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.     

Relationship of the reduction-oxidation state to protein degradation in skeletal and atrial muscle

Changes in proteolysis were correlated with the cell reduction-oxidation state in rat diaphragm and atrium. Protein degradation was measured in the presence of cycloheximide as the linear release of tyrosine into the medium. Intracellular ratios of lactate/pyruvate, total $\text{NADH}\text{NAD}$, and malate/pyruvate were used as indicators of the muscle reduction-oxidation state. Incubation of diaphragms with leucine (0.5–2.0 mm) or its transamination product, sodium α-ketoisocaproate (0.5 mm), resulted in a lower rate of proteolysis and a higher ratio of lactate/pyruvate and $\text{NADH}\text{NAD}$. These effects of leucine could be abolished by inhibiting its transamination with l-cycloserine. Unlike leucine, neither isoleucine nor valine alone produced any change in these parameters. Incubation of diaphragms with glucose (20 mm) or atria with sodium lactate (2 mm) produced a diminution of tyrosine release from the muscles and a rise in the ratio of total $\text{NADH}\text{NAD}$. Similarly, in incubated diaphragms of fasted rats, the anabolic effects of insulin, epinephrine and isoproterenol on protein degradation were associated with a higher malate/pyruvate ratio. In catabolic states, such as fasting, cortisol treatment of fasted, adrenalectomized rats or traumatization, enhanced muscle proteolysis was observed. Fresh-frozen diaphragms from these rats had both lower lactate/pyruvate and malate/pyruvate ratios than did muscles from control animals. These data show that diminution of proteolysis in diaphragm is accompanied by an increase of the $\text{NAD(P)H}\text{NAD(P)}$ ratios. In contrast to these findings, chymostatin and leupeptin, which inhibit directly muscle proteinases, caused a decrease of the lactate/pyruvate and malate/pyruvate ratios. These results suggest that protein degradation in diaphragm and atrium is linked to the cellular redox state.     

The pancreatic beta-cell recognition of insulin secretagogues. Comparisons of glucose with glyceraldehyde isomers and dihydroxyacetone

d-Glyceraldehyde stimulated the release of insulin from pancreatic islets of Umeå-$\text{ob}\text{ob}$-mice whether or not glucose was present in the medium. Like the action of glucose, that of d-glyceraldehyde was biphasic in time, exhibited a sigmoidal dose-response relationship, was potentiated by theophylline, arginine, iodoacetamide, or l-glyceraldehyde, and was inhibited by epinephrine, 2,4-dinitrophenol, or Ca²⁺ deficiency. Half-maximum and maximum stimulations were produced by about 3 mm and 10 mm d-glyceraldehyde. Positive interactions were observed between 5 mm d-glyceraldehyde and 5 mm glucose and between 10 mm d-glyceraldehyde and 10 mm leucine. Mannoheptulose (10 mm) or glucosamine (10 mm) did not inhibit but potentiated the effect of 10 mm d-glyceraldehyde. Dihydroxyacetone (2.5–20 mm) also initiated insulin release in the absence of glucose. On the other hand, 5–10 mm l-glyceraldehyde did not initiate secretion but potentiated the effects of 5 mm glucose or 5 mm d-glyceraldehyde. d-Glyceraldehyde or dihydroxyacetone reduced the production of ¹⁴CO₂ from d-[U-¹⁴C]glucose; l-glyceraldehyde had a smaller and statistically insignificant effect. The results suggest that by being phosphorylated and entering glycolysis in the β-cells, d-glyceraldehyde and dihydroxyacetone act as functional analogues of glucose as secretory stimulus. Initiation of insulin release by glucose, d-glyceraldehyde, or dihydroxyacetone may thus depend on the production of a metabolic signal at or below the triose phosphate level.     

Effect of dichloroacetate and glucagon on the incorporation of labeled substrates into glucose and on pyruvate dehydrogenase in hepatocytes from fed and starved rats

