Short bowel syndrome remains a condition of high morbidity and mortality, and current therapeutic options carry significant side effects. To identify new treatments we focused on postresection changes in microRNAs—short noncoding RNAs, which suppress target genes—and suggest a previously undiscovered role for microRNA-125a (mir-125a) in intestinal adaptation.
Rats underwent either 80% massive small bowel resection or transection and were harvested after 48 hours. Jejunum was harvested for microRNA microarrays, laser capture microdissection, and RNA and protein analysis. Mir-125a was overexpressed in intestinal epithelium–6 (crypt-derived) cells (IEC-6) and effects on proliferation and apoptosis determined using MTS and flow cytometry. Expression of potential targets of mir-125a in rat jejunum and IEC-6 cells was determined using quantitative real-time polymerase chain reaction (RNA) and Western blotting (protein).
Resection upregulated mir-125a and mir-214 by 2.4-folds and 3.2-folds, respectively. Highest levels of expression were noted in the crypt fraction. Mir-125a overexpression induced apoptosis and resultant growth arrest in IEC-6 cells. The expression of the prosurvival Bcl-2 family member Mcl-1 was downregulated in both mir-125a-overexpressing IEC-6 cells and in jejunum of resected rats, confirming Mcl-1 as a previously undiscovered target of mir-125a.
Upregulation of mir-125a suppresses the prosurvival protein Mcl1, producing the increase in apoptosis known to accompany the proliferative changes characteristic of intestinal adaptation. Our data highlight a potential role for microRNAs as mediators of the adaptive process and may facilitate the development of new therapeutic options for short bowel syndrome.
Intestinal drug efflux proteins play a major role in the pharmacokinetics of many drugs. We assessed diurnal rhythmicity in the expression of ten major drug transporters.
We acquired male Sprague-Dawley rats and harvested jejunal mucosa at 3-hourly intervals across a 24-hour period. qPCR was performed for ten transporters: MDR1-3, MRP1, MRP3, MCT1, BRCP, PepT1, OCTN2, OATP-B. Rhythmicity was assessed with the cosinor procedure.
Diurnal rhythmicity was observed for MDR1, MCT1, MRP2, PepT1, and BCRP (1.6–5.4 fold-changes). Mesors occurred during fasting hours.
We conclude many drug transporters display profound diurnal rhythms in transcription, which may underlie diurnal rhythms in drug pharmacokinetics.
Diurnal rhythm; intestinal; drug efflux transporter
Intestinal nutrient absorptive capacity shows a circadian rhythm synchronized with eating patterns. Studies have shown that disrupting these normally coordinated rhythms, e.g. with shift work, may contribute to metabolic disease. While circadian expression of many nutrient transporters has been studied in health, their rhythms in obesity and metabolic disorders is not known. We studied the circadian expression and function of intestinal glucose transporter SGLT1, a major glucose transporter, in a rodent model of obesity and diabetes.
We compared obese Zucker Diabetic Fatty (ZDF) rats to lean ZDF littermates. Temporal feeding patterns were assessed, then rats were harvested at Zeitgeber (ZT, ZT0=7am) 3, 9, or 15 to measure insulin resistance, SGLT1 (Sodium glucose co-transporter type 1) mRNA expression and intestinal glucose absorption capacity. Known regulators of SGLT1 expression (intestinal sweet taste receptor T1R2/3; clock genes) were also measured to elucidate underlying mechanisms.
Both ZDF groups exhibited altered circadian food intake. Obese ZDF rats lost circadian rhythmicity of SGLT1 mRNA expression and functional activity. Lean ZDF rats had glucose levels less than half of the obese rats but were still hyperglycemic. Rhythmicity of mRNA expression was maintained but that of functional glucose uptake was blunted. Circadian rhythms of intestinal clock genes were maintained in both groups while there was no discernible rhythm of intestinal glucose transporter gene GLUT2 expression or of the T1R2 component of the sweet taste receptor in either group. In summary, lean and obese ZDF rats exhibited similar disruptions in circadian feeding pattern. Glucose intolerance was evident in lean rats, but only obese ZDF rats further developed diabetes and exhibited disrupted circadian rhythmicity of both SGLT1 mRNA expression and functional activity.
