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Effects of nutrition on the cell survival and gene expression of transplanted fat tissues in mice
BBRC Biochemical and Biophysical Research Communications 295 (2002) 630–635 www.academicpress.com
Effects of nutrition on the cell survival and gene expression of transplanted fat tissues in mice F. Matsumoto,a H. Bujo,b,* D. Kuramochi,a K. Saito,a M. Shibasaki,c K. Takahashi,c S Yoshimoto,a M. Ichinose,a and Y. Saitoc a b
Department of Plastic, Reconstructive and Esthetic Surgery (J4), Graduate School of Medicine, Chiba University, Chiba 260-8670, Japan Department of Genome Research and Clinical Application (M6), Graduate School of Medicine, Chiba University, Chiba 260-8670, Japan c Department of Clinical Cell Biology (F5), Graduate School of Medicine, Chiba University, Chiba 260-8670, Japan Received 14 June 2002
Abstract Fat tissue transplantation is a useful and common clinical technique in the plastic and reconstructive surgeries. To know the nutritional effects on the survival and maintenance of fat grafts, the weights of tissues and cell sizes, and the gene expressions in the fat tissues were analyzed 14 days after transplantation. The body weight and the plasma insulin level in high nutritional group (HNG) were significantly higher (p < 0:05) than those in low nutritional group (LNG), respectively. The measurements of cell size showed that there were 32.5% distributed in the diameter less than 2 lm in LNG, significantly higher than 28.5% in HNG. There were 7.5% distributed in the diameter more than 6 lm in LNG, significantly lower than 10.0% in HNG. The mRNA levels of leptin, lipoprotein lipase, and b3 -adrenergic receptor were 2.0-, 1.5-, and 1.7-fold higher in HNG than those in LNG, respectively. The levels of hormone sensitive lipase and hexokinase 2 transcripts were not significantly different in both groups. These results show that the systemic nutritional status in host causes the changes of cell size and tissue weight as well as gene expression in the transplanted fat using mice model. The nutritional condition is probably important for the fat graft clinically both as lipid-storage and functional cells. Ó 2002 Elsevier Science (USA). All rights reserved. Keywords: Transplantation; Fat; Mouse; Gene expression; Adipocyte
Fat tissue is becoming a common material for the soft tissue reconstruction using the autologous transplantation techniques in plastic and reconstructive surgery [1,2]. However, the frequently occurred unpredictable short survival of transplanted fat tissues raises the issues to be dissolved how its volume retention after transplantation can be further stable. The establishment of method for the increased fat survival is expected to decrease the need for multiple operations to meet the recipient site volume and shape requirements. There are many potential immunological, nutritional, surgical and anatomical problems to be addressed for the long survival of the transplanted fat tissues. It has been studied how some materials and bioactive peptides can improve the graft maintenance using ani-
*
Corresponding author. Fax: +81-43-226-2095. E-mail address: [email protected] (H. Bujo).
mal models as well as humans. The addition of dextran beads with basic fibroblast growth factor (bFGF) has been shown to cause the improvement of postoperative graft weight maintenance [3]. In animal models, prolonged survival of fat transplants has been demonstrated with the addition of nutrients such as vitamin E and fetal bovine serum (FBS) [4]. It seems that addition of the nutrients enriched with anabolic hormones enabled the survival and take off more adipose cells in the graft. These studies of assessing adipose viability have been based on histological studies or evaluation by photography and other imaging techniques. The nutritional and metabolic effects on the survival of adipocytes as functional cells have not yet been assessed in detail. It remains unknown that the functional activities of transplanted adipocytes are alive in the regulation of systemic whole body as other native adipocytes, such as the responsiveness of gene expressions under the various nutritional and metabolic stresses.
0006-291X/02/$ - see front matter Ó 2002 Elsevier Science (USA). All rights reserved. PII: S 0 0 0 6 - 2 9 1 X ( 0 2 ) 0 0 7 1 1 - 8
F. Matsumoto et al. / Biochemical and Biophysical Research Communications 295 (2002) 630–635
Recent clinical advance of imaging techniques has clarified that the accumulated fat tissues are rather sensitive to the nutritional conditions, such as food restriction and exercise [5,6]. These measurements revealed that the accumulated fat tissues change their volumes in the early phase of nutritional environments, such as fasting conditions. The analysis of the effect of nutritional conditions on the expression of lipid metabolism-related genes such as adrenaline-response gene seems to be important to know how transplanted fat tissues can survive for long time. During calorie restriction, lipid mobilization is induced and a series of physiological changes happen, and several lipid metabolism genes take part in these changes [7]. In this investigation, we first analyzed the effects of two different nutritional conditions on the early changes of transplanted fat volumes and cell sizes using nude mice. Then, to know the mechanism of difference of survival and cell sizes in the transplanted fat cells, we analyzed the gene expressions in the transplanted fat tissues.
