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• W2022840080 abstract "Following loss of functional small bowel surface area due to surgical resection, the remnant gut undergoes an adaptive response characterized by increased crypt cell proliferation and enhanced villus height and crypt depth, resulting in augmented intestinal nutrient absorptive capacity. Previous studies showed that expression of the immediate early gene tis7 is markedly up-regulated in intestinal enterocytes during the adaptive response. To study its role in the enterocyte, transgenic mice were generated that specifically overexpress TIS7 in the gut. Nucleotides -596 to +21 of the rat liver fatty acid-binding protein promoter were used to direct abundant overexpression of TIS7 into small intestinal upper crypt and villus enterocytes. TIS7 transgenic mice had increased total body adiposity and decreased lean muscle mass compared with normal littermates. Oxygen consumption levels, body weight, surface area, and small bowel weight were decreased. On a high fat diet, transgenic mice exhibited a more rapid and proportionately greater gain in body weight with persistently elevated total body adiposity and increased hepatic fat accumulation. Bolus fat feeding resulted in a greater increase in serum triglyceride levels and an accelerated appearance of enterocytic, lamina propria, and hepatic fat. Changes in fat homeostasis were linked to increased expression of genes involved in enterocytic triglyceride metabolism and changes in growth with decreased insulin-like growth factor-1 expression. Thus, TIS7 overexpression in the intestine altered growth, metabolic rate, adiposity, and intestinal triglyceride absorption. These results suggest that TIS7 is a unique mediator of nutrient absorptive and metabolic adaptation following gut resection. Following loss of functional small bowel surface area due to surgical resection, the remnant gut undergoes an adaptive response characterized by increased crypt cell proliferation and enhanced villus height and crypt depth, resulting in augmented intestinal nutrient absorptive capacity. Previous studies showed that expression of the immediate early gene tis7 is markedly up-regulated in intestinal enterocytes during the adaptive response. To study its role in the enterocyte, transgenic mice were generated that specifically overexpress TIS7 in the gut. Nucleotides -596 to +21 of the rat liver fatty acid-binding protein promoter were used to direct abundant overexpression of TIS7 into small intestinal upper crypt and villus enterocytes. TIS7 transgenic mice had increased total body adiposity and decreased lean muscle mass compared with normal littermates. Oxygen consumption levels, body weight, surface area, and small bowel weight were decreased. On a high fat diet, transgenic mice exhibited a more rapid and proportionately greater gain in body weight with persistently elevated total body adiposity and increased hepatic fat accumulation. Bolus fat feeding resulted in a greater increase in serum triglyceride levels and an accelerated appearance of enterocytic, lamina propria, and hepatic fat. Changes in fat homeostasis were linked to increased expression of genes involved in enterocytic triglyceride metabolism and changes in growth with decreased insulin-like growth factor-1 expression. Thus, TIS7 overexpression in the intestine altered growth, metabolic rate, adiposity, and intestinal triglyceride absorption. These results suggest that TIS7 is a unique mediator of nutrient absorptive and metabolic adaptation following gut resection. The small intestine contains a dynamic epithelium that can rapidly adapt to changes in its luminal environment. Following loss of functional small bowel surface area resulting from small bowel resection, crypt cell proliferation is stimulated in the remnant gut, and this contributes to an adaptive response characterized by enhanced villus height and crypt depth and increased nutrient absorptive capacity. To identify the mechanisms responsible for adaptation, we cloned intestinal genes that are differentially expressed in a rat small bowel resection model of adaptation (1Dodson B.D. Wang J.L. Swietlicki E.A. Rubin D.C. Levin M.S. Am. J. Physiol. 