Dallas J. Legare

Quantitation of the Hepatic Arterial Buffer Response to Graded Changes in Portal Blood Flow

Hepatic arterial blood flow changes inversely in response to altered portal blood flow. The hepatic arterial capacity to buffer portal flow changes was studied over a wide range of portal flow with arterial pressure held steady (the active buffer response) or uncontrolled. The active component of the buffer response led to nearly full dilation of the hepatic artery at low portal flows as shown by inability to dilate further in response to adenosine infusion; at high portal flows the hepatic artery was nearly fully constricted as shown by lack of further constriction to norepinephrine. With pressure uncontrolled, active and passive effects combined to produce an increased compensation with similar efficiency (44% +/- 4%) over the full range of portal blood flows. Thus, although the active component of the hepatic arterial buffer response becomes less efficient at very high and low portal flows, the combination of active and passive effects leads to a larger buffer capacity which is equally efficient over a wide range of portal blood flow changes.

Rapid insulin sensitivity test (RIST)

A rapid insulin sensitivity test (RIST) was recently introduced to assess insulin action in vivo (H. Xie, L. Zhu, Y.L. Zhang, D.J. Legare, and W.W. Lautt. J. Pharmacol. Toxicol. Methods, 35: 77-82. 1996). This technical report describes the current recommended standard operating procedure for the use of the RIST in rats based upon additional experience with approximately 100 tests. We describe the manufacture and use of an arterial-venous shunt that allows rapid multiple arterial samples and intravenous administration of drugs. The RIST procedure involves determination of a stable arterial glucose baseline to define the ideal euglycemic level to be maintained following a 5-min infusion of insulin, with the RIST index being the amount of glucose required to be infused to maintain euglycemia over the test period. Insulin administration by a 5-min infusion is preferable to a 30-s bolus administration. No significant difference was determined between the use of Toronto pork-beef or human insulin. Four consecutive RISTs were carried out in the same animal over 4-5 h with no tendency for change with time. The RIST index is sufficiently sensitive and reproducible to permit establishment of insulin dose-response curves and interference of insulin action by elimination of hepatic parasympathetic nerves, using atropine. This technical report provides the current recommended standard operating procedure for the RIST.

HISS-dependent insulin resistance (HDIR) in aged rats is associated with adiposity, progresses to syndrome X, and is attenuated by a unique antioxidant cocktail

The hypotheses were: HISS-dependent insulin resistance (HDIR) accounts for insulin resistance that occurs with aging; HDIR is the initiating metabolic defect that leads progressively to type 2 diabetes and the metabolic syndrome; a synergistic antioxidant cocktail in chow confers protection against HDIR, subsequent symptoms of diabetes, and the metabolic syndrome. Male Sprague Dawley rats were tested at 9, 26, and 52 weeks to determine their dynamic response to insulin, the HISS (hepatic insulin sensitizing substance)-dependent component of insulin action, and the HISS-independent (direct) insulin action using a dynamic insulin sensitivity test. In young rats, the HISS component accounted for 52.3+/-2.1% of the response to a bolus of insulin (50mU/kg) which decreased to 29.8+/-3.4% at 6 months and 17.0+/-2.7% at 12 months. HISS action correlated negatively with whole body adiposity and all regional fat depots (r(2) = 0.67-0.87). The antioxidants (vitamin C, vitamin E, and S-adenosylmethionine) conferred protection of HISS action, fat mass at all sites, blood pressure, postprandial insulin and glucose. Data are consistent with the hypotheses. Early detection and therapy directed towards treatment of HDIR offers a novel therapeutic target.

Meal-induced insulin sensitization in conscious and anaesthetized rat models comparing liquid mixed meal with glucose and sucrose

We have recently shown that meal-induced insulin sensitization (MIS) occurs after feeding and decreases progressively to insignificance after 24 h of fasting and is caused by action of a hepatic insulin sensitizing substance (HISS). In order to carry out quantitative studies of MIS, some standardized meal intake is required. Our objective was to establish animal models to be tested in both the conscious and anaesthetized state using intragastric injection of liquid meals in order to quantify MIS. Insulin sensitivity was assessed before and 90 min after the meal using the rapid insulin sensitivity test (RIST) which is a transient euglycaemic clamp. Rats tested in the conscious state were instrumented under anaesthesia 6-9 d prior to testing with catheters in the carotid artery, jugular vein and stomach. Meals, injected into the stomach, consisted of a liquid mixed meal, sucrose, glucose or water. The glucose sequestration in response to insulin increased by 90 % and 61 % following the liquid mixed meal (10 ml/kg) in conscious and anaesthetized rats, respectively. Glucose, sucrose and water did not effectively activate MIS. MIS was completely reversed in the conscious model by atropine and completely prevented from developing in the anaesthetized model that had previously undergone hepatic denervation. Gastric administration of a liquid mixed meal but not glucose or sucrose is capable of activating MIS for purposes of mechanistic studies and quantification of the MIS process. The feeding signal is mediated by the hepatic parasympathetic nerves.

