Lærke S. Gasbjerg

The Gluco- and Liporegulatory and Vasodilatory Effects of Glucose-Dependent Insulinotropic Polypeptide (GIP) Are Abolished by an Antagonist of the Human GIP Receptor

A truncated form of human glucose-dependent insulinotropic polypeptide (GIP), GIP(3–30)NH2, was recently identified as an antagonist of the human GIP receptor. This study examined the ability of GIP(3–30)NH2 to antagonize the physiological actions of GIP in glucose metabolism, subcutaneous abdominal adipose tissue blood flow (ATBF), and lipid metabolism in humans. Eight lean subjects were studied by measuring arteriovenous concentrations of metabolites and ATBF on three different occasions during hyperglycemic-hyperinsulinemic clamps with concomitant infusions of GIP, GIP(3–30)NH2, or both GIP and GIP(3–30)NH2. During infusion of GIP(3–30)NH2 alone and in combination with GIP, insulin levels and the total glucose amount infused to maintain the clamp were lower than during GIP alone. In addition, ATBF remained constant during the antagonist and increased only slightly in combination with GIP, whereas it increased fivefold during GIP alone. Adipose tissue triacylglyceride (TAG) and glucose uptake decreased, and the free fatty acid/glycerol ratio increased during the antagonist alone and in combination with GIP. The changes in glucose infusion rates and plasma insulin levels demonstrate an inhibitory effect of the antagonist on the incretin effect of GIP. In addition, the antagonist inhibited GIP-induced increase in ATBF and decreased the adipose tissue TAG uptake, indicating that GIP also plays a crucial role in lipid metabolism.

GIP(3–30)NH2 is a potent competitive antagonist of the GIP receptor and effectively inhibits GIP-mediated insulin, glucagon, and somatostatin release

ABSTRACT Alternative processing of the precursor protein pro‐GIP results in endogenously produced GIP(1–30)NH2, that by DPP‐4 cleavage in vivo results in the metabolite GIP(3–30)NH2. We showed previously that GIP(3–30)NH2 is a high affinity antagonist of the human GIPR in vitro. Here we determine whether it is suitable for studies of GIP physiology in rats since effects of GIP agonists and antagonists are strictly species‐dependent. Transiently transfected COS‐7 cells were assessed for cAMP accumulation upon ligand stimulation or assayed in competition binding using human 125I‐GIP(1–42) as radioligand. In isolated perfused rat pancreata, insulin, glucagon, and somatostatin‐releasing properties were evaluated. Competition binding demonstrated that on the rat GIP receptor (GIPR), rat GIP(3–30)NH2 bound with high affinity (Ki of 17 nM), in contrast to human GIP(3–30)NH2 (Ki of 250 nM). In cAMP studies, rat GIP(3–30)NH2 inhibited GIP(1–42)‐induced rat GIPR activation and schild‐plot analysis showed competitive antagonism with a pA2 of 13 nM and a slope of 0.9 ± 0.09. Alone, rat GIP(3–30)NH2 displayed weak, low‐potent partial agonistic properties (EC50 > 1 &mgr;M) with an efficacy of 9.4% at 0.32 &mgr;M compared to GIP(1–42). In perfused rat pancreata, rat GIP(3–30)NH2 efficiently antagonized rat GIP(1–42)‐induced insulin, somatostatin, and glucagon secretion. In summary, rat GIP(3–30)NH2 is a high affinity competitive GIPR antagonist and effectively antagonizes GIP‐mediated G protein‐signaling as well as pancreatic hormone release, while human GIP(3–30)NH2, despite a difference of only one amino acid between the two (arginine in position 18 in rat GIP(3–30)NH2; histidine in human), is unsuitable in the rat system. This underlines the importance of species differences in the GIP system, and the limitations of testing human peptides in rodent systems.

