Beta cell protection to alloxan necrosis by anomers of D-glucose.

Various concentrations of either the alpha or beta anomers of D-glucose were injected into fasted rats prior to a diabetogenic dose of alloxan. Plasma concentrations of glucose were measured 24 hours later. There was a significantly greater protection of the pancreatic beta cells by the alpha anomer of D-glucose as compared to the beta anomer, which was evidenced by concentrations of glucose in the plasma, and morphology of beta cells.

Alanine: key role in gluconeogenesis.

Of 20 amino acids measured, alanine is the principal amino acid released by forearm muscle of man, in accord with its being the principal amino acid extracted by liver for gluconeogenesis. This occurs in both the postabsorptive state and after 4 to 6 weeks of starvation, when total amino acid release is markedly diminished.

Anomeric specificty of 3-0-methyl-D-glycopyranose against alloxan diabetes.

The individual alpha and beta anomers of the nonmetabolized glucose analog 3-O-methyl-D-glucopyranose (3MG) were studied as protective agents against the alloxan toxicity to pancreatic beta cells in an in vivo rat model. The alpha 3MG provides greater protection than either the beta or the equilibrated compound, as indicated by plasma glucose concentrations 24 hours after the experiment. This specificity suggests that the beta cell membrane is extremely stereospecific, and that glucose or 3MG provide protection against alloxan injury directly by an interaction with the cell membrane and not by subsequent metabolism of the protecting compound.

Metabolic effects of exogenous glucocorticoids in fasted man.

The influence of administering excessive amounts of glucocorticoids on circulating substrates and hormones and on urinary excretion of nitrogenous compounds and ketone bodies was examined in man after prolonged starvation. After 35 days of total caloric deprivation the administration of high physiologic doses of glucocorticoids increased circulating glucose and insulin levels without intensifying total urinary nitrogen excretion. The increased blood glucose seemed to be due to diminished peripheral uptake rather than augmented gluconeogenesis. A small, transient increase in circulating plasma amino acids was observed. However, the secondary rise in serum insulin seemed to block the proteolytic effect(s) of glucocorticoids, preventing them from mobilizing body protein stores during starvation. There was no change in circulating free fatty acids or glycerol. Thus, it appeared that the potential catabolic action of excessive glucocorticoids was offset by the anabolic effect of insulin, and a new state of homeostasis was established.An additional effect of glucocorticoid administration was a marked diminution of renal excretion of ketone bodies.

Metabolic response to human growth hormone during prolonged starvation.

The metabolic response to human growth hormone (HGH) was studied in five obese subjects in the fed state and during prolonged (5-6 wk) starvation. In the fed state (three subjects), HGH induced an elevation in basal serum insulin concentration, a minimal increase in blood and urine ketone levels, and a marked reduction in urinary nitrogen and potassium excretion resulting in positive nitrogen and potassium balance. In prolonged fasting (four subjects), HGH administration resulted in a 2- to 3-fold increase in serum insulin which preceded a 50% elevation in blood glucose. Persistence of the lipolytic effects of HGH was indicated by a rise in free fatty acids and glycerol. The response differed markedly from the fed state in that blood beta-hydroxybutyrate and acetoacetate levels rose by 20-40%, resulting in total blood ketone acid concentrations of 10-12 mmoles/liter, ketonuria of 150-320 mmoles/day, and increased urinary potassium loss. The subjects complained of nausea, vomiting, weakness, and myalgias. Despite a 50% reduction in urea excretion during HGH administration, total nitrogen loss remained unchanged as urinary ammonia excretion rose by 50% and correlated directly with the degree of ketonuria. It is concluded that in prolonged starvation (a) HGH may have a direct insulinotropic effect on the beta cell independent of alterations in blood glucose concentration, (b) persistence of the lipolytic action of HGH results in severe exaggeration of starvation ketosis and interferes with its anticatabolic action by necessitating increased urinary ammonia loss, and (c) failure of HGH to reduce net protein catabolism in starvation suggests that this hormone does not have a prime regulatory role in conserving body protein stores during prolonged fasting.

Diffusion of glucose, insulin, inulin, and Evans blue protein into thoracic duct lymph of man.

Immunoreactive insulin, like inulin, quickly equilibrates with interstitial fluid, as evidenced by recovery in thoracic duct lymph in man. Insulin-like activity not accounted for by immunoreactive insulin behaves as a large protein and is confined to the vascular compartment.

