all protocols and experiments were approved by the washington university animals studies committee ( protocol 20150285 ) and followed national institutes of health animal care guidelines .
male c57bl/6 mice , tsc1 and raptor mice , were purchased from the jackson laboratory ( bar harbor , me ) .
villin cre mice were obtained via a generous donation from sylvie robine ( curie institute , paris , france ) .
intestinal epithelial - specific tsc1 and raptor knockout mice were generated by crossing villin cre mice with tsc1 or raptor , respectively .
wild - type littermates villin cre (  ) ; tsc1 or villin cre(- ) ; raptor were used as control mice .
louis , mo ) was dissolved in sunflower oil at 10 mg / ml and injected intraperitoneally at 50 g per gram of body weight for 3 consecutive days to induce deletion of gene expression .
mice were kept in the animal holding area with a 12-hour light - dark cycle and given rodent chow ad libitum after weaning .
the intestinal resections were performed by transecting the bowel 1- to 2-cm distal from the ligament of treitz and at 12-cm proximal to the ileocecal junction , followed by removal of the intervening segment .
intestinal continuity was re - established by an end - to - end primary anastomosis using interrupted 9 - 0 monofilament sutures .
mice then were fed with a standard liquid diet ( pmi micro - stabilized rodent liquid diet ld 101 ; testdiet , richmond , in ) until death . at the time of death
, a midline laparotomy was performed and the entire small intestine was flushed with ice - cold phosphate - buffered saline containing protease inhibitors ( 0.2 nmol / l phenylmethylsulfonyl fluoride , 5 g / ml aprotinin , 1 mol / l benzamidine , 1 mmol / l sodium orthovanadate , and 2 mol / l cantharidin ) . a 2-cm segment of bowel distal to the anastomosis was fixed in 10% neutral - buffered formalin for histology . the remainder of the distal segment was used to isolate crypt and villus .
protein extracted from isolated crypt or villus was used for western blot assay , and rna was used for real - time polymerase chain reaction ( pcr ) assays .
intestinal tissue was paraffin - embedded and cut into 5-m  thick longitudinal sections followed by h&e staining .
villus height and crypt depth were measured with a video - assisted computer program ( nis elements ar 4 ; nikon , melville , ny ) .
crypt depth was measured as the length between the bottom of the crypts and the crypt - villus junction , and villus height was determined by the length between the crypt  villi junction and the top of the villus . at least 20 well - oriented crypts and villi were counted per slide .
crypts were counted only if the crypt  villus junctions on both sides of the crypt were intact and if paneth cells were present at the base of the crypt .
villi were counted only if the central lymphatic channel extended from the villus base to the tip and if the mucosal surface was in continuity with an intact crypt .
alkaline phosphatase activity was detected via immunofluorescence using a vector red alkaline phosphatase substrate kit ( vector laboratories , burlingame , ca ) .
antigen retrieval was performed using diva decloaking solution ( biocare medical , concord , ca ) ( 120c for 2 minutes using a high - pressure cooker ) .
slides were blocked with avidin - pink and biotin - blue ( biocare medical ) , treated with a specific antibody in davinci green ( biocare medical ) , incubated overnight at 4c , and visualized with biotinylated goat anti - rabbit igg ( jackson immunoresearch , inc , west grove , pa ) , followed by streptavidin 
horseradish peroxidase ( jackson immunoresearch laboratories , inc , west grove , pa ) , diaminobenzidine ( sigma - aldrich , st .
p - histone h3 antibody ( 9701 , 1:400 ; cell signaling technology , danvers , ma ) for cell proliferation ; mucin-2 ( 15334 , 1:1000 ; santa cruz , dallas , tx ) for goblet cell differentiation ; chromogranin a ( 20085 , 1:800 ; immunostar , hudson , wi ) for enteroendocrine cell differentiation ; matrix metalloproteinase ( mmp)-7 ( 3801 , 1:100 ; cell signaling technology , danvers , ma ) ; and lysozyme ( rp 028 - 05 , 1:100 ; diagnostic biosystems , pleasanton , ca ) for paneth cell differentiation .
isolated crypt and villus samples were lysed with sodium dodecyl sulfate sample buffer ( 50 mmol / l tris - hcl , ph 6.8 , 2% sodium dodecyl sulfate , 10% glycerol , and 5% mercaptoethanol ) .
the lysate then was heated for 5 minutes at 100c , and the protein concentration was determined using the rc - dc kit ( bio - rad , hercules , ca ) .
antibodies used in this study were as follows : tsc1 ( # 6935 ) , tsc2 ( # 4308 ) ,  raptor ( # 2280 ) , ps6k ( # 9234 ) , s6k ( # 2708 ) , ps6 ( s235/236 ; # 4858 ) , ps6 ( s240/244 ; # 5364 ) , s6 ( # 2217 ) , glyceraldehyde-3-phosphate dehydrogenase ( # 5174 ) , and mmp-7 ( # 3801 ) ( all from cell signaling technology ) .
the proteins were detected using the bio - rad chemidoc xrs+ system with image lab software ( bio - rad ) .
isolated crypts and villi were homogenized in lysis buffer and rna was extracted according to the manufacturer s protocol ( rnaqueous kit ; ambion , austin tx ) .
the total rna concentration was determined using a nanodrop spectrophotometer ( nd-1000 ; nanodrop technologies , wilmington , de ) .
tsc1 , tsc2 , mmp-7 , and lysozyme primers were obtained from life technologies ( carlsbad , ca ) .
