Direct effect of insulin on 45calcium uptake in human erythrocytes

Abstract. Objective. High blood pressure is prevalent in obesity and non-insulin dependent diabetes mellitus: both conditions, with insulin resistance and essential hypertension, have been associated with increasing intra-erythrocytic levels of calcium ions. We tested the hypothesis of whether insulin itself might be responsible for the abnormal red cell cytosolic free calcium. Design. The ionic effects of insulin were studied on the kinetics of 45calcium uptake in vitro in normal human erythrocytes. Setting. The study was performed in the outpatient clinic of a central hospital. Subjects. Sixteen healthy, normotensive individuals with normal body mass index were recruited for the study. Main outcome measures. Blood from eight individuals was used for time-dependent studies of ⁴⁵calcium uptake in erythrocytes and blood from another eight individuals was used for dose-dependent studies of insulin effect. Results. The rate of ⁴⁵calcium influx in red blood cells has two components, a fast component (0–10 min), which measures the initial rate of ⁴⁵calcium influx, and a slow component (10–60 min) probably reflecting a relatively large backflux of calcium (calcium efflux), which accordingly determines an apparent low rate of ⁴⁵calcium influx between 10–60 min. The uptake was linear with time between 10–120 min regardless of insulin being present or not. Insulin at a concentration of 120 mUL^-1 significantly decreased the ⁴⁵calcium uptake in a timedependent fashion between 10–120 min. The uptake was 508 (±59) at 60 min in the presence of insulin vs. a control value of 529 (±59) pmol mL red blood cells^-1 (P < 0.001). The corresponding figures at 120 min were 742 (±log) and 767 (±127), respectively (P = 0.02). Inconsistent results were obtained on ⁴⁵calcium uptake at 60 min by varying insulin concentrations from 40–640 mUL^-1 and a dual effect of insulin on ⁴⁵calcium uptake could not be excluded, one at a fairly low concentration of insulin (40–120 mUL^-1) and another at a high concentration (160–640 mUL^-1). Conclusion. The data indicate a direct role of insulin in the transport process of calcium into normal human erythrocytes.     

Direct effects of rapid-acting insulin analogues on insulin signaling in human pancreatic islets in vitro

Aims

Studies suggest that insulin-signaling molecules are present in the pancreatic islets. For this reason, the effects of insulin glulisine, insulin aspart and regular human insulin (RHI) on the function and molecular features of isolated human pancreatic islets were investigated.

Methods

Human pancreatic islets were prepared by collagenase digestion and density-gradient purification of pancreata from multiple organ donors. Islets were then cultured for 48 h in the presence of 5.5 (normal) or 22.2 (high) mmol/L of glucose with and without glulisine, aspart and RHI (10 or 100 nmol/L). Functional (glucose-stimulated insulin secretion) and molecular (quantitative RT-PCR and immunoblot) studies were performed at the end of the different incubation conditions.

Results

Glucose-stimulated insulin secretion was blunted in islets cultured in 22.2 mmol/L of glucose, with no significant effects from the exogenous added insulins. In islets maintained at 5.5 mmol/L of glucose, insulin receptor (IR) expression was reduced by low RHI, while phosphatidylinositol-3 kinase p110-alpha (PI3K) was enhanced by both concentrations of glulisine and aspart, and by high RHI. In islets preexposed to high glucose, IR expression was increased by both concentrations of aspart and RHI, but not by glulisine. Glulisine at high concentration significantly (P < 0.05) increased PI3K expression. Glulisine and RHI significantly increased IRS-2 phosphorylation compared with control and aspart (P < 0.05).

Conclusion

Insulin analogues have differential effects on the expression of insulin-signaling molecules in human pancreatic islets that are also dependent on the degree of glucose exposure.     

Insulin glulisine, insulin lispro and regular human insulin show comparable end-organ metabolic effects: an exploratory study

