Sotagliflozin, a Dual SGLT1 and SGLT2 Inhibitor, as Adjunct Therapy to Insulin in Type 1 Diabetes.

OBJECTIVE: To assess the safety and efficacy of dual sodium-glucose cotransporter (SGLT) 1 and SGLT2 inhibition with sotagliflozin as adjunct therapy to insulin in type 1 diabetes. RESEARCH DESIGN AND METHODS: We treated 33 patients with sotagliflozin, an oral dual SGLT1 and SGLT2 inhibitor, or placebo in a randomized, double-blind trial assessing safety, insulin dose, glycemic control, and other metabolic parameters over 29 days of treatment. RESULTS: In the sotagliflozin-treated group, the percent reduction from baseline in the primary end point of bolus insulin dose was 32.1% (P = 0.007), accompanied by lower mean daily glucose measured by continuous glucose monitoring (CGM) of 148.8 mg/dL (8.3 mmol/L) (P = 0.010) and a reduction of 0.55% (5.9 mmol/mol) (P = 0.002) in HbA1c compared with the placebo group that showed 6.4% reduction in bolus insulin dose, a mean daily glucose of 170.3 mg/dL (9.5 mmol/L), and a decrease of 0.06% (0.65 mmol/mol) in HbA1c. The percentage of time in target glucose range 70-180 mg/dL (3.9-10.0 mmol/L) increased from baseline with sotagliflozin compared with placebo, to 68.2% vs. 54.0% (P = 0.003), while the percentage of time in hyperglycemic range >180 mg/dL (10.0 mmol/L) decreased from baseline, to 25.0% vs. 40.2% (P = 0.002), for sotagliflozin and placebo, respectively. Body weight decreased (1.7 kg) with sotagliflozin compared with a 0.5 kg gain (P = 0.005) in the placebo group. CONCLUSIONS: As adjunct to insulin, dual SGLT1 and SGLT2 inhibition with sotagliflozin improved glycemic control and the CGM profile with bolus insulin dose reduction, weight loss, and no increased hypoglycemia in type 1 diabetes.

Genetic deletion of Mst1 alters T cell function and protects against autoimmunity.

Mammalian sterile 20-like kinase 1 (Mst1) is a MAPK kinase kinase kinase which is involved in a wide range of cellular responses, including apoptosis, lymphocyte adhesion and trafficking. The contribution of Mst1 to Ag-specific immune responses and autoimmunity has not been well defined. In this study, we provide evidence for the essential role of Mst1 in T cell differentiation and autoimmunity, using both genetic and pharmacologic approaches. Absence of Mst1 in mice reduced T cell proliferation and IL-2 production in vitro, blocked cell cycle progression, and elevated activation-induced cell death in Th1 cells. Mst1 deficiency led to a CD4+ T cell development path that was biased toward Th2 and immunoregulatory cytokine production with suppressed Th1 responses. In addition, Mst1-/- B cells showed decreased stimulation to B cell mitogens in vitro and deficient Ag-specific Ig production in vivo. Consistent with altered lymphocyte function, deletion of Mst1 reduced the severity of experimental autoimmune encephalomyelitis (EAE) and protected against collagen-induced arthritis development. Mst1-/- CD4+ T cells displayed an intrinsic defect in their ability to respond to encephalitogenic antigens and deletion of Mst1 in the CD4+ T cell compartment was sufficient to alleviate CNS inflammation during EAE. These findings have prompted the discovery of novel compounds that are potent inhibitors of Mst1 and exhibit desirable pharmacokinetic properties. In conclusion, this report implicates Mst1 as a critical regulator of adaptive immune responses, Th1/Th2-dependent cytokine production, and as a potential therapeutic target for immune disorders.

Keratinocyte migration in the developing eyelid requires LIMK2.

In vitro studies have identified LIMK2 as a key downstream effector of Rho GTPase-induced changes in cytoskeletal organization. LIMK2 is phosphorylated and activated by Rho associated coiled-coil kinases (ROCKs) in response to a variety of growth factors. The biochemical targets of LIMK2 belong to a family of actin binding proteins that are potent modulators of actin assembly and disassembly. Although numerous studies have suggested that LIMK2 regulates cell morphology and motility, evidence supportive of these functions in vivo has remained elusive. In this study, a knockout mouse was created that abolished LIMK2 biochemical activity resulting in a profound inhibition of epithelial sheet migration during eyelid development. In the absence of LIMK2, nascent eyelid keratinocytes differentiate and acquire a pre-migratory phenotype but the leading cells fail to nucleate filamentous actin and remain immobile causing an eyes open at birth (EOB) phenotype. The failed nucleation of actin was associated with significant reductions in phosphorylated cofilin, a major LIMK2 biochemical substrate and potent modulator of actin dynamics. These results demonstrate that LIMK2 activity is required for keratinocyte migration in the developing eyelid.

