Restoration of direct pathway glycogen synthesis flux in the STZ-diabetes rat model by insulin administration.

Type 1 diabetes subjects are characterized by impaired direct pathway synthesis of hepatic glycogen that is unresponsive to insulin therapy. Since it is not known whether this is an irreversible defect of insulin-dependent diabetes, direct and indirect pathway glycogen fluxes were quantified in streptozotocin (STZ)-induced diabetic rats and compared with STZ rats that received subcutaneous or intraperitoneal insulin (I-SC or I-IP). Three groups of STZ rats were studied at 18 days post-STZ treatment. One group was administered I-SC and another I-IP as two daily injections of short-acting insulin at the start of each light and dark period for days 9-18. A third group did not receive any insulin, and a fourth group of nondiabetic rats was used as control. Glycogen synthesis via direct and indirect pathways, de novo lipogenesis, and gluconeogenesis were determined over the nocturnal feeding period using deuterated water. Direct pathway was residual in STZ rats, and glucokinase activity was also reduced significantly from control levels. Insulin administration restored both net glycogen synthesis via the direct pathway and glucokinase activity to nondiabetic control levels and improved the lipogenic pathway despite an inefficient normalization of the gluconeogenic pathway. We conclude that the reduced direct pathway flux is not an irreversible defect of insulin-dependent diabetes.

Evaluation of hepatic glucose metabolism via gluconeogenesis and glycogenolysis after oral administration of insulin nanoparticles.

Nanoparticles were designed to promote insulin intestinal absorption via the oral route, to increase portal insulin levels to better mimic the physiological pathway, providing enhanced glucose control through glycogenolysis and gluconeogenesis. Nanoparticles were formulated with insulin encapsulated in the core material consisting of alginate and dextran sulfate, associated with poloxamer and subsequently coated with chitosan then albumin. A spherical and slightly rough core was observed in electron micrographs with the appearance of a concentration gradient of the polysaccharide structure toward the periphery of the nanoparticle. Atomic force microscopy showed that the fully formed nanoparticles are about 200 nm in diameter with smooth and spherical morphology. Histopathological analysis of organs and tissues of diabetic rats dosed daily for 15 days with insulin nanoparticles was used to evaluate toxicological issues. No morphological or pathological alterations were observed in rat liver, spleen, pancreas, kidney or intestinal sections. Following, the effect of nanoencapsulated insulin on inhibiting hepatic gluconeogenesis was evaluated after a single insulin administration and oral glucose tolerance test, which represents a significant metabolic challenge to the liver. Alterations in the hepatic glucose metabolism of fasted streptozotocin-diabetic rats were determined by the percent contribution of glycogenolysis and gluconeogenesis, measured by using metabolic tracers, however similar gluconeogenesis contribution to the hepatic metabolism was observed between diabetic rats receiving nanoencapsulated insulin or insulin solution. The metabolic results may be explained by the inability of a single dose in shifting the gluconeogenesis/glycogenolysis contributions, sampling time, fasting period or by influence of the kidney enzymes and impairment in insulin signaling observed in stz-diabetic rats.

Effects of an oral insulin nanoparticle administration on hepatic glucose metabolism assessed by ¹³C and ²H isotopomer analysis.

The purpose of this study was to evaluate hepatic glucose metabolism of diabetic induced rats after a daily oral load of insulin nanoparticles over 2 weeks. After the 2-week treatment, an oral glucose tolerance test was performed with [U-¹³C] glucose and ²H2O. Plasma glucose ²H and ¹³C enrichments were quantified and the contribution of glycogenolysis and gluconeogenesis to overall glucose production were estimated. Animals with the insulin nanoparticles displayed the lowest glycemia before the oral glucose tolerance test. In all animals, 75% of the total glucose production was from gluconeogenesis and glycogen synthesis was only detected in some animals. Gluconeogenic pathway was an active contributor to hepatic glucose production and the treatment with oral delivered insulin nanoparticles did not alter this contribution, suggesting that under this treatment, protocol hepatic glucose metabolism is not the most relevant target of insulin action but instead a more generalised effect in peripheral tissues.

