Abstract
Obesity is an important independent risk factor for type 2 diabetes, cardiovascular diseases and many other chronic diseases. Adipose tissue inflammation is a critical link between obesity and insulin resistance and type 2 diabetes and a contributor to disease susceptibility and progression. The objective of this study was to determine the role of response gene to complement 32 (RGC32) in the development of obesity and insulin resistance. WT and RGC32 knockout (Rgc32−/− (Rgcc)) mice were fed normal chow or high-fat diet (HFD) for 12 weeks. Metabolic, biochemical, and histologic analyses were performed. 3T3-L1 preadipocytes were used to study the role of RGC32 in adipocytes in vitro. Rgc32−/− mice fed with HFD exhibited a lean phenotype with reduced epididymal fat weight compared with WT controls. Blood biochemical analysis and insulin tolerance test showed that RGC32 deficiency improved HFD-induced dyslipidemia and insulin resistance. Although it had no effect on adipocyte differentiation, RGC32 deficiency ameliorated adipose tissue and systemic inflammation. Moreover, Rgc32−/− induced browning of adipose tissues and increased energy expenditure. Our data indicated that RGC32 plays an important role in diet-induced obesity and insulin resistance, and thus it may serve as a potential novel drug target for developing therapeutics to treat obesity and metabolic disorders.
Introduction
Obesity is an important independent risk factor for type 2 diabetes and cardiovascular diseases, and it is associated with high morbidity and mortality (Friedman 2003). Although a range of therapies have been developed, these pharmaceutical therapies often have adverse side effects or limited efficacy (Bandyopadhyay 2009, Distefano & Watanabe 2010). Therefore, a continued effort is required to identify novel drug targets and develop more effective therapeutics for the treatment of obesity.
Adipose tissue plays a critical role in energy homeostasis, not only in storing triglycerides but also in secreting hormones and biologically active molecules that regulate cellular lipid storage capacity, tissue and systemic insulin sensitivity, and metabolic energy balance (Chatterjee et al. 2014). Although the underlying mechanism is not fully understood, adipose tissue inflammation has been acknowledged as a critical link between obesity and insulin resistance and type 2 diabetes, and as a contributor to disease susceptibility and progression (Mori et al. 2010). Adipose tissue inflammation, characterized by increased secretion of proinflammatory cytokines and chemokines including interleukin 6 (IL6), tumor necrosis factor α (TNFα), and IL12, contributes to low-grade systemic inflammation, insulin resistance, and metabolic disorders (Xu et al. 2003, Hotamisligil 2006). Blocking the function of proinflammatory cytokines or chemokines results in improved insulin sensitivity and glucose homeostasis (Uysal et al. 1997, Kanda et al. 2006). Therefore, genes or molecules that control obesity or adipose tissue inflammation are promising therapeutic targets for insulin resistance, type 2 diabetes, and cardiovascular complications.
Response gene to complement 32 (RGC32) is expressed in numerous human organs and tissues including placenta, kidney, liver, heart, and brain (Badea et al. 1998). Functionally, RGC32 plays an important role in cell proliferation, differentiation (Li et al. 2007, Fosbrink et al. 2009, Wang et al. 2011), fibrosis (Li et al. 2011a), and cancer (Fosbrink et al. 2005, Vlaicu et al. 2010, Kim et al. 2011). Recently, we have generated RGC32 knockout (Rgc32−/− (Rgcc)) mice and found that Rgc32−/− mice were born smaller than their WT littermates because of the impaired placental angiogenesis (Cui et al. 2013). However, it is unknown if RGC32 plays a role in postnatal metabolism. In this study, we demonstrate that high-fat diet (HFD) dramatically induces RGC32 expression in the adipose tissue. RGC32 deficiency attenuates HFD-induced obesity and insulin resistance in mice. The beneficial effect of RGC32 deficiency is due to the decreased adipose tissue content and systemic inflammation and increased energy expenditure of adipose tissue. This is the first report showing that RGC32 deficiency prevents diet-induced obesity and insulin resistance in mice. Therefore, RGC32 may serve as a potential novel drug target for preventing obesity and type 2 diabetes.
