Wnt10b Inhibits Obesity in ob/ob and Agouti Mice

All studies were approved by the university committee on use and care of animals, and daily care of mice was overseen by the unit for laboratory animal medicine. Transgenic mice that express mouse Wnt10b under control of the FABP4 promoter (FABP4-Wnt10b) were described previously (14). FABP4-Wnt10b mice bred to C57BL/6 mice (N2-N3) were crossed with B6.Cg-A^y/J mice (The Jackson Laboratory, Bar Harbor, ME), and F1 progeny were used for analyses. Likewise, FABP4-Wnt10b transgenic mice were crossed with B6.V-Lep^ob/J heterozygous mice (The Jackson Laboratory), and the resulting progeny were interbred and F2 mice used for analyses. Mice had ad libitum access to water and rodent chow (5001 rodent chow; Test Diets, Brentwood, MO).

To test glucose tolerance, mice were given an intraperitoneal injection of 1.5 mg glucose/g body wt after a 16-h fast. Blood glucose was determined at the indicated times from tail blood using the OneTouch Ultra glucometer (Lifescan, Burnaby, Canada). To determine insulin sensitivity, mice were given an intraperitoneal injection of 1.0 unit insulin/kg body wt after a 16-h fast. Tail blood samples were collected at the indicated times, and glucose concentrations were determined as described above.

White adipose tissue was dissected and fixed in 10% neutral buffered formalin for at least 48 h. After embedding in paraffin, adipose tissue sections were stained with hematoxylin and eosin. Images were taken on an Olympus BX-51 microscope with an Olympus DP70 color digital camera. Adobe Photoshop was used to enhance the contrast of the images before analysis. MetaMorph software (version 6.1; Molecular Devices, Downingtown, PA) was used to calculate cellular area and the number of adipocytes per field with integrated morphometry analysis. Treatment differences were analyzed by ANOVA.

Quantitative real-time PCR was performed as described (14,34). Briefly, RNA was isolated from tissues using RNA Stat 60 (Tel-Test, Friendswood, TX) and further purified using an RNeasy kit (Qiagen, Valencia, CA). RNA was treated with DNase I, and cDNA was synthesized using the TaqMan system (Applied Biosystems, Foster City, CA) and random hexamer primers. SYBR Green I dye (Molecular Probes, Madison, WI) was used to monitor amplification of cDNA on the I-Cycler and IQ real-time PCR detection system (Bio-Rad Laboratories, Hercules, CA). 18S rRNA was the reference gene used to normalize cDNA. All primer sequences were validated as described (35) and are reported in Table 1.

Fat cells were isolated by a procedure modified from that of Rodbell (36). Gonadal adipose tissue was dissected, weighed, placed in ice-cold Krebs-Ringer HEPES buffer, and minced. Adipose tissue was then digested in 5 ml digestion buffer (1X Krebs-Ringer HEPES containing 1–2 mg/ml Collagenase II, C-6885; Sigma Chemical, St. Louis, MO) at 37°C for 45–60 min with continuous shaking (∼150 rpm) (4,36). Undigested tissue was removed by filtering through a 100-μm mesh. Adipocytes were separated from stromal vascular cells by low-speed centrifugation. RNA was prepared and analyzed as described above.

Measurement of oxygen consumption (Vo₂) with indirect calorimetry was performed on mice at 1 year of age. Animals were maintained on 12-h light and dark cycles beginning at 6:00 a.m. and 6:00 p.m., respectively. Mice were acclimated in measuring chambers for 2 weeks before recording. Measurements of Vo₂ were recorded every 24 min over 4 days using the Oxymax System (Columbus Instruments, Columbus, OH). After 3 days, chow was removed and measurements continued for another day.

Mice were housed individually in microisolator cages with wire mesh flooring and acclimated for at least 7 days. Food consumption from 5 consecutive days was used to calculate average daily food intake.

Mice were anesthetized using inhaled isoflurane and scanned using the pDEXA Sabre X-ray bone densitometer (Norland Medical Systems, Fort Atkinson, WI). System software estimated fat, lean, and bone mineral mass for each mouse.

