Liudmyla Lozinska

Fructose and glucose differentially affect aging and carbonyl/oxidative stress parameters in Saccharomyces cerevisiae cells

Fructose is commonly used as an industrial sweetener and has been excessively consumed in human diets in the last decades. High fructose intake is causative in the development of metabolic disorders, but the mechanisms underlying fructose-induced disturbances are under debate. Fructose compared to glucose has been found to be a more potent initiator of the glycation reaction. Therefore, we supposed that glucose and fructose might have different vital effects. Here we compare the effects of glucose and fructose on yeast cell viability and markers of carbonyl/oxidative stress. Analysis of the parameters in cells growing on glucose and fructose clearly reveals that yeast growing on fructose has higher levels of carbonyl groups in proteins, α-dicarbonyl compounds and reactive oxygen species. This may explain the observation that fructose-supplemented growth as compared with growth on glucose resulted in more pronounced age-related decline in yeast reproductive ability and higher cell mortality. The results are discussed from the point of view that fructose rather than glucose is more extensively involved in glycation and ROS generation in vivo, yeast aging and development of carbonyl/oxidative stress. It should be noted that carbohydrate restriction used in this study does not reveal a significant difference between markers of aging and carbonyl/oxidative stress in yeasts cultivated on glucose and fructose.

Fructose protects baker's yeast against peroxide stress: potential role of catalase and superoxide dismutase

The negative effects of fructose due to its chronic consumption are well documented, while short-term application of fructose is found to protect different types of cells against oxidative stress. Reactive oxygen species (ROS) are suggested to mediate both the cytotoxic and defensive effects. Here, we compare the influence of glucose and fructose on yeast under H(2)O(2)-induced stress. Under control conditions, fructose-grown comparing with glucose-grown yeast demonstrated higher metabolic activity and ROS level. Therefore, fructose was suggested to provoke a mild stress that resulted in the acquisition of cellular resistance to lethal challenges, which explained the higher survival of fructose-grown yeast under H(2)O(2)-induced shock. Exposure to H(2)O(2) increased ROS level in glucose-grown cells, whereas it decreased the ROS level in fructose-grown cells. Hydrogen peroxide activated superoxide dismutase (SOD) and catalase in both the cell types studied, but glucose-grown cells demonstrated a sharp rise of the activities, while cells grown on fructose showed a broad peak of activation. Thus, fructose is likely to protect the antioxidant enzymes against their inactivation by H(2)O(2). Despite a different type of the enzyme activation in both the studied cell types (glucose- and fructose-grown), a strong positive correlation between SOD and catalase was found. The physiological meaning of this relationship and possible mechanisms of the fructose protective effect are discussed.

Fructose compared with glucose is more a potent glycoxidation agent in vitro, but not under carbohydrate-induced stress in vivo: potential role of antioxidant and antiglycation enzymes

The contribution of carbohydrates to non-enzymatic processes such as glycation/autoxidation has been extensively investigated over the last decades. This may be attributed to either beneficial or detrimental effects of reducing carbohydrates, and most studies in the field of glycoxidation are focused on glucose. Non-enzymatic reactions of fructose have not been as thoroughly investigated as those of glucose. To compare glucose and fructose involvement in the generation of glycoxidation products under experimental conditions close to the physiological situation, we used intact Saccharomyces cerevisiae cells as in vivo model and cell-free extracts prepared from whole yeast cells as in vitro model. Both intact cells and cell-free extracts were incubated with glucose or fructose. It was shown that: (i) in vitro fructose was more reactive than glucose and produced higher level of autoxidation and glycation products; (ii) no substantive differences were observed for the effect of glucose and fructose on the intracellular level of glycoxidation products, when intact yeast cells were exposed to the high concentration of hexoses; (iii) the activity of defensive enzymes (superoxide dismutase, catalase, glyoxalases, and glutathione reductase) was increased in both glucose- and fructose-stressed yeasts, indicating the development of oxidative/carbonyl stress; (iv) glucose-6-phosphate dehydrogenase activity significantly dropped in yeast exposed to both hexoses, demonstrating its high sensitivity to reactive oxygen and carbonyl species; and (v) fructose more markedly activated glyoxalases than glucose. Involvement of glucose and fructose in the glycoxidation reactions as well as potential role of antioxidant and antiglycation enzymes in yeast protection against glycoxidation are discussed.

Decreased insulin secretion and glucose clearance in exocrine pancreas-insufficient pigs.

