Decreased islet sensitivity to insulin in hamsters with dietary-induced insulin resistance

In the present study, we evaluated the autocrine modulatory effect of insulin on glucose metabolism and glucose-induced insulin secretion in islets isolated from hamsters with insulin resistance (IR) induced by administration of a sucrose-rich diet (SRD) during 5 weeks. We used an approach of two metabolic pathways (glucose oxidation and utilization) based on the measurement of 14CO2 and 3H2O production from D-[U-14C]-glucose and D-[5-(3)H]-glucose, respectively, in isolated islets incubated with 3.3 and 16.7 mM glucose alone, or with 5 or 15 mU/ml insulin, anti-insulin guinea-pig serum (1:500), 25 microM nifedipine, or 150 nM wortmannin. Insulin release was measured by radioimmunoassay in islets incubated with 3.3 or 16.7 mM glucose, with or without 75, 150, and 300 nM wortmannin. Results showed that the stimulatory effect of insulin upon 14CO2 and 3H2O production in control islets was not observed in SRD islets. Addition of anti-insulin serum, nifedipine or wortmannin to the medium with 16.7 mM glucose decreased 14CO2 and 3H2O production in control but not in SRD islets. Whereas wortmannin did not decrease insulin release induced by 16.7 mM glucose in SRD hamsters, it did in controls. We can conclude that the autocrine stimulatory effect of insulin upon glucose metabolism observed in normal islets is attenuated or even absent in islets from IR animals. Such decreased islet sensitivity to insulin did not prevent the compensatory secretion of insulin from maintaining glucose homeostasis, suggesting that, at least in this model, the islets can put forward alternative mechanisms to overcome such defect.     

Palmitate acutely raises glycogen synthesis in rat soleus muscle by a mechanism that requires its metabolization (Randle cycle)

The acute effect of palmitate on glucose metabolism in rat skeletal muscle was examined. Soleus muscles from Wistar male rats were incubated in Krebs-Ringer bicarbonate buffer, for 1 h, in the absence or presence of 10 mU/ml insulin and 0, 50 or 100 microM palmitate. Palmitate increased the insulin-stimulated [(14)C]glycogen synthesis, decreased lactate production, and did not alter D-[U-(14)C]glucose decarboxylation and 2-deoxy-D-[2,6-(3)H]glucose uptake. This fatty acid decreased the conversion of pyruvate to lactate and [1-(14)C]pyruvate decarboxylation and increased (14)CO(2) produced from [2-(14)C]pyruvate. Palmitate reduced insulin-stimulated phosphorylation of insulin receptor substrate-1/2, Akt, and p44/42 mitogen-activated protein kinases. Bromopalmitate, a non-metabolizable analogue of palmitate, reduced [(14)C]glycogen synthesis. A strong correlation was found between [U-(14)C]palmitate decarboxylation and [(14)C]glycogen synthesis (r=0.99). Also, palmitate increased intracellular content of glucose 6-phosphate in the presence of insulin. These results led us to postulate that palmitate acutely potentiates insulin-stimulated glycogen synthesis by a mechanism that requires its metabolization (Randle cycle). The inhibitory effect of palmitate on insulin-stimulated protein phosphorylation might play an important role for the development of insulin resistance in conditions of chronic exposure to high levels of fatty acids.     

Effect of short-chain fatty acids on cyclic 3',5'-guanosine monophosphate-mediated colonic secretion