Materials and methods Animals. Male ICR mice (for donor, 6–8 weeks) and male ICR nude mice [CD-1 (ICR)-m] (for recipient, 4–6 weeks) were purchased from Charles River Japan (Yokohama, Japan). Fat transplantation. Donor mice and recipient mice were injected 0.5% pentobarbital sodium (0.1 ml/10 g/body weight) into the abdominal cavity. Transplant was harvested en bloc as the epididymal fat from ICR mice. Minimal skin incision and undermining were performed in bilateral lateral abdomen of recipient mice, and transplant was implanted into the subcutaneous space as described previously [8]. Nutrition. Two types of nutritional group were set up. In high nutritional group, ICR nude mice were fed standard rodent chow (protein 25.4%; fat 4.4%, soluble non-nitrogen 50.3%) and 20% sucrose in water everyday (total calories; 188 kcal/day). In low nutritional group, ICR nude mice were fed standard rodent chow with restricted calories in two weeks with pure water (total calories; 66 kcal/day). VEGF treatment. We injected vascular endothelial growth factor (VEGF) (10 ng/ml, 0.1 ml) into w-lateral side of the subcutaneous tissue around the transplant every two or three days. In control, PBS (ad 0.1% BSA, 0.1 ml) was injected into contra-lateral side of the subcutaneous tissue, as same as VEGF-injected side. Nutrition was standard rodent chow and pure water in all cases. Evaluation of transplanted fat mass. Transplanted fat tissues were extirpated after anesthesia. Skin incision was performed in square to surround on the transplanted lesion. Undermining was performed to preserve vessels. Mass of extirpated fat tissue was evaluated by appearance macroscopically, appearance microscopically, and cell size (identified floating cells). Floating cells were prepared by centrifugation at 1500 rpm for 5 min, after incubation at 37 °C for 30 min with 0.0005% type I collagenase (Sigma, Japan) [9]. The sizes of 100 cells were measured microscopically using hemocytometer. Gene expression in transplant. Gene expressions in the transplanted fat tissues were evaluated by reverse transcription-polymerase chain reaction (RT-PCR) as previously described [10]. Two weeks after fat tissue transplantation, fat tissues were extirpated from the mice. Total RNA was isolated with an RNA Extraction Kit from Qiagen (Tokyo, Japan). To quantitate the mRNA levels of leptin, lipoprotein lipase (LPL), hormone sensitive lipase (HSL), b3 -adrenegic receptor (b3 -AR), fatty acid synthase (FAS), and hexokinase 2 (HK2), 2:0 lg of total
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RNA was reverse-transcripted and amplified using GeneAmp Gold RNA PCR Reagent Kit from Biosystems (Tokyo, Japan) and the specific primers as described below. The amplification was performed for 35 cycles, 95 °C for 1 min, annealing 54 °C for 1 min, 72 °C for 1 min, and a final extension period of 72 °C for 8 min. The suitable condition for the quantification was determined based on the results of linear amplifications of the products in 30, 35, and 40 cycles. Ten ll of each PCR product was electrophoresed on 2.0% agarose gel, and stained with ethidium bromide. The relative signal intensities of the PCR products were determined with luminescent image analyzer LAS1000 image analyzer (Fuji Photo Film, Tokyo, Japan). mRNA amounts were normalized to levels of b-actin mRNA, which served as endogenous standard. Primers. Primers synthesized for b-actin were 50 -TGGAATCCTGT GGCATCCATGAAAC-30 and 50 -TAAAACGCAGCTCAGTAACA GTCCG-30 according to known cDNA sequence (GenBank code: NM007393). Primers for leptin were 50 -CCTGTGGCTTTGGTCCTA TCTG-30 and 50 -AGGCAGGCTGGTGAGGACCTG-30 (GenBank code: NM008493). Primers for LPL were 50 -TTCCATTACCAAGTC AAGATTCAC-30 and 50 -TCAGCCAGACTTCTTCAGAGACTT-30 (GenBank code: NM008509). Primers for HSL were 50 -ACCTGAG GCCTTTGAGATGCCACTC-30 and 50 -CACTCCATAGGCTGCTG CCCGAAG-30 (GenBank code: U40001). Primers for b3 -AR were 50 -CATCGCCCGCACGCCGAGACT-30 and 50 -CTTGGTAACCAG CGTGCCGTA-30 (GenBank code: S56152). Primers for FAS were 50 -ATTGGGCACTCCTTGGGAGA-30 and 50 -CTCAGGGATAGA GGTGCTGA-30 (GenBank code: NM 017332). Primers for HK2 were 50 -GGAAACTCAGCCCAGAGCTC-30 and 50 -TCACAATCGGGC ACCAGCCT-30 (GenBank code: NM013820). Statistical analysis. Statistical analysis was performed with t test. All the results reported herein were confirmed by repeating the experiments with different occasions.
Results Metabolic and biochemical changes in mice Table 1 shows the body weight and plasma levels of triglyceride (TG), free fatty acids (FFA), insulin, glucose, and leptin in fasting plasma at the 14th day after transplantation. These plasma levels were not significantly different between the fat-transplanted mice (n ¼ 6) and the Sham-operated mice (n ¼ 4) in high nutritional group (HNG) and low nutritional group (LNG), respectively. The body weight in HNG was significantly higher than that in LNG (p < 0:05). There were no significant differences between HNG and LNG
Table 1 Metabolic markers of mice in different nutritional conditions
BW (g) TG (mg/dl) FFA (mg/dl) Glucose (mg/dl) Insulin (pg/ml) Leptin (ng/ml)
HNG
LNG
p value
33 2:5 57 17 1149 278 257 63 5818 1585 3:5 0:7
24 2 68 28 875 150 256 59 873 69 1:7 0:4
p < 0:05 N.S. N.S. N.S. p < 0:05 N.S.
Data are means SD (n ¼ 6). BW, body weight; TG, triglyceride; FFA, free fatty acids. TG, FFA, and insulin are the serum levels; and glucose and leptin are the plasma levels, respectively.
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in TG, FFA, glucose, and leptin, although the leptin level was 105% higher in HNG, compared to that in LNG. The plasma insulin level in HNG was significantly higher than that in LNG (p < 0:05). These results suggest that the difference (122 kcal/day) of nutritional supply in both groups have rather chronic and mild effects on the metabolic status of mice with the modification of their adaptation mechanism. Macroscopic appearance of transplanted fat tissues Twenty-four transplanted fat tissues were extirpated from subcutaneous connective tissue of mice 14 days after transplantation (Fig. 1A, panel a). Extirpation was performed under magnification not to include unnecessary tissues manually. As shown in panel b, the transplanted fat tissues were mildly attached to connective tissues and abdominal wall under skin. The color of
transplanted tissues was fresh yellow and its surface was smooth without any degeneration. And notably, new arteries from the subcutaneous area were grown around the exogenous fat tissues and into the grafts in all mice. Isolated transplanted tissues were almost round and soft with expanded vessels (panel c). There were no obvious difference of the observation above described on their appearance between HNG and LNG. Fig. 1B shows the comparison of tissue weights of isolated grafts between HNG and LNG. The averaged weight of extirpated fat tissues was decreased compared with that before transplantation in LNG. In HNG, averaged weight of extirpated tissues was heavier than that before transplantation. The mean ratio of weight in the extirpation and before transplantation was 1.08 and 0.91 in HNG and LNG, respectively, indicating significant difference in both groups (p < 0:05). Histological changes in the fat grafts We next examined the histological appearance of transplants in both groups. The microscopical structure of fat tissues was shown in Fig. 2. Each adipocyte was kept with its characteristic features, such as large lipidstorage and small cytosol with nucleus. Dilated vessels in low magnification were clearly observed (Fig. 2A). There were no obvious necrotic regions in the transplanted tissues, although some inflammatory cells were observed to infiltrate into the intercellular lesion (Fig. 2B). There was no clear difference in histological observations between LNG and HNG. Comparison of the diameter of isolated single adipocyte We therefore examined the size of adipocyte in detail. Maximum diameters of isolated single adipocytes were measured as floating cells on the hemocytometer (Fig. 3). The counted floating cells in both groups were classified of different diameters to four ranges (Table 2).