Aims:  To compare the end-organ metabolic effects of insulin glulisine (glulisine), insulin lispro (lispro) and regular human insulin (RHI) in patients with type 1 diabetes mellitus.
Methods:  Eighteen patients with type 1 diabetes mellitus (mean age 36.9 ± 8.6 years, BMI 23.6 ± 2.8 kg/m², haemoglobin A_1c 7.4 ± 0.9%) were randomized in this single-centre, double-blind, three-period cross-over, standard Latin-square, euglycaemic glucose clamp trial. Patients received sequential, primed stepwise intravenous infusions of glulisine, lispro or RHI (infusion rates were increased in a stepwise manner from an initial rate of 0.33 [180 min] to 0.66 [180 min] and 1.00 [180 min] mU/kg/min). The primary variables were the suppression of endogenous glucose production ( S _EGP) and glucose uptake (GU).
Results:  Mean basal endogenous glucose production (EGP) was 1.88, 2.12 and 2.12 mg/kg/min for glulisine, lispro and RHI respectively. Mean (±s.e.) maximum absolute S _EGP (adjusted for basal EGP) was −1.64 ± 0.06, −1.72 ± 0.05 and −1.56 ± 0.05 mg/kg/min respectively. Mean (±s.e.) maximum absolute increase in GU (adjusted for basal GU) was 6.46 ± 0.26, 6.23 ± 0.24 and 6.72 ± 0.24 mg/kg/min respectively. There were no clinically relevant differences between the three insulin treatments with respect to serum insulin, free fatty acid (FFA), glycerol or lactate levels. No serious adverse events and no episodes of severe hypoglycaemia were reported.
Conclusions:  This study shows that glulisine, lispro and RHI have similar effects on S _EGP, GU, FFA, glycerol and lactate levels, providing evidence for similar end-organ metabolic effects.     

A comparison of the pharmacokinetics and metabolic effects of human regular and NPH insulin mixtures.

The effects of three human premixes (Mixtard, Actraphane, Humulin M3), syringe mixed 30% regular and 70% NPH insulin, regular insulin alone and NPH insulin alone, on intermediary metabolism, plasma free insulin levels and action profiles were compared using the euglycemic clamp technique. Seven normal volunteers received 20 IU of each insulin subcutaneously in a randomized fashion on separate days. The first and last 60 min of the 6 h clamp were chosen as summary measures of clinical importance. Significantly elevated plasma free insulin levels were found with all treatments compared to NPH insulin alone during the first hour, although by the final hour only Mixtard produced significantly higher levels compared to NPH (19.4 +/- 1.2, 10.5 +/- 0.3 mU/l P less than 0.01, respectively). Analysis of area under the incremental insulin absorption curve demonstrated that Mixtard produced significantly increased levels compared to syringe-mixed regular: NPH (7.6 +/- 0.8), Actraphane (9.6 +/- 1.0) and Humulin M3 (9.0 +/- 0.8 mU/l all P less than 0.05). Mixtard also resulted in significantly higher glucose infusion rates compared to the other premixes. No difference in action was found between regular and pre- or syringe-mixed human insulins during the first hour of the studies. The effects on intermediary carbohydrate and lipid metabolism were similar for syringe and premixed insulins. We conclude that: (1) fixed human insulin mixtures with NPH cause no blunting of the action of the soluble component. (2) Actraphane and Humulin M3 are similar but Mixtard may have a greater effect on some aspects of insulin action. (3) In clinical practice, fixed human insulin mixtures will be as efficacious as syringe-mixed preparations but may be easier and more convenient to use.     

A comparison of the effects of semisynthetic human insulin and porcine insulin on transmembrane ion shifts and glucose metabolism during euglycaemic clamping

Disturbances of potassium, calcium, phosphate and magnesium homeostasis in diabetes mellitus are well documented. We have compared the effects of semisynthetic human and pancreatic porcine insulin on transmembrane shifts of these ions, and on glucose metabolism, at two insulin infusion rates, 20 and 50 mU/kg/h, during euglycaemic clamping for 2 h in 6 normal volunteers. The glucose requirements and the changes in blood metabolite concentrations were not significantly different during the porcine and human insulin infusions. Serum potassium levels, however, showed a significant greater decline with infusions of porcine insulin (4.2 +/- 0.1 to 3.5 +/- 0.1 mmol/l) compared with human insulin (4.2 +/- 0.1 to 3.7 +/- 0.1 mmol/l) at 50 mU/kg/h (P less than 0.05). Potassium levels were significantly lower during the porcine insulin infusion at 105 and 120 min and at 15 and 30 min after stopping the infusion. Electrocardiographic T-wave voltage decreased during the porcine and human insulin infusion by 0.13 +/- 0.02 and 0.10 +/- 0.01 mV, respectively (P less than 0.02). Changes in serum levels of magnesium, calcium, phosphate, and red blood cell concentrations of magnesium and 2,3-DPG, were not significantly different between the insulins. Thus a small but significant greater decline in potassium levels with similar glucose requirements was found during iv administration of porcine insulin compared with human insulin.     

