DHEA-pretreatment attenuates oxidative stress in kidney-cortex and liver of diabetic rabbits and delays development of the disease.

In view of reported discrepancies concerning antioxidant activity of dehydroepiandrosterone (DHEA), a widely used dietary supplement, the current investigation was undertaken to evaluate the antioxidant properties of DHEA in both kidney-cortex and liver of alloxan (ALX)-induced diabetic rabbits, as this diabetogenic compound exhibits the ROS-dependent action. ALX was injected to animals following 7 days of DHEA administration. Four groups of rabbits were used in the experiments: control, DHEA-treated control, diabetic and DHEA-treated diabetic. Our results show for the first time, that in kidney-cortex DHEA resulted in normalization of hydroxyl free radicals (HFR) levels and restoration of catalase (CAT) and glutathione peroxidase (GPx) activities to near the control values, while in liver DHEA prevented the malondialdehyde (MDA) accumulation and normalized glutathione reductase (GR) and glucose-6-phosphate dehydrogenase (G6PDH) activities. Moreover, in both kidney-cortex and liver DHEA supplementation prevented GSSG elevation accompanied by a decrease in GSH/GSSG ratio. Although DHEA attenuated oxidative stress in both kidney-cortex and liver of ALX-induced diabetic rabbits and significantly delayed the onset of diabetes in time, it did not protect against the final development of diabetes. In conclusion, the current investigation underscores the complexity of the antioxidant action of DHEA. The data are of clinical interest since DHEA supplementation could prevent the deleterious effects of ROS and delay, or even prevent the onset of many diseases. However, in view of the reported pro-oxidant effects of high DHEA doses, the potential use of this agent as a supplement needs a careful evaluation.

Hypoxia increases the rate of renal gluconeogenesis via hypoxia-inducible factor-1-dependent activation of phosphoenolpyruvate carboxykinase expression.

Although up to 25% of glucose released into circulation in the postabsorptive state comes from renal gluconeogenesis, the regulatory mechanisms of this process are still poorly recognized, comparing to hepatic ones. The aim of the present study was to examine if hypoxia-inducible factor-1 (HIF-1) might be involved in the regulation of glucose de novo synthesis in kidneys. It was found that HK-2 cells (immortalized human kidney proximal tubules, capable of gluconeogenesis/glycogen synthesis) cultured with gluconeogenic substrates either in hypoxia (1% O2) or in the presence of DMOG (an inhibitor of HIF-1α degradation) exhibited increased glycogen content. This phenomenon was not correlated with augmented glucose intake and the effects were reversed by echinomycin (an inhibitor of HIF-1 binding to HRE sequence). As concluded from the measurement of the intracellular content of gluconeogenic intermediates followed by Western blot analysis, under conditions of hypoxia/increased HIF-1 level the activity of phosphoenolpyruvate carboxykinase (PEPCK) was elevated, as a result of increased expression of the cytosolic isoform of PEPCK (PEPCK-C). Chromatin immunoprecipitation (ChIP) analysis proved HIF-1 ability to bind to the promoter region of PEPCK-C gene. The final conclusion that hypoxia/HIF-1 accelerates the rate of renal glucogenesis via the mechanism engaging activation of PEPCK-C expression might be useful in terms of e.g. diabetes treatment, as it is commonly accepted that under diabetic conditions kidneys and liver seem to be equally important sources of glucose synthesized de novo.

Biosynthesis of Carnosine and Related Dipeptides in Vertebrates.

Carnosine (ß-alanyl-L-histidine) and its methylated derivatives: anserine (ß-alanyl-Nπ- methyl-L-histidine) and balenine (ß-alanyl-Nτ-methyl-L-histidine) are abundant constituents of excitable tissues of vertebrates. While carnosine and anserine are present at high concentrations and in variable proportions in skeletal muscle and brain of most vertebrates, balenine appears to be rather more abundant in marine mammals and certain reptilian species. Since the discovery of these compounds at the beginning of 20th century, numerous studies have been devoted to identification of the biochemical and physiological properties of carnosine and related dipeptides. These led to the discovery of the pHbuffering, metal-chelation and antioxidant, capabilities of carnosine and anserine, although no definitive ideas concerning their physiological role has yet been formulated. Only recently the molecular identities of the enzymes catalyzing synthesis of carnosine (carnosine synthase, EC 6.3.2.11) and anserine (carnosine N-methyltransferase, EC 2.1.1.22) have been elucidated, which has given a new insight into their metabolism in vertebrates. These findings have opened new research areas and provide authentic opportunities for understanding the biological function of these "enigmatic" dipeptides. This review aims to summarize recent advances in our knowledge concerning enzymes responsible for the biosynthesis of carnosine and related dipeptides and to evaluate their importance in vertebrate physiology.

