The dual GCGR/GLP-1R agonist survodutide: Biomarkers and pharmacological profiling for clinical candidate selection.

AIM: To describe the biomarker strategy that was applied to select survodutide (BI 456906), BI 456908 and BI 456897 from 19 dual glucagon receptor (GCGR)/ glucagon-like peptide-1 receptor (GLP-1R) agonists for in-depth pharmacological profiling, which led to the qualification of survodutide as the clinical development candidate. MATERIALS AND METHODS: Potencies to increase cyclic adenosine monophosphate (cAMP) were determined in Chinese hamster ovary (CHO)-K1 cells stably expressing human GCGR and GLP-1R. Agonism for endogenously expressed receptors was investigated in insulinoma cells (MIN6) for mouse GLP-1R, and in rat primary hepatocytes for the GCGR. In vivo potencies to engage the GLP-1R or GCGR were determined, measuring improvement in oral glucose tolerance (30 nmol/kg) and increase in plasma fibroblast growth factor-21 (FGF21) and liver nicotinamide N-methyltransferase (NNMT) mRNA expression (100 nmol/kg), respectively. Body weight- and glucose-lowering efficacies were investigated in diet-induced obese (DIO) mice and diabetic db/db mice, respectively. RESULTS: Upon acute dosing in lean mice, target engagement biomarkers for the GCGR and GLP-1R demonstrated a significant correlation (Spearman correlation coefficient with p < 0.05) to the in vitro GCGR and GLP-1R potencies for the 19 dual agonists investigated. Survodutide, BI 456908 and BI 456897 were selected for in-depth pharmacological profiling based on the significant improvement in acute oral glucose tolerance achieved (area under the curve [AUC] of 54%, 57% and 60% vs. vehicle) that was comparable to semaglutide (AUC of 45% vs. vehicle), while showing different degrees of in vivo GCGR engagement, as determined by hepatic NNMT mRNA expression (increased by 15- to 17-fold vs. vehicle) and plasma FGF21 concentrations (increased by up to sevenfold vs. vehicle). In DIO mice, survodutide (30 nmol/kg/once daily), BI 456908 (30 nmol/kg/once daily) and BI 456897 (10 nmol/kg/once daily) achieved a body weight-lowering efficacy from baseline of 25%, 27% and 26%, respectively. In db/db mice, survodutide and BI 456908 (10 and 20 nmol/kg/once daily) significantly lowered glycated haemoglobin (0.4%-0.6%); no significant effect was observed for BI 456897 (3 and 7 nmol/kg/once daily). CONCLUSIONS: Survodutide was selected as the clinical candidate based on its balanced dual GCGR/GLP-1R pharmacology, engaging the GCGR for robust body weight-lowering efficacy exceeding that of selective GLP-1R agonists, while achieving antidiabetic efficacy that was comparable to selective GLP-1R agonism. Survodutide is currently being investigated in Phase 3 clinical trials in people living with obesity.

Perspectives in weight control in diabetes - Survodutide.

Glucagon-like peptide-1 receptor (GLP-1R) agonists are approved treatments for Type 2 diabetes mellitus, with liraglutide and semaglutide also approved for the treatment of obesity. The natural gut hormone oxyntomodulin is a weak dual agonist of the glucagon receptor (GCGR) and GLP-1R. Development of poly-agonists mimicking oxyntomodulin, such as the novel dual GCGR/GLP-1R agonist survodutide, represents an important step towards a more effective treatment for people with Type 2 diabetes mellitus and obesity. Survodutide is a 29-amino acid peptide derived from glucagon, with the incorporation of potent GLP-1 activities. It contains a C18 diacid which mediates binding to albumin, thereby prolonging the half-life to enable once-weekly subcutaneous dosing. The utilisation of GCGR agonism aims to enhance body weight-lowering effects by increasing energy expenditure in addition to the anorectic action of GLP-1R agonists. Glucose-lowering efficacy of survodutide has been demonstrated in a Phase II trial in patients with Type 2 diabetes mellitus and obesity and was associated with clinically meaningful body weight loss. These data highlight the potential of dual GCGR/GLP-1R agonism for reducing glycated haemoglobin and body weight in patients with Type 2 diabetes mellitus, and for greater therapeutic efficacy compared with GLP-1R agonism alone.

BI 456906: Discovery and preclinical pharmacology of a novel GCGR/GLP-1R dual agonist with robust anti-obesity efficacy.