Hepatic disposal of biosynthetic human insulin and porcine C-peptide in humans

To examine the fate of insulin across the liver bed, biosynthetic human insulin was infused in increasing amounts in six healthy men. Trapping of insulin by the liver was determined by means of the hepatic venous catheter technique. To minimize any possible error in the estimation of insulin removal as a result of endogenous insulin, pancreatic insulin secretion was suppressed by intravenous administration of somatostatin (500 micrograms/h). Infusion rates of human insulin were 30, 60, and 150 pmol/m2 X min (corresponding to 0.25, 0.5, and 1.25 U/m2 X h) for 70 minutes each. Steady-state insulin levels were within the physiologic range, ie, 69 +/- 2, 135 +/- 3, and 342 +/- 10 pmol/L, respectively. Euglycemia was maintained throughout the study by a variable glucose infusion. The output of C-peptide from the splanchnic bed was reduced by somatostatin by about 90%, indicating that endogenous insulin production only minimally contributed to total insulin levels achieved during infusion of exogenous human insulin. Fractional extraction of insulin by the liver (63 +/- 6%, 71 +/- 4%, and 74 +/- 5%) and hepatic insulin clearance (201 +/- 19, 235 +/- 23, and 245 +/- 29 mL/m2 X min) did not differ significantly during the three insulin infusion studies. The hepatic uptake of insulin rose with increasing insulin infusion rates, constituting 40% to 60% of total-body insulin removal. No change in the total metabolic clearance rate of insulin was observed between the groups (451 +/- 8 mL/m2 X min). To study the extraction rate of C-peptide by the liver, porcine C-peptide was also infused at the same increasing rates.(ABSTRACT TRUNCATED AT 250 WORDS)     

Immunogenicity of semisynthetic human insulin in man. Long-term comparison with porcine monocomponent insulin

Summary The levels of circulating IgG-insulin antibodies were determined in two groups of diabetic patients before and at 3-month intervals after starting insulin treatment either with monocomponent porcine insulin (n=17) or with human semisynthetic insulin (SHI) (n=16). Patients were followed during 15.1±1.0 and 19.9±1.1 months, respectively (m ± SEM). In addition, the quality of metabolic control and residual B-cell function were evaluated in the group under treatment with SHI. The percentage of patients who remained antibody-free after 12.21 months of treatment was 67–75% in the human insulin-treated group and only 25–43% in the one receiving porcine insulin (p<0.01). Moreover, insulin antibody titers, when present, were usually lower in subjects treated with human insulin. In SHI-treated patients: (1) metabolic control was excellent during the first months of treatment as evidenced by values of mean daily blood glucose (7.3±0.6 mmol/l), M-index according to Schlichtkrull (7.4±2.4) and Hb_lc (6.8±0.6%); (2) residual B-cell function, evaluated at 3-month intervals by a circadian profile of plasma C-peptide did not decrease throughout the study; and (3) a significant deterioration of blood glucose control occurred after 18 months of treatment, which might have been due to a less intensive supervision of the patients by the physicians and/or less careful attention by the patients themselves. This observation confirms the need for a continuous education of the patients regardless of the type of insulin used. Deceased     

Factors influencing the hemodynamic and metabolic effects of insulin in muscle

Insulin mediates its own access and that of glucose to muscle by capillary recruitment and an increase in bulk blood flow. In addition, insulin resistance of muscle may result in part from an impaired hemodynamic action of insulin. The present review examines some of the factors that influence the effects of insulin both at the level of hemodynamics and metabolism in muscle. Factors include fatty acids, the inflammatory cytokine TNFalpha, vasodilators that relax the blood vessels and increase bulk flow, and elevated blood pressure that may be mediated by endothelin, a potent locally released vasoconstrictor, or other vasoconstrictor influences.     

Acute effects of insulin on cardiovascular function and noradrenaline uptake and release

Non-enzymatic glycosylation of tissue and haemolysate proteins has been studied in normal and diabetic rats by reduction with tritiated sodium borohydride (NaB3H4) alone or in combination with chromatography on m-aminophenylboronic acid coupled to Biogel P-6. With NaB3H4 reduction alone, there was a linear relationship between plasma glucose and tritium incorporation into haemolysate protein. However, increased non-enzymatic glycosylation of tissue protein could not be demonstrated with NaB3H4 reduction alone. Tritiated glycosylated amino acids could be selectively removed by m-aminophenylboronic acid immobilized on Biogel P-6, then eluted by acidification and the radioactivity in the acidic peak used to estimate non-enzymatic glycosylation. Using the combined techniques, an increase in non-enzymatic glycosylation was observed in heart, kidney and liver obtained from rats with diabetes of 18 weeks duration.     

