METABOLIC CHANGES ASSOCIATED WITH ISO-OSMOTIC REGULATION IN BRAIN CORTEX SLICES

Abstract— The metabolism of single, first cortical rat brain slices was studied in response to incubation in media of various osmolalities. There was an inverse relationship between osmolality and the magnitude of the increase in tissue water content, and a direct relationship between osmolality and inulin space. Brain sodium varied directly with the media sodium, but hyperosmolal sucrose and glucose resulted in a drop in brain sodium. Brain potassium was constant in hyperosmolal sodium media, but it fell in hypo-osmolal media and hyperosmolal sucrose and glucose media.
Hypo-osmolality depressed the oxidation of [¹⁴C]glucose to ¹⁴CO₂ whereas hyperosmolality obtained with sodium, sucrose, or glucose stimulated glucose oxidation. Lactate production was enhanced only by sodium hyperosmolality. Hypo-osmolality caused a 70% drop in phosphocreatine and a minor decrease in energy charge potential, analagous to the effects of hypoxia. Hyperosmolality had no effect on energy metabolism.
The total amino acid nitrogen released into the media was suppressed by hypo-osmolality but was increased by hyperosmolal incubation. Hyperosmolality also increased production of ammonia fourfold.
The time course of the change in tissue osmolality and ion content following incubation in hyperosmolal sodium media showed the tissue osmolality reached the media osmolality within 5 min, but it took 30 min for the tissue sodium to reach equilibrium with the media sodium. This indicates that unidentified or'idiogenic osmoles'are induced transiently by hyperosmolality associated with changes in amino acid and ammonia metabolism.     

HYPEROSMOLALITY-INDUCED GABA RELEASE FROM RAT BRAIN SLICES: STUDIES OF CALCIUM DEPENDENCY AND SOURCES OF RELEASE

Abstract— The effects of hyperosmolal superfusion upon the release of preloaded, radio-labeled GABA has been studied, using both first cortical and first pontine brain slices. GABA release was stimulated with either hyperosmolal Na⁺ or sucrose superfusion in cortical slices. This stimulated release of radio-labeled GABA was partially Ca²⁺-dependent in cortical slices. When barium ions replaced Ca²⁺ in hyperosmolal medium, a similar effect was seen. High concentration of magnesium in Ca²⁺ -free hyperosmolal medium did not induce stimulation. The increased release of α-aminoisobutyric acid (AIBA), a non-metabolized amino acid induced by hyperosmolality, was not Ca²⁺-dependent.
GABA release was also stimulated with hyperosmolal sucrose superfusion in pontine slices. The effect of pre-treatment of cortical and pontine slices with β-alanine or L-2,4-diaminobutyric acid (DABA) was used to study the source of exogenous GABA release induced by hyperosmolality. In cortical slices, β-alanine blocked the hyperosmolal release of GABA and also slightly inhibited GABA uptake. DABA did not change hyperosmolal GABA release, although it inhibited GABA uptake. In pontine slices, both DABA and β-alanine inhibited GABA uptake, but were unable to inhibit the hyperosmolal release of GABA.
The data suggest that hyperosmolality causes increased release of GABA from neurons, analogous to that seen with K⁺-depolarization. AIBA, unlike GABA, is released from brain cells as a non-Ca²⁺ -dependent response to osmotic equilibration. The observation that pre-treatment with β-alanine inhibits the hyperosmolal release of GABA suggests that hyperosmolality alters glial cell function.     

Cyclic AMP, cyclic GMP and glucose utilization by diabetic rat fat cells

Fat cells from epididymal adipose tissue from normal and streptozotocin-diabetic rats were studied to determine glucose utilization and cyclic nucleotide levels. Diabetic rat fat cells present a higher cAMP content (P less than 0.05) compared with controls. Addition of insulin decreases within 10-min incubation the cAMP content in both normal and diabetic cells (P less than 0.05). However, the value obtained in the latter remains by 25% higher than that of normal cells not exposed to insulin. No changes in cGMP were detected. Pretreatment of the diabetic animals during two days with propranolol (1 mg kg body wt-1 day-1) induces the decrease to normal levels of the fat cell cAMP content. However, it persists the impairment on glucose utilization observed in fat cells from diabetic animals. It seems that the increase in the intracellular amount of cAMP found in fat cells from diabetic rats is not involved, at least directly, to the impaired glucose utilization found in the diabetic state. Furthermore, through an unknown mechanism, pretreatment with propranolol can induce a drop in fat tissue cAMP toward normal values without normalizing glucose utilization.     

