Increased β-Oxidation but No Insulin Resistance or Glucose Intolerance in Mice Lacking Adiponectin

Previous reports showed that recombinant fragments of adiponectin (adipo) displayed pharmacological effects when injected into rodents, but the relevance of these observations to the physiological function of adipo is unclear. We generatedAdipo −/− mice by gene targeting.Adipo −/− mice are fertile with normal body and fat pad weights. Plasma glucose and insulin levels ofAdipo −/− and Adipo +/+mice are similar under fasting conditions and during an intraperitoneal glucose tolerance test (GTT). Insulin tolerance test (ITT) also produces similar plasma glucose and insulin levels in the two groups of mice. Hyperinsulinemic-euglycemic clamp analysis showed thatAdipo −/− and Adipo +/+mice have similar glucose infusion rates to maintain a similar serum glucose. High-fat diet feeding for 7 months led to similar weight gain and similar GTT and ITT responses. We next measured β-oxidation and found it to be significantly increased in muscle and liver ofAdipo −/− mice. In conclusion, our study indicates that absence of adipo causes increased β-oxidation but does not cause glucose intolerance or insulin resistance in mice. Previous reports showed that recombinant fragments of adiponectin (adipo) displayed pharmacological effects when injected into rodents, but the relevance of these observations to the physiological function of adipo is unclear. We generatedAdipo −/− mice by gene targeting.Adipo −/− mice are fertile with normal body and fat pad weights. Plasma glucose and insulin levels ofAdipo −/− and Adipo +/+mice are similar under fasting conditions and during an intraperitoneal glucose tolerance test (GTT). Insulin tolerance test (ITT) also produces similar plasma glucose and insulin levels in the two groups of mice. Hyperinsulinemic-euglycemic clamp analysis showed thatAdipo −/− and Adipo +/+mice have similar glucose infusion rates to maintain a similar serum glucose. High-fat diet feeding for 7 months led to similar weight gain and similar GTT and ITT responses. We next measured β-oxidation and found it to be significantly increased in muscle and liver ofAdipo −/− mice. In conclusion, our study indicates that absence of adipo causes increased β-oxidation but does not cause glucose intolerance or insulin resistance in mice. Adiponectin (adipo, also known as Acrp30, adipoQ, GBP28, and apM1) 1The abbreviations used are: adipo, adiponectin; GTT, glucose tolerance test; ITT, insulin tolerance test; HF/HS, high-fat high-sucrose. is a major adipocyte secretory protein of unknown function (1Scherer P.E. Williams S. Fogliano M. Baldini G. Lodish H.F. J. Biol. Chem. 1995; 270: 26746-26749Abstract Full Text Full Text PDF PubMed Scopus (2760) Google Scholar, 2Das K. Lin Y. Widen E. Zhang Y. Scherer P.E. Biochem. Biophys. Res. Commun. 2001; 280: 1120-1129Crossref PubMed Scopus (68) Google Scholar, 3Hu E. Liang P. Spiegelman B.M. J. Biol. Chem. 1996; 271: 10697-10703Abstract Full Text Full Text PDF PubMed Scopus (1901) Google Scholar). Injection of full-length and partial-length fragments of recombinant adipo in rodents was shown to suppress hepatic glucose production (4Combs T.P. Berg A.h. Obici S. Scherer P.E. Rossett L. J. Clin. Invest. 2001; 108: 1875-1881Crossref PubMed Scopus (799) Google Scholar, 5Berg A.H. Combs T.P. Du x. Brownlee M. Scherer P.E. Nat. Med. 2001; 7: 947-953Crossref PubMed Scopus (2218) Google Scholar) and increase fatty acid oxidation in muscle (6Fruebis J. Tsao T.