Insulin Withdrawal Research Articles

The endosomal compartment is an insulin-sensitive recruitment site for GLUT4 and GLUT1 glucose transporters in cardiac myocytes.

In nonstimulated cardiomyocytes, the glucose transporter GLUT4 is confined to intracellular vesicles forming at least two populations: a storage pool enriched in GLUT4 (pool 1) and an endosomal pool containing both GLUT4 and GLUT1 (pool 2). We have now studied the dynamics of these pools in response to insulin or the mitochondrial inhibitor rotenone in rat cardiomyocytes. Rotenone recruited GLUT4 and GLUT1 to the cell surface from endosomal pool 2 without affecting pool 1. Kinetic experiments were consistent with rotenone acting on an intracellular compartment that is in close connection with the plasma membrane. In contrast, insulin caused rapid, complete depletion of GLUT4 from pool 1 and reduced the GLUT1 content of pool 2 by approximately 50%, whereas, surprisingly, no net decrease in GLUT4 occurred in this pool. Subsequent insulin withdrawal resulted in slow replenishment of pool 2 with GLUT1 and of pool 1 with GLUT4. When pool 1 was still largely depleted of GLUT4, a second insulin challenge did reduce GLUT4 in pool 2 and stimulated glucose transport to the same extent as the first insulin treatment. In conclusion, the storage pool is the primary source of GLUT4 in response to insulin, but not to rotenone. In addition, the endosomal compartment is an important recruitment site of both GLUT1 and GLUT4 when the storage pool is either unaffected (rotenone) or depleted (by a previous insulin challenge). GLUT4 mobilized by insulin from the storage pool may pass through an intermediary (possibly endosomal) compartment on its way to the cell surface.

Intrasteric inhibition of ATP binding is not required to prevent unregulated autophosphorylation or signaling by the insulin receptor.

Receptor tyrosine kinases may use intrasteric inhibition to suppress autophosphorylation prior to growth factor stimulation. To test this hypothesis we made an Asp1161Ala mutant in the activation loop that relieved intrasteric inhibition of the unphosphorylated insulin receptor (IR) and its recombinant cytoplasmic kinase domain (IRKD) without affecting the activated state. Solution studies with the unphosphorylated mutant IRKD demonstrated conformational changes and greater catalytic efficiency from a 10-fold increase in k(cat) and a 15-fold-lower K(m ATP) although K(m peptide) was unchanged. Kinetic parameters of the autophosphorylated mutant and wild-type kinase domains were virtually identical. The Asp1161Ala mutation increased the rate of in vitro autophosphorylation of the IRKD or IR at low ATP concentrations and in the absence of insulin. However, saturation with ATP (for the IRKD) or the presence of insulin (for the IR) yielded equivalent rates of autophosphorylation for mutant versus wild-type kinases. Despite a biochemically more active kinase domain, the mutant IR expressed in C2C12 myoblasts was not constitutively autophosphorylated. However, it displayed a 2.5-fold-lower 50% effective concentration for insulin stimulation of autophosphorylation and was dephosphorylated more slowly following withdrawal of insulin than wild-type IR. In tests of the regulation of the unphosphorylated basal state, these results demonstrate that neither intrasteric inhibition against ATP binding nor suppression of kinase activity is required to prevent premature autophosphorylation of the IR. Finally, the lower rate of dephosphorylation suggests invariant residues of the activation loop such as Asp1161 may function at multiple junctures in cellular regulation of receptor tyrosine kinases.

Linoleic acid metabolism in the spontaneously diabetic rat: delta6-desaturase activity vs. product/precursor ratios.