Acid-base alterations and renal gluconeogenesis: effect of pH, bicarbonate concentration, and PCO2.

In previous studies it was found that renal cortical slices from rats with induced metabolic acidosis have an increased capacity to produce glucose, whereas cortical slices from rats with metabolic alkalosis manifest decreased gluconeogenesis. To evaluate the relative influence of extracellular fluid pH, [HCO(3) (-)], and carbon dioxide tension on renal gluconeogenesis, we observed glucose production by cortex from rats with induced respiratory acidosis, and by cortex taken from normal animals and incubated in acid and alkaline media. We found glucose production to be increased in cortex from rats with respiratory acidosis, as is the case in metabolic acidosis. Glucose production by slices from normal rats was increased in media made acidic by reducing [HCO(3) (-)], and decreased in media made alkaline by raising [HCO(3) (-)]. These effects were evident whether the gluconeogenic substrate employed was glutamine, glutamate, alpha-ketoglutarate, or oxalacetate. Glucose production was also increased in media made acidic by raising CO(2) tension and decreased in media made alkaline by reducing CO(2) tension. These data indicate that both in vivo and in vitro, pH, rather than CO(2) tension or [HCO(3) (-)], is the most important acid-base variable affecting renal gluconeogenesis. The findings suggest that a decrease in extracellular fluid pH enhances renal gluconeogenesis through direct stimulation of one of the rate-limiting reactions involved in the conversion of oxalacetate to glucose. We hypothesize that the resultant increase in the rate of removal of glutamate, a precursor of oxalacetate, may constitute an important step in the mechanism by which acidosis increases renal ammonia production.

Amino acid balance across tissues of the forearm in postabsorptive man. Effects of insulin at two dose levels.

Amino acid balance across skeletal muscle and across subcutaneous adipose tissue plus skin of the forearm has been quantified in postabsorptive man before and after insulin infusion into the brachial artery. Skeletal muscle released significant amounts of alpha amino nitrogen after an overnight fast. Most individual amino acids were released. Alanine output was by far the greatest. The pattern of release probably reflects both the composition of muscle protein undergoing degradation and de novo synthesis of alanine by transamination. A significant correlation was observed between the extent of release of each amino acid and its ambient arterial concentration. Elevation of forearm insulin in eight subjects from postabsorptive (12 muU/ml) to high physiologic levels (157 muU/ml) in addition to stimulating muscle glucose uptake blocked muscle alpha amino nitrogen release by 74%. Consistent declines in output were seen for leucine, isoleucine, tyrosine, phenylalanine, threonine, glycine, and alpha-aminobutyric acid. Alanine output was insignificantly affected. Doubling forearm insulin levels (from 10 to 20 muU/ml) in eight subjects increased muscle glucose uptake in three and blocked alpha amino nitrogen output in two although both effects were seen concurrently in only one subject. Changes in net amino acid balance after insulin could be accounted for by increased transport of amino acids into muscle cells or retardation of their exit. It is likely that ambient arterial amino acid concentrations are established and maintained primarily by the extent of muscle amino acid release. The individual amino acids whose outputs from forearm muscle decline after forearm insulinization correspond well with those whose levels fall systematically after systemic insulinization. This suggests that declines in amino acid levels after systemic insulinization are due to inhibition of muscle release. Doubling basal insulin approaches the threshold both for blockade of muscle amino acid output and stimulation of glucose uptake, effects which appear to occur independently.

Amino acid metabolism during prolonged starvation.

Plasma concentration, splanchnic and renal exchange, and urinary excretion of 20 amino acids were studied in obese subjects during prolonged (5-6 wk) starvation. Splanchnic amino acid uptake was also investigated in postabsorptive and briefly (36-48 hr) fasted subjects.A transient increase in plasma valine, leucine, isoleucine, methionine, and alpha-aminobutyrate was noted during the 1st wk of starvation. A delayed, progressive increase in glycine, threonine, and serine occurred after the 1st 5 days. 13 of the amino acids ultimately decreased in starvation, but the magnitude of this diminution was greatest for alanine which decreased most rapidly during the 1st week of fasting. In all subjects alanine was extracted by the splanchnic circulation to a greater extent than all other amino acids combined. Brief fasting resulted in an increased arterio-hepatic venous difference for alanine due to increased fractional extraction. After 5-6 wk of starvation, a marked falloff in splanchnic alanine uptake was attributable to the decreased arterial concentration. Prolonged fasting resulted in increased glycine utilization by the kidney and in net renal uptake of alanine. It is concluded that the marked decrease in plasma alanine is due to augmented and preferential splanchnic utilization of this amino acid in early starvation resulting in substrate depletion. Maintenance of the hypoalaninemia ultimately serves to diminish splanchnic uptake of this key glycogenic amino acid and is thus an important component of the regulatory mechanism whereby hepatic gluconeogenesis is diminished and protein catabolism is minimized in prolonged fasting. The altered renal extraction of glycine and alanine is not due to increased urinary excretion but may be secondary to the increased rate of renal gluconeogenesis observed in prolonged starvation.