OBJECTIVE: Obesity and its associated comorbidities represent a global health challenge with a need for well-tolerated, effective, and mechanistically diverse pharmaceutical interventions. Oxyntomodulin is a gut peptide that activates the glucagon receptor (GCGR) and glucagon-like peptide-1 receptor (GLP-1R) and reduces bodyweight by increasing energy expenditure and reducing energy intake in humans. Here we describe the pharmacological profile of the novel glucagon receptor (GCGR)/GLP-1 receptor (GLP-1R) dual agonist BI 456906. METHODS: BI 456906 was characterized using cell-based in vitro assays to determine functional agonism. In vivo pharmacological studies were performed using acute and subchronic dosing regimens to demonstrate target engagement for the GCGR and GLP-1R, and weight lowering efficacy. RESULTS: BI 456906 is a potent, acylated peptide containing a C18 fatty acid as a half-life extending principle to support once-weekly dosing in humans. Pharmacological doses of BI 456906 provided greater bodyweight reductions in mice compared with maximally effective doses of the GLP-1R agonist semaglutide. BI 456906's superior efficacy is the consequence of increased energy expenditure and reduced food intake. Engagement of both receptors in vivo was demonstrated via glucose tolerance, food intake, and gastric emptying tests for the GLP-1R, and liver nicotinamide N-methyltransferase mRNA expression and circulating biomarkers (amino acids, fibroblast growth factor-21) for the GCGR. The dual activity of BI 456906 at the GLP-1R and GCGR was supported using GLP-1R knockout and transgenic reporter mice, and an ex vivo bioactivity assay. CONCLUSIONS: BI 456906 is a potent GCGR/GLP-1R dual agonist with robust anti-obesity efficacy achieved by increasing energy expenditure and decreasing food intake.

Challenges in tackling energy expenditure as obesity therapy: From preclinical models to clinical application.

BACKGROUND: A chronic imbalance of energy intake and energy expenditure results in excess fat storage. The obesity often caused by this overweight is detrimental to the health of millions of people. Understanding both sides of the energy balance equation and their counter-regulatory mechanisms is critical to the development of effective therapies to treat this epidemic. SCOPE OF REVIEW: Behaviors surrounding ingestion have been reviewed extensively. This review focuses more specifically on energy expenditure regarding bodyweight control, with a particular emphasis on the organs and attractive metabolic processes known to reduce bodyweight. Moreover, previous and current attempts at anti-obesity strategies focusing on energy expenditure are highlighted. Precise measurements of energy expenditure, which consist of cellular, animal, and human models, as well as measurements of their translatability, are required to provide the most effective therapies. MAJOR CONCLUSIONS: A precise understanding of the components surrounding energy expenditure, including tailored approaches based on genetic, biomarker, or physical characteristics, must be integrated into future anti-obesity treatments. Further comprehensive investigations are required to define suitable treatments, especially because the complex nature of the human perspective remains poorly understood.

The glucose-dependent insulinotropic polypeptide (GIP) regulates body weight and food intake via CNS-GIPR signaling.

Uncertainty exists as to whether the glucose-dependent insulinotropic polypeptide receptor (GIPR) should be activated or inhibited for the treatment of obesity. Gipr was recently demonstrated in hypothalamic feeding centers, but the physiological relevance of CNS Gipr remains unknown. Here we show that HFD-fed CNS-Gipr KO mice and humanized (h)GIPR knockin mice with CNS-hGIPR deletion show decreased body weight and improved glucose metabolism. In DIO mice, acute central and peripheral administration of acyl-GIP increases cFos neuronal activity in hypothalamic feeding centers, and this coincides with decreased body weight and food intake and improved glucose handling. Chronic central and peripheral administration of acyl-GIP lowers body weight and food intake in wild-type mice, but shows blunted/absent efficacy in CNS-Gipr KO mice. Also, the superior metabolic effect of GLP-1/GIP co-agonism relative to GLP-1 is extinguished in CNS-Gipr KO mice. Our data hence establish a key role of CNS Gipr for control of energy metabolism.

SGLT2 is not expressed in pancreatic α- and ß-cells, and its inhibition does not directly affect glucagon and insulin secretion in rodents and humans.