What is the central question of this study? Does the exocrine pancreas have an impact on endocrine pancreatic function and peripheral nutrient utilization? What is the main finding and its importance? In an exocrine pancreas‐insufficient pig model, the insulin response to a glucose load was delayed. Oral enzyme supplementation did not improve the insulin release but facilitated blood glucose clearance. These results suggest an acino‐insular axis communication affecting islet function and an impact of gut pancreatic enzymes on blood glucose utilization.

Enteral Pancreatic-like Enzymes of Microbial Origin affect Insulin Release during an Intravenous Glucose Tolerance Test

We have previously shown that the presence of pancreatic enzymes in the gut lumen of exocrine pancreatic insufficient pigs influences blood glucose and insulin levels during an intravenous glucose tolerance test (IVGTT). The present study aims to highlight the effects of orally applied pancreatic-like enzymes on blood glucose and plasma insulin levels during an IVGTT in young intact pigs. Five, 7-week old pigs were fed with pancreatic-like enzymes of microbial origin, a proteinase (from Aspergillus melleus), α-amylase (from Aspergillus oryzae) or lipase (from Burkholderia cepacia) alone or in combination with the Ca/Na salts of α-ketoglutaric acid (AKG). One hour following administration of the various supplements an IVGTT was performed. Blood samples were withdrawn during the 2 hours of IVGTT for glucose and insulin analyses. Blood glucose during the IVGTT was identical following administration of all combinations of the various enzymes or enzyme mixtures. Enteral loading of amylase or any amylase containing mixture resulted in reduced insulin secretion while administration of proteinase or any proteinase containing mixture resulted in enhanced insulin secretion during IVGTT, as compared to the control water vehicle. Lipase or AKG and lipase or AKG containing mixtures did not affect insulin secretion. Thus, it can be suggested that host amylase/protease ratio and their amount in pancreatic juice can participate in regulation of insulin release, thus, possibly affecting development of obesity and diabetes type 2.

Experiments suggesting extra-digestive effects of enteral pancreatic amylase and its peptides on glucose homeostasis in a pig model

The studies presented were designed to highlight the impact of pancreatic enzymes on glycemic control and insulin response. Blood glucose and plasma insulin levels were monitored after intravenous, oral or direct gut glucose tolerance tests (GTT) in 6 pigs with an intact gastrointestinal tract and in 12 pigs following duodenal-jejunal bypass (DJB) surgery. In the intact pigs, pancreatic enzymes (Creon®) given orally 1 h prior to the GTT, lowered the blood glucose levels during the oral and meal GTT and reduced the plasma insulin response during the intravenous and meal GTT. In DJB pigs, blood glucose and plasma insulin levels were higher following glucose loading into the by-passed biliopancreatic limb as compared to that following glucose loading orally or into the common intestinal limb. Infusion of amylase or amylase peptides together with glucose into the biliopancreatic limb lowered blood glucose levels in DJB pigs. These preliminary data suggest new, extra-digestive, actions of enteral pancreatic enzymes – probably amylase or its peptides – on glucose homeostasis, with an reduction in net glucose absorption into the blood and in insulin response. This ability of digestive enzymes (amylase) to reduce post-prandial hyperglycaemia in an insulin-independent manner could aid in preventing the development of obesity and diabetes.

The inverse relationship between blood amylase and insulin levels in pigs during development, bariatric surgery, and intravenous infusion of amylase

The purpose of this research is to explore the link between plasma amylase and insulin levels in growing pigs. Blood was obtained from piglets ranging in age from preterm (8 days to full gestation period), up to postnatal day 90 (2 months post-weaning) that underwent either duodenal-jejunal bariatric interposition surgery or a sham-operation. Plasma amylase activities in preterm and full-term neonates ranged between 500–600 U/L and were decreased by 50% two months post-weaning. Preprandial insulin and C-peptide levels in neonate piglets were not detectable, however they rose gradually after weaning. An increase in plasma amylase activity was observed in the young pigs that underwent duodenal-jejunum bypass (metabolic) surgery. The increase in blood pancreatic amylase activity after an intravenous amylase infusion lowered the subsequent glucose-stimulated insulin/C-peptide release. We suggest a role for blood amylase in the regulation of glucose homeostasis after observing high blood amylase levels in neonate pigs, in pigs that underwent metabolic surgery, and as a result of the reduced glucose-stimulated insulin response following intravenous amylase administration. Blood amylase level is a dynamic physiological parameter, which is not merely a consequence of exocrine pancreatic digestive enzyme production, but rather a regulated factor involved in glucose assimilation and prandial insulin regulation.

