The fate of labelled glucose molecules in the rat adipocyte: Dependence on glucose concentration

Isolated rat adipocytes were incubated with 15 nM [3-³H]glucose or 100 nM [U-¹⁴C]glucose with or without insulin and in the absence or presence of unlabelled glucose. Following a 2 h incubation with 15 nM [3-³H]glucose, about two thirds of the cell-associated ³H-labelled metabolic products were hydrophilic largely anionic intermediates and about one third was lipids. The equivalent values were 40 and 60%, respectively, when using 100 nM [U-¹⁴C]glucose. The only ¹⁴C-labelled metabolite escaping to the incubation medium was ¹⁴CO₂, which accounted for about 15% of the rate of metabolism. Therefore, the rate of incorporation of 100 nM [U-¹⁴C]glucose into the cell-associated metabolites was quite a good measure of its net influx rate. The conversion of the two tracers to the sum of the metabolic products in cells treated with a maximally stimulating insulin concentration remained constant with glucose concentrations up to about 100 μM and then decreased progressively. The incorporation of radioactivity into the different metabolites varied markedly over the glucose concentration range 0–100 μM, presumably due to the saturation of different metabolic pools at different glucose concentrations. This variation was much less in cells not stimulated with insulin. Consequently, the maximal effect of insulin on the incorporation of the tracers into a given metabolite (e.g., labelled lipids) varied over the entire glucose concentration range. In addition, the apparent sensitivity (ED₅₀) with respect to the incorporation into a given metabolite was also dependent on the glucose concentration.     

Regulatory effects of insulin and experimental diabetes on neutral amino acid transport in the perfused rat exocrine pancreas. Kinetics of unidirectional l-serine influx and efflux at the basolateral plasma membrane

Relatively little is known about the hormonal regulation of amino acid transport in the normal and diabetic exocrine pancreas. In this study unidirectional influx and tracer efflux of l-serine at the basolateral interface of the rat pancreatic epithelium was investigated in the perfused exocrine pancreas using a rapid (< 30 s) paired-tracer dilution technique. In the non-diabetic pancreas l-serine influx was saturable and stimulated by perfusion with exogenous bovine insulin (100 μU/ml). Transport of l-serine and methylaminoisobutyric acid was markedly elevated in pancreata isolated from streptozotocin diabetic rats and insulin partially reversed the stimulation of l-serine transport induced by experimental diabetes. These results suggest that insulin and diabetes modulate the epithelial transport activity for small neutral amino acids in the intact exocrine pancreas.     

Endotoxin-induced alterations in glucose transport in isolated adipocytes

2-Deoxyglucose and $\text{3-O-}\text{methyglucose}$ were used to assess endotoxin-induced changes in glucose transport in rat adipocytes. 6 h after Escherichia coli endotoxin injection insulin-stimulated 2-deoxyglucose uptake was significantly depressed ($\text{V}\text{decreased}\text{, K}{}_{\mathrm{<mtext>m</mtext>}}\text{unaltered}$), phosphorylation of 2-deoxyglucose was seemingly unimpaired; basal 3-methylglucose entry was significantly increased, insulin-stimulated uptake was unaltered. Insulin significantly reduced $\text{K}{}_{\mathrm{<mtext>m</mtext>}}$ in control and endotoxin-treated cells. Cytochalasin B-insensitive uptake of both 2-deoxyglucose and 3-methylglucose, a small fraction of total transport, increased significantly in endotoxic cells. Endotoxin reduced spermine- and insulin-stimulated 2-deoxyglucose uptake to a similar extent. Results are consistent with the hypotheses that (1) a site of endotoxin-induced insulin resistance is at the cell membrane level and may reflect a decrease in number or activity of effective carrier units, rather than alterations in affinity, (2) endotoxin does not compromise the hexokinase system, (3) the cell membrane-localized effect of endotoxin on hexose transport is not necessarily mediated by the insulin receptor and (4) the entry of 2-deoxyglucose and 3-methylglucose may involve two separate transport systems.     