Short chain fatty acids (SCFA) prevent and reverse cyclic 3',5'-adenosine monophosphate (cAMP) but not Ca(2+)-mediated Cl- secretion. Mucosal [HCO3-]i has an opposite effect on these secretagogues. We examined whether SCFA and [HCO3-]i affect cyclic 3',5'-guanosine monophosphate (cGMP)-induced secretion. Stripped segments of male Sprague-Dawley rat (Rattus norvegicus) proximal and distal colon, and cultured T84 cells were studied in Using chambers, and pHi and [HCO3-]i were determined. Mucosal [cGMP] was measured in proximal colon. In T84 cells, the increase in Cl- secretion (measured as Isc) induced by mucosal 0.25 microM Escherichia coli heat-stable enterotoxin (STa) was prevented/reversed by bilateral 50 mM Na+ butyrate (71%/73%), acetate (58%/76%), propionate (68%/73%) and (poorly metabolized) isobutyrate (80%/79%). In proximal colon in HCO3- Ringer, basal Cl- secretion was not affected by [HCO3-]i or 25 mM butyrate. Mucosal 0.25 microM STa decreased net Na+ and Cl- absorption. Bilateral but not mucosal 25 mM SCFA reversed STa-induced effects on Na+ absorption and Cl- secretion. Bilateral and mucosal 25 mM SCFA but not [HCO3-]i prevented STa-induced Cl- secretion and increases in mucosal [cGMP]. STa did not produce Cl- secretion in distal colon. It was concluded that SCFA but not [HCO3-]i can prevent and reverse cGMP-induced colonic Cl- secretion.     

Autocrine regulation of glucose metabolism in pancreatic islets

We evaluated the possible autocrine modulatory effect of insulin on glucose metabolism and glucose-induced insulin secretion in islets isolated from normal hamsters. We measured 14CO2 and 3H2O production from d-[U-14C]glucose and d-[5-3H]glucose, respectively, in islets incubated with 0.6, 3.3, 8.3, and 16.7 mM glucose alone or with 5 or 15 mU/ml insulin, anti-insulin guinea pig serum (1:500), 25 microM nifedipine, or 150 nM wortmannin. Insulin release was measured (radioimmunoassay) in islets incubated with 3.3 or 16.7 mM glucose with or without 75, 150, and 300 nM wortmannin. Insulin significantly enhanced 14CO2 and 3H2O production with 3.3 mM glucose but not with 0.6, 8.3, or 16.7 mM glucose. Addition of anti-insulin serum to the medium with 8.3 and 16.7 mM glucose decreased 14CO2 and 3H2O production significantly. A similar decrease was obtained in islets incubated with 8.3 and 16.7 mM glucose and wortmannin or nifedipine. This latter effect was reversed by adding 15 mU/ml insulin to the medium. Glucose metabolism was almost abolished when islets were incubated in a Ca2+-deprived medium, but this effect was not reversed by insulin. No changes were found in 14CO2 and 3H2O production by islets incubated with 3.3 mM glucose and anti-insulin serum, wortmannin, or nifedipine in the media. Addition of wortmannin significantly decreased insulin release induced by 16.7 mM glucose in a dose-dependent manner. Our results suggest that insulin exerts a physiological autocrine stimulatory effect on glucose metabolism in intact islets as well as on glucose-induced insulin release. Such an effect, however, depends on the glucose concentration in the incubation medium.     

Chronic high glucose lowers pyruvate dehydrogenase activity in islets through enhanced production of long chain acyl-CoA: prevention of impaired glucose oxidation by enhanced pyruvate recycling through the malate-pyruvate shuttle

In islet beta-cells, the high expression of pyruvate carboxylase and the functional importance of the downstream anaplerosis pathways result in a unique characteristic whereby high glucose and fatty acids both increase production of a key fatty acid metabolite, long chain acyl-CoA, for signaling and enzyme regulation in beta-cells. We showed previously in islets that pyruvate dehydrogenase (PDH) activity is lowered by excess fatty acids (the so-called Randle effect). We have now investigated PDH activity and pyruvate metabolism in islets after 48-h culture at 16.7 mmol/liter glucose. Active PDH V(max) was lowered 65% by 48 h of high glucose, and this effect was markedly attenuated by co-culture with triacsin C, which inhibits acyl-CoA synthase. Despite the large reduction in PDH activity, glucose oxidation was twice normal. The reason was continued metabolism of pyruvate through pyruvate carboxylase (V(max), 83% of control) and diversion of flux through the pyruvate-malate shuttle. The result was a 3-fold increase of the pyruvate concentration that overcame the lowered PDH activity by mass action as shown by glucose oxidation measured with [6-(14)C]glucose being twice normal. In addition, glucose-induced insulin secretion was 3-fold increased after 48 h of high glucose, and this effect was totally blocked by co-culture with triacsin C. These results show that a unique feature of islet beta-cells is not only fatty acids but also excess glucose that impairs PDH activity. Also, a specialized trait of beta-cells is a long chain acyl-CoA-mediated defense mechanism that prevents a reduction in glucose oxidation and consequently in insulin secretion.     