DHEA supplementation to dexamethasone-treated rabbits alleviates oxidative stress in kidney-cortex and attenuates albuminuria.

Our recent study has shown that dehydroepiandrosterone (DHEA) administered to rabbits partially ameliorated several dexamethasone (dexP) effects on hepatic and renal gluconeogenesis, insulin resistance and plasma lipid disorders. In the current investigation, we present the data on DHEA protective action against dexP-induced oxidative stress and albuminuria in rabbits. Four groups of adult male rabbits were used in the in vivo experiment: (1) control, (2) dexP-treated, (3) DHEA-treated and (4) both dexP- and DHEA-treated. Administration of dexP resulted in accelerated generation of renal hydroxyl free radicals (HFR) and malondialdehyde (MDA), accompanied by diminished superoxide dismutase (SOD) and catalase activities and a dramatic rise in urinary albumin/creatinine ratio. Treatment with DHEA markedly reduced dexP-induced oxidative stress in kidney-cortex due to a decline in NADPH oxidase activity and enhancement of catalase activity. Moreover, DHEA effectively attenuated dexP-evoked albuminuria. Surprisingly, dexP-treated rabbits exhibited elevation of GSH/GSSG ratio, accompanied by a decrease in glutathione peroxidase (GPx) and glutathione-S-transferase (GST) activities as well as an increase in glucose-6-phosphate dehydrogenase (G6PDH) activity. Treatment with DHEA resulted in a decline in GSH/GSSG ratio and glutathione reductase (GR) activity, accompanied by an elevation of GPx activity. Interestingly, rabbits treated with both dexP and DHEA remained the control values of GSH/GSSG ratio. As the co-administration of DHEA with dexP resulted in (i) reduction of oxidative stress in kidney-cortex, (ii) attenuation of albuminuria and (iii) normalization of glutathione redox state, DHEA might limit several undesirable renal side effects during chronic GC treatment of patients suffering from allergies, asthma, rheumatoid arthritis and lupus. Moreover, its supplementation might be particularly beneficial for the therapy of patients with glucocorticoid-induced diabetes.

DHEA-induced modulation of renal gluconeogenesis, insulin sensitivity and plasma lipid profile in the control- and dexamethasone-treated rabbits. Metabolic studies.

In view of antidiabetic and antiglucocorticoid effects of dehydroepiandrosterone (DHEA) both in vitro and in vivo studies were undertaken: (i) to elucidate the mechanism of action of both dexamethasone phosphate (dexP) and DHEA on glucose synthesis in primary cultured rabbit kidney-cortex tubules and (ii) to investigate the influence of DHEA on glucose synthesis, insulin sensitivity and plasma lipid profile in the control- and dexP-treated rabbits. Data show, that in cultured kidney-cortex tubules dexP significantly stimulated gluconeogenesis by increasing flux through fructose-1,6-bisphosphatase (FBPase). DexP-induced effects were dependent only upon glucocorticoid receptor. DHEA decreased glucose synthesis via inhibition of glucose-6-phosphatase (G6Pase) and suppressed the dexP-induced stimulation of renal gluconeogenesis. Studies with the use of inhibitors of DHEA metabolism in cultured renal tubules showed for the first time that DHEA directly affects renal gluconeogenesis. However, in view of analysis of glucocorticoids and DHEA metabolites levels in urine, it seems likely, that testosterone may also contribute to DHEA-evoked effects. In dexP-treated rabbits, plasma glucose level was not altered despite increased renal and hepatic FBPase and G6Pase activities, while a significant elevation of both plasma insulin and HOMA-IR was accompanied by a decline of ISI index. It thus appears that increased insulin levels were required to maintain normoglycaemia and to compensate the insulin resistance. DHEA alone affected neither plasma glucose nor lipid levels, while it increased insulin sensitivity and diminished both renal and hepatic G6Pase activities. Surprisingly, DHEA co-administrated with dexP did not alter insulin sensitivity, while it partially suppressed the dexP-induced elevation of renal G6Pase activity and plasma cholesterol and triglyceride contents. As (i) gluconeogenic pathway in rabbit is similar to that in human, and (ii) DHEA counteracts several dexP-evoked effects, it seems likely, that its supplementation might be beneficial to patients treated with glucocorticoids.