Materials and methods
Animals and diets
Rgc32−/− mice on the C57BL/6 background were generated and genotyped as described previously (Cui et al. 2013). The parallel line WT C57BL/6 mice were purchased from the Jackson Laboratory (Bar Harbor, ME, USA). The age-matched WT and Rgc32−/− male mice were maintained on normal chow for 8 weeks, after which they were fed with either normal chow (25% protein, 62% carbohydrate, and 13% fat; 3.07 kcal/g; 5053, LabDiet, St. Louis, MO, USA) or HFD (20% protein, 40% carbohydrate, and 40% fat; 4.5 kcal/g; D12108C, Research Diets, New Brunswick, NJ, USA) for an additional 12 weeks. The mice were fasted overnight and anesthetized (2.0% isoflurane), and blood was collected by direct cardiac puncture. Epididymal fat was carefully removed and weighed. A portion of the epididymal fat was fixed in 4% paraformaldehyde for histological analysis, whereas the other portion was stored at −80 °C for RNA and protein preparation. All animals were housed under conventional conditions in the animal care facilities and received humane care in compliance with the Principles of Laboratory Animal Care formulated by the National Society for Medical Research and the Guide for the Care and Use of Laboratory Animals. All experimental procedures were approved by Institutional Animal Care and Use Committee (IACUC) of University of Georgia.
Body weight and metabolic studies
Mice were weighed every 4 weeks. For metabolic studies, the mice were housed individually in metabolic cages (model 3600M021, Techniplast, Buguggiate, VA, Italy) with free access to food and water. After a 5-day period of acclimatization, daily food and water intakes were recorded, and fecal pellets and urine were weighed. The mice were observed for 3 days in the metabolic cage and then were put back in their original cages. To assess the energy expenditure, the mice were fasted overnight and weighed. This body weight was used as a baseline. The mice were fasted for an additional 8 h and weighed, and then the percentage change in the body weight from the baseline was calculated.
Glucose and insulin tolerance tests
For the glucose tolerance test (GTT), mice were fasted overnight followed by an i.p. glucose injection (1 g/kg body weight). Blood glucose was measured by tail bleeding using the One-Touch AccuChek Glucometer (Roche) at indicated times. For the insulin tolerance test (ITT), mice without fast were injected intraperitoneally with insulin (Sigma–Aldrich) at a dose of 1.5 IU/kg body weight, and blood glucose was measured at indicated times.
Blood biochemical analysis
Serum samples were analyzed for adiponectin, leptin, insulin, triglyceride, and cholesterol using the Adiponectin Mouse ELISA Kit (Abcam, Cambridge, MA, USA), Leptin Mouse ELISA Kit (Abcam), Rat/Mouse Insulin ELISA Kit (Millipore, Billerica, MA, USA), Triglyceride Quantification Kit (Abcam), and HDL and LDL/VLDL Cholesterol Assay Kit (Abcam).
Cytometric bead array immunoassay
Serum from individual mouse was assayed for inflammatory markers using the Cytometric Bead Array Mouse Inflammation Kit (BD Biosciences, San Jose, CA, USA). Data were collected by FACSCalibur flow cytometer (Becton Dickinson, Franklin Lakes, NJ, USA).
3T3-L1 cell culture and differentiation
3T3-L1 preadipocytes (ATCC, Manassas, VA, USA) were maintained in DMEM (Invitrogen) supplemented with 10% FBS (Sigma). At confluence, adipocyte differentiation was induced by treatment with 1 μmol/l dexamethasone (Sigma), 500 μmol/l isobutylmethylxanthine (Sigma), 10 μmol/l pioglitazone, and 100 nmol/l insulin (Sigma) for 2 days. The cells were then incubated in 100 nmol/l insulin-containing medium for 6 days to induce lipid accumulation. The medium was replaced every other day.