Low protein diet confers resistance to the inhibitory effects of interleukin 1beta on insulin secretion in pancreatic islets*

High protein content in the diet during childhood and adolescence has been associated to the onset insulin-dependent diabetes mellitus. We investigated the effect of interleukin-1beta (IL-1beta) on insulin secretion, glucose metabolism, and nitrite formation by islets isolated from rats fed with normal protein (NP, 17%) or low protein (LP, 6%) after weaning. Pretreatment of islets with IL-1beta for 1 h or 24 h inhibited the insulin secretion induced by glucose in both groups, but it was less marked in LP than in NP group. Islets from LP rats exhibited a decreased IL-1beta-induced nitric oxide (NO) production, lower inhibition of D-[U(14)C]-glucose oxidation to (14)CO(2) and less pronounced effect of IL-1beta on alpha-ketoisocaproic acid-induced insulin secretion than NP islets. However, when the islets were stimulated by high concentrations of K(+) the inhibitory effect of IL-1beta on insulin secretion was not different between groups. In conclusion, protein restriction protects beta-cells of the deleterious effect of IL-1beta, apparently, by decreasing NO production. The lower NO generation in islets from protein deprived rats may be due to increased free fatty acids oxidation and consequent alteration in Ca(2+) homeostasis.     

Studies on the role of beta-cell metabolism in the insulinotropic effect of alpha-ketoisocaproic acid.

alpha-Ketoisocaproic acid has been shown to be a potent insulin secretagogue but the mechanism has not been elucidated. To define the role of beta-cell metabolism in the insulinotropic activity of alpha-ketoisocaproic acid the utilization of glucose and the oxidation of alpha-ketoisocaproic and isovaleric acid by incubated islets of obese hyperglycemic mice were measured. Glucose metabolism was never enhanced by alpha-ketoisocaproic acid. The same 14CO2 amounts were released from the non-secretagogue [1-14C]isovaleric acid (10 mM) or from alpha-keto[2-14C]isocaproic acid (5--20 mM). Pyruvate (20 mM) did not inhibit alpha-ketoisocaproic acid-induced insulin secretion in spite of reduction of decarboxylation of alpha-ketoisocaproic acid by more than 40%. The results indicate that stimulated insulin release in response to alpha-ketoisocaproic acid is not mediated by an indirect increase in glucose metabolism and further suggest that isovaleryl-CoA and following CoA-esters in alpha-ketoisocaproic acid degradation are not likely recognized as signals. The possibility, however, remains that enhanced intramitochondrial production of reducing equivalents elicits insulin secretion.     

Hexose metabolism in pancreatic islets. Regulation of aerobic glycolysis and pyruvate decarboxylation.

1. D-Glucose (0.5-16.7 mM) preferentially stimulates aerobic glycolysis and D-[3,4-14C]glucose oxidation, relative to D-[5-3H]glucose utilization in rat pancreatic islets, the concentration dependency of such a preferential effect displaying a sigmoidal pattern. 2. Inorganic and organic calcium antagonists, as well as Ca2+ deprivation, only cause a minor decrease in the ratio between D-[3,4-14C]glucose oxidation and D-[5-3H]glucose utilization in islets exposed to a high concentration of the hexose (16.7 mM). 3. Non-glucidic nutrient secretagogues such as 2-aminobicyclo[2,2,1]heptane-2-carboxylate (BCH), 2-ketoisocaproate and 3-phenylpyruvate fail to stimulate aerobic glycolysis and D-[3,4-14C]glucose oxidation in islets exposed to 6.0 mM D-glucose. Nevertheless, BCH augments [1-14C]pyruvate and [2-14C]pyruvate oxidation. 4. The glucose-induced increment in the paired ratio between D-[3,4-14C]glucose oxidation and D-[5-3H]glucose utilization is impaired in the presence of either cycloheximide or ouabain. 5. These findings suggest that the preferential effect of D-glucose upon aerobic glycolysis and pyruvate decarboxylation is not attributable solely to a Ca(2+)-induced activation of FAD-linked glycerophosphate dehydrogenase and/or pyruvate dehydrogenase, but may also involve an ATP-modulated regulatory process.     