OBJECTIVE: Sodium-glucose cotransporter 2 (SGLT2) inhibitors (SGLT2i), or gliflozins, are anti-diabetic drugs that lower glycemia by promoting glucosuria, but they also stimulate endogenous glucose and ketone body production. The likely causes of these metabolic responses are increased blood glucagon levels, and decreased blood insulin levels, but the mechanisms involved are hotly debated. This study verified whether or not SGLT2i affect glucagon and insulin secretion by a direct action on islet cells in three species, using multiple approaches. METHODS: We tested the in vivo effects of two selective SGLT2i (dapagliflozin, empagliflozin) and a SGLT1/2i (sotagliflozin) on various biological parameters (glucosuria, glycemia, glucagonemia, insulinemia) in mice. mRNA expression of SGLT2 and other glucose transporters was assessed in rat, mouse, and human FACS-purified α- and ß-cells, and by analysis of two human islet cell transcriptomic datasets. Immunodetection of SGLT2 in pancreatic tissues was performed with a validated antibody. The effects of dapagliflozin, empagliflozin, and sotagliflozin on glucagon and insulin secretion were assessed using isolated rat, mouse and human islets and the in situ perfused mouse pancreas. Finally, we tested the long-term effect of SGLT2i on glucagon gene expression. RESULTS: SGLT2 inhibition in mice increased the plasma glucagon/insulin ratio in the fasted state, an effect correlated with a decline in glycemia. Gene expression analyses and immunodetections showed no SGLT2 mRNA or protein expression in rodent and human islet cells, but moderate SGLT1 mRNA expression in human α-cells. However, functional experiments on rat, mouse, and human (29 donors) islets and the in situ perfused mouse pancreas did not identify any direct effect of dapagliflozin, empagliflozin or sotagliflozin on glucagon and insulin secretion. SGLT2i did not affect glucagon gene expression in rat and human islets. CONCLUSIONS: The data indicate that the SGLT2i-induced increase of the plasma glucagon/insulin ratio in vivo does not result from a direct action of the gliflozins on islet cells.

Glucose-dependent insulinotropic polypeptide (GIP) and cardiovascular disease.

Accumulating evidence suggests that glucose-dependent insulinotropic polypeptide (GIP) in addition to its involvement in type 2 diabetic pathophysiology may be involved in the development of obesity and the pathogenesis of cardiovascular disease. In this review, we outline recent preclinical and clinical cardiovascular-related discoveries about GIP. These include chronotropic and blood pressure-lowering effects of GIP. Furthermore, GIP has been suggested to control vasodilation via secretion of nitric oxide, and vascular leukocyte adhesion and inflammation via expression and secretion of endothelin 1. Also, GIP seems to regulate circulating lipids via effects on adipose tissue uptake and metabolism of lipids. Lastly, we discuss how dysmetabolic conditions such as obesity and type 2 diabetes may shift the actions of GIP in an atherogenic direction, and we provide a perspective on the therapeutic potential of GIP receptor agonism and antagonism in cardiovascular diseases. We conclude that GIP actions may have implications for the development of cardiovascular disease, but also that the potential of GIP-based drugs for the treatment of cardiovascular disease currently is uncertain.

The role of GIP and pancreatic GLP-1 in the glucoregulatory effect of DPP-4 inhibition in mice.

AIMS/HYPOTHESIS: Glucagon-like peptide-1 (GLP-1) and glucose-dependent insulinotropic polypeptide (GIP) are two peptides that function to promote insulin secretion. Dipeptidyl peptidase-4 (DPP-4) inhibitors increase the bioavailability of both GLP-1 and GIP but the dogma continues to be that it is the increase in GLP-1 that contributes to the improved glucose homeostasis. We have previously demonstrated that pancreatic rather than intestinal GLP-1 is necessary for improvements in glucose homeostasis in mice. Therefore, we hypothesise that a combination of pancreatic GLP-1 and GIP is necessary for the full effect of DPP-4 inhibitors on glucose homeostasis. METHODS: We have genetically engineered mouse lines in which the preproglucagon gene (Gcg) is absent in the entire body (GcgRAΔNull) or is expressed exclusively in the intestine (GcgRAΔVilCre) or pancreas and duodenum (GcgRAΔPDX1Cre). These mice were used to examine oral glucose tolerance and GLP-1 and GIP responses to a DPP-4 inhibitor alone, or in combination with incretin receptor antagonists. RESULTS: Administration of the DPP-4 inhibitor, linagliptin, improved glucose tolerance in GcgRAΔNull mice and control littermates and in GcgRAΔVilCre and GcgRAΔPDX1Cre mice. The potent GLP-1 receptor antagonist, exendin-[9-39] (Ex9), blunted improvements in glucose tolerance in linagliptin-treated control mice and in GcgRAΔPDX1Cre mice. Ex9 had no effect on glucose tolerance in linagliptin-treated GcgRAΔNull or in GcgRAΔVilCre mice. In addition to GLP-1, linagliptin also increased postprandial plasma levels of GIP to a similar degree in all genotypes. When linagliptin was co-administered with a GIP-antagonising antibody, the impact of linagliptin was partially blunted in wild-type mice and was fully blocked in GcgRAΔNull mice. CONCLUSIONS/INTERPRETATION: Taken together, these data suggest that increases in pancreatic GLP-1 and GIP are necessary for the full effect of DPP-4 inhibitors on glucose tolerance.