Hyperosmotic stress induces autophagy and apoptosis in recombinant Chinese hamster ovary cell culture

During recombinant Chinese hamster ovary (rCHO) cell culture, various events, such as feeding with concentrated nutrient solutions or the addition of base to maintain an optimal pH, increase the osmolality of the medium. To determine the effect of hyperosmotic stress on two types of programmed cell death (PCD), apoptosis and autophagy, of rCHO cells, two rCHO cell lines, producing antibody and erythropoietin, were subjected to hyperosmotic stress resulting from NaCl addition (310–610 mOsm/kg). For both rCHO cell lines, hyperosmolality up to 610 mOsm/kg increased cleaved forms of PARP, caspase‐3, caspase‐7, and fragmentation of chromosomal DNA, confirming the previous observation that apoptosis was induced by hyperosmotic stress. Concurrently, hyperosmolality increased the level of accumulation of LC3‐II, a widely used autophagic marker, which was determined by Western blot analysis and confocal microscopy. When glucose and glutamine concentrations were measured during the cultures, glucose and glutamine concentrations in the culture medium at various osmolalities (310–610 mOsm/kg) showed no significant differences. This result suggests that induction of PCD by hyperosmotic stress occurred independently of nutrient depletion. Taken together, autophagy as well as apoptosis was observed in rCHO cells subjected to hyperosmolality. Biotechnol. Bioeng. 2010;105: 1187–1192. © 2009 Wiley Periodicals, Inc.     

Effects of 2-deoxy-D-glucose, oligomycin and theophylline on in vitro glycerol metabolism in rat adipose tissue: response to insulin and epinephrine.

The effects of 2-deoxy-D-glucose (2DG), oligomycin and theophylline on the in vitro production and metabolism of glycerol and its response to insulin and epinephrine were studied in epididymal fat pads from fed rats. 2-DG failed to affect basal or epinephrine stimulated glycerol production but it decreased the uptake of 1-14 C-glycerol by the tissue and its conversion to glyceride-glycerol. Oligomycin also failed to affect the basal production of glycerol but it inhibited the effect of epinephrine on this parameter as well as the uptake and utilization of 1-14-C-glycerol. Theophylline enhanced the production of glycerol by the tissue and this effect was not further augmented by epinephrine. Theophyline also inhibited the uptake and utilization of 1-14C-glycerol; the most pronounced effect of theophylline was observed in the formation of 14C-fatty acids from 1-14C-glycerol in the presence of glucose. Insulin, but not epinephrine, decreased the inhibitory effect of theophylline on glycerol utilization. It is concluded that these compounds affect more intensely the ability of adipose tissue to metabolize glycerol than to release it through lipolysis. The pathway for glycerol utilization in adipose tissue appears to be more sensitive to changes in the availability of ATP than the mechanisms responsible for the release of glycerol from the tissue.     

Phenylarsine oxide inhibits insulin-dependent glucose transport activity in rat soleus muscles

Phenylarsine oxide (PAO) is known to block insulin-stimulated glucose transport activity in 3T3 L1 adipocytes at a post-receptor step. Herein, we demonstrate that, at right concentration, PAO also inhibits insulin activation of glucose uptake in rat soleus muscles but does not affect basal level of uptake. In control experiments, insulin stimulation of 2-deoxy-D-glucose uptake is about 400% of that of the control level. After PAO treatment, the stimulation reduces to 150% of the control. Since the intracellular level of ATP remains unchanged after PAO treatment, when measured by phosphorus-31 nmr spectroscopy, this inhibition is not due to depletion of ATP pool size. Moreover, PAO does not affect autophosphorylation of the insulin receptors purified from rat soleus muscles, implying that the PAO blockage of insulin-dependent glucose uptake in soleus muscles also may be post-receptor.     