Insulin sensitivity tested with a modified euglycemic technique in cats and rats.

A new insulin sensitivity test (IST) is described using a modified euglycemic clamp in cats and rats. The IST uses the amount of glucose required to be infused to maintain euglycemia over a 30-min period in rats and 60 min in cats following a bolus administration of insulin as the index of insulin sensitivity. Glucose levels are determined at short time intervals (2-5 min), and variable glucose infusion is used to hold glucose levels within a few percentage points of the basal pre-test glucose level. A new blood sampling procedure is described that allows each IST to be carried out using a total of only 0.5 mL of blood. The IST is sensitive and allows clear insulin dose effects to be demonstrated with 100 mU/kg requiring 355.0 +/- 14.3 mg/kg over 30 min and 50 mU/kg requiring 198.7 +/- 11.1 mg/kg. Five consecutive tests were reproducibly carried out (%CV = 3.0 +/- 0.5) over a 12-hr period in the cat with insulin, glucagon, and glucose levels remaining stable prior to each IST. Glucagon and norepinephrine plasma concentrations do not change significantly during the IST. The IST is sufficiently sensitive to allow demonstration of dose-response relationships for atropine-induced insulin resistance. The IST is thus sensitive, reproducible, and able to demonstrate acute insulin resistance in anesthetized cats and rats. The test is demonstrated in fed (rats) and fasted (cats) state.

Effect of hepatic nerves, norepinephrine, angiotensin, and elevated central venous pressure on postsinusoidal resistance sites and intrahepatic pressures in cats

Portal venous pressure was controlled by resistance localized to specific sites in hepatic lobar veins in cats. All of the pressure drop from the portal vein to the vena cava occurred across postsinusoidal vessels; portal pressure, lobar venous pressure, and, therefore, sinusoidal pressure were not significantly different. Norepinephrine and angiotensin infusions (intraportal) caused elevation in portal pressure due to constriction of hepatic venous resistance sites as well as some constriction of presinusoidal (portal or sinusoidal) resistance sites. At low doses of norepinephrine presinusoidal constriction dominated whereas at higher doses the postsinusoidal constriction increased proportionately more. Hepatic nerve stimulation produced a similar response measured at an early time (1 min), but by 3 min the presinusoidal constriction showed complete escape so that elevated portal pressure was entirely due to hepatic venous constriction. The same site that provided basal vascular resistance also provided the increased hepatic venous resistance with nerve stimulation and infusion of angiotensin and norepinephrine. Rapid elevation of central venous pressure (CVP) caused elevated sinusoidal pressure. At high CVP (16 mm Hg), 75% of a rise in CVP was transmitted whereas at normal CVP (less than 4.5 mm Hg) less than 20% transmission occurred. The presence of a high resistance in the hepatic veins protected intrahepatic pressure from the effects of normal fluctuation of CVP.

Attenuation of age- and sucrose-induced insulin resistance and syndrome X by a synergistic antioxidant cocktail: the AMIS syndrome and HISS hypothesis.

Absence of meal-induced insulin sensitization (AMIS) results in a predictable progression of dysfunctions, including postprandial hyperglycemia, compensatory hyperinsulinemia, resultant hyperlipidemia, increased oxidative stress, and obesity, progressing to syndrome X and diabetes. To one year of age, rats show a slow development of AMIS, but this can be potentiated by addition of a low-dose sucrose supplement to the diet. Provision of a synergistic antioxidant cocktail consisting of S-adenosylmethionine, vitamin E, and vitamin C (Samec) attenuates the rate and extent of development of AMIS in both normal aging animals and in aging animals on the sucrose diet. Adiposity, assessed from weighed regional fat masses and from bioelectrical impedance to estimate whole-body adiposity, correlated strongly with AMIS (r2 = 0.7-0.8). Rats given the sucrose supplement had accelerated AMIS and developed fasting hyperinsulinemia and postprandial hyperglycemia, hyperlipidemia, hyperinsulinemia, and adiposity. Samec completely compensated for the negative impact of this sucrose supplement and attenuated development of the associated dysfunctions. AMIS is explained by the HISS (hepatic insulin-sensitizing substance) hypothesis, which is outlined in the paper.