The activity of delta6-desaturase of linoleic acid, a rate-limiting step in the formation of arachidonic acid, is decreased in animal models of severe, uncontrolled diabetes. The aim of the study was to measure the activity of liver microsomal delta6-desaturase of spontaneously diabetic BioBreeding/Edinburgh rats receiving subcutaneous insulin daily and of genetically related nondiabetic animals. The activity of delta6-desaturase was then compared with indices of activity (plasma lipid fatty acid product/precursor ratios) frequently used in human studies. Diabetic rats treated with insulin had 75 +/- 8% of the activity of microsomal delta6-desaturase of nondiabetic controls (P < 0.05). Insulin withdrawal tended to reduce the activity further (61% of control), although the activity did not differ from insulin-treated diabetic rats. The ratio of plasma phospholipid or cholesteryl ester gamma-linolenic over linoleic acid was not decreased in insulin-treated diabetic rats. By contrast, the ratio of gamma-linolenic over linoleic acid of microsomes was almost three-fold higher in insulin-treated diabetic rats (P < 0.05). The gamma-linolenic over linoleic acid ratio as an index of activity gave inconsistent results in insulin-deprived rats. The ratio of gamma-linolenic over linoleic acid of cholesteryl esters did not differ between control and diabetic rats, nor did it correlate with microsomal delta6-desaturase activity. Furthermore, the index of delta6-desaturase activity, derived from the fatty acid composition of microsomal phospholipids, did not correlate with microsomal delta6-desaturase activity. Diabetes, even when controlled by regular insulin injections, reduces the metabolism of linoleic acid, but the effect is less than previously published. The fatty acid compositions of plasma and liver microsomal lipids are not reliable indices of delta6-desaturase activity in diabetes.

Regulation of pulsatile luteinizing hormone secretion by insulin in the diabetic male lamb.

This study tested the hypothesis that LH secretion is modulated by insulin and that the responsiveness to hypoinsulinemia is enhanced by sex steroids. The model was the developing male lamb (12-26 wk of age) rendered diabetic by chemically induced necrosis of insulin-secreting tissue (streptozotocin). Our approach was to monitor LH secretion under diabetic conditions, with or without insulin supplementation, either in the presence or in the absence of gonadal steroids. The first experiment determined if chronic insulin supplementation could sustain LH secretion in diabetic lambs. After documentation of the induced diabetic condition, twice-daily treatment with a long-acting insulin preparation (Lente) minimized diabetes-induced hyperglycemia, sustained growth, and maintained LH pulse frequency at levels comparable to pre-diabetic conditions. A second experiment evaluated the acute regulation of LH secretion by insulin. Twenty-four hours of insulin withdrawal decreased LH pulse frequency, increased circulating glucose levels, increased the concentration of plasma non-esterified fatty acids (NEFAs), and increased urinary output of ketones. LH pulse frequency continued to decline after 96 h of insulin withdrawal. By contrast, 24 h of insulin re-supplementation increased LH pulse frequency, reduced circulating glucose and NEFA concentrations, decreased plasma cortisol, and reduced urinary output of ketones. After 96 h of insulin re-supplementation, LH pulse frequency increased further, to levels comparable with those before insulin withdrawal. A third experiment determined if the effects of insulin withdrawal on LH secretion are influenced by the presence of gonadal steroids. The same individuals were treated with a physiologic dose of estradiol (Silastic capsule, s.c.) and subsequently monitored for changes in LH secretion in the presence and in the absence of exogenous insulin. Prior to insulin withdrawal, estradiol decreased both LH pulse frequency and pulse amplitude. Moreover, after 96 h of insulin withdrawal, estradiol potentiated the decline in LH pulse frequency (47% reduction in LH pulse frequency in the presence of estradiol versus 26% reduction in LH pulse frequency in the absence of estradiol). These findings support the contention that insulin and/or insulin-dependent changes in glucose availability modulate LH(GnRH) pulse frequency, and that such effects are potentiated by, but not dependent upon, gonadal steroids.

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) inhibits growth factor withdrawal-induced apoptosis in the human mammary epithelial cell line, MCF-10A.

