Portal venous hyperinsulinemia does not stimulate gut glucose absorption in the conscious dog

The purpose of the present study was to assess whether physiological portal vein hyperinsulinemia stimulates gut glucose absorption in vivo. Chronically catheterized (femoral artery, portal vein, inferior vena cava, and proximal and distal duodenum) and instrumented (Doppler flow probe on portal vein) insulin (INS, 2 mU.kg(-1).min(-1), n = 6) or saline (SAL, n = 5) infused dogs were studied during basal (30 minutes) and experimental (90 minutes) periods. Arterial and portal vein plasma insulin were 3.3- and 3.2-fold higher, respectively, throughout the study in INS compared to SAL. An intraduodenal glucose infusion of 8 mg.kg(-1).min(-1) was initiated at t = 0 minutes. At t = 20 and 80 minutes, a bolus of 3-O-[3H]methylglucose (MG) and L-[14C]glucose (L-GLC) was injected intraduodenally. Phloridzin, an inhibitor of the Na+ -dependent glucose transporter (SGLT1), was infused from t = 60 to 90 minutes in the presence of a peripheral isoglycemic clamp. Net gut glucose output (NGGO) was 5.2 +/- 0.6 and 4.6 +/- 0.8 mg.kg(-1).min(-1) in INS and SAL, respectively, from t = 20 to 60 minutes. Transporter-mediated absorption was 87% +/- 2% of NGGO in both INS and SAL. Passive gut glucose absorption was 13% +/- 2% of NGGO in both INS and SAL. Phloridzin-induced inhibition of transporter-mediated absorption completely abolished passive absorption of L-GLC in both groups. This study shows that under physiological conditions, a portal vein insulin infusion that results in circulating hyperinsulinemia does not increase either transporter-mediated or passive absorption of an intraduodenal glucose load.     

Laryngeal muscle and diaphragmatic activities in conscious dog pups

A new chronic animal preparation (i.e. puppies) for the study of respiratory laryngeal muscle activity is described. Posterior cricoarytenoid (PCA), thyroarytenoid (TA) and diaphragmatic activities were monitored in conscious puppies (13-43 days old) using chronically implanted electrodes. PCA activity was prominent during inspiration in all behavioral states. TA and post-inspiratory diaphragmatic activities were consistently high during expiration in quiet wakefulness and quiet sleep, but expiratory TA activity decreased dramatically and post-inspiratory diaphragmatic activity became highly variable in rapid eye movement sleep. There was also a tendency for these expiratory muscle activities to become less pronounced with increasing postnatal age. When a chronically implanted, low resistance T-tube tracheal cannula was suddenly opened, thus effectively eliminating the effects of upper airway resistance on airflow, respiratory frequency dramatically increased. These results indicate that the puppy exhibits muscle activities consistent with the maintenance of an elevated end-expiratory lung volume achieved by the retardation of expiratory airflow using the larynx and diaphragm.     

Net hepatic lactate balance following mixed meal feeding in the four-day fasted conscious dog

The present experiments were undertaken to determine whether four days of fasting and marked hepatic glycogen depletion would alter the effect of mixed meal feeding on net hepatic lactate balance in the conscious dog. Dogs were fasted for four days and were then fed a mixed meal over a ten-minute period. Net hepatic glucose and lactate balance were monitored for the next eight hours using the A-V difference technique. The arterial plasma glucose level rose to a maximum of 121 +/- 3 mg/dL three hours after feeding and then decreased. Net hepatic glucose output declined to 0.44 +/- 0.44 mg/kg/min but the liver never became a net consumer of glucose. The arterial blood lactate level rose from 678 +/- 71 to 1000 +/- 158 mumol/L as the liver switched from net lactate uptake (12.2 +/- 2.0 mumol/kg/min) to net lactate production (4.3 +/- 1.7 mumol/kg/min). Over the course of the eight-hour postprandial period 25 g of glycogen were deposited in the liver. The net hepatic uptake of the gluconeogenic amino acids rose from 6.1 +/- 1.2 mumol/kg/min to a peak of 15.4 +/- 4.3 mumol/kg/min one hour after feeding. Net hepatic uptake of glycerol fell from 3.0 +/- 0.3 mumol/kg/min to an average of 1.5 +/- 0.4 mumol/kg/min. The plasma insulin level increased from 13 +/- 2 microU/mL at 3.5 hours and fell to 32 +/- 7 microU/mL by 8 hours. The plasma glucagon level rose from 22 +/- 3 pg/mL to 93 +/- 12 pg/mL 1.5 hours after feeding and fell to 68 +/- 6 pg/mL 8 hours after feeding.(ABSTRACT TRUNCATED AT 250 WORDS)     

