Effects of exogenous GIP and GLP-2 on bone turnover in individuals with type 2 diabetes.

CONTEXT: Individuals with type 2 diabetes (T2D) have an increased risk of bone fractures despite normal or increased bone mineral density (BMD). The underlying causes are not well understood but may include disturbances in the gut-bone axis, in which both glucose-dependent insulinotropic polypeptide (GIP) and glucagon-like peptide-2 (GLP-2) are regulators of bone turnover. Thus, in healthy fasting participants, both exogenous GIP and GLP-2 acutely reduce bone resorption. OBJECTIVE: The objective of this study was to investigate the acute effects of subcutaneously administered GIP and GLP-2 on bone turnover in individuals with T2D. METHODS: We included 10 men with T2D. Participants met fasting in the morning on three separate test days and were injected subcutaneously with GIP, GLP-2, or placebo in a randomized crossover design. Blood samples were drawn at baseline and regularly after injections. Bone turnover was estimated by circulating levels of collagen type 1 C-terminal telopeptide (CTX), procollagen type 1 N-terminal propeptide (P1NP), sclerostin, and PTH. RESULTS: GIP and GLP-2 significantly reduced CTX to (mean ± SEM) 66 ± 7.8% and 74 ± 5.9% of baseline, respectively, compared with after placebo (p = 0.001). In addition, P1NP and sclerostin increased acutely after GIP whereas a decrease in P1NP was seen after GLP-2. PTH levels decreased to 67 ± 2.5% of baseline after GLP-2 and to only 86 ± 3.4% after GIP. CONCLUSION: Subcutaneous GIP and GLP-2 affect CTX and P1NP in individuals with T2D to the same extent as previously demonstrated in healthy individuals.

Long-acting agonists of human and rodent GLP-2 receptors for studies of the physiology and pharmacological potential of the GLP-2 system.

BACKGROUND AND PURPOSE: Glucagon-like peptide-2 (GLP-2) is secreted postprandially from enteroendocrine Lcells and has anabolic action on gut and bone. Short-acting teduglutide is the only approved GLP-2 analog for the treatment of short-bowel syndrome (SBS). To improve the therapeutic effect, we created a series of lipidated GLP-2R agonists. EXPERIMENTAL APPROACH: Six GLP-2 analogs were studied in vitro for cAMP accumulation, ß-arrestin 1 and 2 recruitment, affinity, and internalization. The trophic actions on intestine and bone were examined in vivo in rodents. KEY RESULTS: Lipidations at lysines introduced at position 12, 16, and 20 of hGLP-2(1-33) were well-tolerated with less than 2.2-fold impaired potency and full efficacy at the hGLP-2R in cAMP accumulation. In contrast, N- and C-terminal (His1 and Lys30) lipidations impaired potency by 4.2- and 45-fold and lowered efficacy to 77% and 85% of hGLP-2, respectively. All variants were similarly active on the rat and mouse GLP-2Rs and the three most active variants displayed increased selectivity for hGLP-2R over hGLP-1R activation, compared to native hGLP-2. Impact on arrestin recruitment and receptor internalization followed that of Gαs-coupling, except for lipidation in position 20, where internalization was more impaired, suggesting desensitization protection. A highly active variant (C16 at position 20) with low internalization and a half-life of 9.5 h in rats showed improved gut and bone tropism with increased weight of small intestine in mice and decreased CTX levels in rats. CONCLUSION AND IMPLICATION: We present novel hGLP-2 agonists suitable for in vivo studies of the GLP-2 system to uncover its pharmacological potential.

Expression Profile of the GLP-1 Receptor in the Gastrointestinal Tract and Pancreas in Adult Female Mice.