Previous studies have demonstrated that 2,3,7, 8-tetrachlorodibenzo-p-dioxin (TCDD) increases cell recovery in the human mammary epithelial cell line MCF-10A grown under growth factor-restricted conditions. TCDD was also found to mimic growth factor signaling pathways by stimulating the tyrosine phosphorylation of numerous effector molecules, and increased phosphatidylinositol 3-kinase (PI3K) activity in the absence of exogenously added growth factors. In the present studies, we have expanded on these initial results to show that TCDD (3-30 nM) increases cell recovery on days 2-6 by as much as 80% when insulin or epidermal growth factor (EGF) was removed from the media. The mechanism for this effect appears to be complex as TCDD inhibited apoptosis stimulated by EGF, or EGF and insulin, withdrawal by almost 80% as determined by Annexin V binding. However, withdrawal of insulin alone did not induce apoptosis even though TCDD did increase cell number in its absence. These results were corroborated by immunoblot analysis of poly(ADP-ribose) polymerase cleavage. Since TCDD stimulates PI3K activity, the phosphorylation status of Akt, a serine/threonine kinase that mediates PI3K-dependent inhibition of apoptosis, was examined. Immunoblot analysis revealed that TCDD causes a transient increase in the phosphorylated form of Akt that peaks at 6 h and disappears by 12 h. It appears that EGF stimulates an anti-apoptotic pathway, while insulin signals a pro-mitogenic pathway. By stimulating or mimicking one or both of these pathways TCDD may alter tightly regulated growth pathways in the MCF-10A cell line.

Classification Of Type 2 Diabetes By Clinical Response To Metformin-Troglitazone Combination And C-Peptide Criteria

ObjectiveTo assess the ability of basal and stimulated C-peptide levels and stimulated glucose values after oral administration of glucose to predict a successful response to metformin and troglitazone combination therapy after discontinuation of insulin therapy. MethodsAt the onset of the study, plasma glucose and C-peptide levels were measured in a group of 64 obese patients with insulin-treated type 2 diabetes while they were fasting and 2 hours after a challenge of 100 g of glucose administered orally. Then combination metformin-troglitazone treatment was initiated while insulin therapy was gradually tapered over 8 to 12 weeks. Subjects who successfully tolerated insulin withdrawal after the metformin-troglitazone combination were categorized as non-insulin-requiring responders, whereas those who needed insulin to obtain glycemic control were categorized as insulin-requiring nonresponders. Basal and glucose-stimulated C-peptide levels as well as stimulated glucose values were contrasted in the responder versus nonresponder groups. In a second protocol, eight obese patients with insulin-treated type 2 diabetes who successfully stopped insulin therapy were reassessed for C-pep-tide and glucose variables during the 2-hour oral glucose tolerance test. This reassessment followed a 12-week period of therapy to determine whether treatment of insulin resistance with the combination of metformin and troglitazone could normalize the impaired glucose tolerance in type 2 diabetes. ResultsAfter metformin-troglitazone therapy, 48 study subjects (75%) could later be managed without insulin, whereas 16 (25%) needed insulin to achieve acceptable glycemic control. In a comparison of the noninsulin-requiring responder and insulin-requiring nonresponder groups, the responder group had significantly higher glucose-stimulated C-peptide levels and much lower stimulated glucose levels. The mean basal plasma C-peptide level was higher in the responder than in the nonresponder group, but a small degree of overlap was found between the two groups. Combination treatment with metformin and troglitazone for 12 weeks resulted in a significant reduction in the C-peptide response and glucose variables after the glucose load. ConclusionThe results of this study indicate that stimulated C-peptide and glucose levels may be useful criteria to identify whether combination metformin-troglitazone treatment can successfully replace insulin therapy in the management of obese patients with type 2 diabetes. Inability to normalize glucose intolerance after restoring insulin resistance with insulin sensitizers is supportive of the presence of both disturbed beta-cell function and insulin resistance in patients with type 2 diabetes. (Endocr Pract. 1999;5:305-313)

Changes in free insulin-like growth factor-1 and leptin concentrations during acute metabolic decompensation in insulin withdrawn patients with type 1 diabetes.