2-Oxocarboxylic acids and function of pancreatic islets in obese–hyperglycaemic mice. Insulin secretion in relation to 45Ca uptake and metabolism

The effects of aliphatic 2-oxocarboxylic acids, at concentrations of up to 40mm, on the function of pancreatic islets from ob/ob (obese–hyperglycaemic) mice were investigated. 1. 2-Oxopentanoate, dl-3-methyl-2-oxopentanoate, 4-methyl-2-oxopentanoate and 2-oxohexanoate all induced insulin release by isolated incubated islets and a biphasic insulin-secretory pattern in perfused mouse pancreas. The last two substances were similar in potency to glucose. Pyruvate, 2-oxobutyrate, 3-methyl-2-oxobutyrate and 2-oxo-octanoate did not induce insulin release significantly. 2. 2-Oxocarboxylic acids with significant insulin-secretory potency also induced significant ⁴⁵Ca uptake by isolated incubated islets. 3. The rates of decarboxylation of [1-¹⁴C]pyruvate, 3-methyl-2-oxo[1-¹⁴C]butyrate and 4-methyl-2-oxo[1-¹⁴C]pentanoate were twice as high as the rates of oxidation of the corresponding U-¹⁴C-labelled compounds. However, whereas the rates of metabolism of labelled pyruvate and 3-methyl-2-oxobutyrate steadily increased over the concentration range 1–40mm, those of labelled 4-methyl-2-oxopentanoate and d-[U-¹⁴C]glucose levelled off at concentrations above 10mm. 4. Omission of ⁴⁰CaCl₂ from the incubation medium reduced the rate of oxidation of the insulin secretagogue [U-¹⁴C]4-methyl-2-oxopentanoate, but left that of the non-(insulin secretagogue) [U-¹⁴C]3-methyl-2-oxobutyrate unaffected. 5. Only glucose, and not pyruvate, 3-methyl-2-oxobutyrate and 4-methyl-2-oxopentanoate, significantly inhibited oxidation of endogenous fatty acids. 6. It is suggested that stimulus–secretion coupling and the resulting exocytosis of insulin in pancreatic β-cells may modulate both fuel oxidation and ⁴⁵Ca uptake.     

Stimulation of proinsulin biosynthesis and insulin release by pyruvate and lactate.

Increasing concentrations of pyruvate failed to stimulate proinsulin biosynthesis and insulin release in freshly isolated islets. Glycolytic flux (3H2O from [5-3H]glucose) decreased by 80-85%, but decarboxylation of [1(-14)C]pyruvate was unaffected in islets tested immediately after alloxan exposure. This strongly suggested that in freshly isolated islets, beta-cells, in relation to other islet cells, hardly contribute to the decarboxylation of pyruvate. Non-alloxan-treated cultured islets decarboxylated 2-2.5 times as much pyruvate as did alloxan-treated islets cultured for 15-18h. Thus the contribution of beta-cells to the metabolism of pyruvate after culturing markedly increased. Concomitantly beta-cells became responsive to pyruvate. At 20mM-pyruvate, release of prelabelled proinsulin and insulin and incorporation of [3H]leucine into proinsulin reached values approximately half of those obtained with 20mM-glucose. Lactate was as effective as pyruvate in inducing responses in cultured islets. The experiments indicate that a critical degree of substrate utilization is necessary for the generation of signals for insulin release and proinsulin biosynthesis.     

