Reduced access to insulin-sensitive tissues in dogs with obesity secondary to increased fat intake.

Physiological hyperinsulinemia provokes hemodynamic actions and augments access of macromolecules to insulin-sensitive tissues. We investigated whether induction of insulin resistance by a hypercaloric high-fat diet has an effect on the extracellular distribution of macromolecules to insulin-sensitive tissues. Male mongrel dogs were randomly selected into two groups: seven dogs were fed an isocaloric control diet ( approximately 3,900 kcal, 35% from fat), and six dogs were fed a hypercaloric high-fat diet ( approximately 5,300 kcal, 54% from fat) for a period of 12 weeks. During hyperinsulinemic-euglycemic clamps, we determined transport parameters and distribution volumes of [(14)C]inulin by applying a three-compartment model to the plasma clearance data of intravenously injected [(14)C]inulin (0.8 microCi/kg). In another study with direct cannulation of the hindlimb skeletal muscle lymphatics, we investigated the effect of physiological hyperinsulinemia on the appearance of intravenously injected [(14)C]inulin in skeletal muscle interstitial fluid and compared the effect of insulin between control and high-fat diet groups. The hypercaloric high-fat diet resulted in significant weight gain (18%; P<0.001) associated with marked increases of subcutaneous (140%; P<0.001) and omental (83%; P<0.001) fat depots, as well as peripheral insulin resistance, measured as a significant reduction of insulin-stimulated glucose uptake during clamps (-35%; P<0.05). Concomitantly, we observed a significant reduction of the peripheral distribution volume of [(14)C]inulin (-26%; P<0.05), whereas the vascular distribution volume and transport and clearance parameters did not change as a cause of the diet. The second study directly confirmed our findings, suggesting a marked reduction of insulin action to stimulate access of macromolecules to insulin-sensitive tissues (control diet 32%, P<0.01; high-fat diet 18%, NS). The present results indicate that access of macromolecules to insulin-sensitive tissues is impaired during diet-induced insulin resistance and suggest that the ability of insulin itself to stimulate tissue access is diminished. We speculate that the observed diet-induced defects in stimulation of tissue perfusion contribute to the development of peripheral insulin resistance.

Physiological hyperinsulinemia in dogs augments access of macromolecules to insulin-sensitive tissues.

Pharmacological doses of insulin increase limb blood flow and enhance tissue recruitment for small solutes such as glucose. We investigated whether elevating insulin within the physiological range (68 +/- 6 vs. 425 +/- 27 pmol/l) can influence tissue recruitment of [(14)C]inulin, an inert diffusionary marker of molecular weight similar to that of insulin itself. During hyperinsulinemic-euglycemic clamps, transport parameters and distribution volumes of [(14)C]inulin were determined in conscious dogs by applying a three-compartment model to the plasma clearance data of intravenously injected [(14)C]inulin (0.8 microCi/kg). In a second set of experiments in anesthetized dogs with direct cannulation of the hindlimb skeletal muscle lymphatics, we measured a possible effect of physiological hyperinsulinemia on the response of the interstitial fluid of skeletal muscle to intravenously injected [(14)C]inulin and compared this response with the model prediction from plasma data. Physiological hyperinsulinemia caused a 48 +/- 10% (P < 0.005) and a 35 +/- 15% (P < 0.05) increase of peripheral and splanchnic interstitial distribution volumes for [(14)C]inulin. Hindlimb lymph measurements directly confirmed the ability of insulin to enhance the access of macromolecules to the peripheral interstitial fluid compartment. The present results show that physiological hyperinsulinemia will enhance the delivery of a substance of similar molecular size to insulin to previously less intensively perfused regions of insulin-sensitive tissues. Our data suggest that the delivery of insulin itself to insulin-sensitive tissues could be a mechanism of insulin action on cellular glucose uptake independent of and possibly synergistic with either enhanced blood flow distribution or GLUT4 transporter recruitment to enhance glucose utilization. Because of the differences between inulin and insulin itself, whether delivery of the bioactive hormone is increased remains speculative.

Endurance training delays age of decline in leg strength and muscle morphology.