Fig. 1. Fat transplantation into mice. (A) Macroscopic appearance of transplanted fat tissues. Transplant was lining in subcutaneous connective tissue of bilateral abdomen in mice (a). Transplanted fat tissues at the extirpation (b). New vessels from the subcutaneous area were grown around transplant. Extirpated transplanted fat tissue, obvious expanded vessels were recognized in the transplant (c). (B) Comparison of extirpated tissue weights between HNG and LNG. Data were presented as the mean ratios of weight in the extirpation and before transplantation. Data are means SD (n ¼ 24), * p < 0:05.
Fig. 2. Microscopic appearance of the extirpated fat tissues. Slices were stained with hematoxylin–eosin. Magnifications are 20 (A) and 100 (B).
F. Matsumoto et al. / Biochemical and Biophysical Research Communications 295 (2002) 630–635
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Fig. 4. Comparison of weights of extirpated fat tissues between VEGFinjected group (1) and control group (2). Data were presented as means SD (n ¼ 20). Fig. 3. Isolated single adipocytes. (A) Microscopic appearance of single cells in HNG (a) and LNG (b). Magnifications are 100. (B) The comparison of proportion of small (<2 lm) and large (>6 lm) cells. (1) HNG, (2) LNG.
Table 2 Size distribution of isolated adipocytes from transplanted tissues
<2.0 (lm) 2.0–4.0 (lm) 4.0–6.0 (lm) 6.0< (lm)
HNG (%)
LNG (%)
28.5 36.5 25.0 10.0
32.5 35.0 25.0 7.5
Data were presented as average of three different measurements.
There were 32.5% distributed in the diameter less than 2 mm in LNG, significantly higher than 28.5% in HNG. There were 7.5% distributed in the diameter more than 6 mm in LNG, significantly lower than 10.0% in HNG (Fig. 3B). These results suggest that the different cell sizes responding to the nutritional conditions cause the difference of transplanted fat tissues in both groups, as shown in Fig. 1.
Gene expressions in the transplanted fat tissues The functional abilities of the transplanted fat tissues were finally examined both in HNG and LNG. Fat metabolism-related gene expressions in the transplanted fat tissues were examined as degeneration of its cell function after transplantation. Transplanted fat tissues were harvested at 14th day, the total RNA was isolated from the harvested fat tissues, and gene expression of leptin, LPL, b3 -AR, HSL, FAS, and HK2 were examined using RT-PCR (Fig. 5). b-Actin mRNA was examined to normalize the amounts of mRNA. Leptin, LPL, b3 -AR, HSL, and HK2 mRNA were clearly detected 14 days after transplantation in both groups. FAS mRNA was not detectable in both groups. The levels of leptin, LPL, and b3 -AR were 2.0-, 1.5-, 1.7-fold higher in HNG than those in LNG, respectively. The levels of HSL and HK2 transcripts were not significantly different
Effect of VEGF injection The observation of new vessels shown in Fig. 1 suggests that the angiogenic factors might improve the survival or conditions of transplanted cells with the induced nutritional supplies via new vessels. We examined the effect of VEGF on the weights of fat grafts in the standard nutrition conditions. Twenty numbers of transplanted fat tissues were harvested from mice (10 were VEGF-injected, the others were controls). Each weight was measured and compared in both groups (Fig. 4). There was no significant difference in both groups. These results suggest that the angiogenic effect of VEGF is not the major determinant for the survival of transplanted tissues.
Fig. 5. Gene expression in the transplanted fat tissues. The results are at 14th day after transplantation. (A) Representative photographs of PCR-products on the gel. (1) LNG, (2) HNG. (B) Comparison of intensities of PCR-products in HNG and LNG. The data are shown as the ratios of HNG and LNG.
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in both groups. Taken together, the different mRNA levels in the fat grafts of both groups suggest that the maintenance of sensitivities of gene expressions in the transplanted fat tissues for the different nutritional conditions.