To determine the effect of acute insulin withdrawal and its subsequent replacement on components of the insulin-like growth factor (IGF)-1 binding protein system and on circulating leptin levels in patients with type 1 diabetes. Seventeen patients (age 31 yr +/-10) with type 1 diabetes treated with continuous subcutaneous insulin infusion (HbA1c 7.6% +/-1.0) were studied. The protocol consisted of two phases: acute insulin withdrawal of up to 8 h followed by a further 2-h period of insulin replacement. For the first phase the basal insulin infusion was stopped (at 0300 h), and for the second a single dose of either regular human or insulin lispro was given subcutaneously (0.2 U/kg). Plasma insulin, glucose, growth hormone, glucagon, IGF-1, free IGF-1, IGFBP-1, -2, -3 and leptin were measured. After interruption of the basal insulin infusion, plasma free insulin levels fell from 60+/-12.0 pmol/L to 10.8+/-4.2 pmol/L, and plasma glucose rose from 5.6+/-0.4 mmol/L to 14.8+/-1.2 mmol/L (P< 0.01). During insulin withdrawal, IGFBP-1 increased by more than 6-fold (from 32+/-8 to 205+/-17 ng/mL, P<0.001), IGFBP-3 increased significantly (from 2631+/-118 to 3053+/-101 ng/mL, P<0.001), and total IGF-1 levels declined modestly (from 226+/-33 to 182+/-26 ng/mL, P<0.001). In contrast, free IGF-1 concentrations (0.72+/-0.22 ng/mL at baseline) were markedly suppressed during insulin withdrawal to values below the detection limit of the assay (0.08 ng/mL) in 15 of the 17 patients (P<0.001). Circulating plasma leptin declined markedly in females from 20+/-3 ng/mL to 11+/-2 ng/mL (P<0.0001) and in males from 10+/-2 ng/mL to 7+/-2 ng/mL (P<0.02). Within 2 h of insulin replacement, the changes in circulating concentrations of IGFBP-1 and IGFBP-3 were partially reversed, and free IGF-1 levels rebounded to 0.54+/-0.22 ng/mL (P<0.1 vs. insulin withdrawal). Growth hormone, glucagon, and IGFBP-2 levels did not change significantly throughout the study. Despite the rapid restoration of plasma insulin and substrate levels, circulating leptin levels continued to fall in the 2-h period after insulin replacement in both females and males. The marked reduction in circulating free IGF-1 after insulin withdrawal and its increase after insulin administration suggest that acute changes in IGFBP concentrations induced by insulin are important regulators of IGF-1 bioavailability in patients with type 1 diabetes. In both males and females, the rapid induction of severe insulin deficiency is associated with a consistent fall in plasma leptin levels.

Changes in Free Insulin-Like Growth Factor-1 and Leptin Concentrations during Acute Metabolic Decompensation in Insulin Withdrawn Patients with Type 1 Diabetes

Changes in Free Insulin-Like Growth Factor-1 and Leptin Concentrations during Acute Metabolic Decompensation in Insulin Withdrawn Patients with Type 1 Diabetes

Transplantation of pancreatic islets contained in minimal volume microcapsules in diabetic high mammalians.

To minimize technical problems relating to excessive size (600-800 mu in diameter) of standard alginate microcapsules (CSM) for pancreatic islet graft immunoisolation, we have developed two novel minimal volume, chemically identical, capsule prototypes (MVC): 1) coherent microcapsules (CM), and 2) medium-size microcapsules (300-400 mu, MSM). CM, which envelop each individual islet within a thin alginate hydrogel cast, are prepared by emulsification, whereas MSM are made by atomizing the islet-alginate suspension through a special microdroplet generator. Upon graft into diabetic rodents, CM have shown to immunoprotect both allo- and xenogeneic nondiscordant islets, and restored normoglycemia. In higher mammals, at subtherapeutic doses, CM fully immunoprotected islet allografts (pig-->pig), but only temporarily xenografts (dog-->pig). We then used MSM to immunoisolate canine islet allografts in the peritoneal cavity of dogs with spontaneous insulin-dependent diabetes. Of three grafted dogs, two showed full remission of hyperglycemia with insulin withdrawal. MSM could represent an intermediate solution between CSM and CM for peritoneal immunoisolated islet transplants.