Oil Red O staining
Culture plates were washed by PBS, and cells were fixed in 4% formaldehyde for 1 h, followed by staining with Oil Red O (Sigma) for 1 h. Oil Red O was prepared by diluting a stock solution (0.5% in isopropanol) with water (60:40 vol/vol) followed by filtration. After staining, the plates were washed twice in water and photographed. The dye was then extracted with 100% isopropanol, and the absorbance was determined at 520 nm.
RNA extraction and real-time quantitative RT-PCR
Quantitative RT-PCR (qPCR) was performed in a Mx3005P qPCR machine using SYBR Green master mix (Agilent Technologies, Santa Clara, CA, USA) as described previously (Li et al. 2011b). Each sample was amplified in triplicate. The expression of each gene was normalized with cyclophilin. Primer sequences are summarized in Table 1.
Primer sequences utilized for qPCR evaluation of gene expression
| Gene | Primer sequence |
|---|---|
| Cyclophilin | F: 5′-GTG GTC TTT GGG AAG GTG AA-3′ |
| R: 5′-TTA CAG GAC ATT GCG AGC AG-3′ | |
| Rgc32 | F: 5′-CCG ATC TGG ACA GGA CCT TA-3′ |
| R: 5′-AGC TTC ACT CTC CGA ACT GC-3′ | |
| Pparγ | F: 5′-ATCCCTGGTTTCATTAACCT-3′ |
| R: 5′-GCTCCATAAAGTCACCAAAG-3′ | |
| Pparα | F: 5′-GGATGTCACACAATGCAATTCG-3′ |
| R: 5′-TCACAGAACGGCTTCCTCAGGT-3′ | |
| C/Ebpα | F: 5′-CAAGAACAGCAACGAGTACCG-3′ |
| R: 5′-GTCACTGGTCAACTCCAGCAC-3′ | |
| Adiponectin | F: 5′-TGTTCCTCTTAATCCTGCCCA-3′ |
| R: 5′-CCAACCTGCACAAGTTCCCTT-3′ | |
| Leptin | F: 5′-GTGGCTTTGGTCCTATCTGTC-3′ |
| R: 5′-CGTGTGTGAAATGTCATTGATCC-3′ | |
| Lipe | F: 5′-AAGGACTTGAGCAACTCAGA-3′ |
| R: 5′-TTGACTATGGGTGACGTGTA-3′ | |
| Pgc1α | F: 5′-ATACCGCAAAGAGCACGAGAA-3′ |
| R: 5′-CTCAAGAGCAGCGAAAGCGTCACA-3′ | |
| Il6 | F: 5′-CTGCAAGAGACTTCCATCCAG-3′ |
| R: 5′-AGTGGTATAGACAGGTCTGTTGG-3′ | |
| Il12 | F: 5′-TGGTTTGCCATCGTTTTGCTG-3′ |
| R: 5′-ACAGGTGAGGTTCACTGTTTCT-3′ | |
| Tnfα | F: 5′-CAGGCGGTGCCTATGTCTC-3′ |
| R: 5′-CGATCACCCCGAAGTTCAGTAG-3′ | |
| Ucp1 | F: 5′-TATCATCACCTTCCCGCTG-3′ |
| R: 5′-GTCATATGTTACCAGCTCTG-3′ | |
| Prdm16 | F: 5′-AGCCCTCGCCCACAACTTGC-3′ |
| R: 5′-TGACCCCCGGCTTCCGTTCA-3′ |
Western blotting analysis
Western blotting was performed as described previously (Cui et al. 2012). Antibodies against hormone-sensitive lipase (LIPE, Cell Signaling Technology, 4107S, Danvers, MA, USA), peroxisome proliferator-activated receptor α (PPARα), (Abcam, ab8934), PPAR gamma coactivator 1α (PGC1α) (Abcam, ab54481), RGC32, and α-tubulin (Cell Signaling Technology, 9099S) were used. All the antibodies were applied at 1:1000 dilution. Protein expressions were detected using an enhanced chemiluminescence kit (Millipore).