Discussion In this study, our results showed that the volumes of transplanted fat tissues in mice are dependent on the nutritional conditions of hosts for 14 days. One of the possible factors for the maintenance of fat volume is cell size in the tissues: the averaged size of adipocytes in low nutrition states was decreased compared with that in high nutrition states. The functional activities, such as the expression of genes related to fat metabolism, depend on the nutritional conditions in the host as well as the volume of transplanted tissues and cells. These results suggest that the exogenous fat tissues survive as the collection of functional adipocytes at least for 2 weeks, and the nutritional conditions in host are capable to change the volume of transplanted tissues and cells as well as the cell functions. Many studies on the establishment of efficient methods for long survival of transplanted, autologous fat tissues have been performed using animal models. It has been reported that some materials and bioactive peptides can improve the graft maintenance. Eppley et al. [3] has been shown that the addition of dextran beads with bFGF improves postoperative graft weight maintenance. The addition of nutrients, such as vitamin E and FBS, was effective on the long survival of transplanted tissue [4]. However, the nutritional and metabolic effects on the survival of adipocytes, particularly on the functional maintenance, have not yet been assessed. Our study firstly revealed that the nutritional difference in host causes the different volumes of transplanted fat tissues and cells. It is most likely that the difference of volumes is associated with the gene expression related with the lipid metabolism in adipocytes. In the course of study, the early angiogenesis from surrounding areas into the fat grafts were observed (Fig. 1A, panel b). One possible elucidation for the difference in volumes of transplanted tissues is nutritional supply from the host via new vessels into the fat tissues. However, there was no obvious improvement of volume change in the tissues with injection of VEGF, a potent angiogenic factor [11], for 2 weeks (Fig. 3). These results suggest that the angiogenic activities for the transplanted tissues are enough with the given nutritional conditions, although further study is needed. The expressions of leptin, LPL, and b3 -AR genes were higher in HNG than those in LNG. The only significant metabolic difference in both groups is plasma insulin levels, together with the difference in BW, suggesting that
the supplied calories were enough at least for the developmental growth, but not for the daily calorie intake for fat storage in the mice. The resultant difference in the levels of plasma leptin, although not significant, indicates that the systemic fat volume in the body is different in both groups, as plasma leptin level is known to be a sensitive marker for the fat volume in mice [12]. The increased leptin mRNA levels in the fat grafts of HNG is inconsistent with the larger size of isolated adipocytes. The high levels of LPL and b3 -AR may indicate the better functional abilities of the transplanted adipocytes in the HNG, because of the sensitive regulatory expressions to nutritions have been reported in rodents. In this context, the metabolic responses of transplanted fat tissues to the systemic conditions, such as low TG and high insulin and FFA levels in plasma, should be considered. Ong et al. [13] have reported that insulin increases LPL mRNA levels by 200% and rates of LPL synthesis by 300% in cultured rat adipocytes. Among the b-adrenergic receptor family, the b3 -AR plays a central role in the regulation of lipolysis in rodent white and brown adipose tissue [14]. b3 -AR mRNA levels are regulated in adipose tissues by the nutritional conditions in mice [15]. On the other hand, the HSL and HK2 mRNA levels were not changed in the same nutritional status. The expression of HSL, the most critical enzyme during lipid mobilization, is believed to be activated by catacholamines through cAMP-dependent phosphorylation, rather than the transcription level [16]. The gene regulation of HSL and HK2 may be different from other genes here in the given calorie-restricted status. In summary, our study shows that, in the transplanted fat tissues, the cell sizes and tissue weights are better kept with the nutrition-rich condition, and the gene expressions in the transplanted tissues are also affected by the systemic metabolic states. Thus, the nutritional condition in hosts is probably important for the transplanted fat tissues both as lipid-storage and functional cells. Further analysis is needed to clarify the long-term effects of nutrition on the survival of transplanted adipocytes.
Acknowledgments These studies were supported by grants from the Japanese Ministry of Education, Science and Culture to Y.S. and H.B.
References [1] K.S. Pinski, Fat transplantation and autologous collagen: a decade of experience, Am. J. Cosmet. Syrg. 16 (1999) 217–223. [2] M.S. Kaminer, N.E. Omura, Autologous fat transplantation, Arch Dermatol. 137 (2001) 812–814. [3] B.L. Eppley, R.V. Snyders Jr., T. Winkelmann, J.J. Delfino, Autologous facial fat transplantation: improved graft mainte-
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[4]
[5]
[6]
[7]
[8]
[9]
nance by microbead bioactivation, J. Oral Maxillofac. Surg. 50 (1992) 477–482. Y. Ullmann, M. Hyams, Y. Ramon, D. Beach, I.J. Peled, E.S. Lindenbaum, Enhancing the survival of aspirated human fat injected into nude mice, Plast. Reconstr. Surg. 101 (1998) 1940– 1944. T. Nakamura, K. Tokunaga, I. Shimomura, M. Nishida, S. Yoshida, K. Kotani, A.H. Islam, Y. Keno, T. Kobatake, Y. Nagai, S. Fujioka, S. Tarui, Y. Matsuzawa, Contribution of visceral fat accumulation to the development of coronary artery disease in non-obese women, Atherosclerosis 107 (1994) 239–246. R. Suzuki, S. Watanabe, Y. Hirai, K. Akiyama, T. Nishide, Y. Matsushima, H. Murayama, H. Ohshima, M. Shinomiya, K. Shirai, Y. Saito, Abdominal wall fat index, estimated by ultrasonography, for assessment of the ratio of visceral fat to subcutaneous fat in the abdomen, Am. J. Med. 95 (1993) 309–314. J.S. Samra, Sir David Cuthbertson Medal Lecture. Regulation of lipid metabolism in adipose tissue, Proc. Nutr. Soc. 59 (2000) 441– 446. T. Shibasaki, K. Takahashi, T. Itou, S. Miyazawa, M. Ito, J. Kobayashi, H. Bujo, Y. Saito, Alterations of insulin sensitivity by the implantation of 3T3-L1 cells in nude mice. A role for TNF-a? Diabetologia, 45 (2002) 518–526. H. Inadera, Y. Ishikawa, K. Shirai, Y. Saito, S. Yoshida, Proliferation and triglyceride synthesizing activities of fibroblastlike cells derived from epididymal and subcutaneous adipose tissues of rats, Scand. J. Clin. Lab Invest. 53 (1993) 225–229.
635
[10] T. Kanaki, N. Morisaki, H. Bujo, K. Takahashi, I. Ishii, Y. Saito, The regulatory expression of procollagen COOH-terminal proteinase enhancer in the proliferation of vascular smooth muscle cells, Biochem. Biophys. Res. Commun. 270 (2000) 1049–1054. [11] M. Toi, T. Matsumoto, H. Bando, Vascular endothelial growth factor: its prognostic, predictive, and therapeutic implications, Lancet Oncol. 2 (2001) 667–673. [12] P.J. Havel, Control of energy homeostasis and insulin action by adipocyte hormones: leptin, acylation stimulating protein, and adiponectin, Curr. Opin. Lipidol. 13 (2002) 51–59. [13] J.M. Ong, T.G. Kirchgessner, M.C. Schotz, P.A. Kern, Insulin increases the synthetic rate and messenger RNA level of lipoprotein lipase in isolated rat adipocytes, J. Biol. Chem. 263 (1988) 12933–12938. [14] K. El Hadri, B. Feve, J. Pairault, Developmental expression and functional activity of beta 1- and beta 3-adrenoceptors in murine 3T3-F442A differentiating adipocytes, Eur. J. Pharmacol. 297 (1996) 107–119. [15] B.A. Evans, M. Papaioannou, F. Anastasopoulos, R.J. Summers, Differential regulation of beta3-adrenoceptors in gut and adipose tissue of genetically obese (ob/ob) C57BL/6J-mice, Br. J. Pharmacol. 124 (1998) 763–771. [16] P. Stralfors, P. Bjorgell, P. Belfrage, Hormonal regulation of hormone-sensitive lipase in intact adipocytes: identification of phosphorylated sites and effects on the phosphorylation by lipolytic hormones and insulin, Proc. Natl. Acad. Sci. USA 81 (1984) 3317–3321.