The GLP-1 Analogs Liraglutide and Semaglutide Reduce Atherosclerosis in ApoE-/- and LDLr-/- Mice by a Mechanism That Includes Inflammatory Pathways.

The glucagon-like peptide-1 receptor agonists (GLP-1RAs) liraglutide and semaglutide reduce cardiovascular risk in type 2 diabetes patients. The mode of action is suggested to occur through modified atherosclerotic progression. In this study, both of the compounds significantly attenuated plaque lesion development in apolipoprotein E-deficient (ApoE-/-) mice and low-density lipoprotein receptor-deficient (LDLr-/-) mice. This attenuation was partly independent of weight and cholesterol lowering. In aortic tissue, exposure to a Western diet alters expression of genes in pathways relevant to the pathogenesis of atherosclerosis, including leukocyte recruitment, leukocyte rolling, adhesion/extravasation, cholesterol metabolism, lipid-mediated signaling, extracellular matrix protein turnover, and plaque hemorrhage. Treatment with semaglutide significantly reversed these changes. These data suggest GLP-1RAs affect atherosclerosis through an anti-inflammatory mechanism.

Generation of a highly diverse panel of antagonistic chicken monoclonal antibodies against the GIP receptor.

Raising functional antibodies against G protein-coupled receptors (GPCRs) is challenging due to their low density expression, instability in the absence of the cell membrane's lipid bilayer and frequently short extracellular domains that can serve as antigens. In addition, a particular therapeutic concept may require an antibody to not just bind the receptor, but also act as a functional receptor agonist or antagonist. Antagonizing the glucose-dependent insulinotropic polypeptide (GIP) receptor may open up new therapeutic modalities in the treatment of diabetes and obesity. As such, a panel of monoclonal antagonistic antibodies would be a useful tool for in vitro and in vivo proof of concept studies. The receptor is highly conserved between rodents and humans, which has contributed to previous mouse and rat immunization campaigns generating very few usable antibodies. Switching the immunization host to chicken, which is phylogenetically distant from mammals, enabled the generation of a large and diverse panel of monoclonal antibodies containing 172 unique sequences. Three-quarters of all chicken-derived antibodies were functional antagonists, exhibited high-affinities to the receptor extracellular domain and sampled a broad epitope repertoire. For difficult targets, including GPCRs such as GIPR, chickens are emerging as valuable immunization hosts for therapeutic antibody discovery.

TUSC5 regulates insulin-mediated adipose tissue glucose uptake by modulation of GLUT4 recycling.

OBJECTIVE: Failure to properly dispose of glucose in response to insulin is a serious health problem, occurring during obesity and is associated with type 2 diabetes development. Insulin-stimulated glucose uptake is facilitated by the translocation and plasma membrane fusion of vesicles containing glucose transporter 4 (GLUT4), the rate-limiting step of post-prandial glucose disposal. METHODS: We analyzed the role of Tusc5 in the regulation of insulin-stimulated Glut4-mediated glucose uptake in vitro and in vivo. Furthermore, we measured Tusc5 expression in two patient cohorts. RESULTS: Herein, we report that TUSC5 controls insulin-stimulated glucose uptake in adipocytes, in vitro and in vivo. TUSC5 facilitates the proper recycling of GLUT4 and other key trafficking proteins during prolonged insulin stimulation, thereby enabling proper protein localization and complete vesicle formation, processes that ultimately enable insulin-stimulated glucose uptake. Tusc5 knockout mice exhibit impaired glucose disposal and TUSC5 expression is predictive of glucose tolerance in obese individuals, independent of body weight. Furthermore, we show that TUSC5 is a PPARγ target and in its absence the anti-diabetic effects of TZDs are significantly blunted. CONCLUSIONS: Collectively, these findings establish TUSC5 as an adipose tissue-specific protein that enables proper protein recycling, linking the ubiquitous vesicle traffic machinery with tissue-specific insulin-mediated glucose uptake into adipose tissue and the maintenance of a healthy metabolic phenotype in mice and humans.