PURPOSE: It has been reported that maximal strength peaks at approximately 30 yr of age, plateaus, and remains relatively stable for the next 20 yr, with an age-related decline in strength becoming significant after age 50. Much of the research attributes this decrease in peak force to age-associated reductions in muscle mass, with a selective atrophy and reduction in Type II fiber area and number being the primary factors. The influence that chronic endurance training has upon age-associated changes in muscular strength and muscle morphology has been largely undetermined. The purpose of this investigation was to examine the influence of chronic endurance training and age on leg extensor strength, and muscle fiber size and type distribution. METHODS: Male master runners (N = 107, age range = 40-88 yr) were tested for maximal strength of the leg extensor muscles. A subgroup of 30 master athletes participated in muscle biopsy testing. The effects of age were addressed by subdividing the sample into five cohorts. RESULTS: Peak isokinetic concentric torque did not differ between age groups until after age 70 yr. Regression analysis revealed a significant (P < 0.05, r(2) = 0.1838) age-associated decrease in relative strength (N.m.kg(-1) lean body mass). Type I and Type II fiber area and distribution did not differ between age groups through the eighth decade. CONCLUSIONS: These data suggest that chronic endurance training can delay the age of significant decline in peak torque and changes in muscle morphology characteristics of the vastus lateralis.

Mechanism of action in dogs of slow-acting insulin analog O346.

We compared metabolic effects as well as plasma and interstitial fluid kinetics of fatty acid-acylated insulin, Lys(B29)(N(epsilon)-omega-carboxynonadecanoyl)-des(B30) human insulin (O346), with previously determined kinetics of native insulin and insulin detemir. Euglycemic clamps with iv injection of O346 (90 pmol/kg) or saline control were performed in 10 male mongrel dogs under inhalant anesthesia. The t(1/2) for the clearance of O346 from plasma was 375.7 +/- 26.7 min; the t(1/2) for the appearance of O346 in interstitial fluid was 137 +/- 20 min (mean +/- SEM). Glucose disposal with O346 injection was increased 4-fold (t = 480 min, 8.3 +/- 1.42 mg/min/kg) compared with preinjection (t = 0 min, 2.1 +/- 0.13 mg/min/kg; P < 0.05) or saline control (t = 480 min, 2.09 +/- 0.22 mg/min/kg; P < 0.05). O346 plasma elimination and transendothelial transport were 0.3% and 3.5% of regular insulin and 3% and 50% of insulin detemir, respectively. Combination of in vivo results and compartmental modeling suggests that the duration of action of O346 after iv injection is about 25-fold and 10-fold longer compared with regular human insulin and insulin detemir, respectively. This study demonstrates that O346 stimulates glucose disposal very slowly, but when injected iv, its effect may be maintained for as long as 48 h as estimated from simulation analysis. The data suggest that O346 bound to albumin in plasma acts as a storage compartment for O346 from which the analog is slowly released to insulin-sensitive tissues. Reduced liver clearance of O346 is suggested to be the major mechanism for the protracted action.

Burst-like control of lipolysis by the sympathetic nervous system in vivo.

Rapid oscillations of visceral lipolysis have been reported. To examine the putative role of the CNS in oscillatory lipolysis, we tested the effects of beta(3)-blockade on pulsatile release of FFAs. Arterial blood samples were drawn at 1-minute intervals for 120 minutes from fasted, conscious dogs (n = 7) during the infusion of saline or bupranolol (1.5 micro g/kg/min), a high-affinity beta(3)-blocker. FFA and glycerol time series were analyzed and deconvolution analysis was applied to estimate the rate of FFA release. During saline infusion FFAs and glycerol oscillated in phase at about eight pulses/hour. Deconvolution analysis showed bursts of lipolysis (nine pulses/hour) with time-dependent variation in burst frequency. Bupranolol completely removed rapid FFA and glycerol oscillations. Despite removal of lipolytic bursts, plasma FFAs (0.31 mM) and glycerol (0.06 mM) were not totally suppressed and deconvolution analysis revealed persistent non-oscillatory lipolysis (0.064 mM/min). These results show that lipolysis in the fasting state consists of an oscillatory component, which appears to be entirely dependent upon sympathetic innervation of the adipose tissue, and a non-oscillatory, constitutive component, which persists despite beta(3)-blockade. The extinction of lipid fuel bursts by beta(3)-blockade implies a role for the CNS in the maintenance of cyclic provision of lipid fuels.

Mode of transcapillary transport of insulin and insulin analog NN304 in dog hindlimb: evidence for passive diffusion.