Histological analysis
After fixing in 4% paraformaldehyde overnight, the epididymal fat was dehydrated and embedded in paraffin. The sections (5 μm) were cut with a microtome. The sections were deparaffinized and stained with hematoxylin and eosin (H&E). Images were captured by a Nikon microscope. For quantitative analysis of adipocyte area, eight images of H&E-stained sections were acquired from each animal, and the cross-sectional area of each adipocyte was measured using ImageJ software.
Statistical analyses
Data are presented as means±s.d., and the numbers of independent experiments are indicated for each data set. For statistical analysis, the two groups were compared using two-tailed Student's t-tests, and the four groups were evaluated by two-way ANOVA followed by Bonferroni post hoc tests for multiple comparisons, whereas the three and five groups were evaluated by one-way ANOVA followed by Tukey's multiple comparisons using GraphPad Prism 5.0 software (La Jolla, CA, USA). P<0.05 was considered statistically significant.
Results
RGC32 deficiency prevented HFD-induced obesity
Our previous study has shown that the body size of Rgc32−/− mice is smaller compared with their WT littermates when they are born (Cui et al. 2013). Interestingly, the difference is not as dramatic as they age, suggesting that RGC32 has little effect on the post-natal growth on the regular chow diet. It is unknown, however, whether RGC32 affects HFD-induced obesity. To test this, we fed WT mice with HFD for 12 weeks and then detected RGC32 expression in adipose tissue. We found that RGC32 expression was dramatically upregulated by the HFD (Fig. 1A). To investigate the potential role of RGC32 in obesity, the WT and Rgc32−/− mice were fed with HFD for 12 weeks. The HFD-fed WT mice gained significantly more weight than the normal chow controls. However, Rgc32−/− appeared to diminish the weight gain (Fig. 1B). The weight of epididymal fat pads was also markedly lower in HFD-fed Rgc32−/− mice as compared to HFD-fed WT mice, although it was increased compared with the normal chow controls (Fig. 1C). Histological analysis of epididymal fat showed that HFD induced a significant adipocyte hypertrophy (more than fivefold) in WT mice. However, this effect was significantly reduced in Rgc32−/− mice (Fig. 1D and E). To determine whether the lean phenotype of Rgc32−/− mice was due to a reduced energy intake, we housed the mice individually in metabolic cages and monitored the food intake. As shown in Fig. 1F, the energy intake of WT and Rgc32−/− mice fed with HFD was increased compared with the normal chow controls, while there was no difference between WT and Rgc32−/− mice fed on either normal chow or HFD. There were also no significant differences in the water intake, urine, and feces (data not shown). To assess the energy expenditure, we measured the body weight before and after an 8-h fast. In the absence of energy intake, greater loss of body weight indicates increased energy expenditure. After fasting, although there was no significant difference between WT and Rgc32−/− mice under chow conditions, HFD-fed Rgc32−/− mice lost more body weight than HFD-fed WT mice (Fig. 1G), suggesting that the energy expenditure was increased in HFD-fed Rgc32−/− mice, which may, at least partially, be responsible for the lean phenotype of HFD-fed Rgc32−/− mice.
RGC32 deficiency prevented HFD-induced obesity. (A) RGC32 expression in adipose tissue of WT mice fed with normal chow or a 12-week high-fat diet (HFD) were detected by western blotting and normalized to α-tubulin (n=3). (B) Body weight of WT and Rgc32−/− mice fed with normal chow (n=6) or HFD (n=10). (C) Weights of epididymal fat from WT and Rgc32−/− mice fed on normal chow and HFD. (D) Representative H&E-stained images of epididymal fat from WT and Rgc32−/− mice fed on normal chow and HFD. (E) Quantitative analysis of the mean adipocyte area. The areas were normalized to the mean adipocyte area of WT mice fed on normal chow. (F) Energy intake of WT and Rgc32−/− mice fed on normal chow and HFD. (G) Body weight change of WT and Rgc32−/− mice after an 8-h fast. **P<0.01 compared with WT chow group, ##P<0.01 compared with WT HFD group. A full colour version of this figure is available at http://dx.doi.org/10.1530/JOE-14-0548.