Liver and kidney metabolism during prolonged starvation.

This study quantifies the concentrations of circulating insulin, growth hormone, glucose, free fatty acids, glycerol, beta-hydroxybutyrate, acetoacetate, and alpha amino nitrogen in 11 obese subjects during prolonged starvation. The sites and estimated rates of gluconeogenesis and ketogenesis after 5-6 wk of fasting were investigated in five of the subjects. Blood glucose and insulin concentrations fell acutely during the 1st 3 days of fasting, and alpha amino nitrogen after 17 days. The concentration of free fatty acids, beta-hydroxybutyrate, and acetoacetate did not reach a plateau until after 17 days. Estimated glucose production at 5-6 wk of starvation is reduced to approximately 86 g/24 hr. Of this amount the liver contributes about one-half and the kidney the remainder. Approximately all of the lactate, pyruvate, glycerol, and amino acid carbons which are removed by liver and kidney are converted into glucose, as evidenced by substrate balances across these organs.

Brain metabolism during fasting.

Catheterization of cerebral vessels in three obese patients undergoing 5-6 wk of starvation demonstrated that beta-hydroxybutyrate and acetoacetate replaced glucose as the predominant fuel for brain metabolism. A strikingly low respiratory quotient was also observed, suggesting a carboxylation mechanism as a means of disposing of some of the carbon of the consumed substrates.

Glucagon levels and metabolic effects in fasting man.

The role of glucagon in the metabolic adaptation to prolonged fasting in man has been examined. Plasma immunoreactive glucagon was determined during 6-wk fasts and during infusion of exogenous glucagon using an assay which minimized nonpancreatic immunoreactivity. Plasma glucagon concentrations rose twofold to a peak on the 3rd day of fasting and then declined thereafter to a level maintained at or above postabsorptive. Insulin concentration declined to a plateau by the 3rd day. Thus a persisting altered relationship of glucagon and insulin concentrations characterized the fasted state. A synergism of low insulin and relative or absolute elevation of glucagon levels is viewed as a hormonal mechanism controlling the rate of hepatic substrate extraction for gluconeogenesis. Glucagon was infused systemically into 4-6 wk fasted subjects at three dose levels. A marked sensitivity of individual plasma free amino acids to the induced elevations of plasma glucagon within the physiologic range was demonstrated. At higher concentrations, equivalent to those present in the portal vein, stimulation of hepatic gluconeogenesis occurred, and the effects on glucose, insulin, and growth hormone levels and on ketone metabolism were induced.

Hormone-fuel concentrations in anephric subjects. Effect of hemodialysis (with special reference to amino acids).

Arterial blood concentrations of insulin, glucagon, and various substrates were determined in six anephric subjects in the postabsorptive state and immediately after hemodialysis. Plasma glucose and serum insulin concentrations were normal, and declined during dialysis. Plasma glucagon was elevated and remained unchanged. There was moderate hypertriglyceridemia before dialysis, but this decreased significantly after administration of heparin just before the start of dialysis, and at the end of dialysis was lowered further into the normal range. Comparison of postabsorptive whole blood concentrations of amino acids with those in normal, healthy adults revealed striking differences. Glutamine, proline, citrulline, glycine and both 1- and 3-methyl-histidines were increased, while serine, glutamate, tyrosine, lysine, and branched-chain amino acids were decreased. The glycine/serine ratio was elevated to 300% and tyrosine/phenylalanine ratio was lowered to 60% of normal. To investigate the potential role of blood cells in amino acid transport, the distribution of individual amino acids in plasma and blood cell compartments was studied. Despite a markedly diminished blood cell mass (mean hematocrit, 20.6 +/- 1.4%), there was no significant decrease in the fraction of most amino acids present in the cell compartment, and this was explained by increases of several amino acids in cellular water. None were decreased. Furthermore, during dialysis, whole blood and plasma amino acids declined by approximately 30% and 40%, respectively, whereas no significant change was observed in the cell compartment. Alanine was the only amino acid whose concentration declined in the cells as well as in plasma. The results indicate (a) significant alterations in the concentrations of hormones and substrates in patients on chronic, intermittent hemodialysis; (b) removal of amino acids during hemodialysis, predominantly from the plasma compartment, with no significant change in cell content; and (c) a redistribution of amino acids in plasma and blood cell compartments with increased gradients of most of the amino acids per unit cell water, by mechanism(s) as yet undetermined.