Normal flux through ATP-citrate lyase or fatty acid synthase is not required for glucose-stimulated insulin secretion

It has been proposed that de novo synthesis of long-chain acyl-CoA (LC-CoA) is a signal for glucose-stimulated insulin secretion (GSIS). Key enzymes involved in synthesis of fatty acids from glucose include ATP-citrate lyase (CL) and fatty acid synthase (FAS). An inhibitor of CL, hydroxycitrate (HC), has been reported to inhibit insulin secretion in some laboratories but not in others. Here we show that high concentrations of NaCl created during preparation of HC by standard methods explain the inhibition of GSIS, and that removal of the excess NaCl prevents the effect. To further investigate the role of CL, two small interfering RNA adenoviruses (Ad-siCL2 and Ad-siCL3) were generated. Ad-siCL3 reduced CL mRNA levels by 92 +/- 6% and CL protein levels by 75 +/- 4% but did not affect GSIS in 832/13 cells compared with cells treated with a control adenovirus (Ad-siControl). Similar results were obtained with Ad-siCL2. Ad-siCL3-treated cells also exhibited a 52 +/- 7% reduction in cytosolic oxaloacetate, an 83 +/- 4% reduction in malonyl-CoA levels, and inhibition of [U-(14)C]glucose incorporation into lipid by 43 +/- 4%, all expected metabolic out-comes of CL suppression. Similarly, treatment of 832/13 cells with a recombinant adenovirus specific to FAS (Ad-siFAS) reduced FAS mRNA levels by 81 +/- 2% in 832/13 cells, resulting in a 59 +/- 4% decrease in [U-(14)C]glucose incorporation into lipid, without affecting GSIS. Finally, treatment of primary rat islets with Ad-siCL3 or Ad-siFAS reduced CL and FAS mRNA levels by 65 +/- 4% and 52 +/- 3%, respectively, but had no effect on GSIS relative to Ad-siControl-treated islets. These findings demonstrate that a normal rate of flux of glucose carbons through CL and FAS is not required for GSIS in insulinoma cell lines or rat islets.     

Effects of lactate on pancreatic islets. Lactate efflux as a possible determinant of islet-cell depolarization by glucose.

The secretion of insulin from perifused rat pancreatic islets was stimulated by raising the glucose concentration from 5.6 to 20 mM or by exposure to tolbutamide. The addition of sodium lactate (40 mM) to islets perifused in the presence of glucose (5.6 mM) resulted in a small, transient, rise in the rate of secretion. The subsequent removal of lactate, but not glucose or tolbutamide, from the perifusate produced a dramatic potentiation of insulin release. The rate of efflux of 45Ca2+ was also increased when islets were exposed to a high concentration of glucose or lactate or to tolbutamide, and again subsequently upon withdrawal of lactate. Efflux of 86Rb+ was modestly inhibited upon addition of lactate and markedly enhanced by the subsequent withdrawal of lactate from islets. The output of [14C]lactate from islets incubated in the presence of [U-14C]glucose increased linearly with increasing concentrations of glucose (1-25 mM). It is proposed that the activation of islets by the addition or withdrawal of lactate is not due to increased oxidative flux, but occurs as a result of the electrogenic passage of lactate ions across the plasma membrane, resulting in islet-cell depolarization, Ca2+ entry and insulin secretion. The production of lactate via the glycolytic pathway, and the subsequent efflux of lactate from the islet cells with concomitant exchange of H+ for Na+, could be a major determinant of depolarization and hence insulin secretion, in response to glucose.     