The apical sorting signal for human GLUT9b resides in the N-terminus.

The two splice variants of human glucose transporter 9 (hGLUT9) are targeted to different polarized membranes. hGLUT9a traffics to the basolateral membrane, whereas hGLUT9b traffics to the apical region. This study examines the sorting mechanism of these variants, which differ only in their N-terminal domain. Mutating a di-leucine motif unique to GLUT9a did not affect targeting. Chimeric proteins were made using GLUT1, a basolaterally targeted transporter, and GLUT3, an apically targeted protein whose signal lies in the C-terminus. Overexpression of the chimeric proteins in polarized cells demonstrates that the N-terminus of hGLUT9b contains a signal capable of redirecting GLUT1 to the apical membrane. The N-terminus of hGLUT9a, however, does not contain a basolateral signal sufficient enough to redirect GLUT3. Portions of the GLUT9a N-terminus were substituted with corresponding portions of the GLUT9b N-terminus to determine the motif responsible for apical targeting. The first 16 amino acids were not found to be a sufficient apical signal. The last ten amino acids of the N-termini differ only in amino-acid class at one location. In the B-form, leucine, a hydrophobic residue, is substituted for lysine, a basic residue, found in the A-form. However, mutation of the leucine in hGLUT9b to a lysine resulted in retention of the apical signal. We therefore believe the apical signal exists as an interplay between the final ten amino acids of the N-terminus and another motif within the protein such as the intracellular loop or other motifs within the N-terminus.

GTPase ARFRP1 is essential for normal hepatic glycogen storage and insulin-like growth factor 1 secretion.

The GTPase ADP-ribosylation factor-related protein 1 (ARFRP1) is located at the trans-Golgi compartment and regulates the recruitment of Arf-like 1 (ARL1) and its effector golgin-245 to this compartment. Here, we show that liver-specific knockout of Arfrp1 in the mouse (Arfrp1(liv-/-)) resulted in early growth retardation, which was associated with reduced hepatic insulin-like growth factor 1 (IGF1) secretion. Accordingly, suppression of Arfrp1 in primary hepatocytes resulted in a significant reduction of IGF1 release. However, the hepatic secretion of IGF-binding protein 2 (IGFBP2) was not affected in the absence of ARFRP1. In addition, Arfrp1(liv-/-) mice exhibited decreased glucose transport into the liver, leading to a 50% reduction of glycogen stores as well as a marked retardation of glycogen storage after fasting and refeeding. These abnormalities in glucose metabolism were attributable to reduced protein levels and intracellular retention of the glucose transporter GLUT2 in Arfrp1(liv-/-) livers. As a consequence of impaired glucose uptake into the liver, the expression levels of carbohydrate response element binding protein (ChREBP), a transcription factor regulated by glucose concentration, and its target genes (glucokinase and pyruvate kinase) were markedly reduced. Our data indicate that ARFRP1 in the liver is involved in the regulation of IGF1 secretion and GLUT2 sorting and is thereby essential for normal growth and glycogen storage.

Functional characterisation of human SGLT-5 as a novel kidney-specific sodium-dependent sugar transporter.

Sodium glucose cotransporters (SGLT) actively catalyse carbohydrate transport across cellular membranes. Six of the 12 known SGLT family members have the capacity to bind and/or transport monosaccharides (SGLT-1 to 6); of these, all but SGLT-5 have been characterised. Here we demonstrate that human SGLT-5 is exclusively expressed in the kidney. Four splice variants were detected and the most abundant SGLT-5-mRNA was functionally characterised. SGLT-5 mediates sodium-dependent [(14)C]-α-methyl-D-glucose (AMG) transport that can be inhibited by mannose, fructose, glucose, and galactose. Uptake studies using demonstrated high capacity transport for mannose and fructose and, to a lesser extent, glucose, AMG, and galactose. SGLT-5 mediated mannose, fructose and AMG transport was weakly (µM potency) inhibited by SGLT-2 inhibitors. In summary, we have characterised SGLT-5 as a kidney mannose transporter. Further studies are warranted to explore the physiological role of SGLT-5.