Therapies based on glucagon-like peptide-1 receptor (GLP-1R) agonism are highly effective in treating type 2 diabetes and obesity, but the localization of GLP-1Rs mediating the antidiabetic and other possible actions of GLP-1 is still debated. The purpose with this study was to identify sites of GLP-1R mRNA and protein expression in the mouse gastrointestinal system by means of GLP-1R antibody immunohistochemistry, Glp1r mRNA fluorescence in situ hybridization, and 125I-exendin (9-39) autoradiography. As expected, GLP-1R staining was observed in almost all ß-cells in the pancreatic islets, but more rarely in α- and δ-cells. In the stomach, GLP-1R staining was found exclusively in the gastric corpus mucous neck cells, known to protect the stomach mucosa. The Brunner glands were strongly stained for GLP-1R, and pretreatment with GLP-1 agonist exendin-4 caused internalization of the receptor and mucin secretion, while pretreatment with phosphate-buffered saline or antagonist exendin (9-39) did not. In the intestinal mucosa, GLP-1R staining was observed in intraepithelial lymphocytes, lamina propria lymphocytes, and enteroendocrine cells containing secretin, peptide YY, and somatostatin, but not cholecystokinin. GLP-1R staining was seen in nerve fibers within the choline acetyl transferase- and nitric oxide-positive myenteric plexuses from the gastric corpus to the distal large intestine being strongest in the mid- and hindgut area. Finally, intraperitoneal administration of radiolabeled exendin (9-39) strongly labeled myenteric fibers. In conclusion, this study expands our knowledge of GLP-1R localization and suggests that GLP-1 may serve an important role in modulating gastrointestinal health and mucosal protection.

Antagonizing somatostatin receptor subtype 2 and 5 reduces blood glucose in a gut- and GLP-1R-dependent manner.

Somatostatin (SS) inhibits glucagon-like peptide-1 (GLP-1) secretion in a paracrine manner. We hypothesized that blocking somatostatin subtype receptor 2 (SSTR2) and 5 (SSTR5) would improve glycemia by enhancing GLP-1 secretion. In the perfused mouse small intestine, the selective SSTR5 antagonist (SSTR5a) stimulated glucose-induced GLP-1 secretion to a larger degree than the SSTR2 antagonist (SSTR2a). In parallel, mice lacking the SSTR5R showed increased glucose-induced GLP-1 secretion. Both antagonists improved glycemia in vivo in a GLP-1 receptor-dependent (GLP-1R-dependent) manner, as the glycemic improvements were absent in mice with impaired GLP-1R signaling and in mice treated with a GLP-1R-specific antagonist. SSTR5a had no direct effect on insulin secretion in the perfused pancreas, whereas SSTR2a increased insulin secretion in a GLP-1R-independent manner. Adding a dipeptidyl peptidase 4 inhibitor (DPP-4i) in vivo resulted in additive effects on glycemia. However, when glucose was administered intraperitoneally, the antagonist was incapable of lowering blood glucose. Oral administration of SSTR5a, but not SSTR2a, lowered blood glucose in diet-induced obese mice. In summary, we demonstrate that selective SSTR antagonists can improve glucose control primarily through the intestinal GLP-1 system in mice.

Glucagon acutely regulates hepatic amino acid catabolism and the effect may be disturbed by steatosis.

OBJECTIVE: Glucagon is well known to regulate blood glucose but may be equally important for amino acid metabolism. Plasma levels of amino acids are regulated by glucagon-dependent mechanism(s), while amino acids stimulate glucagon secretion from alpha cells, completing the recently described liver-alpha cell axis. The mechanisms underlying the cycle and the possible impact of hepatic steatosis are unclear. METHODS: We assessed amino acid clearance in vivo in mice treated with a glucagon receptor antagonist (GRA), transgenic mice with 95% reduction in alpha cells, and mice with hepatic steatosis. In addition, we evaluated urea formation in primary hepatocytes from ob/ob mice and humans, and we studied acute metabolic effects of glucagon in perfused rat livers. We also performed RNA sequencing on livers from glucagon receptor knock-out mice and mice with hepatic steatosis. Finally, we measured individual plasma amino acids and glucagon in healthy controls and in two independent cohorts of patients with biopsy-verified non-alcoholic fatty liver disease (NAFLD). RESULTS: Amino acid clearance was reduced in mice treated with GRA and mice lacking endogenous glucagon (loss of alpha cells) concomitantly with reduced production of urea. Glucagon administration markedly changed the secretion of rat liver metabolites and within minutes increased urea formation in mice, in perfused rat liver, and in primary human hepatocytes. Transcriptomic analyses revealed that three genes responsible for amino acid catabolism (Cps1, Slc7a2, and Slc38a2) were downregulated both in mice with hepatic steatosis and in mice with deletion of the glucagon receptor. Cultured ob/ob hepatocytes produced less urea upon stimulation with mixed amino acids, and amino acid clearance was lower in mice with hepatic steatosis. Glucagon-induced ureagenesis was impaired in perfused rat livers with hepatic steatosis. Patients with NAFLD had hyperglucagonemia and increased levels of glucagonotropic amino acids, including alanine in particular. Both glucagon and alanine levels were reduced after diet-induced reduction in Homeostatic Model Assessment for Insulin Resistance (HOMA-IR, a marker of hepatic steatosis). CONCLUSIONS: Glucagon regulates amino acid metabolism both non-transcriptionally and transcriptionally. Hepatic steatosis may impair glucagon-dependent enhancement of amino acid catabolism.