The pentose cycle and insulin release in mouse pancreatic islets

1. Rates of insulin release, glucose utilization (measured as [(3)H]water formation from [5-(3)H]glucose) and glucose oxidation (measured as (14)CO(2) formation from [1-(14)C]- or [6-(14)C]-glucose) were determined in mouse pancreatic islets incubated in vitro, and were used to estimate the rate of oxidation of glucose by the pentose cycle pathway under various conditions. Rates of oxidation of [U-(14)C]ribose and [U-(14)C]xylitol were also measured. 2. Insulin secretion was stimulated fivefold when the medium glucose concentration was raised from 3.3 to 16.7mm in the absence of caffeine; in the presence of caffeine (5mm) a similar increase in glucose concentration evoked a much larger (30-fold) increase in insulin release. Glucose utilization was also increased severalfold as the intracellular glucose concentration was raised over this range, particularly between 5 and 11mm, but the rate of oxidation of glucose via the pentose cycle was not increased. 3. Glucosamine (20mm) inhibited glucose-stimulated insulin release and glucose utilization but not glucose metabolism via the pentose cycle. No evidence was obtained for any selective effect on the metabolism of glucose via the pentose cycle of tolbutamide, glibenclamide, dibutyryl 3':5'-cyclic AMP, glucagon, caffeine, theophylline, ouabain, adrenaline, colchicine, mannoheptulose or iodoacetamide. Phenazine methosulphate (5mum) increased pentose-cycle flux but inhibited glucose-stimulated insulin release. 4. No formation of (14)CO(2) from [U-(14)C]ribose could be detected: [U-(14)C]xylitol gave rise to small amounts of (14)CO(2). Ribose and xylitol had no effect on the rate of oxidation of glucose; ribitol and xylitol had no effect on the rate of glucose utilization. Ribose, ribitol and xylitol did not stimulate insulin release under conditions in which glucose produced a large stimulation. 5. It is concluded that in normal mouse islets glucose metabolism via the pentose cycle does not play a primary role in insulin-secretory responses.     

Studies on the role of β-cell metabolism in the insulinotropic effect of α-ketoisocaproic acid

α-Ketoisocaproic acid has been shown to be apotent insulin secretagogue but the mechanism has not been elucidated. To define the role of β-cell metabolism in the insulinotropic activity of α-ketoisocaproic acid the utilization of glucose and the oxidation of α-ketoisocaproic and isovaleric acid by incubated islets of obese hyperglycemic mice were measured.Glucose metabolism was never enhanced by α-ketoisocaproic acid. The same ¹⁴CO₂ amounts were released from the non-secretagogue [1-¹⁴C]isovaleric acid (10 mM) or from α-keto [2-¹⁴C]isocaproic acid (5–20 mM). Pyruvate (20 mM) did not inhibit α-ketoisocaproic acid-induced insulin secretion in spite of reduction of decarboxylation of α-ketoisocaproic acid by more than 40%.The results indicate that stimulated insulin release in response to α-ketoisocaproic acid is not mediated by an indirect increase in glucose metabolism and further suggest that isovaleryl-CoA and following CoA-esters in α-ketoisocaproic acid degradation are not likely recognized as signals. The possibility, however, remains that enhanced intramitochondrial production of reducing equivalents elicits insulin secretion.     

Glucose metabolism in mouse pancreatic islets

1. Rates of glucose oxidation, lactate output and the intracellular concentration of glucose 6-phosphate were measured in mouse pancreatic islets incubated in vitro. 2. Glucose oxidation rate, measured as the formation of (14)CO(2) from [U-(14)C]glucose, was markedly dependent on extracellular glucose concentration. It was especially sensitive to glucose concentrations between 1 and 2mg/ml. Glucose oxidation was inhibited by mannoheptulose and glucosamine but not by phlorrhizin, 2-deoxyglucose or N-acetylglucosamine. Glucose oxidation was slightly stimulated by tolbutamide but was not significantly affected by adrenaline, diazoxide or absence of Ca(2+) (all of which may inhibit glucose-stimulated insulin release), by arginine or glucagon (which may stimulate insulin release) or by cycloheximide (which may inhibit insulin synthesis). 3. Rates of lactate formation were dependent on the extracellular glucose concentration and were decreased by glucosamine though not by mannoheptulose; tolbutamide increased the rate of lactate output. 4. Islet glucose 6-phosphate concentration was also markedly dependent on extracellular glucose concentration and was diminished by mannoheptulose or glucosamine; tolbutamide and glucagon were without significant effect. Mannose increased islet fructose 6-phosphate concentration but had little effect on islet glucose 6-phosphate concentration. Fructose increased islet glucose 6-phosphate concentration but to a much smaller extent than did glucose. 5. [1-(14)C]Mannose and [U-(14)C]fructose were also oxidized by islets but less rapidly than glucose. Conversion of [1-(14)C]mannose into [1-(14)C]glucose 6-phosphate or [1-(14)C]glucose could not be detected. It is concluded that metabolism of mannose is associated with poor equilibration between fructose 6-phosphate and glucose 6-phosphate. 6. These results are consistent with the idea that glucose utilization in mouse islets may be limited by the rate of glucose phosphorylation, that mannoheptulose and glucosamine may inhibit glucose phosphorylation and that effects of glucose on insulin release may be mediated through metabolism of the sugar.     