-actin was used as the endogenous control ( applied biosystems , foster city , ca ) .
the applied biosystems 7500 fast real - time pcr system was used to obtain relative rna expression .
statistical analysis was performed using the student t test to compare the 2 experimental groups .
all protocols and experiments were approved by the washington university animals studies committee ( protocol 20150285 ) and followed national institutes of health animal care guidelines .
male c57bl/6 mice , tsc1 and raptor mice , were purchased from the jackson laboratory ( bar harbor , me ) .
villin cre mice were obtained via a generous donation from sylvie robine ( curie institute , paris , france ) .
intestinal epithelial - specific tsc1 and raptor knockout mice were generated by crossing villin cre mice with tsc1 or raptor , respectively .
wild - type littermates villin cre (  ) ; tsc1 or villin cre(- ) ; raptor were used as control mice .
louis , mo ) was dissolved in sunflower oil at 10 mg / ml and injected intraperitoneally at 50 g per gram of body weight for 3 consecutive days to induce deletion of gene expression .
mice were kept in the animal holding area with a 12-hour light - dark cycle and given rodent chow ad libitum after weaning .
the intestinal resections were performed by transecting the bowel 1- to 2-cm distal from the ligament of treitz and at 12-cm proximal to the ileocecal junction , followed by removal of the intervening segment .
intestinal continuity was re - established by an end - to - end primary anastomosis using interrupted 9 - 0 monofilament sutures .
mice then were fed with a standard liquid diet ( pmi micro - stabilized rodent liquid diet ld 101 ; testdiet , richmond , in ) until death . at the time of death ,
a midline laparotomy was performed and the entire small intestine was flushed with ice - cold phosphate - buffered saline containing protease inhibitors ( 0.2 nmol / l phenylmethylsulfonyl fluoride , 5 g / ml aprotinin , 1 mol / l benzamidine , 1 mmol / l sodium orthovanadate , and 2 mol / l cantharidin ) . a 2-cm segment of bowel distal to the anastomosis
protein extracted from isolated crypt or villus was used for western blot assay , and rna was used for real - time polymerase chain reaction ( pcr ) assays .
intestinal tissue was paraffin - embedded and cut into 5-m  thick longitudinal sections followed by h&e staining .
villus height and crypt depth were measured with a video - assisted computer program ( nis elements ar 4 ; nikon , melville , ny ) .
crypt depth was measured as the length between the bottom of the crypts and the crypt - villus junction , and villus height was determined by the length between the crypt  villi junction and the top of the villus . at least 20 well - oriented crypts and villi were counted per slide .
crypts were counted only if the crypt  villus junctions on both sides of the crypt were intact and if paneth cells were present at the base of the crypt .
villi were counted only if the central lymphatic channel extended from the villus base to the tip and if the mucosal surface was in continuity with an intact crypt .
alkaline phosphatase activity was detected via immunofluorescence using a vector red alkaline phosphatase substrate kit ( vector laboratories , burlingame , ca ) .
antigen retrieval was performed using diva decloaking solution ( biocare medical , concord , ca ) ( 120c for 2 minutes using a high - pressure cooker ) .
slides were blocked with avidin - pink and biotin - blue ( biocare medical ) , treated with a specific antibody in davinci green ( biocare medical ) , incubated overnight at 4c , and visualized with biotinylated goat anti - rabbit igg ( jackson immunoresearch , inc , west grove , pa ) , followed by streptavidin 
horseradish peroxidase ( jackson immunoresearch laboratories , inc , west grove , pa ) , diaminobenzidine ( sigma - aldrich , st .
p - histone h3 antibody ( 9701 , 1:400 ; cell signaling technology , danvers , ma ) for cell proliferation ; mucin-2 ( 15334 , 1:1000 ; santa cruz , dallas , tx ) for goblet cell differentiation ; chromogranin a ( 20085 , 1:800 ; immunostar , hudson , wi ) for enteroendocrine cell differentiation ; matrix metalloproteinase ( mmp)-7 ( 3801 , 1:100 ; cell signaling technology , danvers , ma ) ; and lysozyme ( rp 028 - 05 , 1:100 ; diagnostic biosystems , pleasanton , ca ) for paneth cell differentiation .
isolated crypt and villus samples were lysed with sodium dodecyl sulfate sample buffer ( 50 mmol / l tris - hcl , ph 6.8 , 2% sodium dodecyl sulfate , 10% glycerol , and 5% mercaptoethanol ) .
the lysate then was heated for 5 minutes at 100c , and the protein concentration was determined using the rc - dc kit ( bio - rad , hercules , ca ) .
antibodies used in this study were as follows : tsc1 ( # 6935 ) , tsc2 ( # 4308 ) ,  raptor ( # 2280 ) , ps6k ( # 9234 ) , s6k ( # 2708 ) , ps6 ( s235/236 ; # 4858 ) , ps6 ( s240/244 ; # 5364 ) , s6 ( # 2217 ) , glyceraldehyde-3-phosphate dehydrogenase ( # 5174 ) , and mmp-7 ( # 3801 ) ( all from cell signaling technology ) .
the proteins were detected using the bio - rad chemidoc xrs+ system with image lab software ( bio - rad ) .
isolated crypts and villi were homogenized in lysis buffer and rna was extracted according to the manufacturer s protocol ( rnaqueous kit ; ambion , austin tx ) .
the total rna concentration was determined using a nanodrop spectrophotometer ( nd-1000 ; nanodrop technologies , wilmington , de ) .
tsc1 , tsc2 , mmp-7 , and lysozyme primers were obtained from life technologies ( carlsbad , ca ) .
