Incretin effect due to increased secretion and decreased clearance of insulin in normal humans

To assess the contribution of changes in insulin secretion and clearance to the incretin effect (greater insulinemia after oral than after intravenous glucose), 10 healthy subjects were studied after oral glucose (1 g/kg body wt) and again when glucose was infused intravenously at rates to match arterialized plasma glucose concentrations after oral glucose. Although basal and integrated plasma glucose did not differ between oral and intravenous glucose, integrated responses of insulin (3.3 +/- 0.5 vs. 1.8 +/- 0.4 mU ml-1.240 min-1, P less than .001), C-peptide (456.5 +/- 58.5 vs. 327.9 +/- 46.3 ng.ml-1.240 min-1, P = .002), gastric inhibitory polypeptide, (16.8 +/- 3.5 vs. -2.8 +/- 1.0 micrograms.ml-1.240 min-1, P less than .001), and insulin secretion (6.6 +/- 1.1 vs. 4.7 +/- 0.7 U.240 min-1, P = .003) were greater with oral than intravenous glucose. However, insulin clearance, whether calculated as the molar ratio of integrated C-peptide to integrated insulin responses (6.9 +/- 0.7 vs. 14.2 +/- 3.8, P = .005) or from the formula insulin clearance equals insulin secretion divided by integrated insulin responses (1.1 +/- 0.2 vs. 2.5 +/- 0.7 L.min-1.m-2, respectively, P = .002), was less for oral than for intravenous glucose. Therefore, the incretin effect is mediated both by increased secretion and decreased clearance of insulin.     

Somatostatin impairs clearance of exogenous insulin in humans

Somatostatin has been widely used to suppress endogenous pancreatic hormone secretion in research studies. Many of these studies required the simultaneous infusion of a hormone together with somatostatin. A critical assumption for its use in metabolic investigation is that somatostatin has no effect on the action or clearance of a concomitantly infused hormone. To test whether clearance of an exogenously infused hormone is affected, we infused insulin with or without somatostatin in two sets of studies. Insulin (40 mU X kg-1 X h-1) was infused for 100 min (n = 6). Plasma glucose levels fell to 55 +/- 4.1 mg/dl with insulin alone and significantly lower, to 44 +/- 1.9 mg/dl, when somatostatin (250 micrograms/h) was also infused (P less than .01). Plasma immunoreactive insulin (IRI) rose to 57 +/- 12.5 microU/ml with insulin alone, which was significantly different from 88 +/- 15 microU/ml when insulin was infused together with somatostatin (P less than .01). When a smaller dose of insulin (30 mU X kg-1 X h-1) was infused for 100 min (n = 4), similar results were observed. When somatostatin was infused together with insulin, plasma glucose fell to lower levels (41 +/- 4.2 vs. 62 +/- 9.5 mg/dl; P less than .01) and plasma IRI rose higher (39 +/- 8.5 vs. 27 +/- 5.9 microU/ml; P less than .01) than when insulin was infused alone. C-peptide was equally suppressed by hypoglycemia regardless of whether somatostatin was administered, indicating suppression of endogenous insulin during these studies. We conclude that somatostatin infusion impairs the clearance of exogenous insulin.(ABSTRACT TRUNCATED AT 250 WORDS)     

Acute effect of inhaled bradykinin on tracheobronchial clearance in normal humans.

BACKGROUND: Bradykinin, a nonapeptide that contributes as a mediator to the pathogenesis of asthma, may affect lung mucociliary clearance, as it has been shown to be a potent secretagogue in canine airways and in human nasal mucosa in vivo. To evaluate this possibility the effect of inhaled bradykinin on mucociliary clearance has been studied in 10 healthy volunteers. METHODS: Subjects attended the laboratory on two occasions to take part in tracheobronchial clearance studies using a non-invasive radioisotopic technique. Inhalation of radioaerosol was followed 30 minutes later by inhalation of either bradykinin (8 mg/ml) or vehicle placebo in a randomised, double blind fashion. After each inhalation the number of coughs was recorded. Whole lung radioactivity was measured every half hour for six hours with two collimated scintillation counters, and a tracheobronchial clearance curve was plotted for each subject on each occasion. RESULTS: Mucociliary clearance, expressed as the area under the tracheobronchial radioaerosol retention curve calculated for the first six hours (AUC0-6h), was greater in nine out of 10 subjects after inhalation of bradykinin than after placebo. The median values (range) for AUC0-6h were significantly reduced from 126% (78-232%)/h with placebo to 87% (51-133%)/h with bradykinin. CONCLUSION: It is concluded that acute exposure to inhaled bradykinin accelerates tracheobronchial clearance in normal human airways.     

