Department of Internal Medicine I, University of Regensburg, 93042 Regensburg, Germany
Submitted 7 October 2003 ; accepted in final form 8 January 2004
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ABSTRACT |
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insulin resistance; high-fat diet; liver perfusion
Clinical experience has associated insulin-resistant states, such as the metabolic syndrome, with fat accumulation in the liver over a long period of time (21). In patients with hepatic steatosis, but without known diabetes, insulin resistance as measured by homeostasis models is a common trait (8); impaired glucose tolerance as a hallmark of insulin resistance can be expected in 30% of such patients (31, 38). Clamp experiments in humans reveal an impaired suppression of HGO by insulin in patients with hepatic steatosis before the onset of impaired glucose tolerance or overt type 2 diabetes (32). Animal studies have similarly described negative correlations between perturbed hepatic insulin action and liver fat content (19).
Although these data clearly show that hepatic steatosis is commonly associated with obesity, insulin resistance, and a decreased insulin-induced suppression of HGO, they do not prove a causal relationship between hepatic fat accumulation and impaired insulin action on the liver. After all, the latter could also result indirectly from systemic factors influencing the liver's insulin sensitivity in vivo. To our knowledge, studies proving a defect in insulin action on isolated fatty livers without single genetic defects of glucose or lipid metabolism, which would allow us to define the role of direct insulin action on steatotic livers in insulin-resistant states, do not exist at present.
This issue obviously cannot be studied in humans. We therefore used the Wistar rat high-fat-diet model of hepatic steatosis and insulin resistance (7) to quantify direct hepatic insulin action in fatty livers. For this, the effects of insulin on HGO and on the insulin-dependent activation of hepatic glycogen synthase were analyzed using a liver perfusion system. Our results show sustained hepatic insulin action in high-fat-fed animals despite a sevenfold elevation of hepatic triglyceride content; they therefore argue against a simple equation of hepatic steatosis with direct hepatic insulin resistance.
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MATERIALS AND METHODS |
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Experimental design. Obesity and insulin resistance were induced in male Wistar rats by a 6-wk HF diet; controls were fed with standard chow (SC). The model was characterized metabolically by measuring basal fasting plasma insulin, glucose, triglyceride, and free fatty acids, and intraperitoneal insulin tolerance tests were performed. Retroperitoneal, epididymal, and perirenal fat pads were removed and weighed, and liver triglyceride content was measured. Fed animals were used for liver perfusion experiments to analyze the hepatic glucose output with or without exposure to epinephrine and insulin or the insulin stimulation of glycogen synthase activity, as we will describe in detail. Unless otherwise stated, all reagents were purchased from Sigma (St. Louis, MO) or Merck Eurolab (Darmstadt, Germany) at the highest purity grade available.
HF diet. A premixed HF diet (no. 157j) was purchased from Altromin (Lage, Germany). In terms of percentage by weight, it consisted of 20% fat (mainly lard), 42% carbohydrate, and 21% protein.
Liver perfusion experiments. Krebs-Ringer-Henseleit (KRB) buffer (7) [containing 4 mmol/l glucose and 2 mmol/l lactate and oxygenated by continuous gassing with carbogen (95% O2-5% CO2) at 37°C, and a pH of 7.4 with additions as will be described] was used as perfusate. The experiments were performed between 8:00 and 10:00 AM with fed HF or SC rats anesthetized with pentobarbital sodium (50 mg/kg body wt). Single-pass liver perfusion was then performed. For this, a 16-gauge catheter was inserted into the portal vein and secured by a ligature, and perfusion was started at a flow rate of 34 ml·min1·g liver1. To avoid an elevation of portal venous pressure, the vena cava inferior was immediately cut after the start of perfusion. A 16-gauge outflow catheter was inserted into the vena cava superior and a 23-gauge catheter into the common bile duct. After a stable perfusate flow was established with an adequate liver perfusion, assayed by homogenous color change and liver temperature as well as a portal venous pressure <5 cmH2O, the vena cava inferior was ligated, and the equilibration phase started. Glucose output was measured every 5 min with a portable glucose monitor (Roche Diagnostics, Mannheim, Germany). After 30 min, when a stable glucose output had been established, an epinephrine infusion (50 nmol/l) was started by use of a continuous pump to stimulate glycogenolysis (9). Glucose output was monitored as described above for 30 min. In another set of experiments, insulin was added to the perfusate from the start at 600 pmol/l, equaling a low physiological portal insulin concentration (5); epinephrine infusion and glucose monitoring were performed as described above. Appropriate control experiments were performed without insulin and/or epinephrine.