Our findings suggest that disrupted circadian feeding rhythms contribute to glucose intolerance, but additional factors (genetics, changes in nutrient sensing/ transport) are needed to lead to full diabetes.
circadian; intestinal glucose sensing; metabolic disease; SGLT1
ALT is used to detect NAFLD and has been associated with increased risk of metabolic syndrome and T2DM. Bariatric procedures result in significant weight-loss and a rapid resolution of T2DM. We aimed to study the impact of bariatric interventions on ALT levels in patients with or without T2DM, and compare this effect between different types of weight-loss procedures.
We reviewed 756 patients undergoing bariatric surgery. Demographics, co-morbidities, baseline and postoperative ALT and HbA1C levels, weight-loss data and diabetes status were recorded. ALT levels were compared between different procedures and between diabetics and non-diabetic patients. Chi-square test, ANOVA and t-test were used to evaluate outcomes.
Males and diabetics had significantly higher ALT at baseline. Both RYGB and LAGB resulted in significant reduction in ALT levels beginning at the 3rd postoperative month (20% and 17% respectively compared to baseline, p<0.001). ALT remained at the new low level up to year-3 after surgery. The degree of reduction was similar for both procedures and independent of the degree of weight-loss. In diabetics, ALT reduction was associated with improvement in disease but in T2DM patients who remained on insulin, ALT remained elevated.
RYGB and LAGB decrease ALT levels to the same degree and independent of weight loss. Our data confirm higher ALT in diabetics and demonstrate a rapid normalization after bariatric surgery with a simultaneous decrease in HbA1C. These results suggest that ALT may be used as a marker of metabolic improvement after bariatric surgery.
Bariatric surgery; T2DM; RYGB; LAP-BAND; metabolic syndrome
Laparoscopic cholecystectomy (LC) is the gold standard procedure for gallbladder removal. Conversion to an open procedure is sometimes deemed necessary, especially in complex cases in which a prolonged laparoscopic operative time is anticipated. A prolonged LC case is thought to be associated with increased complications and cost and therefore generally discouraged. The purpose of this study was to test this assumption, and compare outcomes and cost of converted and prolonged LC cases.
By using institutional National Surgical Quality Improvement Program and financial databases, we retrospectively reviewed and compared prolonged laparoscopic cases (Long-LC) with converted (CONV) procedures. Surgical times, length of stay (LOS), 30-day complications, operative room, and total hospital charges were compared between the 2 groups.
A total of 101 Long-LC and 66 CONV cases met our inclusion criteria. Long-LC cases were 19 minutes longer than CONV cases (123 vs 104 min; P <.01). No differences in postoperative complications were found between the 2 groups (P > .05). When Poisson regression was used, we found that LOS was significantly shorter in the Long-LC compared with CONV group (1 day vs 4 days; P < .01). Long-LC cases had greater operative charges ($15,278 vs $13,128; P < .01). However, hospital charges for Long-LC cases were 26% less than for CONV cases ($23,946 vs $32,446; P < .01).
Conversion is associated with a 3-day increase in LOS. Long-LC cases have greater operative room charges, but overall hospital charges were 26% less than CONV cases. Our data suggest that decision making regarding conversion should focus on safety and not time considerations.
The intestine demonstrates profound circadian rhythmicity in glucose absorption in rodents, mediated entirely by rhythmicity in the transcription, translation and function of the sodium glucose co-transporter SGLT1 (Slc5a1). Clock genes are rhythmic in the intestine and have been implicated in the regulation of rhythmicity of other intestinal genes, however their role in the regulation of SGLT1 is unknown. We investigated the effects of one clock gene, PER1, on SGLT1 transcription in vitro.
Caco-2 cells were stably transfected with knockdown vectors for PER1 and mRNA expression of clock genes and SGLT1 determined using quantitative polymerase chain reaction (qPCR). Chinese hamster ovary (CHO) cells were transiently cotransfected with combinations of the PER1 expression vectors and the wild-type SGLT1-luciferase promoter construct or the promoter with mutated E-box sequences.
