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Deficiency Does Not Alter Insulin Sensitivity in Mice Maintained on Regular or High-Fat Diet: Hyperinsulinemic-Euglycemic Clamp Studies
Third Department of Medicine (M.H.), First Faculty of Medicine, Charles University, 128 08 Prague 2, Czech Republic; and Mouse Metabolism Laboratory (O.G.) and Diabetes Branch (D.L.), National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, Maryland 20892-1758
Address all correspondence and requests for reprints to: Martin Haluzik, Third Department of Medicine, First Faculty of Medicine, Charles University, U nemocnice 1, 128 08, Prague 2, Czech Republic. E-mail: mhalu{at}lf1.cuni.cz.
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Abstract |
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activation improves glucose metabolism in rodent models of insulin resistance and diabetes; however, PPAR- deficiency was also reported to protect against high-fat diet (HFD)-induced insulin resistance. The aim of this study was to clarify the role of PPAR- in the development of insulin resistance using PPAR- knockout (KO) mice and wild-type controls (WT). Both WT and PPAR- KO mice on HFD gained significantly more weight relative to chow-fed groups and displayed an increase in insulin levels and a decrease in adiponectin levels. Hyperinsulinemic-euglycemic clamp performed in the nonfasting state demonstrated that HFD caused a marked reduction in whole body, muscle, and white and brown adipose tissue glucose uptake in both WT and PPAR- KO mice relative to chow-fed groups. Suppression of endogenous glucose production during the clamp was markedly blunted in both WT and PPAR- KO HFD-fed mice, indicating liver insulin resistance. The magnitude of HFD-induced changes in the clamp parameters of insulin sensitivity was comparable in PPAR- KO and WT mice. In conclusion, these data show that PPAR- deficiency does not alter insulin sensitivity in mice fed normal chow diet and does not protect against HFD-induced insulin resistance as measured by hyperinsulinemic-euglycemic clamp in nonfasted state. |
Introduction |
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plays a pivotal role in the control of mitochondrial ß-oxidation of fatty acids (2, 3). This process is the most important metabolic pathway by which fatty acids are used providing energy primarily for the heart and skeletal muscles. The ability to oxidize fatty acids is necessary for the normal response to fasting as demonstrated by severe hypoglycemia with early death in fasted PPAR--deficient mice (2). The metabolism of lipids and carbohydrates is closely interconnected markedly affecting each other on multiple levels (4, 5). In 1963 the so-called glucose fatty acid cycle was proposed by Randle et al. (6), suggesting that there is a reciprocal metabolic relationship between glucose and fatty acid utilization. Although the general view on the mechanism by which fatty acids affect glucose metabolism has changed substantially (7, 8), there is growing evidence suggesting that both the circulating fatty acids and the deposition of triglycerides and/or other lipid metabolites in nonadipose tissues can induce insulin resistance (9, 10, 11). Moreover, a number of experimental and clinical studies (12, 13, 14) demonstrated a negative correlation between tissue triglyceride content and insulin sensitivity. For example, increased lipid deposition in the muscle and the liver in the face of complete lack of adipose tissue leads to a severe insulin resistance and type 2 diabetes in both lipoatrophic A-ZIP/F-1 mice and the patients with severe lipodystrophies (15). Interventions decreasing lipid deposition in nonadipose tissues such as leptin treatment of lipodystrophic patients or fat transplantation in lipoatrophic mice markedly improved insulin sensitivity and reduced the decompensation to diabetes (16, 17, 18).
Several experimental studies showed that the activation of PPAR- receptors by PPAR- agonists such as fibrates decrease adiposity and subsequently insulin resistance in rodent models of obesity and insulin resistance by promoting lipid depletion in both the adipose and nonadipose tissues (19, 20). PPAR- agonist also improved insulin sensitivity in the lipoatrophic A-ZIP/F-1 mice (21) and MKR mice with impaired insulin and IGF-1 signaling in skeletal muscle (22). In both cases improvements of tissue insulin sensitivity were accompanied by decreased lipid content in the muscle and liver tissues.