Regulation of whole-body leucine metabolism with insulin during mixed-meal absorption in normal and diabetic humans

To determine the effects of insulin on dietary and endogenous leucine metabolism, five normal subjects, seven insulin-insufficient insulin-dependent (IDDM) diabetic patients, and five diabetic patients controlled with continuous subcutaneous insulin infusion (CSII) were studied before and for 8 h after ingestion of a chemically defined elemental test meal (10 cal/kg) containing crystalline amino acids. L-[1-14C]leucine was included in the meal to trace the entry and oxidation of the dietary leucine. Total (meal + endogenous) entry of leucine into the circulation was estimated with a constant infusion of [2H3]leucine. Postabsorptive and meal-related increases in the plasma leucine concentration were greater (P less than .05) in the insulin-insufficient IDDM than in the normal subjects but returned to near-normal values with CSII. Baseline leucine flux was approximately 40% greater in the insulin-insufficient IDDM than in normal subjects (2.17 +/- 0.17 vs. 1.55 +/- 0.15 mumol.kg-1.min-1, respectively; .05 less than P less than .01) but were near normal during CSII treatment (1.85 +/- 0.25 mumol.kg-1.min-1). Furthermore, total leucine entry during meal absorption was greater in the insulin-insufficient IDDM (1.41 +/- 0.10 mmol.kg-1.8 h-1) than in either normal (0.96 +/- 0.08 mmol.kg-1.8 h-1, P less than .01) or IDDM subjects during CSII treatment (1.09 +/- 0.11 mmol.kg-1.8 h-1, P less than .05). Fractional oxidation (approximately 40-50%) and entry of dietary leucine were similar in all three groups.(ABSTRACT TRUNCATED AT 250 WORDS)     

Intestinal calcium absorption during hyperinsulinemic euglycemic glucose clamp in healthy humans

Summary The influence of postprandial-like plasma insulin levels on intestinal calcium absorption (CaA) was studied in 9 health men. On separate occasions, they received either an i.v. infusion of 40 mU/m² minute synthetic human insulin as well as a variable glucose infusion in order to clamp the plasma glucose at the baseline level (=glucose clamp), or insulin- and glucose-free vehicle infusions (=vehicle). During these infusions, an oral load containing 326 mg Ca in the form of Ca chloride was administered and CaA was determined thereafter with a⁴⁷Ca/⁸⁵Sr double tracer method. During glucose clamp, mean plasma insulin was 172 ±(1 SEM) 10 as compared to 6±1 μU/ml during vehicle infusions. During the clamp, 3-hour cumulative CaA rose significantly by 14% as compared to vehicle (39.2±2.5 vs. 34.4±2%,P<0.02). At the same time, serum potassium and phosphorus dropped significantly, whereas serum parathyroid hormone (PTH) and 1,25(OH)₂D levels were unchanged as compared to vehicle. The urinary excretions of potassium, sodium, and inorganic phosphorus as well as the urinary specific activity of⁴⁷Ca, dropped significantly during glucose clamp, whereas the urinary excretion of cAMP was unchanged as compared to vehicle. The results suggest that, under the conditions of euglycemic hyper-insulinemic clamp, insulin stimulates CaA of healthy humans in a PTH- and 1,25(OH)₂D-independent manner. Insulin may thus possibly be regarded as a factor participating in the regulation of CaA in humans.     