Citation: Journal of Endocrinology 224, 2; 10.1530/JOE-14-0548
RGC32 deficiency improved metabolic homeostasis in HFD-fed mice
Diet-induced obesity is typically accompanied by dyslipidemia and insulin resistance. Therefore, we measured serum triglyceride and cholesterol concentrations. No difference was observed between WT and Rgc32−/− mice on normal chow (Fig. 2A and B). However, on HFD, WT mice exhibited significantly increased serum concentrations of triglyceride, HDL cholesterol, and LDL/VLDL cholesterol (Fig. 2A and B). Importantly, Rgc32−/− mice appeared to be resistant to the HFD-induced increase in serum triglyceride and cholesterol. The serum triglyceride and LDL/VLDL cholesterol concentrations in Rgc32−/− mice were not altered by the HFD feeding and thus were much lower compared with the HFD-fed WT control. HDL cholesterol was slightly lower in HFD-fed Rgc32−/− mice than the WT control, although it was increased compared with Rgc32−/− mice fed with normal chow (Fig. 2A and B).
RGC32 deficiency improved metabolic homeostasis in HFD-fed mice. (A) Serum triglyceride (TG), (B) HDL cholesterol, and LDL/VLDL cholesterol concentrations in WT and Rgc32−/− mice fed on normal chow and HFD (n=6). (C) Fasting blood glucose, (D) insulin concentration, and (E) homeostasis model assessment-insulin resistance (HOMA-IR=fasting glucose×fasting insulin/22.5) in WT and Rgc32−/− mice fed on normal chow and HFD (n=6). **P<0.01 compared with WT chow group, ##P<0.01 compared with WT HFD group.
Citation: Journal of Endocrinology 224, 2; 10.1530/JOE-14-0548
To determine whether RGC32 affects insulin sensitivity, blood glucose and serum insulin levels were detected. Rgc32−/− mice showed similar fasting blood glucose and insulin levels compared with WT mice fed with normal chow (Fig. 2C and D). Thus, the homeostasis model assessment-insulin resistance (HOMA-IR) scores had no difference (Fig. 2E). HFD significantly increased fasting blood glucose level, insulin concentration, and HOMA-IR score in WT mice but not in Rgc32−/− mice (Fig. 2C, D, and E), suggesting that insulin sensitivity is improved due to RGC32 deficiency. Improved insulin sensitivity in HFD-fed Rgc32−/− mice was also confirmed using ITT, although there was no difference between WT and Rgc32−/− mice fed with normal chow (Fig. 3A, B, and C). Furthermore, GTT showed that on normal chow, blood glucose levels appeared to be improved at 15 and 30 min (Fig. 3D), and the area under the curve (AUC) was significantly lower in Rgc32−/− mice compared with WT controls (Fig. 3F), indicating that the glucose tolerance was improved in Rgc32−/− mice under chow conditions. HFD-fed WT mice developed severe glucose intolerance, whereas Rgc32−/− mice showed a significantly improved glucose tolerance compared with the HFD-fed WT mice (Fig. 3E and F). These data demonstrate that RGC32 deficiency improves glucose tolerance and protects mice from HFD-induced insulin resistance.
RGC32 deficiency prevents HFD-induced insulin resistance in mice. (A and B) Insulin tolerance test (ITT) in WT and Rgc32−/− mice fed on normal chow (A) and HFD (B) (n=6). (C) Inverse area under the curve (AUC) of ITT. (D and E) Glucose tolerance test (GTT) in WT and Rgc32−/− mice fed on normal chow (D) and HFD (E) (n=6). (F) Quantification of the AUC of GTT. *P<0.05, **P<0.01 compared with WT chow group, #P<0.05, ##P<0.01 compared with WT HFD group.