Interestingly, there were two reports demonstrating that complete PPAR- deficiency might be protective against high-fat diet (HFD)-induced insulin resistance and glucose intolerance (23, 24). Insulin sensitivity in both of the above-mentioned studies has been assessed in fasted mice (23, 24). The results thus may have been influenced by the fact that PPAR- knockout (KO) mice cannot oxidize fatty acids and therefore are unable to normally respond to fasting. Thus, the ultimate role of PPAR- in the regulation of insulin sensitivity is not completely established. To prevent such a possibility we performed a hyperinsulinemic-euglycemic clamp study in nonfasted animals to directly address the role of PPAR- deficiency in the regulation of insulin sensitivity. We demonstrate that after elimination of the confounding effects of fasting, PPAR- KO mice have comparable susceptibility to the HFD-induced insulin resistance as the wild-type (WT) controls.
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Materials and Methods |
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KO mice and control mice were purchased from Jackson Laboratory (PPAR- KO mice, strain name 129S4/SvJae-Pparatm1Gonz, stock number 003580; control mice, strain name 129S1/SvImJ, stock number 002448). Animals were maintained on a 12-h light/12-h dark cycle (0600 h/1800 h) and fed either standard chow diet (NIH-07, 5% fat) or HFD (45% fat cat. no. D12451, Research Diets, Inc., Mount Prospect, NJ) for 14 wk. Experiments were performed using mice at 22 wk of age. The blood glucose, serum insulin, and adiponectin concentrations measurements were repeated in two independent sets of animals, with results similar to those described bellow. Muscle triglycerides were measured after solvent extraction as previously described (25) using a triglyceride kit from ThermaDMA Ltd. (Melbourne, Australia, catalog no. TR22421). Livers were fixed in 4% paraformaldehyde and processed by American Histolabs (Gaithersburg, MD) for histological study. Liver sections were stained by hematoxylin and eosin. All experiments were performed in accordance with NIH guidelines and with the approval of the Animal Care and Use Committee of the National Institute of Diabetes and Digestive and Kidney Diseases.
Hyperinsulinemic-euglycemic clamps
Clamp procedure was in detail described previously (9, 26). In brief, catheters were implanted under ketamine and xylazine anesthesia. The silastic catheter (ID 0.30 mm, OD 0.64 mm, 508–001, Dow Corning, Midland, MI), filled with heparin solution (100 USP U/ml in 0.9% NaCl), was inserted via a right lateral neck incision, advanced into the superior vena cava via the right internal jugular vein and sutured in place (procedure adapted from MacLeod and Shapiro (27). The distal end of the catheter was knotted, tunneled subcutaneously, exteriorized first at the dorsal cervical midline, and then further tunneled subcutaneously and exteriorized in the dorsal midline, 2 cm above the tail. A silk suture was fastened around the catheter at the neck site. The clamps were then performed 4–5 d later after complete recovery of the animals from the operation.
Clamps began at 0700 h and were performed in nonfasted mice. Mice were placed into a restrainer (552-BSRR, Plas-Labs, Lansing, MI) and the catheter was externalized. The tip of tail was cut before the start of the first infusion, and all subsequent blood drawings were carried out using this site. Blood was collected into heparinized microhematocrit capillary tubes (Fisher Scientific, Pittsburgh, PA) and centrifuged for 10 sec to obtain plasma. Basal endogenous glucose production was estimated by continuously infusing [3-3H]glucose (3 µCi bolus, then 0.02 µCi/min, 740 GBq/mmol; NET 331C, NEN Life Science Products, Boston, MA) for 2 h. Samples for determination of plasma [3-3H]glucose concentration were taken after 90 and 115 min of basal infusion. Basal glucose and insulin concentrations were measured in the sample taken 90 min after the start of basal infusion. After 120 min of basal [3-3H]glucose infusion the hyperinsulinemic-euglycemic clamp was begun with a prime continuous infusion of human insulin (bolus 300 mU/kg over 3 min and then 2.5 mU/kg/min; Humulin R, Eli Lilly, Indianapolis, IN). Plasma glucose was measured at 15-min intervals and 20% glucose was infused at a rate that was adjusted to keep the plasma glucose at approximately 110 mg/dl. Insulin-stimulated whole-body glucose uptake was measured using a prime continuous infusion of [3-3H]glucose (10 µCi bolus, 0.1 µCi/min) throughout the clamps.