Growth factor and Bcl‐2 mediated survival during abortive proliferation of hybridoma cell line

Cultures of the CRL-1606 hybridoma (ATCC) have been reported to undergo continuous proliferation with simultaneous death during nutrient limited fed-batch fermentations. The bcl-2 proto-oncogene has been shown to prevent cell death under a variety of otherwise death inducing conditions. We were interested in elucidating the nature of the massive death observed in cultures of CRL-1606, specifically with respect to the possible environmental causes, and the ability of overexpressed human bcl-2 (hbcl-2) to mitigate cell death. Abortive proliferation, or continuous proliferation in the presence of continuous death, could be induced in serum free cultures of CRL-1606 through the withdrawal of insulin provided the culture was competent for cell proliferation. Culture competency for proliferation was found to be solely determined by the presence of cell culture nutrients. Abortive proliferation was defective in cultures transfected with hbcl-2 and the enhanced viability observed resulted from an increased viable cell population and at the expense of the nonviable cell population normally found in untransfected cultures. Abortive proliferation was also observed in serum containing cultures upon serum shiftdowns. Like the insulin-supplemented serum free culture system, hbcl-2 transfected cultures exhibited defects in the abortive proliferation process. These results suggest that the massive death observed during nutrient-limited fed-batch fermentation originate, in part, from growth or survival factor limitations. Hence, approaches to design cell culture media that account for the cell's proliferation requirements without accounting for the cell's survival requirements may represent a cell death sentence. Given the transformed nature of the hybridomas, we conclude that the abortive proliferation of CRL-1606 is a consequence of inappropriate cell cycle entry in a survival factor limited environment. © 1998 John Wiley & Sons, Inc. Biotechnol Bioeng 57: 164–171, 1998.

Growth factor and Bcl-2 mediated survival during abortive proliferation of hybridoma cell line

Cultures of the CRL-1606 hybridoma (ATCC) have been reported to undergo continuous proliferation with simultaneous death during nutrient limited fed-batch fermentations. The bcl-2 proto-oncogene has been shown to prevent cell death under a variety of otherwise death inducing conditions. We were interested in elucidating the nature of the massive death observed in cultures of CRL-1606, specifically with respect to the possible environmental causes, and the ability of overexpressed human bcl-2 (hbcl-2) to mitigate cell death. Abortive proliferation, or continuous proliferation in the presence of continuous death, could be induced in serum free cultures of CRL-1606 through the withdrawal of insulin provided the culture was competent for cell proliferation. Culture competency for proliferation was found to be solely determined by the presence of cell culture nutrients. Abortive proliferation was defective in cultures transfected with hbcl-2 and the enhanced viability observed resulted from an increased viable cell population and at the expense of the nonviable cell population normally found in untransfected cultures. Abortive proliferation was also observed in serum containing cultures upon serum shiftdowns. Like the insulin-supplemented serum free culture system, hbcl-2 transfected cultures exhibited defects in the abortive proliferation process. These results suggest that the massive death observed during nutrient-limited fed-batch fermentation originate, in part, from growth or survival factor limitations. Hence, approaches to design cell culture media that account for the cell's proliferation requirements without accounting for the cell's survival requirements may represent a cell death sentence. Given the transformed nature of the hybridomas, we conclude that the abortive proliferation of CRL-1606 is a consequence of inappropriate cell cycle entry in a survival factor limited environment.

Biochemical and physiological manifestations of acute insulin withdrawal.

When insulin is withdrawn acutely from a severely diabetic patient, a sequence of intricately interconnected events begins which, without intervention, can result in coma and death. These events involve not only carbohydrate metabolism, but protein and lipid metabolism, electrolyte and fluid balance as well. Acute insulin withdrawal permits the unopposed action of the counter-regulatory hormones (which are attempting to elevate the plasma glucose concentration), namely glucagon, cortisol, growth hormone, and epinephrine. This article details interrelationships existing between the many biochemical and physiological events that are occurring more or less simultaneously in the animal following acute insulin withdrawal.