1996; 271: G347-G356Crossref PubMed Google Scholar). Murine tis7 (homologous to rat PC4) was one of several genes that showed increased mRNA expression in the remnant small bowel during the intestinal adaptive response following surgical resection in rodents. The TIS7/PC4 orthologue is expressed in the adapting gut in upper crypt and villus-associated small bowel enterocytes (2Rubin D.C. Swietlicki E.A. Wang J.L. Levin M.S. Am. J. Physiol. 1998; 275: G506-G513PubMed Google Scholar). The tis7/PC4 orthologue is an immediate early gene that is up-regulated in response to growth factors (3Tirone F. Shooter E.M. Proc. Natl. Acad. Sci. U. S. A. 1989; 86: 2088-2092Crossref PubMed Scopus (74) Google Scholar, 4Herschman H.R. Kujubu D.A. Fletcher B.S. Ma Q. Varnum B.C. Gilbert R.S. Reddy S.T. Prog. Nucleic Acid Res. Mol. Biol. 1994; 47: 113-148Crossref PubMed Scopus (27) Google Scholar, 5Varnum B.C. Lim R.W. Herschman H.R. Oncogene. 1989; 4: 1263-1265PubMed Google Scholar). It encodes a plasma membrane-bound protein that translocates to the nucleus upon growth factor stimulation (6Guardavaccaro D. Montagnoli A. Ciotti M.T. Gatti A. Lotti L. Di Lazzaro C. Torrisi M.R. Tirone F. J. Neurosci. Res. 1994; 37: 660-674Crossref PubMed Scopus (22) Google Scholar) and following c-Jun activation in epithelial cells (7Vietor I. Vadivelu S.K. Wick N. Hoffman R. Cotten M. Seiser C. Fialka I. Wunderlich W. Haase A. Korinkova G. Brosch G. Huber L.A. EMBO J. 2002; 21: 4621-4631Crossref PubMed Scopus (40) Google Scholar). We have shown previously that its expression increases in intestinal epithelial cells in response to a variety of growth factors, including glucagon-like peptide 2 and epidermal growth factor (8Swietlicki E. Iordanov H. Fritsch C. Yi L. Levin M.S. Rubin D.C. JPEN (J. Parenter. Enteral Nutr.). 2003; 27: 123-131Crossref PubMed Scopus (19) Google Scholar). TIS7 has been shown in cultured cells to function as a transcriptional co-regulator (7Vietor I. Vadivelu S.K. Wick N. Hoffman R. Cotten M. Seiser C. Fialka I. Wunderlich W. Haase A. Korinkova G. Brosch G. Huber L.A. EMBO J. 2002; 21: 4621-4631Crossref PubMed Scopus (40) Google Scholar, 9Wick N. Schleiffer A. Huber L.A. Vietor I. J. Mol. Biol. 2004; 336: 589-595Crossref PubMed Scopus (13) Google Scholar), and TIS7-null mice have defective muscle regeneration after injury (10Vadivelu S.K. Kurzbauer R. Dieplinger B. Zweyer M. Schafer R. Wernig A. Vietor I. Huber L.A. Mol. Cell. Biol. 2004; 24: 3514-3525Crossref PubMed Scopus (38) Google Scholar). By 24 months of age, these mice have significantly decreased body weight due, at least in part, to decreased skeletal muscle mass. No specific intestinal or other phenotype was noted, but intestinal morphology and response to injury and/or resection were not examined. To investigate the role of TIS7 in the normal and adapting murine intestine, we generated transgenic mice that overexpress this gene in enterocytes of the small intestine and colon, thus mimicking the pattern of gene expression seen during adaptation to resection. We used promoter sequences from the rat liver fatty acid-binding protein gene (Fabpl-596 to +21) to direct high levels of target gene expression to the crypt and villus epithelium of the proximal small bowel with lower levels of transgene expression in the distal small bowel and proximal colonic epithelium (11Simon T.C. Roth K.A. Gordon J.I. J. Biol. Chem. 1993; 268: 18345-18358Abstract Full Text PDF PubMed Google Scholar, 12Stappenbeck T.S. Gordon J.I. Development. 