Physiological hyperinsulinemia stimulates p70(S6k) phosphorylation in human skeletal muscle

Using tracer methods, insulin stimulates muscle protein synthesis in vitro, an effect not seen in vivo with physiological insulin concentrations in adult animals or humans. To examine the action of physiological hyperinsulinemia on protein synthesis using a tracer-independent method in vivo and identify possible explanations for this discrepancy, we measured the phosphorylation of ribosomal protein S6 kinase (P70(S6k)) and eIF4E-binding protein (eIF4E-BP1), two key proteins that regulate messenger ribonucleic acid translation and protein synthesis. Postabsorptive healthy adults received either a 2-h insulin infusion (1 mU/min.kg; euglycemic insulin clamp; n = 6) or a 2-h saline infusion (n = 5). Vastus lateralis muscle was biopsied at baseline and at the end of the infusion period. Phosphorylation of P70(S6k) and eIF4E-BP1 was quantified on Western blots after SDS-PAGE. Physiological increments in plasma insulin (42 +/- 13 to 366 +/- 36 pmol/L; P: = 0.0002) significantly increased p70(S6k) (P: < 0.01), but did not affect eIF4E-BP1 phosphorylation in muscle. Plasma insulin declined slightly during saline infusion (P: = 0.04), and there was no change in the phosphorylation of either p70(S6k) or eIF4E-BP1. These findings indicate an important role of physiological hyperinsulinemia in the regulation of p70(S6k) in human muscle. This finding is consistent with a potential role for insulin in regulating the synthesis of that subset of proteins involved in ribosomal function. The failure to enhance the phosphorylation of eIF4E-BP1 may in part explain the lack of a stimulatory effect of physiological hyperinsulinemia on bulk protein synthesis in skeletal muscle in vivo.     

Skeletal muscle is a major site of lactate uptake and release during hyperinsulinemia.

During conditions of increased glucose disposal, plasma lactate concentrations increase due to an increase in plasma lactate appearance. The tissue sites of the elevated lactate production are controversial. Although skeletal muscle would be a logical source of this lactate, studies using the limb net balance technique have failed to demonstrate a major change in net lactate output when plasma glucose disposal is increased. Because the limb balance technique underestimates production of a substrate when the limb not only produces but also consumes that substrate, we infused 3-14C-lactate basally and during a hyperinsulinemic euglycemic clamp in seven normal volunteers to determine plasma lactate appearance, forearm lactate fractional extraction, and forearm lactate uptake and release. After 3 hours of hyperinsulinemia, glucose and lactate turnovers increased from basal values of 11.8 +/- 0.13 and 12.2 +/- 0.59 to 32.6 +/- 3.4 and 16.5 +/- 1.07 mumol/(min.kg), accompanied by an increase in plasma lactate from 0.88 +/- 0.07 to 1.16 +/- 0.09 mmol/L (P less than .05). Forearm lactate extraction increased from 27% +/- 2% to 38% +/- 2% (P less than .001), resulting in an increase in forearm lactate uptake from 0.65 +/- 0.09 to 1.18 +/- 0.08 mumol/(min.100 mL tissue) (P less than .001). Although forearm lactate net output decreased during hyperinsulinemia, forearm lactate production increased from 1.04 +/- 0.12 basally to 1.69 +/- 0.13 mumol/(min.100 mL). When forearm data was extrapolated to whole body, muscle could account for 41% +/- 4% of systemic lactate appearance basally and 45% +/- 4% during hyperinsulinemia.(ABSTRACT TRUNCATED AT 250 WORDS)     

