Non-invasive quantification of stem cell-derived islet graft size and composition.

AIMS/HYPOTHESIS: Stem cell-derived islets (SC-islets) are being used as cell replacement therapy for insulin-dependent diabetes. Non-invasive long-term monitoring methods for SC-islet grafts, which are needed to detect misguided differentiation in vivo and to optimise their therapeutic effectiveness, are lacking. Positron emission tomography (PET) has been used to monitor transplanted primary islets. We therefore aimed to apply PET as a non-invasive monitoring method for SC-islet grafts. METHODS: We implanted different doses of human SC-islets, SC-islets derived using an older protocol or a state-of-the-art protocol and SC-islets genetically rendered hyper- or hypoactive into mouse calf muscle to yield different kinds of grafts. We followed the grafts with PET using two tracers, glucagon-like peptide 1 receptor-binding [18F]F-dibenzocyclooctyne-exendin-4 ([18F]exendin) and the dopamine precursor 6-[18F]fluoro-L-3,4-dihydroxyphenylalanine ([18F]FDOPA), for 5 months, followed by histological assessment of graft size and composition. Additionally, we implanted a kidney subcapsular cohort with different SC-islet doses to assess the connection between C-peptide and stem cell-derived beta cell (SC-beta cell) mass. RESULTS: Small but pure and large but impure grafts were derived from SC-islets. PET imaging allowed detection of SC-islet grafts even <1 mm3 in size, [18F]exendin having a better detection rate than [18F]FDOPA (69% vs 44%, <1 mm3; 96% vs 85%, >1 mm3). Graft volume quantified with [18F]exendin (r2=0.91) and [18F]FDOPA (r2=0.86) strongly correlated with actual graft volume. [18F]exendin PET delineated large cystic structures and its uptake correlated with graft SC-beta cell proportion (r2=0.68). The performance of neither tracer was affected by SC-islet graft hyper- or hypoactivity. C-peptide measurements under fasted or glucose-stimulated conditions did not correlate with SC-islet graft volume or SC-beta cell mass, with C-peptide under hypoglycaemia having a weak correlation with SC-beta cell mass (r2=0.52). CONCLUSIONS/INTERPRETATION: [18F]exendin and [18F]FDOPA PET enable non-invasive assessment of SC-islet graft size and aspects of graft composition. These methods could be leveraged for optimising SC-islet cell replacement therapy in diabetes.

Metformin increases the uptake of glucose into the gut from the circulation in high-fat diet-fed male mice, which is enhanced by a reduction in whole-body Slc2a2 expression.

OBJECTIVES: Metformin is the first line therapy recommended for type 2 diabetes. However, the precise mechanism of action remains unclear and up to a quarter of patients show some degree of intolerance to the drug, with a similar number showing poor response to treatment, limiting its effectiveness. A better understanding of the mechanism of action of metformin may improve its clinical use. SLC2A2 (GLUT2) is a transmembrane facilitated glucose transporter, with important roles in the liver, gut and pancreas. Our group previously identified single nucleotide polymorphisms in the human SLC2A2 gene, which were associated with reduced transporter expression and an improved response to metformin treatment. The aims of this study were to model Slc2a2 deficiency and measure the impact on glucose homoeostasis and metformin response in mice. METHODS: We performed extensive metabolic phenotyping and 2-deoxy-2-[18F]fluoro-d-glucose ([18F]FDG)-positron emission tomography (PET) analysis of gut glucose uptake in high-fat diet-fed (HFD) mice with whole-body reduced Slc2a2 (Slc2a2+/-) and intestinal Slc2a2 KO, to assess the impact of metformin treatment. RESULTS: Slc2a2 partial deficiency had no major impact on body weight and insulin sensitivity, however mice with whole-body reduced Slc2a2 expression (Slc2a2+/-) developed an age-related decline in glucose homoeostasis (as measured by glucose tolerance test) compared to wild-type (Slc2a2+/+) littermates. Glucose uptake into the gut from the circulation was enhanced by metformin exposure in Slc2a2+/+ animals fed HFD and this action of the drug was significantly higher in Slc2a2+/- animals. However, there was no effect of specifically knocking-out Slc2a2 in the mouse intestinal epithelial cells. CONCLUSIONS: Overall, this work identifies a differential metformin response, dependent on expression of the SLC2A2 glucose transporter, and also adds to the growing evidence that metformin efficacy includes modifying glucose transport in the gut. We also describe a novel and important role for this transporter in maintaining efficient glucose homoeostasis during ageing.

