Clinical Study | Open Access
Short Bowel Patients Treated for Two Years with Glucagon-Like Peptide 2: Effects on Intestinal Morphology and Absorption, Renal Function, Bone and Body Composition, and Muscle Function
Background and aims. In a short-term study, Glucagon-like peptide 2 (GLP-2) has been shown to improve intestinal absorption in short bowel syndrome (SBS) patients. This study describes longitudinal changes in relation to GLP-2 treatment for two years. Methods. GLP-2, 400 micrograms, s.c.,TID, were offered, to eleven SBS patients keeping parenteral support constant. 72-hour nutritional balance studies were performed at baseline, weeks 13, 26, 52 during two years intermitted by an 8-week washout period. In addition, mucosal morphometrics, renal function (by creatinine clearance), body composition and bone mineral density (by DEXA), biochemical markers of bone turnover (by s-CTX and osteocalcin, PTH and vitamin D), and muscle function (NMR, lungfunction, exercise test) were measured. Results. GLP-2 compliance was 93%. Three of eleven patients did not complete the study. In the remaining 8 patients, GLP-2 significantly reduced the fecal wet weight from approximately 3.0 to approximately 2.0 kg/day. This was accompanied by a decline in the oral wet weight intake, maintaining intestinal wet weight absorption and urinary weight constant. Renal function improved. No significant changes were demonstrated in energy intake or absorption, and GLP-2 did not significantly affect mucosal morphology, body composition, bone mineral density or muscle function. Conclusions. GLP-2 treatment reduces fecal weight by approximately 1000 g/d and enables SBS patients to maintain their intestinal fluid and electrolyte absorption at lower oral intakes. This was accompanied by a 28% improvement in creatinine clearance.
GLP-2 is cosecreted with GLP-1 from the enteroendocrine L cells following nutrient ingestion . In animal and human studies, GLP-2 decreases gastric acid secretion , inhibits antral gastric emptying [3, 4] and upregulates intestinal blood-flow . In addition, GLP-2 has been demonstrated to have trophic effects on the intestinal mucosa [6–9] and positive effects on the absorptive function [10–12]. Furthermore, GLP-2 decreases bone resorption .
Theoretically, treatment with GLP-2 could reduce the rapid gastric emptying and hypersecretion and increase the intestinal absorption in short bowel syndrome (SBS) patients. In addition, GLP-2 could decelerate bone losses and diminish osteoporosis often described in these patients . Therefore, native GLP-2 and a dipeptidyl-peptidase IV (DPP-IV) degradation-resistant gly-2 GLP-2 analog , Teduglutide, have been evaluated in short-term, “proof of concept” studies in the treatment of SBS patients [15, 16]. The positive effects on intestinal morphology were confirmed by increases in small intestinal villus heights and crypt depths, and the positive absorptive effects by increases in the wet weight, and to a minor degree, in the energy absorption. Positive effects on bone mineral content were also described . However, the long-term effects of GLP-2 treatment in SBS patients remain to be evaluated.
This open-label study describes the effects of GLP-2, 400 mcg offered for subcutaneous injection TID, to eleven patients for two consecutive years intermitted by an 8-week washout period. As a part of the study design, parenteral support was kept constant during the two years in order to evaluate longitudinal changes in the intestinal absorption and dietary intake in relation to GLP-2 treatment. Mucosal morphology, renal function, body composition, bone mineral density and muscle function were also recorded.