Dichloroacetate (2 mm) stimulated the conversion of [1-¹⁴C]lactate to glucose in hepatocytes from fed rats. In hepatocytes from rats starved for 24 h, where the mitochondrial $\text{NADH}\text{NAD}{}^{\mathrm{+}}$ ratio is elevated, dichloroacetate inhibited the conversion of [1-¹⁴C]lactate to glucose. Dichloroacetate stimulated ¹⁴CO₂ production from [1-¹⁴C]lactate in both cases. It also completely activated pyruvate dehydrogenase and increased flux through the enzyme. The addition of β-hydroxybutyrate, which elevates the intramitochondrial $\text{NADH}\text{NAD}{}^{\mathrm{+}}$ ratio, changed the metabolism of [1-¹⁴C]lactate in hepatocytes from fed rats to a pattern similar to that seen in hepatocytes from starved rats. Thus, the effect of dichloroacetate on labeled glucose synthesis from lactate appears to depend on the mitochondrial oxidation-reduction state of the hepatocytes. Glucagon (10 nm) stimulated labeled glucose synthesis from lactate or alanine in hepatocytes from both fed and starved rats and in the absence or presence of dichloroacetate. The hormone had no effect on pyruvate dehydrogenase activity whether or not the enzyme had been activated by dichloroacetate. Thus, it appears that pyruvate dehydrogenase is not involved in the hormonal regulation of gluconeogenesis. Glucagon inhibited the incorporation of 10 mm [1-¹⁴C]pyruvate into glucose in hepatocytes from starved rats. This inhibition has been attributed to an inhibition of pyruvate dehydrogenase by the hormone (Zahlten et al., 1973, Proc. Nat. Acad. Sci. USA70, 3213–3218). However, dichloroacetate did not prevent the inhibition of glucose synthesis. Nor did glucagon alter the activity of pyruvate dehydrogenase in homogenates of cells that had been incubated with 10 mm pyruvate in the absence or presence of dichloroacetate. Thus, the inhibition by glucagon of pyruvate gluconeogenesis does not appear to be due to an inhibition of pyruvate dehydrogenase.     

Substrate control of glycogen levels in isolated hepatocytes from fed rats.

Isolated parenchymal cells from fed rat liver rapidly lose glycogen when incubated with glucose. The addition of glycerol or fructose but not insulin prevents much of the breakdown. When cells are incubated with glycerol and glucose, more glycogen is retained with the further addition of xylitol than of fructose or pyruvate. Oleate stimulates glycogen breakdown. The results indicate that glycerol may play an important physiological role in the control of glycogen synthesis in the liver, possibly by esterifying fatty acids. Furthermore, the results support the concept that the main effect of insulin on liver glycogen levels $\text{in vivo}$ may be the results of diminished flow of free fatty acids to the liver.     

Stimulation of pyruvate dehydrogenase activity by insulin-dextran complex in mouse adipose tissue.

Soluble and stable insulin-dextran complex was prepared. Pyruvate dehydrogenase activity, as assayed by ¹⁴CO₂ formation from [1-¹⁴C]-pyruvate in crude mitochondria of mouse adipose tissue, was increased after incubation of fat pads with native insulin or insulin-dextran. The direct addition of insulin or insulin-dextran to mitochondria was without effect. At submaximal stimulation, insulin-dextran was 10 times less effective than native insulin but the degree of maximal stimulation and the time course of activation by insulin and insulin-dextran were similar. The results favor the concept that the activation of pyruvate dehydrogenase in fat cells does not need the entry of insulin into cells.     

The relationship between the transport of glucose and cations across cell membranes in isolated tissues XI. The effect of vanadate on 45Ca-efflux and sugar transport in adipose tissue and skeletal muscle

(1) The effects of vanadate of hexose transport, ⁴⁵Ca-exchange and (Na⁺, K⁺)-contents have been characterized in isolated adipose tissue and skeletal muscles of the rat. (2) In whole epididymal fat pads, vanadate (0.5–5.0 mM) markedly stimulated the uptake of 2-deoxyl[¹⁴C]glucose as well as the efflux of $\text{3-O-[}{}^{14}\text{C}\text{]}\text{methylglucose}$. (3) Within the same concentration range, vanadate induced an early increase in ⁴⁵Ca-washout from preloaded fat pads. The maximum increases in the fractional losses of $\text{3-O-[}{}^{14}\text{C}\text{]}\text{methylglucose}$ and ⁴⁵Ca were significantly correlated ($\text{P < 0.001}$, $\text{r = 0.98}$). (4) In extensor digitorum longus and soleus muscles, vanadate (0.5–5.0 mM) stimulated the efflux of $\text{3-O-[}{}^{14}\text{C}\text{]}\text{methylglucose}$ and this effect was preceded by a rise in the washout of ⁴⁵Ca. The maximum increases in the fractional losses of $\text{3-O-[}{}^{14}\text{C}\text{]}\text{methyglucose}$ and ⁴⁵Ca were significantly correlated ($\text{P < 0.005}$, $\text{r = 0.98}$). (5) In extensor digitorum longus and soleus muscles, vanadate increased K⁺-contents and decreased Na⁺ contents. (6) The stimulation of ⁴⁵Ca-washout presumably reflects an increase in the cytoplasmic Ca²⁺ level, brought about by an inhibitory effect of vanadate on the Ca²⁺-sensitive ATPase of the sarcoplasmic or the endoplasmic reticulum. As demonstrated for most other insulin-like agents (Sørensen, S.S., Christensen, F. and Clausen, T. (1980) Biochim. Biophys. Acta 602, 433–445), the stimulating effect of vanadate on glucose transport appears to be associated with or mediated by a rise in the cytoplasmic Ca²⁺ level.     