-actin was used as the endogenous control ( applied biosystems , foster city , ca ) .
the applied biosystems 7500 fast real - time pcr system was used to obtain relative rna expression .
statistical analysis was performed using the student t test to compare the 2 experimental groups .
to determine whether mtorc1 is involved in intestinal adaptation after resection , we first performed 50% proximal sbr on male c57bl/6 mice followed by systemic inhibition of mtorc1 via rapamycin .
small bowel resection is a major abdominal surgery with a significant physiological impact on the mice in the first 48 hours after surgery . in addition , we see a significant decrease in oral intake during this period despite the rapid regeneration process occurring at the molecular level .
rapamycin injections have a much smaller , but similar , effect on oral intake and frailty in the mice .
when these 2 procedures are compounded , the postsurgery survival rate decreases sharply . to accommodate , we allowed the resected mice 48 hours to recover from surgery before randomization .
mice received a daily 200-l intraperitoneal injection of either vehicle ( 5% tween-80 , 5% polyethylene glycol 400 ( peg-400 ) in phosphate - buffered saline ) or rapamycin ( dissolved in vehicle ) for 1 week at a dose of 4 mg / kg .
, crypts , villi , and the adjacent mesenchyme were separated for detecting mtorc1 activity in response to rapamycin inhibition .
one week of rapamycin treatment resulted in a diminished adaptive response as shown by significantly decreased villus height ( figure  1a and d ) .
crypt cell proliferation , as measured by phosphorylated histone h3 ( p - hh3 , mitosis marker ) ihc staining , also was decreased significantly in rapamycin - treated sbr mice ( figure  1c and e ) .
the successful inhibition of mtorc1 activity across all layers of small intestine was confirmed by measuring phosphorylation of ribosomal protein s6 , which is a common surrogate of mtorc1 activity.15 , 21 in crypts , western blot assay showed a dramatic decrease in ribosomal protein s6 phosphorylation at both ser235/236 and ser240/244 , indicating decreased mtorc1 signaling ( figure  1f ) .
because s6 phosphorylation is similar at ser235/236 and ser240/244 , only ser235/236 phosphorylation was recorded for subsequent experiments .
ihc staining of small intestine with ps6 antibody confirmed a significant reduction of ps6 signal in rapamycin - treated mice ( figure  1b ) .
when body weight was measured , rapamycin - treated mice weighed significantly less when compared with vehicle - treated mice ( figure  1 g ) .
these data indicate that systemic mtorc1 signaling is important for crypt cell proliferation and adaptation after sbr . to determine whether the effects of rapamycin inhibition were specific for the adapting bowel , we also treated unoperated c57bl/6 mice with the same dose of rapamycin for the same time period .
rapamycin treatment of these mice did not affect intestinal structure , crypt cell proliferation , or weight gain ( figure  2 ) .
these findings verify that the mtorc1 signaling pathway is involved specifically in the resection - induced adaptation process . although treatment of mice with rapamycin showed that systemic mtorc1 signaling is critical for intestinal adaptation after massive sbr , our ultimate goal was to enhance adaptation responses after sbr .
because of the ubiquitous nature of mtorc1 signaling throughout the body , tissue - specific contributions to adaptation may require targeted mtorc1 activation . because adaptation is believed to be an event involving stimulated rates of enterocyte proliferation , we focused on genetic manipulation of intestinal epithelial cells . when bred with rosa26 reporter mouse line , the villin cre line has been shown to target intestinal stem cells after tam injection .
we therefore crossed the villin cre transgenic mouse with the tsc1 mouse line to generate inducible intestinal epithelial cell  specific tsc1 null mice ( i - tsc1-/- ) .
when mice reached 68 weeks of age , tam was injected intraperitoneally for 3 consecutive days .
after 2 weeks , the distal ileum was excised and the deletion of tsc1 in i - tsc1-/- mice was confirmed by real - time pcr using isolated epithelial cells ( data not shown ) .
western blot then was used to verify the efficient deletion of tsc1 protein ( figure  3a ) .
as expected , in tsc1-deficient epithelial cells , the well - established mtorc1 kinase target s6k and s6 were highly phosphorylated .
in addition , protein expression of tsc2 , the binding partner of tsc1 , was degraded ( figure  3a ) .
in contrast , tsc1 protein expression in smooth muscle was not changed in i - tsc1-/- mice ( data not shown ) .
when crypt depth and villus height were measured , a small , yet significant , increase ( 17.6% ) in crypt depth was seen in unoperated i - tsc1 null mice ( figure  3b and d ) .
the villus height , however , was not changed ( figure  3b and d ) .
when the paraffin - embedded slides were stained with p - hh3specific antibody to measure crypt cell proliferation , we found 34% more mitotically active crypt cells in i - tsc1 null mice ( figure  3c ) .
the literature shows several differing approaches to determine the role of mtorc1 in intestinal epithelial cell differentiation .
however , these studies have yielded many different results based on the approach used.16 , 21 systemic inhibition of mtor or raptor has been shown to diminish the activity of alkaline phosphatase . in this study , we found no change in alkaline phosphatase activity within villi between wild - type ( wt ) and i - tsc1-/- small intestine ( figure  4a ) , which indicates normal absorptive cell lineage differentiation in the absence of tsc1 .
secretory cell differentiation was investigated ( paneth , goblet , and enteroendocrine cells ) with ihc staining using lysozyme , mucin-2 , and chromogranin a , respectively .