Blood was collected from the tail or orbital plexus and allowed to clot at room temperature for 30 min. Serum was then prepared and stored at −80°C until assayed. Insulin, leptin, resistin, and adiponectin were simultaneously measured on a Luminex 100 machine using the Lincoplex mouse serum adipokine kit according to the manufacturer’s protocol (Linco Research, St. Charles, MO). Insulin was assayed in mice at 1 year of age using the Ultra Sensitive Rat Insulin ELISA kit (Crystal Chem, Downers Grove, IL).

Concentrations of serum nonesterified fatty acids were determined using the Free fatty acid, Half-micro test according to manufacturer’s protocol (Roche Diagnostics, Mannheim, Germany). Serum triacylglycerol was determined according to the manufacturer’s instructions with the Infinity Triglyceride Reagent kit (Sigma Chemical), using glycerol as the standard. Serum cholesterol was determined using the Infinity Cholesterol Reagent kit (Sigma Chemical).

We reported previously that FABP4-Wnt10b transgenic mice resist diet-induced obesity (14). To determine if Wnt10b also inhibits obesity due to leptin deficiency, the FABP4-Wnt10b transgene was transferred onto the ob/ob background. While ob/ob mice demonstrate early-onset obesity with a 50% increase in body weight compared with wild-type mice at 8 weeks of age, Wnt10b-ob/ob mice show a modest, yet statistically insignificant, 11% increase in body weight compared with Wnt10b alone (Fig. 1A). As described previously (14), FABP4-Wnt10b mice weigh more than wild-type mice due to increased thickness of skin (Fig. 1A). Inspection reveals that Wnt10b-ob/ob mice have a remarkable decrease in the epididymal and perirenal adipose depots compared with ob/ob mice (Fig. 1B). Quantification of tissue weight reveals a 65–70% reduction in visceral and subcutaneous adipose tissue. Although weights of heart, lung, kidneys, and spleen were not different (data not shown), increased weight of pancreas and liver was observed in Wnt10b-ob/ob mice (Fig. 1C). Profound hepatic steatosis was observed in both ob/ob and Wnt10b-ob/ob mice (data not shown). Taken together, these data indicate that obesity due to leptin deficiency is inhibited by expression of Wnt10b in adipose tissue.

To further investigate the ability of Wnt10b to avert genetic obesity, we crossed FABP4-Wnt10b and lethal yellow agouti (A^y) mice. Ectopic expression of agouti in A^y mice causes mild hyperphagia, late-onset obesity and severe hyperinsulinemia (32,33). In our studies, A^y and Wnt10b-A^y mice had similar body weights at 4 months of age. However, analysis of body composition by dual-energy X-ray absorptiometry (DEXA) revealed a >50% decrease in fat mass in the Wnt10b-A^y mice, with a concomitant increase in lean mass (Fig. 2A). Quantification of adipose depots revealed that reduction in total body lipid in Wnt10b-A^y mice was accounted for by decreased white adipose tissue. Weights of visceral and subcutaneous adipose depots in Wnt10b-A^y mice were reduced by 55 and 70%, respectively (Fig. 2B and C). Average adipocyte size was not different between genotypes (data not shown), consistent with a large body of data indicating that Wnt signaling impairs adipogenesis. Reduced adipose tissue in Wnt10b-A^y was not due to decreased energy intake, as food consumption was similar between genotypes (data not shown). As in ob/ob mice (Fig. 1C), expression of Wnt10b in A^y mice increases weight of pancreas and liver (Fig. 2C). Histological analysis suggests that both genotypes have hepatic steatosis (not shown). Thus, Wnt10b inhibits accumulation of adipose tissue in two distinct genetic models of obesity.

