Subclinical thiamine deficiency results in failed reproduction in Arctic foxes.

Thiamine deficiency can result in life-threatening physiological and neurological complications. While a thiamine-deficient diet may result in the onset of such symptoms, the presence of thiaminase - an enzyme that breaks down thiamine - is very often the cause. In such instances, thiaminase counteracts the bioavailability and uptake of thiamine, even when food-thiamine levels are adequate. Here, we report on a case of failed reproduction in seven Arctic fox (Vulpes lagopus) breeding pairs kept at a captive breeding facility, including the presentation of severe thiamine deficiency symptoms in two male foxes. Symptoms included ataxia, obtundation, truncal sway, star-gazing and visual impairment. Blood tests were inconclusive, yet symptoms resolved following treatment with a series of thiamine hydrochloride injections, thereby verifying the diagnosis. A fish-dominated feed, which for the first time had been frozen for a prolonged period, was identified as the likely source of thiaminase and subsequent deterioration in the animals' health. Symptoms in the two males arose during the annual mating period. All seven breeding pairs at the captive breeding station failed to reproduce - a phenomenon never recorded during the captive breeding facility's preceding 17-year operation. Relating our findings to peer-reviewed literature, the second part of this case report assesses how thiamine deficiency (due to thiaminase activity) likely resulted in subclinical effects that impaired the production of reproduction hormones, and thereby led to a complete breeding failure. While previous work has highlighted the potentially lethal effects of thiamine deficiency in farmed foxes, this is, to our knowledge the first study showing how subclinical effects in both males and females may inhibit reproduction in foxes in general, but specifically Arctic foxes. The findings from our case report are not only relevant for captive breeding facilities, but for the welfare and management of captive carnivorous animals in general.

Cellular localization of guanylin and uroguanylin mRNAs in human and rat duodenal and colonic mucosa.

Guanylin (GUCA2A/Guca2a/GN) and uroguanylin (GUCA2B/Guca2b/UGN) are expressed in the gastrointestinal tract and have been implicated in ion and fluid homeostasis, satiety, abdominal pain, growth and intestinal barrier integrity. Their cellular sources are debated and include goblet cells, entero-/colonocytes, enteroendocrine (EE) cells and tuft cells. We therefore investigated the cellular sources of GN and UGN mRNAs in human and rat duodenal and colonic epithelium with in situ hybridization (ISH) to determine co-expression with Chromogranin A (CHGA/Chga/CgA; enterochromaffin [EC] cells), defensin alpha 6 (DEFA6/Defa6; Paneth cells), mucin 2 (MUC2/Muc2; goblet cells) and selected tuft cell markers. GUCA2A/Guca2a expression was localized to goblet cells and colonocytes in human and rat colon. In human duodenum, GUCA2A was expressed in Paneth cells and was scarce in villous epithelial cells. In rat duodenum, Guca2a was only localized to goblet cells. Guca2b was focally expressed in rat colon. In human and rat duodenum and in human colon, GUCA2B/Guca2b was expressed in dispersed solitary epithelial cells, some with a tuft cell-like appearance. Neither GUCA2A nor GUCA2B were co-expressed with CHGA in human duodenal cells. Consequently, EC cells are probably not the major source of human GN or UGN but other EE cells as a source of GN or UGN are not entirely excluded. No convincing overlap with tuft cell markers was found. For the first time, we demonstrate the cellular expression of GUCA2B in human duodenum. The specific cellular distribution of both GN and UGN differs between duodenum and colon and between human and rat intestines.

The guanylate cyclase-C signaling pathway is down-regulated in inflammatory bowel disease.

OBJECTIVE: Activation of membrane receptor guanylate cyclase-C (GC-C) is implicated in gastrointestinal fluid and electrolyte balance, preservation of intestinal barrier integrity, anti-trophic effects and inhibition of pain sensation. To evaluate GC-C signaling, we examined the regulation of GC-C (GUCY2C/Gucy2c) and its endogenous ligands guanylin (GN/GUCA2A/Guca2a) and uroguanylin (UGN/GUCA2B/Guca2b) in colonic Crohn's disease (CD), ulcerative colitis (UC) and in rats with 2,4,6-Trinitrobenzene sulphonic acid (TNBS) colitis. Correlation analyses between expression of GUCA2A and GUCY2C and expression of inflammatory cytokines (IL1A, IL1B, TNFA and IFNG) were conducted. Additionally, expression of transcription factors for GUCA2A and GUCY2C, and the GC-C signaling pathway, were examined. MATERIAL AND METHODS: Biopsies from active UC/CD, un-inflamed UC/CD and healthy controls, and inflamed and healthy rat colon were investigated with gene expression microarray, immunohistochemistry (IHC) and in situ hybridization (ISH). RESULTS: GUCA2A/Guca2a, GUCA2B, GUCY2C/Gucy2c, transcription factors, as well as several cyclic guanosine-3',5'-monophosphate downstream mediators were all significantly down-regulated in both inflamed colonic inflammatory bowel disease (IBD) mucosa and TNBS colitis. Expression of GUCA2A and GUCY2C negatively correlated to expression of inflammatory cytokines. IHC and ISH confirmed microarray results for GUCA2A/Guca2a and GUCY2C/Gucy2c in inflamed samples. We identified a highly significant positive correlation between the expression of the transcription factor caudal type homeobox 2 (CDX2) and the expression of the downstream target gene GUCY2C. CONCLUSIONS: GUCA2A, GUCA2B and GUCY2C as well as several steps of the GC-C signaling pathway are down-regulated in IBD. This may have implications in IBD pathogenesis.