we found that mtorc1 activation through tsc1 ablation in epithelial cells had no effect on differentiation of goblet and enteroendocrine cells ( figure  4b  e ) . because paneth cell turnover time is much longer ( 3 to 8 weeks ) than other cell lineages,24 , 25
, 26 we investigated the long - term ( 4 weeks ) effects of tsc1 deficiency on paneth cells .
lysozyme - positive paneth cells in wt mice showed a typical clustered pattern on the bottom of the crypt base .
however , lysozyme - positive cells in i - tsc1-/- crypts were less uniform , yet remained confined to the crypt compartment ( figure  5a ) . despite the aberrant location of lysozyme - positive cells ,
lysozyme is one of many antimicrobial peptides within the granules of paneth cells . to see whether tsc1 affects the overall paneth cells ,
indeed , the granule - containing paneth cells often were not clustered on the bottom of i - tsc1-/- crypts ( figure  5b ) . to further delineate the effects of tsc1 deficiency on paneth cell function , a metalloproteinase ( mmp-7 ) that regulates intestinal -defensin activation was measured.27 , 28 , 29 similar to lysozyme staining , mmp-7positive paneth cells also clustered on the bottom of wt crypts , but were disorganized in tsc1-deficient crypts ( figure  5c ) .
the expression level of mmp-7 protein as shown via western blot assay was not affected in tsc1-deficient crypts ( figure  5e ) .
these data indicate that acute activation of mtorc1 in adult mice affects crypt cell proliferation and paneth cell organization , whereas goblet , enteroendocrine , and absorptive cell lineage differentiation remain undisturbed .
next , we sought to investigate whether the increased proliferation seen with increased mtorc1 activation would promote structural adaptation after sbr .
this study was designed to test whether acute enhancement of mtorc1 activity may have therapeutic benefit in the postoperative phase after massive sbr .
therefore , we performed sbr on villin - cre(- ) ; tsc1 or villin - cre(+ ) ; tsc1 mice first . after a 2-day recovery period
, we followed up with tam injection for 3 consecutive days to activate mtorc1 as a means of intervention during the adaptation process .
wt mice showed a typical adaptation response although the response of the i - tsc1-/- mice was much stronger ( 20% greater villus growth ) ( figure  6a and b ) .
crypt proliferation in i - tsc1-/- mice , as shown by p - hh3 ihc staining , was 44% more than in the wt mice ( figure  6c ) .
the activation of mtorc1 signaling in resected mice was confirmed by decreased tsc2 expression and increased phosphorylation of ps6 and s6k in epithelial cells ( figure  6d ) . despite the increased structural intestinal adaptation
, the i - tsc1-/- mice did not gain more weight than wt mice at 2 weeks after resection ( figure  6e ) .
we wanted to confirm that the proliferative and adaptive responses seen after sbr were owing to mtorc1 activation secondary to tsc1 inhibition rather than an alternate function of tsc1 protein . to test this , we injected mice with 3 doses of tam to delete tsc1 first , waited for a week , and then performed sbr procedures on i - tsc1-/- mice .
the modified research design was to focus on the role of tsc1 and simultaneously avoid the stress of injecting both tam and rapamycin in postsurgical mice .
after a 2-day recovery , the resected mice were randomized and treated with either vehicle or rapamycin ( to inhibit mtorc1 ) for 2 weeks .
we found that adaptation as well as resection - induced crypt cell proliferation was diminished by rapamycin treatment in i - tsc1-/- mice ( figure  7a  c ) .
the inhibition of mtorc1 in the rapamycin - treated mice was confirmed by western blot ( figure  7d ) .
these data clearly verify that the enhanced proliferation and adaptation seen in tsc1-null mice after sbr is owing to the activated mtorc1 signaling pathway within intestinal epithelial cells .
it also suggests that resection - induced crypt cell proliferation could be modulated by enhancing intestinal epithelial cell mtorc1 activity .
we have established that intestinal epithelial - specific activation of mtorc1 is capable of enhancing adaptation after sbr .
however , it was not clear after the systemic rapamycin inhibition study whether mtorc1 signaling in intestinal epithelium is necessary for normal adaptation to occur . to investigate this , we bred villin cre mice with raptor mice to delete raptor protein as a means of inactivating mtorc1 specifically in epithelial cells . the same experimental design in tsc1 null mice was adopted for this study .
we first confirmed the deletion of raptor protein in epithelial cells ( figure  8a ) .
the downstream protein kinase s6k was phosphorylated at baseline but was undetectable when raptor was deleted .
in addition , ps6 phosphorylation was greatly reduced in the absence of raptor . despite the lack of mtorc1 signaling in the i - raptor-/- mice , the structure of the small intestine did not change ( figure  8b and d ) .
rates of crypt cell proliferation were the same as in wt mice ( figure  8c ) , which is consistent with previously published data .
in addition , alkaline phosphatase activity was not changed , verifying that enterocyte maturation was not altered ( figure  9a ) . when secretory cell lineage differentiation was analyzed via ihc staining using cell differentiation specific markers
, we found that goblet cells were decreased , whereas enteroendocrine cells were increased in the absence of intestinal epithelial - specific raptor ( figure  9b  e ) .
lysozyme - positive paneth cells were greatly reduced after 4 weeks ( figure  10a ) .
the granule - containing paneth cells also were diminished in raptor - deficient crypts in h&e - stained tissue ( figure  10b ) .
moreover , the expression of mmp-7 also was reduced dramatically in raptor - deficient crypts ( figure  10c and f ) .