The effect of metabolites,papaverine, and probenecid on cyclic AMP efflux from isolated rat islets of Langerhans

The process of cyclic AMP efflux from rat islets of Langerhans has been studied. The dynamics of glucose-induced cyclic AMP efflux closely resembled the pattern of glucose-induced insulin release. Thus, both processes were dose-dependent for glucose having the same threshold concentrations (4–8 mmol/l glucose), with the time course of cyclic AMP efflux and insulin release from 0–60 min being very similar. Galactose did not affect insulin release, cyclic AMP efflux and intra-islet cyclic AMP accumulation. On the other hand, inosine, N-acetylglucosamine, α-ketoisocaproic acid, L-leucine and xylitol all promoted insulin release and cyclic AMP efflux. Except for L-leucine, all these substances enhanced the intracellular accumulation of cyclic AMP. The phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine, greatly augmented all these parameters in the presence of glucose whereas in the absence of glucose, insulin release was not enhanced, while both cyclic AMP efflux and cyclic AMP accumulation were elevated. The drug, probenecid, did not alter either insulin release or intra-islet cyclic AMP levels, while cyclic AMP efflux was markedly reduced (though not abolished). Papaverine inhibited both insulin release and cyclic AMP efflux, but was found to augment the intra-islet cyclic AMP levels. The efflux of cyclic AMP correlates more closely with insulin release than with the cyclic AMP accumulation in most instances. The efflux is independent of either insulin secretory granule extrusion or intracellular fluctuations of the nucleotide, though it is not yet known whether cyclic AMP efflux may have some regulatory significance in insulin release.     

First phase release coefficient of insulin in subjects with normal glucose tolerance on glucose infusion analyzed by computer simulation

We report here a mathematical model using computer simulation to solve the phase fractionation coefficient (f) of instantaneous insulin release on glucose infusion. By extensive model testing with the cited parameters obtained from the literature, the values of the factor f were shown to lie in range of 0.93+/-0.02 (mean+/-2S.D., n=15), indicating that the high pulsatile bolus of glucose by i.v. infusion may trigger acute insulin release (AIR) corresponding to a fraction of more than 90% of the stored insulin release in the first phase from the secretory granules of pancreatic beta cells. In addition, the value of the factor f was shown to be independent of both the glucose infusion method and the non-insulin-dependent uptake of glucose.     

Structural changes of isolated hepatocytes during treatment with digitonin

The structural changes accompanying digitonin-induced release of enzymes and metabolites from isolated hepatocytes have been studied by scanning and transmission electron microscopy. In the initial phase, characterized by total release of the cytosolic marker enzyme, lactate dehydrogenase, the plasma membrane was immediately damaged, rapidly followed by extensive damage to the endoplasmic reticulum. The shape of the cell, however, was maintained, and the mitochondria and nucleus remained tightly held together by the cytoskeleton. Mitochondria remained intact initially, whereas the cytosol became less electron dense and the nuclear chromatin was more dispersed. An intermediate phase was characterized by total release of adenylate kinase and most of the glucose-6-phosphatase, marker enzymes for the mitochondrial intermembrane space and the endoplasmic reticulum, respectively. The outer mitochondrial membrane was ruptured, but mitochondria maintained their normal matrix electron density. In the final phase, characterized by the beginning of citrate synthase release from the mitochondrial matrix space, the mitochondria became swollen, and only the nucleus, inner and outer mitochondrial membranes, and the cytoskeleton could be clearly distinguished. Although the plasma membrane could not be readily discerned in electron micrographs after the initial phase, the plasma membrane marker enzyme 5′-nucleotidase remained associated with digitonin-treated hepatocytes. Acetyl-CoA carboxylase was released much more slowly than lactate dehydrogenase, indicating some severe restriction on its release. The release of acetyl-CoA carboxylase closely paralleled the release of glucose-6-phosphatase. The controlled exposure of hepatocytes to digitonin, therefore, leads to the sequential release of soluble, compartmentalized cellular components and some membrane-bound components, but the mitochondrial membrane, cytoskeleton and the nucleoskeleton survive even long-term digitonin treatment.     