Transcriptome analysis identifies activated signaling pathways and regulated ABC transporters and solute carriers after hyperosmotic stress in renal MDCK I cells

Hyperosmolality is found under physiological conditions in the kidneys, whereas hyperosmolality in other tissues may be associated with pathological conditions. In such tissues an association between inflammation and hyperosmolality has been suggested. During hyperosmotic stress, an important phenomenon is upregulation of solute carriers (SLCs). We hypothesize that hyperosmolality affects the expression of many SLCs as well as ABC transporters. Through RNA-sequencing and topological pathway analysis, the cell cycle, the cytokine-cytokine receptor interaction pathway, and the chemokine-signaling pathway were significantly activated in MDCK I cells after hyperosmotic treatment (Δ200 mOsm) with raffinose or NaCl. 9065, 8052 and 5018 genes were significantly regulated by raffinose, NaCl or urea supplementation (500 mOsm), respectively, compared to control (300 mOsm). Cytokines, that have not previously been associated with hyperosmolality, were identified. We further provide an overview of transport proteins that could be of relevance in tissues exposed to hyperosmolality. Especially Slc5a8 was found highly up-regulated.     

Effects of insulin on glucose metabolism in isolated human fat cells

Isolated fat cells were used for the study of in vitro effects of insulin on glucose metabolism in human and rat adipose tissue. In human subcutaneous fat cells, effects of insulin could be detected at concentrations of glucose in the medium from 1 to 10 micro moles/ml. Cellular responsiveness was inversely proportional to the glucose level. At a constant concentration of 6 micro moles of glucose per ml, the effects of insulin at various concentrations up to 500 micro U/ml were investigated. At the highest concentration, which gave the maximal response, there was a 100% increase in the conversion of glucose-U-(14)C to glyceride-glycerol and a 40% increase in glucose oxidation. The dose-response curve was steepest between 2 and 20 micro U/ml. Rat epididymal fat cells were much more responsive to insulin. Glucose lipogenesis and pentose cycle activity could also be demonstrated in rat cells, whereas these activities could not be shown in fat cells from human omental and subcutaneous tissue. The findings for human cells are attributed to changes in cellular activity during preparation.     

No in vitro effects of fatty acids on glucose uptake, lipolysis or insulin signaling in rat adipocytes.

Elevated plasma levels of free fatty acids (FFA) can produce insulin resistance in skeletal muscle tissue and liver and, together with alterations in beta-cell function, this has been referred to as lipotoxicity. This study explores the effects of FFAs on insulin action in rat adipocytes. Cells were incubated 4 or 24 h with or without an unsaturated FFA, oleate or a saturated FFA, palmitate (0.6 and 1.5 mM, respectively). After the culture period, cells were washed and insulin effects on glucose uptake and lipolysis as well as cellular content of insulin signaling proteins (IRS-1, PI3-kinase, PKB and phosphorylated PKB) and the insulin regulated glucose transporter GLUT4 were measured. No significant differences were found in basal or insulin-stimulated glucose uptake in FFA-treated cells compared to control cells, regardless of fatty acid concentration or incubation period. Moreover, there were no significant alterations in the expression of IRS-1, PI3-kinase, PKB and GLUT4 following FFA exposure. Insulin's ability to stimulate PKB phosphorylation was also left intact. Nor did we find any alterations following FFA exposure in basal or cAMP-stimulated lipolysis or in the ability of insulin to inhibit lipolysis. The results indicate that oleate or palmitate does not directly influence insulin action to stimulate glucose uptake and inhibit lipolysis in rat fat cells. Thus, lipotoxicity does not seem to occur in the fat tissue itself.     

Short-term fatty acid effects on adipocyte glucose uptake: Mechanistic insights

The modulation of insulin sensitivity in visceral fat tissue could be important in the treatment of Type 2 diabetes mellitus. Selected fatty acids may impact on insulin-stimulated and basal glucose uptake in adipocytes, thus isolated rat epididymal adipocytes were exposed to 100 μM oleic, arachidonic, eicosapentaenoic, docosahexaenoic or stearic acids and insulin (15 nM) or vehicle for 30 min. Glucose uptake was quantified by measuring uptake of ³H-deoxyglucose/mg adipocyte protein/min. Where appropriate, inhibitors were included to elucidate the mechanisms involved.In this model, insulin stimulated glucose uptake with 62±7%. All fatty acids tested, except for stearic acid, depressed insulin-stimulated glucose uptake by an average of 33±4.2%. On the other hand, all fatty acids tested except stearic and arachidonic acids, stimulated basal glucose uptake with an average of 34±8.1%. Inhibitor studies showed the involvement of prostaglandins, lipoxins, protein kinase C and tyrosine kinase in these processes.     