surprisingly , neither lysozyme nor mmp-7 mrna expression levels were affected ( figure  10d and e ) , suggesting that raptor protein may have a role in the translational regulation of lysozyme and mmp-7 expression during paneth cell maturation .
sampson et  al previously showed that mtorc1 activity is required for crypt regeneration after irradiation - induced intestinal injury .
we therefore sought to determine whether mtorc1 activity is necessary for resection - stimulated crypt cell proliferation .
a similar strategy was used in which sbr was performed on villin cre(- ) ; raptor mice and villin cre(+ ) ; raptor mice .
two days after surgery tam was given via intraperitoneal injection for 3 consecutive days to inactivate the mtorc1 pathway in the intestinal epithelium .
remarkably , crypt depth and villus height in i - raptor-/- mice was the same as in wt mice ( figure  11a and b ) .
crypt cell proliferation , as measured by p - hh3 ihc , also was not changed ( figure  11c ) .
blocked mtorc1 signaling in raptor - deficient mice was confirmed ( figure  11d ) . despite the normal structural adaptation , the raptor - deficient mice had significantly more weight loss after surgery than the wt mice ( figure  11e ) . in summary , our results show that mtorc1 activity in intestinal epithelial cells is not required for crypt proliferation and has different effects on enterocyte differentiation .
furthermore , resection - induced crypt proliferation and intestinal adaptation requires systemic , but not intestinal epithelial - specific , mtorc1 activity . however , stimulation of mtorc1 in intestinal epithelium increases crypt cell proliferation , which further enhances adaptation after sbr ( figure  12 ) .
the long - term functional significance of manipulating mtorc1 signaling in intestinal epithelial cells after small bowel resection remains unclear and will require further studies .
to determine whether mtorc1 is involved in intestinal adaptation after resection , we first performed 50% proximal sbr on male c57bl/6 mice followed by systemic inhibition of mtorc1 via rapamycin .
small bowel resection is a major abdominal surgery with a significant physiological impact on the mice in the first 48 hours after surgery . in addition , we see a significant decrease in oral intake during this period despite the rapid regeneration process occurring at the molecular level .
rapamycin injections have a much smaller , but similar , effect on oral intake and frailty in the mice .
when these 2 procedures are compounded , the postsurgery survival rate decreases sharply . to accommodate , we allowed the resected mice 48 hours to recover from surgery before randomization .
mice received a daily 200-l intraperitoneal injection of either vehicle ( 5% tween-80 , 5% polyethylene glycol 400 ( peg-400 ) in phosphate - buffered saline ) or rapamycin ( dissolved in vehicle ) for 1 week at a dose of 4 mg / kg .
, crypts , villi , and the adjacent mesenchyme were separated for detecting mtorc1 activity in response to rapamycin inhibition .
one week of rapamycin treatment resulted in a diminished adaptive response as shown by significantly decreased villus height ( figure  1a and d ) .
crypt cell proliferation , as measured by phosphorylated histone h3 ( p - hh3 , mitosis marker ) ihc staining , also was decreased significantly in rapamycin - treated sbr mice ( figure  1c and e ) .
the successful inhibition of mtorc1 activity across all layers of small intestine was confirmed by measuring phosphorylation of ribosomal protein s6 , which is a common surrogate of mtorc1 activity.15 , 21 in crypts , western blot assay showed a dramatic decrease in ribosomal protein s6 phosphorylation at both ser235/236 and ser240/244 , indicating decreased mtorc1 signaling ( figure  1f ) .
because s6 phosphorylation is similar at ser235/236 and ser240/244 , only ser235/236 phosphorylation was recorded for subsequent experiments .
ihc staining of small intestine with ps6 antibody confirmed a significant reduction of ps6 signal in rapamycin - treated mice ( figure  1b ) .
when body weight was measured , rapamycin - treated mice weighed significantly less when compared with vehicle - treated mice ( figure  1 g ) .
these data indicate that systemic mtorc1 signaling is important for crypt cell proliferation and adaptation after sbr . to determine whether the effects of rapamycin inhibition were specific for the adapting bowel , we also treated unoperated c57bl/6 mice with the same dose of rapamycin for the same time period .
rapamycin treatment of these mice did not affect intestinal structure , crypt cell proliferation , or weight gain ( figure  2 ) .
these findings verify that the mtorc1 signaling pathway is involved specifically in the resection - induced adaptation process .
although treatment of mice with rapamycin showed that systemic mtorc1 signaling is critical for intestinal adaptation after massive sbr , our ultimate goal was to enhance adaptation responses after sbr .
because of the ubiquitous nature of mtorc1 signaling throughout the body , tissue - specific contributions to adaptation may require targeted mtorc1 activation . because adaptation is believed to be an event involving stimulated rates of enterocyte proliferation , we focused on genetic manipulation of intestinal epithelial cells . when bred with rosa26 reporter mouse line , the villin cre line has been shown to target intestinal stem cells after tam injection .
we therefore crossed the villin cre transgenic mouse with the tsc1 mouse line to generate inducible intestinal epithelial cell  specific tsc1 null mice ( i - tsc1-/- ) .
when mice reached 68 weeks of age , tam was injected intraperitoneally for 3 consecutive days .
after 2 weeks , the distal ileum was excised and the deletion of tsc1 in i - tsc1-/- mice was confirmed by real - time pcr using isolated epithelial cells ( data not shown ) .
western blot then was used to verify the efficient deletion of tsc1 protein ( figure  3a ) .