A mouse knockout library for secreted and transmembrane proteins.

Large collections of knockout organisms facilitate the elucidation of gene functions. Here we used retroviral insertion or homologous recombination to disrupt 472 genes encoding secreted and membrane proteins in mice, providing a resource for studying a large fraction of this important class of drug target. The knockout mice were subjected to a systematic phenotypic screen designed to uncover alterations in embryonic development, metabolism, the immune system, the nervous system and the cardiovascular system. The majority of knockout lines exhibited altered phenotypes in at least one of these therapeutic areas. To our knowledge, a comprehensive phenotypic assessment of a large number of mouse mutants generated by a gene-specific approach has not been described previously.

GPIHBP1 stabilizes lipoprotein lipase and prevents its inhibition by angiopoietin-like 3 and angiopoietin-like 4.

Glycosylphosphatidylinositol-anchored HDL-binding protein (GPIHBP1) binds both LPL and chylomicrons, suggesting that GPIHBP1 is a platform for LPL-dependent processing of triglyceride (TG)-rich lipoproteins. Here, we investigated whether GPIHBP1 affects LPL activity in the absence and presence of LPL inhibitors angiopoietin-like (ANGPTL)3 and ANGPTL4. Like heparin, GPIHBP1 stabilized but did not activate LPL. ANGPTL4 potently inhibited nonstabilized LPL as well as heparin-stabilized LPL but not GPIHBP1-stabilized LPL. Like ANGPTL4, ANGPTL3 inhibited nonstabilized LPL but not GPIHBP1-stabilized LPL. ANGPTL3 also inhibited heparin-stabilized LPL but with less potency than nonstabilized LPL. Consistent with these in vitro findings, fasting serum TGs of Angptl4(-/-)/Gpihbp1(-/-) mice were lower than those of Gpihbp1(-/-) mice and approached those of wild-type littermates. In contrast, serum TGs of Angptl3(-/-)/Gpihbp1(-/-) mice were only slightly lower than those of Gpihbp1(-/-) mice. Treating Gpihbp1(-/-) mice with ANGPTL4- or ANGPTL3-neutralizing antibodies recapitulated the double knockout phenotypes. These data suggest that GPIHBP1 functions as an LPL stabilizer. Moreover, therapeutic agents that prevent LPL inhibition by ANGPTL4 or, to a lesser extent, ANGPTL3, may benefit individuals with hyperlipidemia caused by gene mutations associated with decreased LPL stability.

Identification of a new functional domain in angiopoietin-like 3 (ANGPTL3) and angiopoietin-like 4 (ANGPTL4) involved in binding and inhibition of lipoprotein lipase (LPL).

Angiopoietin-like 3 (ANGPTL3) and angiopoietin-like 4 (ANGPTL4) are secreted proteins that regulate triglyceride (TG) metabolism in part by inhibiting lipoprotein lipase (LPL). Recently, we showed that treatment of wild-type mice with monoclonal antibody (mAb) 14D12, specific for ANGPTL4, recapitulated the Angptl4 knock-out (-/-) mouse phenotype of reduced serum TG levels. In the present study, we mapped the region of mouse ANGPTL4 recognized by mAb 14D12 to amino acids Gln(29)-His(53), which we designate as specific epitope 1 (SE1). The 14D12 mAb prevented binding of ANGPTL4 with LPL, consistent with its ability to neutralize the LPL-inhibitory activity of ANGPTL4. Alignment of all angiopoietin family members revealed that a sequence similar to ANGPTL4 SE1 was present only in ANGPTL3, corresponding to amino acids Glu(32)-His(55). We produced a mouse mAb against this SE1-like region in ANGPTL3. This mAb, designated 5.50.3, inhibited the binding of ANGPTL3 to LPL and neutralized ANGPTL3-mediated inhibition of LPL activity in vitro. Treatment of wild-type as well as hyperlipidemic mice with mAb 5.50.3 resulted in reduced serum TG levels, recapitulating the lipid phenotype found in Angptl3(-/-) mice. These results show that the SE1 region of ANGPTL3 and ANGPTL4 functions as a domain important for binding LPL and inhibiting its activity in vitro and in vivo. Moreover, these results demonstrate that therapeutic antibodies that neutralize ANGPTL4 and ANGPTL3 may be useful for treatment of some forms of hyperlipidemia.

Large-scale gene trapping in C57BL/6N mouse embryonic stem cells.