Caffeine ingestion decreases glucose disposal during a hyperinsulinemic-euglycemic clamp in sedentary humans

The purpose of this investigation was to examine the effect of caffeine (an adenosine receptor antagonist) on whole-body insulin-mediated glucose disposal in resting humans. We hypothesized that glucose disposal would be lower after the administration of caffeine compared with placebo. Healthy, lean, sedentary (n = 9) men underwent two trial sessions, one after caffeine administration (5 mg/kg body wt) and one after placebo administration (dextrose) in a double-blind randomized design. Glucose disposal was assessed using a hyperinsulinemic-euglycemic clamp. Before the clamp, there were no differences in circulating levels of methylxanthines, catecholamines, or glucose. Euglycemia was maintained throughout the clamp with no difference in plasma glucose concentrations between trials. The insulin concentrations were also similar in the caffeine and placebo trials. After caffeine administration, glucose disposal was 6.38 +/- 0.76 mg/kg body wt compared with 8.42 +/- 0.63 mg/kg body wt after the placebo trial. This represents a significant (P < 0.05) decrease (24%) in glucose disposal after caffeine ingestion. In addition, carbohydrate storage was 35% lower (P < 0.05) in the caffeine trial than in the placebo trial. Furthermore, even when the difference in glucose disposal was normalized between the trials, there was a 23% difference in the amount of carbohydrate stored after caffeine administration compared with placebo administration. Caffeine ingestion also resulted in higher plasma epinephrine levels than placebo ingestion (P < 0.05). These data support our hypothesis that caffeine ingestion decreases glucose disposal and suggests that adenosine plays a role in regulating glucose disposal in resting humans.     

Insulin sensitivity and exogenous insulin clearance in Graves' disease. Measurement by the glucose clamp technique and continuous indirect calorimetry

Insulin sensitivity was measured in a group of seven thyrotoxic patients and in a group of seven normal subjects by means of the glucose clamp technique. Infusion of insulin at a rate of 0.80 +/- 0.05 mU/kg X min in the hyperthyroid patients and of 0.55 +/- 0.04 mU/kg X min in the control group was performed to obtain a steady-state plasma insulin concentration of approximately 50 microU/ml. Substrate oxidation rates were measured in the postabsorptive state and during the 2 h of the clamp by means of continuous indirect calorimetry. In the postabsorptive state, hyperthyroid patients presented a preferential oxidation of lipids. During the period 60-120 min of the clamp, mean plasma glucose (92 +/- 2 versus 93 +/- 2 mg/dl), insulin (50 +/- 5 versus 58 +/- 3 microU/ml), and total glucose metabolism (5.8 +/- 0.7 versus 6.1 +/- 0.3 mg/kg X min) were similar in the hyperthyroid patients and the control subjects. The rate of glucose oxidation was higher in hyperthyroid patients than in control subjects (4.3 +/- 0.5 versus 2.2 +/- 0.2 mg/kg X min, P less than 0.001), while that of lipid oxidation was similar in both groups (0.6 +/- 0.2 versus control 0.7 +/- 0.1 mg/kg X min). The calculated metabolic clearance rate of insulin was markedly higher in the hyperthyroid patients (1144 +/- 132 ml/min) than in the normal subjects (812 +/- 56 ml/min, P less than 0.025). It is concluded that insulin sensitivity is not altered in the thyrotoxic state. The major route of insulin-stimulated glucose disposal in the hyperthyroid patients appears to be glucose oxidation.     

Vitamin D, insulin secretion, sensitivity, and lipids: results from a case-control study and a randomized controlled trial using hyperglycemic clamp technique

OBJECTIVE

Vitamin D deficiency is associated with an unfavorable metabolic profile in observational studies. The intention was to compare insulin sensitivity (the primary end point) and secretion and lipids in subjects with low and high serum 25(OH)D (25-hydroxyvitamin D) levels and to assess the effect of vitamin D supplementation on the same outcomes among the participants with low serum 25(OH)D levels.

RESEARCH DESIGN AND METHODS

Participants were recruited from a population-based study (the Tromsø Study) based on their serum 25(OH)D measurements. A 3-h hyperglycemic clamp was performed, and the participants with low serum 25(OH)D levels were thereafter randomized to receive capsules of 20,000 IU vitamin D₃ or identical-looking placebo twice weekly for 6 months. A final hyperglycemic clamp was then performed.

