Plasma glucose and insulin responses to oral and intravenous glucose in cold-exposed humans

Although glucose tolerance and skeletal muscle glucose uptake are markedly improved by cold exposure in animals, little is known about such responses in humans. This study used two variations of a glucose tolerance test (GTT) to investigate changes in carbohydrate metabolism in healthy males during nude exposure to cold. In experiment 1, an oral GTT was performed in the cold and in the warm (3 h at 10 or 29 degrees C). To bypass the gastrointestinal tract, and to suppress hepatic glucose output, a second experiment was carried out as described above, using an intravenous GTT. Even though cold exposure raised metabolic rate greater than 2.5 times, plasma glucose and insulin responses to an oral GTT remained unaltered. In contrast, cold exposure reduced the entire plasma glucose profile as a function of time during the intravenous GTT (P less than 0.05), as plasma glucose was returned to basal levels within 1 h in comparison to a full 2 h in the warm, despite low insulin levels. The results of the intravenous GTT demonstrate that even with low insulin levels, carbohydrate metabolism is increased in cold-exposed males. This effect could be masked in the oral GTT by gastrointestinal factors and a high hepatic glucose output. Cold exposure may enhance insulin sensitivity and/or responsiveness for glucose uptake, mainly in shivering skeletal muscles.     

Phospholipase C mimics insulin action on pyruvate dehydrogenase and insulin mediator generation but not glucose transport or utilization

This study investigated the extent to which a purified phosphatidylinositol-specific and a commercial non-specific phospholipase C mimicked acute insulin action in rat adipocytes. The enzymes mimicked insulin stimulation of pyruvate dehydrogenase (PDH) and breakdown of a glycophospholipid proposed as a precursor for an intracellular mediator of insulin action, but were much less effective in stimulating glucose transport and utilization. These observations corroborate recent suggestions that insulin may activate a phospholipase C to generate a mediator that can account for insulin activation of PDH from a mediator precursor with a phosphatidylinositol anchor. This mediator precursor is probably an outer membrane component since effects were obtained with intact cells. It is unlikely that this mechanism accounts fully for insulin action since phosphatidylinositol-specific and commercial phospholipase C stimulation of glucose transport was significantly less than that elicited by insulin.     

The generation of inositolglycan mediators from rat liver plasma membranes: the role of guanine nucleotide binding proteins.

The guanine nucleotide dependence for the generation of inositolglycan second messengers from rat liver plasma membranes has been investigated. Plasma membranes, when treated with insulin release a soluble mediator substance which activates pyruvate dehydrogenase (PDH). Guanosine 5'-[3-thio]triphosphate (GTP gamma S) was found to be as potent as insulin in stimulating mediator release. The stimulatory effects of GTP gamma S required the presence of magnesium and following preincubation of membranes with guanosine 5'-[2-thio]diphosphate (GDP beta S) the stimulation of mediator release by either insulin or GTP gamma S was blocked. The activation of PDH by mediator fractions produced in response to either insulin or GTP gamma S was abolished following treatment of the fractions with anti-inositolglycan antibodies. The significance of these observations with respect to the possible involvement of a regulatory guanine-nucleotide binding protein (G-protein) in the generation of insulin mediators is discussed.     

MuRF1-dependent regulation of systemic carbohydrate metabolism as revealed from transgenic mouse studies

