[The mechanism of insulin resistance in peripheral tissues]. / Mechanizm powstawania opornosci na insuline w tkankach obwodowych.

Chronic metabolic and cardiovascular diseases, described as the epidemics of XXI century, are connected to the resistance of peripheral tissues, such as liver, muscle and fat, to insulin. Insulin resistance, which precedes the development of type 2 diabetes by several years, is difficult to diagnose, mainly because of practical limitations to the use of "gold standard" hyperinsulinemic euglycemic clamp technique for screening. It is also begins a certain vicious circle, in which insulin resistant peripheral tissues force pancreatic beta cells to increased insulin release, and sustained high concentrations of insulin cause further development of insulin resistance. Currently, there are two major hypotheses describing the mechanism of insulin resistance: one relating to the "lipid overload" in liver and muscle cells as the key factor and another one emphasizing the role of lipid accumulation in adipocytes, which leads to the overgrowth of fatty tissue and chronic local inflammation.

[Nuclear receptors PPAR as a drug target in metabolic disorders]. / Receptory jadrowe PPAR jako miejsce dzialania leków w zaburzeniach metabolicznych.

Nuclear receptors regulate many basic cellular processes and their malfunction can lead to serious consequences including metabolic disorders, obesity and type 2 diabetes. Among many nuclear receptor families, the best known for their therapeutic use are the PPARs. These are key transcription factors determining, proper cellular metabolism of glucose and lipids, tissue sensitivity to insulin, appropriate immune responses including inflammatory processes and finally cell division and differentiation. Currently two types of PPAR activators are in medical use: in the therapy of type 2 diabetes--thiazolidinediones (TZDs), which act via PPARgamma receptors and in the treatment of dyslipidemia-fibrates, which act via PPARalpha receptors. The search for new drugs acting through PPAR mechanism consists in the design of new molecules with tissue specific proprieties, which would selectively bind and modulate the activity of appropriate receptors, thus reducing the number of adverse events typically observed with the use of full agonists. These molecules have been named selective nuclear receptor modulators (SNuRMs).

PPAR-gamma-independent inhibitory effect of rosiglitazone on glucose synthesis in primary cultured rabbit kidney-cortex tubules.

Therapeutic effect of rosiglitazone has been reported to result from an improvement of insulin sensitivity and inhibition of glucose synthesis. As the latter process occurs in both liver and kidney cortex the aim of this study was to elucidate the rosiglitazone action on glucose formation in both tissues. Primary cultured cells of both liver and kidney cortex grown in defined medium were use throughout. To identify the mechanism responsible for drug-induced changes, intracellular gluconeogenic intermediates and enzyme activities were determined. In contrast to hepatocytes, the administration of a 10 micromol/L concentration of rosiglitazone to renal tubules resulted in about a 70% decrease in the rate of gluconeogenesis, accompanied by an approximately 75% decrease in alanine utilization and a 35% increase in lactate synthesis. The effect of rosiglitazone was not abolished by GW9662, the PPAR-gamma irreversible antagonist, indicating that this action is not dependent on PPAR-gamma activation. In view of rosiglitazone-induced changes in gluconeogenic intermediates and a diminished incorporation of 14CO2 into pyruvate, it is likely that the drug causes a decline in flux through pyruvate carboxylase and (or) phosphoenolpyruvate carboxykinase. It is likely that the hypoglycemic action of rosiglitazone is PPAR-gamma independent and results mainly from its inhibitory effects on renal gluconeogenesis.

Melatonin is more effective than taurine and 5-hydroxytryptophan against hyperglycemia-induced kidney-cortex tubules injury.

The antioxidative effects of melatonin (Mel), 5-hydroxytryptophan (5-HTP) and taurine (TAU) on hyperglycemia-induced oxidative stress was investigated in primary cultures of kidney-cortex tubule cells grown in metabolically and hormonally defined medium. In the presence of 30 mm glucose (hyperglycemic conditions), cell viability was decreased by about 35% in comparison with that estimated in the glucose-depleted medium probably as a result of induction of apoptosis, as concluded from: (i) chromatin condensation and DNA fragmentation assays, (ii) a significant enhancement of reactive oxygen species (ROS) production, (iii) 8-hydroxydeoxyguanosine (8-OHdG) generation, (iv) an increased protein peroxidation and (v) a decline of reduced glutathione (GSH) levels leading to a disturbed glutathione redox state. The addition of 100 microm Mel to the hyperglycemic medium resulted in a twofold decrease in both 8-OHdG accumulation and protein peroxidation as well as restoration of the control intracellular ROS levels accompanied by a substantial increase in GSH/oxidized glutathione (GSSG) ratio due to a decline in GSSG content. ROS elimination was also achieved in the presence of 1 mm TAU which diminished protein and DNA injuries by about 25% and 30%, respectively. On the contrary, the action of 100 microm 5-HTP on ROS level, 8-OHdG generation, protein peroxidation and GSH/GSSG ratio was negligible. Thus, in contrast to 5-HTP and TAU, Mel might be considered as beneficial for diabetes therapy, particularly in terms of reduction of hyperglycemia-induced kidney injury.