RESULTS

The 52 participants with high serum 25(OH)D levels (85.6 ± 13.5 nmol/L [mean ± SD]) had significantly higher insulin sensitivity index (ISI) and lower HbA_1c and triglycerides (TGs) than the 108 participants with low serum 25(OH)D (40.3 ± 12.8 nmol/L), but the differences in ISI and TGs were not significant after adjustments. After supplementation, serum 25(OH)D was 142.7 ± 25.7 and 42.9 ± 17.3 nmol/L in 49 of 51 completing participants randomized to vitamin D and 45 of 53 randomized to placebo, respectively. At the end of the study, there were no statistically significant differences in the outcome variables between the two groups.

CONCLUSIONS

Vitamin D supplementation to apparently healthy subjects with insufficient serum 25(OH)D levels does not improve insulin sensitivity or secretion or serum lipid profile.Type 2 diabetes is a chronic condition associated with increased risk of micro- and macrovascular morbidity (1). The underlying pathophysiological mechanisms include insulin resistance combined with a relative deficit of insulin secretion from the pancreas, usually accompanied by systemic inflammation (2). The number of people suffering from the disease is increasing globally (2). Effective preventive means are therefore needed, and modifiable risk factors should be identified and explored.Vitamin D insufficiency, which is reported to be highly prevalent (3), might be such a factor. The vitamin D receptor (4) and the enzyme 1-α hydroxylase (5), which is necessary for the production of the active form of the hormone 1,25(OH)₂D (1,25-dihydroxyvitamin D), are present in pancreatic β-cells. Accordingly, vitamin D has been reported to increase glucose-mediated insulin secretion in animal studies (6). In vitro, 1,25(OH)₂D increases the expression of the insulin receptor and enhances insulin-mediated glucose transport (7). Although less explored, the anti-inflammatory effects of vitamin D might also affect diabetes development (8).Consistent with this, observational data from a number of epidemiological studies show an inverse association between serum 25(OH)D (25-hydroxyvitamin D) and glucose levels (9–12), insulin resistance (11–18), and prevalence of type 2 diabetes (18–20). However, to demonstrate a causal relation between vitamin D and glucose metabolism, evidence from randomized and adequately powered placebo-controlled intervention trials is needed. As recently reviewed, the studies published thus far are heterogeneous regarding dose and formulation of vitamin D treatment, duration, and inclusion criteria; most use indirect measures of insulin secretion and sensitivity; and the results are inconsistent (21).In the sixth Tromsø Study in 2008, serum 25(OH)D was measured in nearly 12,000 subjects. On the basis of these measurements, we invited subjects with low or high serum 25(OH)D levels to a follow-up study where insulin sensitivity and secretion were evaluated with the hyperglycemic clamp technique. Thereafter, the subjects with low serum 25(OH)D levels were invited to a 6-month intervention study to compare the effect of vitamin D₃ 20,000 IU twice per week with placebo on the same measures. As we previously have found cross-sectional and longitudinal associations between serum 25(OH)D levels and serum lipids (22), measurements of serum lipids were also included.     

Effect of epinephrine on lidocaine clearance in vivo: a microdialysis study in humans.

BACKGROUND: Local anesthetic nerve block prolonged by epinephrine is thought to result from local vasoconstriction and consequent decreased local anesthetic clearance from the injection site. However, no study has yet confirmed this directly in humans by measuring tissue concentrations of local anesthetic over time. In addition, recent studies have shown that the alpha2-adrenergic receptor agonist, clonidine, also prolongs nerve block without altering local anesthetic clearance. Because epinephrine is also an alpha2-adrenergic receptor agonist, it is possible that epinephrine prolongs local anesthetic block by a pharmacodynamic mechanism and not a pharmacokinetic one. This study was designed to address this issue. METHODS: Microdialysis probes were placed adjacent to the superficial peroneal nerve in both feet of eight volunteers. Plain lidocaine (1%) was injected along one peroneal nerve and lidocaine with epinephrine (2.5 microg/ml) was injected along the other nerve in a double-blinded, randomized manner. The concentration of lidocaine in tissue was measured at 5-min intervals, and sensory block and cutaneous blood flow were assessed by laser Doppler at 10-min intervals for 5 h. The resulting data for lidocaine concentration versus time were fit to a two-compartment model using modeling software. RESULTS: Epinephrine prolonged sensory block by decreasing local blood flow and slowing clearance. There was no evidence of a pharmacodynamic effect of epinephrine. CONCLUSION: Although epinephrine activates alpha2-adrenergic receptors, its mechanism for prolonging the duration of local anesthetic block rests on its ability to decrease local anesthetic clearance and not on a pharmacodynamically mediated potentiation of local anesthetic effect.     