Under various pathophysiological muscle-wasting conditions, such as diabetes and starvation, a family of ubiquitin ligases, including muscle-specific RING-finger protein 1 (MuRF1), are induced to target muscle proteins for degradation via ubiquitination. We have generated transgenic mouse lines over-expressing MuRF1 in a skeletal muscle-specific fashion (MuRF1-TG mice) in an attempt to identify the in vivo targets of MuRF1. MuRF1-TG lines were viable, had normal fertility and normal muscle weights at eight weeks of age. Comparison of quadriceps from MuRF1-TG and wild type mice did not reveal elevated multi-ubiquitination of myosin as observed in human patients with muscle wasting. Instead, MuRF1-TG mice expressed lower levels of pyruvate dehydrogenase (PDH), a mitochondrial key enzyme in charge of glycolysis, and of its regulator PDK2. Furthermore, yeast two-hybrid interaction studies demonstrated the interaction of MuRF1 with PDH, PDK2, PDK4, PKM2 (all participating in glycolysis) and with phosphorylase β (PYGM) and glycogenin (both regulating glycogen metabolism). Consistent with the idea that MuRF1 may regulate carbohydrate metabolism, MuRF1-TG mice had twofold elevated insulin blood levels and lower hepatic glycogen contents. To further examine MuRF1's role for systemic carbohydrate regulation, we performed glucose tolerance tests (GTT) in wild type and MuRF1-TG mice. During GTT, MuRF1-TG mice developed striking hyperinsulinaemia and hepatic glycogen stores, that were depleted at basal levels, became rapidly replenished. Taken together, our data demonstrate that MuRF1 expression in skeletal muscle re-directs glycogen synthesis to the liver and stimulates pancreatic insulin secretion, thereby providing a regulatory feedback loop that connects skeletal muscle metabolism with the liver and the pancreas during metabolic stress.     

Coexistence of insulin resistance and increased glucose tolerance in pregnant rats: a physiological mechanism for glucose maintenance

AimThe contribution of insulin resistance (IR) and glucose tolerance to the maintenance of blood glucose levels in non diabetic pregnant Wistar rats (PWR) was investigated.Main methodsPWR were submitted to conventional insulin tolerance test (ITT) and glucose tolerance test (GTT) using blood sample collected 0, 10 and 60 min after intraperitoneal insulin (1 U/kg) or oral (gavage) glucose (1 g/kg) administration. Moreover, ITT, GTT and the kinetics of glucose concentration changes in the fed and fasted states were evaluated with a real-time continuous glucose monitoring system (RT-CGMS) technique. Furthermore, the contribution of the liver glucose production was investigated.Key findingsConventional ITT and GTT at 0, 7, 14 and 20 days of pregnancy revealed increased IR and glucose tolerance after 20 days of pregnancy. Thus, this period of pregnancy was used to investigate the kinetics of glucose changes with the RT-CGMS technique. PWR (day 20) exhibited a lower (p < 0.05) glucose concentration in the fed state. In addition, we observed IR and increased glucose tolerance in the fed state (PWR-day 20 vs. day 0). Furthermore, our data from glycogenolysis and gluconeogenesis suggested that the liver glucose production did not contribute to these changes in insulin sensitivity and/or glucose tolerance during late pregnancy.SignificanceIn contrast to the general view that IR is a pathological process associated with gestational diabetes, a certain degree of IR may represent an important physiological mechanism for blood glucose maintenance during fasting.     

Insulin mediator stimulation of pyruvate dehydrogenase phosphatases.

A two stage assay for detecting insulin mediator based upon its stimulation of soluble pyruvate dehydrogenase (PDH) phosphatase to activate soluble pyruvate dehydrogenase complex (PDC) has been developed. This coupled assay determines the activation of PDC by monitoring production of [14C]CO2 from [1-14C]pyruvic acid. In addition to being more sensitive than the rat liver mitoplast assay previously used, it allows for the separation and investigation of the effects of mediator on the PDH phosphatases individually. It has been previously shown that the insulin mediator stimulates the most abundant PDH phosphatase, the divalent cation dependent PDH phosphatase, by decreasing the phosphatase's metal requirement (1). A metal independent PDH phosphatase has been found in bovine heart mitochondria. This phosphatase is not immunoprecipitated by antiphosphatase 2A antibody, it is not inhibited by okadaic acid, and it is not stimulated by spermine. However, it is stimulated (more than threefold) by insulin mediator prepared from isolated rat liver membranes. It is inhibited by Mg-ATP, with half-maximal inhibition at 0.3 mM; however, this inhibition is overcome by the insulin mediator.     