Effects of nutrition on the cell survival and gene expression of transplanted fat tissues in mice F. Matsumoto,a H. Bujo,b,* D. Kuramochi,a K. Saito,a M. Shibasaki,c K. Takahashi,c S Yoshimoto,a M. Ichinose,a and Y. Saitoc a b
Department of Plastic, Reconstructive and Esthetic Surgery (J4), Graduate School of Medicine, Chiba University, Chiba 260-8670, Japan Department of Genome Research and Clinical Application (M6), Graduate School of Medicine, Chiba University, Chiba 260-8670, Japan c Department of Clinical Cell Biology (F5), Graduate School of Medicine, Chiba University, Chiba 260-8670, Japan Received 14 June 2002
Abstract Fat tissue transplantation is a useful and common clinical technique in the plastic and reconstructive surgeries. To know the nutritional effects on the survival and maintenance of fat grafts, the weights of tissues and cell sizes, and the gene expressions in the fat tissues were analyzed 14 days after transplantation. The body weight and the plasma insulin level in high nutritional group (HNG) were significantly higher (p < 0:05) than those in low nutritional group (LNG), respectively. The measurements of cell size showed that there were 32.5% distributed in the diameter less than 2 lm in LNG, significantly higher than 28.5% in HNG. There were 7.5% distributed in the diameter more than 6 lm in LNG, significantly lower than 10.0% in HNG. The mRNA levels of leptin, lipoprotein lipase, and b3 -adrenergic receptor were 2.0-, 1.5-, and 1.7-fold higher in HNG than those in LNG, respectively. The levels of hormone sensitive lipase and hexokinase 2 transcripts were not significantly different in both groups. These results show that the systemic nutritional status in host causes the changes of cell size and tissue weight as well as gene expression in the transplanted fat using mice model. The nutritional condition is probably important for the fat graft clinically both as lipid-storage and functional cells. Ó 2002 Elsevier Science (USA). All rights reserved. Keywords: Transplantation; Fat; Mouse; Gene expression; Adipocyte
Fat tissue is becoming a common material for the soft tissue reconstruction using the autologous transplantation techniques in plastic and reconstructive surgery [1,2]. However, the frequently occurred unpredictable short survival of transplanted fat tissues raises the issues to be dissolved how its volume retention after transplantation can be further stable. The establishment of method for the increased fat survival is expected to decrease the need for multiple operations to meet the recipient site volume and shape requirements. There are many potential immunological, nutritional, surgical and anatomical problems to be addressed for the long survival of the transplanted fat tissues. It has been studied how some materials and bioactive peptides can improve the graft maintenance using ani-
*
Corresponding author. Fax: +81-43-226-2095. E-mail address: [email protected] (H. Bujo).
mal models as well as humans. The addition of dextran beads with basic fibroblast growth factor (bFGF) has been shown to cause the improvement of postoperative graft weight maintenance [3]. In animal models, prolonged survival of fat transplants has been demonstrated with the addition of nutrients such as vitamin E and fetal bovine serum (FBS) [4]. It seems that addition of the nutrients enriched with anabolic hormones enabled the survival and take off more adipose cells in the graft. These studies of assessing adipose viability have been based on histological studies or evaluation by photography and other imaging techniques. The nutritional and metabolic effects on the survival of adipocytes as functional cells have not yet been assessed in detail. It remains unknown that the functional activities of transplanted adipocytes are alive in the regulation of systemic whole body as other native adipocytes, such as the responsiveness of gene expressions under the various nutritional and metabolic stresses.
0006-291X/02/$ - see front matter Ó 2002 Elsevier Science (USA). All rights reserved. PII: S 0 0 0 6 - 2 9 1 X ( 0 2 ) 0 0 7 1 1 - 8
F. Matsumoto et al. / Biochemical and Biophysical Research Communications 295 (2002) 630–635
Recent clinical advance of imaging techniques has clarified that the accumulated fat tissues are rather sensitive to the nutritional conditions, such as food restriction and exercise [5,6]. These measurements revealed that the accumulated fat tissues change their volumes in the early phase of nutritional environments, such as fasting conditions. The analysis of the effect of nutritional conditions on the expression of lipid metabolism-related genes such as adrenaline-response gene seems to be important to know how transplanted fat tissues can survive for long time. During calorie restriction, lipid mobilization is induced and a series of physiological changes happen, and several lipid metabolism genes take part in these changes [7]. In this investigation, we first analyzed the effects of two different nutritional conditions on the early changes of transplanted fat volumes and cell sizes using nude mice. Then, to know the mechanism of difference of survival and cell sizes in the transplanted fat cells, we analyzed the gene expressions in the transplanted fat tissues.