Insulin insensitivity and altered glucose utilization in cultured rat adipose tissue

Glucose utilization was studied in isolated fat cells prepared from rat adipose tissue which had been cultured for 18 hr in TC 199 medium. When 1% bovine serum albumin (BSA) was in the culture medium, basal rates of (14)CO(2) and [(14)C]triglyceride production from [1-(14)C]glucose were markedly depressed and there was no effect of insulin. With 4% BSA, basal (14)CO(2) production was the same as in cells prepared from fresh tissue and basal triglyceride production was greatly increased. Insulin effect on these cells was minimal. One-minute uptake of [(14)C]2-deoxyglucose was stimulated by 800-1000% in fresh cells and 300-500% in cells cultured with either 1% or 4% BSA. Oxidation of [U-(14)C]glucose showed a much smaller impairment in cultured cells than for [1-(14)C]glucose, suggesting that the pentose phosphate shunt was more severely impaired than glycolysis. Glyceride-glycerol production was increased in cultured cells relative to preculture (fresh) cells. There was no effect of insulin in the culture medium in any of these systems. Rates of free fatty acid and glycerol release were markedly increased in cultured cells, especially when insulin was present in the culture medium. The acute antilipolytic effect of insulin was retained, so that insulin in the test incubation decreased lipolysis by 40-80%. Nevertheless, cell-associated fatty acids were increased in cultured cells and FFA/albumin ratios in the medium often reached potentially toxic levels. The reduction in pentose phosphate shunt activity, lipogenesis, and insulin effect resembles other models of insulin insensitivity. The impaired metabolism is probably due to an intracellular defect. A possible toxic role of either intracellular or extracellular fatty acids cannot be excluded. This system should be a useful model in which to study the cellular mechanisms of insulin insensitivity in adipocytes.-Bernstein, R. S. Insulin insensitivity and altered glucose utilization in cultured rat adipose tissue.     

Effect of insulin on glucose transport and metabolism in isolated fat-cells of gonadal adipose tissue from mature age-matched male and female rats.

Insulin action on glucose transport and metabolism was studied in paraovarian adipocytes from 3-month-old female rats and compared with insulin action in epididymal adipocytes from closely age-matched males. At maximal insulin concentrations the stimulations of 2-deoxyglucose uptake (4-fold the basal value) and of [U-14C]glucose incorporation into CO2 and total lipids (3- and 2-fold the basal values respectively) were similar in adipocytes from rats of both sexes. At submaximal insulin concentrations (less than 0.2 nM) the ability of paraovarian adipocytes to transport and to metabolize glucose was higher than that of epididymal adipocytes; accordingly an increase in insulin binding was observed in paraovarian adipocytes as compared with epididymal adipocytes. These results show that paraovarian adipocytes from mature female rats were highly responsive to insulin, and exhibited a higher sensitivity to the hormone than did epididymal adipocytes from male rats of the same age.     

Relationship of lipoprotein lipase activity to triglyceride uptake in adipose tissue

Fasted rats injected with actinomycin or fed glucose show increased lipoprotein lipase activity of epididymal adipose tissue. Data from the actinomycin-treated animals showed a direct correlation between the lipoprotein lipase activity and the uptake of lipoprotein triglyceride by the epididymal fat pad in vitro and in vivo. Data from the animals fed glucose confirmed these findings in vitro. These data strongly suggest that lipoprotein lipase plays a major role in triglyceride deposition in adipose tissue.     