Insulin-stimulated glucose uptake in muscle and white and brown adipose tissues was measured using a bolus injection of 2-deoxy-D-[1-14C] glucose (10 µCi in 5 µl of 0.9% saline, 2.1 GBq/mmol; NEC 495, NEN Life Science Products) at 70 min after the start of the insulin infusion. Blood samples (20 µl) were withdrawn at 80, 85, 90, 100, 110, and 120 min after start of the insulin infusion for the measurement of plasma 3H and 14C. Clamp insulin levels were measured in 5 µl of plasma from the 110-min point. All infusions were performed using a microdialysis pump (model CMA 102; CMA/Microdialysis, Acton, MA). Hamilton Gastight syringes (10 µl; Hamilton Co., Reno, NV) were used for the bolus injections. After 120 min of insulin infusion, animals were anesthetized with ketamine/xylazine solution. Within 5 min, gastrocnemius and quadriceps muscles and white and brown adipose tissues were removed, immediately frozen in liquid nitrogen, and stored at -70 C.
In vivo glucose flux analysis
The determination of plasma [3-3H]glucose and 2-deoxy-D-[1-14C] glucose concentrations and tissue 2-deoxy-D-[1-14C] glucose-6-phosphate were performed as described previously (28).
Calculations
Basal endogenous glucose production was calculated as the ratio of the preclamp [3-3H]glucose infusion rate (disintegrations per minute/minute) to the specific activity of the plasma glucose (mean of the values in the 90 and 120 min of basal preclamp period in disintegrations per minute per micromoles). Clamp whole-body glucose uptake was calculated as the ratio of the [3-3H]glucose infusion rate (disintegrations per minute/minute) to the specific activity of plasma glucose (disintegrations per minute per micromoles) during the last 30 min of the clamp (mean of the 90- to 120-min samples). Clamp endogenous glucose production was determined by subtracting the average glucose infusion rate in the last 30 min of clamp from the whole-body glucose uptake. Muscle and white and brown adipose tissue glucose uptake was calculated from the plasma 2-deoxy-D-[1-14C] glucose concentration profile (using plasma 14C counts at 80–120 min, the area under the curve was calculated by trapezoidal approximation) and tissue 2-deoxy-D-[1-14C] glucose-6-phosphate content as described previously (28).
Biochemical and hormonal assays.
Glucose was measured using a Glucometer Elite (Bayer, Elkhart, IN). Insulin and adiponectin (no. SRI-13K and no. MADP-60HK, respectively, Linco Research, St. Charles, MO), triglycerides (no. 337-B, Sigma, St. Louis, MO), and nonesterified fatty acids (no. 13831175, Roche Molecular Biochemicals, Indianapolis, IN) were quantitated with the indicated kits.
Statistical analysis
Data are expressed means ± SE. Statistical significance between the groups was determined with SigmaStat (SPSS Inc., Chicago, IL) using two-way ANOVA or t test as appropriate.
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Results |
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KO mice and WT mice fed chow and HFD KO mice on chow diet were comparable with those of WT mice. HFD feeding for 14 wk led to a significantly accelerated weight gain in both PPAR- KO and WT mice relative to chow-fed animals (Table 1
). Body weight in HFD-fed PPAR- KO and HFD-fed WT animals did not differ. The same was true for the fat pad weight of inguinal and brown adipose tissues (Table 1). In contrast, gonadal fat pad weight on HFD was significantly higher in PPAR- KO relative to HFD-fed WT animals. Muscle triglyceride content in WT mice on either diet and PPAR- KO mice on chow diet did not differ significantly (Table 1). Muscle triglycerides in PPAR- KO mice on HFD were significantly higher relative to WT groups on both chow and HFD (Table 1).
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KO and WT animals on normal chow did not differ. Liver weights of HFD-fed animals were higher relative to chow-fed animals of the same genotype (Table 1
). This increase was potentiated by PPAR- deficiency. The livers of PPAR- KO mice were 56% heavier than those of HFD-fed WT mice. The difference in liver weight was paralleled by a difference in liver fat content as revealed by histological examination (Fig. 1). Whereas no triglyceride deposition or a very slight one was detected in livers of chow-fed WT mice and PPAR- KO mice, respectively, HFD feeding led to a focal liver steatosis in WT and a diffuse one in PPAR- KO mice (Fig. 1).