2000; 127: 2629-2642Crossref PubMed Google Scholar, 13Stappenbeck T.S. Gordon J.I. Development. 2001; 128: 2603-2614Crossref PubMed Google Scholar). TIS7 transgenic mice exhibited a distinct phenotype. As early as postnatal day 4, they weighed less than wild type littermates. By 1 month, they exhibited decreased total body surface area but had increased adiposity and decreased lean body mass. TIS7 overexpression also decreased small bowel length and villus height. Despite these changes, triglyceride absorption was enhanced in the transgenic gut, and following small bowel resection, the transgenic mice exhibited an intact morphologic adaptive response. Our data indicate that TIS7, induced in the gut epithelium in response to nutrient stress and deprivation, has direct effects on enterocytic fat absorption and thus acts to enhance functional adaptation following resection. These results also suggest that modulation of fat metabolism in the gut can affect whole body metabolic rate and adiposity/obesity. FVB/N mice were housed in Washington University School of Medicine animal facilities and were maintained on a strict 12 h:12 h light/dark cycle. Animals were fed a standard rodent chow diet (Picolab 20, Ralston Purina, St. Louis, MO) containing 4.5, 20.0, and 36.8 g/100 g from fat, protein, and carbohydrate, respectively) except as described below for the fat feeding and small intestinal resection experiments. The Animal Studies Committee of the Washington University School of Medicine approved all animal experimentation. Mice were sacrificed at various time points; small intestines were collected, rinsed, and weighed; and length was measured by suspension with a fixed weight. Intestines were then divided into four segments including duodenum, proximal jejunum, distal jejunum, and ileum. All other organs were harvested and weighed. Tissues were frozen in liquid nitrogen for RNA and protein isolation and placed in optimal cutting temperature solution and in formalin for histochemical and immunohistochemical analysis. Nucleotides -596 to +21 of the liver fatty acid-binding protein (Fabpl) gene (a gift of Jeffrey Gordon, Washington University) were utilized to direct abundant expression of TIS7 into the crypt and villus epithelial cells of rodent small intestine (11Simon T.C. Roth K.A. Gordon J.I. J. Biol. Chem. 1993; 268: 18345-18358Abstract Full Text PDF PubMed Google Scholar). Fabpl-596 to +21 was fused to the full-length mouse tis7 cDNA generated by RT 4The abbreviations used are: RT, reverse transcription; GH, growth hormone; hGH, human growth hormone; DEXA, dual energy x-ray absorptiometry; WT, wild type; TG, transgenic; NS, non-significant; DGAT, diacylglycerol acyltransferase; MTP, microsomal triglyceride transfer protein; C/EBP, CCAAT/enhancer-binding protein. -PCR of mouse intestinal RNA (Fig. 1A). The tis7 cDNA sequence was confirmed by automated sequencing analysis performed by the Protein Chemistry Facility of Washington University School of Medicine. The full-length cDNA was inserted into the BamHI site of the vector pLPNDon. Twenty spacer nucleotides were placed between the Fabpl promoter and the ATG of tis7 to ensure efficient transcription. In addition, nucleotides +3 to +2150 of the human growth hormone (hGH) gene were linked to the 3′ end of tis7 cDNA to ensure appropriate processing of the transgenic mRNA. The insertion of three termination codons between the tis7 cDNA and hGH+3 to +2150 sequences and the lack of an initiation codon and internal ribosomal reentry site in the hGH+3 to +2150 sequence prevents translation of hGH transcripts. The transgene