Leucine supplementation in rats induced a delay in muscle IR/PI3K signaling pathway associated with overall impaired glucose tolerance

Although activation of the mammalian target of rapamycin complex/p70 S6 kinase (S6K1) pathway by leucine is efficient to stimulate muscle protein synthesis, it can also exert inhibition on the early steps of insulin signaling leading to insulin resistance. We investigated the impact of 5-week leucine supplementation on insulin signaling and sensitivity in 4-month old rats fed a 15% protein diet supplemented (LEU) or not (C) with 4.5% leucine. An oral glucose tolerance test was performed in each rat at the end of the supplementation and glucose transport was measured in vitro using isolated epitrochlearis muscles incubated with 2-deoxy-d-[³H]-glucose under increasing insulin concentrations. Insulin signaling was assessed on gastrocnemius at the postabsorptive state or 30 and 60 min after gavage with a nutrient bolus. Tyrosine phosphorylation of IRβ, IRS1 and PI3 kinase activity were reduced in LEU group 30 min after feeding (−36%, −36% and −38% respectively, P<.05) whereas S6K1, S6rp and 4EBP1 phosphorylations were similar. Overall glucose tolerance was reduced in leucine-supplemented rats and was associated with accumulation of perirenal adipose tissue (+27%, P<.05). Conversely, in vitro insulin-response of muscle glucose transport tended to be improved in leucine-supplemented rats. In conclusion, dietary leucine supplementation in adult rats induced a delay in the postprandial stimulation in the early steps of muscle insulin signaling without muscle resistance on insulin-induced glucose uptake. However, it resulted in overall glucose intolerance linked to increased local adiposity. Further investigations are necessary to clearly define the beneficial and/or deleterious effects of chronic dietary leucine supplementation in healthy subjects.     

A role for calcium/calmodulin kinase in insulin stimulated glucose transport

Previous research has shown that the CAMK (calcium/calmodulin dependent protein kinase) inhibitor, KN62, can lead to reductions in insulin stimulated glucose transport. Although controversial, an L-type calcium channel mechanism has also been hypothesized to be involved in insulin stimulated glucose transport. The purpose of this report was to determine if 1) L-type calcium channels and CAMK are involved in a similar signaling pathway in the control of insulin stimulated glucose transport and 2) determine if insulin induces an increase in CAMKII phosphorylation through an L-type calcium channel dependent mechanism. Insulin stimulated glucose transport was significantly (p<0.05) inhibited to a similar extent ( approximately 30%) by both KN62 and nifedipine in rat soleus and epitrochelaris muscles. The new finding of these experiments was that the combined inhibitory effect of these two compounds was not greater than the effect of either inhibitor alone. To more accurately determine the interaction between CAMK and L-type calcium channels, we measured insulin induced changes in CAMKII phosphorylation using Western blot analysis. The novel finding of this set of experiments was that insulin induced an increase in phosphorylated CAMKII ( approximately 40%) in rat soleus muscle that was reversed in the presence of KN62 but not nifedipine. Taken together these results suggest that a CAMK signaling mechanism may be involved in insulin stimulated glucose transport in skeletal muscle through an L-type calcium channel independent mechanism.     

Regulation of glycogenolysis in the liver of the newborn rat in vivo inhibitory effect of glucose

Newborn rats were injected immediately after delivery with glucose or glucose plus mannoheptulose, and the time-courses of liver glycogen, plasma glucose, insulin and glucagon concentration were studied. The administration of glucose prevented both liver glycogenolysis and the increase in plasma glucagon concentration which normally occurs immediately after delivery. In addition, the administration of glucose prevented the decrease of plasma glucose and insulin concentration which normally occurs during the first hour of extrauterine life. Supplementation of glucose with mannoheptulose prevented the increase of plasma insulin concentrations caused by the administration of glucose; liver glycogenolysis, however, was not stimulated in these circumstances. The increase in the rate of glycogenolysis caused by the administration of glucagon was prevented in newborn rats previously treated with glucose. These results suggest that glucose exerts an inhibitory effect on the stimulation of neonatal liver glycogenolysis by glucagon.     

Exogenous secretory proteins do not inhibit carbamylcholine-stimulated release of pulse-labelled secretory proteins from rat pancreatic tissue slices

Experiments were conducted to test the concept that the results of secretion studies employing pancreatic tissue slices are significantly biased by the distribution of exocrine secretory proteins between the tissue and the incubation medium. The findings demonstrate (1) that the kinetics of release of pulse-labelled secretory proteins from cholinergically-stimulated tissue slices are independent of the concentration of exogenous exocrine secretory proteins and (2) that if there are bidirectional fluxes of secretory proteins across the cell membrane of pancreatic exocrine cells, these fluxes do not account for the fractional release of pulse-labelled secretory proteins.     

