Co-products of beef processing enhance non-haem iron absorption in an in vitro digestion/caco-2 cell model

Beef processing produces high volumes of protein rich, low value, ‘waste’ co‐products such as offal. Beef improves uptake of low bioavailable non‐haem iron (found in vegetables, fortificants, supplements) and this effect is dubbed the ‘meat‐factor’, although the underlying mechanism is not fully understood. Here, we investigate whether bovine co‐products (kidney, lung, heart) not previously studied share this enhancing potential. This was determined by coupled in vitro digestion of co‐products and subsequent caco‐2 cell ferritin formation (an intracellular iron storage protein). In this study we show that bovine co‐products significantly increase caco‐2 cells’ response to non‐haem iron from infant rice cereal. The presence of these co‐products, (kidney, lung and heart), increased relative uptake (by 207.13%, 171.21%, 265.28%, respectively), to a greater extent than beef (30.23%). Our findings present a novel function for co‐products of beef processing that may have potential as food ingredients to improve non‐haem iron bioavailability, thus adding value

Adenosine triphosphate is co-secreted with glucagon-like peptide-1 to modulate intestinal enterocytes and afferent neurons.

Enteroendocrine cells are specialised sensory cells located in the intestinal epithelium and generate signals in response to food ingestion. Whilst traditionally considered hormone-producing cells, there is evidence that they also initiate activity in the afferent vagus nerve and thereby signal directly to the brainstem. We investigate whether enteroendocrine L-cells, well known for their production of the incretin hormone glucagon-like peptide-1 (GLP-1), also release other neuro-transmitters/modulators. We demonstrate regulated ATP release by ATP measurements in cell supernatants and by using sniffer patches that generate electrical currents upon ATP exposure. Employing purinergic receptor antagonists, we demonstrate that evoked ATP release from L-cells triggers electrical responses in neighbouring enterocytes through P2Y2 and nodose ganglion neurones in co-cultures through P2X2/3-receptors. We conclude that L-cells co-secrete ATP together with GLP-1 and PYY, and that ATP acts as an additional signal triggering vagal activation and potentially synergising with the actions of locally elevated peptide hormone concentrations.Wellcome Trust joint investigator award (106262/Z/14/Z and 106263/Z/14/Z); MRC programme within the Metabolic Diseases Unit (MRC_MC_UU_12012/3); MRC Metabolic Diseases Unit [MRC_MC_UU_12012/5] ; Wellcome Trust Strategic Award [100574/Z/12/Z

Functional protein rich extracts from bovine and porcine hearts using acid or alkali solubilisation and isoelectric precipitation

Alkali solubilisation (ALS) was compared with acid solubilisation (ACS) for preparation of protein rich extracts from bovine and porcine hearts. ACS and ALS recovered 51.53%–55.74% of the total protein from bovine and porcine hearts. All extracts were rich in myofibrillar proteins with both treatments resulting in reductions in fat, collagen and cholesterol contents compared with starting materials. At 0% NaCl, ACS and ALS extracts had good gelling properties with the ALS gels having lower % cook loss. While treatments did not affect gel hardness, ACS extracts formed gel networks with higher storage modulus after heating and cooling. At 2% NaCl gel hardness, % cook loss and storage modulus values increased, with greater increases occurring for ACS extracts. The results show that ALS‐ and ACS‐based processes have potential to produce functional ingredients for processed meat products

Mechanisms underlying monosaccharide stimulated incretin hormone secretion from the intestine