High-fat diet results in postprandial insulin resistance that involves parasympathetic dysfunction

Different diets have distinct impacts on glucose homoeostasis, for which insulin sensitivity (IS) after a meal (postprandial IS) is highly relevant. Postprandial IS depends upon hepatic parasympathetic activation and glutathione content elevation. We tested the hypothesis that postprandial IS is compromised in high-fat diet (HFD)-induced obesity. Sprague-Dawley rats were fed a standard diet (STD, n 10), 1-week HFD (n 9) or 4-week HFD (n 8). IS was tested in postprandial state using the rapid IS test (RIST) before and after the blockade of the parasympathetic nerves (atropine, 1 mg/kg); parasympathetic-dependent IS was obtained from the difference between control and post-atropine RIST. Fasting IS was also assessed in the STD-fed rats (n 4) and 4-week HFD-fed rats (n 3) using the RIST. Whole-body fat and regional fat pads were heavier in the 1-week HFD-fed rats (79.8 (SE 7.9) and 23.7 (SE 1.0) g, respectively) or 4-week HFD-fed rats (106.5 (SE 6.1) and 30.1 (SE 1.4) g, respectively) than in the STD-fed rats (32.5 (SE 3.7) and 13.7 (SE 1.0) g, respectively; P < 0.001). Fasted-state IS was similar between the groups studied. Postprandial IS was higher in the STD-fed rats (185.8 (SE 5.6) mg glucose/kg body weight (bw)) than in both the 1-week HFD-fed rats (108.8 (SE 2.9) mg glucose/kg bw; P < 0.001) and 4-week HFD-fed rats (69.3 (SE 2.6) mg glucose/kg bw; P < 0.001). Parasympathetic-dependent IS was impaired in both HFD-fed groups (STD, 108.9 (SE 3.9) mg glucose/kg bw; 1-week HFD, 38.6 (SE 4.2) mg glucose/kg bw; 4-week HFD, 5.4 (SE 1.7) mg glucose/kg bw; P < 0.001). Total (postprandial) and parasympathetic-dependent IS correlated negatively with whole-body fat (R² 0.81 and 0.87) and regional adiposity (R² 0.85 and 0.79). In conclusion, fat accumulation induced by HFD is associated with postprandial insulin resistance, but not with fasting insulin resistance. HFD-associated postprandial insulin resistance is largely mediated by impairment of parasympathetic-dependent insulin action, which correlates with adiposity.

Absence of meal-induced insulin sensitization (AMIS) in aging rats is associated with cardiac dysfunction that is protected by antioxidants

We have previously demonstrated that progressive development of absence of meal-induced insulin sensitization (AMIS) leads to postprandial hyperglycemia, compensatory hyperinsulinemia, resultant hyperlipidemia, increased oxidative stress, and obesity, progressing to syndrome X in aging rats. The present study tested the hypothesis that progressive development of AMIS in aging rats further resulted in deterioration in cardiac performance. Anesthetized male Sprague-Dawley rats were tested at 9, 26, and 52 wk to determine their dynamic response to insulin and cardiac function. Dynamic insulin sensitivity was determined before and after atropine to quantitate hepatic insulin sensitizing substance (HISS)-dependent and -independent insulin action. Cardiac performance was evaluated using a Millar pressure-volume conductance catheter system. AMIS developed with age, as demonstrated by significant decrease in HISS-dependent insulin action, and this syndrome was increased by sucrose supplementation and inhibited by the antioxidant treatment. Associated with progressive development of AMIS, aging rats showed impaired cardiac performance, including the reduction in cardiac index, heart rate, dP/dt(max), dP/dt(min), ejection fraction and decreased slope of left ventricular end-systolic pressure-volume relationship, and increased relaxation time constant of left ventricular pressure as well as increased left ventricular end-diastolic pressure. Total peripheral vascular resistance also increased with age. Sucrose supplementation and antioxidant treatment, respectively, potentiated and attenuated cardiac dysfunction associated with age. In addition, poor cardiac performance correlated closely with the development of AMIS. These results indicate that AMIS is the first metabolic defect that leads to homeostatic disturbances and dysfunctions, including cardiovascular diseases.

Insulin sensitization by voluntary exercise in aging rats is mediated through hepatic insulin sensitizing substance (HISS)

BACKGROUND Food in the upper gastrointestinal tract potentiates the glucose uptake response to insulin. Meal-induced insulin sensitization (MIS) occurs as a result of insulin-mediated release of hepatic insulin sensitizing substance (HISS) that increases glucose uptake in peripheral tissues. HISS release decreases with age, and exercise causes metabolic improvements in aging, therefore it is important to analyze the effect of exercise on age-associated decline in HISS-action. OBJECTIVES The aim of this study is to test the hypothesis that improved insulin sensitivity, by voluntary running-wheel exercise in aging rats, is attained by preserving the HISS response. We also investigate the possible association of exercise-mediated beneficial changes in metabolic conditions and body composition with improved HISS-action. METHODS We measured insulin- and HISS-sensitivity in 9, 14 and 21-week old rats with/without exercise. Metabolic markers were also determined in age-matched control and exercised rats. RESULTS Exercise significantly improved HISS-dependent glucose uptake in all age groups. The direct action of insulin was minimally altered by age or exercise. Body composition and metabolic conditions were beneficially changed with exercise-induced improvements in the HISS response. CONCLUSION The therapeutic efficacy of voluntary exercise against insulin resistance in aging rats is achieved mainly through restoration of HISS-action.