A defect in transcapillary transport of insulin in skeletal muscle and adipose tissue has been proposed to play a role in the insulin resistance that leads to type 2 diabetes, yet the mechanism of insulin transfer across the capillary endothelium from plasma to interstitium continues to be debated. This study examined in vivo the interstitial appearance of insulin in hindlimb using the fatty acid acylated insulin analog Lys(B29)-tetradecanoyl des-(B30) human insulin, or NN304, as a marker for insulin transport. If the insulin transport were a saturable process, then "swamping" the capillary endothelial insulin receptors with native insulin would suppress the subsequent appearance in interstitial fluid of the insulin analog NN304. This analog binds to insulin receptors with an affinity of about 50% of native insulin. Experimental conditions established a physiologic NN304 dose in the absence or presence of pharmacologic and saturating concentrations of regular human insulin. Euglycemic clamps were performed in dogs under inhalant anesthesia with deep hindlimb lymphatic sampling, representative of skeletal muscle interstitial fluid (ISF). In group 1 (n = 8), NN304 alone was infused (3.6 pmol center dot min(-1) center dot kg(-1)) from 60 to 360 min. In group 2 (n = 6), starting at time 0, human insulin was infused at a pharmacologic dose (60 pmol center dot min(-1) center dot kg(-1)) with the addition of NN304 infusion (3.6 pmol center dot min(-1) center dot kg(-1)) from 60 to 360 min. In group 3 (n = 4), the human insulin infusion was increased to a saturating dose (120 pmol center dot min(-1) center dot kg(-1)). Pharmacologic insulin infusion (group 2) established steady-state human insulin concentrations of 6,300 plus minus 510 pmol/l in plasma and 5,300 plus minus 540 pmol/l in ISF. Saturating insulin infusion (group 3) achieved steady-state human insulin concentrations of 22,000 plus minus 1,800 pmol/l in plasma and 19,000 plus minus 1,500 pmol/l in ISF. Total (bound and unbound) NN304 plasma concentrations rose from a steady state of 1,900 plus minus 110 (group 1) to 2,400 plus minus 200 pmol/l (group 2) and 3,100 plus minus 580 pmol/l (group 3), consistent with a competition-driven decline in NN304 clearance from plasma as the human insulin level increased (P < 0.05 by ANOVA). Steady-state interstitial NN304 concentrations also rose with increasing human insulin levels but did not achieve significance in comparison with analog alone (162 plus minus 15 vs. 196 plus minus 22 and 241 plus minus 53 pmol/l for group 1 versus groups 2 and 3, respectively; P = 0.20), yet the steady-state plasma:ISF ratio for NN304 remained essentially unchanged in the absence and presence of elevated human insulin levels (12.6 plus minus 1.2 vs. 12.4 plus minus 0.5 and 13.1 plus minus 1.5 for group 1 versus groups 2 and 3, respectively; P = 0.93). Last, NN304 rate of appearance in interstitial fluid (i.e., half-time to steady state) was similar between groups; mean half-time of 92 plus minus 4 min (NS between groups). In conclusion, appearance of the insulin analog NN304 in skeletal muscle interstitial fluid was constant whether in the absence or presence of human insulin concentrations sufficient to saturate the endothelial insulin receptors. These findings support the hypothesis, provided that the mechanism of insulin and NN304 transcapillary transport is similar, that transcapillary transport of insulin in skeletal muscle occurs primarily via a nonsaturable process such as passive diffusion via a paracellular or transcellular route.

Albumin binding of acylated insulin (NN304) does not deter action to stimulate glucose uptake.

NN304 [Lys(B29)-tetradecanoyl des(B30) human insulin] is a potentially therapeutic insulin analog designed to exhibit protracted glucose-lowering action. In dogs with infusion rates similar to insulin itself, NN304 exhibits similar glucose uptake (R(d)) stimulation with delayed onset of action. This compartmental modeling study was to determine if NN304 action could be accounted for by the approximately 2% unbound NN304 concentration. NN304 (or human insulin) (n = 6 each) was infused at 10.2 pmol center dot min(-1) center dot kg(-1) under euglycemic clamp conditions in anesthetized dogs. NN304 appearance in lymph, representing interstitial fluid (ISF), was slow compared with insulin (t(1/2) = 70 +/- 7 vs. 14 +/- 1 min, P < 0.001). R(d) was highly correlated with the ISF concentration for insulin and NN304 (r = 0.86 and 0.93, respectively), suggesting that slow transendothelial transport (TET) is responsible for sluggish NN304 action. Insulin and NN304 concentration data were fit to a two-compartment (plasma and ISF) model. NN304 plasma elimination and TET were reduced to 10 and 7% of insulin, respectively. Thus, there was reduction of NN304 transport, but not to the degree expected. In ISF, there was no reduction in NN304 elimination. Thus, this acylated insulin analog demonstrates blunted kinetics in plasma, and full efficacy in the compartment of action, ISF.