In addition to obesity, other hallmarks of the lethal yellow agouti phenotype are glucose intolerance, hyperinsulinemia, and insulin resistance (32,37,38). Given the well-known relationship between adiposity and insulin resistance, as well as the reduced amounts of adipose tissue and increased liver and pancreas weights in Wnt10b-A^y mice, we performed experiments to investigate glucose homeostasis in these mice. Random-fed Wnt10b-A^y and A^y littermates were both hyperinsulinemic and exhibited similar blood glucose concentrations (Fig. 3A). To assess glucose tolerance, mice were subjected to an intraperitoneal glucose challenge (Fig. 3B). While A^y mice are glucose intolerant, Wnt10b-A^y mice had significantly lower blood glucose levels at fasted baseline and at every time point thereafter. Although both A^y and Wnt10b-A^y mice were relatively insensitive to insulin, blood glucose levels in Wnt10b-A^y mice were lower than in A^y mice at each time point after administration of insulin (Fig. 3C). While blood glucose from A^y mice failed to fall below baseline during the assay, littermate Wnt10b-A^y mice showed a decline in blood glucose observed 45 min after intraperitoneal insulin challenge (Fig. 3C). Therefore, expression of Wnt10b in adipose tissue improves whole-body glucose homeostasis in genetically obese mice, similar to results observed with diet-induced obesity (14).

To gain insight into potential causes of improved glucose tolerance and insulin sensitivity, we evaluated the levels of key serum adipokines. Consistent with previous results in nonagouti mice (14), Wnt10b-A^y mice had a 22% reduction in total adiponectin compared with A^y mice (Fig. 3D). In addition, Wnt10b-A^y mice showed a 63% reduction in leptin, correlating with reduced adipose tissue mass. Notably, resistin levels in serum and expression of resistin in adipose tissue were decreased in Wnt10b-A^y mice (Fig. 3D and E), possibly contributing to improved glucose tolerance.

It has been proposed that obesity-related insulin resistance is secondary to chronic inflammation in adipose tissue (39–43). Thus, we next examined expression of proinflammatory genes in epididymal adipose tissue of A^y and Wnt10b-A^y mice. Interleukin-6 and TNF-α are two proinflammatory cytokines commonly elevated in obesity and both have been implicated in insulin resistance (39). While interleukin-6 was similarly expressed in Wnt10b-A^y mice (data not shown), expression of TNF-α mRNA was reduced by ∼50% compared with A^y mice (Fig. 3E). Obesity is accompanied by increased expression of the chemokine MCP-1 in adipose tissue, where MCP-1 plays a roll in recruiting macrophages and stimulating insulin resistance (44). Our assessment of mRNA levels of MCP-1 revealed that expression of MCP-1 is decreased in Wnt10b-A^y mice. While only a trend toward reduced expression of F4/80 was also noted (not shown), iNOS expression, one possible mediator of inflammation-associated insulin resistance (43,45), is reduced by 73% in white adipose tissue of Wnt10b-A^y mice (Fig. 3E). Thus, improved glucose tolerance and insulin sensitivity in Wnt10b-A mice may be due, in part, to reduced recruitment and/or activation of resident macrophage and decreased inflammation in adipose tissue.

Expansion of adipose tissue mass in agouti mice occurs relatively gradually, with severe obesity observed only later in life. To assess whether Wnt10b continues to provide protection against obesity with age, we compared phenotypes of female A^y and Wnt10b-A^y mice at 1 year of age. Whereas agouti mice gained >20 g of weight between 5 and 12 months of age, weights of Wnt10b-A^y mice remained relatively constant, with Wnt10b-A^y weighing ∼40% less than A^y littermates by 12 months of age (Fig. 4A). The reduction in body weight is largely due to a striking decrease in visceral and subcutaneous adipose depots, with 80–90% less ovarian, perirenal, and dorso-lumbar adipose tissue (Fig. 4A). These data indicate that Wnt10b-A^y mice resist late-onset obesity associated with the agouti mutation.

Histomorphometric analysis was used to establish that the decrease in adipose tissue weight (Fig. 4A) was not due to decreased adipocyte size (Fig. 4B). Furthermore, molecular characterization of adipocytes isolated from Wnt10b-A^y mice revealed that many adipocyte genes are expressed normally, with the notable exception of resistin, which was decreased by 85% (Fig. 4C). Taken together, these data suggest that protection against genetic obesity by Wnt10b is through a reduction in adipocyte number rather than altered adipocyte metabolism and or function.