Effect of clonidine on lidocaine clearance in vivo: a microdialysis study in humans.

BACKGROUND: The addition of clonidine to local anesthetics has been shown to prolong both peripheral and central neuraxial local anesthetic blocks. Whether clonidine prolongs local anesthetic block by a pharmacokinetic effect or a pharmacodynamic effect is unclear. By directly measuring lidocaine tissue concentrations at the site of injection in the presence and absence of clonidine, this study was designed to address this question. METHODS: Microdialysis probes were placed adjacent to the superficial peroneal nerve in both feet of seven volunteers. Plain lidocaine (1%) was injected along one nerve, and lidocaine with clonidine (10 microg/ml) was injected along the other nerve in a double-blind, randomized manner. The extracellular fluid was then sampled for lidocaine concentration at 5-min intervals using microdialysis, cutaneous blood flow was assessed by laser Doppler at 10-min intervals, and sensory block was assessed every 10 min until resolution. RESULTS: Consistent with previous studies, clonidine prolonged lidocaine sensory block. Blood flow increased in both groups but was significantly lower in the clonidine group, especially during the first 60 min. Consistent with the lower blood flow, the area under the lidocaine concentration-versus-time curve was significantly greater in the clonidine group during the first 60 min. CONCLUSION: When added to lidocaine, clonidine prolonged peripheral nerve block. The pharmacokinetic data suggest that the mechanism of prolongation is at least in part pharmacokinetic.     

Demonstration of glycated insulin in human diabetic plasma and decreased biological activity assessed by euglycemic-hyperinsulinemic clamp technique in humans

The presence and biological significance of circulating glycated insulin has been evaluated by high-pressure liquid chromatography (HPLC), electrospray ionization mass spectrometry (ESI-MS), radioimmunoassay (RIA), receptor binding, and hyperinsulinemic-euglycemic clamp techniques. ESI-MS analysis of an HPLC-purified plasma pool from four male type 2 diabetic subjects (HbA(1c) 8.1 +/- 0.2%, plasma glucose 8.7 +/- 1.3 mmol/l [means +/- SE]) revealed two major insulin-like peaks with retention times of 14-16 min. After spectral averaging, the peak with retention time of 14.32 min exhibited a prominent triply charged (M+3H)(3+) species at 1,991.1 m/z, representing monoglycated insulin with an intact M(r) of 5,970.3 Da. The second peak (retention time 15.70 min) corresponded to native insulin (M(r) 5,807.6 Da), with the difference between the two peptides (162.7 Da) representing a single glucitol adduct (theoretical 164 Da). Measurement of glycated insulin in plasma of type 2 diabetic subjects by specific RIA gave circulating levels of 10.1 +/- 2.3 pmol/l, corresponding to approximately 9% total insulin. Biological activity of pure synthetic monoglycated insulin (insulin B-chain Phe(1)-glucitol adduct) was evaluated in seven overnight-fasted healthy nonobese male volunteers using two-step euglycemic-hyperinsulinemic clamps (2 h at 16.6 micro g x kg(-1) x min(-1), followed by 2 h at 83.0 micro g x kg(-1) x min(-1); corresponding to 0.4 and 2.0 mU x kg(-1) x min(-1)). At the lower dose, the exogenous glucose infusion rates required to maintain euglycemia during steady state were significantly lower with glycated insulin (P < 0.01) and approximately 70% more glycated insulin was required to induce a similar rate of insulin-mediated glucose uptake. Maximal responses at the higher rates of infusion were similar for glycated and control insulin. Inhibitory effects on endogenous glucose production, insulin secretion, and lipolysis, as indicated by measurements of C-peptide, nonesterified free fatty acids, and glycerol, were also similar. Receptor binding to CHO-T cells transfected with human insulin receptor and in vivo metabolic clearance revealed no differences between glycated and native insulin, suggesting that impaired biological activity is due to a postreceptor effect. The present demonstration of glycated insulin in human plasma and related impairment of physiological insulin-mediated glucose uptake suggests a role for glycated insulin in glucose toxicity and impaired insulin action in type 2 diabetes.     