Mechanistic comparison between gastric bypass vs. duodenal switch with sleeve gastrectomy in rat models.

BACKGROUND: Both gastric bypass (GB) and duodenal switch with sleeve gastrectomy (DS) have been widely used as bariatric surgeries, and DS appears to be superior to GB. The aim of this study was to better understand the mechanisms leading to body weight loss by comparing these two procedures in experimental models of rats. METHODS: Animals were subjected to GB, DS or laparotomy (controls), and monitored by an open-circuit indirect calorimeter composed of comprehensive laboratory animal monitoring system and adiabatic bomb calorimeter. RESULTS: Body weight loss was greater after DS than GB. Food intake was reduced after DS but not GB. Energy expenditure was increased after either GB or DS. Fecal energy content was increased after DS but not GB. CONCLUSION: GB induced body weight loss by increasing energy expenditure, whereas DS induced greater body weight loss by reducing food intake, increasing energy expenditure and causing malabsorption in rat models.

Relevance of TNBS-colitis in rats: a methodological study with endoscopic, histologic and Transcriptomic [corrected] characterization and correlation to IBD.

BACKGROUND: Rectal instillation of trinitrobenzene sulphonic acid (TNBS) in ethanol is an established model for inflammatory bowel disease (IBD). We aimed to 1) set up a TNBS-colitis protocol resulting in an endoscopic and histologic picture resembling IBD, 2) study the correlation between endoscopic, histologic and gene expression alterations at different time points after colitis induction, and 3) compare rat and human IBD mucosal transcriptomic data to evaluate whether TNBS-colitis is an appropriate model of IBD. METHODOLOGY/PRINCIPAL FINDINGS: Five female Sprague Daley rats received TNBS diluted in 50% ethanol (18 mg/0.6 ml) rectally. The rats underwent colonoscopy with biopsy at different time points. RNA was extracted from rat biopsies and microarray was performed. PCR and in situ hybridization (ISH) were done for validation of microarray results. Rat microarray profiles were compared to human IBD expression profiles (25 ulcerative colitis Endoscopic score demonstrated mild to moderate colitis after three and seven days, but declined after twelve days. Histologic changes corresponded with the endoscopic appearance. Over-represented Gene Ontology Biological Processes included: Cell Adhesion, Immune Response, Lipid Metabolic Process, and Tissue Regeneration. IL-1α, IL-1ß, TLR2, TLR4, PRNP were all significantly up-regulated, while PPARγ was significantly down-regulated. Among genes with highest fold change (FC) were SPINK4, LBP, ADA, RETNLB and IL-1α. The highest concordance in differential expression between TNBS and IBD transcriptomes was three days after colitis induction. ISH and PCR results corresponded with the microarray data. The most concordantly expressed biologically relevant pathways included TNF signaling, Cell junction organization, and Interleukin-1 processing. CONCLUSIONS/SIGNIFICANCE: Endoscopy with biopsies in TNBS-colitis is useful to follow temporal changes of inflammation visually and histologically, and to acquire tissue for gene expression analyses. TNBS-colitis is an appropriate model to study specific biological processes in IBD.

Development of obesity is associated with increased calories per meal rather than per day. A study of high-fat diet-induced obesity in young rats.

BACKGROUND: We challenge the current belief that obesity is a result of overnutrition by studying a rodent model of human obesity. METHODS: Male Sprague-Dawley rats at 3 weeks of age were fed with a mixed normal diet of 10% fat and high-fat diet of 60% fat (50:50) for 2 weeks and then turned to 100% high-fat diet until 43 weeks of age. Body weight gain was recorded, and food intake, eating behavior, and metabolic variables were measured by a comprehensive laboratory animal monitoring system. Body composition was determined by dual-energy X-ray absorptiometry. Ghrelin/obestatin-producing A-like cells in the stomach were analyzed by immunohistochemistry. RESULTS: Rats on high-fat diet were overweight at 9 weeks of age and later became obese characterized by increased body weight and excess fat deposition. There were no obesity-prone, obesity middle tertile, and obesity-resistant subgroups in rats on high-fat diet. The young rats on high-fat diet, even before becoming overweight (i.e., 8 weeks), consumed larger portion of meal (kilocalorie per meal) and ate faster but less frequent than the rats on normal diet. Obese rats had reduced food intake (expressed as gram per 100-g body weight per 24 h), unchanged calorie intake (kilocalorie per 100-g body weight per 24 h), and energy expenditure (kilocalorie per hour per 100-g body weight), and increased number of A-like cells in the stomach. CONCLUSION: Large size of meal, but not overnutrition, appears to be responsible for high-fat diet-induced obesity in rats. We propose a consideration that prevention strategies for obesity epidemic should strongly focus on meal size at early childhood and adolescence.

