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.

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The hypothesis tested was that the hemodynamic consequence of partial hepatectomy (PHX) triggers the cascade of events that leads to liver regeneration. After PHX, all the portal flow must go through the remaining vascular bed, thus producing increased shear stress and release of nitric oxide (NO), which then initiates the next stages of the regeneration process. As an index of triggering of the regeneration cascade, we used an in vitro bioassay detecting the appearance of proliferating factors (PFs; various growth factors, cytokines, and hormones) in plasma 4 h after two-thirds PHX in rats. PF levels, assessed using proliferation of cultured hepatocytes, were elevated in two-thirds PHX rats, fully blocked by the NO synthase inhibitor N(G)-nitro-L-arginine methyl ester (L-NAME), and restored by L-arginine. L-NAME inhibited liver weight restoration at 48 h but resulted in high mortality. L-NAME lacked toxic effects in non-PHX rats. NO was directly antiproliferative on cultured cells, suggesting that the proliferative effect of NO in vivo was secondary to the activation of other proliferative stimuli. The data support the hypothesis that vascular shear stress induced release of NO following PHX serves as a primary trigger to initiate the regeneration process.

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Data are reviewed that are consistent with the following working hypothesis that proposes a novel mechanism regulating insulin sensitivity, which when nonfunctional, leads to severe insulin resistance. Postprandial elevation in insulin levels activates a hepatic parasympathetic reflex release of a putative hepatic insulin-sensitizing substance (HISS), which activates glucose uptake at skeletal muscle. Insulin causes HISS release in fed but not fasted animals. The reflex is mediated by acetylcholine and involves release of nitric oxide in the liver. Interruption of the release of HISS is achieved by surgical denervation of the anterior hepatic nerve plexus, muscarinic receptor blockade, or nitric oxide synthase antagonism and leads to immediate severe insulin resistance. The nitric oxide donor, SIN-1, reverses L-NAME-induced insulin resistance. Denervation-induced insulin resistance is reversed by intraportal but not intravenous administration of acetylcholine or SIN-1. Liver disease is often associated with insulin resistance; the bile duct ligation model of liver disease results in parasympathetic neuropathy and insulin resistance that is reversed by intraportal acetylcholine. Possible relevance of this HISS-dependent control of insulin action to insulin resistance in diabetes, liver disease, and obesity is discussed.

We tested the hypothesis that nerve-induced constriction is modulated by nitric oxide only if shear stress is allowed to increase. Shear stress is the effect of moving fluid producing distortion of endothelial cells. Blood flow to the superior mesenteric artery in anesthetized cats was controlled using a perfusion circuit. Shear stress was increased by holding blood flow constant during vasoconstriction induced by nerve stimulation (2 and 10 Hz) or infusion of norepinephrine (0.5 microgram.kg-1.min-1). The increase in perfusion pressure in response to nerve stimulation when shear stress was allowed to rise was 24.8 +/- 4.7 mmHg (1 mmHg = 133.3 Pa) (2 Hz) and 100.0 +/- 17.8 mmHg (10 Hz). After NO synthase blockade using NG-nitro-L-arginine methyl ester (L-NAME) (2.5 mg/kg i.v.) potentiation occurred (74.0 +/- 21.6 mmHg at 2 Hz and 151.9 +/- 14.1 mmHg at 10 Hz). Potentiation was reversed after L-arginine (75 mg/kg i.v.). When shear stress was held constant, L-NAME did not affect the responses to nerve stimulation. In contrast, the vasoconstriction to norepinephrine was not affected by L-NAME. The data are compatible with the hypothesis that if vasoconstriction leads to increased shear stress, release of nitric oxide produces vasodilation subsequent to decreased sympathetic nerve activity. The absence of a similar effect with norepinephrine suggests that the shear-dependent release of nitric oxide at the site of constriction does not act directly on the vascular smooth muscle in the small resistance vessels.

A preparation is described by which hepatic arterial blood flow and portal venous blood flow can be accurately and continuously measured while simultaneously providing a method by which multiple blood samples can be taken from the hepatic artery, portal vein, and hepatic vein without disrupting hepatic hemodynamics or causing hemodilution. By this means hepatic uptake or release of blood-borne substances can be measured in situ and correlated with hemodynamic parameters. In 13 splenectomized cats, oxygen uptake by the denervated liver was 4.5 +/- 0.3 ml . min-1. 100 g-1 of tissue, representing 54% of total oxygen removed by the splanchnic bed. The hepatic hemodynamics determined by this method are similar to those reported by others in vivo and the metabolic state of the liver remained stable for at least 2 h during which an average of 29 blood samples were taken. Advantages of this preparation over other methods of obtaining similar data are discussed.