Incretion of Insulin by Pancreas and Its Inactivation in Women during Long-Term Head-down Tilt Bed Rest

An increase in insular activity in response to hyperglycemia when performing a glucose tolerance test (GTT) during normal physical activity in eight women volunteers was found to be associated with increased blood concentrations of insulin provided by an increase in secretion rate and a decrease in inactivation rate. After 81 days of stay in long-term –6° head-down tilt bed rest (bed rest), flattened glycemic curves (with low hyperglycemic lifting) observed during the GTT, what was indicated high insular activity. An increase in the insular activity was evidencing also by a smaller peak of the blood concentration of glucose from 5 min after intravenous nfusion of 20 mL of 40% glucose solution at 120 minute of the GTT. In bed rest on empty stomach no significant changes in the concentrations of insulin and C-peptide were observed. The dynamics of hormone during the GTT was showed that the appearance of flattened glycemic curves when performing GTT during bed rest was associated with more pronounced, than under the usual conditions, an increase in insulin concentration in response to hyperglycemia. An increase in the blood insulin concentration when performing GTT in bed rest was mainly provided by increased hormone secretion rate in the pancreas, but not by a decrease in its inactivation rate. If you use regular exercises for prevention of hypokinesia under conditions of bed rest, an increase in insulin concentration during GTT was mainly provided by a decrease in its inactivation rate and not by an increase in secretion rate.     

Improvement of glucose tolerance with immunomodulators in type 2 diabetic animals

Cytokine-inducers prevent insulin-dependent diabetes mellitus (IDDM) in animal models. We extended this therapy to non-insulin-dependent diabetes mellitus (NIDDM), because it was reported that diabetes of KK-Ay mice, a model for NIDDM, was recovered by allogenic bone-marrow transplantation that also prevented IDDM in animal models. An i.p. or i.v. injection of streptococcal preparation (OK 432) lowered fasting blood glucose (FBG) levels and markedly improved glucose tolerance test (GTT) in KK-Ay mice for more than 32 h regardless of the glucose loading routes (oral, i.v. or i.p.), while an i.v. injection of BCG improved FBG and GTT for more than 4 wks without body weight loss. The improvement of FBG and GTT with OK-432 was brought about in other NIDDM animals, GK rats and Wistar fatty rats. Among various cytokines possibly induced by OK-432 and BCG, IL-1α, TNFα and lymphotoxin significantly improved FBG and GTT in KK-Ay mice, whereas IL-2 and IFN_γ did not. There were no differences between the OK-432-treated KK-Ay mice and control in histology of the pancreas, degree of insulin-induced decrease in blood glucose levels, and muscle glycogen synthase activities. As to insulin secretion, there is a tendency that the OK-432-treatment less than 1 week did not affect insulin levels during GTT, whereas the treatment more than 2 weeks increased the insulin levels. Thus, cytokine-inducers improved FBG and glucose tolerance of NIDDM animals probably via cytokines. The results imply a role of the cytokines in glucose tolerance of NIDDM, although precise immune and metabolic mechanisms remain to be elucidated.     

Glucose-dependent insulinotropic polypeptide (GIP) and the enteroinsular axis in equines (Equus caballus).

To investigate the enteroinsular axis (EIA) in equines oral (oGTT) and intravenous (i.v.GTT) glucose tolerance tests (5.6 and 1 mmol glucose/kg BW, respectively) were performed with healthy, normal weight large horses and Shetland ponies. Plasma was analysed for concentrations of glucose, glucose-dependent insulinotropic polypeptide (GIP) and insulin. In all equines plasma GIP concentrations only increased significantly when glucose was administered orally. The insulin glucose ratio (IGR) was significantly higher during the oGTT than during the i.v.GTT in both races. Basal plasma glucose levels were significantly higher in large horses than in ponies in both experiments. During the oGTT maximum glucose values were significantly higher in ponies. Ponies tended to a higher insulin secretion but the IGRs were identical in both races after oral and intravenous glucose administration. One clinically inconspicuous pony showed hyperinsulinaemia and, in case of the oGTT, insulin resistance, glucose intolerance, and GIP hypersecretion. The results of this study indicate the existence of an EIA in equines due to the higher IGRs during the oGTT. Furthermore, the similarity of plasma GIP levels and IGRs in ponies and large horses suggest a comparable activity of the EIA in both races. Regarding the elevated plasma GIP concentrations of the insulin resistant pony the EIA appears to participate in equine hyperinsulinaemia.     