Metformin treatment significantly enhances intestinal glucose uptake in patients with type 2 diabetes: Results from a randomized clinical trial.

AIMS: Metformin therapy is associated with diffuse intestinal 18F-fluoro-deoxyglucose (FDG) accumulation in clinical diagnostics using routine FDG-PET imaging. We aimed to study whether metformin induced glucose uptake in intestine is associated with the improved glycaemic control in patients with type 2 diabetes. Therefore, we compared the effects of metformin and rosiglitazone on intestinal glucose metabolism in patients with type 2 diabetes in a randomized placebo controlled clinical trial, and further, to understand the underlying mechanism, evaluated the effect of metformin in rats. METHODS: Forty-one patients with newly diagnosed type 2 diabetes were randomized to metformin (1g, b.i.d), rosiglitazone (4mg, b.i.d), or placebo in a 26-week double-blind trial. Tissue specific intestinal glucose uptake was measured before and after the treatment period using FDG-PET during euglycemic hyperinsulinemia. In addition, rats were treated with metformin or vehicle for 12weeks, and intestinal FDG uptake was measured in vivo and with autoradiography. RESULTS: Glucose uptake increased 2-fold in the small intestine and 3-fold in the colon for the metformin group and associated with improved glycemic control. Rosiglitazone increased only slightly intestinal glucose uptake. In rodents, metformin treatment enhanced intestinal FDG retention (P=0.002), which was localized in the mucosal enterocytes of the small intestine. CONCLUSIONS: Metformin treatment significantly enhances intestinal glucose uptake from the circulation of patients with type 2 diabetes. This intestine-specific effect is associated with improved glycemic control and localized to mucosal layer. These human findings demonstrate directs effect of metformin on intestinal metabolism and elucidate the actions of metformin. Clinical trial number NCT02526615.

Low kidney uptake of GLP-1R-targeting, beta cell-specific PET tracer, 18F-labeled [Nle14,Lys40]exendin-4 analog, shows promise for clinical imaging.

BACKGROUND: Several radiometal-labeled, exendin-based tracers that target glucagon-like peptide-1 receptors (GLP-1R) have been intensively explored for ß cell imaging. The main obstacle has been the high uptake of tracer in the kidneys. This study aimed to develop a novel GLP1-R-specific tracer, with fluorine-18 attached to exendin-4, to label ß cells for clinical imaging with PET (positron emission tomography). We hypothesized that this tracer would undergo reduced kidney uptake. 18F-labeled [Nle14,Lys40]exendin-4 analog ([18F]exendin-4) was produced via Cu-catalyzed click chemistry. The biodistribution of [18F]exendin-4 was assessed with ex vivo organ γ-counting and in vivo PET imaging. We also tested the in vivo stability of the radiotracer. The localization of 18F radioactivity in rat and human pancreatic tissue sections was investigated with autoradiography. Receptor specificity was assessed with unlabeled exendin-3. Islet labeling was confirmed with immunohistochemistry. The doses of radiation in humans were estimated based on biodistribution results in rats. RESULTS: [18F]exendin-4 was synthesized with high yield and high specific activity. Results showed specific, sustained [18F]exendin-4 uptake in pancreatic islets. In contrast to previous studies that tested radiometal-labeled exendin-based tracers, we observed rapid renal clearance of [18F]exendin-4. CONCLUSIONS: [18F]exendin-4 showed promise as a tracer for clinical imaging of pancreatic ß cells, due to its high specific uptake in native ß cells and its concomitant low kidney radioactivity uptake.

The Cannabinoid Receptor-1 Is an Imaging Biomarker of Brown Adipose Tissue.

UNLABELLED: Recently, the existence of significant deposits of brown adipose tissue (BAT) in human adults was confirmed. Its role in the human metabolism is unknown but could be substantial. Inhibition of the cannabinoid receptor-1 (CB1) by the antagonist rimonabant (SR141716) has been associated with activation of BAT thermogenesis and weight loss in mice and rats. The role of peripheral and central CB1 in the activation of BAT merits further investigation. Here we developed a technique for quantifying CB1 in BAT by PET. METHODS: Sections of rat BAT and subcutaneous white adipose tissue (WAT) were stained for CB1 and uncoupling protein-1 by immunofluorescent staining. Binding of the radiolabeled CB1 antagonist (3R,5R)-5-(3-(18F-fluoromethoxy)phenyl)-3-(((R)-1-phenylethyl)amino)-1-(4-(trifluoromethyl)-phenyl)pyrrolidin-2-one ((18)F-FMPEP-d2) to BAT in vivo and in vitro was assessed in rats by PET. RESULTS: We found that CB1 was colocalized with uncoupling protein-1 in BAT, but neither protein was found in WAT. Binding of the radiotracer to BAT sections (but not WAT) in vitro was high and displaceable by pretreatment with rimonabant. Deposits of BAT in rats had significant binding of (18)F-FMPEP-d2 in vivo, indicating high CB1 density. WAT deposits were negative for (18)F-FMPEP-d2, consistent with the immunofluorescent staining and in vitro results. CONCLUSION: (18)F-FMPEP-d2 PET can quantify CB1 density noninvasively in vivo in rats. CB1 is therefore a promising surrogate imaging biomarker for assessing the presence of BAT deposits as well as for elucidating the mechanism of CB1 antagonist-mediated weight loss.