Obesity and type II diabetes (T2D) are healthcare crises with continually rising prevalence worldwide. Drugs which mimic the gut hormone glucagon-like peptide-1 (GLP-1), e.g. Semaglutide, are currently the most effective obesity and T2D therapeutics, activating GLP-1 receptors in the pancreas and brain to improve blood glucose and appetite control. Due to the therapeutic benefits of GLP-1 signalling, understanding the factors that regulate its release are of continued interest. Gut hormones are produced by enteroendocrine cells (EECs) of the intestinal epithelium and are released in response to a variety of intestinal signals, with diverse roles in the control of metabolism. The gut hormones GLP-1 and glucose-dependent insulinotropic polypeptide (GIP), known as incretins due to their insulinotropic properties, are produced and released by the EEC sub types L-cells and K-cells respectively. The monosaccharide glucose is a trigger for incretin secretion, which requires glucose uptake via the sodium-dependent glucose transporter 1 (SGLT1) expressed by L-cells and K-cells, as demonstrated by *in vitro* cell based studies. SGLT1 is expressed throughout the intestinal epithelium and additionally facilitates glucose absorption to the bloodstream. In contrast to *in vitro* experiments, *in vivo* studies where humans or animal models lack functional SGLT1, show that GLP-1 is still secreted in response to oral glucose, while GIP secretion is absent. In this thesis, we aimed to explore potential SGLT1 independent mechanisms by which the presence of unabsorbed glucose in the intestine triggers GLP-1, but not GIP, secretion *in vivo*. We first confirmed that an oral glucose challenge resulted in GLP-1, but not GIP secretion in Sglt1 knock out (*Sglt1*-/-) mice, while the secretion of both hormones was observed in wild-type mice. We then showed that SGLT1 substrates (α-MDG (α-Methyl-D-Glucose), D-Glucose) stimulated GLP-1 secretion from ileal organoids, but that this GLP-1 secretion was absent in experiments using *Sglt1*-/- ileal organoids. These experiments confirmed the requirement of SGLT1 for *in vitro*, but not *in vivo*, glucose-stimulated GLP-1 secretion. Using metabolomic techniques we subsequently determined that fermentation of unabsorbed glucose by the gut microbiome generated short chain fatty acids (SCFAs) in the colon of *Sglt1*-/- mice, which have the potential to stimulate GLP-1 release. However, through studies using a panel of glucose analogues which are not microbiome substrates (3-OMG (3-O-Methyl-D-Glucose), L-Glucose), antibiotic mediated microbially depleted *Sglt1*-/- mice and metabolomic techniques, we demonstrated that the microbiota and SCFA synthesis were not essential for SGLT1 independent glucose-stimulated GLP-1 release. We observed that GLP-1 secretion triggered by an unabsorbable intestinal glucose load occurs alongside intestinal distension in both microbially intact and depleted mice, which may exert pressure on the intestinal epithelium. To explore whether increased intra-intestinal pressure might trigger GLP-1 release from L-cells, we initially used RNAsequencing techniques to generate transcriptomic datasets of EECs. These datasets facilitated the investigation of EEC expression of “mechanosensors”, which could convert mechanical stimuli, such as pressure, to hormone release. We described expression of the mechanosensitive G protein-coupled receptor AT1aR (angiotensin II receptor type 1a) and the mechanosensitive ion channel Piezo2 by L-cells, but not K-cells, presenting potential mechanisms by which an unabsorbed glucose load in the intestine may trigger GLP-1, but not GIP, secretion *in vivo*. Investigations into the functional relevance of mechanosensors expressed by L-cells may lead to pharmacological targets which could be explored for the development of novel therapeutics to treat obesity and T2D by stimulating endogenous GLP-1 release from the intestine

Stimulating intestinal GIP release reduces food intake and body weight in mice.

OBJECTIVE: Glucose dependent insulinotropic polypeptide (GIP) is well established as an incretin hormone, boosting glucose-dependent insulin secretion. However, whilst anorectic actions of its sister-incretin glucagon-like peptide-1 (GLP-1) are well established, a physiological role for GIP in appetite regulation is controversial, despite the superior weight loss seen in preclinical models and humans with GLP-1/GIP dual receptor agonists compared with GLP-1R agonism alone. METHODS: We generated a mouse model in which GIP expressing K-cells can be activated through hM3Dq Designer Receptor Activated by Designer Drugs (DREADD, GIP-Dq) to explore physiological actions of intestinally-released GIP. RESULTS: In lean mice, Dq-stimulation of GIP expressing cells increased plasma GIP to levels similar to those found postprandially. The increase in GIP was associated with improved glucose tolerance, as expected, but also triggered an unexpected robust inhibition of food intake. Validating that this represented a response to intestinally-released GIP, the suppression of food intake was prevented by injecting mice peripherally or centrally with antagonistic GIPR-antibodies, and was reproduced in an intersectional model utilising Gip-Cre/Villin-Flp to limit Dq transgene expression to K-cells in the intestinal epithelium. The effects of GIP cell activation were maintained in diet induced obese mice, in which chronic K-cell activation reduced food intake and attenuated body weight gain. CONCLUSIONS: These studies establish a physiological gut-brain GIP-axis regulating food intake in mice, adding to the multi-faceted metabolic effects of GIP which need to be taken into account when developing GIPR-targeted therapies for obesity and diabetes

Peptidomics of enteroendocrine cells and characterisation of potential effects of a novel preprogastrin derived-peptide on glucose tolerance in lean mice.