Materials and methods Animals. Male ICR mice (for donor, 6–8 weeks) and male ICR nude mice [CD-1 (ICR)-m] (for recipient, 4–6 weeks) were purchased from Charles River Japan (Yokohama, Japan). Fat transplantation. Donor mice and recipient mice were injected 0.5% pentobarbital sodium (0.1 ml/10 g/body weight) into the abdominal cavity. Transplant was harvested en bloc as the epididymal fat from ICR mice. Minimal skin incision and undermining were performed in bilateral lateral abdomen of recipient mice, and transplant was implanted into the subcutaneous space as described previously [8]. Nutrition. Two types of nutritional group were set up. In high nutritional group, ICR nude mice were fed standard rodent chow (protein 25.4%; fat 4.4%, soluble non-nitrogen 50.3%) and 20% sucrose in water everyday (total calories; 188 kcal/day). In low nutritional group, ICR nude mice were fed standard rodent chow with restricted calories in two weeks with pure water (total calories; 66 kcal/day). VEGF treatment. We injected vascular endothelial growth factor (VEGF) (10 ng/ml, 0.1 ml) into w-lateral side of the subcutaneous tissue around the transplant every two or three days. In control, PBS (ad 0.1% BSA, 0.1 ml) was injected into contra-lateral side of the subcutaneous tissue, as same as VEGF-injected side. Nutrition was standard rodent chow and pure water in all cases. Evaluation of transplanted fat mass. Transplanted fat tissues were extirpated after anesthesia. Skin incision was performed in square to surround on the transplanted lesion. Undermining was performed to preserve vessels. Mass of extirpated fat tissue was evaluated by appearance macroscopically, appearance microscopically, and cell size (identified floating cells). Floating cells were prepared by centrifugation at 1500 rpm for 5 min, after incubation at 37 °C for 30 min with 0.0005% type I collagenase (Sigma, Japan) [9]. The sizes of 100 cells were measured microscopically using hemocytometer. Gene expression in transplant. Gene expressions in the transplanted fat tissues were evaluated by reverse transcription-polymerase chain reaction (RT-PCR) as previously described [10]. Two weeks after fat tissue transplantation, fat tissues were extirpated from the mice. Total RNA was isolated with an RNA Extraction Kit from Qiagen (Tokyo, Japan). To quantitate the mRNA levels of leptin, lipoprotein lipase (LPL), hormone sensitive lipase (HSL), b3 -adrenegic receptor (b3 -AR), fatty acid synthase (FAS), and hexokinase 2 (HK2), 2:0 lg of total
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RNA was reverse-transcripted and amplified using GeneAmp Gold RNA PCR Reagent Kit from Biosystems (Tokyo, Japan) and the specific primers as described below. The amplification was performed for 35 cycles, 95 °C for 1 min, annealing 54 °C for 1 min, 72 °C for 1 min, and a final extension period of 72 °C for 8 min. The suitable condition for the quantification was determined based on the results of linear amplifications of the products in 30, 35, and 40 cycles. Ten ll of each PCR product was electrophoresed on 2.0% agarose gel, and stained with ethidium bromide. The relative signal intensities of the PCR products were determined with luminescent image analyzer LAS1000 image analyzer (Fuji Photo Film, Tokyo, Japan). mRNA amounts were normalized to levels of b-actin mRNA, which served as endogenous standard. Primers. Primers synthesized for b-actin were 50 -TGGAATCCTGT GGCATCCATGAAAC-30 and 50 -TAAAACGCAGCTCAGTAACA GTCCG-30 according to known cDNA sequence (GenBank code: NM007393). Primers for leptin were 50 -CCTGTGGCTTTGGTCCTA TCTG-30 and 50 -AGGCAGGCTGGTGAGGACCTG-30 (GenBank code: NM008493). Primers for LPL were 50 -TTCCATTACCAAGTC AAGATTCAC-30 and 50 -TCAGCCAGACTTCTTCAGAGACTT-30 (GenBank code: NM008509). Primers for HSL were 50 -ACCTGAG GCCTTTGAGATGCCACTC-30 and 50 -CACTCCATAGGCTGCTG CCCGAAG-30 (GenBank code: U40001). Primers for b3 -AR were 50 -CATCGCCCGCACGCCGAGACT-30 and 50 -CTTGGTAACCAG CGTGCCGTA-30 (GenBank code: S56152). Primers for FAS were 50 -ATTGGGCACTCCTTGGGAGA-30 and 50 -CTCAGGGATAGA GGTGCTGA-30 (GenBank code: NM 017332). Primers for HK2 were 50 -GGAAACTCAGCCCAGAGCTC-30 and 50 -TCACAATCGGGC ACCAGCCT-30 (GenBank code: NM013820). Statistical analysis. Statistical analysis was performed with t test. All the results reported herein were confirmed by repeating the experiments with different occasions.
Results Metabolic and biochemical changes in mice Table 1 shows the body weight and plasma levels of triglyceride (TG), free fatty acids (FFA), insulin, glucose, and leptin in fasting plasma at the 14th day after transplantation. These plasma levels were not significantly different between the fat-transplanted mice (n ¼ 6) and the Sham-operated mice (n ¼ 4) in high nutritional group (HNG) and low nutritional group (LNG), respectively. The body weight in HNG was significantly higher than that in LNG (p < 0:05). There were no significant differences between HNG and LNG
Table 1 Metabolic markers of mice in different nutritional conditions
BW (g) TG (mg/dl) FFA (mg/dl) Glucose (mg/dl) Insulin (pg/ml) Leptin (ng/ml)
HNG
LNG
p value
33 2:5 57 17 1149 278 257 63 5818 1585 3:5 0:7
24 2 68 28 875 150 256 59 873 69 1:7 0:4
p < 0:05 N.S. N.S. N.S. p < 0:05 N.S.
Data are means SD (n ¼ 6). BW, body weight; TG, triglyceride; FFA, free fatty acids. TG, FFA, and insulin are the serum levels; and glucose and leptin are the plasma levels, respectively.
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in TG, FFA, glucose, and leptin, although the leptin level was 105% higher in HNG, compared to that in LNG. The plasma insulin level in HNG was significantly higher than that in LNG (p < 0:05). These results suggest that the difference (122 kcal/day) of nutritional supply in both groups have rather chronic and mild effects on the metabolic status of mice with the modification of their adaptation mechanism. Macroscopic appearance of transplanted fat tissues Twenty-four transplanted fat tissues were extirpated from subcutaneous connective tissue of mice 14 days after transplantation (Fig. 1A, panel a). Extirpation was performed under magnification not to include unnecessary tissues manually. As shown in panel b, the transplanted fat tissues were mildly attached to connective tissues and abdominal wall under skin. The color of
transplanted tissues was fresh yellow and its surface was smooth without any degeneration. And notably, new arteries from the subcutaneous area were grown around the exogenous fat tissues and into the grafts in all mice. Isolated transplanted tissues were almost round and soft with expanded vessels (panel c). There were no obvious difference of the observation above described on their appearance between HNG and LNG. Fig. 1B shows the comparison of tissue weights of isolated grafts between HNG and LNG. The averaged weight of extirpated fat tissues was decreased compared with that before transplantation in LNG. In HNG, averaged weight of extirpated tissues was heavier than that before transplantation. The mean ratio of weight in the extirpation and before transplantation was 1.08 and 0.91 in HNG and LNG, respectively, indicating significant difference in both groups (p < 0:05). Histological changes in the fat grafts We next examined the histological appearance of transplants in both groups. The microscopical structure of fat tissues was shown in Fig. 2. Each adipocyte was kept with its characteristic features, such as large lipidstorage and small cytosol with nucleus. Dilated vessels in low magnification were clearly observed (Fig. 2A). There were no obvious necrotic regions in the transplanted tissues, although some inflammatory cells were observed to infiltrate into the intercellular lesion (Fig. 2B). There was no clear difference in histological observations between LNG and HNG. Comparison of the diameter of isolated single adipocyte We therefore examined the size of adipocyte in detail. Maximum diameters of isolated single adipocytes were measured as floating cells on the hemocytometer (Fig. 3). The counted floating cells in both groups were classified of different diameters to four ranges (Table 2).