Proinsulin C-peptide potentiates the inhibitory action of insulin on glucose synthesis in primary cultured rabbit kidney-cortex tubules: metabolic studies.

Effects of equimolar concentrations of proinsulin C-peptide and insulin on glucose synthesis were studied in primary cultures of rabbit kidney-cortex tubules grown in the presence of alanine, glycerol, and octanoate. The rhodamine-labeled C-peptide entered renal tubular cells and localized in nuclei, both in the presence and absence of insulin; preincubations with the unlabeled compound inhibited internalization. C-peptide did not affect glucose formation when added alone but potentiated the inhibitory action of insulin by about 20% due to a decrease in flux through glucose-6-phosphate isomerase (GPI) and (or) glucose-6-phosphatase (G6Pase). GPI inhibition was caused by: (i) increased intracellular contents of fructose-1,6-bisphosphate and fructose-1-phosphate, inhibitors of the enzyme and (ii) reduced level of the phosphorylated GPI, which exhibits higher enzymatic activity in the presence of casein kinase 2. A decrease in flux through G6Pase, due to diminished import of G6P by G6P-transporter from the cytoplasm into endoplasmic reticulum lumen, is also suggested. The data show for the first time that in the presence of insulin and C-peptide, both GPI and G6P-ase may act as regulatory enzymes of renal gluconeogenic pathway.

[The role of gut microbiota in the pathogenesis of obesity and diabetes]. / Rola mikroflory jelitowej w patogenezie otylosci i cukrzycy.

The rapidly increasing number of people with obesity and type 2 diabetes is one of the most serious problems of the contemporary world. Until recently, it was thought that the main cause of this phenomenon is the change of lifestyle and dietary habits. According to recent reports, the gut microbiota may also play an important role in the "epidemic" of obesity and diabetes. Changes in its composition have been observed in people suffering from these diseases. In addition, the fact that the intestinal microbiota may affect body weight, insulin sensitivity or sugar and lipid metabolism has led to the hypothesis that these changes may contribute to the pathogenesis of obesity and diabetes. Scientists, using antibacterial drugs, pro- and prebiotics, are trying to modify the intestinal flora and thus affect its interaction with the host. The results are very promising, lead to further analysis and indicate gut microbiota as a potential therapeutic target for obesity and diabetes treatment.

[Influence of bariatric surgery on remission of type 2 diabetes]. / Wplyw operacji bariatrycznych na ustepowanie cukrzycy typu 2.

The plague of obesity afflicts an increasing group of people. Moreover type 2 diabetes, which is the most serious illness accompanying excessive weight, is becoming more and more common. Traditional methods of obesity treatment, such as diet and physical exercise, fail. This applies especially to people with class III obesity. The only successful way of treating obesity in their case is bariatric surgery. There are three types of bariatric surgery: restrictive procedures (reducing stomach volume), malabsorptive procedures, and mixed procedures, which combine both methods. In spite of the risk connected with the surgery and complications after it, bariatric procedures are advised to patients with class III obesity and class II with an accompanying illness which increases the probability of death. It has been proved that bariatric surgery not only eliminates obesity but also very frequently (in 90% of cases) leads to the remission of type 2 diabetes. Moreover, the remission occurs very fast--it takes place a long time before the patients reduce their weight, even within a few days after surgery. Detailed studies have shown that the remission of diabetes is caused mostly by the change of the gastro-intestinal hormones' profile, resulting from the surgery. These hormones include GLP-1, GIP, peptide YY, ghrelin and oxyntomodulin. Additionally, the change of the amount of adipose tissue after the surgery influences the level of adipokines, i.e. the hormones of the adipose tissue, among which the most important are leptin, adiponectin and resistin. Thus, bariatric surgery not only changes the shape of the gastrointestinal tract but it also modulates the hormonal activity. Bariatric surgery is considered as therapy not only for the obese but also for diabetic patients.

[Physiology and molecular mechanism of glucocorticoid action]. / Fizjologia i molekularny mechanizm dzialania glikokortykoidów.