Gastric bypass surgery causes body weight loss without reducing food intake in rats.

BACKGROUND: It is a common dogma that gastric bypass (GB) induces early satiety and consequent reductions in food intake and nutrient absorption. The aim of the present study was to analyze feeding behavioral and metabolic changes in rats after GB. METHODS: Male Sprague-Dawley rats at the ages of 23 and 42 weeks were placed in metabolic cages connected with a comprehensive laboratory animal monitoring system. At the age of 48 weeks they were subjected to either GB or sham operation, and then placed in metabolic cages at 51 and 62 weeks (or 3 and 14 weeks postoperatively). RESULTS: GB rats lost 20% of the body weight within 2-3 weeks and remained at this lower level until the end of the study at 14 weeks postoperatively. Satiety ratio was higher during daytime than nighttime in both sham-operated and GB rats, but was not significantly different between the two groups. Neither daily accumulated food intake nor food intake per 100 g of body weight was different between sham-operated and GB rats. Apparently, GB rats ate more frequently during daytime and had smaller meal size during nighttime at 3 weeks postoperatively. These changes were not present at 14 weeks postoperatively. Energy density in the feces was the same in GB and sham-operated rats postoperatively. Energy expenditure declined with age, but increased in GB rats compared with age-matched sham-operated rats. CONCLUSIONS: GB reduced the body weight without causing early satiety, reducing food intake or inducing malabsorption. It did, however, increase energy expenditure.

Characterization of obestatin- and ghrelin-producing cells in the gastrointestinal tract and pancreas of rats: an immunohistochemical and electron-microscopic study.

Both ghrelin and obestatin are derived from preproghrelin by post-translational processing. We have morphologically characterized the cells that produce obestatin and ghrelin in new-born and adult Sprague-Dawley rats that were freely fed, fasted, or subjected to gastric bypass surgery or reserpine treatment. Tissue samples collected from the gastrointestinal tract and pancreas were examined by double-immunofluorescence staining, immunoelectron microscopy, and conventional electron microscopy. Obestatin was present in the stomach, duodenum, jejunum, colon, and pancreas. In the stomach, differences were noted in the development of obestatin- and preproghrelin-immunreactive (IR) cells on the one hand and ghrelin-IR cells on the other, particularly 2 weeks after birth. Preproghrelin- and obestatin-IR cells were more numerous than ghrelin-IR cells in the stomach, suggesting the lack of ghrelin in some A-like cells. Most obestatin-producing cells in the stomach were distributed in the basal part of the oxyntic mucosa; these cells co-localized with chromogranin A (pancreastatin) and vesicle monoamine transporters type 1 and 2, but not with serotonin or histidine decarboxylase. Immunoelectron microscopy revealed the obestatin- and ghrelin-producing cells to be A-like cells, characterized by numerous highly electron-dense granules containing ghrelin and obestatin. Some granules exhibited an even electron density with thin electron-lucent halos, suggestive of monoamines. Feeding status, gastric bypass surgery, and reserpine treatment had no obvious effect on the A-like cells. In the pancreas, obestatin was present in the peripheral part of the islets, with a distribution distinct from that of glucagon-producing A cells, insulin-producing beta cells, and cells producing pancreatic polypeptide Y. Thus, obestatin and ghrelin co-localize with an anticipated monoamine in A-like cells in the stomach, and obestatin is found in pancreatic islets.

Mechanism of gastric bypass-induced body weight loss: one-year follow-up after micro-gastric bypass in rats.

Bariatric surgery (Roux-en-Y or mini-gastric bypass) is designed to limit food intake by creating a small gastric pouch and to reduce nutrient absorption by bypassing the long limb of the intestine. We report 1-year follow-up results after micro-gastric bypass in rats. Micro-gastric bypass was performed by anastomosis of the esophagus and the proximal jejunum. Body weight, body composition, bone mineral density, food intake, and serum levels of ghrelin and obestatin were measured. Growing rats had a 40% weight reduction 2 months after micro-gastric bypass surgery compared to 20% after gastrectomy and 30% after stomach bypass (anastomosis of the esophagus and duodenal bulb). Six months after micro-gastric bypass surgery, the rats stopped growing compared to controls that gained continuously due to expansion of the fat compartment. Adult rats (600 g) lost 30% of their body weight 5 months after the micro-gastric bypass, while food intake was not reduced. Serum levels of obestatin (but not ghrelin) were reduced in rats with micro-gastric bypass. The results suggest that micro-gastric bypass efficiently reduced body weight, particularly fat mass; loss of the weight after micro-gastric bypass was not due to reduced food intake; and lean tissue and bone development were impaired in growing subjects after gastric bypass.