Fig. 1. Fat transplantation into mice. (A) Macroscopic appearance of transplanted fat tissues. Transplant was lining in subcutaneous connective tissue of bilateral abdomen in mice (a). Transplanted fat tissues at the extirpation (b). New vessels from the subcutaneous area were grown around transplant. Extirpated transplanted fat tissue, obvious expanded vessels were recognized in the transplant (c). (B) Comparison of extirpated tissue weights between HNG and LNG. Data were presented as the mean ratios of weight in the extirpation and before transplantation. Data are means SD (n ¼ 24), * p < 0:05.
Fig. 2. Microscopic appearance of the extirpated fat tissues. Slices were stained with hematoxylin–eosin. Magnifications are 20 (A) and 100 (B).
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Fig. 4. Comparison of weights of extirpated fat tissues between VEGFinjected group (1) and control group (2). Data were presented as means SD (n ¼ 20). Fig. 3. Isolated single adipocytes. (A) Microscopic appearance of single cells in HNG (a) and LNG (b). Magnifications are 100. (B) The comparison of proportion of small (<2 lm) and large (>6 lm) cells. (1) HNG, (2) LNG.
Table 2 Size distribution of isolated adipocytes from transplanted tissues
<2.0 (lm) 2.0–4.0 (lm) 4.0–6.0 (lm) 6.0< (lm)
HNG (%)
LNG (%)
28.5 36.5 25.0 10.0
32.5 35.0 25.0 7.5
Data were presented as average of three different measurements.
There were 32.5% distributed in the diameter less than 2 mm in LNG, significantly higher than 28.5% in HNG. There were 7.5% distributed in the diameter more than 6 mm in LNG, significantly lower than 10.0% in HNG (Fig. 3B). These results suggest that the different cell sizes responding to the nutritional conditions cause the difference of transplanted fat tissues in both groups, as shown in Fig. 1.
Gene expressions in the transplanted fat tissues The functional abilities of the transplanted fat tissues were finally examined both in HNG and LNG. Fat metabolism-related gene expressions in the transplanted fat tissues were examined as degeneration of its cell function after transplantation. Transplanted fat tissues were harvested at 14th day, the total RNA was isolated from the harvested fat tissues, and gene expression of leptin, LPL, b3 -AR, HSL, FAS, and HK2 were examined using RT-PCR (Fig. 5). b-Actin mRNA was examined to normalize the amounts of mRNA. Leptin, LPL, b3 -AR, HSL, and HK2 mRNA were clearly detected 14 days after transplantation in both groups. FAS mRNA was not detectable in both groups. The levels of leptin, LPL, and b3 -AR were 2.0-, 1.5-, 1.7-fold higher in HNG than those in LNG, respectively. The levels of HSL and HK2 transcripts were not significantly different
Effect of VEGF injection The observation of new vessels shown in Fig. 1 suggests that the angiogenic factors might improve the survival or conditions of transplanted cells with the induced nutritional supplies via new vessels. We examined the effect of VEGF on the weights of fat grafts in the standard nutrition conditions. Twenty numbers of transplanted fat tissues were harvested from mice (10 were VEGF-injected, the others were controls). Each weight was measured and compared in both groups (Fig. 4). There was no significant difference in both groups. These results suggest that the angiogenic effect of VEGF is not the major determinant for the survival of transplanted tissues.
Fig. 5. Gene expression in the transplanted fat tissues. The results are at 14th day after transplantation. (A) Representative photographs of PCR-products on the gel. (1) LNG, (2) HNG. (B) Comparison of intensities of PCR-products in HNG and LNG. The data are shown as the ratios of HNG and LNG.
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in both groups. Taken together, the different mRNA levels in the fat grafts of both groups suggest that the maintenance of sensitivities of gene expressions in the transplanted fat tissues for the different nutritional conditions.
Discussion In this study, our results showed that the volumes of transplanted fat tissues in mice are dependent on the nutritional conditions of hosts for 14 days. One of the possible factors for the maintenance of fat volume is cell size in the tissues: the averaged size of adipocytes in low nutrition states was decreased compared with that in high nutrition states. The functional activities, such as the expression of genes related to fat metabolism, depend on the nutritional conditions in the host as well as the volume of transplanted tissues and cells. These results suggest that the exogenous fat tissues survive as the collection of functional adipocytes at least for 2 weeks, and the nutritional conditions in host are capable to change the volume of transplanted tissues and cells as well as the cell functions. Many studies on the establishment of efficient methods for long survival of transplanted, autologous fat tissues have been performed using animal models. It has been reported that some materials and bioactive peptides can improve the graft maintenance. Eppley et al. [3] has been shown that the addition of dextran beads with bFGF improves postoperative graft weight maintenance. The addition of nutrients, such as vitamin E and FBS, was effective on the long survival of transplanted tissue [4]. However, the nutritional and metabolic effects on the survival of adipocytes, particularly on the functional maintenance, have not yet been assessed. Our study firstly revealed that the nutritional difference in host causes the different volumes of transplanted fat tissues and cells. It is most likely that the difference of volumes is associated with the gene expression related with the lipid metabolism in adipocytes. In the course of study, the early angiogenesis from surrounding areas into the fat grafts were observed (Fig. 1A, panel b). One possible elucidation for the difference in volumes of transplanted tissues is nutritional supply from the host via new vessels into the fat tissues. However, there was no obvious improvement of volume change in the tissues with injection of VEGF, a potent angiogenic factor [11], for 2 weeks (Fig. 3). These results suggest that the angiogenic activities for the transplanted tissues are enough with the given nutritional conditions, although further study is needed. The expressions of leptin, LPL, and b3 -AR genes were higher in HNG than those in LNG. The only significant metabolic difference in both groups is plasma insulin levels, together with the difference in BW, suggesting that
the supplied calories were enough at least for the developmental growth, but not for the daily calorie intake for fat storage in the mice. The resultant difference in the levels of plasma leptin, although not significant, indicates that the systemic fat volume in the body is different in both groups, as plasma leptin level is known to be a sensitive marker for the fat volume in mice [12]. The increased leptin mRNA levels in the fat grafts of HNG is inconsistent with the larger size of isolated adipocytes. The high levels of LPL and b3 -AR may indicate the better functional abilities of the transplanted adipocytes in the HNG, because of the sensitive regulatory expressions to nutritions have been reported in rodents. In this context, the metabolic responses of transplanted fat tissues to the systemic conditions, such as low TG and high insulin and FFA levels in plasma, should be considered. Ong et al. [13] have reported that insulin increases LPL mRNA levels by 200% and rates of LPL synthesis by 300% in cultured rat adipocytes. Among the b-adrenergic receptor family, the b3 -AR plays a central role in the regulation of lipolysis in rodent white and brown adipose tissue [14]. b3 -AR mRNA levels are regulated in adipose tissues by the nutritional conditions in mice [15]. On the other hand, the HSL and HK2 mRNA levels were not changed in the same nutritional status. The expression of HSL, the most critical enzyme during lipid mobilization, is believed to be activated by catacholamines through cAMP-dependent phosphorylation, rather than the transcription level [16]. The gene regulation of HSL and HK2 may be different from other genes here in the given calorie-restricted status. In summary, our study shows that, in the transplanted fat tissues, the cell sizes and tissue weights are better kept with the nutrition-rich condition, and the gene expressions in the transplanted tissues are also affected by the systemic metabolic states. Thus, the nutritional condition in hosts is probably important for the transplanted fat tissues both as lipid-storage and functional cells. Further analysis is needed to clarify the long-term effects of nutrition on the survival of transplanted adipocytes.
