Our aim was to characterize the hepatic-dependent and -independent components of whole-body insulin action in an obesity animal model, the Zucker rat (OZR). The results reported herein suggest that the IR observed in the OZR is due to the impairment of both of these components, which are decreased exactly to the same extent. The metabolic defect responsible for the severe IR observed in the OZR is located downstream from HPN stimulation and hepatic NO synthesis and seems to be common to both components of insulin action.
To assess the IS, we used the modified euglycemic clamp RIST because it is the test that better fits within the experimental protocol design for this study. It avoids induction of hypoglycemia and does not alter the circulating catecholamines or glucagon levels (14). Moreover, both insulinemia and glycemia always return to basal levels after each RIST (14). The RIST technique can be reproduced several times in each experiment (up to four consecutive RISTs), without causing significant interference with the animal's physiological conditions, while simultaneously maintaining the high sensitivity of the test (14). Furthermore, our results show that the RIST is a reproducible tool for measuring IS also in the Zucker rat model because there was no change between the first and second control RISTs (control experiments). Results obtained using the RIST are similar to those obtained using the insulin tolerance test, although somewhat different from those obtained with the hyperinsulinemic euglycemic clamp (15). This may be related to the vagal impairment induced by the hyperinsulinemic euglycemic clamp technique (16), making it hard to detect the hepatic-dependent component of insulin action, which requires hepatic parasympathetic stimulation. Most methods used to assess IS are based on the steady-state glucose and insulin concentrations achieved after an overnight fast. However, the hepatic-dependent component of peripheral insulin action is maximal in the postprandial state. By refeeding the animals after an overnight fast, as it was done, we ensured that hepatic-dependent insulin action was maximal at the time of the test.
IR in the Obese Rats (OZR)
Our results show that total peripheral IS is significantly diminished in OZR when compared with their lean litter mates (LZR). OZRs present a 70% to 75% deficit of IS compared with the LZR, which is in accordance with reports of marked IR in obese subjects, both rats and humans (12, 13, 17, 18, 19, 20). In the fasted state, the glucose disposal is due only to the insulin action per se (hepatic-independent component) because the hepatic pathway contribution for the overall insulin action is absent in this state. Therefore, because most studies concerning the assessment of the OZR IS in vivo were performed in the fasted state (17), they only show the impairment of the hepatic-independent component, which we also confirmed in the present report.
The skeletal muscle still represents the majority of glucose uptake in OZR, although the weight ratio of fat to skeletal muscle is increased in these animals (13), and because this tissue is primarily affected by IR in the OZR model (13, 18), abnormalities at this level will reflect in the overall insulin-stimulated glucose disposal. The deficiency in insulin action in OZR is also supported by several in vitro studies, in which multiple defects in the insulin-stimulated glucose uptake in the skeletal muscle of these animals have been described and can lead to the observed hepatic-independent IR. These reports include the impairment of insulin responsiveness due to a decreased binding to the plasma membrane (21, 22) and to postreceptor abnormalities. These include excessive serine/threonine phosphorylation of the insulin receptor (23), leading to interruption of the insulin signaling, or the impairment of phosphatidylinositol 3 kinase activity (24), which is associated with reduced glucose transporter (GLUT) 4 translocation (18, 25, 26, 27, 28).
As we mentioned above, the normal peripheral IS depends also on the action of the hepatic pathway that starts with the meal-induced activation of the HPNs, followed by stimulation of the hepatic M1 muscarinic receptors and NO synthesis. These events lead to the release of HISS, the liver-derived factor that acts selectively in the skeletal muscle, enhancing insulin action. The impairment of the hepatic pathway results in the decrease of total insulin action, as previously observed in several other animal models of IR, such as the spontaneously hypertensive rat, sucrose-fed rats, aging rats, adult offspring of fetal alcohol exposure, and liver disease induced by chronic bile duct ligation (6, 29, 30). In all these models, the impairment of the total IS was due only to a deficient hepatic-dependent component, whereas the hepatic-independent component remained unchanged (6, 29, 30). On the contrary, the OZR present an impairment of the hepatic-dependent component, which is accompanied by a proportional decrease of the hepatic-independent component of insulin action, as we report herein.