Identification of two forms of glutamine synthetase in barley (Hordeum vulgare).

Hepatocytes of 14-day-old rats have no detectable glucokinase activity $\text{in}$$\text{vivo}$, but it was induced by insulin (10^?8M) in primary cultures of these hepatocytes. The glucokinase induced by insulin was separated by electrophoresis on a cellulose acetate membrane and identified by its low affinity for glucose. This precocious induction of glucokinase was completely prevented by the presence of either actinomycin D or cycloheximide. Glucagon also inhibited its induction by insulin. Dexamethasone and testosterone, which alone had no inductive effect, strongly enhanced the induction by insulin. When hepatocytes of 14-day-old rats were cultured with 10^?7M insulin, 10^?6M dexamethasone and 10^?7M testosterone for 48 hr, their glucokinase activity increased to the non-induced level in hepatocytes of adult rats. Estrogen, thyroxine or growth hormone did not induce glucokinase precociously. Testosterone did not enhance induction of glucokinase by insulin in cultured hepatocytes of adult rats.     

Gluconeogenesis from L-serine in rat liver.

Livers of fed and fasted rats were perfused $\text{in situ}$ in the presence and absence of 4.8 mM quinolinate, an $\text{in vivo}$ inhibitor of phosphoenolpyruvate carboxykinase. An assay of the hepatic activities of serine dehydratase and serine pyruvate transaminase and a comparison of the $\text{in vivo}$ incorporation of radioactivity from serine 3-¹⁴C and serine U-¹⁴C into blood glucose were also carried out in the above nutritional states. Our results demonstrate that gluconeogenesis from L-serine proceeds through two pathways. One, involving the reversal of the biosynthetic route of serine, bypasses conversion to pyruvate phosphoenolpyruvate and oxaloacetate and is not inhibited by quinolinate. This pathway appears to be the only one active in the fed state but produces a very insignificant amount of glucose. The other involves serine dehydratase mediated conversion of serine to pyruvate, is inhibited by quinolinate and becomes predominant during starvation.     

Futile cycles in isolated perfused rat liver and in isolated rat liver parenchymal cells.

Isolated livers from fed rats were perfused with a medium containing glucose labeled uniformly with ¹⁴C and specifically with ³H. There was considerable formation of glucose from endogenous sources but simultaneously uptake of about half of the ¹⁴C in glucose. After 2 hours the ${}^3\text{H}{}^{14}\text{C}$ ratios in perfusate glucose decreased by 55–60% with (2-³H, U-¹⁴C), 40–50% with (5-³H, U-¹⁴C), 25–30% with (3-³H or 4-³H, U-¹⁴C) and by 10–15% with (6-³H, U-¹⁴C) glucose. Qualitatively comparable patterns were obtained with rat hepatocytes. These results demonstrate recycling of carbon between glucose and pyruvate. Superimposed upon this there is an extensive futile cycle between glucose and glucose 6-P. There is also futile cycling between fructose 6-P and fructose 1,6 P₂ and to a small extent between phosphoenol pyruvate and pyruvate.     

Accumulation of 2-deoxyglucose against its concentration gradient in rat adipocytes