-S. Javorschi S. Ebbets-Reed D. Erickson M.R. Yen F.T. Bihain B.E. Lodish H.F. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 2005-2010Crossref PubMed Scopus (1770) Google Scholar, 7Yamauchi T. Kamon J. Waki H. Terauchi Y. Kubota N. Hara K. Mori Y. Ide T. Murakami K. Tsuboyama-Kasaoka N. Ezaki O. Akanuma Y. Gavrilova O. Vinson C. Reitman M.L. Kagechika H. Shudo K. Yoda M. Nakano Y. Tobe K. Nagai R. Kimura S. Tomita M. Froguel P. Kadowaki T. Nat. Med. 2001; 7: 941-946Crossref PubMed Scopus (4099) Google Scholar). The plasma concentration of adipo is reduced in obesity and type 2 diabetes (8Weyer C. Funahashi T. Tanaka S. Hotta K. Matsuzawa Y. Pratley R.E. Tataranni P.A. J. Clin. Endocrinol. Metab. 2001; 86: 1930-1935Crossref PubMed Scopus (2631) Google Scholar). These observations led to the proposal that adipo may play important roles in glucose homeostasis and insulin resistance. To elucidate the in vivo role of adipo, we generatedAdipo −/− mice by gene targeting. We found that these mice had no glucose intolerance or insulin resistance under basal conditions or even after they were fed a high-fat diet for 7 months. Unexpectedly, we found that Adipo −/− mice had increased β-oxidation in muscle and liver. This study sheds light on the function of native adipo in vivo. We used a replacement-type targeting vector constructed from a mouse 129/Sv strain bacterial artificial chromosome genomic clone and R1 ES cell line for gene targeting (Fig. 1 A). Transfection and ES cell clone selection were as described (9Chang B.H.-J. Liao W., Li, L. Nakamuta M. Mack D. Chan L. J. Biol. Chem. 1999; 274: 6051-6055Abstract Full Text Full Text PDF PubMed Scopus (118) Google Scholar). Nine positive ES clones were injected into blastocysts of C57BL/6J, and chimeric mice obtained were mated with C57BL/6J mice. Germ line transmission was confirmed by Southern blots using tail DNA. Northern blots were performed on total RNA isolated from white and brown adipose tissue using a 32P-labeled mouse full-length cDNA probe as described (9Chang B.H.-J. Liao W., Li, L. Nakamuta M. Mack D. Chan L. J. Biol. Chem. 1999; 274: 6051-6055Abstract Full Text Full Text PDF PubMed Scopus (118) Google Scholar, 10Lau P.P. Villanueva H. Kobayashi K. Nakamuta M. Chang B.H.J. Chan L. J. Biol. Chem. 2001; 276: 46445-46452Abstract Full Text Full Text PDF PubMed Scopus (40) Google Scholar). Glyceraldehyde 3-phosphate dehydrogenase cDNA was used as a control. Polyclonal rabbit antibody was raised against a C-terminal fragment of mouse adipo (residues 110–247) fused to glutathione S-transferase. Immunoblotting were performed as described previously using 3 μl of plasma (9Chang B.H.-J. Liao W., Li, L. Nakamuta M. Mack D. Chan L. J. Biol. Chem. 1999; 274: 6051-6055Abstract Full Text Full Text PDF PubMed Scopus (118) Google Scholar). Plasma-free fatty acid, glycerol, triglyceride, total cholesterol, and leptin levels were determined by a NEFA-C test (Wako Chemicals, Neuss, Germany), Triglyceride GPO-Trinder, total cholesterol INFINITY (Sigma) and Quantikine M kit (R 274: 6051-6055Abstract Full Text Full Text PDF PubMed Scopus (118) Google Scholar, 11Martinez-Botas J. Anderson J.B. Tessier D. Lapillonne A. Chang B.H.J. Quast M.J. Gorenstein D. Chen K.-H. Chan L. Nat. Genet. 2000; 26: 474-479Crossref PubMed Scopus (495) Google Scholar). We measured in vivo glucose utilization by the hyperinsulinemic-euglycemic clamp method as described previously (12Fujita Y. Kojima H. Hidaka H. Fujimiya M. Kashiwagi A. Kikkawa R. Diabetologia. 