Sodium hydrosulfide improves the protective potential of the cardioplegic histidine buffer solution.

Since H(2)S has an emerging role as a cardioprotector, we hypothesized that NaHS addition to the new cardioplegic histidine buffer solution (HBS) could improve its cardioprotective potential. Male Wistar-Han rat hearts were divided in 4 groups: i) control, ii) perfusion control (perfusion only), iii) 6h ischemia in HBS or in a modified-HBS with 100 µM of NaHS, a H(2)S donor, (HBSM) and iv) as iii followed by 30 min reperfusion. During ischemia, aliquots of the cardioplegic solution were collected for NMR analysis. Heart mitochondria respiration and transmembrane potential were measured after ischemia or after ischemia followed by reperfusion. Proteins involved in the apoptotic signaling pathway were also quantified in both mitochondrial and tissue samples. Cardiac mechanic performance was evaluated by measuring the heart rate and the left ventricular pressure. In HBSM-preserved hearts, a) glucose consumption increased as well as lactate and alanine production during ischemia, b) heart mitochondria presented an improved phosphorylative efficiency, including decreased phosphorylative lag phase for complex I and complex II substrates, c) mitochondrial and tissue p53, Bax and caspase-9 were lower and d) there was a more positive atrial chronotropic response than in HBS-preserved hearts. We concluded that the addition of NaHS to HBS enhances glycolysis during ischemia, decreases mitochondrial dysfunction, especially by preserving the phosphorylative system, prevents apoptosis and during ischemia/reperfusion.

Effects of galactose on direct and indirect pathway estimates of hepatic glycogen synthesis.

Hepatic glycogen is formed by direct and indirect pathways whose activities reflect altered nutrition or disease. Direct/indirect pathway measurements often involve test meals where ∼ 10% of carbohydrate is galactose, but its effects on direct/indirect pathway estimates are unknown. Therefore, direct/indirect pathway contributions in 24-h fasted rats given 2g/kg 100% glucose (GLU, n=6) or 90% glucose-10% galactose (GLU+GAL, n=6) were measured by [U-(13)C]glucose dilution and by position-5/position-2 glycogen enrichment (H5/H2) from 2H(2)O. For GLU+GAL, galactose glycogenesis was independently measured with [1-(13)C]galactose. Glycogenesis was equivalent in both groups but for GLU+GAL, 23 ± 4% of glycogen was derived from galactose. [U-(13)C]glucose reported a 30 ± 3% direct pathway contribution to glycogenesis for GLU but only 20 ± 3% for GLU+GAL (p=0.012 vs. GLU). H5/H2 yielded identical direct pathway estimates (32 ± 3% GLU, 29 ± 6% GLU+GAL). Thus, galactose glycogenesis was undetected by H5/H2 while [U-(13)C]glucose reported a reduced direct/indirect pathway ratio. With [1-(13)C]galactose also present, correct glycogenic source contributions were obtained.

Quantifying hepatic glycogen synthesis by direct and indirect pathways in rats under normal ad libitum feeding conditions.

Hepatic glycogen synthesis from intact hexose (direct pathway) relative to that from gluconeogenic precursors (indirect pathway) was quantified in ad libitum-fed rats. Following (2)H(2)O administration and overnight feeding, the livers were removed and glycogen (2)H-enrichment was measured by (2)H NMR. Six controls and six rats rendered hyperglycemic by streptozotocin (STZ; fasting blood glucose = 385 +/- 31 mg/dl) were studied. The indirect pathway contribution, estimated as glycogen hydrogen 5 relative to hydrogen 2 enrichment, was 54% +/- 4% for control rats-similar to values from healthy, meal-fed humans. In STZ-treated rats, the indirect pathway contribution was significantly higher (68% +/- 4%, P < 0.05 vs. controls), similar to that of Type 1 diabetic (T1D) patients. In conclusion, sources of hepatic glycogen synthesis in rats during ad libitum nocturnal feeding were quantified by analysis of glycogen enrichment from (2)H(2)O. STZ caused alterations resembling the pathophysiology of hepatic glycogen synthesis in T1D patients.