Rikke Kaae Kirk

GLP-1 Receptor Localization in Monkey and Human Tissue: Novel Distribution Revealed With Extensively Validated Monoclonal Antibody

Glucagon-like peptide 1 (GLP-1) analogs are increasingly being used in the treatment of type 2 diabetes. It is clear that these drugs lower blood glucose through an increase in insulin secretion and a lowering of glucagon secretion; in addition, they lower body weight and systolic blood pressure and increase heart rate. Using a new monoclonal antibody for immunohistochemistry, we detected GLP-1 receptor (GLP-1R) in important target organs in humans and monkeys. In the pancreas, GLP-1R was predominantly localized in β-cells with a markedly weaker expression in acinar cells. Pancreatic ductal epithelial cells did not express GLP-1R. In the kidney and lung, GLP-1R was exclusively expressed in smooth muscle cells in the walls of arteries and arterioles. In the heart, GLP-1R was localized in myocytes of the sinoatrial node. In the gastrointestinal tract, the highest GLP-1R expression was seen in the Brunners gland in the duodenum, with lower level expression in parietal cells and smooth muscle cells in the muscularis externa in the stomach and in myenteric plexus neurons throughout the gut. No GLP-1R was seen in primate liver and thyroid. GLP-1R expression seen with immunohistochemistry was confirmed by functional expression using in situ ligand binding with (125)I-GLP-1. In conclusion, these results give important new insight into the molecular mode of action of GLP-1 analogs by identifying the exact cellular localization of GLP-1R.

Acute neurological signs as the predominant clinical manifestation in four dogs with Angiostrongylus vasorum infections in Denmark

Four dogs with acute neurological signs caused by haemorrhages in the central nervous system were diagnosed with Angiostrongylus vasorum infection as the underlying aetiology. Two dogs presented with brain lesions, one dog with spinal cord lesions and one with lesions in both the brain and spinal cord. Only one dog presented with concurrent signs of classical pulmonary angiostrongylosis (respiratory distress, cough), and only two dogs displayed overt clinical signs of haemorrhages. Results of coagulation assays were inconsistent. Neurological signs reflected the site of pathology and included seizures, various cranial nerve deficits, vestibular signs, proprioceptive deficits, ataxia and paraplegia. One dog died and three were euthanised due to lack of improvement despite medical treatment. This emphasises canine angiostrongylosis as a potential cause of fatal lesions of the central nervous system and the importance of including A. vasorum as a differential diagnosis in young dogs with acute neurological signs in Denmark.

Modulating the Gut Microbiota Improves Glucose Tolerance, Lipoprotein Profile and Atherosclerotic Plaque Development in ApoE-Deficient Mice

The importance of the gut microbiota (GM) in disease development has recently received increased attention, and numerous approaches have been made to better understand this important interplay. For example, metabolites derived from the GM have been shown to promote atherosclerosis, the underlying cause of cardiovascular disease (CVD), and to increase CVD risk factors. Popular interest in the role of the intestine in a variety of disease states has now resulted in a significant proportion of individuals without coeliac disease switching to gluten-free diets. The effect of gluten-free diets on atherosclerosis and cardiovascular risk factors is largely unknown. We therefore investigated the effect of a gluten-free high-fat cholesterol-rich diet, as compared to the same diet in which the gluten peptide gliadin had been added back, on atherosclerosis and several cardiovascular risk factors in apolipoprotein E-deficient (Apoe-/-) mice. The gluten-free diet transiently altered GM composition in these mice, as compared to the gliadin-supplemented diet, but did not alter body weights, glucose tolerance, insulin levels, plasma lipids, or atherosclerosis. In parallel, other Apoe-/- mice fed the same diets were treated with ampicillin, a broad-spectrum antibiotic known to affect GM composition. Ampicillin-treatment had a marked and sustained effect on GM composition, as expected. Furthermore, although ampicillin-treated mice were slightly heavier than controls, ampicillin-treatment transiently improved glucose tolerance both in the absence or presence of gliadin, reduced plasma LDL and VLDL cholesterol levels, and reduced aortic atherosclerotic lesion area. These results demonstrate that a gluten-free diet does not seem to have beneficial effects on atherosclerosis or several CVD risk factors in this mouse model, but that sustained alteration of GM composition with a broad-spectrum antibiotic has beneficial effects on CVD risk factors and atherosclerosis. These findings support the concept that altering the microbiota might provide novel treatment strategies for CVD.

In Situ Phosphorylation of Akt and ERK1/2 in Rat Mammary Gland, Colon, and Liver Following Treatment with Human Insulin and IGF-1

High doses of insulin and the insulin analog AspB10 have been reported to increase mammary tumor incidence in female rats likely via receptor-mediated mechanisms, possibly involving enhanced IGF-1 receptor activation. However, insulin and IGF-1 receptor functionality and intracellular signaling in the rat mammary gland in vivo is essentially unexplored. The authors investigated the effect of a single subcutaneous dose of 600 nmol/kg human insulin or IGF-1 on Akt and ERK1/2 phosphorylation in rat liver, colon, and mammary gland. Rat tissues were examined by Western blotting and immunohistochemistry by phosphorylation-specific antibodies. Insulin as well as IGF-1 caused Akt phosphorylation in mammary epithelial cells, with myoepithelial and basal epithelial cells being most sensitive. IGF-1 caused stronger Akt phosphorylation than insulin in mammary gland epithelial cells. Phosphorylation of ERK1/2 was not influenced by insulin or IGF-1. Rather, in liver and mammary gland P-ERK1/2 appeared to correlate with estrous cycling, supporting that ERK1/2 has important physiological roles in these two organs. In short, these findings supported that the rat mammary gland epithelium expresses functional insulin and IGF-1 receptors and that phosphorylation of Akt as well as ERK1/2 may be of value in understanding the effects of exogenous insulin in the rat mammary gland and colon.