Acknowledgments These studies were supported by grants from the Japanese Ministry of Education, Science and Culture to Y.S. and H.B.
References [1] K.S. Pinski, Fat transplantation and autologous collagen: a decade of experience, Am. J. Cosmet. Syrg. 16 (1999) 217–223. [2] M.S. Kaminer, N.E. Omura, Autologous fat transplantation, Arch Dermatol. 137 (2001) 812–814. [3] B.L. Eppley, R.V. Snyders Jr., T. Winkelmann, J.J. Delfino, Autologous facial fat transplantation: improved graft mainte-
F. Matsumoto et al. / Biochemical and Biophysical Research Communications 295 (2002) 630–635
[4]
[5]
[6]
[7]
[8]
[9]
nance by microbead bioactivation, J. Oral Maxillofac. Surg. 50 (1992) 477–482. Y. Ullmann, M. Hyams, Y. Ramon, D. Beach, I.J. Peled, E.S. Lindenbaum, Enhancing the survival of aspirated human fat injected into nude mice, Plast. Reconstr. Surg. 101 (1998) 1940– 1944. T. Nakamura, K. Tokunaga, I. Shimomura, M. Nishida, S. Yoshida, K. Kotani, A.H. Islam, Y. Keno, T. Kobatake, Y. Nagai, S. Fujioka, S. Tarui, Y. Matsuzawa, Contribution of visceral fat accumulation to the development of coronary artery disease in non-obese women, Atherosclerosis 107 (1994) 239–246. R. Suzuki, S. Watanabe, Y. Hirai, K. Akiyama, T. Nishide, Y. Matsushima, H. Murayama, H. Ohshima, M. Shinomiya, K. Shirai, Y. Saito, Abdominal wall fat index, estimated by ultrasonography, for assessment of the ratio of visceral fat to subcutaneous fat in the abdomen, Am. J. Med. 95 (1993) 309–314. J.S. Samra, Sir David Cuthbertson Medal Lecture. Regulation of lipid metabolism in adipose tissue, Proc. Nutr. Soc. 59 (2000) 441– 446. T. Shibasaki, K. Takahashi, T. Itou, S. Miyazawa, M. Ito, J. Kobayashi, H. Bujo, Y. Saito, Alterations of insulin sensitivity by the implantation of 3T3-L1 cells in nude mice. A role for TNF-a? Diabetologia, 45 (2002) 518–526. H. Inadera, Y. Ishikawa, K. Shirai, Y. Saito, S. Yoshida, Proliferation and triglyceride synthesizing activities of fibroblastlike cells derived from epididymal and subcutaneous adipose tissues of rats, Scand. J. Clin. Lab Invest. 53 (1993) 225–229.
635
[10] T. Kanaki, N. Morisaki, H. Bujo, K. Takahashi, I. Ishii, Y. Saito, The regulatory expression of procollagen COOH-terminal proteinase enhancer in the proliferation of vascular smooth muscle cells, Biochem. Biophys. Res. Commun. 270 (2000) 1049–1054. [11] M. Toi, T. Matsumoto, H. Bando, Vascular endothelial growth factor: its prognostic, predictive, and therapeutic implications, Lancet Oncol. 2 (2001) 667–673. [12] P.J. Havel, Control of energy homeostasis and insulin action by adipocyte hormones: leptin, acylation stimulating protein, and adiponectin, Curr. Opin. Lipidol. 13 (2002) 51–59. [13] J.M. Ong, T.G. Kirchgessner, M.C. Schotz, P.A. Kern, Insulin increases the synthetic rate and messenger RNA level of lipoprotein lipase in isolated rat adipocytes, J. Biol. Chem. 263 (1988) 12933–12938. [14] K. El Hadri, B. Feve, J. Pairault, Developmental expression and functional activity of beta 1- and beta 3-adrenoceptors in murine 3T3-F442A differentiating adipocytes, Eur. J. Pharmacol. 297 (1996) 107–119. [15] B.A. Evans, M. Papaioannou, F. Anastasopoulos, R.J. Summers, Differential regulation of beta3-adrenoceptors in gut and adipose tissue of genetically obese (ob/ob) C57BL/6J-mice, Br. J. Pharmacol. 124 (1998) 763–771. [16] P. Stralfors, P. Bjorgell, P. Belfrage, Hormonal regulation of hormone-sensitive lipase in intact adipocytes: identification of phosphorylated sites and effects on the phosphorylation by lipolytic hormones and insulin, Proc. Natl. Acad. Sci. USA 81 (1984) 3317–3321.