Orally compensated short bowel patients are thin, potentially malnourished but rarely sarcopenic.

BACKGROUND AND AIM: In short bowel syndrome, insufficient absorptive capacity of the remnant bowel may lead to metabolic and nutritional consequences including electrolyte disturbances, severe diarrhea and malnutrition. While intestinal failure requires parenteral nutrition, short bowel patients with intestinal insufficiency (SB/II) have achieved oral autonomy. The aim of this exploratory study was to assess the nutritional, muscular and functional status of orally compensated SB/II patients. METHODS: 28 orally compensated SB/II patients with a mean of 46 months after termination of parenteral nutrition and 56 age- and sex-matched healthy controls (HC) were compared regarding anthropometric parameters, body composition using bioelectrical impedance analysis, handgrip strength and gait speed, blood parameters as well as nutritional intake and physical activity using validated questionnaires. Malnutrition and sarcopenia were diagnosed according to the criteria of the GLIM or EWGSOP2. RESULTS: SB/II patients had lower body mass index (BMI) and anthropometric parameters than HC but were within the normal weight range. The GLIM algorithm operationally diagnosed malnutrition in 39% (n = 11) of SB/II patients. Reduced skeletal muscle mass index and phase angle were rarely accompanied by a reduction of handgrip strength below cut-off values and the subsequent diagnosis of sarcopenia in SB/II patients (15%, n = 4). Compared to 11% of HC, 37% of SB/II patients had low physical activity level. Female SB/II patients had higher caloric and macronutrient intake. Caloric intake negatively correlated with body weight indicating compensatory hyperphagia in patients with lower body weight. Some of the SB/II patients showed signs of dehydration. CONCLUSIONS: Orally compensated SB/II patients are thinner than HC but have mostly normal BMI. Malnutrition is frequently diagnosed but may be overestimated due to the underlying malabsorption and its interplay with hyperphagia. Muscle mass is often reduced but is rarely accompanied by functional impairment leading to sarcopenia diagnosis. Thus, SB/II patients long term after termination of parenteral support may be malnourished but usually do not develop sarcopenia.

Uncoupling protein 2 deficiency of non-cancerous tissues inhibits the progression of pancreatic cancer in mice.

BACKGROUND: Pancreatic ductal adenocarcinoma (PDAC) is a disease of the elderly mostly because its development from preneoplastic lesions depends on the accumulation of gene mutations and epigenetic alterations over time. How aging of non-cancerous tissues of the host affects tumor progression, however, remains largely unknown. METHODS: We took advantage of a model of accelerated aging, uncoupling protein 2-deficient (Ucp2 knockout, Ucp2 KO) mice, to investigate the growth of orthotopically transplanted Ucp2 wild-type (WT) PDAC cells (cell lines Panc02 and 6606PDA) in vivo and to study strain-dependent differences of the PDAC microenvironment. RESULTS: Measurements of tumor weights and quantification of proliferating cells indicated a significant growth advantage of Panc02 and 6606PDA cells in WT mice compared to Ucp2 KO mice. In tumors in the knockout strain, higher levels of interferon-γ mRNA despite similar numbers of tumor-infiltrating T cells were observed. 6606PDA cells triggered a stronger stromal reaction in Ucp2 KO mice than in WT animals. Accordingly, pancreatic stellate cells from Ucp2 KO mice proliferated at a higher rate than cells of the WT strain when they were incubated with conditioned media from PDAC cells. CONCLUSIONS: Ucp2 modulates PDAC microenvironment in a way that favors tumor progression and implicates an altered stromal response as one of the underlying mechanisms.

Analysis of ESPEN and GLIM algorithms reveals specific drivers for the diagnosis of malnutrition in patients with chronic gastrointestinal diseases.