Importance of neonatal immunoglobulin transfer for hippocampal development and behaviour in the newborn pig

Neurological disorders are among the main clinical problems affecting preterm children and often result in the development of communication and learning disabilities later in life. Several factors are of importance for brain development, however the role of immunoglobulins (passive immunity transfer) has not yet been investigated. Piglets are born agammaglobulinemic, as a result of the lack of transfer of maternal immunoglobulins in utero, thus, they serve as an ideal model to mimic the condition of immunoglobulin deficiency in preterm infants. Thirty six, unsuckled newborn piglets were fed an infant formula or colostrum and supplemented orally or intravenously with either species-specific or foreign immunoglobulin and then compared to both newborn and sow-reared piglets. Two days after the piglets were born behavioural tests (novel recognition and olfactory discrimination of conspecifics scent) were performed, after which the piglets were sacrificed and blood, cerebrospinal fluid and hippocampi samples were collected for analyses. Both parameters of neuronal plasticity (neuronal maturation and synapse-associated proteins) and behavioural test parameters appeared to be improved by the appearance of species-specific porcine immunoglulin in the circulation and cerebrospinal fluid of the piglets. In conclusion, we postulate possible positive clinical effects following intravenous infusion of human immunoglobulin in terms of neuronal plasticity and cognitive function in preterm infants born with low blood immunoglobulin levels.

Monitoring changes in plasma levels of pancreatic and intestinal enzymes in a model of pancreatic exocrine insufficiency – induced by pancreatic duct-ligation – in young pigs

PURPOSE Plasma levels of pancreatic and intestinal enzymes were measured after pancreatic duct ligation (PDL) to monitor pancreatic exocrine insufficiency (PEI) in a model using young pigs. MATERIAL/METHODS Five, 6 week-old pigs (10.9±0.2kg), underwent PDL while age-matched, un-operated pigs were used as controls. Plasma levels of immunoreactive cationic trypsinogen (IRCT), amylase, lipase, and diamine oxidase (DAO) activities were analyzed for 48 days after PDL, including 1 week of oral pancreatic enzyme supplementation (PES) with Creon(®). RESULTS PDL resulted in an arrested body growth and a rapid surge of pancreatic enzymes (IRCT, amylase and lipase) into the plasma. Nine days after PDL, the plasma levels of these pancreatic enzymes had decreased. IRCT then remained below the level in un-operated pigs while amylase only fell below control at 25 days. The intestinally derived marker DAO and plasma protein levels were unaffected by PDL but DAO decreased slightly with time in PEI pigs. One-week of oral PES restored body growth, but had little effect on pancreatic enzyme plasma levels, except for a tendency towards increased DAO. CONCLUSIONS The study showed that PEI developed within 1-2 weeks after PDL and that only IRCT is a reliable plasma enzyme marker for this. The reduced plasma DAO indicated that PEI also affected the intestines, while PES therapy restored growth of the PDL pigs and slightly increased plasma DAO, suggesting an improved intestinal function.

Oral uricase eliminates blood uric acid in the hyperuricemic pig model

An elevated level of serum uric acid—hyperuricemia, is strongly associated with the development of gout and chronic kidney disease (CKD) which is often accompanied by a significantly reduced glomerular filtration rate (GFR). In the present study, we investigated the extra-renal elimination of uric acid via the intestine in a healthy pig model and the effect of oral uricase therapy on plasma uric acid concentrations in pigs with induced hyperuricemia and CKD. The experiment was conducted on eleven, ten-week-old pigs (n = 11). The porcine model of CKD was developed by performing 9/10 nephrectomy surgery on eight pigs. A stable model of hyperuricemia was established in only five of the eight nephrectomized pigs by frequent injections of uric acid (UA) into the jugular vein. All pigs (three healthy pigs and five CKD pigs) were operated for implantation of jugular vein catheters and the three healthy pigs also had portal vein catheters inserted. Blood uric acid concentrations were measured spectrophotometrically, using the Uric Acid Assay Kit (BioAssay Systems, Hayward, USA). The piglets with CKD received orally administered uricase (treatment) and served as their own controls (without uricase supplementation). Oral uricase therapy significantly decreased plasma uric acid concentrations in pigs with CKD, whereas hyperuricemia was observed in the pigs whilst not being treated with uricase. Urinary uric acid excretion was similar during both the treatment and control periods during the first 8 h and 24 h after UA infusions in the CKD pigs. To demonstrate the elimination of UA via the intestine, the healthy pigs were infused with UA into the jugular vein. The blood collected from the jugular vein represents circulating UA concentrations and the blood collected from the portal vein represents the concentration of UA leaving the intestine. The final (after 2 h) concentration of UA was significantly lower in blood collected from the portal vein compared to that collected from the jugular vein (3.34 vs. 2.43 mg/dL, respectively, p = 0.024). The latter allows us to suggest that UA is eliminated from the blood via the gut tissue.