Knockdown of PER1 increased native SGLT1 expression in Caco-2 enterocytes, while promoter studies confirmed that the inhibitory activity of PER1 on SGLT1 occurs via the proximal 1kb of the SGLT1 promoter. E-box sites exerted a suppressive effect on the SGLT1 promoter, however mutation of E-boxes had little effect on the inhibitory activity of PER1 on the SGLT1 promoter suggesting that the actions of PER1 on SGLT1 are independent of E-boxes.
Our findings suggest that PER1 exerts an indirect suppressive effect on SGLT1, possibly acting via other clock-controlled genes binding to non-E-box sites on the SGLT1 promoter. Understanding the regulation of rhythmicity of SGLT1 may lead to new treatments for the modulation of SGLT1 expression in conditions such as malabsorption, diabetes and obesity.
Clock genes; SGLT1; circadian; PER1; intestine
Laparoscopic cholecystectomy (LC) is the gold standard procedure for gallbladder removal. However, conversion to open surgery is sometimes needed. The factors underlying a surgeon's decision to convert a laparoscopic case to an open case are complex and poorly understood. With decreasing experience in open cholecystectomy, this procedure is however no longer the “safe” alternative it once was. With such an impending paradigm shift, this study aimed to identify the main reasons for conversion and ultimately to develop guidelines to help reduce the conversion rates.
Using the National Surgical Quality Improvement Program (NSQIP) database and financial records, the authors retrospectively reviewed 1,193 cholecystectomies performed at their institution from 2002 to 2009 and identified 70 conversions. Two independent surgeons reviewed the operative notes and determined the reasons for conversion. The number of ports at the time and the extent of dissection before conversion were assessed and used to create new conversion categories. Hospital length of stay (LOS), 30-day complications, operative times and charges, and hospital charges were compared between the new groups.
In 91% of conversion cases, the conversion was elective. In 49% of these conversions, the number of ports was fewer than four. According to the new conversion categories, most conversions were performed after minimal or no attempt at dissection. There were no differences in LOS, complications, operating room charges, or hospital charges between categories. Of the six emergent conversions (9%), bleeding and concern about common bile duct (CBD) injury were the main reasons. One CBD injury occurred.
In 49% of the cases, conversion was performed without a genuine attempt at laparoscopic dissection. Considering this new insight into the circumstances of conversion, the authors recommend that surgeons make a genuine effort at a laparoscopic approach, as reflected by placing four ports and trying to elevate the gallbladder before converting a case to an open approach.
Conversion; Gallbladder removal; Laparoscopic cholecystectomy
The surgical learning curve persists for years after training, yet existing CME efforts targeting this are limited. We describe a pilot study of a scalable video-based intervention, providing individualized feedback on intra-operative performance.
Four complex operations performed by surgeons of varying experience – a chief resident accompanied by the operating senior surgeon, a surgeon with <10 years in practice, another with 20–30 years, and a surgeon with >30 years of experience – were video-recorded. Video playback formed the basis of 1-hour coaching sessions with a peer-judged surgical expert. These sessions were audio-recorded, transcribed, and thematically coded.
The sessions focused on operative technique, both technical aspects and decision-making. With increasing seniority, more discussion was devoted to the optimization of teaching and facilitation of the resident’s technical performance. Coaching sessions with senior surgeons were peer-to-peer interactions, with each discussing his preferred approach. The coach alternated between directing the session (asking probing questions) and responding to specific questions brought by the surgeons, depending on learning style. At all experience levels, video review proved valuable in identifying episodes of failure-to-progress and troubleshooting alternative approaches. All agreed this tool is a powerful one. Inclusion of trainees seems most appropriate when coaching senior surgeons; it may restrict the dialogue of more junior attendings.
Video-based coaching is an educational modality that targets intra-operative judgment, technique, and teaching. Surgeons of all levels found it highly instructive. This may provide a practical, much needed approach for continuous professional development.
Background and aims
The intestine exhibits profound diurnal rhythms in function and morphology, in part due to changes in enterocyte proliferation. The regulatory mechanisms behind these rhythms remain largely unknown. We hypothesized that microRNAs are involved in mediating these rhythms, and studied the role of microRNAs specifically in modulating intestinal proliferation.