as expected , in tsc1-deficient epithelial cells , the well - established mtorc1 kinase target s6k and s6 were highly phosphorylated .
in addition , protein expression of tsc2 , the binding partner of tsc1 , was degraded ( figure  3a ) .
in contrast , tsc1 protein expression in smooth muscle was not changed in i - tsc1-/- mice ( data not shown ) .
these results confirm that we successfully established an inducible intestinal epithelial cell  specific mtorc1 activation system for further characterization of mtorc1 signaling in the gut .
when crypt depth and villus height were measured , a small , yet significant , increase ( 17.6% ) in crypt depth was seen in unoperated i - tsc1 null mice ( figure  3b and d ) .
the villus height , however , was not changed ( figure  3b and d ) .
when the paraffin - embedded slides were stained with p - hh3specific antibody to measure crypt cell proliferation , we found 34% more mitotically active crypt cells in i - tsc1 null mice ( figure  3c ) .
the literature shows several differing approaches to determine the role of mtorc1 in intestinal epithelial cell differentiation .
however , these studies have yielded many different results based on the approach used.16 , 21 systemic inhibition of mtor or raptor has been shown to diminish the activity of alkaline phosphatase . in this study , we found no change in alkaline phosphatase activity within villi between wild - type ( wt ) and i - tsc1-/- small intestine ( figure  4a ) , which indicates normal absorptive cell lineage differentiation in the absence of tsc1 .
secretory cell differentiation was investigated ( paneth , goblet , and enteroendocrine cells ) with ihc staining using lysozyme , mucin-2 , and chromogranin a , respectively .
we found that mtorc1 activation through tsc1 ablation in epithelial cells had no effect on differentiation of goblet and enteroendocrine cells ( figure  4b  e ) . because paneth cell turnover time is much longer ( 3 to 8 weeks ) than other cell lineages,24 , 25
, 26 we investigated the long - term ( 4 weeks ) effects of tsc1 deficiency on paneth cells .
lysozyme - positive paneth cells in wt mice showed a typical clustered pattern on the bottom of the crypt base .
however , lysozyme - positive cells in i - tsc1-/- crypts were less uniform , yet remained confined to the crypt compartment ( figure  5a ) . despite the aberrant location of lysozyme - positive cells ,
lysozyme is one of many antimicrobial peptides within the granules of paneth cells . to see whether tsc1 affects the overall paneth cells ,
indeed , the granule - containing paneth cells often were not clustered on the bottom of i - tsc1-/- crypts ( figure  5b ) . to further delineate the effects of tsc1 deficiency on paneth cell function , a metalloproteinase ( mmp-7 ) that regulates intestinal -defensin activation was measured.27 , 28 , 29 similar to lysozyme staining , mmp-7positive paneth cells also clustered on the bottom of wt crypts , but were disorganized in tsc1-deficient crypts ( figure  5c ) .
the expression level of mmp-7 protein as shown via western blot assay was not affected in tsc1-deficient crypts ( figure  5e ) .
these data indicate that acute activation of mtorc1 in adult mice affects crypt cell proliferation and paneth cell organization , whereas goblet , enteroendocrine , and absorptive cell lineage differentiation remain undisturbed .
next , we sought to investigate whether the increased proliferation seen with increased mtorc1 activation would promote structural adaptation after sbr .
this study was designed to test whether acute enhancement of mtorc1 activity may have therapeutic benefit in the postoperative phase after massive sbr .
therefore , we performed sbr on villin - cre(- ) ; tsc1 or villin - cre(+ ) ; tsc1 mice first .
after a 2-day recovery period , we followed up with tam injection for 3 consecutive days to activate mtorc1 as a means of intervention during the adaptation process .
wt mice showed a typical adaptation response although the response of the i - tsc1-/- mice was much stronger ( 20% greater villus growth ) ( figure  6a and b ) .
crypt proliferation in i - tsc1-/- mice , as shown by p - hh3 ihc staining , was 44% more than in the wt mice ( figure  6c ) .
the activation of mtorc1 signaling in resected mice was confirmed by decreased tsc2 expression and increased phosphorylation of ps6 and s6k in epithelial cells ( figure  6d ) . despite the increased structural intestinal adaptation
, the i - tsc1-/- mice did not gain more weight than wt mice at 2 weeks after resection ( figure  6e ) .
we wanted to confirm that the proliferative and adaptive responses seen after sbr were owing to mtorc1 activation secondary to tsc1 inhibition rather than an alternate function of tsc1 protein . to test this , we injected mice with 3 doses of tam to delete tsc1 first , waited for a week , and then performed sbr procedures on i - tsc1-/- mice .
the modified research design was to focus on the role of tsc1 and simultaneously avoid the stress of injecting both tam and rapamycin in postsurgical mice .
after a 2-day recovery , the resected mice were randomized and treated with either vehicle or rapamycin ( to inhibit mtorc1 ) for 2 weeks .
we found that adaptation as well as resection - induced crypt cell proliferation was diminished by rapamycin treatment in i - tsc1-/- mice ( figure  7a  c ) .
the inhibition of mtorc1 in the rapamycin - treated mice was confirmed by western blot ( figure  7d ) .
these data clearly verify that the enhanced proliferation and adaptation seen in tsc1-null mice after sbr is owing to the activated mtorc1 signaling pathway within intestinal epithelial cells .
it also suggests that resection - induced crypt cell proliferation could be modulated by enhancing intestinal epithelial cell mtorc1 activity .