Circulating Glucagon 1-61 Regulates Blood Glucose by Increasing Insulin Secretion and Hepatic Glucose Production

Summary Glucagon is secreted from pancreatic α cells, and hypersecretion (hyperglucagonemia) contributes to diabetic hyperglycemia. Molecular heterogeneity in hyperglucagonemia is poorly investigated. By screening human plasma using high-resolution-proteomics, we identified several glucagon variants, among which proglucagon 1-61 (PG 1-61) appears to be the most abundant form. PG 1-61 is secreted in subjects with obesity, both before and after gastric bypass surgery, with protein and fat as the main drivers for secretion before surgery, but glucose after. Studies in hepatocytes and in β cells demonstrated that PG 1-61 dose-dependently increases levels of cAMP, through the glucagon receptor, and increases insulin secretion and protein levels of enzymes regulating glycogenolysis and gluconeogenesis. In rats, PG 1-61 increases blood glucose and plasma insulin and decreases plasma levels of amino acids in vivo. We conclude that glucagon variants, such as PG 1-61, may contribute to glucose regulation by stimulating hepatic glucose production and insulin secretion.

Glucose-dependent insulinotropic polypeptide (GIP) receptor antagonists as anti-diabetic agents

Graphical abstract Figure. No caption available. HighlightsN‐ and C‐terminally truncated peptides are potent GIPR antagonists with high affinity.There are remarkable species differences in the GIP system (rodent‐human).GIPR antagonists have therapeutic potential as anti‐diabetic and anti‐obesity agents.The first studies with a GIPR antagonist in humans are recently published.For now, no studies are published with a GIPR antagonist in patients with T2D or obesity. ABSTRACT Glucose‐dependent insulinotropic polypeptide (GIP) is an intestinal hormone with a broad range of physiological actions. In the postprandial state, the hormone stimulates insulin secretion and during eu‐ and hypoglycemia, it stimulates glucagon secretion. In addition, GIP increases triacylglycerol (TAG) uptake in adipose tissue and decreases bone resorption. However, the importance of these actions in humans are not clearly understood as a specific GIP receptor (GIPR) antagonist – an essential tool to study GIP physiology – has been missing. Several different GIPR antagonists have been identified comprising both peptides, vaccines against GIP, GIP antibodies or antibodies against the GIPR. However, most of these have only been tested in rodents. In vitro, N‐ and C‐terminally truncated GIP variants are potent and efficacious GIPR antagonists. Recently, GIP(3–30)NH2, a naturally occurring peptide, was shown to block the GIPR in humans and decrease GIP‐induced insulin secretion as well as adipose tissue blood flow and TAG uptake. So far, there are no studies with a GIPR antagonist in patients with type 2 diabetes (T2D), but because the elevations in fasting plasma glucagon and paradoxical postprandial glucagon excursions, seen in patients with T2D, are aggravated by GIP, a GIPR antagonist could partly alleviate this and possibly improve the fasting and postprandial glycemia. Since the majority of patients with T2D are overweight, inhibition of GIP‐induced fat deposition may be beneficial as well. Here we summarize the studies of GIPR antagonists and discuss the therapeutic potential of the GIP system in humans.

The bile acid‐sequestering resin sevelamer eliminates the acute GLP‐1 stimulatory effect of endogenously released bile acids in patients with type 2 diabetes

Discovery of the specific bile acid receptors farnesoid X receptor (FXR) and Takeda G protein‐coupled receptor 5 (TGR5) in enteroendocrine L cells has prompted research focusing on the impact of bile acids on glucagon‐like peptide‐1 (GLP‐1) secretion and glucose metabolism. The aim of the present study was to assess the GLP‐1 secretory and gluco‐metabolic effects of endogenously released bile, with and without concomitant administration of the bile acid‐sequestering resin, sevelamer, in patients with type 2 diabetes.