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.

Melatonin-induced modulation of glucose metabolism in primary cultures of rabbit kidney-cortex tubules.

The effect of melatonin on glucose metabolism in the presence and absence of insulin has been investigated in the primary cultures of renal tubules grown in a defined medium. In the absence of glucose in the medium containing 5 microg/mL of insulin and 2 mm alanine + 5 mm glycerol + 0.5 mm octanoate, 100 nm melatonin stimulated both glucose and lactate synthesis, while in the medium devoid of insulin melatonin action was negligible. Melatonin-induced increase in glucose and lactate synthesis was accompanied by an enhancement of alanine and glycerol consumption. In view of measurements of [U-14C]L-alanine and [U-14C]L-glycerol incorporation into glucose, it is likely that melatonin increased alanine utilization for glucose production, while accelerated lactate synthesis was because of an enhanced glycerol consumption. As (i) 10 nm luzindole attenuated the stimulatory action of melatonin on glucose formation and (ii) the indole induced a decrease in intracellular cAMP level, it seems likely that in renal tubules melatonin binds to ML1 membrane receptor subtype. In view of a decline of intracellular fructose-1,6-bisphosphate content accompanied by a significant rise in hexose-6-phosphate and glucose levels, melatonin might result in an acceleration of flux through fructose-1,6-bisphosphatase probably because of an increase in the active, dephosphorylated form of this enzyme. Thus, the administration of melatonin in combination with insulin might be beneficial for diabetic therapy because of protection against hypoglycemia.

The role of intracellular cAMP in renal gluconeogenesis in view of differential action of various cAMP analogues.

Effects of various cAMP analogues on gluconeogenesis in isolated rabbit kidney tubules have been investigated. In contrast to N(6),2'-O-dibutyryladenosine-3',5'-cyclic monophosphate (db-cAMP) and cAMP, which accelerate renal gluconeogenesis, 8-bromoadenosine-3',5'-cyclic monophosphate (Br-cAMP) and 8-(4-chlorophenylthio)-cAMP (pCPT-cAMP) inhibit glucose production. Stimulatory action of cAMP and db-cAMP may be evoked by butyrate and purinergic agonists generated during their extracellular and intracellular metabolism resulting in an increase in flux through fructose-1,6-bisphosphatase and in consequence acceleration of the rate of glucose formation. On the contrary, Br-cAMP is poorly metabolized in renal tubules and induces a fall of flux through glyceraldehyde-3-phosphate dehydrogenase. The contribution of putative extracellular cAMP receptors to the inhibitory Br-cAMP action is doubtful in view of a decline of glucose formation in renal tubules grown in the primary culture supplemented with forskolin. The presented data indicate that in contrast to hepatocytes, in kidney-cortex tubules an increased intracellular cAMP level results in an inhibition of glucose production.

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.

Purinergic regulation of glucose and glutamine synthesis in isolated rabbit kidney-cortex tubules.

The effects of extracellular purinergic agonists and their breakdown products on glucose and glutamine synthesis in rabbit kidney-cortex tubules incubated with aspartate + glycerol or alanine + glycerol + octanoate were investigated. A rapid extracellular degradation of ATP was accompanied by an accumulation of AMP, inosine, and hypoxanthine. Extracellular ATP and its breakdown products accelerated glucose synthesis in renal tubules, while ammonium released from adenine-containing compounds enhanced glutamine synthesis and diminished the degree of gluconeogenesis stimulation. In contrast to AMP and inosine, ATP evoked calcium signals, while both ATP and inosine decreased intracellular cAMP content and accelerated the flux through fructose-1,6-bisphosphatase as concluded from changes in gluconeogenic intermediates. Since (i) the activity of partially purified renal fructose-1,6-bisphosphatase was increased upon protein phosphatase-1 treatment and decreased following treatment of previously dephosphorylated enzyme with protein kinase A catalytic subunit and (ii) both 8-bromoadenosine 3',5'-cyclic monophosphate and 8-(4-chlorophenyltio)-cAMP inhibited renal glucose synthesis, it seems likely that in rabbit renal tubules ATP and inosine stimulate gluconeogenesis via cAMP decrease, which favors the appearance of a more active, dephosphorylated form of fructose-1,6-bisphosphatase, a key gluconeogenic enzyme.