Effect of insulin on muscle glutamate uptake. Whole blood versus plasma glutamate analysis.

For decades, investigators concerned with protein metabolism in man have performed detailed amino acid analyses of human plasma obtained under a wide range of experimental situations. A large body of information has been used to calculated rates of protein synthesis and proteolysis. During the course of an investigation of the effect of intrabrachial artery infusion of insulin (70 muU/min per kg body weight) on glutamate uptake by human forearm muscle, it was discovered that plasma arterio-deep venous glutamate difference analysis failed to document any increase in the uptake of this amino acid, suggesting that insulin had little influence on glutamate uptake by muscle. However, whole blood glutamate analyses, performed on the same blood samples, revealed that (a) the resting muscle uptake of glutamate is smaller than previously reported and (b) insulin is capable of markedly increasing glutamate uptake by muscle from whole blood. Since the hematocrit was obtained on all samples, detailed analyses of the various compartments in which glutamate could be found were performed. It was determined that circulating blood cells have a dynamic role in glutamate transport. These data underscore the need for both whole blood and plasma amino acid analysis in investigations concerned with protein synthesis and/or amino acid flux, for analysis of plasma samples alone could be misleading as illustrated in the present study.

Muscle and splanchnic glutmine and glutamate metabolism in postabsorptive andstarved man.

Arterio-venous differences across forearm muscle in man in both prolonged starvation and in the postabsorptive state, show an uptake of glutamate and a relatively greater production of glutamine. Splanchnic arteriovenous differences in the postabsorptive state show a net uptake of glutamine and lesser rate of glutamate production. These data suggest that muscle is a major site of glutamine synthesis in man, and that the splanchnic bed is a site of its removal. The relative roles of liver and other tissues in the splanchnic circuit were not directly assessed, only the net balance. These data in man are in conflict with most previous studies in other species attributing the major proportion of glutamine production to the liver and, pari passu, to the splanchnic bed.

Plasma and cerebrosponal fluid amino acid levels in diabetic ketoacidosis before and after corrective therapy.

To evaluate the effect of insulin-saline-bicarbonate therapy on amino acid metabolism in diabetic ketoacidosis, arterial and venous blood samples as well as cerebrospinal fluid (CSF) were obtained from six patients before and after initiation of corrective therapy. Levels of CSF glutamine were decreased while alanine alpha-amino-n-butyrate, valine, isoleucine and leucine were increased significantly compared to a control group composed of six normal, postabsorptive adults free of any neurologic disease. Following therapy, CSF levels of alanine, alpha-amino-n-butyrate, valine, isoleucine, and leucine declined while glutamine levels did not change. Admission arterial plasma levels of the glycogenic amino acids were lower than normal while the branched-chain amino acids were elevated. Plasma alanine and glutamine arterio-venous (A-V) differences across forearm tissue were larger. After four hours of corrective therapy, arterial plasma levels of most of the amino acids had declined sharply and A-V differences for glutamine and alanine were markedly reduced (p smaller than.025 and p smaller than.01, paired t, respectively). Coincident with the decrease in A-V amino acid differences, plasma glucagon and free fatty acid levels declined significantly. These data suggest that the effect exerted by insulin-saline-bicarbonate therapy on amino acid metabolism is manifested by diminished A-V plasma alanine and glutamine differences across forearm tissue. Thus, the role played by the splanchnic bed both before and following corrective measures may be secondary to substrate availability.

Studies of alloxan toxicity on the beta cell.