Dietary fish oil reverse epididymal tissue adiposity, cell hypertrophy and insulin resistance in dyslipemic sucrose fed rat model small star, filled

The present work was designed to assess the possible benefits of (7% w/w) dietary fish oil in reversing the morphological and metabolic changes present in the adipose tissue of rats fed an SRD for a long time. With this purpose, in the epididymal fat tissue, we investigated the effect of dietary fish oil upon: i) the number, size and distribution of cells, ii) the basal and stimulated lipolysis, iii) the lipoprotein lipase (LPL) and the glucose 6-phosphate dehydrogenase activities, and iv) the antilipolytic action of insulin. The study was conducted on rats fed an SRD during 120 days with fish oil being isocaloric substituted for corn oil for 90-120 days in half the animals. Permanent hypertriglyceridemia, insulin resistance and abnormal glucose homeostasis were present in the rats before the source of fat in the diet was replaced. The major new findings of this study are the following: i) Dietary fish oil markedly reduced the fat pads mass, the hypertrophy of fat cells and improved the altered cell size distribution. ii) The presence of fish oil in the diet corrected the inhibitory effect of high sucrose diet upon the antilipolytic action of insulin, reduced the "in vitro" enhanced basal lipolysis and normalized isoproterenol-stimulated lipolysis. Fat pads lipoprotein lipase activity decreased reaching values similar to those observed in age-matched controls fed a control diet (CD). These effects were not accompanied by any change in rat body weight. All these data suggest that the dyslipemic rats fed a moderate amount of dietary fish oil constitute a useful animal model to study diet-regulated insulin action.     

Effects of SH-reagents of different molecular size upon glucose metabolism in isolated rat fat cells.

To study the role of membrane SH-groups in glucose transport of isolated rat fat cells we compared the effects of a small organic mercurial reagent p-CMB with those of a large p--CMB-derivative -- p-CMB-Dextran, MW 10.000 --. It could be shown that both compounds were of almost identical reactivity on fat cell homogenate metabolism. When applied to intact fat cells uncoupled p--CMB showed an (1) insulin like enhancement of 14C incorporation from (U-14C) glucose into CO2 and triglyceride, (2) inhibition of the insulin-stimulatory effect on these parameters and (3) inhibition of basal glucose uptake dependent on the concentrations used. Identical concentrations of p-CMB-Dextran, however, failed to influence basal glucose uptake as well as the insulin mediated increase in glucose metabolism.     

A portal-arterial glucose concentration gradient as a signal for an insulin-dependent net glucose uptake in perfused rat liver

Since in the usual perfusion of isolated rat liver via the portal vein an insulin-dependent increase of hepatic glucose uptake could not be demonstrated, the possibility was considered that hepatic glucose uptake might not be a function of the absolute concentration of this substrate but of its concentration gradient between the portal vein and the hepatic artery. Therefore a new method was established for the simultaneous perfusion of isolated rat liver via both the hepatic artery (20-35% flow) and the portal vein (80-65% flow). When glucose was offered in a concentration gradient, 9.5 mM in the portal vein and 6 mM in the hepatic artery, insulin given via both vessels caused a shift from net glucose release to uptake. This insulin-dependent shift was not observed when glucose was offered without a gradient or with an inverse gradient, 6 mM in the portal vein and 9.5 mM in the hepatic artery. Using a portal-arterial glucose gradient as a signal the liver might be able to differentiate between endogenous and exogenous glucose.     

Function of rabbit kidneys in vitro at normothermia following equilibration with 3.0 M Me2SO and removal by hypertonic washout at 10 degrees C

In the present study, rabbit kidneys were assayed for function on a 37 °C in vitro perfusion system after perfusion on a 10 °C perfusion system which permits the slow introduction and removal of cryoprotectant. The final concentration of 3.0 M Me₂SO was introduced slowly at two different rates. The washout was achieved by perfusion with Me₂SO-free solutions made hypertonic with mannitol. Two regimens of washout were used: 800, 700, 600, 500, and 400 mOsm/kg; and 600, 500, and 400 mOsm/kg.During perfusion at 37 °C, the glomerular filtration rate was similar in all groups and this increased significantly in all groups with time. Protein leakage was minimal. All three Me₂SO groups showed a depressed Na reabsorption capacity, but the 800 mOsm group was the most severely affected. This was also found with glucose reabsorption. We concluded that rabbit kidneys will function well with the cryoprotectant Me₂SO up to 3 M concentration when introduced slowly and washed out with hypertonic mannitol beginning at 600 mOsm/kg. When 800 mOsm is used at the initial step, the proximal tubular function is severely affected.     