Absorption of subcutaneously administered regular human and porcine insulin in different concentrations

The influence of the insulin concentration on its absorption from the subcutaneous tissues was studied by direct measurement of the appearance of insulin in the circulation after its subcutaneous administration. 10 units of regular insulin of semisynthetic human and porcine origin in strength of 15 units/ml, 40 units/ml, and 100/units/ml were administered as bolus to 9 healthy male volunteers, using switched-off, manually handled insulin pumps. Insulinaemia rose significantly higher (p less than 0.05) and insulin levels peaked earlier after the delivery of the 15 units/ml insulin solutions compared to the 100 units/ml insulin solutions. These findings might be of interest for type-I diabetic patients using insulin pumps that are to be loaded with regular insulin in varying concentrations.     

Impaired splanchnic and peripheral glucose uptake in liver cirrhosis.

BACKGROUND/AIM: Patients with liver cirrhosis are insulin-resistant and frequently glucose-intolerant. Although peripheral glucose uptake has been shown to be impaired in liver cirrhosis, little is known about the significance of splanchnic (hepatic) glucose uptake after oral glucose load. METHODS/RESULTS: We performed an oral glucose tolerance test and euglycemic hyperinsulinemic clamp with oral glucose load for eight patients with liver cirrhosis and eight patients with chronic active hepatitis. The patients with liver cirrhosis had higher plasma glucose levels 2 h after glucose load than those with chronic active hepatitis (228+/-22 mg/dl vs. 102+/-9 mg/dl, p<0.01). Using the euglycemic hyperinsulinemic clamp with oral glucose load, we simultaneously measured peripheral and splanchnic glucose uptake. Peripheral glucose uptake in liver cirrhosis was 6.1+/-0.7 mg x kg(-1) x min(-1), which was lower than that in healthy volunteers (10.5+/-0.9 mg x kg(-1) x min(-1), p<0.05) and in chronic active hepatitis (8.4+/-0.3 mg x kg(-1) x min(-1), p<0.05). Furthermore, splanchnic glucose uptake in liver cirrhosis was much lower (20.1+/-3.4%) than in healthy volunteers (36.0+/-4.0%, p<0.05) and in chronic active hepatitis (37.2+/-3.1%, p<0.05). CONCLUSION: These results suggest that glucose intolerance in patients with liver cirrhosis is caused by a defect of the glucose uptake of both splanchnic and peripheral tissues.     

The effect of insulin and glucagon on splanchnic oxygen consumption

The purpose of these experiments was to measure the influence of insulin and glucagon on the splanchnic oxygen consumption. Two experiments were performed. METHODS: In one experiment, the influence of hyperinsulinaemia was investigated in six healthy subjects, who were studied during a euglycaemic hyperinsulinaemic clamp. In another experiment, the influence of glucagon was investigated in seven healthy subjects, who were studied twice during a pancreatic islet clamp with either supplementation of insulin and glucagon, or of insulin alone. In both situations the measurements were performed during euglycaemia. Splanchnic oxygen consumption and net substrate balances were studied by the arterio-hepatic venous catheterisation technique and measurement of splanchnic blood flow in all experiments. RESULTS: During the euglycaemic hyperinsulinaemic clamp, the splanchnic blood flow increased significantly and the splanchnic oxygen consumption decreased by about 20%, while the net splanchnic glucose output reversed to a net uptake. In the pancreatic islet clamp experiments there was a significant difference between the net splanchnic glucose outputs whether glucagon and insulin or only insulin was supplemented. In spite of this, the splanchnic oxygen consumption decreased by about 20% in both situations, i.e. independent of glucagon supplementation. In both experiments there was a pronounced inhibition of lipolysis, which led to decreased fatty acids availability to the liver. This resulted in a concomitant decrease in hepatic ketone body formation. This decrease could account for about 30% of the decrease in splanchnic oxygen consumption. CONCLUSION: The reduction in splanchnic oxygen consumption can be explained by decreased ketogenesis, decreased protein synthesis and changes in splanchnic fuel selection, while changes in the rate of gluconeogenesis does not seem to play a significant role.     