Inhibition by L-valine and L-norleucine of 3-phenylpyruvate-induced insulin release

Insulin release induced by 3-phenylpyruvate in isolated rat pancreatic islets was inhibited by L-valine, L-norleucine or aminooxyacetate. The inhibitory effect of these three agents coincided with a lesser stimulation by 3-phenylpyruvate of 14CO2 output from islets prelabelled with L-[U-14C] glutamine. Conversely, 3-phenylpyruvate augmented the rate of conversion of L-valine to 2-ketoisovalerate and that of L-norleucine to 2-ketocaproate. However, 3-phenylpyruvate, which increased 2-ketoisovalerate oxidative decarboxylation, inhibited 14CO2 production by islets exposed to D, L-[1-14C] norleucine. These findings reveal that distinct nutrient secretagogues (e.g. 3-phenylpyruvate and L-norleucine), which are each able to stimulate insulin release, may act antagonistically upon the secretory process when used in combination. The present results also emphasize the relevance of both mitochondrial oxidation and intracellular transfer of reducing equivalents as determinants of the secretory response to such nutrients as 3-phenylpyruvate and norleucine.     

Aromatic-L-amino-acid decarboxylase activity in mouse pancreatic islets

Aromatic-L-amino-acid decarboxylase activity has been measured in intact or homogenised pancreatic islets of ob/ob mice (Ume? ob/ob). The method used involves the trapping and measuring of the 14CO2 released from L-[1-14C]dihydroxyphenylalanine (L-dopa). Islets showed a decarboxylase activity which was dependent on pyridoxal phosphate and inhibitable by 0.1 mM benserazide or 0.1 mM alpha-monofluoromethyldopa. Maximum activity in intact islets was about 330 mmol/kg dry islet per h with an apparent Km of 3.3 mM. Islet homogenates had a Vmax of about 120 mmol/kg per h with a Km of 0.3 mM. L-5-Hydroxytryptophan, m-tyrosine and o-tyrosine interfered with the decarboxylation of L-dopa in a way that suggested a high activity also towards those substrates. L-Phenylalanine, L-tyrosine and D-glucose had no effect. At 0.05 mM L-dopa islet homogenates showed a much higher activity than homogenates of liver, kidney, or spleen. Islet uptake of L-[3H]dopa was well in excess of the decarboxylation rate and thus probably not rate-limiting. It is concluded that mouse pancreatic islets have a high activity of aromatic-L-amino-acid decarboxylase. This is in accordance with previous suggestions of a stimulatory effect of this enzyme on insulin secretion.     

Participation of endogenous fatty acids in the secretory activity of the pancreatic B-cell.