Endogenous glucocorticoids (GCs) are secreted into the systemic circulation from the adrenal cortex. This release is under the control of the circadian clock and can be enhanced at any time in response to a stressor. The levels of circulating GC are regulated systemically by the hypothalamo-pituitary-adrenal axis and locally by access to target cells and pre-receptor metabolism by 11beta-hydroxysteroids dehydrogenase enzymes. GCs mediate their genomic action by binding to two different ligand-inducible transcription factors: high-affinity mineralocorticoid receptor (MR) and 10-fold lower affinity glucocorticoid receptors (GRs). Responses to GCs vary among individuals, cells, and tissues. The diversity and specificity in the steroid hormone's response in the cell is controlled at different levels, including receptor translocation, interaction with specific transcription factors and coregulators, and the regulation of receptor protein levels by microRNA. Moreover, multiple GR isoforms are generated from one single GR gene by alternative splicing and alternative translation initiation. These isoforms all have unique tissue distribution patterns and transcriptional regulatory profiles. Furthermore, each is subjected to various post-translational modifications that affect receptor function. Deciphering the molecular mechanisms of GC action is further complicated by the realization that GCs can induce rapid, non-genomic effects within the cytoplasm. A tight regulation of GC secretion and their cell-specific activity is essential for proper organism function. This is particularly seen under conditions of GC deficiency or excess, as in Addison's disease and Cushing's syndrome, respectively.

Differential action of methylselenocysteine in control and alloxan-diabetic rabbits.

Antidiabetic action of inorganic selenium compounds is commonly accepted. Since in diet selenium mainly exists as selenoamino acids, potential hypoglycemic properties of methylselenocysteine (MSC) were investigated in four groups of rabbits: untreated and MSC-treated control animals as well as alloxan-diabetic and MSC-treated diabetic rabbits. MSC (at a dose of 1mg/kg body weight) was administered daily for 3 weeks via intraperitoneal injection. The data show, that in MSC-treated control animals plasma glucose concentration was diminished, while plasma urea and creatinine levels as well as urine albumin content were elevated and necrotic changes occurred in kidney-cortex. Decreased GSH/GSSG ratios in blood, liver and kidney-cortex were accompanied by increased glutathione peroxidase and glutathione reductase activities and a diminished renal gamma-glutamylcysteine synthetase activity. Death of 50% of control animals was preceded by a dramatic decline in blood glucose concentration. Surprisingly, in MSC-treated diabetic rabbits, plasma glucose levels were either normalized or significantly decreased. Blood and liver GSH/GSSG ratios were increased and renal functions were markedly improved, as indicated by a diminished albuminuria and attenuated histological changes characteristic of diabetes. However, after administration of MSC to diabetic rabbits plasma urea and creatinine levels as well as renal GSH/GSSG ratios were not altered. In view of MSC-induced marked accumulation of selenium in kidneys and liver of control rabbits, accompanied by a decline in blood glucose level, disturbance of glutathione homeostasis and kidney-injury, application of MSC in chemotherapy needs a careful evaluation. On the contrary, MSC supplementation might be beneficial for diabetes therapy due to an improvement of both glycemia and renal function.

Differential effects of selenium compounds on glucose synthesis in rabbit kidney-cortex tubules and hepatocytes. In vitro and in vivo studies.

Although selenium is taken with diet mainly as selenoamino acids, its hypoglycaemic action on hepatic gluconeogenesis has been studied with the use of inorganic selenium derivatives. The aim of the present investigation was to compare relative efficacies of inorganic and organic selenium compounds in reducing glucose synthesis in hepatocytes and renal tubules, significantly contributing to the glucose homeostasis. In contrast to hepatocytes, both selenite and methylselenocysteine inhibited renal gluconeogenesis by about 40-45% in control rabbits. Selenate did not affect this process, whereas selenomethionine inhibited gluconeogenesis by about 20% in both hepatocytes and renal tubules. In contrast to methylselenocysteine, selenite decreased intracellular ATP content, glutathione reduced/glutathione oxidized (GSH/GSSG) ratio and pyruvate carboxylase, PEPCK and FBPase activities, while methylselenocysteine diminished PEPCK activity due to elevation of intracellular 2-oxoglutarate and GSSG, inhibitors of this enzyme. Experiments in vivo indicate that in 3 of 9 alloxan-diabetic rabbits treated for 14 days with methylselenocysteine (0.182mg/kg body weight) blood glucose level was normalized, whereas in all diabetic rabbits plasma creatinine and urea levels decreased from 2.52+/-0.18 and 87.4+/-9.7 down to 1.63+/-0.11 and 39.0+/-2.8, respectively. In view of these data selenium supplementation might be beneficial for protection against diabetes-induced nephrotoxicity despite selenium accumulation in kidneys and liver.