Citation: Journal of Endocrinology 224, 2; 10.1530/JOE-14-0548
RGC32 deficiency attenuated adipose tissue and systemic inflammation in HFD-fed mice
Since adipose tissue inflammation and low levels of systemic inflammation are important factors contributing to the development of obesity, we detected adipose tissue and serum inflammatory status of the mice. RGC32 deficiency appeared to decrease Il6 and Tnfα mRNA expression in the fat tissue under chow conditions, while had no effect on other adipokines such as adiponectin, leptin, and IL12 (Fig. 4A and B). HFD dramatically decreased adiponectin, an anti-inflammatory adipokine, while increased the expression of pro-inflammatory adipokines leptin, IL6, TNFα, and IL12 in the fat tissue of WT mice. However, these effects were alleviated in Rgc32−/− mice (Fig. 4A and B). Similar results were observed with the circulating adipokines (Fig. 4C and D), although the circulating IL6, TNFα, and IL12 were undetectable under chow conditions (data not shown). These data demonstrate that RGC32 deficiency attenuates adipose tissue and systemic inflammation in HFD-fed mice.
RGC32 deficiency attenuated adipose tissue and systemic inflammation in HFD-fed mice. (A and B) mRNA expression of adiponectin, leptin, interleukin 6 (IL6), tumor necrosis factor α (TNFα), and IL12 in epididymal adipose tissue from WT and Rgc32−/− mice (n=6) was measured by qPCR. (C and D) Protein concentration of adiponectin, leptin, IL6, TNFα, and IL12 in the serum from WT and Rgc32−/− mice (n=6) was measured by cytometric bead array immunoassay. *P<0.05, **P<0.01 compared with WT chow group, #P<0.05, ##P<0.01 compared with WT HFD group.
Citation: Journal of Endocrinology 224, 2; 10.1530/JOE-14-0548
RGC32 deficiency increased the expression of metabolic genes in adipose tissues
Since HFD-fed Rgc32−/− mice had reduced fat mass and improved adipose tissue inflammation, we further assessed the expression of metabolic genes related to β-oxidation, lipolysis, and thermogenesis in adipose tissue. As shown in Fig. 5A, RGC32 deficiency increased the mRNA expression of Pparα, Lipe, and Pgc1α in epididymal fat tissue as compared with WT controls both under normal chow and HFD conditions. Since the alterations in HFD groups were greater than those in the normal chow groups, and the body weight difference was observed in HFD groups, we confirmed the protein expression of these genes in the epididymal adipose tissue of HFD-fed mice (Fig. 5B and C).
RGC32 deficiency increased the expression of metabolic genes in adipose tissues. (A) mRNA expression of Pparα, Lipe, and Pgc1α in the epididymal adipose tissues of WT and Rgc32−/− mice (n=6). (B and C) PPARα, LIPE, and PGC1α protein expression in the epididymal adipose tissues of WT and Rgc32−/− mice were detected by western blotting analysis and normalized to α-tubulin (n=6). (D and E) mRNA expression of Pgc1α, Ucp1, and Prdm16 in the interscapular (D) and inguinal (E) fat tissues of WT and Rgc32−/− mice (n=6). *P<0.05, **P<0.01 compared with WT chow group, #P<0.05, ##P<0.01 compared with WT HFD group.
Citation: Journal of Endocrinology 224, 2; 10.1530/JOE-14-0548
It is known that PGC1α can induce browning of subcutaneous adipose tissue through regulation of uncoupling protein 1 (UCP1) (Bostrom et al. 2012). We further determined adipose tissue browning in interscapular and inguinal fat tissues of WT and Rgc32−/− mice. As shown in Fig. 5D and E, PGC1α, UCP1, and PRDM16 were all increased in both interscapular and inguinal fat tissues of Rgc32−/− mice compared with WT controls under both normal chow and HFD conditions. These data suggest that RGC32 deficiency induces browning of adipose tissues, leading to increased energy expenditure.