Dose-response study of sodium cromoglycate in exercise-induced asthma.

Ten patients with exercise-induced asthma participated in a single-blind dose-response study comparing the protective effect of inhaled sodium cromoglycate in increasing concentrations from 2 to 40 mg/ml. Saline was used as a control. Effects were assessed from the mean maximal percentage fall in forced expiratory volume in one second (FEV1) after the patients had run on a treadmill for eight minutes. There was slight bronchodilation evident from the increase in baseline FEV1 after inhalation of sodium cromoglycate, the difference reaching statistical significance with the highest concentration (5.7%, p less than 0.05). After exercise the maximal percentage falls in FEV1 (means and SEM) after saline and after sodium cromoglycate at 2, 10, 20, and 40 mg/ml were 37.3 +/- 4.7, 17.3 +/- 4.1, 10 +/- 3.3, 7.6 +/- 2.4, and 12 +/- 2.9. Sodium cromoglycate inhibited the exercise-induced fall in FEV1 at all the concentrations used in the study (p less than 0.001) and its inhibitory effect increased from 2 to 20 mg/ml. The mean FEV1 returned to baseline values within 15 minutes at higher concentrations of sodium cromoglycate (20 and 40 mg/ml) and a small bronchodilator effect was noted at 30 minutes. The findings suggest that the protective effect of sodium cromoglycate in exercise asthma is dose related. At higher concentration the drug suppresses chemical mediator release from the lung mast cells and may also modify the bronchial reactivity to release mediators.     

Glucose homeostasis and insulin secretion during isoflurane anesthesia in humans

The effect of isoflurane-air anesthesia on glucose tolerance in humans was investigated using two successive intravenous glucose tolerance tests (IVGTT). After a first IVGTT while awake, patients received a second IVGTT either while awake (group I), during anesthesia with isoflurane-air and pancuronium without surgical stimulation (group II), or during the same anesthetic technique but combined with surgery (group III). Isoflurane seemed to induce glucose intolerance (glucose disappearance rate K10-60 min = 1.628 +/- 0.462% min-1 [control] versus 1.086 +/- 0.920% min-1 [anesthesia], P less than 0.05) partly due to a decreased glucose induced insulin response. Growth hormone and norepinephrine levels were also increased during anesthesia. Epinephrine levels were lowered by isoflurane anesthesia. Although glucose intolerance was marked during surgery (K10-60 min = 0.892 +/- 0.286% min-1), the glucose-induced insulin response remained similar to that observed in patients in group II, while growth hormone, cortisol, epinephrine, and norepinephrine concentrations increased significantly. These known stress factors thus seemed to enhance glucose intolerance through a diminished response to insulin action and/or an enhanced hepatic glucose output, rather than by further impairing pancreatic insulin secretion.     

Beta-endorphin-induced inhibition and stimulation of insulin secretion in normal humans is glucose dependent

This study evaluated the effect of human beta-endorphin on pancreatic hormone levels and their responses to nutrient challenges in normal subjects. Infusion of 0.5 mg/h beta-endorphin caused a significant rise in plasma glucose concentrations preceded by a significant increase in peripheral glucagon levels. No changes occurred in the plasma concentrations of insulin and C-peptide. Acute insulin and C-peptide responses to intravenous pulses of different glucose amounts (0.33 g/kg and 5 g) and arginine (3 g) were significantly reduced by beta-endorphin infusion (P less than .01). This effect was associated with a significant reduction of the glucose disappearance rates, suggesting that the inhibition of insulin was of biological relevance. beta-Endorphin also inhibited glucose suppression of glucagon levels and augmented the glucagon response to arginine. To verify whether the modification of prestimulus glucose level could be important in these hormonal responses to beta-endorphin, basal plasma glucose concentrations were raised by a primed (0.5 g/kg) continuous (20 mg kg-1.min-1) glucose infusion. After stabilization of plasma glucose levels (350 +/- 34 mg/dl, t = 120 min), beta-endorphin infusion caused an immediate and marked increase in plasma insulin level (peak response 61 +/- 9 microU/ml, P less than .01), which remained elevated even after the discontinuation of opioid infusion. Moreover, the acute insulin response to a glucose pulse (0.33 g/kg i.v.) given during beta-endorphin infusion during hyperglycemia was significantly higher than the response obtained during euglycemia (171 +/- 32 vs. 41 +/- 7 microU/ml, P less than .01).(ABSTRACT TRUNCATED AT 250 WORDS)     

Dose-response relationship between lymph insulin and glucose uptake reveals enhanced insulin sensitivity of peripheral tissues.