Diurnal rhythmicity of microRNAs in rat jejunum was analyzed by microarrays and validated by qPCR. Temporal expression of diurnally rhythmic mir-16 was further quantified in intestinal crypts, villi, and smooth muscle using laser capture microdissection and qPCR. Morphological changes in rat jejunum were assessed by histology and proliferation by immunostaining for bromodeoxyuridine. In IEC-6 cells stably overexpressing mir-16, proliferation was assessed by cell counting and MTS assay, cell cycle progression and apoptosis by flow cytometry, and cell cycle gene expression by qPCR and immunoblotting.
mir-16 peaked 6 hours after light onset (HALO 6) with diurnal changes restricted to crypts. Crypt depth and villus height peaked at HALO 13-14 in antiphase to mir-16. Overexpression of mir-16 in IEC-6 cells suppressed specific G1/S regulators (Cyclins D1-3, Cyclin E1 and Cyclin-dependent kinase 6) and produced G1 arrest. Protein expression of these genes exhibited diurnal rhythmicity in rat jejunum, peaking between HALO 11-17 in antiphase to mir-16.
This is the first report of circadian rhythmicity of specific microRNAs in rat jejunum. Our data provide a link between anti-proliferative mir-16 and the intestinal proliferation rhythm and point to mir-16 as an important regulator of proliferation in jejunal crypts. This function may be essential to match proliferation and absorptive capacity with nutrient availability.
microRNA; diurnal; proliferation; enterocyte
Increasing emphasis is being placed on health care quality measurement and improvement in the US. Within general surgery, several sophisticated quality-measurement and outcomes systems have been developed. These include the National Surgical Quality Improvement Program, the use of selective referral and centers of excellence, the Surgical Care Improvement Project, and the World Health Organization Surgical Safety Checklist. This article reviews each of these quality-improvement initiatives, highlights their relative contributions, and discusses future directions of quality improvement within general surgery.
The intestinal sodium-glucose cotransporter SGLT1 is responsible for all secondary active transport of dietary glucose, and thus presents a potential therapeutic target for obesity and diabetes. SGLT1 expression varies with a profound diurnal rhythm, matching expression to nutrient intake. The mechanisms entraining this rhythm remain unknown. We investigated the role of local nutrient signals in diurnal SGLT1 entrainment.
Male Sprague-Dawley rats, acclimatized to a 12: 12 light: dark cycle, underwent laparotomy with formation of isolated proximal jejunal loops (Thiry-Vella loops). Animals were recovered for 10 days before harvesting at four six-hourly intervals (Zeitgeber times ZT3, ZT9, ZT15, ZT21 where ZT0 is lights-on; n=6–8). SGLT1 expression was assessed in protein and mRNA extracts of mucosa harvested from both isolated loops (LOOP) and remnant jejunum (JEJ).
Isolated loops were healthy but atrophic, with minimal changes to villus architecture. A normal anticipatory rhythm was observed in Sglt1 transcription in both LOOP and JEJ, with peak signal at ZT9 (2.7-fold, p<0.001). Normal diurnal rhythms were also observed in protein signal, with peak expression in both LOOP and JEJ at ZT9 to 15 (2.1-fold, p<0.05). However, an additional more mobile polypeptide band was also observed in all LOOP samples, but not in JEJ samples (61kDa versus 69kDa). Enzymatic deglycosylation suggested this to be deglycosylated SGLT1.
Persistence of SGLT1 rhythmicity in isolated loops indicates that diurnal induction is independent of local luminal nutrient delivery, and suggests reliance on systemic entrainment pathways. However, local luminal signals may regulate glycosylation and therefore post-translational handling of SGLT1.