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KO mice on chow diet did not differ from those of WT mice on the same chow. Serum glucose levels were not affected significantly by HFD feeding (Fig. 2). In contrast, serum insulin levels in HFD-fed groups were significantly higher relative to chow-fed groups, indicating insulin resistance (Fig. 2). No significant effect of PPAR- deficiency on HFD-induced increase of insulin levels was found. The same was true for circulating adiponectin levels, which slightly, but significantly declined in HFD-fed groups of both genotypes (Fig. 2).
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KO mice relative to WT controls (Fig. 2). HFD feeding did not affect circulating fatty acids but decreased serum triglycerides in both WT and PPAR- KO mice relative to respective chow-fed groups (Fig. 2).
In summary, PPAR- deficiency led to higher circulating lipid levels on chow diet and did not affect either weight gain or increase in insulin levels in HFD-fed animals. The only differences found in HFD-fed PPAR- KO mice relative to HFD-fed WT mice were slightly higher gonadal fat pad weights and accelerated liver steatosis.
Hyperinsulinemic-euglycemic clamp analysis
Increased insulin levels in HFD-fed mice of both genotypes indicated the development of insulin resistance. To study whole-body and tissue insulin sensitivity in detail, we performed hyperinsulinemic-euglycemic clamps.
It was previously demonstrated that PPAR- KO mice are incapable of normal response to fasting due to defective fatty acid oxidation (2). As a result, PPAR- KO mice normally become hypoglycemic after a relative short time fasting and subsequently die from severe hypoglycemia (1, 2). To prevent those effects of fasting on insulin sensitivity, the clamps were performed in nonfasted mice (in contrast to normally used overnight fasting). Because the clamp was preceded by 2-h period of basal [3-3H]glucose infusion in saline, at the time of the start of the clamp the mice were in fact fasted for 2 h. The sample for basal glucose and insulin determination was thus taken after 90 min of fasting.
Even after such short-term fasting, PPAR--deficient mice on both diets had significantly lower blood glucose levels relative to respective WT groups (Table 2). This finding contrasted with normal glucose levels in nonfasting state as described above (Fig. 2). Insulin levels tended to follow the same pattern, but the differences did not reach statistical significance (Table 2). Basal endogenous glucose production was not affected either by diet or genotype (Table 2).
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). Whole-body glucose uptake was decreased to a comparable extent in both HFD-fed WT and PPAR- KO mice. Glucose uptake measured directly in the muscle and white and brown adipose tissues, respectively, was markedly reduced in HFD-fed animals regardless of PPAR- deficiency (Fig. 4).
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KO mice fed by chow diet, in which clamp endogenous glucose production was close to zero (Fig. 3). In contrast, clamp endogenous production in HFD-fed groups remained high, reflecting liver insulin resistance (Fig. 3). The extent of liver insulin resistance in HFD-fed PPAR- KO mice was comparable with that of HFD-fed WT mice.
In summary, hyperinsulinemic-euglycemic clamp analysis showed that PPAR- deficiency did not affect insulin sensitivity on normal chow diet and did not modify the development of HFD-induced insulin resistance.
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Discussion |
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deficiency on the HFD-induced insulin resistance. We show that after 14 wk of HFD feeding both the WT and PPAR- KO mice display a comparable degree of insulin resistance in the liver, muscle, and fat as evidenced by impaired suppression of clamp endogenous glucose production and glucose uptake in respective tissues.
The complete lack of PPAR- does not affect body composition or glucose metabolism profoundly under normal conditions (29). Consistent with the previous reports (29), we found a tendency toward higher body fat content in PPAR- KO mice on normal chow and a significantly higher gonadal fat pad weights in HFD-fed PPAR- KO mice relative to respective WT group. A possible explanation of this difference may be an increased lipid availability and storage in the adipose tissue due to a lack of fatty acid oxidation in the skeletal muscle of PPAR- KO mice.
Similarly as in the previous reports (2, 23, 29), we found no difference in nonfasting glucose or insulin levels between PPAR- KO and WT mice. However, even relatively short-term fasting led to a rapid decrease of blood glucose of PPAR- KO mice due to their inability to use fatty acids (2). The importance of fasting vs. nonfasting conditions was further underlined by a lower preclamp basal glucose levels in PPAR- KO relative to WT animals after only 90 min of fasting (Table 2). Because the clamps have been performed in nonfasted conditions, the results could have been somewhat affected by additional glucose absorption from the gut during the clamp. However, the control animals were subjected to the same nonfasting conditions, and it is therefore unlikely that this effect was different in PPAR- KO vs. control animals.