2. Material and Methods
Eleven SBS patients (3 female, 8 male; years; remnant small bowel cm; 2 with a colon; 7 had intestinal failure, 3 receiving parenteral fluids and electrolytes exclusively and 4 receiving parenteral nutrition; 4 had intestinal insufficiency and did not need parenteral nutrition or fluid) were recruited to the study based on a fecal energy excretion exceeding 2.0 MJ/d (measured at a previous admission) or a remnant small bowel of 200 cm or less (measured perioperatively from the ligament of Treitz) (Table 1). One patient had previously received native GLP-2 (OBJ) and two teduglutide (LM and HRM) in the short-term experiments, whereas the remaining 8 patients were GLP-2 treatment naïve. All patients, except FL and LM, took antidiarrheal medication (codein, loperamide or opium) and 6 of eleven patients (HM, OB, EFP, JE, JHJ and UDJ) took antisecretory agents (omeprazole).
|M: male, F: female, CD: Crohns disease, UC compl.: Ulcerative colitis complications, Abd. Pain: Abdominal pain, Unrel. Feedback: Unreliable feedback.|
2.2. Study Protocol
Over the two years, the patients were admitted at least eight times to the hospital for 72-hours evaluations (Table 2). After the baseline evaluations, the patients were given native GLP-2, 400 mcg TID, subcutaneous, for one year. For these studies we employed synthetic human GLP-2, as described previously . During the first year, the patients were scheduled for readmissions at 13, 26 and 52 weeks (abbreviated Y1-W13, Y1-W26, Y1-W52, resp.,). After the first readmission at week 13, the patients were given an option to test a double dose of GLP-2, 800 mcg TID for 3 weeks. The patients, who accepted, were readmitted for an extra 72-hour nutrient balance study at week 17 (Y1-W17). After completing this evaluation, the original GLP-2 dose was reintroduced. GLP-2 treatment was discontinued for 8 weeks after the first 52 weeks of treatment. After a 72-hour washout evaluation, the GLP-2 treatment, 400 mcg TID, was reintroduced and evaluations were repeated during admission at 13, 26 and 52 weeks during the second year of treatment (abbreviated Y2-W13, Y2-W26, Y2-W52, resp.,). In relation to the week 26 readmission, during the second year of GLP-2 treatment, the patients were given cholylsarcosine bile acid replacement therapy, 2 grams TID, two days prior to the admission and during the 72-hour balance studies [18, 19]. Cholylsarcosine was supplied as the water soluble sodium salt, >99% pure by HPLC and thin-layer chromatography, and was packed into gelatine capsules (250 mg/capsule) . Cholylsarcosine was taken in relation to the three main meals and in conjunction with subcutaneous GLP-2 injections.
*Cholylsarcosine was given two days prior to the admission and during the 72-hour balance studies.|
2.2.1. Morphological Analysis
At least two small bowel biopsy specimens were obtained before GLP-2 treatment at baseline and repeated at week 52, year 1, in 6 of 7 patients with a jejunostomy. Histologic sections of the biopsies were analyzed by morphometric methods (Image pro plus) as described previously .
2.2.2. Fluid, Electrolyte and Nutrient Balance Studies
The study—and collection—period began at 9 o’clock on the first day of admission, where patients were requested to urinate, defecate or empty their stoma-bags. During the 72-hour balance periods, all ad libitum oral intakeand stomal output were weighed, and the contents of energy (bomb calorimetry), carbohydrate (Englyst’s method), nitrogen (Kjeldahl’s method), fat (gas liquid chromatography), sodium and potassium (flame photometry), calcium and magnesium (atomic absorption spectrophotometry) were determined as previously described [21, 22]. The absolute intestinal absorption was calculated as the difference between the ingested and excreted and the relative as the absolute absorption divided by oral intake.
The medication and parenteral supplements were fixed according to the status at baseline.
2.2.3. Urine Creatinine Excretion and Creatinine Clearence
Urinary creatinine was measured at 505 nm as a pikrat-creatinine complex using a standard hospital analytical technique according to the method of Jaffe and the 72-hour output calculated. The creatinine clearance was calculated by diving daily urinary creatine excretion by the plasma creatinine concentration.
2.2.4. Assessment of Body Weight, Body Composition and Bone Mineral Density
The fasting body weights were measured every morning after emptying of the bladder and stoma-bags, before breakfast, using a leveled platform scale, and were calculated as the mean for 4 consecutive days. Body composition (BC) and bone mineral density (BMD) of the posterior-anterior spine, hip and total body were measured by Dual-energy X-ray Absorptiometry (Norland XR-36 DXA densitometer, Norland Corp., Fort Atkinson, WI., USA).