Enteroendocrine K Cells Exert Complementary Effects to Control Bone Quality and Mass in Mice.

The involvement of a gut-bone axis in controlling bone physiology has been long suspected, although the exact mechanisms are unclear. We explored whether glucose-dependent insulinotropic polypeptide (GIP)-producing enteroendocrine K cells were involved in this process. The bone phenotype of transgenic mouse models lacking GIP secretion (GIP-GFP-KI) or enteroendocrine K cells (GIP-DT) was investigated. Mice deficient in GIP secretion exhibited lower bone strength, trabecular bone mass, trabecular number, and cortical thickness, notably due to higher bone resorption. Alterations of microstructure, modifications of bone compositional parameters, represented by lower collagen cross-linking, were also apparent. None of these alterations were observed in GIP-DT mice lacking enteroendocrine K cells, suggesting that another K-cell secretory product acts to counteract GIP action. To assess this, stable analogues of the known K-cell peptide hormones, xenin and GIP, were administered to mature NIH Swiss male mice. Both were capable of modulating bone strength mostly by altering bone microstructure, bone gene expression, and bone compositional parameters. However, the two molecules exhibited opposite actions on bone physiology, with evidence that xenin effects are mediated indirectly, possibly via neural networks. Our data highlight a previously unknown interaction between GIP and xenin, which both moderate gut-bone connectivity. © 2020 American Society for Bone and Mineral Research.

Why is it so difficult to measure glucagon-like peptide-1 in a mouse?

AIMS/HYPOTHESIS: In humans, glucagon-like peptide-1 (GLP-1) is rapidly degraded by dipeptidyl peptidase-4 to a relatively stable metabolite, GLP-1(9-36)NH2, which allows measurement of GLP-1 secretion. However, little is known about the kinetics of the GLP-1 metabolite in mice. We hypothesised that the GLP-1 metabolite is rapidly degraded in this species by neutral endopeptidase(s) (NEP[s]). METHODS: We administered glucose, mixed meal or water orally to 256 mice, and took blood samples before and 2, 6, 10, 20, 30, 60 or 90 min after stimulation. To study the metabolism of the GLP-1 metabolite, i.v. GLP-1(9-36)NH2 (800 fmol) or saline (154 mmol/l NaCl) was administered to 160 mice, some of which had a prior injection of a selective NEP 24.11 ± inhibitor (candoxatril, 5 mg/kg) or saline. Blood was collected before and 1, 2, 4 and 12 min after GLP-1/saline injection. Plasma GLP-1 levels were analysed using a customised single-site C-terminal ELISA, two different two-site ELISAs and MS. RESULTS: GLP-1 secretion profiles after oral glucose administration differed markedly when assayed by C-terminal ELISA compared with sandwich ELISAs, with the former showing a far higher peak value and AUC. In mice injected with GLP-1(9-36)NH2, immunoreactive GLP-1 plasma levels peaked at approximately 75 pmol/l at 1 min when measured with sandwich ELISAs, returning to baseline (~20 pmol/l) after 12 min, but remained elevated using the C-terminal ELISA (~90 pmol/l at 12 min). NEP 24.11 inhibition by candoxatril significantly attenuated GLP-1(9-36)NH2 degradation in vivo and in vitro. MS identified GLP-1 fragments consistent with NEP 24.11 degradation. CONCLUSIONS/INTERPRETATION: In mice, the GLP-1 metabolite is eliminated within a few minutes owing to endoproteolytic cleavage by NEP 24.11. Therefore, accurate measurement of GLP-1 secretion in mice requires assays for NEP 24.11 metabolites. Conventional sandwich ELISAs are inadequate because of endoproteolytic cleavage of the dipeptidyl peptidase-4-generated metabolite.