was excised from the vector by NotI/SalI digestion, and the purified construct was injected into the pronuclei of FVB/N oocytes, courtesy of the Animal Models Core of the Digestive Diseases Research Core Center. Four transgenic founders were generated, and experiments were performed using two independent transgenic lines derived from two founders (lines 2412 and 2454). The presence of the transgene was determined by RT-PCR of tail genomic DNA to detect a portion of the hGH gene. The forward 5′ hGH oligonucleotide primer is 5′-CTG CAC CAG CTG GCG TTT GAC ACC TAC CAG-3′. The reverse 3′ hGH oligo primer is 5′-TTT CTG TTG TGT TTC CTC CCT GTT GGA GGG-3′. RT-PCR was performed using KlenTaq LA polymerase. For analysis of growth rates, transgenic mice and their normal littermates were weighed every 2-3 days after birth. Body composition analyses were performed on anesthetized mice by dual energy x-ray absorptiometry (DEXA) using a small animal densitometer (PIXImus, Lunar Instruments, Fitchburg, WI) as described in Ref. 14Bernal-Mizrachi C. Weng S. Li B. Nolte L.A. Feng C. Coleman T. Holloszy J.O. Semenkovich C.F. Arterioscler. Thromb. Vasc. Biol. 2002; 22: 961-968Crossref PubMed Scopus (67) Google Scholar. Body surface area was calculated by the DEXA measurement. Animals were studied following sedation, at rest and in a fed state, in a single chamber indirect calorimetry Oxymax system to measure oxygen consumption and carbon dioxide production (Columbus Instruments, Columbus, OH) according to Refs. 14Bernal-Mizrachi C. Weng S. Li B. Nolte L.A. Feng C. Coleman T. Holloszy J.O. Semenkovich C.F. Arterioscler. Thromb. Vasc. Biol. 2002; 22: 961-968Crossref PubMed Scopus (67) Google Scholar and 15Li B. Nolte L.A. Ju J.S. Han D.H. Coleman T. Holloszy J.O. Semenkovich C.F. Nat. Med. 2000; 6: 1115-1120Crossref PubMed Scopus (265) Google Scholar. Transgenic mice and normal littermates were studied at room temperature. Quantitative Food Consumption Study—Mice from both transgenic lines 2412 and 2454 and normal littermates were caged individually in metabolic cages containing inserts that permit collection of wasted food. Six-week-old and 2-month-old transgenic mice and wild type littermates were studied. The quantity of food ingested per mouse was measured every 2 days for 10-14 days (n = 4 per group). High Fat Feeding Experiment: Chronic—Transgenic mice and normal littermates were maintained for 14 days on a synthetic, high fat diet (21% (w/w) with 42% of total calories from fat; Adjusted Calories Diet TD 88137, Harlan Teklad, Madison, WI). Mice were weighed on days 2, 5, 10, and 14 after beginning the high fat diet and sacrificed on day 14 (n = 4 per group for males and females). Livers and intestines were harvested for Oil Red O staining. Intestinal Triglyceride Absorption in Vivo: Acute High Fat Feeding— Experiments were performed as described previously (16Narisawa S. Huang L. Iwasaki A. Hasegawa H. Alpers D.H. Millan J.L. Mol. Cell. Biol. 2003; 23: 7525-7530Crossref PubMed Scopus (169) Google Scholar). Transgenic mice and normal littermates were fasted overnight, and serum was removed for fasting triglyceride and free fatty acid levels. Mice received a bolus (500 μl by gavage) of corn oil and were sacrificed at either 1 h (wild type (WT), n = 4; transgenic (TG), n = 3)or 3 h (WT, n = 5; TG, n = 3) after gavage feeding. Serum triglyceride, free fatty acid, and cholesterol levels were measured, and tissues were harvested for histochemical analysis. Routine hematoxylin and eosin staining, Oil Red O staining to detect fat, and electron microscopy were performed. Mice were caged individually in metabolic cages with inserts that permit collection and prevent ingestion of fecal material. Mice were maintained either on a normal chow diet or high fat diet