we have established that intestinal epithelial - specific activation of mtorc1 is capable of enhancing adaptation after sbr . however , it was not clear after the systemic rapamycin inhibition study whether mtorc1 signaling in intestinal epithelium is necessary for normal adaptation to occur . to investigate this , we bred villin cre mice with raptor mice to delete raptor protein as a means of inactivating mtorc1 specifically in epithelial cells .
we first confirmed the deletion of raptor protein in epithelial cells ( figure  8a ) .
the downstream protein kinase s6k was phosphorylated at baseline but was undetectable when raptor was deleted .
in addition , ps6 phosphorylation was greatly reduced in the absence of raptor . despite the lack of mtorc1 signaling in the i - raptor-/- mice , the structure of the small intestine did not change ( figure  8b and d ) .
rates of crypt cell proliferation were the same as in wt mice ( figure  8c ) , which is consistent with previously published data . in addition , alkaline phosphatase activity was not changed , verifying that enterocyte maturation was not altered ( figure  9a ) . when secretory cell lineage differentiation was analyzed via ihc staining using cell differentiation specific markers
, we found that goblet cells were decreased , whereas enteroendocrine cells were increased in the absence of intestinal epithelial - specific raptor ( figure  9b  e ) .
lysozyme - positive paneth cells were greatly reduced after 4 weeks ( figure  10a ) .
the granule - containing paneth cells also were diminished in raptor - deficient crypts in h&e - stained tissue ( figure  10b ) . moreover
, the expression of mmp-7 also was reduced dramatically in raptor - deficient crypts ( figure  10c and f ) .
surprisingly , neither lysozyme nor mmp-7 mrna expression levels were affected ( figure  10d and e ) , suggesting that raptor protein may have a role in the translational regulation of lysozyme and mmp-7 expression during paneth cell maturation .
sampson et  al previously showed that mtorc1 activity is required for crypt regeneration after irradiation - induced intestinal injury .
we therefore sought to determine whether mtorc1 activity is necessary for resection - stimulated crypt cell proliferation .
a similar strategy was used in which sbr was performed on villin cre(- ) ; raptor mice and villin cre(+ ) ; raptor mice .
two days after surgery tam was given via intraperitoneal injection for 3 consecutive days to inactivate the mtorc1 pathway in the intestinal epithelium .
remarkably , crypt depth and villus height in i - raptor-/- mice was the same as in wt mice ( figure  11a and b ) .
crypt cell proliferation , as measured by p - hh3 ihc , also was not changed ( figure  11c ) .
blocked mtorc1 signaling in raptor - deficient mice was confirmed ( figure  11d ) . despite the normal structural adaptation , the raptor - deficient mice had significantly more weight loss after surgery than the wt mice ( figure  11e ) . in summary , our results show that mtorc1 activity in intestinal epithelial cells is not required for crypt proliferation and has different effects on enterocyte differentiation . furthermore , resection - induced crypt proliferation and intestinal adaptation requires systemic , but not intestinal epithelial - specific , mtorc1 activity . however , stimulation of mtorc1 in intestinal epithelium increases crypt cell proliferation , which further enhances adaptation after sbr ( figure  12 ) .
the long - term functional significance of manipulating mtorc1 signaling in intestinal epithelial cells after small bowel resection remains unclear and will require further studies .
the mtor signaling pathway has been studied extensively because it is a master regulator of cell proliferation , growth , and metabolism.3 , 4 , 6 , 30 historically , the majority of these studies were based in cell culture .
however , more recently , studies have emerged with tissue / organ - specific research that has shown varied physiological functions of mtor in adult tissues and during development.12 , 13 , 15 , 16 , 31 in intestinal tissue , several groups have used various strategies to manipulate mtor signaling and have yielded dissimilar results . for example , using a similar strategy to constitutively delete mtor protein specifically in intestinal epithelial cells,11 , 16 one group clearly showed a dilated small bowel with a reduced body weight . on the other hand
, another group found that the mtor - deficient mice have a shorter small bowel but a similar body weight .
there is also some controversy regarding the role of mtor signaling in intestinal cell differentiation .
one report found that intestinal epithelial - specific mtor signaling is required for proper lysozyme transcription in paneth cells .
however , other studies found that mtor activation via a constitutive systemic tsc2 mutation also impaired lysozyme expression in paneth cells . by using an inducible cre system to manipulate mtor signaling in adult intestinal epithelial cells , we found that paneth cell differentiation was indeed regulated by mtorc1 signaling in intestinal epithelial cells , but via a different mechanism .
mtorc1 inactivation does not inhibit lysozyme or mmp-7 transcription , rather , it appears to regulate lysozyme or mmp-7 translation . activating mtorc1 in intestinal epithelium , on the other hand , appears to affect paneth cell clustering on the base of crypts .
maturation of enterocytes also has been found to be disrupted in mtor or raptor - deficient cells based on alkaline phosphatase activity . in our acute ablation model , we did not see any alteration in the expression of alkaline phosphatase in raptor- or tsc - deficient mice .
these seemingly conflicting studies may be explained by the different methods used in each study to manipulate mtor signaling and may represent different aspects of regulation during enterocyte differentiation . in the tsc2 mutant model , mtorc1 activation caused by loss of the functional tsc2 was not confined to the intestine .
the mesenchyme , as well as other tissues , also would show mtorc1 activity , confounding the autonomous role of mtor in intestinal epithelial cells . in the enterocyte mtor - deficient model
, villin cre was activated from embryonic day 12.5 , which suggests that mtorc1 activity may be required during the development of paneth cells as well as absorptive enterocyte differentiation .