The parasympathetic dysfunction is usually associated with the obesity condition (9) and could explain the observed hepatic-dependent IR. However, although such parasympathetic dysfunction could contribute to the impairment of the hepatic-dependent insulin action in the obese animals, it fails to explain why the hepatic-independent component is also affected. Some authors have suggested that a decrease of the parasympathetic tone can lead to the impairment of insulin secretion (31), but this may be a controversial issue because the atropine-induced decrease in insulin secretion seems to be attenuated in obese subjects (31). Moreover, OZR have been described as hyperinsulinemic (13). On the other hand, even when what is evaluated is the insulin action through the administration of exogenous insulin, we observed an impaired glucose disposal, suggesting that the major defect in OZR does not seem to concern the insulin production, but rather the insulin peripheral action.
Hepatic NO synthesis is also an important intermediate step of the pathway initiated with the HPN stimulation and that culminates with the synthesis of HISS (hepatic-dependent pathway). However, impairment of the hepatic NO synthase activity is unlikely to account for the observed hepatic-dependent IR. Although NO synthase activity is known to be significantly altered in several tissues in the OZR, such as the gastric fundus, central nervous system, and skeletal muscle (10, 19), there are no reports of NO synthase activity deficiency in the liver of the OZR. Even if we extrapolate these studies and consider that the hepatic NO synthase activity is also decreased in the OZR, such impairment would affect only the hepatic-dependent component and not the hepatic-independent component, which we observed to be equally impaired.
The similar impairment of both hepatic-dependent and -independent components of insulin action in OZR suggests that the deficiency of insulin action lies downstream from insulin secretion, HPN stimulation, and NO synthesis, probably at a metabolic site common to hepatic-dependent and -independent components. Alternatively, insulin action may be impaired both in the ability to stimulate glucose uptake and to stimulate HISS release.
Impairment of Both Components of Insulin Action in the OZR: the Same Metabolic Deficiency
The OZR is an animal model characterized by deficiencies at the leptin receptor (11, 12, 13). Under physiological conditions, leptin inhibits insulin secretion (32) and stimulates endothelial NO release in what seems to be a compensatory mechanism to counteract the pressor effect of sympathoexcitation induced by leptin (11). In obesity, however, the leptin action deficiency results in decreased production of NO, which could only explain the impairment of the hepatic-dependent insulin sensitization observed, as discussed before. On the other hand, leptin metabolism deficiencies cannot account for the decrease in the hepatic-independent component because the major effect of a deficient leptin action, as it occurs in OZR, is the over-production of insulin, which may be responsible for the observed hyperinsulinemia. Furthermore, others show that leptin has no direct effect on glucose uptake in rat skeletal muscle (33). Therefore, although the role of leptin in obesity and IR is worthy of study, deficiencies at the level of its metabolism by themselves do not explain the observed IR (hepatic-dependent plus -independent) in OZR.
As mentioned, the skeletal muscle is the tissue primarily affected by IR in OZR. Indeed, deficiencies concerning both the insulin receptor, which leads to a deficient binding of insulin, and the GLUT4 translocation have been reported in the skeletal muscle of OZR (18, 21, 23, 25, 26, 27, 28). These deficiencies may affect not only the insulin action per se (hepatic-independent component) but also the hepatic-dependent component because this pathway enhances the glucose uptake, mainly in the skeletal muscle. Hypothesizing that HISS may exert its action primarily by interacting with the insulin receptor in the cell membrane, resulting in the initiation of the signal transduction pathway or in its own receptor requiring GLUT4 translocation to the cell surface to promote glucose uptake, seem to be two essential steps of the insulin intracellular signaling cascade where the confluence between the two pathways can occur. Therefore, because insulin binding and GLUT4 translocation are known to be impaired in OZR, neither insulin nor HISS are capable of inducing proper glucose uptake; thus, the impairment of the hepatic-dependent and -independent action is similar because it results from the same defect. Further studies are required to elucidate these questions.
In conclusion, our results confirm that the obesity animal model OZR is considerably insulin resistant in comparison with its lean litter mates (LZR). Furthermore, we show for the first time that in the OZR, both the hepatic-dependent and -independent components of insulin action are decreased. The fact that both components are decreased to the same extent suggests a defect in a pathway common to both components, giving us an indication regarding the hepatic-dependent component and its role in the control of glucose uptake in the skeletal muscle that should be pursued through further studies.