Deleterious effects of increased heart rate on infarct size in the conscious dog

This study was designed to determine whether augmentation of heart rate influences infarct size after coronary occlusion in unanesthetized dogs, some of which had experimentally induced atrioventricular (A-V) block. Coronary occlusion was produced in 27 conscious dogs by constriction of an externalized coronary arterial snare placed 3 to 5 days earlier. Heart rate was augmented at selected intervals after coronary occlusion by ventricular pacing or administration of isoproterenol or atropine, and left atrial pressure was monitored in selected animals in each group. Infarct size was determined from analysis of serial serum creatine phosphokinase (CPK) changes and verified by myocardial CPK analysis. After coronary occlusion alone, release of CPK from myocardial tissue ceased within approximately 14 hours, and calculated infarct size therefore became constant. Augmentation of heart rate at that time by ventricular pacing or administration of isoproterenol or atropine led to extension of infarction with additional myocardial necrosis averaging 40, 72 and 40 percent, respectively, of the initial infarct size in the 3 groups. Stepwise increments of heart rate led to progressively smaller increments in infarct size, and even modest increases in heart rate (from 60 to 90 beats/min) in dogs with A-V block led to marked increases in infarct size averaging 73 percent ± 17 (SE, no. = 4). Augmentation of heart rate with isoproterenol as late as 72 hours after coronary occlusion led to marked extension of infarction. Thus, acceleration of heart rate after coronary occlusion consistently and markedly increased the extent of myocardial necrosis.     

Intraportal hyperinsulinemia decreases insulin-stimulated glucose uptake in the dog

Hyperinsulinemia and insulin resistance are commonly seen in obese and non-insulin-dependent diabetes mellitis (NIDDM) patients. While it is known that chronic exposure to severe hyperinsulinemia can lead to an insulin-resistant state and mild hyperinsulinemia for rather short durations (20 to 40 hours) and can also lead to insulin resistance, it is less clear whether mild hyperinsulinemia for a more prolonged duration can lead to insulin resistance. In the present study we determined the effects of chronic (28 days) exposure to mild hyperinsulinemia on insulin-stimulated glucose use. Chronic hyperinsulinemia was produced by an intraportal infusion of porcine insulin (425 microU/kg/min), which raised the basal peripheral insulin levels by approximately 50%. Insulin responsiveness was assessed using the euglycemic hyperinsulinemic clamp (2 mU/kg/min) in dogs before the induction of chronic hyperinsulinemia (day 0), after 28 days of hyperinsulinemia (day 28), and 28 days after discontinuation of the chronic insulin infusion (day 56). The amount of glucose (M) required to maintain euglycemia during the euglycemic hyperinsulinemic clamp was decreased (relative to day 0) 39% +/- 3% on day 28 and 18% +/- 3% on day 56 (P less than .05). In control animals that received a chronic infusion of saline for the 28-day period the glucose infusion rate (M) was not changed significantly (decreasing 2% +/- 5% and 5% +/- 10% on days 28 and 56, respectively). In conclusion insulin resistance can be produced by a mild hypersecretion of insulin and discontinuation of the chronic insulin infusion tends to reverse the resistance.     

Oxidation of palmitate and lactate by beating myocytes isolated from adult dog heart.

Myocytes were isolated from hearts of adult dogs by repeated digestion with collagenase and hyaluronidase in Ca²⁺-free phosphate buffer followed by centrifugation in the presence of Ficoll. The isolated myocytes were free of other tissue elements and were contracting autorhythmically. The oxidation of palmitate and lactate were studied by incubating cell preparation with [1-¹⁴C]palmitate or [U-¹⁴C]lactate either in the presence or absence of CaCl₂ for 30 min at 37°C. [¹⁴C]O₂ was collected by KOH-soaked filter paper. Cardiac myocytes oxidized 0.1 to 0.18 nmol/mg protein min of palmitate and 1.5 to 2.2 nmol/mg protein min of lactate. The oxidation was linear for 75 min. Maximal rate of oxidation was obtained at substrate concentrations of 0.2 mm palmitate or 5.0 mm lactate. Adenine or pyridine nucleotides had no effect on the oxidation of these substrates. Palmitate oxidation was enhanced by carnitine and also by an increase in the molar ratio of palmitate/albumin, but was depressed (15 to 50%) by CaCl₂ (0.025 to 1.0 mm). CaCl₂ (0.05 to 1.0 mm) increased the rate of lactate oxidation by 50 to 68%. Lactate oxidation was also enhanced (68 to 87%) in the presence of palmitate (0.4 to 0.8 mm). These findings demonstrate the suitability of isolated adult heart myocytes for the study of cardiac metabolism.     