OBJECTIVES: Disease-related malnutrition (MN) is common in patients with liver cirrhosis (LC), short bowel syndrome (SBS), and chronic pancreatitis (CP). Different MN risk screening tools and diagnostic criteria of the European Society for Clinical Nutrition and Metabolism (ESPEN) and Global Leadership Initiative on Malnutrition (GLIM) algorithms were analyzed for their diagnostic accuracy and role as specific drivers to diagnose MN in patients with LC, SBS, and CP. METHODS: A total of 187 patients with LC, SBS, and CP, as well as control patients were prospectively recruited in a multicenter cross-sectional study. MN risk was screened using Nutritional Risk Screening 2002 (NRS-2002), the Malnutrition Universal Screening Tool (MUST), and the Royal Free Hospital Nutritional Prioritizing Tool (RFH-NPT), and diagnosed using the ESPEN, GLIM, and GLIMCRP+ (GLIM incorporating C-reactive protein [CRP] >5 mg/L) algorithms. For each of the individual diagnostic criteria, relative frequency, sensitivity, specificity, as well as positive and negative predictive values were calculated. RESULTS: NRS-2002 was only sensitive in conjunction with ESPEN, while MUST was sensitive additionally with the GLIM algorithm. RFH-NPT worked the best for LC. GLIM and GLIMCRP+ diagnosed MN more frequently than the ESPEN algorithm. Diagnostic criteria were detected at remarkably different relative frequencies starting with reduced food intake/malabsorption and chronic disease/inflammation, followed by weight loss, reduced fat-free mass index, low body mass index, and body mass index <18.5 kg/m². Relative frequencies differed between LC, SBS, and CP. Weight loss in LC and CP and reduced fat-free mass index and food intake in SBS had good diagnostic accuracy, suggesting that these criteria act as specific drivers for MN. CONCLUSIONS: RFH-NPT and MUST performed better in conjunction with the GLIM algorithm than NRS-2002. MN was diagnosed more frequently by GLIM than the ESPEN algorithm in LC, SBS, and CP. Individual criteria acted as specific drivers in MN in chronic gastrointestinal diseases.

Effects of Bile Duct Ligation and Ghrelin Treatment on the Colonic Barrier and Microbiome of Mice.

INTRODUCTION: Cholestatic liver disease (CLD) is associated with intestinal barrier dysfunction. The peptide hormone ghrelin may exert both hepatoprotective and barrier-strengthening effects. Here, we have evaluated these effects under the conditions of experimental cholestasis. METHODS: C57BL/6J mice with bile duct ligation (BDL) or sham surgery were treated with ghrelin or solvent for 9 days. Liver injury was assessed by histological and laboratory analyses. Paracellular macromolecule permeability and transmural electrical resistance (TMER) of colonic tissues were measured using a Ussing chamber. Expression of tight junction (TJ) genes was quantified by real-time PCR. Amplicon metagenomic sequencing was employed to analyze bacterial 16S rRNA from colonic stool samples. RESULTS: Mice with BDL exhibited weight loss and signs of severe liver injury. These changes were unaffected by ghrelin treatment. FITC-4-kDa-dextran flux was increased and TMER decreased after BDL. Treatment with ghrelin tended to reduce these effects. Furthermore, application of ghrelin was associated with higher mRNA levels of claudin-4, occludin, and ZO-1 in colonic tissues of mice with BDL. Reduced alpha-diversity of the microbiome was observed in solvent-treated mice with BDL but not in ghrelin-treated animals. CONCLUSION: Ghrelin treatment did not improve weight loss and liver damage but increased gene expression of colonic TJ proteins and restored the alpha-diversity of the microbiome. Since protective effects of ghrelin might be masked by the severity of the model, we suggest follow-up studies in models of milder CLD.

In silico investigation of molecular networks linking gastrointestinal diseases, malnutrition, and sarcopenia.