The ability of sugars to protect the beta cell from alloxan diabetes is highly stereospecific. The alpha anomer, which is present in equilibrium in both glucose and 3-O-methyl glucose (3-OMG) at approximately 34 per cent, provides greater protection than the beta anomer. The greater protection of the alpha anomer of glucose is present fifteen seconds between its administration and alloxan, but there is no difference in protection following a thirty-second interval. The nonmetabolized analogue, 3-OMG, provides even greater protection than glucose, and this higher affinity is expressed both by the lower dose necessary to provide protection, as well as by the higher dose of mannoheptulose needed to remove thr protection. Mannoheptulose not only removes the protection provided by exogenous glucose but sensitizes the beta cell to the toxic effects of alloxan in the fasting state, probably by inhibiting the protection provided by endogenous glucose. Mannoheptulose is able to remove glucose protection before, with, or after the administration of glucose prior to alloxan injection. Finally, the protective effect of both glucose and 3-OMG is time-related, and the protection not only is due to absolute concentration but also appears to be affected by a changing concentration.

Pancreatic beta cell toxicity by streptozotocin anomers.

D-glucose in the pyranose (ring) form exists as two anomers. The alpha-anomer is more effective than the beta-anomer in promoting insulin secretion, suppressing that of glucagon, and protecting beta-cells against alloxan toxicity. Streptozotocin (SZ), a beta cell toxin, is composed of a cytotoxic moiety, 1-methyl 1-nitrosourea, attached to carbon-2 of glucose and exists as either of two anomers in the pyranose form. In 24-hour-fasted male rats, predominantly alpha- or predominantly beta-SZ was injected intravenously and plasma glucose levels were obtained 48 hours later. The alpha-anomer produced significantly greater beta-cell necrosis at doses of 30, 35, and 40 mg./kg. body weight. At higher doses, no differences between the alpha and beta anomers were observed. 3-O-Methyl glucose (3-OMG) protected against both SZ anomers; however, the alpha-SZ remained more toxic. Larger doses of glucose protected against the lower doses of SZ and, under such conditions, the individual glucose anomers appeared equally potent. Finally, mannitol at comparable molar concentrations was ineffective in protecting against the SZ toxicity. This study suggests that streptozotocin's beta cell toxicity is mediated through recognition by the beta cell. In addition, 3-OMG and, to a lesser but significant extent, glucose were shown to protect against the streptozotocin toxicity, whereas mannitol did not.

Effect of alanine and glycine on glucagon secretion in postabsorptive and fasting obese man.

Changes in blood levels of glucagon, insulin and glucose in response to infusions of alanine and glycine have been studied in postabsorptive and fasting obese human subjects. Four-to-five-fold elevations of baseline plasma alanine levels stimulated glucagon secretion significantly. Supraphysiological plasma levels of glycine had a small but insignificant stimulatory effect on glucagon secretion. The glucose increase (6 to 10 mg per 100 ml) observed within 30 min of a supraphysiologic alanine infusion in subjects fasted for 2 or more weeks may be secondary to glucagonmediated glycogen breakdown. However, despite equivalent glucagon rises in the other two study periods, no significant rise in blood sugar was noted during the period of infusion.

The redox state and regulation of amino acid metabolism in man.

Traditionally, regulation of amino acid metabolism in both postabsorptive and prolonged-fasted man has been generally regarded as being hormonal in nature. In particular, insulin, and to a lesser extent glucagon, have been nominated for key roles in this process. More recently, however, reconsideration of previous studies involving insulin, glucagon, and protein meals as well as previously unreported studies (cortisol and tri-iodothyronine) from this laboratory, have suggested another means of regulating amino acid metabolism in fasting man. This new hypothesis is centered on the redox state of muscle of fasting man, which is remarkably reduced in both cytosolic and mitochondrial compartments. It was found that insulin, and to a lesser extent glucagon, when infused into fasting subjects (1) rendered muscle significantly more reduced, and (2) resulted in a diminution in urinary nitrogen excretion. In contrast, when either tri-iodothyronine or cortisol were administered to fasting individuals (1) muscle was found to become more oxidized when compared with the control period, and (2) increased urinary nitrogen excretion was observed in both cases. It was noteworthy that the ingestion of a protein meal by a nitrogen-depleted individual was followed by a dramatic change in muscle redox state (the muscle became more reduced), together with marked uptakes of a variety of amino acids. It is therefore proposed that the protein conservation evidenced by fasting man may be dependent on the reduced state of muslce tissue.