Analysis of blood glucose and serum insulin at 12 months showed no difference in the fed state; however, a slight decrease in blood glucose and a substantial 60% decrease in serum insulin in the fasted state suggest that Wnt10b-A^y mice remain more sensitive to insulin as they age (Fig. 4D). Analysis of adipokines revealed a 30–50% reduction in serum leptin, an ∼98% decrease in serum resistin, and a 55–70% reduction in total adiponectin in Wnt10b-A^y mice. Analyses of serum lipids showed no statistical change in triglyceride or free fatty acid concentrations (Table 2). As observed at 6 months of age, Wnt10b-A^y mice show a dramatic decrease in expression of MCP-1, F4/80, and iNOS mRNAs in perirenal adipose tissue at 12 months of age, although altered TNF-α was not observed (Fig. 4E). Thus, decreased inflammation in adipose tissue of Wnt10b-A^y mice is a possible basis for improved insulin sensitivity in these mice as they age.

We next investigated energy balance in A^y and Wnt10b-A^y mice to determine if altered food intake or oxygen consumption contributed to the ability of Wnt10b-A^y mice to resist expansion of their adipose tissues with age. As expected based on analysis of younger A^y and Wnt10b-A^y mice, as well as prior analysis of FABP4-Wnt10b mice (14), food consumption was not altered by the Wnt10b transgene (Fig. 5A; not shown), although a trend toward reduced intake was observed. We then used indirect calorimetry to investigate oxygen consumption and respiratory quotient. There was no significant change in total oxygen consumption under random-fed or fasted conditions (Fig. 5B). Furthermore, respiratory quotient (Fig. 5B) under random-fed and fasted conditions was similar between genotypes. By DEXA, Wnt10b-A^y mice have far less adipose mass but have a surprising increase in lean mass that is not accounted for by weights of individual muscles or other tissues/organs (Fig. 5C and Table 2). Taken together, it appears likely that differences in body weight and accumulation of adipose tissue are due to very small, insensible changes in energy balance as these mice age.

In addition to decreasing adiposity, activation of Wnt signaling in Wnt10b-A^y greatly improves glucose homeostasis due to improved insulin sensitivity (Fig. 3). Similar findings were observed with FABP4-Wnt10b mice on a chow or high-fat diet (14). Based on analysis of glucose homeostasis in mice with tissue-selective loss of insulin receptors, it is likely that improved insulin sensitivity decreases hepatic glucose production in Wnt10b-A^y mice; however, euglycemic-hyperinsulinemic clamp studies will be required to confirm this hypothesis. Surprisingly, phosphorylation of Akt in liver and muscle 5 min after administration of insulin to wild-type or FABP4-Wnt10b mice is not altered (data not shown), suggesting a mechanism downstream of Akt. While at 1 year of age, absolute weights of liver and pancreas are not different between genotypes, Wnt10b-A^y mice have larger livers and pancreata when expressed relative to total body weight. This idea is supported by increased size of these organs when the Wnt10b transgene is expressed on a mixed genetic background (14) or in the absence of leptin (Fig. 1). As observed in mice deficient for lipin (46), hepatocytes in FABP4-Wnt10b mice transiently accumulate neutral lipid in the postnatal period (not shown); however, our analysis of liver glycogen, lipid content, total protein, and tissue morphology later in life did not reveal altered composition (not shown). Thus, it may be that some of the improved glucose homeostasis comes from increased metabolic capacity of liver and pancreas. Whether Wnt signaling influences the chalone that regulates size of liver or pancreas will require additional study.