Malnutrition (MN) is a common primary or secondary complication in gastrointestinal diseases. The patient's nutritional status also influences muscle mass and function, which can be impaired up to the degree of sarcopenia. The molecular interactions in diseases leading to sarcopenia are complex and multifaceted, affecting muscle physiology, the intestine (nutrition), and the liver at different levels. Although extensive knowledge of individual molecular factors is available, their regulatory interplay is not yet fully understood. A comprehensive overall picture of pathological mechanisms and resulting phenotypes is lacking. In silico approaches that convert existing knowledge into computationally readable formats can help unravel mechanisms, underlying such complex molecular processes. From public literature, we manually compiled experimental evidence for molecular interactions involved in the development of sarcopenia into a knowledge base, referred to as the Sarcopenia Map. We integrated two diseases, namely liver cirrhosis (LC), and intestinal dysfunction, by considering their effects on nutrition and blood secretome. We demonstrate the performance of our model by successfully simulating the impact of changing dietary frequency, glycogen storage capacity, and disease severity on the carbohydrate and muscle systems. We present the Sarcopenia Map as a publicly available, open-source, and interactive online resource, that links gastrointestinal diseases, MN, and sarcopenia. The map provides tools that allow users to explore the information on the map and perform in silico simulations.

YAP activates pancreatic stellate cells and enhances pancreatic fibrosis.

BACKGROUND: Pancreatic stellate cells (PSCs) foster the progression of pancreatic adenocarcinoma and chronic pancreatitis (CP) by producing a dense fibrotic stroma. However, the incomplete knowledge of PSCs biology hampers the exploration of antifibrotic therapies. Here, we explored the role of the Hippo pathway in the context of PSCs activation and experimental CP. METHODS: CP model was created in rats with the tail vein injection of dibutyltin dichloride (DBTC). The expression of Yes-associated protein (YAP) in CP tissue was assessed. Primary and immortalized rats PSCs were treated with the YAP-inhibitor verteporfin. Furthermore, YAP siRNA was employed. Subsequently, DNA synthesis, cell survival, levels of α-smooth muscle actin (α-SMA) protein, presence of lipid droplets and PSCs gene expression were evaluated. Upstream regulators of YAP signaling were studied by reporter gene assays. RESULTS: In DBTC-induced CP, pronounced expression of YAP in areas of tubular structures and periductal fibrosis was observed. Verteporfin diminished DNA replication in PSCs in a dose-dependent fashion. Knockdown of YAP reduced cell proliferation. Primary cultures of PSCs were characterized by a decrease of lipid droplets and increased synthesis of α-SMA protein. Both processes were not affected by verteporfin. At the non-cytotoxic concentration of 100 nmol/L, verteporfin significantly reduced mRNA levels of transforming growth factor-ß1 (Tgf-ß1) and Ccn family member 1 (Ccn1). YAP signaling was activated by TGF-ß1, but repressed by interferon-γ. CONCLUSIONS: Activated YAP enhanced PSCs proliferation. The antifibrotic potential of Hippo pathway inhibitors warrants further investigation.

Malnutrition Is Highly Prevalent in Patients With Chronic Pancreatitis and Characterized by Loss of Skeletal Muscle Mass but Absence of Impaired Physical Function.

Background/Aims: Patients with chronic pancreatitis (CP) have an increased risk of malnutrition, a condition linked to reduced muscle mass and physical performance. We have investigated the risk factors, phenotypic presentation, and health implications associated with malnutrition in CP. Materials and Methods: In a multicenter cross-sectional study we recruited patients with confirmed CP and healthy volunteers as a control group. Malnutrition was diagnosed according to the criteria proposed by the Global Leadership Initiative on Malnutrition. We performed detailed examinations of body composition and physical function as well as testing of routine blood parameters and markers of inflammation. Results: We included 66 patients [mean (±SD) age: 56.0 (±14.5) years; 51 males] and an equal number of age- and sex-matched controls. Moderate malnutrition was diagnosed in 21% (n = 14) and severe malnutrition in 42% (n = 28) of patients. Besides weight loss malnourished patients showed lower fat and skeletal muscle mass compared to both non-malnourished subjects and healthy controls. Only in severe malnutrition, blood parameters reflected elevated inflammation and reduced muscle reserves. Handgrip strength in patients did not differ by nutritional status but there was a significant correlation (rho = 0.705, p < 0.001) with skeletal muscle mass. Although 20 patients (30%) had pathologically reduced skeletal muscle mass, only two individuals (3%) had sarcopenia with concomitantly reduced handgrip strength. Conclusion: Malnutrition is a frequent complication of CP characterized by loss of skeletal muscle mass. As this condition becomes evident only at an advanced stage, regular testing for altered body composition is recommended. Suitable biomarkers and the link between loss of muscle mass and physical function require further investigation. Clinical Trial Registration: [https://clinicaltrials.gov/ct2/show/NCT04474743], identifier [NCT04474743].