the multitude of differing results illustrates the complexity of the mtorc1 signaling pathway in regulating development and maintenance of the small intestine . in this study , we investigated the role of mtorc1 during normal intestinal homeostasis as well as during the adaptive response after massive sbr .
although inactivating mtorc1 signaling in intestinal epithelium has no effect on intestinal structure or the adaptive response after resection , we found that stimulating mtorc1 resulted in enhanced crypt proliferation in adult mice .
in addition , we showed that after massive sbr , adaptation and crypt proliferation were enhanced further when mtorc1 was activated in remnant small bowel . the notion that the function of mtorc1 differs during homeostasis and regeneration
after injury has been established.15 , 16 , 32 constitutively ablating mtor or raptor in intestinal epithelial cells has little effect on crypt cell proliferation and wnt activity in the normal intestine .
however , when mtorc1 signaling was absent , crypt regeneration after irradiation injury was compromised . in apc mice , mtorc1 activity is required for wnt - mediated intestinal polyp formation , likely via regulating the translational elongation rate.15 , 32 our results support the notion that mtorc1 regulates intestinal homeostasis and recovery after injury by different mechanisms . however , they also provide a clear distinction between the mechanism behind adaptation and that of wnt - mediated crypt growth .
raptor deficiency alone is not sufficient to alter small intestine proliferation or adaptation after massive sbr , which indicates pathways other than the wnt pathway may be crucial for regulating adaptation . inhibiting mtorc1 via rapamycin systemically , but not by raptor deficiency in intestinal epithelial cells ,
it is possible that rapamycin may have other unknown targets aside from mtorc1 that regulate crypt cell proliferation .
alternatively , it must be considered that intestinal epithelial cells possess a redundant system to compensate for the loss of mtorc1 signaling to maintain proliferation and growth . however
, this theory contradicts the findings that raptor - deficient crypts do not grow into organoid outside of the intestine niche in  vitro .
the more plausible mechanism to consider is that mtor signals originating from the mesenchyme or the surrounding tissues play a critical role in this process , given that mtor functions in a non  cell - autonomous manner .
the critical role of mesenchyme subepithelial fibroblasts in maintaining intestinal niche function was established clearly in a recent report .
it appears that non  cell - autonomous mtorc1 signaling is crucial for the small bowel adaptive response after resection .
it is well known that nutrients and growth factors can enhance adaptation after resection.18 , 22 , 34 we previously showed that a high - protein diet improves postoperative weight gain in resected mice , an effect possibly owing to the higher glutamine levels in the high - protein diet .
our laboratory previously has shown that epidermal growth factor ( egf ) treatment will increase the adaptive response after resection . because egf stimulation activates phosphatidylinositol 3 kinase , it may destabilize the tsc1/tsc2 complex , causing increased mtorc1 activation and enhanced adaptation .
interestingly , we recently discovered that the intestinal epithelium - specific egf receptor is not necessary for adaptation .
these egf and mtorc1 studies provide a similar pattern for how adaptation might be regulated .
the crypt niche is crucial to provide cues for adaptation to occur ; when the cues are present , adaptation can be manipulated in an enterocyte - dependent manner .
 background & aimsintestinal adaptation is a compensatory response to the massive loss of small intestine after surgical resection . 
 we investigated the role of intestinal epithelial cell  specific mammalian target of rapamycin complex 1 ( i - mtorc1 ) in intestinal adaptation after massive small bowel resection ( sbr).methodswe performed 50% proximal sbr on mice to study adaptation . to manipulate i - mtorc1 activity , 
 villin - creer transgenic mice were crossed with tuberous sclerosis complex ( tsc)1flox / flox or raptorflox / flox mice to inducibly activate or inactivate i - mtorc1 activity with tamoxifen . 
 western blot was used to confirm the activity of mtorc1 . 
 crypt depth and villus height were measured to score adaptation . 
 immunohistochemistry was used to investigate differentiation and rates of crypt proliferation.resultsafter sbr , mice treated with systemic rapamycin showed diminished structural adaptation , blunted crypt cell proliferation , and significant body weight loss . activating i - mtorc1 via tsc1 deletion 
 induced larger hyperproliferative crypts and disorganized paneth cells without a significant change in villus height . 
 after sbr , ablating tsc1 in intestinal epithelium induced a robust villus growth with much stronger crypt cell proliferation , but similar body weight recovery . 
 acute inactivation of i - mtorc1 through deletion of raptor did not change crypt cell proliferation or  mucosa structure , but significantly reduced lysozyme / matrix metalloproteinase-7positive paneth cell and goblet cell numbers , with increased enteroendocrine cells . 
 surprisingly , ablation of intestinal epithelial cell  specific raptor after sbr did not affect adaptation or crypt proliferation , but dramatically reduced body weight recovery after surgery.conclusionssystemic , but not intestinal - specific , mtorc1 is important for normal adaptation responses to sbr . 
 although not required , forced enterocyte mtorc1 signaling after resection causes an enhanced adaptive response . 
Materials and Methods
Mice
Small Bowel Resection and Harvest
Histology
Intestinal Epithelial Cell Proliferation and Differentiation Analysis
Western Blot
Real-Time Quantitative PCR
Statistical Analysis
Results
Resection-Induced Intestinal Adaptation Is Blunted by Rapamycin
mTORC1 Activation in Intestinal Epithelium Promotes Adaptation After Resection
mTORC1 Inactivation in Intestinal Epithelium Does Not Affect Adaptation
Discussion