Amino-acid-dependent, differential effects of ethanol on glucose production in rabbit kidney-cortex tubules.

AIMS: The effect of ethanol on glucose synthesis in kidney-cortex tubules of control and diabetic rabbits has been investigated. METHODS: Both freshly isolated and grown in primary cultures, kidney-cortex tubules were incubated with alanine or aspartate plus lactate or glycerol plus octanoate in the absence and presence of 100 mmol/l ethanol. RESULTS: In freshly isolated renal tubules incubated in the presence of alanine plus lactate or glycerol plus octanoate, and in tubules grown in primary culture in the medium containing alanine plus lactate plus octanoate alcohol, resulted in about 30% decrease in glucose formation. A diminished glucose production in freshly isolated tubules was accompanied by: (i) a decrease in alanine utilization, (ii) an increase in lactate or glycerol consumptions and (iii) a decline in GSH:GSSG ratio. The ethanol action was not abolished by 4-methylpyrazole, an inhibitor of alcohol dehydrogenase (ADH). In view of ethanol-induced changes in gluconeogenic intermediates it is likely that in the presence of alanine plus glycerol plus octanoate ethanol causes a decline in flux through phosphoenolpyruvate carboxykinase, probably due to either an increase in intracellular content of 2-oxoglutarate, inhibitor of this key gluconeogenic enzyme and/or an enhanced flux through pyruvate kinase, as concluded from an increased lactate formation in the presence of glycerol in the incubation medium. In renal tubules grown in primary cultures in the presence of alanine plus lactate plus octanoate a decrease in GSH:GSSG ratio was accompanied by elevated generation of reactive oxygen species (ROS). Upon replacement of alanine by aspartate ethanol affected neither glucose production, substrate uptake, ROS accumulation nor GSH:GSSG ratio. CONCLUSIONS: In the presence of alanine ethanol-induced decrease in glucose production and elevation of ROS might cause a limited NADPH generation resulting in a decrease in the intracellular GSH:GSSG ratio. On the contrary, aspartate might protect against ROS generation, so intensive gluconeogenesis supports NADPH generation and in consequence high values of the intracellular GSH:GSSG ratio are maintained.

Inhibition of gluconeogenesis by vanadium and metformin in kidney-cortex tubules isolated from control and diabetic rabbits.

Effect of vanadyl acetylacetonate (VAc) and metformin on gluconeogenesis has been studied in isolated hepatocytes and kidney-cortex tubules of rabbit. Glucose formation from alanine+glycerol+octanoate, pyruvate or dihydroxyacetone was inhibited by 50-80% by 100 microM VAc or 500 microM metformin in renal tubules of control and alloxan-diabetic animals, while the inhibitory action of these compounds in hepatocytes was less pronounced (by about 20-30%). In contrast to VAc, metformin increased the rate of lactate formation by about 2-fold in renal tubules incubated with alanine+glycerol+octanoate. In view of VAc-induced changes in intracellular gluconeogenic intermediates and gluconeogenic enzyme activities, it is likely that this compound may decrease fluxes through pyruvate carboxylase, phosphoenolpyruvate carboxykinase, fructose-1,6-bisphosphatase and glucose-6-phosphatase. In contrast to VAc, metformin-induced decrease in renal gluconeogenesis may result from a decline of cytosolic oxaloacetate level and consequently PEPCK activity. Following 6 days of VAc administration (1.275 mg Vkg(-1) body weight daily) the blood glucose level in alloxan-diabetic rabbits was normalised while blood glucose changes in control animals were not observed. On the contrary, in diabetic animals treated for 6 days with metformin (200 mg kg(-1) body weight day(-1)) a high blood glucose level was maintained. Unfortunately, VAc-treated control and diabetic rabbits exhibited elevated serum urea and creatinine levels. In VAc-treated animals vanadium was accumulated in kidney-cortex up to 7.6+/-0.6 microg Vg(-1) dry weight. In view of a potential vanadium nephrotoxicity a therapeutic application of vanadium compounds needs a critical re-evaluation.