Use of anesthesia dramatically alters the oral glucose tolerance and insulin secretion in C57Bl/6 mice.

Evaluation of the impact of anesthesia on oral glucose tolerance in mice. Anesthesia is often used when performing OGTT in mice to avoid the stress of gavage and blood sampling, although anesthesia may influence gastrointestinal motility, blood glucose, and plasma insulin dynamics. C57Bl/6 mice were anesthetized using the following commonly used regimens: (1) hypnorm/midazolam repetitive or single injection; (2) ketamine/xylazine; (3) isoflurane; (4) pentobarbital; and (5) A saline injected, nonanesthetized group. Oral glucose was administered at time 0 min and blood glucose measured in the time frame -15 to +150 min. Plasma insulin concentration was measured at time 0 and 20 min. All four anesthetic regimens resulted in impaired glucose tolerance compared to saline/no anesthesia. (1) hypnorm/midazolam increased insulin concentrations and caused an altered glucose tolerance; (2) ketamine/xylazine lowered insulin responses and resulted in severe hyperglycemia throughout the experiment; (3) isoflurane did not only alter the insulin secretion but also resulted in severe hyperglycemia; (4) pentobarbital resulted in both increased insulin secretion and impaired glucose tolerance. All four anesthetic regimens altered the oral glucose tolerance, and we conclude that anesthesia should not be used when performing metabolic studies in mice.

Dynamics of glucagon secretion in mice and rats revealed using a validated sandwich ELISA for small sample volumes.

Glucagon is a metabolically important hormone, but many aspects of its physiology remain obscure, because glucagon secretion is difficult to measure in mice and rats due to methodological inadequacies. Here, we introduce and validate a low-volume, enzyme-linked immunosorbent glucagon assay according to current analytical guidelines, including tests of sensitivity, specificity, and accuracy, and compare it, using the Bland-Altman algorithm and size-exclusion chromatography, with three other widely cited assays. After demonstrating adequate performance of the assay, we measured glucagon secretion in response to intravenous glucose and arginine in anesthetized mice (isoflurane) and rats (Hypnorm/midazolam). Glucose caused a long-lasting suppression to very low values (1-2 pmol/l) within 2 min in both species. Arginine stimulated secretion 8- to 10-fold in both species, peaking at 1-2 min and returning to basal levels at 6 min (mice) and 12 min (rats). d-Mannitol (osmotic control) was without effect. Ketamine/xylazine anesthesia in mice strongly attenuated (P < 0.01) α-cell responses. Chromatography of pooled plasma samples confirmed the accuracy of the assay. In conclusion, dynamic analysis of glucagon secretion in rats and mice with the novel accurate sandwich enzyme-linked immunosorbent assay revealed extremely rapid and short-lived responses to arginine and rapid and profound suppression by glucose.

Acute disruption of glucagon secretion or action does not improve glucose tolerance in an insulin-deficient mouse model of diabetes.