Our analysis of energy balance in A^y and Wnt10b-A^y mice led to paradoxical findings. While a trend toward reduced food intake was observed, there was no significant change in total oxygen consumption under random-fed or fasted conditions (Fig. 5B). In addition, Wnt10b-A^y mice consumed similar amounts of oxygen throughout the day (not shown), providing further evidence that Wnt10b-A^y mice do not differentially enter torpor while in the postabsorptive state, nor that the lack of brown adipose tissue in FABP4-Wnt10b mice influences oxygen consumption under these conditions (14). Average respiratory quotient (Fig. 5B) under random-fed and fasted conditions was similar between genotypes, suggesting that oxidation of fuels is regulated normally in Wnt10b-A^y mice. However, the normal fluctuations over a 24-h period were blunted in Wnt10b-A^y mice (not shown), perhaps due to increased nibbling throughout the day or decreased metabolic flexibility. Although expressing oxygen consumption relative to body mass^0.75 suggests that Wnt10b-A^y mice have reduced body weight due to increased metabolic rate, this may not be appropriate in light of the large differences in body composition (Fig. 5C). However, given the large increase in apparent lean mass, normalizing oxygen consumption to lean body mass gives the paradoxical finding that Wnt10b-A^y mice have reduced metabolic rate (Fig. 5C). While the profound alterations in fat content of the Wnt10b-A^y mice as assessed by DEXA are closely aligned with our measurements of adipose tissue weights, the source of increased lean mass in Wnt10b-A^y mice remains elusive as the additional 10 g of “nonfat nonmineral” mass cannot be explained by the weights of individual muscles or other tissues/organs (Fig. 5C and Table 2), perhaps suggesting that Wnt signaling in this context alters hemodynamics to increase total water content. Some of the lean mass may also be from weight of skin, which although not measured in this experiment, is increased in FABP4-Wnt10b mice (14).

Our analyses of adipokine expression suggests that while expression of adiponectin and leptin are not changed on a per-adipocyte basis (Fig. 4C), circulating levels of both of these factors are decreased in serum (Figs. 3 and 4). Based on these experiments, improved glucose tolerance is unlikely to be associated with either adiponectin or leptin, although differential sensitivity to these factors cannot be ruled out. A caveat of this experiment is that we measured total adiponectin; thus, we do not have a measure of the more biologically active higher-order forms (47). Additionally, increased insulin sensitivity could be due, in part, to the striking decrease in expression of resistin in adipocytes and adipose tissue and the corresponding lower levels in circulation (48) (Figs. 3 and 4). Although resistin may contribute to increased insulin sensitivity in Wnt10b-A^y mice, improved glucose homeostasis is more likely associated with decreased inflammation in adipose tissue, as assessed by expression of F4/80 and iNOS (Figs. 3 and 4). Fewer macrophages could be secondary to inhibition of obesity (39,40,49), and the corresponding decrease in expression of MCP-1 (Figs. 3 and 4) from preadipocytes, endothelial cells, and adipocytes (42). Reduced MCP-1 in Wnt10b-A^y mice may also influence insulin sensitivity independent of macrophage recruitment, as exposure of differentiated 3T3-L1 adipocytes to MCP-1 decreases expression of adipocyte genes, including GLUT4 and peroxisome proliferator–activated receptor γ, and impairs insulin-stimulated glucose uptake (42). Although controversial (50), decreased signaling by the MCP-1 receptor, CCR2 (C-C motif chemokine receptor-2), has been reported to improve insulin sensitivity and glucose homeostasis in obese mice (51). Alternatively, macrophage development or activation could be influenced by expression of Wnt10b in bone marrow, macrophages, or adipose tissue. Along with decreased MCP-1, we observed that TNF-α, an inflammatory cytokine well known for stimulating insulin resistance in adipose tissue, is expressed at lower levels in 6-month-old mice. Consistent with decreased activity of inflammatory cytokines, we observed decreased expression of a downstream target, iNOS, at both 6 months and 1 year of age (Figs. 3 and 4). iNOS is normally elevated in adipose tissues and muscle of genetic and dietary models of obesity (43) and is associated with insulin resistance in muscle. Thus, improved glucose homeostasis in Wnt10b-A^y mice is probably caused by reduced resistin and decreased recruitment and activation of macrophages in adipose tissues. Alternatively, increased Wnt10b signaling in adipose tissue may alter whole-body insulin sensitivity through a unique mechanism.

This work was supported by grants from the National Institutes of Health to O.A.M. (DK51563 and DK62876). Other support was from the Nathan Shock Mutant and Transgenic Rodent Core. Fellowships were from Tissue Engineering and Regeneration Training Grant, Center for Organogenesis, a Rackham predoctoral fellowship, a mentor-based postdoctoral fellowship from the American Diabetes Association, and the Belgian Fonds National de la Recherche Scientifique. This work used the Morphology and Image Analysis Core of the Michigan Diabetes Research and Training Center, which is funded by NIH5P60 DK20572 from the National Institute of Diabetes and Digestive and Kidney Diseases of the National Institutes of Health.