Thiry-vella loop; glucose transport; diurnal rhythm; circadian
Integrin-linked kinase (ILK) facilitates signal transduction between extracellular events and important intracellular survival pathways involving protein kinase B/Akt. We examined the role of ILK in determining pancreatic adenocarcinoma cellular chemoresistance to the nucleoside analogue gemcitabine. Cellular ILK expression was quantified by Western blot analysis. We examined the effects of overexpression of active ILK and of ILK knockdown induced by RNA interference on gemcitabine chemoresistance. We also examined the effects of modulating ILK expression on gemcitabine-induced caspase 3– mediated apoptosis, phosphorylation status of Akt (Ser473) and glycogen synthase kinase. Overexpression of ILK increased cellular gemcitabine chemoresistance, whereas ILK knockdown induced chemosensitization via increased caspase 3– mediated apoptosis. ILK knockdown attenuated Akt Ser473 and glycogen synthase kinase phosphorylation, whereas overexpression of constitutively active myristoylated Akt was sufficient to induce significant recovery in gemcitabine chemoresistance in the presence of ILK knockdown. Levels of ILK expression affect gemcitabine chemoresistance in pancreatic adenocarcinoma cells. This novel finding suggests that therapies directed against ILK and its downstream signaling targets may have the potential to enhance the efficacy of gemcitabine-based chemotherapy.
Our group has previously created a functional neointestine that is capable of restoring absorptive function. However, the endogenous level of vascular endothelial growth factor (VEGF) is markedly reduced in the construct compared to native bowel. Therefore, we wanted to locally deliver VEGF in a sustained fashion to upregulate angiogenesis in the neointestine. Rat recombinant VEGF was encapsulated in poly (lactide-co-glycolide) microspheres by a double emulsion method. Release kinetics and bioactivity were determined in vitro. Tissue-engineered intestine was generated by seeding donor neonatal rat intestinal organoid units onto a biodegradable polyglycolic acid scaffold along with VEGF-containing or empty microspheres, and wrapped in the omentum of recipient rats. After four weeks, the neointestinal cysts were analyzed for morphometry, VEGF levels, epithelial proliferation, and capillary density. Sustained release of biologically active VEGF was confirmed by in vitro studies. Intestinal constructs with VEGF microspheres were significantly larger than those containing empty microspheres. Tissue VEGF levels were significantly higher in neointestine loaded with encapsulated VEGF compared to those without growth factor. Epithelial cellular proliferation and capillary density were significantly increased in the VEGF-containing neointestinal constructs compared to empty constructs. Tissue-engineered intestine responds to sustained delivery of VEGF by upregulating microvasculature and epithelial proliferation.
Ghrelin, an orexigenic 28 amino-acid peptide, has been studied primarily in relation to the control of appetite and fat metabolism. In addition to these well-known functions, ghrelin, and its target receptors, growth hormone secretagogue receptors (GHS-Rs), have been localized to neutrophils, lymphocytes and macrophages suggesting that ghrelin may be involved in immune modulation. To assess the therapeutic role of ghrelin in production of pro-inflammatory and anti-inflammatory cytokines, the effects of exogenous ghrelin administration on the regulation of cytokine release in lipopolysaccharide (LPS)-activated murine RAW 264.7 macrophages were analyzed. Ghrelin and GHS-Rs are expressed in murine macrophages. In addition, exogenous ghrelin inhibited the production of pro-inflammatory cytokines IL-1β & TNF-α in LPS-stimulated murine macrophages in a dose dependent and time-dependent fashion. Exogenous ghrelin pretreatment resulted in a decrease in LPS-induced NFκB activation and was presumably the reason for this ghrelin-mediated effect. In contrast to these findings, exogenous ghrelin significantly augmented the release of the anti-inflammatory cytokine IL-10 in a dose dependent and time-dependent fashion from LPS-stimulated murine macrophages. Ghrelin administration enhanced activation of p38 MAPK, which is known to control the release of IL-10 in macrophages independent of the NFκB pathway. These effects of ghrelin on both pro- and anti-inflammatory cytokines were offset when a specific GHS-R receptor antagonist was added to the culture media. In conclusion, these data suggest that ghrelin has potent anti-inflammatory properties through modulation of secretion of both pro- and anti-inflammatory cytokines from LPS-stimulated macrophages through distinct signaling cascades. Therapeutic utility of ghrelin to control, modulate or treat pathologic inflammatory conditions like endotoxemic shock and ulcerative colitis requires further investigation.
Ghrelin; Macrophages; RAW 264.7; Lipopolysaccharide; TNF-α; IL-1β; IL-10; p38 MAPK; NFκB; Inflammation