AIMS/HYPOTHESIS: Normal glucose metabolism depends on pancreatic secretion of insulin and glucagon. The bihormonal hypothesis states that while lack of insulin leads to glucose underutilisation, glucagon excess is the principal factor in diabetic glucose overproduction. A recent study reported that streptozotocin-treated glucagon receptor knockout mice have normal glucose tolerance. We investigated the impact of acute disruption of glucagon secretin or action in a mouse model of severe diabetes by three different approaches: (1) alpha cell elimination; (2) glucagon immunoneutralisation; and (3) glucagon receptor antagonism, in order to evaluate the effect of these on glucose tolerance. METHODS: Severe diabetes was induced in transgenic and wild-type mice by streptozotocin. Glucose metabolism was investigated using OGTT in transgenic mice with the human diphtheria toxin receptor expressed in proglucagon producing cells allowing for diphtheria toxin (DT)-induced alpha cell ablation and in mice treated with either a specific high affinity glucagon antibody or a specific glucagon receptor antagonist. RESULTS: Near-total alpha cell elimination was induced in transgenic mice upon DT administration and resulted in a massive decrease in pancreatic glucagon content. Oral glucose tolerance in diabetic mice was neither affected by glucagon immunoneutralisation, glucagon receptor antagonism, nor alpha cell removal, but did not deteriorate further compared with mice with intact alpha cell mass. CONCLUSIONS/INTERPRETATION: Disruption of glucagon action/secretion did not improve glucose tolerance in diabetic mice. Near-total alpha cell elimination may have prevented further deterioration. Our findings support insulin lack as the major factor underlying hyperglycaemia in beta cell-deficient diabetes.

Hyperglucagonaemia analysed by glucagon sandwich ELISA: nonspecific interference or truly elevated levels?

AIM/HYPOTHESIS: Hyperglucagonaemia is a characteristic of several clinical conditions (e.g. end-stage renal disease (ESRD), type 2 diabetes, obesity before and after Roux-en-Y gastric bypass (RYGB) and vagotomy with pyloroplasty), but the molecular nature of 'immunoreactive' glucagon is poorly characterised. The specific determination of fully processed, intact glucagon requires a 'sandwich' assay employing a combination of antibodies directed against both N- and C-termini. We compared a novel assay for intact glucagon with a highly sensitive C-terminal RIA (hitherto considered specific) to determine the extent to which the hyperglucagonaemia measured in clinical samples was caused by authentic glucagon. METHODS: We examined the performance of three commercial glucagon 'sandwich' ELISAs. The ELISA with the best overall performance was selected to compare glucagon measurements in clinical samples with an established glucagon RIA. RESULTS: The first assay performed poorly: there was high cross-reactivity with glicentin (22%) and a lack of sensitivity for glucagon. The second and third assays showed minor cross-reactivity (1-5%) with oxyntomodulin and glicentin; however, the second assay had insufficient sensitivity for glucagon in plasma (>10-20 pmol/l). Thus, only the third assay was suitable for measuring glucagon concentrations in clinical samples. The ELISA and RIA measured similar glucagon levels in healthy individuals. Measurements of samples from individuals with abnormally high (type 2 diabetes or obese) or very elevated (post vagotomy with pyloroplasty, post-RYGB) glucagon levels were also similar in both assays. However, glucagon levels in participants with ESRD were much lower when measured by ELISA than by RIA, indicating that the apparent hyperglucagonaemia is not caused by fully processed intact glucagon. CONCLUSIONS/INTERPRETATION: For most purposes, sensitive C-terminal glucagon RIAs are accurate. However, measurements may be spuriously high, at least in patients with renal disease. Trial Registration Samples from type 2 diabetic and normoglucose-tolerant patients before and 1 year after RYGB were from a study by Bojsen-Møller et al (trial registration number NCT 01202526). Samples from vagotomised and control individuals were from a study by Plamboeck et al (NCT01176890). Samples from ESRD patients were from a study by Idorn et al (NCT01327378).

Fructose stimulates GLP-1 but not GIP secretion in mice, rats, and humans.