The quality of the perfusion was checked by monitoring O2 concentration before and after the liver passage, portal vein perfusion pressure (PVPP), bile flow, and liver temperature, as well as lactate levels and alanine aminotransferase (AAT) activity in the outflow. Only perfusion experiments with continuous O2 extraction of >40%, continuous bile flow, and homogenous liver surface temperature of >35°C, and without significant elevations of PPVP (i.e., <5 cmH2O) or lactate and AAT activity in the outflowing perfusate were used for subsequent analysis. All experimental subgroups consisted of at least five independent experiments.
Glycogen synthase activity.
Liver perfusion was initiated as described above. After 30 min of equilibration with oxygenated KRB buffer, a liver sample of 50100 mg was carefully cut out from the right ventral liver lobe and clamp-frozen in liquid nitrogen. Perfusion pressure did not change during this procedure, indicating that no major hemodynamic changes had been induced. Insulin infusion was then started at 600 pmol/l via a continuous pump. Thirty minutes later, a second, similarly sized liver sample was removed from the right ventral liver lobe and immediately clamp-frozen. Control samples were obtained in perfusions performed without addition of insulin. The liver samples were then homogenized in 50 mmol/l Tris·HCl, pH 7.8, 10 mmol/l EDTA, 100 mmol/l NaF, 1 mmol/l dithiothreitol, and 1 mmol/l phenylmethylsulfonyl fluoride. The insoluble matter was removed by centrifugation at 10,000 g for 15 min, and the supernatant was used for assaying insulin-stimulated glycogen synthase (GS) activity, as previously described (36) with slight modifications. The supernatant (60 µl) and 30 µl of assay buffer {50 mmol/l Tris·HCl, pH 7.8, 3.35 mmol/l EDTA, 10 mg/ml glycogen, 0.75 mmol/l uridine diphosphate (UDP)-D-glucose, 1.19 µCi/mol UDP-D-[14C]glucose} were mixed and incubated at 30°C for 20 min in the presence or absence of 10 mmol/l glucose 6-phosphate to quantitate activated and total GS activity. After stopping the reaction with 50 µl of 60% KOH, the samples were spotted onto prelabeled chromatography filter papers (3MM; Whatman, Maidstone, UK). Glycogen was precipitated by immersion in 500 ml of 70% ethanol at 4°C. The filters were washed two times in 250 ml of 70% ethanol for 30 min to remove unincorporated substrate from precipitated glycogen. Filters were air-dried, and incorporated radioactivity was measured by liquid scintillation counting. Activated GS (GS
) was normalized with total GS activity by calculating the ratios of radioactivity incorporated into glycogen with and without addition of glucose 6-phosphate.
Insulin tolerance tests. Experiments were performed with fasted HF and SC rats. Rats were anesthetized with pentobarbital sodium (50 mg/kg body wt). After 30 min, baseline glucose was measured with a hand-held glucometer (AccuTrend, Roche Diagnostics) from whole blood drawn from the tail tip capillary region. The animals were then injected intraperitoneally with 600 pmol/kg body wt insulin (Aventis, Frankfurt, Germany). Whole blood glucose was monitored every 8 min for 60 min.
Liver triglycerides. Tissue triglycerides were determined as described previously, with slight modifications (7). Briefly, frozen liver samples were first powdered under liquid nitrogen. Twenty to fifty milligrams of frozen liver powder were then weighed in 1 ml of a chloroform-methanol mix (2:1 vol/vol) and incubated for 1 h at room temperature with occasional shaking to extract the lipid. After addition of 200 µl of H2O, vortexing, and centrifugation for 5 min at 3,000 g, the lower lipid phase was collected and dried at room temperature. The lipid pellet was redissolved in 60 µl of tert-butanol and 40 µl of a Triton X-114-methanol (2:1 vol/vol) mix, and triglycerides were measured by means of the GPO-triglyceride kit (Sigma) with appropriate triglyceride standards (Sigma).