Evaluation ofin vivo insulin action and glucose metabolism in milk-fed rats

Milk diet has long been recommended in the management of gastrointestinal pathologies. Since milk feeding represents a high fat-low carbohydrate diet and it is acknowledged that insulin resistance is one of the consequences of high fat feeding, it is important to know whether or not chronic milk feeding leads to an impairment of the insulin-mediated glucose metabolism. To examine this question, adult female rats were given raw cow's milk (50% of total calories as lipids) for 18 days. They were compared to rats raised in parallel and fed the standard laboratory diet (15% of total calories as lipids). At the end of the 18 day period, body weight, daily caloric intake, basal plasma glucose and insulin levels in the milk-fed rats were similar to those in the control rats.In vivo insulin action was assessed with the euglycemichyperinsulinemic clamp technique in anesthetized animals. These studies were coupled with the 2-deoxyglucose technique allowing a measurement of glucose utilization by individual tissues. In the milk fed rats: 1) the basal rate of endogenous glucose production was significantly (p<0.01) reduced (by 20%); 2) their hepatic glucose production was however normally suppressed by hyperinsulinemia; 3) their basal glucose utilization rate was significantly (p<0.01) reduced (by 20%); 4) their glucose utilization rate by the whole-body mass or by individual tissues was normally increased by hyperinsulinemia. These results indicate that insulin action in adult rats is not grossly altered after chronic milk-feeding, at least under the present experimental conditions.     

Ghrelin stimulates insulin-induced glucose uptake in adipocytes

The gastric and hypothalamic hormone ghrelin is the endogenous agonist of the growth hormone secretagogue receptor GHS-R1(a). Ghrelin stimulates growth hormone release and appetite via the hypothalamus. However, putative direct peripheral effects of ghrelin remain poorly understood. Rat adipose tissue expresses GHS-R1(a) mRNA, suggesting ghrelin may directly influence adipocyte function. We have investigated the effects of ghrelin on insulin-stimulated glucose uptake in isolated white adipocytes in vitro. RT-PCR confirmed the expression of GHS-R1(a) mRNA in epididymal adipose tissue. However, GHS-R1(a) expression was not detected in the peri-renal fat pads. Ghrelin increased insulin-stimulated deoxyglucose uptake in isolated white adipocytes extracted from the epididymal fat pads of male Wistar rats. Ghrelin 1000 nM significantly increased deoxyglucose uptake by 55% in the presence of 0.1 nM insulin. However, ghrelin administration in the absence of insulin had no effect on adipocyte deoxyglucose uptake, suggesting that ghrelin acts synergistically with insulin. Des-acyl ghrelin, a major circulating non-octanylated form of ghrelin, had no effect on insulin-stimulated glucose uptake. Furthermore, acylated ghrelin had no effect on deoxyglucose uptake in adipocytes from peri-renal fat pads suggesting that ghrelin may influence glucose uptake via the GHS-R1(a). Ghrelin therefore appears to directly potentiate adipocyte insulin-stimulated glucose uptake in selective adipocyte populations. Ghrelin may play a role in adipocyte regulation of glucose homeostasis.     

Reconstitution of insulin action in muscle, white adipose tissue, and brain of insulin receptor knock-out mice fails to rescue diabetes

Type 2 diabetes results from an impairment of insulin action. The first demonstrable abnormality of insulin signaling is a decrease of insulin-dependent glucose disposal followed by an increase in hepatic glucose production. In an attempt to dissect the relative importance of these two changes in disease progression, we have employed genetic knock-outs/knock-ins of the insulin receptor. Previously, we demonstrated that insulin receptor knock-out mice (Insr(-/-)) could be rescued from diabetes by reconstitution of insulin signaling in liver, brain, and pancreatic β cells (L1 mice). In this study, we used a similar approach to reconstitute insulin signaling in tissues that display insulin-dependent glucose uptake. Using GLUT4-Cre mice, we restored InsR expression in muscle, fat, and brain of Insr(-/-) mice (GIRKI (Glut4-insulin receptor knock-in line 1) mice). Unlike L1 mice, GIRKI mice failed to thrive and developed diabetes, although their survival was modestly extended when compared with Insr(-/-). The data underscore the role of developmental factors in the presentation of murine diabetes. The broader implication of our findings is that diabetes treatment should not necessarily target the same tissues that are responsible for disease pathogenesis.