Identification of a potential biomarker for FABP4 inhibition: the power of lipidomics in preclinical drug testing.

The fatty acid binding protein 4 (FABP4) belongs to the family of lipid chaperones that control intracellular fluxes and compartmentalization of their respective ligands (e.g., fatty acids). FABP4, which is almost exclusively expressed in adipocytes and macrophages, contributes to the development of insulin resistance and atherosclerosis in mice. Lack of FABP4 protects against the development of insulin resistance associated with genetic or diet-induced obesity in mice. Furthermore, total or macrophage-specific FABP4 deficiency is protective against atherosclerosis in apolipoprotein E-deficient mice. The FABP4 small-molecule inhibitor BMS309403 has demonstrated efficacy in mouse models for type 2 diabetes mellitus and atherosclerosis, resembling phenotypes of mice with FABP4 deficiency. However, despite the therapeutically attractive long-term effects of FABP4 inhibition, an acute biomarker for drug action is lacking. The authors applied mass spectrometry lipidomics analysis to in vitro and in vivo (plasma and adipose tissue) samples upon inhibitor treatment. They report the identification of a potential biomarker for acute in vivo FABP4 inhibition that is applicable for further investigations and can be implemented in simple and fast-flow injection mass spectrometry assays. In addition, this approach can be considered a proof-of-principle study that can be applied to other lipid-pathway targeting mechanisms.

The protein family of glucose transport facilitators: It's not only about glucose after all.

The protein family of facilitative glucose transporters comprises 14 isoforms that share common structural features such as 12 transmembrane domains, N- and C-termini facing the cytoplasm of the cell, and a N-glycosylation side either within the first or fifth extracellular loop. Based on their sequence homology, three classes can be distinguished: class I includes GLUT1-4 and GLUT14, class II the "odd transporters" GLUT5, 7, 9, 11, and class III the "even transporters" GLUT6, 8, 10, 12 and the proton driven myoinositol transporter HMIT (or GLUT13). With the cloning and characterization of the more recent class II and III isoforms, it became apparent that despite their structural similarities, the different isoforms not only show a distinct tissue-specific expression pattern but also show distinct characteristics such as alternative splicing, specific (sub)cellular localization, and affinities for a spectrum of substrates. This review summarizes the current understanding of the physiological role for the various transport facilitators based on human genetically inherited disorders or single-nucleotide polymorphisms and knockout mice models. The emphasis of the review will be on the potential functional role of the more recent isoforms.

Lysosomal localization of GLUT8 in the testis--the EXXXLL motif of GLUT8 is sufficient for its intracellular sorting via AP1- and AP2-mediated interaction.

The class III sugar transport facilitator GLUT8 co-localizes with the lysosomal protein LAMP1 in heterologous expression systems. GLUT8 carries a [D/E]XXXL[L/I]-type dileucine sorting signal that has been postulated to retain the protein in an endosomal/lysosomal compartment via interactions with clathrin adaptor protein (AP) complexes. However, contradictory findings have been described regarding the subcellular localization of the endogenous GLUT8 and the adaptor proteins that interact with its dileucine motif. Here we demonstrate that endogenous GLUT8 is localized in a late endosomal/lysosomal compartment of spermatocytes and spermatids, and that the adaptor complexes AP1 and AP2, but not AP3 or AP4, interact with its N-terminal intracellular domain (NICD). In addition, fusion of the GLUT8 NICD to the tailless lumenal domain of the IL-2 receptor alpha chain (TAC) protein (interleukin-2 receptor a chain) targeted the protein to intracellular membranes, indicating that its N-terminal dileucine signal is sufficient for endosomal/lysosomal targeting of the transporter. The localization and targeting of GLUT8 show striking similarities to sorting mechanisms reported for lysosomal proteins. Therefore, we suggest a potential role for GLUT8 in the so far unexplored substrate transport across intracellular membranes.

Neuronal functions, feeding behavior, and energy balance in Slc2a3+/- mice.