Plasma measurements. Plasma glucose, triglycerides, and free fatty acids as well as perfusate AAT activity and lactate content were measured with kits from Sigma and Roche Diagnostics. Plasma insulin was measured using rat-specific RIA kits (Linco Research, St. Charles, MO).
Statistical methods.
All results are expressed as means ± SE of at least five independent experiments unless otherwise stated. The area under the curve (AUC) was analyzed by employing the trapezoid rule. Statistical significance was determined by an unpaired Student's t-test by use of the statistics module of Microsoft Excel, version 8.0 (Microsoft, Seattle, WA). P values were corrected for multiple testing according to the Bonferroni method. Statistical significance was assumed at P 0.05.
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RESULTS |
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Activation of GS by insulin in HF and SC rats.
To further analyze the direct hepatic insulin sensitivity in SC and HF rats, we measured the activation of GS in livers perfused with insulin at 100 µmol/l. The results are shown in Fig. 3. Both basal and insulin-stimulated GS activities tended to be higher in HF rats, whereas the relative increment in enzyme activity induced by insulin was similar in the two diet groups (fold increase for SC: 1.68 ± 0.11; for HF: 1.60 ± 0.08, P = 0.65, when diet groups were compared with each other).
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DISCUSSION |
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By using a lard-based high-fat rodent diet, we were able to induce visceral obesity and moderate signs of whole body insulin resistance in normal male Wistar rats. The animals showed a marked rise in hTG content, as previously described (30). Hepatic insulin action was assessed in HF-fed and control rats by using an isolated liver perfusion system that enabled us to look at direct insulin effects. By this method, the multiple confounding factors influencing insulin action in in vivo clamp settings, in which systemic insulin effects on glucose as well as on lipid and amino acid metabolism may influence hepatic glycogenolysis and gluconeogenesis, could be ruled out. Hepatocyte cultures were not employed, because the time-consuming isolation procedure might have led to changes of hTG deposits and also might have influenced the putative insulin-resistant phenotype. We used a cell-free perfusate to avoid changes in lactate concentration originating from erythrocyte metabolism. The perfusions were performed with fed rats to ensure sufficient glycogen deposits. A physiological perfusate glucose concentration was chosen because hyperglycemia is known to attenuate diet-induced insulin resistance (10), whereas low glucose conditions would have increased hepatic glucose production, thereby diminishing glycogen stores and potentially lowering the HGO-suppressing effect of insulin. Relevant hypoxia during the perfusions, which would influence the results, can be ruled out, because the parameters of liver vitality, in particular oxygen extraction, were stable during the perfusions.
Basal HGO, as assessed by the portocaval glucose difference during liver perfusions, did not differ significantly between HF rats and SC controls. These results argue against an increase in basal hepatic glucose production induced solely by chronic hTG elevation, although we cannot rule out that a putative HGO elevation might have been counteracted by concomitantly decreased hepatic glycogen stores, as observed previously in high-fat-fed rats (23). Insulin at a low physiological portal concentration was not able to suppress basal HGO in either experimental group. This is in keeping with previous results, demonstrating that insulin by itself does not change HGO in isolated liver perfusion systems at normal glucose concentrations (41).
The glycogenolytic catecholamine epinephrine effectively stimulated HGO; the extent and duration of epinephrine-induced HGO stimulation were markedly lowered by insulin in SC rats. These results correspond with earlier work demonstrating a strong reduction of adrenergically stimulated glycogenolysis by insulin in comparable perfusion systems (11, 16, 22). In livers of HF rats, insulin also effectively decreased epinephrine-induced HGO stimulation. This indicates that insulin is able to directly suppress adrenergically stimulated hepatic glycogenolysis regardless of liver triglyceride content. To our knowledge, no previous study has measured direct hepatic insulin action in high-fat-fed rats with defined hepatic steatosis by use of an isolated perfusion system. Ikeda and Fujiyama (17) showed insulin resistance in perfused livers of high-fructose-fed rats, but they did not state hepatic triglyceride content, making our results difficult to compare. To confirm the sustained action of insulin in fatty livers with a second experimental parameter, we measured GS activity in livers perfused with and without insulin. We observed clear insulin-induced increments of GS activity, showing that the experimental system was valid for analysis of hepatic insulin action. No clear difference between HF and SC rat livers was found (Fig. 3). Again, to our knowledge, no comparable previous results exist.