Rat adipocytes were incubated at 37°C with 2-deoxy-d-[1-¹⁴C]glucose ([¹⁴C]2dGlc) at various concentrations and the intracellular concentrations of [¹⁴C]2dGlc and deoxy[¹⁴C]glucose phosphate ($\text{[}{}^{14}\text{C}\text{]2}\text{dGlc}\text{P}$) were measured. Using 7 μM extracellular [¹⁴C]2dGlc, the intracellular [¹⁴C]2dGlc concentration approached the extracellular by 5 min in insulin-stimulated cells and by 60 min it exceeded the extracellular concentration by 50-fold. A maximum accumulation ratio of 3.5 was reached by 7 min using 1 mM and a ratio of 1.6 was reached by 1 to 3 min using 10 mM extracellular 2dGlc. The time at which the concentration of intracellular 2dGlc exceeded the extracellular was inversely related to the accumulation of $\text{2}\text{dGlc}\text{P}$. The rate of accumulation of total radioactivity ([¹⁴C]2dGlc plus $\text{[}{}^{14}\text{C}\text{]2}\text{dGlc}\text{P}$ decreased after 20 min using 7 μM extracellular [¹⁴C]2dGlc. This change occurred later at 22°C or in the absence of insulin and sooner at higher concentrations of 2dGlc. Experiments where uptake was stopped by dilution indicated that radioactivity appearing in the medium was [¹⁴C]2dGlc, but radioactivity disappearing from the cells was largerly $\text{[}{}^{14}\text{C}\text{]2}\text{dGlc}\text{P}$. Addition of 10 mM unlabelled 2dGlc or glucose to cells preincubated with 7 μM [¹⁴C]2dGlc resulted in a more rapid loss of accumulated label from the cells, while addition of 10 mM $\text{3-O-}\text{methylglucose}$, a non-metabolizeable sugar analogue with about the same affinity for the transport system as 2dGlc, was without effect. The results show that 2dGlc is accumulated against its concentration gradient. It is suggested that the mechanism involves first, dephosphorylation of $\text{2}\text{dGlc}\text{P}$ and second, the presence of a diffusion barrier between the site of dephosphorylation and the transport site.     

In vivo lipogenesis and enzyme levels in adipose and liver tissues from pair-fed genetically obese and lean rats

Genetically obese Zucker rats pair-fed to lean controls were similar in body weight and food intake, however, epididymal fat pads were considerably larger than lean controls. $\text{In}$$\text{vivo}$ incorporation of acetate-¹⁴C into adipose tissue lipid was not significantly different, however, $\text{in}$$\text{vivo}$ liver lipogenesis was elevated in the obese rat. Characterization of enzyme profiles in both liver and adipose tissues revealed that enzymes normally associated with lipogenesis were elevated in liver tissue from obese rats. Malic enzyme and citrate cleavage enzyme were both depressed in adipose tissue of obese animals. From these data, it appears that the liver may be prominently involved in the development of excessive blood lipid and enlarged fat cells in the Zucker obese rat.     

Amino acid uptake by yeasts: IV. Effect of thiol reagents on l-leucine transport in Saccharomyces cerevisiae

(1) $\text{N-}\text{Ethylmaleimide}$ (a penetrating SH- reagent) inactivated l-[¹⁴C]leucine entrance (binding and translocation) into Saccharomyces cerevisiae, the extent of inhibition depending on the time of preincubation with $\text{N-}\text{ethylmaleimide}$, $\text{N-}\text{ethylmaleimide}$ concentration, the amino acid external and internal concentration, and the energization state of the yeast cells. With d-glucose-energized yeast, $\text{N-}\text{ethylmaleimide}$ inhibited l-[¹⁴C]leucine entrance in all the assayed experimental conditions, but with starved yeast and low (0.1 mM) amino acid concentration, it did not inhibit l-[¹⁴C]leucine binding, except when the cells were preincubated with l-leucine. With the rho^? respiratory-deficient mutant (energized cells), $\text{N-}\text{ethylmaleimide}$ inhibited l[¹⁴C]leucine entrance as with the energized wild-type, though to a lesser extent. (2) Analysis of the $\text{N-}\text{ethylmaleimide}$ effect as a function of l-[¹⁴C]leucine concentration showed a significant decrease of $\text{J}{}_{\mathrm{<mtext>max</mtext>}}$ values of the high- (S₁) and low- (S₂) affinity amino acid transport systems, but $\text{K}{}_{\mathrm{<mtext>T</mtext>}}$ values were not significantly modified. (3) When assayed in the presence of d-glucose, $\text{N-}\text{ethylmaleimide}$ inhibition of d-glucose uptake and respiration contributed significantly to inactivation of l-[¹⁴C]leucine entrance. Pretreatment of yeast cells with 2,4-dinitrophenol enhanced the effect of l-[¹⁴C]leucine binding and translocation. (4) Bromoacetylsulfanilic acid and bromoacetylaminoisophthalic acid, two non-penetrating SH- reagents, did not inactivate l-[¹⁴C]leucine entrance, while $\text{p-}\text{chloromercuribenzoate}$, a slowly penetrating SH- reagent, inactivated it to a limited extent. When compared with the effect of $\text{N-}\text{ethylmaleimide}$, these negative results indicate that thiol groups of the l-[¹⁴C]leucine carrier were not exposed on the outer surface of the yeast cell permeability barrier.     