Human GIP(3-30)NH2 inhibits G protein-dependent as well as G protein-independent signaling and is selective for the GIP receptor with high-affinity binding to primate but not rodent GIP receptors

Graphical abstract Figure. No Caption available. Abstract GIP(3‐30)NH2 is a high affinity antagonist of the GIP receptor (GIPR) in humans inhibiting insulin secretion via G protein‐dependent pathways. However, its ability to inhibit G protein‐independent signaling is unknown. Here we determine its action on arrestin‐recruitment and receptor internalization in recombinant cells. As GIP is adipogenic, we evaluate the inhibitory actions of GIP(3‐30)NH2 in human adipocytes. Finally, we determine the receptor selectivity of GIP(3‐30)NH2 among other human and animal GPCRs. cAMP accumulation and &bgr;‐arrestin 1 and 2 recruitment were studied in transiently transfected HEK293 cells and real‐time internalization in transiently transfected HEK293A and in HEK293A &bgr;‐arrestin 1 and 2 knockout cells. Furthermore, human subcutaneous adipocytes were assessed for cAMP accumulation following ligand stimulation. Competition binding was examined in transiently transfected COS‐7 cells using human 125I‐GIP(3–30)NH2. The selectivity of human GIP(3‐30)NH2 was examined by testing for agonistic and antagonistic properties on 62 human GPCRs. Human GIP(3‐30)NH2 inhibited GIP(1‐42)‐induced cAMP and &bgr;‐arrestin 1 and 2 recruitment on the human GIPR and Schild plot analysis showed competitive antagonism with a pA2 and Hill slope of 16.8 nM and 1.11 ± 0.02 in cAMP, 10.6 nM and 1.15 ± 0.05 in &bgr;‐arrestin 1 recruitment, and 10.2 nM and 1.06 ± 0.05 in &bgr;‐arrestin 2 recruitment. Efficient internalization of the GIPR was dependent on the presence of either &bgr;‐arrestin 1 or 2. Moreover, GIP(3‐30)NH2 inhibited GIP(1‐42)‐induced internalization in a concentration‐dependent manner and notably also inhibited GIP‐mediated signaling in human subcutaneous adipocytes. Finally, the antagonist was established as GIPR selective among 62 human GPCRs being species‐specific with high affinity binding to the human and non‐human primate (Macaca fascicularis) GIPRs, and low affinity binding to the rat and mouse GIPRs (Kd values of 2.0, 2.5, 31.6 and 100 nM, respectively). In conclusion, human GIP(3–30)NH2 is a selective and species‐specific GIPR antagonist with broad inhibition of signaling and internalization in transfected cells as well as in human adipocytes.

The Combination of Fosfomycin, Metronidazole, and Recombinant Human Granulocyte-Macrophage Colony-Stimulating Factor is Stable in vitro and Has Maintained Antibacterial Activity

BACKGROUND Treatment of secondary peritonitis includes surgery and antimicrobial agents. Antimicrobial agents are often administered intravenously, however, the alternative route intraperitoneal administration could be considered. Investigations must be conducted prior to clinical application. Therefore, we aimed to investigate the combination of fosfomycin, metronidazole, and recombinant human granulocyte-macrophage colony-stimulating factor with regard to its chemical properties and the solutions stability. In addition, the antibacterial effect of the mixed drug solution was compared with the effect of the individual antibacterial agents. METHODS The drugs were mixed to an aqueous solution. Basic chemical investigations of pH, precipitation, and calculated osmolarity of the drug combination were conducted. Fosfomycin and metronidazoles chemical stability was investigated using High Pressure Liquid Chromatography-Mass Spectrometry. Microbiological investigations using the agar cup method were carried out to measure the antibacterial effect of fosfomycin and metronidazole. RESULTS The aqueous solution of the combination of the three drugs had a pH of 7.46-7.62, which was stable during 24 h, was without precipitation, and had a calculated osmolarity of 293 mOsm/l. High Pressure Liquid Chromatography-Mass Spectrometry found stable concentrations of fosfomycin and metronidazole both alone and in combination during 24 h. The antibacterial effect of the drug combination solution was similar to the antibacterial effects of fosfomycin and metronidazole alone. CONCLUSION The drug combination had neutral and stable pH, was iso-osmotic, and had stable concentrations during 24 h of storage. The antibacterial effect of fosfomycin and metronidazole were not altered when the drugs were mixed.