In conclusion, no signs of resistance to direct actions of insulin were detected in steatotic rat livers when two different parameters of hepatic insulin action were observed. These results do not support the simple preconception that elevated liver trigyceride deposition by itself necessarily impairs hepatic insulin action, e.g., by influencing binding of insulin to its receptor, insulin receptor activity, or postreceptor signal transduction. This notion is supported by a previous study, which failed to demonstrate clear deteriorating effects of hepatic steatosis when insulin-induced activation of phosphatidylinositol 3-kinase in livers of high-fat-fed rats was analyzed (3). However, other authors have reported a decreased insulin receptor kinase activity (39), so this issue must remain controversial. Also, when interpreting our results, it should be considered that the results we obtained apply only to the insulin suppression of hepatic glycogenolysis. HGO deriving from gluconeogenesis might be regulated differently, and direct insulin action on this part of hepatic glucose metabolism remains to be quantified in fat-laden livers.
Substantial evidence for an impairment of direct insulin action on fatty livers does exist from in vivo studies. Seppala-Lindroos et al. (32) recently demonstrated that basal HGO remains unchanged in subjects with hepatic steatosis despite elevated fasting insulin levels. Basal HGO is regulated by the portal vein insulin concentration in vivo (35). Taken together, these studies suggest that higher insulin concentrations were needed to restrain HGO in these subjects. The fact that steatotic livers show an impairment of insulin action in vivo, but not in our isolated perfusion system, argues for the hypothesis of a secondary origin of hepatic insulin resistance. Extrahepatic factors that regulate aspects of hepatic insulin action, e.g., suppression of HGO, would then be pathologically changed in insulin-resistant states. Previous authors examining hepatic insulin action have come to similar conclusions. The "single gateway theory" proposed by Bergman (4) states that insulin resistance of the adipocyte impairs insulin-induced suppression of lipolysis. This leads to an elevation of free fatty acids, which in turn stimulate hepatic glucose production. Key assumptions of this theory have been confirmed during the last few years (1, 20, 25, 34), but they are challenged by recent findings in liver-specific insulin receptor knockout mice, demonstrating that both the direct and the indirect insulin actions on HGO require an intact insulin-signaling pathway in the liver (12).
Other studies following the single gateway theory have suggested alternative extrahepatic regulators of HGO. New studies show that adipocyte-derived hormones, such as resistin, decrease the hepatic insulin effect (27). Also, it has recently been demonstrated that mice lacking insulin receptors in the central nervous system develop insulin resistance (6), and central insulin antagonism impairs the ability of circulating insulin to inhibit hepatic glucose production (26). Other arguments against hepatic triglyceride content as the main regulator of the insulin effect on the liver come from rodent studies that use different interventions to increase hepatic insulin action. Insulin-induced HGO suppression was reversed to normal after a 3-day diet change, which cannot reasonably be expected to reverse hepatic steatosis, in HF-fed rats (15). Removal of visceral fat in obese rats markedly improved hepatic insulin action over that in controls (13). In summary, a multitude of recent results strongly argues for the existence of an extrahepatic regulation of hepatic insulin action, which might be altered in insulin-resistant and diabetic states.
When our findings are taken together, we demonstrate sustained direct hepatic insulin action in steatotic livers of HF-fed rats in terms of suppression of glycogenolysis. Elevated hTG, therefore, do not necessarily interfere with insulin receptor function or intracellular insulin signaling. The hepatic "insulin resistance" observed in vivo may at least partially result indirectly, i.e., be caused by circulating factors that interfere with insulin signaling and/or hepatic glucose production.
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GRANTS |
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FOOTNOTES |
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The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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REFERENCES |
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