OBJECTIVES: To analyse the peptidomics of mouse enteroendocrine cells (EECs) and human gastrointestinal (GI) tissue and identify novel gut derived peptides. METHODS: High resolution nano-flow liquid chromatography mass spectrometry (LC-MS/MS) was performed on (i) flow-cytometry purified NeuroD1 positive cells from mouse and homogenised human intestinal biopsies, (ii) supernatants from primary murine intestinal cultures, (iii) intestinal homogenates from mice fed high fat diet. Candidate bioactive peptides were selected on the basis of species conservation, high expression/biosynthesis in EECs and evidence of regulated secretionin vitro. Candidate novel gut-derived peptides were chronically administered to mice to assess effects on food intake and glucose tolerance. RESULTS: A large number of peptide fragments were identified from human and mouse, including known full-length gut hormones and enzymatic degradation products. EEC-specific peptides were largely from vesicular proteins, particularly prohormones, granins and processing enzymes, of which several exhibited regulated secretion in vitro. No regulated peptides were identified from previously unknown genes. High fat feeding particularly affected the distal colon, resulting in reduced peptide levels from GCG, PYY and INSL5. Of the two candidate novel peptides tested in vivo, a peptide from Chromogranin A (ChgA 435-462a) had no measurable effect, but a progastrin-derived peptide (Gast p59-79), modestly improved glucose tolerance in lean mice. CONCLUSION: LC-MS/MS peptidomic analysis of murine EECs and human GI tissue identified the spectrum of peptides produced by EECs, including a potential novel gut hormone, Gast p59-79, with minor effects on glucose tolerance.AstraZenec

Proposed declassification of disease categories related to sexual orientation in the International Statistical Classification of Diseases and Related Health Problems

The World Health Organization is developing the 11th revision of the International Statistical Classification of Diseases and Related Health Problems (ICD-11), planned for publication in 2017. The Working Group on the Classification of Sexual Disorders and Sexual Health was charged with reviewing and making recommendations on disease categories related to sexuality in the chapter on mental and behavioural disorders in the 10th revision (ICD-10), published in 1990. This chapter includes categories for diagnoses based primarily on sexual orientation even though ICD-10 states that sexual orientation alone is not a disorder. This article reviews the scientific evidence and clinical rationale for continuing to include these categories in the ICD. A review of the evidence published since 1990 found little scientific interest in these categories. In addition, the Working Group found no evidence that they are clinically useful: they neither contribute to health service delivery or treatment selection nor provide essential information for public health surveillance. Moreover, use of these categories may create unnecessary harm by delaying accurate diagnosis and treatment. The Working Group recommends that these categories be deleted entirely from ICD-11. Health concerns related to sexual orientation can be better addressed using other ICD categories

The updated European Consensus 2009 on the use of Botulinum toxin for children with cerebral palsy

An interdisciplinary European group of clinical experts in the field of movement disorders and experienced Botulinum toxin users has updated the consensus for the use of Botulinum toxin in the treatment of children with cerebral palsy (CP). A problem-orientated approach was used focussing on both published and practice-based evidence. In part I of the consensus the authors have tabulated the supporting evidence to produce a concise but comprehensive information base, pooling data and experience from 36 institutions in 9 European countries which involves more than 10,000 patients and over 45,000 treatment sessions during a period of more than 280 treatment years. In part II of the consensus the Gross Motor Function Measure (GMFM) and Gross Motor Function Classification System (GMFCS) based Motor Development Curves have been expanded to provide a graphical framework on how to treat the motor disorders in children with CP. This graph is named "CP(Graph) Treatment Modalities - Gross Motor Function" and is intended to facilitate communication between parents, therapists and medical doctors concerning (1) achievable motor function, (2) realistic goal-setting and (3) treatment perspectives for children with CP. The updated European consensus 2009 summarises the current understanding regarding an integrated, multidisciplinary treatment approach using Botulinum toxin for the treatment of children with CP