Mechanisms of supersensitivity to sympathomimetic amines in the chronically denervated heart of the conscious dog

Mechanisms of denervation supersensitivity to sympathomimetic amines were studied in conscious animals. Norepinephrine, 0.1 micrograms/kg, increased left ventricular dP/dt significantly more (4208 +/- 828 mm Hg/sec) in dogs with cardiac denervation than in intact dogs (1029 +/- 280 mm Hg/sec), P less than 0.01, whereas responses to isoproterenol were similar in both groups. Denervation supersensitivity to isoproterenol could be demonstrated only after opposing reflex effects were blocked. After ganglionic blockade, norepinephrine still induced 2- to 3-fold greater increases in left ventricular dP/dt and 3- to 7-fold greater increases in heart rate in cardiac-denervated dogs, whereas isoproterenol and prenalterol, not taken up by presynaptic nerve endings, elicited only 40%-50% greater increases in left ventricular dP/dt and heart rate in dogs with cardiac denervation. The density of beta-adrenergic receptors [( 3H]dihydroalprenolol) was elevated (P less than 0.01) in denervated left ventricles (105 +/- 6.9 fmol/mg protein, n = 8) compared to normal left ventricles (70 +/- 6.3 fmol/mg protein, n = 12). This was accompanied by enhanced isoproterenol-mediated adenylate cyclase activity. However, muscarinic cholinergic receptor density, [( 3H]quinuclidinyl benzilate), decreased from control levels of 251 +/- 11 fmol/mg protein (n = 7) to 193 +/- 14 fmol/mg protein (n = 6). Thus, chronic cardiac denervation results in upregulation of the beta-adrenergic receptor and down-regulation of the muscarinic receptor. The increased beta-adrenergic receptor density and adenylate cyclase stimulation correlated well with the amount of denervation supersensitivity to isoproterenol and prenalterol, but accounted for only a minor fraction of denervation supersensitivity to norepinephrine. The major mechanism of denervation supersensitivity to norepinephrine appears to involve lack of the norepinephrine reuptake.     

Physiologic determinants of biliary calcium secretion in the dog

Studies were carried out to define the origins and physiologic determinants of biliary calcium secretion in the dog. Canalicular bile flow was varied by infusions of several bile acids or a canalicular choleretic agent, SC2644. Three natural bile acids (cholyltaurine, chenodeoxycholyltaurine, and ursodeoxycholyltaurine) that have a low critical micellar concentration and a synthetic hydroxy-oxo bile acid (12-dehydrocholyltaurine) with a much higher critical micellar concentration were used. Calcium output, bile acid output, and bile flow were determined; canalicular bile flow was assessed by measurement of erythritol clearance. Calcium output increased linearly with bile acid output for all three natural bile acids (r = 0.94; 0.026-0.035 mumol delta calcium/mumol delta bile acid), but chenodeoxycholyltaurine and ursodeoxycholyltaurine infusions induced a slightly greater secretion of calcium for a given bile acid output and bile flow than cholyltaurine. Calcium output also increased linearly with bile flow, whether bile flow was induced by the canalicular choleretic SC2644 (r = 0.93; 0.0018 mumol delta calcium/microliter delta water) or the hydroxy-oxo bile acid (r = 0.99; 0.0021 mumol delta calcium/microliter delta water). Because the calcium output induced by the three micelle-forming bile acids was greater than that which could be explained by the induced increase in bile flow per se, it was deduced that canalicular calcium has two origins: a micellar component that is dependent on the presence of micelles or micelle-forming bile acids and an osmotic component that is dependent on osmotic forces, e.g., caused by secretion induced by SC2644 or bile acid molecules and possibly bile acid micelles. A small bile acid-independent output of calcium was also detected and should contribute to the osmotic fraction. To determine whether ductular modification of bile flow influenced biliary calcium secretion, ductular secretion was induced by secretin and ductular absorption was induced by somatostatin. Changes in calcium output followed water movements, increasing with ductular secretion and decreasing with ductular absorption. The relationship between changes in ductular bile flow and calcium output was identical during induced ductular secretion (r = 0.83; 0.0016 mumol delta calcium/microliter delta water) or induced ductular absorption (r = 0.91; 0.0019 mumol delta calcium/microliter delta water). Thus calcium movements in relation to water movements were identical for the osmotic component of canalicular bile and the ductular component of bile.(ABSTRACT TRUNCATED AT 400 WORDS)     

Proglumide stimulates basal pancreatic secretion in the conscious rat

The effect of proglumide, a glutaramic acid derivative, on pancreatic secretion was examined in vivo in the conscious rat with and without the return of bile-pancreatic juice (BPJ) to the intestine. Intravenous infusion of both 300 and 60 mg/kg proglumide significantly decreased protein output in a dose-related manner during BPJ diversion, but did not completely abolish the pancreatic hypersecretory response to BPJ diversion. Conversely, during basal secretion with BPJ being returned to the intestine, 300 mg/kg/h of proglumide increased the protein output. Dibutyryl cyclic GMP infused simultaneously with proglumide did not abolish the stimulatory effect of proglumide on basal secretion. It was concluded that proglumide inhibits pancreatic protein output during stimulated secretion by means of the luminal feedback mechanism but increases protein output during basal (BPJ returned) secretion in the conscious rat.     