Dapiglutide, a novel dual GLP-1 and GLP-2 receptor agonist, attenuates intestinal insufficiency in a murine model of short bowel.

BACKGROUND: Extensive intestinal resection may lead to short bowel (SB) syndrome, resulting in intestinal insufficiency or intestinal failure (IF). Intestinal insufficiency and IF involve deficiency of the proglucagon-derived hormones glucagon-like peptide-1 (GLP-1) and GLP-2. Two major problems of SB are epithelial surface loss and accelerated transit. Standard treatment now targets intestinal adaptation with a GLP-2 analogue to enlarge absorptive surface area. It is possible that additional benefit can be gained from a combination of GLP-1 and GLP-2 activity, with the aim to enlarge intestinal surface area and slow intestinal transit. METHODS: The GLP-1- and GLP-2-specific effects of the novel dual GLP-1 receptor (GLP-1R) and GLP-2 receptor (GLP-2R) agonist dapiglutide (rINN) were characterized in rodents. Furthermore, in a murine SB model of intestinal insufficiency with 40% ileocecal resection, the influence of dapiglutide on intestinal growth, body weight, food intake, volume status, and stool water content was tested against vehicle and sham-operated male mice. RESULTS: Dapiglutide significantly improves oral glucose tolerance, reduces intestinal transit time, and promotes intestinal growth. In the SB mouse model, dapiglutide promotes body weight recovery, despite unchanged intake of liquid diet. Dapiglutide promotes significant intestinal growth, as indicated by significantly increased villus height as well as intestinal length. Furthermore, dapiglutide reduces stool water losses, resulting in reduced plasma aldosterone. CONCLUSION: Dapiglutide possesses specific and potent GLP-1R and GLP-2R agonist effects in rodents. In the murine SB model, combined unimolecular GLP-1R and GLP-2R stimulation with dapiglutide potently attenuates intestinal insufficiency and potentially also IF.

Molecular Mechanism Contributing to Malnutrition and Sarcopenia in Patients with Liver Cirrhosis.

Liver cirrhosis is frequently accompanied by disease-related malnutrition (DRM) and sarcopenia, defined as loss of skeletal muscle mass and function. DRM and sarcopenia often coexist in cirrhotic patients and are associated with increased morbidity and mortality. The clinical manifestation of both comorbidities are triggered by multifactorial mechanisms including reduced nutrient and energy intake caused by dietary restrictions, anorexia, neuroendocrine deregulation, olfactory and gustatory deficits. Maldigestion and malabsorption due to small intestinal bacterial overgrowth, pancreatic insufficiency or cholestasis may also contribute to DRM and sarcopenia. Decreased protein synthesis and increased protein degradation is the cornerstone mechanism to muscle loss, among others mediated by disease- and inflammation-mediated metabolic changes, hyperammonemia, increased myostatin and reduced human growth hormone. The concise pathophysiological mechanisms and interactions of DRM and sarcopenia in liver cirrhosis are not completely understood. Furthermore, most knowledge in this field are based on experimental models, but only few data in humans exist. This review summarizes known and proposed molecular mechanisms contributing to malnutrition and sarcopenia in liver cirrhosis and highlights remaining knowledge gaps. Since, in the prevention and treatment of DRM and sarcopenia in cirrhotic patients, more research is needed to identify potential biomarkers for diagnosis and development of targeted therapeutic strategies.