RGC32 had no effect on adipocyte differentiation
Since HFD induced a significant adipocyte hypertrophy in WT mice (Fig. 1D and E), we sought to determine whether RGC32 promotes adipocyte differentiation. RGC32 has been shown to induce smooth muscle cell differentiation (Li et al. 2007). In the differentiation of 3T3-L1 preadipocytes, RGC32 expression was dramatically increased accompanied with elevated expression of adipogenic differentiation specific genes Pparγ and C/Ebpα (Fig. 6A, B, and C). To test RGC32 function in this process, we knocked down or overexpressed RGC32 in 3T3-L1 cells followed by induction of adipocyte differentiation and found that neither knockdown nor overexpression of RGC32 had any effect on the expression of adipogenic-specific genes (Fig. 6D) or lipid accumulation (Fig. 6E and F). These data indicate that the role of RGC32 in obesity was not due to an increased adipocyte differentiation. To confirm the regulation of energy expenditure-related genes by RGC32 as observed in the fat tissue (shown in Fig. 5A, B, and C), we detected the protein expression of PPARα, LIPE, and PGC1α in adipocytes differentiated from 3T3-L1 cells. Consistently, all of these genes were increased when RGC32 was knocked down (Fig. 6G and H). The expression of Lipe and Pgc1α was decreased when RGC32 was overexpressed. Pparα expression appeared not to be dramatically affected by RGC32 overexpression (Fig. 6G and H). These data indicate that RGC32 deficiency prevents mice from developing diet-induced obesity through the upregulation of energy expenditure-related genes in adipose tissues.
RGC32 had no effect on adipocyte differentiation. (A) mRNA expression of Rgc32, Pparγ, and C/Ebpα during 3T3-L1 preadipocyte differentiation at the indicated times. (B and C) RGC32 protein expression during 3T3-L1 preadipocyte differentiation was detected by western blotting analysis and normalized to α-tubulin. *P<0.05, **P<0.01 compared with vehicle-treated group (0 day). (D, E, F, G, and H) 3T3-L1 preadipocyte was transduced with Ad-GFP, Ad-shRGC32, or Ad-RGC32 for 24 h and then was induced for adipocyte differentiation. (D) mRNA expression of Rgc32, Pparγ, and C/Ebpα and (E and F) lipid droplet accumulation was determined at the indicated times. (G and H) PPARα, LIPE, PGC1α, and RGC32 expression was detected by western blotting analysis and normalized to α-tubulin. *P<0.05, **P<0.01 compared with Ad-GFP group. All results are representatives of at least three independent experiments. A full colour version of this figure is available at http://dx.doi.org/10.1530/JOE-14-0548.
Citation: Journal of Endocrinology 224, 2; 10.1530/JOE-14-0548
Discussion
Our present study demonstrates for the first time that RGC32 plays an important role in HFD-induced obesity. RGC32 is strongly upregulated in adipose tissue of HFD-fed mice. RGC32 deficiency prevents the development of HFD-induced obesity and insulin resistance because Rgc32−/− mice fed a HFD gain less weight without change in energy intake and have a reduced fat mass. Interestingly, HFD increases the fat mass of Rgc32−/− mice without altering their body weight, which is probably because the increase in the fat mass in these mice accounts for a relatively small portion of the whole body mass and thus does not cause a significant change in their body weight (as shown in Fig. 1B). HFD-fed Rgc32−/− mice also exhibit significantly improved insulin sensitivity as indicated by improved HOMA-IR and insulin tolerance. Our results are consistent with previous studies showing that HFD causes obesity and insulin resistance (Winzell & Ahren 2004), which is a characteristics of type 2 diabetes and several cardiometabolic syndromes including hypertension and dyslipidemia (Kim et al. 2008). Although adipocyte differentiation is involved in diet-induced obesity (Berry et al. 2012, Federico et al. 2012), it may not be involved in RGC32-mediated obesity because either overexpression or knockdown of RGC32 has no effect on the expression of adipogenic differentiation-specific genes Pparγ and C/Ebpα and lipid accumulation of the adipocytes.