Pancreatic metabolism, blood flow, and ß-cell function in obese humans.

CONTEXT: Glucolipotoxicity is believed to induce pancreatic ß-cell dysfunction in obesity. Previously, it has not been possible to study pancreatic metabolism and blood flow in humans. OBJECTIVE: The objective of the study was to investigate whether pancreatic metabolism and blood flow are altered in obesity using positron emission tomography (PET). In the preclinical part, the method was validated in animals. DESIGN: This was a cross-sectional study. SETTING: The study was conducted in a clinical research center. PARTICIPANTS: Human studies consisted of 52 morbidly obese and 25 healthy age-matched control subjects. Validation experiments were done with rodents and pigs. INTERVENTIONS: PET and magnetic resonance imaging studies using a glucose analog ([(18)F]fluoro-2-deoxy-d-glucose), a palmitate analog [14(R,S)-[(18)F]fluoro-6-thia-heptadecanoic acid], and radiowater ([(15)O]H2O) were performed. In animals, a comparison between ex vivo and in vivo data was performed. MAIN OUTCOME MEASURES: Pancreatic glucose/fatty acid (FA) uptake, fat accumulation, and blood flow parameters of ß-cell function were measured. RESULTS: PET proved to be a feasible method to measure pancreatic metabolism. Compared with healthy participants, obese participants had elevated pancreatic FA uptake (P < .0001), more fat accumulation (P = .0001), lowered glucose uptake both during fasting and euglycemic hyperinsulinemia, and blunted blood flow (P < .01) in the pancreas. Blood flow, FA uptake, and fat accumulation were negatively associated with multiple markers of ß-cell function. CONCLUSIONS: Obesity leads to changes in pancreatic energy metabolism with a substrate shift from glucose to FAs. In morbidly obese humans, impaired pancreatic blood flow may contribute to ß-cell dysfunction and in the pathogenesis of type 2 diabetes.

64Cu- and 68Ga-labelled [Nle(14),Lys(40)(Ahx-NODAGA)NH2]-exendin-4 for pancreatic beta cell imaging in rats.

PURPOSE: Glucagon-like peptide-1 receptor (GLP-1R) is a molecular target for imaging of pancreatic beta cells. We compared the ability of [Nle(14),Lys(40)(Ahx-NODAGA-(64)Cu)NH2]-exendin-4 ([(64)Cu]NODAGA-exendin-4) and [Nle(14),Lys(40)(Ahx-NODAGA-(68)Ga)NH2]-exendin-4 ([(68)Ga]NODAGA-exendin-4) to detect native pancreatic islets in rodents. PROCEDURES: The stability, lipophilicity and affinity of the radiotracers to the GLP-1R were determined in vitro. The biodistribution of the tracers was assessed using autoradiography, ex vivo biodistribution and PET imaging. Estimates for human radiation dosimetry were calculated. RESULTS: We found GLP-1R-specific labelling of pancreatic islets. However, the pancreas could not be visualised in PET images. The highest uptake of the tracers was observed in the kidneys. Effective dose estimates for [(64)Cu]NODAGA-exendin-4 and [(68)Ga]NODAGA-exendin-4 were 0.144 and 0.012 mSv/MBq, respectively. CONCLUSION: [(64)Cu]NODAGA-exendin-4 might be more effective for labelling islets than [(68)Ga]NODAGA-exendin-4. This is probably due to the lower specific radioactivity of [(68)Ga]NODAGA-exendin-4 compared to [(64)Cu]NODAGA-exendin-4. The radiation dose in the kidneys may limit the use of [(64)Cu]NODAGA-exendin-4 as a clinical tracer.

Synthesis and preclinical characterization of [64Cu]NODAGA-MAL-exendin-4 with a Nε-maleoyl-L-lysyl-glycine linkage.