Effect of cyproheptadine administration on insulin secretion in acromegalic, diabetic and normal subjects.

The effect of cyproheptadine (Cypro) and Placebo administration on insulin secretion and glucose utilization following i.v. glucose (IVGTT) was evaluated in 8 normal, 7 diabetic and 8 acromegalic subjects. Five of the diabetic subjects had overt diabetes and two of the diabetic subjects had "chemical" diabetes (oral GTT). One of the acromegalic subjects had overt diabetes, while one had borderline glucose tolerance and six had normal glucose tolerance (oral GTT). Cypro increased insulin secretion in the acromegalic but not in the diabetic or normal subjects. Methysergide (Methyl) increased insulin secretion in acromegalic and diabetic subjects but not in normal subjects. Methy and Cypro both increased insulin secretion in the same acromegalic subjects. None of the three groups of subjects had a modification in insulin secretion following Placebo administration. Neither Placebo, Cypro or Methy altered the glucose utilization rate contant (KG). There was no change in insulin half life or tissue sensitivity to insulin from Cypro (normal and acromegalic subjects) or Methy (normal subjects) administration. Despite their increase in insulin secretion in response to serotonin antagonists, acromegalic subjects have normal urinary 5-hydroxyindoleacetic acid excretion and normal serum serotonin concentrations. Their response cannot therefore be attributed to a generalized overproduction of serotonin.     

Glucose-stimulated insulin secretion does not require activation of pyruvate dehydrogenase: impact of adenovirus-mediated overexpression of PDH kinase and PDH phosphate phosphatase in pancreatic islets

Glucose-stimulated increases in mitochondrial metabolism are generally thought to be important for the activation of insulin secretion. Pyruvate dehydrogenase (PDH) is a key regulatory enzyme, believed to govern the rate of pyruvate entry into the citrate cycle. We show here that elevated glucose concentrations (16 or 30 vs 3 mM) cause an increase in PDH activity in both isolated rat islets, and in a clonal beta-cell line (MIN6). However, increases in PDH activity elicited with either dichloroacetate, or by adenoviral expression of the catalytic subunit of pyruvate dehydrogenase phosphatase, were without effect on glucose-induced increases in mitochondrial pyridine nucleotide levels, or cytosolic ATP concentration, in MIN6 cells, and insulin secretion from isolated rat islets. Similarly, the above parameters were unaffected by blockade of the glucose-induced increase in PDH activity by adenovirus-mediated over-expression of PDH kinase (PDK). Thus, activation of the PDH complex plays an unexpectedly minor role in stimulating glucose metabolism and in triggering insulin release.     

Dietary fibres,fibre analogues,and glucose tolerance: importance of viscosity.

To define the type of dietary fibre of fibre analogue with the greatest potential use in diabetic treatment, groups of four to six volunteers underwent 50-g glucose tolerance tests (GTT) with and without the addition of either guar, pectin, gum tragacanth, methylcellulose, wheat bran, or cholestyramine equivalent to 12 g fibre. The addition of each substance significantly reduced blood glucose concentration at one or more points during the GTT and generally reduced serum insulin concentrations. The greatest flattening of the glucose response was seen with guar, but this effect was abolished when hydrolysed non-viscous guar was used. The reduction in the mean peak rise in blood glucose concentration for each substance correlated positively with its viscosity (r = 0.926; P less than 0.01), as did delay in mouth-to-caecum transit time (r = 0.885; P less than 0.02). Viscous types of dietary fibre are therefore most likely to be therapeutically useful in modifying postprandial hyperglycaemia.     

Insulin stimulates the release from liver plasma membranes of a chemical modulator of pyruvate dehydrogenase

Incubation of a rat liver particulate fraction with physiological concentrations of insulin enhances the production of a small molecular weight substance which modulates adipocyte as well as liver mitochondrial pyruvate dehydrogenase. While low concentrations of insulin enhance production of this activity, levels of greater than 10^?9M produce significantly less. Similarly, while increasing concentrations of mediator cause increased stimulation of pyruvate dehydrogenase activity, higher concentrations no longer exhibit this effect. The putative insulin mediator was partially purified on HPLC and Sephadex G-25 columns. Its molecular weight was about 1000–2000. These results indicate the presence of a chemical mediator of insulin action in liver similar to that observed in other insulin target tissues.     

