regression analyses revealed that CACS significantly correlated with GPx-1 genotypes and age. CONCLUSION: The presence of Pro197Leu substitution of the GPx-1 gene may play a crucial role in determining genetic susceptibility to coronary-arteriosclerosis in T2D. The mechanism may be associated with a decreased ability to scavenge ROS with the variant GPx-1.

In vitro and pathological investigations of MODY5 with the R276X-HNF1beta (TCF2) mutation.

Maturity-onset diabetes of the young type 5 (MODY5) is caused by mutation of hepatocyte nuclear factor 1beta (HNF1 beta) (TCF2) gene, resulting in a wide range of phenotypes including diabetes and renal abnormalities, but little is known about the pathogenesis of the clinical spectrum. We describe a 27-year-old Japanese male with the MODY phenotype including an atrophic kidney and multiple renal cysts. Genetic analysis revealed the patient to be heterozygous for a nonsense mutation in codon 276 of the HNF1beta gene (CGA or Arginine to TGA or stop codon; R276X). To clarify the pathophysiological relevance of this mutation, we conducted an in vitro study monitoring human C-peptide secretion after transfecting both the HNF1beta mutant cDNA and preproinsulin cDNA into a murine beta cell line, MIN6. Functional studies of the transformed MIN6 cells indicated that expression of the R276X caused a significant decrease in glucose-stimulated insulin secretion but no change in either KCl-stimulated or basal insulin secretion. These results suggest that the R276X functions in a negative manner in regard to metabolic responses of insulin secretion in beta cells. Analysis with light and electron microscopy on biopsied kidney specimens suggested that the origin of the cysts might be glomeruli but the primary lesion could be tubules.

Enhanced insulin secretion from engineered 3T3-L1 preadipocytes by induction of cellular differentiation.

We have established insulin-secreting cell line, L1-INS/fur cells, by engineering 3T3-L1 murine preadipocytes with human preproinsulin cDNA. Analysis with HPLC, mass spectrometry and immunological assay identified human insulin in the culture medium. Notably, secretion of insulin from L1-INS/fur cell was increased 8.2 times higher after induction of cellular differentiation. The increment of insulin secretion during differentiation was further enhanced by additive treatment with thiazolidinedione, a promoting agent of adipocyte differentiation. This observation strongly suggests that the enhancement of insulin secretion is tightly associated with cellular differentiation process itself. Expression rate of the insulin transgene was not changed after the additional treatment with thiazolidinedione. On the other hand, furin gene expression by Northern analysis showed an increase, and Western analysis revealed even more reduction in cellular content of proinsulin. These results indicate that mechanism of the observed enhancement in insulin secretion during the differentiation is mainly due to increased capacity of the proinsulin processing by induction of furin. Results of our present study will provide important information on cell-based therapy using undifferentiated progenitors and tissue stem cells.