Anti-IL-21 monoclonal antibody combined with liraglutide effectively reverses established hyperglycemia in mouse models of type 1 diabetes

Immunotherapy for type 1 diabetes (T1D) has previously focused on suppressing the autoimmune response against pancreatic beta cells to preserve endogenous insulin production and regulate glucose levels. With increased attention toward combination therapy strategies, studies indicate the multifunctional cytokine interleukin-21 (IL-21) may be a suitable target as an immuno-modulatory arm, while glucagon-like peptide-1 receptor (GLP-1R) agonists may be appropriate as a beta cell protective arm in combination therapy for T1D. We report here that treatment with anti-IL-21 monoclonal antibody delays diabetes onset in the spontaneous non-obese diabetic (NOD) and NOD.scid adoptive transfer models, while its effect in reversing recent-onset hyperglycemia is limited. However, the combination of anti-IL-21 plus the GLP-1R agonist liraglutide is effective in reversing established disease compared to either monotherapy in both the NOD and rat insulin promotor-lymphocytic choriomeningitis virus glycoprotein (RIP-LCMV-GP) models of autoimmune diabetes. Enhanced efficacy is particularly evident in severely hyperglycemic mice, with return to normoglycemia remaining stable for the majority of mice even after therapy is withdrawn. Importantly, increased beta cell proliferation does not appear to be the predominant mechanism. In conclusion, combination therapy with anti-IL-21 and liraglutide is able to consistently reverse disease in mouse models of T1D. The observed effects rival the most effective experimental disease-modifying treatments tested in preclinical studies.

Spatial distribution of soluble insulin in pig subcutaneous tissue: Effect of needle length, injection speed and injected volume

The spatial distribution of a soluble insulin formulation was visualized and quantified in 3-dimensions using X-ray computed tomography. The drug distribution was visualized for ex vivo injections in pig subcutaneous tissue. Pig subcutaneous tissue has very distinct layers, which could be separated in the tomographic reconstructions and the amount of drug in each tissue class was quantified. With a scan time of about 45min per sample, and a robust segmentation it was possible to analyze differences in the spatial drug distribution between several similar injections. It was studied how the drug distribution was effected by needle length, injection speed and injected volume. For an injected volume of 0.1ml and injection depth of 8mm about 50% of the injections were partly intramuscular. Using a 5mm needle resulted in purely subcutaneous injections with minor differences in the spatial drug distribution between injections. Increasing the injected volume from 0.1ml to 1ml did not increase the intramuscular volume fraction, but gave a significantly higher volume fraction placed in the fascia separating the deep and superficial subcutaneous fat layers. Varying the injection speed from 25l/s up to 300l/s gave no changes in the drug concentration distribution. The method presented gives novel insight into subcutaneous injections of soluble insulin drugs and can be used to optimize the injection technique for subcutaneous drug administration in preclinical studies of rodents.

Immunohistochemical Assessment of Glucagon-like Peptide 1 Receptor (GLP-1R) Expression in the Pancreas of Patients with Type 2 Diabetes

Glucagon‐like peptide‐1 (GLP‐1) is an incretin hormone which stimulates insulin release and inhibits glucagon secretion from the pancreas in a glucose‐dependent manner. Incretin‐based therapies, consisting of GLP‐1 receptor (GLP‐1R) agonists and dipeptidyl peptidase‐4 (DPP‐4) inhibitors, are used for the treatment of type 2 diabetes (T2D). Immunohistochemical studies for GLP‐1R expression have been hampered previously by the use of unspecific polyclonal antibodies. This study aimed to assess the expression levels of GLP‐1R in a set of T2D donor samples obtained via nPOD.

Long-term Western diet fed apolipoprotein E-deficient rats exhibit only modest early atherosclerotic characteristics

In the apolipoprotein E–deficient mouse, the gut microbiota has an impact on the development of atherosclerosis, but whether such correlations are also present in rats requires investigation. Therefore, we studied female SD-Apoetm1sage (Apoe−/−) rats fed either a Western diet or a low-fat control diet with or without gluten, which is known to promote gut microbiota changes, until 20 weeks of age. We hypothesized that the manifestation of atherosclerosis would be more severe in Apoe−/− rats fed the Western high-fat diet, as compared with rats fed the low-fat diet, and that atherosclerosis would be accelerated by gluten. Both Western diet-feeding and gluten resulted in significant changes in gut microbiota, but the microbiota impact of gluten was transient. Compared with Apoe−/− rats fed a low-fat diet, Western diet-fed Apoe−/− rats were heavier and became glucose intolerant with increased levels of oxidative stress. They developed early fatty streak lesions in their aortic sinus, while there was no evidence of atherosclerosis in the thoracic aorta. No conclusions could be made on the impact of gluten on atherosclerosis. Although Western diet-fed Apoe−/− rats exhibited a more human-like LDL dominated blood lipid profile, signs of obesity, type 2 diabetes and cardiovascular disease were modest.