2.2.5. Biochemical Markers of Bone Turnover
Bone resorption was assessed from the concentration of s-CTX (Serum CrossLabs one step ELISA; Nordic Bioscience, Denmark) . Bone formation was assessed from the concentration of s-osteocalcin (Osteocalcin N-MID ELISA assay, Nordic Bioscience, Denmark) .
2.2.6. Evaluation of Lung Function and Maximal Inspiratory and Expiratory Force
Lung function was tested by dynamic and static spirometry and measurement of single-breath diffusion capacity of carbon monoxide (MasterLab plethysmograph, Jaeger, Germany) according to the recommendations of the European Respiratory Society [24, 25]. Results were expressed in absolute values and as percentage of predicted values calculated according to European reference equations. Respiratory muscle strength was assessed by measurement of maximal expiratory and inspiratory pressures .
2.2.7. Evaluation of Maximal Aerobic ATP Turnover of Skeletal Muscle Mitochondria
The 31P NMR spectroscopy investigations were conducted in a short 26 cm bore magnet at 2.9 T as described previously [27–29]. The examination of the forearm flexor muscles and the tibialis anterior muscle of the lower leg was done as two separate experiments on the same day and both protocols involved: a 5 minutes rest, three minutes of dynamic exercise at 50% of maximal voluntary contraction followed by 10 minutes of recovery. The aim of the measurements was to obtain from the pH and PCr recovery a measure of the capacity of aerobic ATP synthesis.
On a separate day, patients performed an exercise test on a modified Krogh cycling ergometer with the upper body in a 45° position . Exercise was maintained at 60 round per minute with an increase in workload by 50 Watt every third minute until exhaustion. Breath-by-breath measurements of pulmonary O2 consumption (VO2) were made with an online gas analyzer (CPX/D, Medical Graphics, St. Paul, MN) and data were averaged every 30 seconds. Heart rate was recorded noninvasively .
The Ethics Committee for Medical Research in Copenhagen, Denmark (KF 01-235/98) approved the protocol. Procedures followed were in accordance with the ethical standards of the Helsinki Declaration of 1975, as revised in 1983. Patients signed informed consent before entrance to the study.
As the vast majority of data was normally distributed, the results are presented as means ± standard deviations. The differences between admissions periods were tested with a Friedman repeated measures analysis of variance on ranks on using the SigmaStat for Windows Version 2.0 (1992–1995, Jandel Corporation, Erkrath, Germany) in patients completing the study. For comparisons of admission periods to the baseline period, the Dunnett method was used as the post hoc test. A value of was considered significant.
Results regarding compliance, safety, adverse events, quality of life and treatment satisfaction are presented separately . In summary, all patients injected more than 93% of the prescribed GLP-2. Three of eleven patients did not complete the study. Two of these patients experienced abdominal pain, and in one patient, the investigator discontinued GLP-2 treatment, since the feedback from the patient regarding the administration of GLP-2 was lacking . Abdominal pain could be a consequence of providing an intestinotrophic agent to patients with a relative stenosis, and caution should probably be taken, when prescribing GLP-2 or analogs to patients with a relative intestinal stenosis, a narrow stoma, or a history of chronic abdominal pain .
3.1. Morphological Analysis
Villus height increased in 4 out of 6 patients, but overall no significant changes were seen (m versus m, ) in relation to GLP-2 treatment. Crypt depth increased in 3 patients, and decreased in 3, but overall no change was seen (m versus m, ).