INTRODUCTION: Renal localization of high radioactivity levels during targeted imaging compromises tissue visualization in the kidney region and limits diagnostic accuracy. Radioiodinated antibody fragments with a renal enzyme-cleavable N(ε)-maleoyl-L-lysyl-glycine (MAL) linkage demonstrated low renal radioactivity levels in mice, from early postinjection times. This study tested the hypothesis whether a (64)Cu-labeled NODAGA-exendin-4 peptide with a MAL linkage ([(64)Cu]NODAGA-MAL-exendin-4) could decrease kidney radioactivity levels in rats, compared to a [(64)Cu]NODAGA-exendin-4 reference, without impairing the radioactivity levels in the target tissue. METHODS: NODAGA-MAL-exendin-4 was synthesized in a two-phase approach using solid support to prepare maleoyl-derivatized NODAGA followed by Michael addition to cysteine-derivatized exendin-4 in solution. Radiolabeling was performed in buffered aqua with [(64)Cu]CuCl2, which was produced via the (64)Ni(p,n)(64)Cu nuclear reaction. The in vitro and in vivo stability, lipophilicity, and distribution kinetics in major rat organs for [(64)Cu]NODAGA-MAL-exendin-4 were studied and compared to [(64)Cu]NODAGA-exendin-4. Labeling of pancreatic islets was assessed using autoradiography. RESULTS: NODAGA-MAL-exendin-4 was synthesized, with an overall yield of 9%, and radiolabeled with (64)Cu with high specific radioactivity. Serum incubation studies showed high stability for [(64)Cu]NODAGA-MAL-exendin-4. Similar tissue distribution kinetics was observed for [(64)Cu]NODAGA-MAL-exendin-4 and [(64)Cu]NODAGA-exendin-4, with high kidney radioactivity levels. CONCLUSIONS: The incorporated MAL linkage in [(64)Cu]NODAGA-MAL-exendin-4 was unable to reduce kidney radioactivity levels, compared to [(64)Cu]NODAGA-exendin-4. The applicability of metabolizable linkages in the design of kidney-saving exendin-4 analogs requires further investigation.

Assessment of islet specificity of dihydrotetrabenazine radiotracer binding in rat pancreas and human pancreas.

UNLABELLED: Vesicular monoamine transporter 2 (VMAT2) is a putative molecular target for the quantitative imaging of pancreatic beta-cell mass by PET. The VMAT2 PET tracer (11)C-dihydrotetrabenazine ((11)C-DTBZ) exhibits high pancreatic uptake that is reduced in type 1 diabetes. The aim of this study was to assess the islet and VMAT2 specificity of DTBZ binding in the pancreas. METHODS: The biodistribution of (11)C-DTBZ in rats was determined 10 and 60 min after injection. The localization of DTBZ radioactivity in rat and human pancreatic tissue sections was investigated by autoradiography. Saturation and competition binding assays were performed with (3)H-DTBZ and sections of rat pancreatic and control tissues. The binding of (11)C-DTBZ in pancreatic sections from rats with streptozotocin-induced diabetes was compared with that in control rats. RESULTS: The values for the pancreatic uptake of (11)C-DTBZ (percentage injected dose per gram of tissue) were 3.0 at 10 min and 2.7 at 60 min. At 10 min, pancreatic radioactivity was heterogeneously distributed, with higher levels toward the head of the pancreas (head-to-tail ratio, 1.7). No such gradient was observed in pancreatic sections incubated with (11)C-DTBZ and (3)H-DTBZ in vitro. In rats, (11)C-DTBZ and (3)H-DTBZ binding in pancreatic islets did not exceed binding in the exocrine pancreas. Saturable (3)H-DTBZ binding was observed in the rat brain striatum (dissociation constant [K(d)], 1.3 nM) and the bovine adrenal medulla (K(d), 3.3 nM), whereas in the rat pancreas, (3)H-DTBZ binding was nonsaturable. Competition binding with (3)H-DTBZ and VMAT2 antagonists also indicated that DTBZ binding in the rat pancreas was nonspecific and did not represent binding to VMAT2. Nonspecific pancreatic (11)C-DTBZ binding was lower in rats with streptozotocin-induced diabetes than in control rats. In sections of human pancreas, a subset of pancreatic islets were weakly but VMAT2-specifically labeled with (3)H-DTBZ. CONCLUSION: The results showed that the pancreatic uptake of (11)C-DTBZ is mainly due to nonspecific binding in the exocrine pancreas and suggested that the reduction in pancreatic (11)C-DTBZ binding observed in type 1 diabetes is not specific for the loss of beta-cell mass.