Impact of diet and genes on murine autoimmune pancreatitis.

The impact of environmental factors, such as diet, and the genetic basis of autoimmune pancreatitis (AIP) are largely unknown. Here, we used an experimental murine AIP model to identify the contribution of diet to AIP development, as well as to fine-map AIP-associated genes in outbred mice prone to develop the disease. For this purpose, we fed mice of an autoimmune-prone intercross line (AIL) three different diets (control, calorie-reduced and western diet) for 6 months, at which point the mice were genotyped and phenotyped for AIP. Overall, 269 out of 734 mice (36.6%) developed AIP with signs of parenchymal destruction, equally affecting mice of both sexes. AIP prevalence and severity were reduced by approximately 50% in mice held under caloric restriction compared to those fed control or western diet. We identified a quantitative trait locus (QTL) on chromosome 4 to be associated with AIP, which is located within a previously reported QTL. This association does not change when considering diet or sex as an additional variable for the mapping. Using whole-genome sequences of the AIL founder strains, we resolved this QTL to a single candidate gene, namely Map3k7. Expression of Map3k7 was largely restricted to islet cells as well as lymphocytes found in the exocrine pancreas of mice with AIP. Our studies suggest a major impact of diet on AIP. Furthermore, we identify Map3k7 as a novel susceptibility gene for experimental AIP. Both findings warrant clinical translation.

Preclinical insights into the gut-skeletal muscle axis in chronic gastrointestinal diseases.

Muscle wasting represents a constant pathological feature of common chronic gastrointestinal diseases, including liver cirrhosis (LC), inflammatory bowel diseases (IBD), chronic pancreatitis (CP) and pancreatic cancer (PC), and is associated with increased morbidity and mortality. Recent clinical and experimental studies point to the existence of a gut-skeletal muscle axis that is constituted by specific gut-derived mediators which activate pro- and anti-sarcopenic signalling pathways in skeletal muscle cells. A pathophysiological link between both organs is also provided by low-grade systemic inflammation. Animal models of LC, IBD, CP and PC represent an important resource for mechanistic and preclinical studies on disease-associated muscle wasting. They are also required to test and validate specific anti-sarcopenic therapies prior to clinical application. In this article, we review frequently used rodent models of muscle wasting in the context of chronic gastrointestinal diseases, survey their specific advantages and limitations and discuss possibilities for further research activities in the field. We conclude that animal models of LC-, IBD- and PC-associated sarcopenia are an essential supplement to clinical studies because they may provide additional mechanistic insights and help to identify molecular targets for therapeutic interventions in humans.

Different characteristics of chronic dibutyltin dichloride-induced pancreatitis and cholangitis in mouse and rat.

BACKGROUND: Current animal models of chronic pancreatitis (CP) often provide only limited pathophysiological insights since they incompletely reflect the human disease. CP induced by injection of dibutyltin dichloride (DBTC-pancreatitis) shares with human CP the important feature of extended fibrosis and would be an even more attractive model if it could be transferred from rats to mice, as recently suggested in the context of combined ethanol and DBTC application. This study aimed to evaluate the effects of DBTC in pancreas and liver of C57BL/6 mice, a strain commonly used to engineer genetic mouse models. METHODS: C57BL/6 mice and Lewis rats were exposed to variable doses of DBTC. After an investigation period of up to 4 weeks, laboratory findings and histopathological changes of pancreas and liver were evaluated. RESULTS: Chronic DBTC-pancreatitis in rats was characterized by acinar cell damage, ductal changes, fibrosis, and inflammatory cell infiltrates. Mice treated with DBTC at 6-8 mg/kg body weight, the standard doses in rats, showed transient increases of lipase activities but no morphological signs of chronic DBTC-pancreatitis 4 weeks after injection of the drug. Increased doses of 10-12 mg/kg DBTC were intolerable due to their high toxicity. In contrast, mice and rats presented with a similar histopathology of the liver that can be characterized as a chronic-proliferative DBTC-cholangitis with predominating damage and proliferation of the small bile ducts as well as secondary portal inflammatory cell infiltrates and a beginning portal fibrosis. CONCLUSIONS: The DBTC-model cannot be transferred from rats to C57BL/6 mice with respect to chronic DBTC-pancreatitis, but might be of interest to study DBTC-cholangitis in both species.