With the exception of a difference in fasting plasma glucose levels, we did not detect any difference between PPAR- and WT mice in any of other parameters measured before and during the clamp either on chow diet or HFD. This suggests that PPAR- deficiency did not significantly alter insulin sensitivity under given experimental conditions. It should be, however, noted that the complete lack of PPAR- function in PPAR- KO mice might be compensated for by other pathways and therefore may not reflect precisely the importance of PPAR- in the regulation of insulin sensitivity in normal animals.
Thus, our data showing no protective effect of PPAR- deficiency against HFD-induced insulin resistance are in disagreement with previously published study by Guerre-Millo et al. (23). In the study by Guerre-Millo, insulin sensitivity and glucose tolerance were assessed by insulin and glucose tolerance tests in halothane anesthetized 2-h fasted mice. Serum chemistry including glucose and insulin were studied in the animals fasted for 4 h. It is possible that the differences in insulin levels and glucose tolerance found in the study by Guerre-Millo reflected rather the impaired response to fasting of PPAR- KO mice than increased insulin sensitivity by itself. The inability of PPAR- KO mice to use fatty acids may lead to preferential use of glucose as a fuel during fasting. Glycogen stores in those animals are thus depleted more rapidly than in WT mice, which may in turn increase their glucose disposal.
The results of our study further underscore the complexity of the role of PPAR- in the regulation of insulin sensitivity. Increased circulating fatty acids and triglycerides normally accompany and/or induce insulin resistance (8, 10, 30). PPAR- KO mice on normal chow have markedly increased circulating fatty acids and triglycerides but demonstrate no signs of insulin resistance. PPAR- thus may be involved in the mechanism of insulin resistance induced by circulating fatty acids. According to the original hypothesis of Randle (6), increased fatty acid availability competitively inhibits glucose oxidation in the muscle. Lack of this effect in PPAR- KO mice incapable of fatty acid oxidation may protect those animals against fatty acid-induced insulin resistance.
HFD feeding in our study decreased circulating lipid levels but led to the development of obesity and insulin resistance in the liver, muscle, and fat, respectively. Our study suggests that the mechanism of long-term high-fat feeding-induced insulin resistance is different from the one induced primarily by high circulating fatty acids. Shulman and others (10, 31, 32, 33, 34) suggested that intracellular triglyceride and/or other lipid metabolites in nonadipose tissues alter insulin signaling on the postreceptor level. According to our data presented herein, this effect appears to be PPAR- independent.
In conclusion, our study showed that PPAR--deficient mice fed chow diet have normal insulin sensitivity despite increased circulating fatty acids and triglycerides. PPAR- deficiency, however, did not protect against insulin resistance induced by the long-term HFD feeding.
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Acknowledgments |
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Footnotes |
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Abbreviations: HFD, High-fat diet; KO, knockout; PPAR, peroxisome proliferator-activated receptor; WT, wild-type.
Received August 7, 2003.
Accepted for publication December 5, 2003.
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References |
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mediates the adaptive response to fasting. J Clin Invest 103:1489–1498[Medline]
activators improve insulin sensitivity and reduce adiposity. J Biol Chem 275:16638–16642 activation lowers muscle lipids and improves insulin sensitivity in high fat-fed rats: comparison with PPAR-
activation. Diabetes 50:411–417 (PPAR) agonist, improves hepatic and muscle steatosis and reverses insulin resistance in lipoatrophic A-ZIP/F-1 mice. J Biol Chem 277:24484–24489 agonist treatment in a transgenic model of type 2 diabetes reverses the lipotoxic state and improves glucose homeostasis. Diabetes 52:1770–1778-null mice are protected from high-fat diet-induced insulin resistance. Diabetes 50:2809–2814 deficiency reduces insulin resistance and atherosclerosis in apoE-null mice. J Clin Invest 107:1025–1034[Medline]
regulates lipid homeostasis, but is not associated with obesity: studies with congenic mouse lines. J Biol Chem 276:39088–39093This article has been cited by other articles:
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