Metabolic effects of acetate in perfused rat liver studies on ketogenesis,glucose output,lactate uptake and lipogenesis

1. Livers from fed male rats were perfused in situ in a non-recirculating system with whole rat blood containing acetate at six concentrations, from 0.04 to 1.5 μmol/ml, to cover the physiological range encountered in the hapatic portal venous blood in vivo. 2. Below a concentration of 0.25 μmol/ml there was net production of acetate by the liver, while above it there was ner uptake with a fractional extraction of 40%. 3.No relationship was observed between blood [acetate] and hepatic ketogenesis, the ration [3-hydroxybutyrate]/[acetoacetate] or glucose output, either at low fatty acid concentration s or during oleate infusion. 4. Following the increase in serum fatty acid concentration, induced by oleate infusion, there were suquential incresase in ketogenesis and the ratio of [3-hydroxybutyrate]/[acetoacetate] while glucose output rose and lactate uptake fell significantly after in redox state. 5. There was a highly significant negative correlation between blood [acetate] and hepatic lactate uptake during oleate infusion. At the highest acetate concentration of 1.5 μmol/ml there was a small net hepatic lactate output. After oleate infusion ceased, lactate uptake increased, but the negative correlation between blood [acetate] and hepatic lactate uptake persisted. 6. Livers were also perfused with iether [1-¹⁴C]acetate or [U-¹⁴C]lactate at a concentration of acetate of either 0.3 or 1.3 μmol/ml of blood. With [1-¹⁴C]acetate, most of the radioactivity was recovered as fatty acids at the lower concentration of blood acetate. At the higher blood [acetate] a considerably smaller proportion of the radioactivity was recovered in lipids. With [U-¹⁴C]lactate the reverse pattern obtained i.e., recovery was greater at the high concentration of acetate and fell at the low concentration. Fatty acid biosynthesis, measured with ³H₂O, was stimulated from 2.4 to 6.6 μmol of fatty acid/g of liver per h by high blood [acetate] although the contribution of (acetate+lactate) to synthesis remained constant at 33–38% of the total. 7. These results emphasize the important role of the liver in regulating blood acetate concentrations and indicate that it can be major hepatic substrate. Acetate taken up by the liver appeared to compete directly with lactate, for lipogenesis and metabolism and acetate uptake was inhibited by raised bloodd [lactate].     

The stimulating effect of 3′,5′-(cyclic)adenosine monophosphate and lipolytic hormones on 3-O-methylglucose transport and 45Ca2+ release in adipocytes and skeletal muscle of the rat

(1) In order to assess the possible role of 3′,5′-(cyclic)adenosine monophosphate (cAMP) in the control of glucose transport, the effect of the nucleotide or agents known to increase its intracellular concentration on sugar transport or ⁴⁵Ca²⁺ washout were characterized in epididymal fat pads, free fat cells and soleus muscles of the rat. (2) When added to the incubation medium, cAMP (0.1–2.0 mM) stimulated $\text{3-O-[}{}^{14}\text{C}\text{]}\text{methylglucose}$ washout from fat pads. This effect was abolished by cytochalasin B, and additive to that induced by submaximal (10–25 μU/ml), but not by supramaximal (10 mU/ml) concentrations of insulin. (3) cAMP (2 mM) stimulated the conversion of [U-¹⁴C]glucose into CO₂ and triacylglycerols. This effect was additive to that of insulin (100 μU/ml). (4) ACTH, glucagon, adrenaline, noradrenaline and salbutamol, which are all known to increase the cAMP content of adipose tissue, stimulated the washout of $\text{3-O-[}{}^{14}\text{C}\text{]}\text{methylglucose}$ and ⁴⁵Ca²⁺ from preloaded fat pads. The fractional losses of the two isotopes were significantly correlated ($\text{P < 0.001}$, $\text{r = 0.73}$). (5) In free fat cells, adrenaline (10^?6 M) and salbutamol (10^?5 M) stimulated the uptake of $\text{3-O-[}{}^{14}\text{C}\text{]}\text{methylglucose}$, and salbutamol (10^?5 M) did not interfere with the stimulating effect of insulin (25 μU/ml) on sugar uptake. (6) In rat soleus muscles, adrenaline and salbutamol produced a dose-dependent stimulation of the washout of $\text{3-O-[}{}^{14}\text{C}\text{]}\text{methylglucose}$ and ⁴⁵Ca²⁺. The effect of adrenaline on sugar efflux was abolished by propranolol. (7) It is concluded that the activation of the glucose transport system by insulin is unlikely to be mediated by a drop in the cellular concentration of cAMP. An increase in cAMP brought about by β-adrenoceptor agonists or lipolytic hormones may induce a mobilization of calcium ions from cellular pools into the cytoplasm, which in turn leads to the activation of the glucose transport system demonstrated in the present as well as in several earlier studies.     