Adoptive transfer of CD3+ T cells and CD4+ CD44high memory T cells induces autoimmune pancreatitis in MRL/MpJ mice.

The immunopathogenesis of autoimmune pancreatitis (AIP) is poorly understood. Here, we have used MRL/MpJ mice, a model of spontaneous AIP, to address the role of cellular autoimmune processes in the initiation and progression of the disease. Therefore, different T cell subpopulations were adoptively transferred from sick to still healthy (but susceptible) MRL/MpJ mice. Unpurified splenocytes and CD3+ T cells both efficiently induced AIP, while CD4+ and CD8+ T cells alone, as well as splenocytes from healthy mice, were insufficient to trigger the disease. Strikingly, CD4+ CD44high memory T cells, although transferred at lower numbers than other T cells, also induced AIP in recipient mice. Employing a modified experimental design, we also evaluated the effects of regulatory T cells (Tregs ) on the progression of AIP in already diseased mice. Under the given experimental conditions, there was no significant suppressive effect of adoptively transferred Tregs on pancreatic histopathology. The results of our studies suggest a key role of T cell-mediated processes in murine AIP. The effects of CD4+ CD44high memory T cells are in accordance with genetic studies of our group, which had previously implicated this cell type into the pathogenesis of AIP. In follow-up studies, we will focus on the interplay of cellular and humoral autoimmunity in the context of AIP.

Antifibrogenic effects of vitamin D derivatives on mouse pancreatic stellate cells.

AIM: To study the molecular effects of three different D-vitamins, vitamin D2, vitamin D3 and calcipotriol, in pancreatic stellate cells (PSCs). METHODS: Quiescent PSCs were isolated from mouse pancreas and activated in vitro by seeding on plastic surfaces. The cells were exposed to D-vitamins as primary cultures (early-activated PSCs) and upon re-culturing (fully-activated cells). Exhibition of vitamin A-containing lipid droplets was visualized by oil-red staining. Expression of α-smooth muscle actin (α-SMA), a marker of PSC activation, was monitored by immunofluorescence and immunoblot analysis. The rate of DNA synthesis was quantified by 5-bromo-2'-deoxyuridine (BrdU) incorporation assays. Real-time PCR was employed to monitor gene expression, and protein levels of interleukin-6 (IL-6) were measured by ELISA. Uptake of proline was determined using 18F-proline. RESULTS: Sustained culture of originally quiescent PSCs induced cell proliferation, loss of lipid droplets and exhibition of stress fibers, indicating cell activation. When added to PSCs in primary culture, all three D-vitamins diminished expression of α-SMA (to 32%-39% of the level of control cells; P < 0.05) and increased the storage of lipids (scores from 1.97-2.15 on a scale from 0-3; controls: 1.49; P < 0.05). No such effects were observed when Dvitamins were added to fully-activated cells, while incorporation of BrdU remained unaffected under both experimental conditions. Treatment of re-cultured PSCs with Dvitamins was associated with lower expression of IL-6 (-42% to -49%; P < 0.05; also confirmed at the protein level) and increased expression of the vitamin D receptor gene (209%-321% vs controls; P < 0.05). There was no effect of Dvitamins on the expression of transforming growth factor-ß1 and collagen type 1 (chain α1). The lowest uptake of proline, a main component of collagen, was observed in calcipotriol-treated PSCs. CONCLUSION: The three D-vitamins inhibit, with similar efficiencies, activation of PSCs in vitro, but cannot reverse the phenotype once the cells are fully activated.