for 14 days, and feces were collected every 2 days. Fecal fat was quantified by extraction with 2:1 chloroform:methanol according to Ref. 17Schwarz M. Lund E.G. Setchell K.D. Kayden H.J. Zerwekh J.E. Bjorkhem I. Herz J. Russell D.W. J. Biol. Chem. 1996; 271: 18024-18031Abstract Full Text Full Text PDF PubMed Scopus (224) Google Scholar. Three-month-old TIS7 transgenic mice and normal littermates were anesthetized with ketamine HCl (87 mg/kg)/xylazine HCl (13 mg/kg) and isoflurane inhalation and underwent 50% bowel resection beginning 2-3 cm distal to the ligament of Treitz and extending to 8 cm proximal to the ileocecal valve as described previously (18Wang L.C. Nassir F. Liu Z.Y. Ling L. Kuo F. Crowell T. Olson D. Davidson N.O. Burkly L.C. Gastroenterology. 2002; 122: 469-482Abstract Full Text Full Text PDF PubMed Scopus (67) Google Scholar). An end-toend anastomosis was formed between the remnant jejunum and ileum using 9-0 silk sutures. All animals received gentamicin (0.2 mg in 0.5 ml of saline intraperitoneally) and buprenorphine (0.03 mg/kg subcutaneously) for analgesia. Mice were sacrificed 2 weeks postoperatively under isoflurane anesthesia by cervical dislocation. The remnant intestines were divided into duodenal-jejunal and ileal segments and were either fixed in formalin or frozen in optimal cutting temperature solution for histochemical and immunohistochemical analysis or placed into liquid nitrogen for RNA and protein isolation. All histologic studies were performed in the Morphology Core of the Digestive Diseases Research Core Center. Oil Red O stains were performed on cryostat sections of flash frozen tissues as described previously (19Tang Y. Swartz-Basile D.A. Swietlicki E.A. Yi L. Rubin D.C. Levin M.S. Gastroenterology. 2004; 126: 220-230Abstract Full Text Full Text PDF PubMed Scopus (54) Google Scholar). Routine hematoxylin and eosin staining was performed on paraffin-embedded tissues for morphometric analyses (2Rubin D.C. Swietlicki E.A. Wang J.L. Levin M.S. Am. J. Physiol. 1998; 275: G506-G513PubMed Google Scholar, 18Wang L.C. Nassir F. Liu Z.Y. Ling L. Kuo F. Crowell T. Olson D. Davidson N.O. Burkly L.C. Gastroenterology. 2002; 122: 469-482Abstract Full Text Full Text PDF PubMed Scopus (67) Google Scholar). Villus heights, crypt depths, and crypt cell numbers were measured in all intestinal segments in at least 10 well oriented, full-length crypt-villus units. Morphometry was performed by AxioVision analysis of digitally acquired images (Zeiss, version 2.0). Crypt cell proliferation was measured by 5-bromodeoxyuridine incorporation into DNA. Mice were injected subcutaneously with a solution of 5-bromodeoxyuridine (8 g/liter) and 5-fluorodeoxyuridine (0.8 g/liter; total dose, 120 mg/kg) and were sacrificed 90 min after injection. 5-BrdUrd was detected with a monoclonal anti-BrdUrd antibody (Zymed Laboratories Inc.) and streptavidin-biotin amplification. Crypt cell proliferation is expressed as the percentage of labeled cells per full-length crypt (number of labeled cells divided by the total number of cells per crypt). Only full-length crypts were evaluated. Quantification of apoptotic cell index was performed by morphologic assessment of tissues stained with hematoxylin and eosin (19Tang Y. Swartz-Basile D.A. Swietlicki E.A. Yi L. Rubin D.C. Levin M.S. Gastroenterology. 