3.1.1. Fluid, Electrolyte and Nutrient Balance Studies (Table 3)
Fecal wet weight was significantly reduced by approximately one liter/d at all time intervals after the initiation of GLP-2 treatment (range g/d to g/d). The average reduction the first year was 811 g/d and not different from the average reduction the second year of 1081 g/d, and no trend of a further increase in the effect on fecal weight beyond the 13th week of treatment was seen (Table 3). The fecal wet weight excretion reverted to baseline levels in the washout period after one year of treatment, but as for the first year the effect on fecal weight was fully regained 13 weeks after treatment was reinstituted the second year. The reduction in fecal wet weight was also seen in the two SBS patients with colon-in-continuity and numerically equaled the findings in the patients with a jejunostomy.
*Friedman repeated measures analysis of variance on ranks in patients completing the study. For comparisons of admission periods to the baseline period, the Dunnett method was used as the post hoc test. . Cholylsarcosine was given two days prior to the admission and during the 72-hour balance studies.|
The reduction in fecal wet weight excretion was accompanied by a reduction in oral intake. However, in contrast to the prompt effect on fecal weight, the effect on oral intake gradually increased over the first year by an average from g/d after 13 weeks of treatment to g/d after 26 weeks to finally g/d after 52 weeks (Y1-W52). Also in contrast to fecal excretions, oral intake was not reversed to baseline values during the eight weeks where treatment was stopped, but was only halved to an intermediate level of g/d. However, in the second year the effect on oral intake was already back to levels of g/d after 13 weeks, comparable to effects it took 52 weeks of treatment to reach the first year. In the second year, no further effect was seen on oral intake beyond week 13, and the effect leveled off at a decreased oral intake of g/d and g/d after 26 and 52 weeks of treatment, respectively. The decrease in fecal output resulted in a nearly comparable decrease in oral intake rendering absolute effect on intestinal absorption unaltered although intestinal absorption in percentage of oral intake increased (Table 3).
Similarly, the numerical increase in urine volume was small and did not reach statistical significance (on average 291 g/d the first year and 238 g/d the second year) rendering the overall urine excretion rather constant throughout the GLP-2 treatment periods in spite of the reduced oral intake. Fecal sodium excretions were reduced at all admissions (on average 53 mmol/day the first year and 58 mmol/day the second year) in relation to GLP-2 treatment and reverted to baseline levels during the washout period. Similarly, urinary sodium excretions increased at most admissions (54 mmol/day the first year and 24 mmol/day the second year). Although the decrease in fecal excretion and the increase in urinary excretion indicate that intestinal sodium absorption must have been increased, the -value for this was not significant (-value of .06 from the data in Table 3). Treatment did not affect oral sodium intake in contrast to oral wet weight intake.
Both dietary intake and fecal excretions of potassium decreased with no significant change in absorption although urinary excretions increased during several sessions of measurements. Consistent changes in dietary intake, fecal excretion and intestinal absorption of calcium and magnesium were not seen. Urinary excretion of calcium did not increase in relation to GLP-2 treatment (), whereas urinary magnesium excretion increased significantly () from a baseline of mmol/d to values ranging from mmol/d to mmol/d.
The results on the energy and macronutrient balances are presented in Table 4. On an average, treatment with GLP-2 numerically decreased oral energy intake by 349 kJ/d in the first year and 1182 kJ/d in the second year, which was less than 3% and 8% of baseline intake and not significant (, Table 4). Fecal excretion of energy numerically decreased on an average by 448 kJ/d in the first year and 1438 kJ/d in the second year. None of these changes were significant. However, the combination of GLP-2 and cholylsarcosine appeared successful in reducing fecal excretions by a total of 1852 kJ/d (31%), which partly appeared to be caused by a cholylsarcosine associated 42% decrease in fecal fat excretion of 28 g/d (1094 kJ/d, ), as GLP-2 alone did not influence fecal fat (Table 4). Nevertheless, the accompanying reduction in dietary intake reduced the overall gain in energy absorption to a negligible amount of less than 100 kJ/d except for the period where cholylsarcosine was added, which resulted in a numerical increase in absorption of 701 kJ/day or 8% of baseline, but still not enough to reach significance.