1998; 41: 1459-1466Crossref PubMed Scopus (64) Google Scholar) with slight modification. Mice received an insulin infusion (10 milliunits/kg/min) for 90 min. The infusion rate of glucose solution (4%) was adjusted to a target plasma glucose level of 100 mg/dl. Plasma glucose was monitored every 3 min for 90 min. Total body glucose infusion rate was calculated as described (12Fujita Y. Kojima H. Hidaka H. Fujimiya M. Kashiwagi A. Kikkawa R. Diabetologia. 1998; 41: 1459-1466Crossref PubMed Scopus (64) Google Scholar). For high-fat diet feeding experiments, F6 C57BL/6JAdipo −/− and Adipo +/+mice were fed a high-fat/high-sucrose (per Kg feed containing 210 g of milk fat and 341 g of sucrose) diet from Harlan Teklad (TD 88137) for 7 months. For quantitation of β-oxidation, we measured 14CCO2 production from 1-14Cpalmitic acid in isolated soleus muscle as described (13Alam N. Saggerson E.D. Biochem. J. 1998; 334: 233-241Crossref PubMed Scopus (115) Google Scholar) and in liver homogenates as described (14Yu X.X. Drackley J.K. Odle J. J. Nutr. 1997; 127: 1814-1821Crossref PubMed Scopus (38) Google Scholar). Targeted mice were produced by the strategy shown in Fig.1, A and B. Germ line transmission was confirmed by Southern blotting (Fig.1 C). Initially, six Adipo −/− lines were obtained and we bred three of the lines into C57BL/6J. Experiments were performed with identical results in two independent lines. Homologous recombination removed exon 2 of Adipo, which contained the translation initiation codon. Adipo mRNA was absent in the knock-out mice (Fig. 1 D), and adipo protein was undetectable in the plasma of knockout mice (Fig. 1 E). As the immunoblot was performed with an antibody against the C-terminal fragment of adipo, together with the Northern blot, it excludes the expression of a truncated adipo from a downstream translation initiation codon. Adipo −/− mice were fertile, and the null allele was transmitted to the progeny in a Mendelian pattern. Adipo −/− mice were backcrossed into C57BL/6 for six generations.Adipo −/− and wild-type mice had identical body weights (data not shown) and similar fat pad weights (percent body weights, mean ± S.E.; male epididymal: −/−, 1.17 ± 0.19; +/+, 0.88 ± 0.19; female parametrial: −/−, 1.05 ± 0.11; +/+, 1.33 ± 0.09), as well as plasma lipids (TableIA) and leptin (mean ± S.E., male: −/−, 2.31 ± 0.34 ng/ml; +/+, 1.95 ± 0.41 ng/ml; female: −/−, 3.22 ± 0.38 ng/ml; +/+, 2.82 ± 0.55 ng/ml).Table ICharacterization of Adipo−/− miceA. Plasma lipid levels at 8 weeksAdipo+/+ (n = 11)Adipo+/− (n = 11)Adipo−/−(n = 8)Cholesterol (mg/dl)84.47 ± 7.3489.35 ± 5.55101.24 ± 8.19Triglyceride (mg/dl)45.43 ± 4.3446.22 ± 2.0943.67 ± 2.92Free fatty acid (nm)0.97 ± 0.100.83 ± 0.050.79 ± 0.08Glycerol (mg/dl)110.79 ± 17.07107.23 ± 11.0093.93 ± 14.60B. Hyperinsulinemic-euglycemic clampAdipo+/+ (n = 13)Adipo−/−(n = 7)Glucose infusion rate (mg/Kg/min)8.02 ± 0.72710.56 ± 1.318Serum insulin during clamp (ng/ml)18.80 ± 2.94923.44 ± 1.398Serum glucose during clamp (mg/dl)105.98 ± 1.180101.72 ± 0.525C. Fatty acid oxidationAdipo+/+Adipo−/−14CPalmitate → CO2(nmol/g/h)Muscle1.58 ± 0.072.33 ± 0.394ap ≤ 0.05 (n = 7, Student's t test, paired).Liver10.32 ± 1.9413.39 ± 2.72bp ≤ 0.01 (n = 9, Student's t test, paired).Data represent mean ± S.E.a p ≤ 0.05 (n = 7, Student's t test, paired).b p ≤ 0.01 (n = 9, Student's t test, paired). Open table in a new tab Data represent mean ± S.E. Intraperitoneal GTT performed on F2 and F3 mice revealed no difference between Adipo +/+ mice