Homozygous deletion of the gene of the neuronal glucose transporter GLUT3 (Slc2a3) in mice results in embryonic lethality, whereas heterozygotes (Slc2a3+/-) are viable. Here, we describe the characterization of heterozygous mice with regard to neuronal function, glucose homeostasis, and, since GLUT3 might be a component of the neuronal glucose-sensing mechanism, food intake and energy balance. Levels of GLUT3 mRNA and protein in brain were reduced by 50% in Slc2a3+/- mice. Electrographic features examined by electroencephalographic recordings give evidence for slightly but significantly enhanced cerebrocortical activity in Slc2a3+/- mice. In addition, Slc2a3+/- mice were slightly more sensitive to an acoustic startle stimulus (elevated startle amplitude and reduced prepulse inhibition). However, systemic behavioral testing revealed no other functional abnormalities, e.g., in coordination, reflexes, motor abilities, anxiety, learning, and memory. Furthermore, no differences in body weight, blood glucose, and insulin levels were detected between wild-type and Slc2a3+/- littermates. Food intake as monitored randomly or after intracerebroventricular administration of 2-deoxyglucose or d-glucose, or food choice for carbohydrates/fat was not affected in Slc2a3+/- mice. Taken together, our data indicate that, in contrast to Slc2a1, a single allele of Slc2a3 is sufficient for maintenance of neuronal energy supply, motor abilities, learning and memory, and feeding behavior.

ADP-ribosylation factor-like GTPase ARFRP1 is required for trans-Golgi to plasma membrane trafficking of E-cadherin.

ADP-ribosylation factor-related protein 1 (ARFRP1) plays a specific role in Golgi function controlling recruitment of GRIP domain proteins and ARL1 to the trans-Golgi. Deletion of the mouse Arfrp1 gene causes embryonic lethality during early gastrulation, because epiblast cells detach from the ectodermal cell layer and do not differentiate to mesodermal tissue. Here we show that in Arfrp1(-/-) embryos E-cadherin is mistargeted to intracellular compartments, whereas in control embryos it is present at the cell surface of trophectodermal and ectodermal cells. In enterocytes of intestine-specific Arfrp1 null mutants (Arfrp1(vil)(-/-)), E-cadherin is associated with intracellular membranes, partially colocalizing with the cis-Golgi marker GM130 or with punctae close to the cell surface. In contrast, in control enterocytes E-cadherin is exclusively located in the lateral membranes. In addition, ARL1 is dislocated from Golgi membranes to the cytosol of Arfrp1(vil)(-/-) enterocytes. Depletion of endogenous ARFRP1 by RNA interference leads to a dislocation of E-cadherin from the cell surface in HeLa cells and to a reduced cell aggregation in Ltk(-)Ecad cells. ARFRP1 was coimmunoprecipitated in a complex with E-cadherin, alpha-catenin, beta-catenin, gamma-catenin, and p120(ctn) from lysates of Madin-Darby canine kidney cells stably expressing myc-ARFRP1. These data indicate that knock-out of Arfrp1 disrupts the trafficking of E-cadherin through the Golgi and suggest an essential role of the GTPase in trans-Golgi network function.

Targeted disruption of Slc2a8 (GLUT8) reduces motility and mitochondrial potential of spermatozoa.

GLUT8 is a class 3 sugar transport facilitator which is predominantly expressed in testis and also detected in brain, heart, skeletal muscle, adipose tissue, adrenal gland, and liver. Since its physiological function in these tissues is unknown, we generated a Slc2a8 null mouse and characterized its phenotype. Slc2a8 knockout mice appeared healthy and exhibited normal growth, body weight development and glycemic control, indicating that GLUT8 does not play a significant role for maintenance of whole body glucose homeostasis. However, analysis of the offspring distribution of heterozygous mating indicated a lower number of Slc2a8 knockout offspring (30.5:47.3:22.1%, Slc2a8(+/+), Slc2a8(+/-), and Slc2a8(-/-) mice, respectively) resulting in a deviation (p=0.0024) from the expected Mendelian distribution. This difference was associated with lower ATP levels, a reduced mitochondrial membrane potential and a significant reduction of sperm motility of the Slc2a8 knockout in comparison to wild-type spermatozoa. In contrast, number and survival rate of spermatozoa were not altered. These data indicate that GLUT8 plays an important role in the energy metabolism of sperm cells.