The protection from diet-induced obesity in Rgc32−/− mice is linked to the attenuated adipose tissue and systemic inflammation and increased adipose tissue energy expenditure. Recent data have shown that adipose tissue inflammation is a key mechanism leading to obesity and insulin resistance (Chakrabarti et al. 2009, Lee et al. 2010). RGC32 is essential for C5b-9-induced cell cycle activation (Fosbrink et al. 2009), indicating an important role in the regulation of inflammation. Indeed, RGC32 deficiency suppresses adipose tissue and systemic inflammation in HFD-fed mice, as evidenced by the reduced production of proinflammatory adipokines including leptin, IL6, TNFα, and IL12 and increased anti-inflammatory adipokine adiponectin. Dietary excess and obesity have been shown to cause lipid accumulation in adipocytes, initiating a state of cellular stress and activation of NFκB signaling pathway (Shoelson et al. 2006), leading to an increased adipocyte production of proinflammatory cytokines. Our previous study has shown that RGC32 activates NFκB in vascular endothelial cells (Cui et al. 2013). Therefore, RGC32 may increase inflammation of adipose tissue through activating NF-κB signaling pathway, which may be studied in the future.
RGC32 appears to regulate obesity development by influencing the thermogenesis. Particularly, RGC32 may suppress the expression of metabolic genes PPARα, Lipe, and PGC1α because dramatic elevation of PPARα, LIPE, and PGC1α is observed in the adipose tissue of HFD-fed Rgc32−/− mice. LIPE is a rate-limiting enzyme to cleave fatty acids from the triglyceride molecule (Schweiger et al. 2006). PPARα promotes uptake, utilization, and catabolism of fatty acids by upregulating the genes involved in fatty acid transport; fatty acid binding and activation; and peroxisomal and mitochondrial fatty acid β-oxidation (Rakhshandehroo et al. 2010). PGC1α enhances thermogenesis and oxidative metabolism of adipose tissue (Liang & Ward 2006, Jun et al. 2014). Indeed, RGC32 deficiency promotes the browning of adipose tissues. Brown adipose tissue is known to dissipate chemical energy and protect against obesity through a process termed as nonshivering thermogenesis (Bi et al. 2014). Active brown adipose tissue burns lipids to produce heat, resulting in an increase in energy expenditure. PGC1α and UCP1 are highly expressed in brown adipose tissue (Fisher et al. 2012), and PRDM16 is a brown adipose determination factor (Seale et al. 2011). Increased expression of these genes in interscapular and inguinal fat tissues may have collectively caused the increased energy expenditure and the lean phenotype of HFD-fed Rgc32−/− mice. Besides adipose tissue, defective energy expenditure of skeletal muscle also contributes to diet-induced obesity and insulin resistance. However, since RGC32 expression is undetectable in skeletal muscle, contribution of skeletal muscle energy expenditure to the lean phenotype of HFD-fed Rgc32−/− mice is likely to be minimal.
In summary, our study demonstrates that RGC32 mediates HFD-induced obesity through enhancing inflammation while decreasing energy expenditure in adipose tissues.
Declaration of interest
The authors declare that there is no conflict of interest that could be perceived as prejudicing the impartiality of the research reported.
Funding
This work was supported by NIH grants HL107526, HL119053, and HL123302 and National Natural Science Foundation of China grant 81328002 (to S-Y C). X-B C is supported by an American Heart Association Postdoctoral Fellowship (14POST20480015).
Author contribution statement
X-B C conceived and carried out the experiments including data collection, analysis and interpretation, and wrote the manuscript. J-N L performed the qPCR, western blotting analysis and adipocyte differentiation experiments. J Y offered critical logistical advice on experimental design and data interpretation and critically read the manuscript. S-Y C is the guarantor of this work and, as such, had full access to all the data in the study and takes responsibility for the integrity of the data and the accuracy of the data analysis. All authors had final approval of the submitted and published versions.
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