Control by thyrotropin of the production by thyroid cells of an inositol phosphate-glycan

The increased turnover of phosphatidylinositol promoted by thyrotropin (TSH) in pig thyroid tissue does not seem to be caused by an increased production of inositol tris-phosphate. We have explored another possibility, the synthesis of an inositol phosphate-glycan (IP-gly). Our results show that thyroid cells in culture produced this substance from a precursor phosphatidylinositol-glycan (Gly-PI). The obtained IP-gly seemed, by its analytical and biological properties, to be identical, or similar, to the previously described insulin mediator.     

The effect of insulin on the uptake and metabolic fate of glucose in isolated perfused hearts of dyslipemic rats

Male Wistar rats chronically (15 weeks) fed a sucrose-rich diet (SRD; 63% w/w) developed hypertriglyceridemia and impaired glucose homeostasis. Hearts from these animals were isolated and perfused using the Langendorff recirculating method. Glucose at levels similar to those found in the animal in vivo was used as the only exogenous substrate. The hearts were perfused for 30 minutes in the presence or absence of insulin (30 mU/mL) in the perfusion medium. In the absence of the hormone, glucose uptake was impaired and the glucose utilization was reduced, with a significant increase of lactate release. Glucose oxidation, which was estimated from the activation state of the enzyme pyruvate dehydrogenase complex (PDHc), was depressed mainly due to both an increase of PDH kinase and a decrease of PDHa (active form of PDHc) activities. Although the addition of insulin in the perfusion medium improved the above parameters, it was unable to normalize them. The present results suggest that at least two different mechanisms might contribute to insulin resistance and to the impaired glucose metabolism in the perfused hearts of the dyslipemic SRD-fed animals: (1) reduced basal and insulin-stimulated glucose uptake and its utilization or (2) increased availability and oxidation of lipids (low PDHa and high PDH kinase activities), which in turn decrease glucose uptake and utilization. Thus, this nutritional experimental model may be useful to study how impaired glucose homeostasis, increases plasma free fatty acid levels and hypertriglyceridemia could contribute to heart tissue malfunction.     

The insulin signal and its effects on the pyruvate dehydrogenase complex in circulating lymphocytes of obese children.

1. Studies have shown that in circulating lymphocytes pyruvate dehydrogenase (PDH) is responsive to insulin. 2. To improve existing knowledge on how insulin influences PDH behaviour, situations in which cell responsiveness to insulin is impaired could be of interest. 3. PDH behaviour in circulating lymphocytes from obese children, with high plasma insulin levels and normal glucose tolerance, was examined. 4. Masking and unmasking processes of insulin receptors on the plasma membrane appear to modulate the enzyme response to insulin.     

"In vitro" effects of insulin on the PDH complex of the isolated perfused heart of rats fed a sucrose-rich diet.

We have recently reported that the "in situ" myocardial concentrations of the active form of the Pyruvate Dehydrogenase Complex (PDHa) were significantly decreased in hearts obtained from normal rats fed for 3 weeks on an isocaloric sucrose rich (63%) diet (SRD) when compared to age matched controls fed on the standard laboratory chow (STD). Since, on the one hand SRD rats present glucose intolerance and impaired "in vivo" insulin action and, on the other hand the effects of insulin on the interconversion of heart PDH remains a controversial matter, we found it relevant to study the effects of insulin on the PDH complex in the "in vitro" perfused (Langendorff technique) heart preparations obtained from SRD rats. After a 35 minute perfusion period with 5.5 mM glucose as the only nutrient in the perfusate, PDHa as a percentage of total PDH was found to remain significantly lower in SRD hearts (M +/- SEM 32.6 +/- 2.3) when compared to STD hearts (68.3 +/- 4.6, P less than 0.05) in spite of comparable total PDH activities in both groups of animals. Although the addition of insulin to the perfusate (20 mu/ml) resulted in a significant increase in the percentage of PDHa (45.8 +/- 3.4) of SRD heart, values attained still remained significantly lower than those obtained in STD controls (67.5 +/- 3.6; P less than 0.05). Simultaneously, the addition of insulin to the perfusate, significantly reduced the Acetyl-CoA/CoASH ratio in SRD hearts although this ratio remained still much higher than those observed in STD controls under the same experimental conditions.     