THE EFFECT OF PROTEIN-SPARING MODIFIED FAST IN OBESE CHILDREN WITH INSULIN DEPENDENT MODY-TYPE DIABETES

Maturity onset diabetes of the youth (MODY) is frequently associated with obesity and often requires high daily amounts of insulin therapy. Weight loss has a beneficial effect on pancreatic endocrine function in these patients. However, the problems of insulin regulation and carbohydrate tolerance frequently complicate dieting in these patients. A protein-sparing modified fast (PSMF) allows effective control of carbohydrate metabolism and hunger, by reducing serum glucose and insulin and increasing free fatty acid and ketone contrations. In a preliminary investigation, we treated two obese insulin dependent MODY patients who were admitted in diabetic ketoacidosis. Patient 1 is an 11 year old black female with a weight of 66.8 kgs (142% ideal body weight (IBW); patient 2 is a 15 year old black male weighing 98.8 kgs (152% IBW). Prior to this admission these patients' maintenace insulin dosages were 60 IU/day and 70 IU/day, respectively. Both patients were started on a PSMF diet and were taken off insulin immediately upon commencing the diet. Blood glucose levels stayed within safe limits (≥170) and none required supplemental exogenous insulin. Both patients could be discharged without insulin while continuing with the PSMF. To our knowledge, this is the first report to investigate the effects of combined PSMF and immediate withdrawal of exogenous insulin in children. These preliminary data suggest that PSMF is a safe and effective treatment for obese pediatric patients with insulin dependent MODY.

Regulation of the murine adipocyte fatty acid transporter gene by insulin.

A cDNA encoding a novel fatty acid transport protein (FATP) was identified recently using expression cloning methodologies. We have studied the expression of FATP in differentiating 3T3-L1 cells and adipose tissue in vivo. When 3T3-L1 preadipocytes are treated with a combination of methylisobutylxanthine, dexamethasone, and insulin to induce differentiation, the abundance of FATP mRNA decreases within 24 h to less than one-third that of preadipocytes and increases subsequently, such that mature adipocytes have 5-7 times more FATP mRNA than fibroblastic precursors. In fully differentiated 3T3-L1 adipocytes, insulin alone is sufficient to down-regulate FATP mRNA levels 10-fold. The concentration of insulin necessary for half-maximal repression (I0.5) is approximately 1 nM and is specific for insulin; insulin-like growth factor I (IGF-I) has little effect at similar concentrations. Kinetic analysis indicates that the reduction in expression of FATP mRNA by insulin is rapid (t1/2 = approximately 4 h) and reversible upon withdrawal of insulin. The half-lives of FATP mRNA are 2.9 h and 4.4 h in the absence and presence of insulin, respectively. The insulin-mediated decrease in FATP steady state mRNA level correlates with a decrease in its transcription rate as measured by nuclear run-on transcription assay. To determine whether physiological conditions that alter insulin concentration in vivo affect adipose FATP levels, feeding/fasting studies are employed. Fasting of C57BL/6J mice for 48 h results in an 11-fold up-regulation of FATP mRNA expression in adipose tissue. Refeeding of fasted animals for 72 h results in a return of FATP mRNA to basal levels. In sum, these results indicate that the expression of FATP gene is negatively regulated by insulin at the transcriptional level in cultured adipocytes and that transporter mRNA expression in murine adipose tissue is altered in a manner consistent with insulin being a negative regulator of gene activity.

2,3,7,8-Tetrachlorodibenzo- p-dioxin blocks the physiological regulation of hepatic phosphoenolpyruvate carboxykinase activity in primary rat hepatocytes

It has been previously reported that TCDD dose-dependently reduces the activity of PEPCK, the rate-limiting enzyme of hepatic gluconeogenesis. To further investigate the mechanism, whereby TCDD decreases PEPCK activity, we studied the effect: of TCDD on PEPCK activity in primary rat hepatocytes (PRH). PRH were isolated from male Sprague-Dawley rate by collagenase perfusion and incubated on collagen-coated culture dishes in medium M199 containing 1 nM insulin. Cells were pretreated with dexamethasone (100 nM) 8 h before PEPCK induction was initiated by addition of glucagon (10 nM) and concurrent withdrawal of insulin. This hormonal treatment induced the enzymatic activity of PEPCK in control cells about 2-fold within 8 h. This PEPCK induction regimen was used to perform two sets of experiments. In the first set of experiments, rats were pretreated with TCDD (125 μg/kg p.o. in corn oil, 4 ml/kg) 4 days prior to isolation of PRH. This resulted in a complete block of the glucagon-dependent induction of PEPCK in PRH from TCDD-pretreated animals. In the second set of experiments, TCDD (100 nM) was added directly to the PRH either 24 or 48 h prior to the induction regimen. Incubation pf PRH with TCDD 24 h prior to initiation of the induction regimen resulted in a slight decrease in the degree of PEPCK induction when compared to controls. However, treatment of PRH with TCDD 48 h prior to initiation of the induction regimen almost completely blocked PEPCK induction. lit is, therefore, suggested that the effect of TCDD on liver PEPCK activity is due to a direct effect on liver cells and is not mediated by factors from outside the liver.