2004; 126: 220-230Abstract Full Text Full Text PDF PubMed Scopus (54) Google Scholar). The number of apoptotic cells per crypt and per 1000 crypt cells was measured. In the acute fat feeding experiments, intestines were fixed at 4 °C in 2.5% glutaraldehyde in 0.1 m sodium cacodylate buffer and were sequentially stained with osmium tetroxide, tannic acid, and uranyl acetate; then dehydrated; and embedded in Polybed 812. Samples were thin sectioned on a Reichert-Jung Ultra-Cut, poststained in 4% uranyl acetate and lead citrate, and viewed on a Zeiss 902 electron microscope. Photographs were recorded with Eastman Kodak Co. electron microscope film. Hepatic lipids were extracted by homogenizing 100-400 mg wet weight of liver in 1.5-2.0 ml of phosphate-buffered saline and adding 0.1% sulfuric acid as well as 5 ml chloroform:methanol (2:1). The organic phase was collected and treated with chloroform containing 1% Triton X-100 in a glass tube, which was then vortexed and incubated at 37 °C for 20 min. Triglyceride and cholesterol content were assessed according to Ref. 21Carr T.P. Andresen C.J. Rudel L.L. Clin. Biochem. 1993; 26: 39-42Crossref PubMed Scopus (483) Google Scholar. Serum total cholesterol and triglyceride concentrations were determined using Cholesterol E and L-type TG-H kits, respectively, and serum free fatty acids were determined using the NEFA C kit (Wako Chemicals, Richmond, VA). Serum levels of mouse growth hormone, insulin-like growth factor-1 (IGF-1), insulin, leptin, and glucagon were measured by Ani Lytics, Inc. (Gaithersburg, MD). Serum for growth hormone levels was collected strictly between 8:00 and 8:30 a.m. For immunoblots, a rabbit polyclonal anti-TIS7 antibody was prepared. TIS7 protein was expressed and purified using the IMPACT-CN system (New England Biolabs, Beverly, MA). The mouse tis7 cDNA was subcloned into the pTYB2 vector and expressed in Escherichia coli ER2566 cells. The intein-TIS7 fusion protein was purified after loading onto a chitin bead column followed by cleavage with dithiothreitol. TIS7 protein was run on an SDS-polyacrylamide gel to check for the adequacy of purification. A rabbit polyclonal antibody to mouse TIS7 protein was generated by BioDesign (Saco, ME). Intestinal extracts were prepared, and protein aliquots were electrophoresed on 10% SDS-polyacrylamide gels and transferred onto polyvinylidene difluoride plus membranes. Immunoblots were incubated with the rabbit polyclonal anti-TIS7 antibody (1:2000) followed by horseradish peroxidase-conjugated anti-IgG antibodies (1:10,000, Amersham Biosciences) and developed with chemiluminescent peroxidase substrate (ECL Western blotting kit, Amersham Biosciences). For Northern blot hybridization, RNA was purified using TRIzol reagent (Invitrogen) and run on agarose gels. Nylon transfer membranes were probed with a radiolabeled [α-32P]dCTP cDNA probe (RediPrime II, Amersham Biosciences). Membranes were visualized either by exposure of Kodak Biomax MS film or by using an Amersham Biosciences phosphor screen analyzed with Amersham Biosciences software. Total RNA from duodenum, proximal jejunum, distal jejunum, ileum, and liver was extracted by TRIzol (Invitrogen). The RNAs were treated with DNase I using the DNA-free kit (Ambion, Austin, TX). First strand cDNA was synthesized from 1 μg of total RNA using Super-Script II reverse transcriptase (Invitrogen) with random hexamer primers (Invitrogen). Real time PCR analysis was performed on an SDS 7000 (Applied Biosystems, Foster City, CA) using 2× Sybr Green Master Mix (Applied Biosystems). Oligonucleotide primers were chosen using the Primer Express software (Applied Biosystems). Primers used in quantitative RT-PCR are as follows: TIS7: forward, 5′-CGC TGT CTG AAA AAA GGA AAG AGT-3′; and reverse, 5′-GGC CCA GCT GAA TAC AAA GAA C-3′; IGF-I: exon 1 forward, 5′-GAT GGG GAA AAT CAG CAG CC-3′; exon 2 forward, 5′-TGC TGT GTA AAC GAC CCG-3′; and common reverse, 5′-CAA CAC TCA TCC ACA ATG CC-3′; apolipoprotein A-IV: forward, 5′-CAA TGC CAA GGA GGC TGT AGA-3′; and reverse, 5′-AGT TTG TCC TTG AAG AGG GTA CTG-3′; diacylglycerol acyltransferase 1: forward, 5′-TCC GCC TCT GGG CAT TC-3′; and reverse, 5′-GAA TCG GCC CAC AAT CCA-3′; diacylglycerol acyltransferase 