Nutrients often stimulate gut hormone secretion, but the effects of fructose are incompletely understood. We studied the effects of fructose on a number of gut hormones with particular focus on glucagon-like peptide 1 (GLP-1) and glucose-dependent insulinotropic polypeptide (GIP). In healthy humans, fructose intake caused a rise in blood glucose and plasma insulin and GLP-1, albeit to a lower degree than isocaloric glucose. Cholecystokinin secretion was stimulated similarly by both carbohydrates, but neither peptide YY3-36 nor glucagon secretion was affected by either treatment. Remarkably, while glucose potently stimulated GIP release, fructose was without effect. Similar patterns were found in the mouse and rat, with both fructose and glucose stimulating GLP-1 secretion, whereas only glucose caused GIP secretion. In GLUTag cells, a murine cell line used as model for L cells, fructose was metabolized and stimulated GLP-1 secretion dose-dependently (EC50 = 0.155 mM) by ATP-sensitive potassium channel closure and cell depolarization. Because fructose elicits GLP-1 secretion without simultaneous release of glucagonotropic GIP, the pathways underlying fructose-stimulated GLP-1 release might be useful targets for type 2 diabetes mellitus and obesity drug development.

Identification and characterization of locus-specific methylation patterns within novel loci undergoing hypermethylation during breast cancer pathogenesis.

INTRODUCTION: Despite similar clinical and pathological features, large numbers of breast cancer patients experience different outcomes of the disease. This, together with the fact that the incidence of breast cancer is growing worldwide, emphasizes an urgent need for identification of new biomarkers for early cancer detection and stratification of patients. METHODS: We used ultrahigh-resolution microarrays to compare genomewide methylation patterns of breast carcinomas (n = 20) and nonmalignant breast tissue (n = 5). Biomarker properties of a subset of discovered differentially methylated regions (DMRs) were validated using methylation-sensitive high-resolution melting (MS-HRM) in a case-control study on a panel of breast carcinomas (n = 275) and non-malignant controls (n = 74). RESULTS: On the basis of microarray results, we selected 19 DMRs for large-scale screening of cases and controls. Analysis of the screening results showed that all DMRs tested displayed significant gains of methylation in the cancer tissue compared to the levels in control tissue. Interestingly, we observed two types of locus-specific methylation, with loci undergoing either predominantly full or heterogeneous methylation during carcinogenesis. Almost all tested DMRs (17 of 19) displayed low-level methylation in nonmalignant breast tissue, independently of locus-specific methylation patterns in cases. CONCLUSIONS: Specific loci can undergo either heterogeneous or full methylation during carcinogenesis, and loci hypermethylated in cancer frequently show low-level methylation in nonmalignant tissue.

A major lineage of enteroendocrine cells coexpress CCK, secretin, GIP, GLP-1, PYY, and neurotensin but not somatostatin.

Enteroendocrine cells such as duodenal cholecystokinin (CCK cells) are generally thought to be confined to certain segments of the gastrointestinal (GI) tract and to store and release peptides derived from only a single peptide precursor. In the current study, however, transgenic mice expressing enhanced green fluorescent protein (eGFP) under the control of the CCK promoter demonstrated a distribution pattern of CCK-eGFP positive cells that extended throughout the intestine. Quantitative PCR and liquid chromatography-mass spectrometry proteomic analyses of isolated, FACS-purified CCK-eGFP-positive cells demonstrated expression of not only CCK but also glucagon-like peptide 1 (GLP-1), gastric inhibitory peptide (GIP), peptide YY (PYY), neurotensin, and secretin, but not somatostatin. Immunohistochemistry confirmed this expression pattern. The broad coexpression phenomenon was observed both in crypts and villi as demonstrated by immunohistochemistry and FACS analysis of separated cell populations. Single-cell quantitative PCR indicated that approximately half of the duodenal CCK-eGFP cells express one peptide precursor in addition to CCK, whereas an additional smaller fraction expresses two peptide precursors in addition to CCK. The coexpression pattern was further confirmed through a cell ablation study based on expression of the human diphtheria toxin receptor under the control of the proglucagon promoter, in which activation of the receptor resulted in a marked reduction not only in GLP-1 cells, but also PYY, neurotensin, GIP, CCK, and secretin cells, whereas somatostatin cells were spared. Key elements of the coexpression pattern were confirmed by immunohistochemical double staining in human small intestine. It is concluded that a lineage of mature enteroendocrine cells have the ability to coexpress members of a group of functionally related peptides: CCK, secretin, GIP, GLP-1, PYY, and neurotensin, suggesting a potential therapeutic target for the treatment and prevention of diabetes and obesity.