Effect of insulin on glucose uptake and metabolism in the human placenta

The effect of insulin on glucose uptake, transfer, and metabolism was investigated in the human placenta perfused in vitro. Insulin concentrations in maternal perfusion medium were varied from 0-1200 microU/ml, whereas the glucose concentration was kept constant in maternal and fetal perfusion media. Despite significant uptake of insulin by the perfused placenta, neither glucose uptake and transfer nor lactate release were significantly modified during a 1-h insulin perfusion. The MCR of insulin by the placenta was 0.29 +/- 0.03 (+/- SEM) ml/min X g at physiological insulin levels. These data suggest that placental glucose transport and metabolism are insensitive to maternal plasma insulin variations and that the low clearance rate of insulin by the placenta is not a major determinant of maternal insulin adjustments during pregnancy.     

Evidence that human and porcine insulin differently affect the human insulin receptor: studies with monoclonal anti-insulin receptor antibodies.

Binding studies have been carried out with radioiodinated monoclonal antibodies directed to various epitopes of the insulin receptor in order to detect differences between human and porcine insulin in the interaction with the human insulin receptor. Human insulin was more effective that porcine insulin at inhibiting the binding of 125I-MA-5 to IM-9 cells, Hep-2 human larynx cells and human placenta membranes. On the contrary, human and porcine insulin showed similar inhibitory effect on the binding of two other labeled anti-insulin receptor monoclonal antibodies, thus ruling out the possibility that results were due to experimental artifacts. Although several interpretations are possible, data reported suggest that human insulin and porcine insulin might differently affect the insulin receptor, even if, the biological significance of these findings remains unknown.     

Direct electrochemistry of human, bovine and porcine cytochrome P450c17

The direct electrochemistry of human, bovine and porcine cytochrome P450c17 (CYP17) has been examined on an edge-oriented pyrolytic graphite electrode. The recombinant protein was immobilized on an electrode modified with a surfactant to simulate the environment of a biological membrane, and hence physiological electron-transfer conditions. The P450 enzymes all retained 'electron-transfer' activity while immobilized at the electrode surface as assessed by the presence of catalytic signals under aerobic conditions. The redox potentials for porcine P450c17 were more positive (anodic) than both the human and bovine forms, perhaps reflecting the differences in substrate specificity for these species. In addition, these enzymes were all influenced by pH, consistent with a single proton associated with the single electron-transfer event. Ionic strength of the buffer medium also shifted the redox potentials towards positive, suggesting that electrostatic forces contribute to the protein environment required for the electron-transfer process. The effect of substrate on the redox potential for each P450c17 was measured in the presence of pregnenolone, progesterone, 17alpha-hydroxypregnenolone and 17alpha-hydroxyprogesterone. However, no influence on the redox parameters was observed.     

A comparison of semisynthetic human NPH insulin and porcine NPH insulin in the treatment of insulin-dependent diabetes mellitus

Twenty-two insulin-dependent diabetic patients participated in a double-blind, cross-over study, where treatment with semisynthetic human NPH insulin (Novo Industri) was compared with porcine NPH insulin (Nordisk). Each treatment period lasted 8 weeks. Blood glucose level, glycosylated haemoglobin, insulin requirements, and frequency of hypoglycaemic events were compared. No difference was found in 24-hour blood glucose profiles. Fasting blood glucose level was 8.3 mmol/l during treatment with human insulin and 8.7 mmol/l during treatment with porcine insulin (p less than 0.1). Mean HbA1c was 7.7% at the end of study compared to 9.5% at baseline (p less than 0.01), but this decline in HbA1c was independent of the treatment regimen. Forty-six hypoglycaemic events occurred during treatment with human insulin compared to 39 events during treatment with porcine insulin. No difference was found regarding insulin requirements during the study. It is concluded that semisynthetic human NPH insulin is indistinguishable from porcine NPH insulin with respect to 24-hour blood glucose profile, HbA1c level and insulin dose requirements.     

Direct effects of protirelin (TRH) on cultured porcine thyrocytes.

In several cases of thyroid adenoma in which no TSH was detectable in the serum, a paradoxical decrease of PB123I and its conversion rate Q (PB123I/serum total radioactivity) was observed after infusion of protirelin (TRH). The possibile direct effect of TRH on thyrocytes was therefore studied in vitro. Porcine thyroid cells were cultured in NCTC-135 medium supplemented with a serum substitute and insulin. TRH and some of its analogs enhanced the incorporation of [3H]thymidine into DNA and the uptake of radioiodide into thyrocytes. In the presence of TSH, TRH did not stimulate radioiodine uptake, whereas incorporation of [3H]thymidine into DNA was not antagonized by TSH. Thus, in this culture system, TRH had a direct effect on both growth and metabolism of thyrocytes. TSH can modulate these effects.