2: forward, 5′-AGA ACC GCA AAG GCT TTG TG-3′; and reverse, 5′-AGG AAT AAG TGG GAA CCA GAT CAG-3′; microsomal triglyceride transfer protein: forward, 5′-AAG ACA GCG TGG GCT ACA AAA-3′; and reverse, 5′-TCA TCA TCA CCA TCA GGA TTC C-3′; intestinal fatty acid-binding protein: forward, 5′-ACT AAT CCA GAC CTA CAC ATA TGA AGG A-3′; and reverse, 5′-GCT CCA GGC TCT GAG AAG TTG A-3′; liver fatty acid-binding protein: forward, 5′-GAA CTT CTC CGG CAA GTA CCA A-3′; and reverse, 5′-GTC CTC GGG CAG ACC TAT TG-3′; cellular retinol-binding protein 2: forward, 5′-ACA TGA AGG CCC TAG ATA TTG ATT TT-3′; and reverse, 5′-AGT GAT GAT CTT CGT CTG AGT CAG A-3′; sodium/glucose cotransporter 1: forward, 5′-CAC CAT CTT GAT CAT CTC CTT CCT-3′; and reverse, 5′-TGC GAT GAC TCC AAC ACA AAC G-3′; and 18 S ribosomal RNA: forward, 5′-CGG CTA CCA CAT CCA AGG AA-3′; and reverse, 5′-GCT GGA ATT ACC GCG GCT-3′. For all primer sets, the kinetics of the PCR was confirmed by serial dilutions of different cDNA preparations. These analyses verified that the efficiencies of amplification were equal for both primer sets, thereby allowing quantification by the comparative Ct method (User Bulletin Number 2, Applied Biosystems). Means were compared between normal and transgenic mice using a Student's t test. Values in the text are means ± S.E. Differences were considered significant at p < 0.05. Generation and Analysis of Transgenic Mice with Intestine-specific Overexpression of TIS7—Transgenic mice were generated that specifically overexpress TIS7 in the intestine as described under “Experimental Procedures.” TIS7 transgenic mRNA levels in gut and other organs were quantified by Northern blot hybridization and quantitative real time RT-PCR assays, and TIS7 protein levels were assessed by Western blotting. Hybridization of total RNA with tis7-specific cDNA probes detected transcripts of sizes varying from 2.3 to ∼4.3 kb in the intestines of transgenic mice, whereas native tis7 mRNAs of 2.3 kb were detected in wild type mice as expected (Fig. 1B). Several higher molecular weight tis7 transcripts were produced by alternative splicing of hGH as found for other transgenic mouse constructs utilizing the hGH gene to ensure appropriate RNA processing. 5T. Simon, personal communication. Intestines of transgenic animals typically presented a cephalocaudal gradient of TIS7 expression; steady state mRNA levels were increased in duodenum, jejunum, and ileum compared with wild type littermates with the greatest increase in expression in duodenum (Fig. 1C). tis7 protein levels were also detected in the small intestine of transgenic mice (Fig. 1D). As reported in other transgenic animals containing this promoter, expression in the liver was increased (11Simon T.C. Roth K.A. Gordon J.I. J. Biol. Chem. 1993; 268: 18345-18358Abstract Full Text PDF PubMed Google Scholar), but we found increased liver expression in only one of the two transgenic lines examined. The phenotype of the mice was the same in both lines irrespective of the liver expression. All experiments described below were performed in both lines. Expression was not increased in the kidney, and tis7 mRNA levels were unchanged in transgenic compared with wild type mice in various other non-digestive organs, including heart, lung, skeletal muscle, white adipose tissue, and brain (data not shown). Reduced Body Weight and Small Bowel Weight and Increased Adiposity in TIS7 Transgenic Mice—Transgenic mice appeared healthy without excess mortality for up to 1 year of observation. However, TIS7 overexpression resulted in several phenotypic changes. Small bowel weight was significantly reduced in TIS7 mice compared with normal littermates, but the weights of other organs were unchanged (Fig. 2A). TIS7 transgenic mice showed reduced total body