andAdipo −/− littermates (data not shown). To exclude the possibility that the uneven mixed genetic background of these early-generation mice might have masked subtle changes in glucose homeostasis, we backcrossed the mice into C57BL/6J and performed the experimental analyses in F6 mice. Like their F6 wild-type littermates, both male and female F6 Adipo −/− mice (with >99% C57BL/6J background) had normal fasting plasma glucose and insulin levels; furthermore, there was no difference in these parameters during a 2-h GTT (Fig. 2,A and B). We next performed an ITT, again detecting no difference in the plasma glucose response to insulin between F6 Adipo −/− mice andAdipo +/+ littermates (Fig. 2 C), which suggests that there was no impairment in insulin sensitivity. The GTT and ITT suggest that Adipo −/− mice have no significant glucose intolerance or insulin resistance. To assess insulin resistance with a more stringent test, we studied Adipo −/− and wild-type mice by the hyperinsulinemic-euglycemic clamp technique. As shown in Table IB, Adipo −/− andAdipo +/+ had similar serum steady-state insulin concentration. To maintain a similar serum glucose, the glucose infusion rate tended to be higher in Adipo −/−mice (suggesting that they tended to be more sensitive to insulin), although the difference betweenAdipo −/− and Adipo +/+mice was not significant. Therefore, compared withAdipo +/+ mice, Adipo −/−mice displayed no insulin resistance. To bring out potential subtle changes in Adipo −/−mice that were not evident under basal conditions, we fed these mice a HF/HS diet for 7 months. Plasma lipids ofAdipo −/− and Adipo +/+mice were similar (mean ± S.E., triglyceride: −/−, 73.08 ± 10.81 mg/dl; +/+, 79.60 ± 9.23 mg/dl; cholesterol: −/−, 238.88 ± 28.84 mg/dl; +/+, 261.71 ± 24.94 mg/dl; free fatty acids: 1.67 ± 0.53 mm; +/+, 1.69 ± 0.24 mm) while they were maintained on this diet. They also had similar body weights throughout the 7-month feeding period (data not shown). Both wild-type and knock-out mice developed mild fasting hyperglycemia and fasting hyperinsulinemia (compare Figs. 2 and3), indicating that the diet-induced weight gain produced similar degrees of mild insulin resistance in mice that produced adipo as well as those that lacked the protein. The plasma glucose and insulin response of both types of mice to GTT was also similar (Fig. 3, A and B). Although the plasma insulin in wild-type mice was higher than that ofAdipo −/− mice during the GTT, the difference was not significant (Fig. 3 B). We next performed an ITT, which revealed that Adipo −/− mice and theirAdipo +/+ littermates had a similar plasma glucose response to insulin (Fig. 3 C). Therefore, there was no difference in insulin sensitivity (or resistance) inAdipo +/+ or Adipo −/−mice. This was true irrespective of whether the comparison was made while the animals were on regular chow or after they were fed a HF/HS diet for 7 months. As recombinant fragments of adipo were reported to stimulate β-oxidation in muscle of rodents (6Fruebis J. Tsao T.-S. Javorschi S. Ebbets-Reed D. Erickson M.R. Yen F.T. Bihain B.E. Lodish H.F. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 2005-2010Crossref PubMed Scopus (1770) Google Scholar, 7Yamauchi T. Kamon J. Waki H. Terauchi Y. Kubota N. Hara K. Mori Y. Ide T. Murakami K. Tsuboyama-Kasaoka N. Ezaki O. Akanuma Y. Gavrilova O. Vinson C. Reitman M.L. Kagechika H. Shudo K. Yoda M. Nakano Y. Tobe K. Nagai R. Kimura S. Tomita M. Froguel P. Kadowaki T. Nat. Med. 2001; 7: 941-946Crossref PubMed Scopus (4099) Google Scholar), we measured the β-oxidation activity of soleus muscle isolated from Adipo −/− andAdipo +/+ mice. Unexpectedly, we found that β-oxidation rate was significantly increased (by ∼47%) in the muscle of Adipo −/− mice compared with wild-type littermate controls (Table IC). The β-oxidation rate in liver homogenates of these animals was also determined and was found to be ∼30% higher in Adipo −/− mice than that in wild-type littermate controls. Therefore, the absence of adipo stimulates β-oxidation. While we were preparing this manuscript for publication, Kubotaet al. (15Kubota N. Terauchi Y. Yamauchi T. Kubota T. Moroi M. Matsui J. Eto K. Yamashita T. Kamon J. Satoh H. Yano W. Froguel P. Nagai R. Kimura S. Kadowaki T. Noda T. J. Biol. Chem. 2002; 277: 25863-25866Abstract Full Text Full Text PDF PubMed Scopus (1192) Google Scholar) reported that F1 and F2 Adipoknock-out mice displayed insulin resistance; they did not examine β-oxidation in their animals. The reason for the difference between our study and that of Kubota et al. (15Kubota N. Terauchi Y. Yamauchi T. Kubota T. Moroi M. Matsui J. Eto K. Yamashita T. Kamon J. Satoh H. Yano W. Froguel P. Nagai R. Kimura S. Kadowaki T. Noda T. J. Biol. Chem. 2002; 277: 25863-25866Abstract Full Text Full Text PDF PubMed Scopus (1192) Google Scholar) is unclear, but may be related to environmental factors or genetic background. We did not detect any significant difference in glucose homeostasis in F2 and F3 mice produced in our laboratory. The variation among F2 and F3 mice was higher than that among F6 C57BL/6J mice, presumably because of the inhomogeneous genetic background in the former groups. None of these differences were found to be significant in large groups. To rule out the mixed genetic background as a factor that might have masked differences between knock-out and wild-type mice, we bred them into C57BL/6J background and re-examined the mice at the F6 generation. Again, there was no difference in glucose tolerance or insulin sensitivity between Adipo −/− andAdipo +/+ mice (Figs. 2 and 3). This lack of a difference was evident in regular chow-fed mice as well as in mice fed a HF/HS diet for 7 months. The high-fat diet did induce a mild, but similar, degree of glucose intolerance and insulin resistance inAdipo −/− and Adipo +/+mice (Fig. 3). Interestingly, both Kubota et al. (15Kubota N. Terauchi Y. Yamauchi T. Kubota T. Moroi M. Matsui J. Eto K. Yamashita T. Kamon J. Satoh H. Yano W. Froguel P. Nagai R. Kimura S. Kadowaki T. Noda T. J. Biol. Chem. 2002; 277: 25863-25866Abstract Full Text Full Text PDF PubMed Scopus (1192) Google Scholar) and we observed no difference between knock-out and wild-type mice in their plasma insulin level in the postabsorptive, i.e. fasting state, or after a glucose load. In both studies the plasma insulin during a GTT tended to be (insignificantly) higher in the wild-type mice than in Adipo −/− mice (Fig. 2 Jin Kubota et al. (15Kubota N. Terauchi Y. Yamauchi T. Kubota T. Moroi M. Matsui J. Eto K. Yamashita T. Kamon J. Satoh H. Yano W. Froguel P. Nagai R. Kimura S. Kadowaki T. Noda T. J. Biol. Chem. 2002; 277: 25863-25866Abstract Full Text Full Text PDF PubMed Scopus (1192) Google Scholar) and Figs. 2 B and3 B in this study). These are unlikely scenarios if significant insulin resistance existed. The absence of insulin resistance in Adipo −/− mice was also confirmed by the hyperinsulinemic-euglycemic clamp method in the current study (Table IB). How can we reconcile the absence of insulin resistance inAdipo −/− mice and the previously reported effects of full-length and partial-length adipo fragments injected into rodents (6Fruebis J. Tsao T.