Effect of chronic psychological stress on insulin release from rat isolated pancreatic islets

Despite documented studies, the exact role of stress on diabetes is still unclear. The present study investigates the effect of chronic psychological stress on insulin release from isolated rat pancreatic islets. Male Wistar rats were divided into two groups of control and stressed (n=8/group). The animals of the stressed group were exposed to restraint stressors (1 h twice daily) for 15 or 30 consecutive days. At the beginning and end of the experimental periods, the animals were weighed and blood samples taken to determine the fasting plasma levels of glucose, insulin and corticosterone. On the following day the pancreatic islets of 5/group of the above animals were isolated and the static release of insulin in the presence of different glucose concentrations (2.8, 5.6, 8.3, 16.7 mM) was assessed. The results showed that in the stressed group, fasting plasma glucose levels were increased significantly on the 15th day as compared to the control group. However there was no significant increase on the 30th day. Fasting plasma insulin was significantly decreased on the 15th and 30th days of the experiment in the stressed group. Stressed rats showed significantly higher fasting plasma corticosterone levels, only on the 15th day, as compared to the control rats. In response to increasing concentrations of glucose, insulin release from islets of the stressed group was increased significantly on the 30th day of the experiment as compared to the control group. We conclude that chronic psychological stress could increase responsiveness of pancreatic beta cells to glucose, in vitro, and thus, low insulin levels of the stressed animals, in vivo, may be due to reason(s) other than the reduction of insulin releasing capacity of pancreatic beta cells.     

Glucose inhibits 45Ca efflux from pancreatic β-cells also in the absence of Na+-Ca2+ countertransport

During perifusion with medium deprived of Ca²⁺, addition of glucose or omission of Na⁺ resulted in prompt and quantitatively similar inhibitions of ⁴⁵Ca efflux from β-cell rich pancreatic islets microdissected from ob / ob mice. Glucose had no additional inhibitory effect when Na⁺ was isoosmotically replaced by sucrose or choline⁺. When K⁺ was used as a substitute for Na⁺, the inhibitory effect of Na⁺ removal on ⁴⁵Ca efflux became additive to that of glucose. The observation that glucose can be equally effective in inhibiting ⁴⁵Ca efflux in the presence or absence of Na⁺ is difficult to reconcile with the postulate that the Na⁺-Ca²⁺ countertransport mechanism is a primary site of action for glucose.     

Cyclin-dependent kinase-5 is a key molecule in tumor necrosis factor-α-induced insulin resistance

The mechanism of TNF-α-induced insulin resistance has remained unresolved with evidence for down-regulation of insulin effector targets effects or blockade of proximal as well as distal insulin signaling events depending upon the dose, time, and cell type examined. To address this issue we examined the acute actions of TNF-α in differentiated 3T3L1 adipocytes. Acute (5-15 min) treatment with 20 ng/ml (~0.8 nm) TNF-α had no significant effect on IRS1-associated phosphatidylinositol 3-kinase. In contrast, TNF-α increased insulin-stimulated cyclin-dependent kinase-5 (CDK5) phosphorylation on tyrosine residue 15 through an Erk-dependent pathway and up-regulated the expression of the CDK5 regulator protein p35. In parallel, TNF-α stimulation also resulted in the phosphorylation and GTP loading of the Rho family GTP-binding protein, TC10α. TNF-α enhanced the depolymerization of cortical F-actin and inhibited insulin-stimulated glucose transporter-4 (GLUT4) translocation. Treatment with the MEK inhibitor, PD98059, blocked the TNF-α-induced increase in CDK5 phosphorylation and the depolymerization of cortical F-actin. Conversely, siRNA-mediated knockdown of CDK5 or treatment with the MEK inhibitor restored the impaired insulin-stimulated GLUT4 translocation induced by TNF-α. Furthermore, siRNA-mediated knockdown of p44/42 Erk also rescued the TNF-α inhibition of insulin-stimulated GLUT4 translocation. Together, these data demonstrate that TNF-α-mediated insulin resistance of glucose uptake can occur through a MEK/Erk-dependent activation of CDK5.