Postinhibitory overshoot of insulin release by gastric inhibitory polypeptide

Following intravenous infusion of somatostatin $\text{in vivo}$ occasionally there is a large rebound overshoot of insulin release. An $\text{in vitro}$ model to simulate this phenomenon was made by perfusing rat pancreas with gastric inhibitory polypeptide (GIP) during simultaneous perfusion with somatostatin. Adding GIP (100 ng/ml) to the perfusate for 2 minutes beginning either 3 or 9 minutes before terminating the somatostatin perfusion produced a large overshoot in insulin release. The magnitude of overshoot was greater when medium contained 300 mg/dl glucose that when it contained 150 mg/dl glucose. Perfusion with GIP for 2 minutes beginning 9 minutes before increasing the glucose concentration of the medium from 30 to 300 mg/dl elicited a large increase in both the acute and second-phase release of insulin. These suggest that post-inhibitory overshoot of insulin release after somatostatin may be produces $\text{in vitro}$ by the suppressed action of stimulatory hormones such as GIP. Prior infusion with GIP can also potentiate glucose-stimulated insulin increase.     

A new test of peripheral insulin sensitivity invivo using artificial beta cell

A new $\text{in}$$\text{vivo}$ test of insulin sensitivity is described. It utilizes closed-loop insulin delivery device (GCIIS, Biostator^®) capable of infusing glucose and insulin according to preselected algorithms. In euglycemic patients, insulin was infused by GCIIS to maintain euglycemia in the face of challenges with gradually increasing doses of intravenously administered glucose. Under the described experimental conditions, the endogenous insulin release was minimized as evidenced by serum C-peptide levels of less than 2 ng/ml, and thus the peripheral disposal of glucose should have depended entirely on the exogenous insulin. The amount of the insulin infused was considered to be a measure of peripheral insulin sensitivity. The test was applied to normal and non diabetic obese individuals, and to diabetics, both insulin dependent and independent. Significant insulin resistance was demonstrated in the obese and diabetic patients. In two obese females, the test was repeated after a prolonged period of starvation, and showed marked increase in insulin sensitivity. In two poorly controlled insulin dependent diabetics, marked increase in insulin sensitivity was also observed, here following a prolonged period of euglycemia (48 hours). It is concluded that the GCIIS controlled insulin sensitivity test is a simple, reliable test of peripheral insulin sensitivity, most convenient for clinical and experimental studies $\text{in}$$\text{vivo}$     

Somatostatin analogs which inhibit glucagon and growth hormone more than insulin release

Three analogs of somatostatin, [$\text{D}$-Cys¹⁴] -, [Ala², $\text{D}$-Cys¹⁴] - and [$\text{D}$-Trp⁸, $\text{D}$-Cys¹⁴] - somatostatin, were synthesized by the solid phase method, characterized by several means, and tested for their effects on the release of insulin, glucagon, and growth hormone. The peptides sharply suppressed the release of growth hormone $\text{in vitro}$ and glucagon $\text{in vivo}$, but had less effect on insulin secretion $\text{in vivo}$. These analogs, particularly [$\text{D}$-Trp⁸, $\text{D}$-Cys¹⁴] - somatostatin, could possibly be useful for the treatment of diabetes mellitus.     

Cytochalasin B inhibition and temperature dependence of 3-O-methylglucose transport in fat cells

The transport of $\text{3-O-}\text{methylglucose}$ in white fat cells was measured under equilibrium exchange conditions at $\text{3-O-}\text{methylglucose}$ concentrations up to 50 mM with a previously described method (Vinten, J., Gliemann, J. and Østerlind, K. (1976) J. Biol. Chem. 251, 794–800). Under these conditions the main part of the transport was inhibitable by cytochalasin B. The inhibition was found to be of competitive type with an inhibition constant of about 2.5 · 10^?7 M, both in the absence and in the presence of insulin (1μM). The cytochalasin B-insensitive part of the $\text{3-O-}\text{methylglucose}$ permeability was about 2 · 10^?9 cm · s^?1, and was not affected by insulin. As calculated from the maximum transport capacity, the half saturation constant and the volume/ surface ratio, the maximum permeability of the fat cell membrane to $\text{3-O-}\text{methylglucose}$ at 37°C and in the presence of insulin was 4.3 · 10^?6 cm · s^?1. From the temperature dependence of the maximum transport capacity in the interval 18–37°C and in the presence of insulin, an Arrhenius activation energy of $\text{14.8 ± 0.44}\text{kcal/mol}$ was found. The corresponding value was $\text{13.9 ± 0.89}$ in the absence of insulin. The half saturating concentration of $\text{3-O-}\text{methylglucose}$ was about 6 mM in the temperature interval used, and it was not affected by insulin, although this hormone increased the maximum transport capacity about ten-fold to 1.7 mmol · s^?1 per 1 intracellular water at 37°C.