Plasma sialic acid in animal models of diabetes mellitus: Evidence for modulation of sialic acid concentrations by insulin deficiency

An elevated circulating sialic acid concentration is a risk factor for cardiovascular disease. Serum sialic acid levels are elevated in NIDDM but not in uncomplicated IDDM. To study why sialic acid is increased in some types of diabetes, we assayed plasma sialic acid in various animal models of diabetes: obese (ob/ob) mice, before and after streptozotocin treatment, neonatal streptozotocin-treated (nSTZ) rats, and diabetic BB rats during and after insulin treatment. In obese mice, which exhibit moderate hyperglycemia and marked hyperinsulinemia, plasma sialic acid was decreased by 45% (fed) and 42% (fasted), compared to lean controls. Fasting reduced plasma glucose and insulin but increased sialic acid in the obese and lean mice. There was a negative correlation (r = −0.84, P < 0.001) between log plasma insulin and sialic acid in the lean and obese mice. The plasma sialic acid:globulin ratio was reduced by 35% in obese mice vs. lean controls, indicating that there may be altered sialylation of glycoproteins in obese mice. Streptozotocin treatment of obese and lean mice reduced plasma insulin but increased sialic acid. In nSTZ rats, hyperglycemia was associated with mild hypoinsulinemia, but not significantly different from control animals, and sialic acid was not altered. In diabetic BB rats, plasma glucose rose from a mean of 4.9 to 23.5 mM 48 hr after insulin withdrawal but sialic acid did not change. We conclude that an elevated plasma sialic acid level is associated with marked insulin deficiency, rather than hyperglycemia per se. The magnitude and speed of this change in sialic acid varies between species.

Evidence Against Dephosphorylation of Insulin-Elicited Phosphotyrosine Proteins in Vivo by the Phosphatase PTP2C

In order to determine whether the tyrosine phosphatase PTP2C dephosphorylates insulin-elicited phosphotyrosine proteins in vivo, we have compared the patterns of protein tyrosine phosphorylation and its reversal in the kidney 293 cell line with those in 293 cell lines overexpressing PTP2C and a catalytically inactive point mutant of PTP2C. In all three cell types insulin caused the rapid tyrosine phosphorylation of a 160 kD protein, which was shown not to be the insulin receptor substrate 1 (IRS-1) and may be the recently described IRS-2 as well as that of a 100 kD polypeptide, which is probably a mixture of the β subunits of the insulin and insulinlike growth factor I receptors. There was no difference among the three cell lines in the extent of tyrosine phosphorylation or in the rate of its reversal upon insulin withdrawal. These results indicate that PTP2C does not function to dephosphorylate these proteins significantly in vivo.

Kinetic of body nitrogen loss during a whole day infusion and withdrawal of glucose and insulin in injured patients