To examine the role of transcapillary insulin transport to peripheral insulin sensitivity in vivo, we performed dose-response experiments in which both plasma and thoracic duct lymph insulin and glucose utilization (Rd) were measured in conscious dogs. Euglycemic clamps (n = 22) consisted of a 3-h activation period in which insulin was infused (rates: "physiological" 3.6, 5.4, 7.2 pmol.min-1.kg-1; "pharmacological" 108 pmol.min-1.kg-1), followed by a 3-h deactivation period. [14C]inulin was also infused as a diffusionary marker. Insulin sensitivity was estimated as the ED50. When based on plasma insulin, ED50 was 480 pM. However, when calculated from lymph (i.e., interstitial) insulin measurements, ED50 was 240 pM. Thus, interstitial insulin measurements reveal that insulin sensitivity of peripheral tissues is approximately twice that estimated from plasma insulin and is similar to sensitivity reported for suppression of hepatic glucose production. Furthermore, although [14C]inulin achieved equilibrium between plasma and lymph within 180 min, within the physiological range, steady state plasma insulin was higher than insulin in lymph (306 +/- 18, 474 +/- 42, and 780 +/- 60 pM vs. 180 +/- 18, 318 +/- 12, and 504 +/- 36 pM; P less than 0.0001); plasma insulin achieved steady state faster than lymph insulin (6 +/- 1, 6 +/- 2, and 11 +/- 3 min vs. 29 +/- 4, 16 +/- 6, and 44 +/- 8 min; P less than 0.01) and disappeared faster (5 +/- 2, 7 +/- 2, and 15 +/- 6 min vs. 37 +/- 8, 32 +/- 4, and 43 +/- 9 min; P less than 0.01). The time course of lymph insulin at each dose was similar to that of Rd, and at each dose, unlike plasma insulin, lymph insulin was strongly correlated with Rd (r = 0.93 or better). At pharmacological hyperinsulinemia (plasma 35232 +/- 5250 pM, lymph 27366 +/- 4380 pM), Rd rose faster than lymph insulin and disappeared more slowly than insulin. Thus, lymph insulin data indicate that the periphery is more sensitive to insulin than previously realized from estimates based solely on plasma hormone. Furthermore, lymph insulin is proportional to Rd within the physiological but not pharmacological range of insulin, indicating that transcapillary insulin transport is rate limiting for insulin action in this range. Finally, based on in vivo lymph (i.e., interstitial) insulin measurements, peripheral tissue is almost twice as sensitive to insulin than previously realized.     

Secretion of insulin in a perifusion system and conversion of proinsulin to insulin by pancreatic islets from hyperglycemic rats.

The secretory pattern of insulin and the rate of conversion of proinsulin to insulin were studied in isolated pancreatic islets from normoglycemic (buffer-infused for 24 hours) and hyperglycemic (glucose-infused for 24 hours) rats. The profiles of insulin secretion obtained during one hour of perifusion were markedly different in the two groups. The rate of insulin secretion by islets from the hyperglycemic rats was initially very high but progressively declined during the late period of the perifusion. The reverse pattern was found with the islets from buffer-infused rats. For the estimation of the rate of proinsulin conversion, islets were pulse-labeled with L-[4,5-3H]-leucine for 15 minutes and "chase"-incubated for 30 and 60 minutes. Labeled rat proinsulin and rat insulins in the medium and in the islet extracts were separated by a validated SDS-urea electrophoretic acrylamide procedure following immunoprecipitation. The conversion rate was estimated from the radioactivity in the insulin band, expressed as a per cent of the radioactivity in the proinsulin + insulin bands. Islets from hyperglycemic rats converted newly synthesized proinsulin to insulin at significantly higher rates than did control islets.