-S. Javorschi S. Ebbets-Reed D. Erickson M.R. Yen F.T. Bihain B.E. Lodish H.F. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 2005-2010Crossref PubMed Scopus (1770) Google Scholar, 7Yamauchi T. Kamon J. Waki H. Terauchi Y. Kubota N. Hara K. Mori Y. Ide T. Murakami K. Tsuboyama-Kasaoka N. Ezaki O. Akanuma Y. Gavrilova O. Vinson C. Reitman M.L. Kagechika H. Shudo K. Yoda M. Nakano Y. Tobe K. Nagai R. Kimura S. Tomita M. Froguel P. Kadowaki T. Nat. Med. 2001; 7: 941-946Crossref PubMed Scopus (4099) Google Scholar)? The overall phenotype ofAdipo −/− mice could be the result of the activation or overexpression of compensatory biochemical pathways or molecules in Adipo −/− mice that reversed the insulin resistance, if indeed adipo is a natural insulin-sensitizing hormone in vivo. The expression of leptin is largely unchanged in these mice, but there are potentially many other molecules involved in insulin resistance that could be activated in the absence of adipo (16Havel P.J. Curr. Opin. Lipid. 2002; 13: 51-59Crossref PubMed Scopus (525) Google Scholar). Another consideration is the fact that the data obtained in many of the experiments involving the injection of recombinant adipo into rodents may not reflect the normal action of the native protein. Adipo normally exists in plasma as trimers that associate noncovalently to form high molecular complexes, and most of the injected fragments were not competent to reassemble into the native multimeric complexes in vivo (17Berg A.H. Combs T.P. Scherer P.E. Trends Endocrinol. Metab. 2002; 13: Full Text Full Text PDF PubMed Scopus Google Scholar). adipo (17Berg A.H. Combs T.P. Scherer P.E. Trends Endocrinol. Metab. 2002; 13: Full Text Full Text PDF PubMed Scopus Google Scholar, C. N. T. K. J. Biol. Chem. 2001; 276: Full Text Full Text PDF PubMed Scopus Google Scholar), which does not in recombinant Adipo acid that to be C. N. T. K. J. Biol. Chem. 2001; 276: Full Text Full Text PDF PubMed Scopus Google recombinant adipo produced in may not this modification. acid have shown to be involved in as well as other Y. M. A. of the Google Scholar, N. H. S. 98: 2005-2010Crossref PubMed Scopus (1770) Google Scholar, 7Yamauchi T. Kamon J. Waki H. Terauchi Y. Kubota N. Hara K. Mori Y. Ide T. Murakami K. Tsuboyama-Kasaoka N. Ezaki O. Akanuma Y. Gavrilova O. Vinson C. Reitman M.L. Kagechika H. Shudo K. Yoda M. Nakano Y. Tobe K. Nagai R. Kimura S. Tomita M. Froguel P. Kadowaki T. Nat. Med. 2001; 7: 941-946Crossref PubMed Scopus (4099) Google Scholar), but also inAdipo −/− mice that lacked native adipo, suggests that the injected fragments might have as molecules that the normal action of native vivo. experiments be to this important While this was under et al. N. K. H. M. H. N. H. M. Y. R. N. S. Y. K. M. S. T. Funahashi T. Matsuzawa Y. Nat. Med. 2002; PubMed Scopus Google Scholar) reported the production of Adipo −/− mice in their laboratory. In with our they observed no of insulin resistance when Adipo −/−mice were fed a regular they found that feeding −/− mice a HF/HS diet for 2 insulin resistance in these animals. The reason for the difference between our which involved a 7-month HF/HS diet feeding and that of et al. N. K. H. M. H. N. H. M. Y. R. N. S. Y. K. M. S. T. Funahashi T. Matsuzawa Y. Nat. Med. 2002; PubMed Scopus Google Scholar) is unclear.