To investigate the kinetics of body nitrogen (N) excretion during 24 h glucose infusion (relating glycemia with insulin supply) and during subsequent 24 h saline infusion in injured patients during a full blown stress reaction. To define the lag time between the start of the withdrawal of glucose and insulin infusion, and the modification in the N loss from the body, and the time span to reach the maximum effect and its size. The knowledge of these variables is mandatory to plan short term studies in critically ill patients, while assuring the stability of the metabolic condition during the study period, and also to assess the possible weaning of the effect on protein breakdown during prolonged glucose and insulin infusion. 24-36 h after injury, patients were fasted ( < 100 g glucose) for 24 h (basal day). Thereafter, a 24 h glucose infusion in amount corresponding to measured fasting energy production rate (EPR), clamping glycemia at normal level with insulin supply followed by 24 h saline infusion, was performed. Total N, urea and 3-methyl-histidine (3-MH) in urine were measures on 4 h samples starting from 20th h of the basal day. Multipurpose ICU in University Hospital. 6 consecutive patients who underwent accidental and/or surgical injury, immediately admitted for respiratory assistance (FIO2 < 0.04). Excluded patients were those with abnormal nutritional status, cardiovascular compromise and organ failures. Patients showed a 33% increase in measured versus predicted fasting EPR and a consistent increase in N and 3-MH urinary loss. An infusion of glucose at 5.95 +/- 0.53 mg/kg x min (97.20 +/- 0.03% of the fasting measured EPR) with 1.22 +/- 0.18 mU/kg x min insulin infusion reduced N and 3-MH loss after a time lag of 12 h. The peak decrease in body N (-36%) and 3-MH loss (-38%) was reached during the first 12 h of glucose withdrawal period. Thereafter, during the following 12 h, the effect completely vanished confirming that it is therapy-dependent and that the metabolic environment of the patients did not change during the three days study period. 24 h glucose withdrawal reduces N and 3-MH loss injured patients, the drug-like effect is maintained during the first 12 h of withdrawal and thereafter disappears. The study suggests that at least a 24 h study period is necessary when planning studies exploring energy-protein metabolism relationship in injured patients, and, again 24 h before changing protocol in a crossover study.

A simulation model for glucose kinetics and estimates of glucose utilization rate in type 1 diabetic patients.

A glucose kinetic model is described that contains insulin-independent and insulin-dependent pathways of glucose uptake and includes a saturating flux term for glucose transport into the cells by the glucose transporters. The rate of glucose utilization was estimated in control subjects and diabetic patients by use of a bolus of [3-3H]glucose. Diabetic patients were studied after three regimens of insulin therapy: 1) after 24 h of insulin withdrawal, 2) after conventional insulin therapy, and 3) after an overnight insulin infusion such that plasma glucose was maintained between 4 and 6 mmol/l. In addition, the model was used to simulate published results from glucose clamp experiments with a range of glycemic levels and insulin concentrations. Estimates of glucose utilization flux in diabetic patients and model simulation suggest that high plasma glucose concentrations in uncontrolled diabetic patients can be attributed to a high glucose production rate by the liver associated with a reduction in glucose metabolic clearance rate due to relative saturation of the available pool of glucose transporters.

Regulation of Postprandial Whole-Body Proteolysis in Insulin-Deprived IDDM

Suppression of tissue proteolysis is an important mechanism of postprandial protein anabolism, and it may be mediated by insulin, hyperaminoacidemia, or both. To evaluate whether insulin is essential in the regulation of this process, we have investigated the effect of mixed-meal ingestion on whole-body protein breakdown in insulin-deprived insulin-dependent diabetes mellitus (IDDM) patients and normal control subjects. Endogenous phenylalanine and leucine rate of appearance (Ra) from proteolysis were measured at steady-state conditions using a multiple stable isotope technique before and after the constant administration of a synthetic mixed meal. In the postabsorptive state, the IDDM patients exhibited accelerated intracellular leucine Ra (IDDM, 2.64 +/- 0.19 mumol.min-1.kg-1; control, 2.02 +/- 0.08 mumol.min-1.kg-1; P < 0.05) and plasma phenylalanine Ra (IDDM, 0.73 +/- 0.03 mumol.min-1.kg-1; control, 0.61 +/- 0.04 mumol.min-1.kg-1; P < 0.05). During meal ingestion, endogenous phenylalanine and leucine Ra values were suppressed in both the insulin-deficient IDDM (P < 0.05) and control subjects (P < 0.05). Although postmeal endogenous leucine and phenylalanine Ra values remained greater (P < 0.05) in IDDM, the delta changes from the basal endogenous leucine Ra (IDDM, -0.56 +/- 0.11 mumol.min-1.kg-1; control, -0.56 +/- 0.09 mumol.min-1.kg-1) and phenylalanine Ra (IDDM, -0.13 +/- 0.01 mumol.min-1.kg-1; control, -0.14 +/- 0.02 mumol.min-1.kg-1) were similar in both groups. In the IDDM patients, the postmeal increases from the basal leucine concentration were onefold greater (P < 0.05) than in the control-subjects.(ABSTRACT TRUNCATED AT 250 WORDS)