Regulation of large coronary arteries by beta-adrenergic mechanisms in the conscious dog

We examined, in conscious dogs, the effects of beta-adrenergic stimulation on measurements of left circumflex coronary arterial diameter and blood flow and on calculations of late diastolic coronary resistance (LDCR) and left circumflex coronary internal cross-sectional area (CSA). Isoproterenol (0.1 microgram/kg) initially decreased mean arterial pressure by 25 +/- 2% (mean +/- SEM), and LDCR by 62 +/- 4%, and increased heart rate by 82 +/- 10%, left ventricular (LV) dP/dt by 79 +/- 12%, and mean coronary blood flow by 85 +/- 5%, while CSA rose slightly. The peak effects on CSA (24 +/- 2%) occurred later, along with decreases in mean arterial pressure (7.4 +/- 1.0%) and LDCR (25 +/-5.3%) and increases in coronary blood flow (14 +/- 2%), LV dP/dt (12 +/- 3%), and heart rate (24 +/- 4%). Pirbuterol (1.0 microgram/kg) induced changes that were qualitatively similar to those induced by isoproterenol. Prenalterol (20 micrograms/kg), a cardioselective beta 1-adrenergic receptor agonist, did not affect mean arterial pressure, but increased heart rate by 40 +/- 5%, LV dP/dt by 72 +/- 10%, mean coronary blood flow by 34 +/- 11%, and CSA by 26 +/- 3%, and decreased LDCR by 29 +/- 5+. Isoproterenol and pirbuterol, but not prenalterol, increased coronary sinus O2 content and decreased A-VO2 difference. After beta 1-adrenergic receptor blockade with atenolol (1 mg/kg), prenalterol no longer induced significant effects, whereas isoproterenol and pirbuterol decreased mean arterial pressure similarly to what was observed prior to blockade, but did not increase LV dP/dt, and induced attenuated increases in mean coronary blood flow, CSA, and decreases in LDCR. Thus, in the intact, conscious animal, large coronary arteries are regulated by beta-adrenergic mechanisms. Surprisingly, a major fraction of large coronary arterial dilation appeared to be either directly or indirectly due to beta 1-adrenergic receptor mechanisms, although beta 2-adrenergic effects were also significant.     

Salvage of ischemic myocardium by ibuprofen during infarction in the conscious dog

Because nonsteroidal anti-inflammatory drugs differ in potency and degree of prostaglandin inhibition, they may have different effects on ischemic myocardium. The effect of ibuprofen, an agent of this type, on myocardial infarct size was measured 2 days after occlusion of the left circumflex coronary artery in conscious dogs. Treatment was randomized in dogs after occlusion: Intravenous infusions of ibuprofen (6.25 mg/kg per hour) were administered to 13 dogs and saline solution (0.9 percent) to 13 control dogs over a period of 6 hours. The boundary of the occluded coronary bed, or anatomic risk region, was defined by postmortem coronary arteriography. Masses of infarct and occluded bed were measured by planimetry of weighed transverse sections of the left ventricle. Ibuprofen decreased infarct size compared with that in control dogs, both as percent of the left ventricle (mean ± standard error of the mean 7.5 ± 1.4 versus 15.2 ± 3.1, p < 0.05) and as percent of the occluded bed (16.3 ± 2.3 versus 38.6 ± 5.7, p < 0.005). Ibuprofen also altered (p < 0.001) the relation between the size of the infarct and the size of the occluded bed, so that hearts with occluded beds of similar size had smaller infarcts than those of control dogs. Morphologically, ibuprofen salvaged myocardium in both lateral and epicardial regions of the occluded bed. Changes in arterial pressure, left atrial pressure and heart rate were similar in the two groups. Changes in regional myocardial blood flow measured with 7 to 9 μm radioactive microspheres were also similar in both groups, with an increase in flow to infarcted regions and borders between 20 seconds to 15 minutes after occlusion, but no further change from 15 minutes to 6 hours. Thus, protection of ischemic myocardium by ibuprofen was not due to changes in collateral flow or myocardial oxygen demands, suggesting that cellular and metabolic effects might be important.