The pancreatic B-cell may represent a fuel-sensor organ, the release of insulin evoked by nutrient secretagogues being attributable to an increased oxidation of exogenous and/or endogenous substrates. The participation of endogenous fatty acids in the secretory response of isolated rat pancreatic islets was investigated. Methyl palmoxirate (McN-3716, 0.1 mM), an inhibitor of long-chain-fatty-acid oxidation, suppressed the oxidation of exogenous [U-14C]palmitate and inhibited 14CO2 output from islets prelabelled with [U-14C]palmitate. Methyl palmoxirate failed to affect the oxidation of exogenous D-[U-14C]glucose or L-[U-14C]glutamine, the production of NH4+ and the output of 14CO2 from islets prelabelled with L-[U-14C]glutamine. In the absence of exogenous nutrient and after a lag period of about 60 min, methyl palmoxirate decreased O2 uptake to 69% of the control value. Methyl palmoxirate inhibited insulin release evoked by D-glucose, D-glyceraldehyde, 2-oxoisohexanoate, L-leucine, 2-aminobicyclo[2.2.1]heptane-2-carboxylate or 3-phenylpyruvate. However, methyl palmoxirate failed to affect insulin release when the oxidation of endogenous fatty acids was already suppressed, e.g. in the presence of pyruvate or L-glutamine. These findings support the view that insulin release evoked by nutrient secretagogues tightly depends on the overall rate of nutrient oxidation, including that of endogenous fatty acids.     

The stimulus–secretion coupling of amino acid-induced insulin release. Insulinotropic action of l-alanine

Available information on the fate and insulinotropic action of l-alanine in isolated pancreatic islets is restricted to data collected in obese hyperglycemic mice. Recent data, however, collected mostly in tumoral islet cells of either the RINm5F line or BRIN-BD11 line, have drawn attention to the possible role of Na⁺ co-transport in the insulinotropic action of l-alanine. In the present study conducted in islets prepared from normal adult rats, l-alanine was found (i) to inhibit pyruvate kinase in islet homogenates, (ii) not to affect the oxidation of endogenous fatty acids in islets prelabelled with [U-¹⁴C]palmitate, (iii) to stimulate ⁴⁵Ca uptake in islets deprived of any other exogenous nutrient, and (iv) to augment insulin release evoked by either 2-ketoisocaproate or l-leucine, whilst failing to significantly affect glucose-induced insulin secretion. The oxidation of l-[U-¹⁴C]alanine was unaffected by d-glucose, but inhibited by l-leucine. Inversely, l-alanine decreased the oxidation of d-[U-¹⁴C]glucose, but failed to affect l-[U-¹⁴C]leucine oxidation. It is concluded that the occurrence of a positive insulinotropic action of l-alanine is restricted to selected experimental conditions, the secretory data being compatible with the view that stimulation of insulin secretion by the tested nutrient(s) reflects, as a rule, their capacity to augment ATP generation in the islet B cells. However, the possible role of Na⁺ co-transport in the secretory response to l-alanine cannot be ignored.     

The role of insulin in the intestinal absorption of glucose in the rat.

1. Acute pre-treatment with either mannoheptulose or streptozotocin--both compounds acting as powerful suppressors of insulin secretion--caused a significant decrease on the in vivo rate of intestinal glucose absorption following an intragastric [U-14C]glucose administration. 2. Mannoheptulose treatment also lowered the rate of whole-body oxidation of the administered tracer. 3. Insulin had no effect on the metabolic fate of [U-14C]glucose by isolated enterocytes. 4. However, the rate of glucose uptake, measured by the oxidation of [1-14C]glucose to 14CO2 in the presence of phenazine methosulphate, was decreased by insulin at concentrations of 50-200 munits/ml. 5. In addition, the rate of transport of [U-14C]glucose by brush-border membrane vesicles was also inhibited by insulin at high concentrations (100-1000 munits/ml). 6. This indicated that insulin acts by inhibiting glucose transport in isolated in vitro preparations. 7. Acute pre-treatment with either mannoheptulose or streptozotocin caused a significant decrease in the rate of gastric emptying, measured as the distribution of [3H]insulin along the gastrointestinal tract, following an intragastric glucose load. 8. It is concluded that insulin secretion modulates intestinal glucose absorption in vivo by enhancing gastric emptying in spite of the inhibitory effects of glucose transport observed with in vitro preparations.