Partial purification of an insulin-releasing activity in human serum

An activity that enhances insulin release from perifused rat pancreatic islets has recently been isolated from human serum fractions (molecular weight 1,000–5,000 daltons). To characterize this activity we have studied the insulin-releasing effect of serum subfractions from obese and non-obese children obtained by reversed-phase high-performance liquid chromatography (HPLC). The serum insulin-releasing activity eluted in the HPLC system at 12–13 minutes, which corresponded to the retention time of the tridecapeptide insulin-glucagon liberin isolated from bovine hypothalamus. Insulin-releasing activity was found in serum subfractions from both obese and normal-weight children. The relative insulin-releasing potency of the active subfractions was higher than that of the original total serum fractions, indicating the presence of some substance(s) which inhibit insulin secretion in the total serum fractions. Oral glucose loading increased the relative insulin-releasing activity in the HPLC subfractions from obese children. This study suggests that the insulin secretagogue in human serum might be identical to hypothalamic insulin-glucagon liberin as these substances behave similarly on reversed-phase HPLC and have parallel insulin-releasing properties.     

Increased beta -oxidation but no insulin resistance or glucose intolerance in mice lacking adiponectin

Previous reports showed that recombinant fragments of adiponectin (adipo) displayed pharmacological effects when injected into rodents, but the relevance of these observations to the physiological function of adipo is unclear. We generated Adipo(-/-) mice by gene targeting. Adipo(-/-) mice are fertile with normal body and fat pad weights. Plasma glucose and insulin levels of Adipo(-/-) and Adipo(+/+) mice are similar under fasting conditions and during an intraperitoneal glucose tolerance test (GTT). Insulin tolerance test (ITT) also produces similar plasma glucose and insulin levels in the two groups of mice. Hyperinsulinemic-euglycemic clamp analysis showed that Adipo(-/-) and Adipo(+/+) mice have similar glucose infusion rates to maintain a similar serum glucose. High-fat diet feeding for 7 months led to similar weight gain and similar GTT and ITT responses. We next measured beta-oxidation and found it to be significantly increased in muscle and liver of Adipo(-/-) mice. In conclusion, our study indicates that absence of adipo causes increased beta-oxidation but does not cause glucose intolerance or insulin resistance in mice.     

The potentiations by insulin-stimulating peptide from bovine serum albumin of the effects of insulin mimickers and insulin in stimulating glucose utilization by rat adipocytes

An insulin-stimulating peptide derived from bovine serum albumin by digestion with trypsin was shown to inhibit insulin degradation. Addition of this peptide (1.2 microM) to the medium of isolated rat adipocytes markedly inhibited the degradation of insulin in the medium, but had a little effect on degradation of cell-associated insulin. Moreover, this peptide did not prevent dissociation of cell-associated insulin, suggesting that it is a bacitracin-type, not a chloroquine-type inhibitor of insulin degradation. The peptide also potentiated the stimulation by insulin mimickers of glucose oxidation by rat adipocytes, strongly indicating that it has some other effects besides inhibition of insulin degradation. Therefore, the effect of the peptide on activation of pyruvate dehydrogenase (PDH), one of the postbinding actions of insulin, was studied. Addition of the peptide (4 microM) to adipocytes was found to activate PDH in the absence or presence of insulin. This stimulatory effect of the peptide on PDH was dose-dependent and was observed in both whole cells and subcellular fractions of rat adipocytes. The peptide also stimulated PDH in a subcellular system of either plasma membranes and mitochondria or mitochondria only. Sodium fluoride, an inhibitor of phosphatase, blocked the action of the peptide almost completely, suggesting that the stimulatory effect of the peptide on PDH activity is at least partly due to its activation of PDH phosphatase. The mechanisms of action of the peptide are discussed. The peptide should be useful in studies on modulation of the action of insulin.