weight from postnatal day 4 through the suckling-weaning transition and into adulthood (Fig. 2, B and C; representative growth curves for males and females from individual litters). Both male and female mice showed body weight differences beginning shortly after birth. Analysis of mice aged 120 days revealed that the weight differences remained constant for males and females (data not shown). To further evaluate the difference in body weight, food consumption was quantified, organs were harvested and weighed, DEXA analyses were performed to determine body composition, and oxygen consumption was measured to determine basal metabolic rates. To examine the temporal regulation of the phenotypic changes in relation to growth, 6-week-, 2-3-month-, and 9-month-old mice were studied. There were no significant differences in consumption of a normal chow diet measured for 10-14 days in 6-8-week-(data not shown) or 2.5-3-month-old male and female transgenic and wild type mice (Fig. 2D). Surprisingly dual energy x-ray absorptiometry showed that although the TIS7 mice weighed less and had decreased body surface area, they had a higher percentage of body fat and reduced lean body mass compared with wild type littermates (Fig. 2E and TABLE ONE). Body composition was significantly different as early as 6 weeks after birth and became even more apparent by 3 months after birth (Fig. 2E and TABLE ONE). The percentage of body fat mass was increased in both male and female transgenic mice (e.g. at 3 months, 16 versus 25% for wild type and transgenic mice, respectively). There was no significant difference related to gender in fat mass in normal or transgenic mice at the ages examined (data not shown). To determine the phenotype in aged mice, DEXA was performed at 270 days; body surface area remained reduced (7.66 versus 9.85 cm2, p < 0.000001), yet adiposity was further increased (35 versus 22%, p < 0.00001), and as a result, weights were no longer significantly different. The calculated body mass indices for these aged mice (measured as g/cm2) were 3.89 for TG mice and 3.14 for wild type mice.TABLE ONEBody composition and oxygen consumption measurements in 3-month-old wild type control and TIS7 transgenic mice Body composition was determined by dual energy x-ray absorptiometry. n = 13-15 control or transgenic mice per group. Oxygen consumption was quantified by indirect calorimetry according to Ref. 14Bernal-Mizrachi C. Weng S. Li B. Nolte L.A. Feng C. Coleman T. Holloszy J.O. Semenkovich C.F. Arterioscler. Thromb. Vasc. Biol. 2002; 22: 961-968Crossref PubMed Scopus (67) Google Scholar. n = 22 wild type, n = 25 transgenic mice. Data are expressed as means ± S.E., and p values were calculated by Student's t test. Control Transgenic p value Weight (g) 25.59 ± 0.81 21.2 ± 0.59 0.00045 (<0.001) Body surface area (cm2) 9.86 ± 0.27 7.07 ± 0.16 8.62 × 10–14 Fat (g) 4.093 ± 0.29 5.4 ± 0.33 0.0069 (<0.01) Fat (%) 16.25 ± 0.73 25.41 ± 1.12 2.85 × 10–7 (<0.000001) Lean (g) 20.78 ± 0.74 15.78 ± 0.53 8.18 × 10–6 (<0.00001) Oxygen consumption (ml/g0.75/h) 12.58 ± 0.08 11.28 ± 0.04 1.68 × 10–16 Open table in a new tab To further characterize the differences in weight and body composition in TIS7 transgenic mice, oxygen consumption was assessed as a measure of basal whole body metabolic rate (14Bernal-Mizrachi C. Weng S. Li B. Nolte L.A. Feng C. Coleman T. Hollos" @default.
• W2022840080 created "2016-06-24" @default.
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• W2022840080 date "2005-10-01" @default.
• W2022840080 modified "2023-09-29" @default.
• W2022840080 title "Targeted Intestinal Overexpression of the Immediate Early Gene tis7 in Transgenic Mice Increases Triglyceride Absorption and Adiposity" @default.
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