We report the construction and analysis of a mouse gene trap mutant resource created in the C57BL/6N genetic background containing more than 350,000 sequence-tagged embryonic stem (ES) cell clones. We also demonstrate the ability of these ES cell clones to contribute to the germline and produce knockout mice. Each mutant clone is identified by a genomic sequence tag representing the exact insertion location, allowing accurate prediction of mutagenicity and enabling direct genotyping of mutant alleles. Mutations have been identified in more than 10,000 genes and show a bias toward the first intron. The trapped ES cell lines, which can be requested from the Texas A&M Institute for Genomic Medicine, are readily available to the scientific community.

High-throughput screening of mouse knockout lines identifies true lean and obese phenotypes.

We developed a high-throughput approach to knockout (KO) and phenotype mouse orthologs of the 5,000 potential drug targets in the human genome. As part of the phenotypic screen, dual-energy X-ray absorptiometry (DXA) technology estimates body-fat stores in eight KO and four wild-type (WT) littermate chow-fed mice from each line. Normalized % body fat (nBF) (mean KO % body fat/mean WT littermate % body fat) values from the first 2322 lines with viable KO mice at 14 weeks of age showed a normal distribution. We chose to determine how well this screen identifies body-fat phenotypes by selecting 13 of these 2322 KO lines to serve as benchmarks based on their published lean or obese phenotype on a chow diet. The nBF values for the eight benchmark KO lines with a lean phenotype were > or =1 s.d. below the mean for seven (perilipin, SCD1, CB1, MCH1R, PTP1B, GPAT1, PIP5K2B) but close to the mean for NPY Y4R. The nBF values for the five benchmark KO lines with an obese phenotype were >2 s.d. above the mean for four (MC4R, MC3R, BRS3, translin) but close to the mean for 5HT2cR. This screen also identifies novel body-fat phenotypes as exemplified by the obese kinase suppressor of ras 2 (KSR2) KO mice. These body-fat phenotypes were confirmed upon studying additional cohorts of mice for KSR2 and all 13 benchmark KO lines. This simple and cost-effective screen appears capable of identifying genes with a role in regulating mammalian body fat.

Mouse Fkbp8 activity is required to inhibit cell death and establish dorso-ventral patterning in the posterior neural tube.

Neural tube defects (NTDs) are birth defects that can be disabling or lethal and are second in their prevalence after cardiac defects among major human congenital malformations. Spina bifida is a NTD where the spinal cord is dysplastic, and the overlying spinal column is absent. At present, the molecular mechanisms underlying the spinal bifida development are largely unknown. In this study, we present a Fkbp8 mouse mutant that has an isolated and completely penetrant spina bifida, which is folate- and inositol-resistant. Fkbp8 mutants are not embryo lethal, but they display striking features of human spina bifida, including a dysplastic spinal cord, open neural canal and disability. The loss of Fkbp8 leads to increased apoptosis in the posterior neural tube, demonstrating that in vivo FKBP8 inhibits cell death. Gene expression analysis of Fkbp8 mutants revealed a perturbation of expression of neural tube patterning genes, suggesting that endogenous FKBP8 activity establishes dorso-ventral patterning of the neural tube. These studies demonstrate that Fkbp8 is not important for embryo survival, but is essential for spinal neural tube patterning, and to block apoptosis, in the developing neural tube. The mutant Fkbp8 allele is a new experimental model which will be useful in dissecting the pathogenesis of spinal NTDs, and enhance our understanding of the etiology of human NTDs.

Lipid-lowering effects of anti-angiopoietin-like 4 antibody recapitulate the lipid phenotype found in angiopoietin-like 4 knockout mice.

We used gene knockout mice to explore the role of Angiopoietin-like-4 (Angptl4) in lipid metabolism as well as to generate anti-Angptl4 mAbs with pharmacological activity. Angptl4 -/- mice had lower triglyceride (TG) levels resulting both from increased very low-density lipoprotein (VLDL) clearance and decreased VLDL production and had modestly lower cholesterol levels. Also, both Angptl4 -/- suckling mice and adult mice fed a high-fat diet showed reduced viability associated with lipogranulomatous lesions of the intestines and their draining lymphatics and mesenteric lymph nodes. Treating C57BL/6J, ApoE -/-, LDLr -/-, and db/db mice with the anti-Angptl4 mAb 14D12 recapitulated the lipid and histopathologic phenotypes noted in Angptl4 -/- mice. This demonstrates that the knockout phenotype reflects not only the physiologic function of the Angptl4 gene but also predicts the pharmacologic consequences of Angptl4 protein inhibition with a neutralizing antibody in relevant models of human disease.

Severe retinal degeneration associated with disruption of semaphorin 4A.

PURPOSE: Semaphorin 4A (Sema4A) is a member of the transmembrane class 4 family of semaphorins. It has recently been shown to participate in cell-cell communication in the immune system. High levels of sema4A are also present in brain and eye, but its function in the central nervous system has not been studied. To investigate the function of Sema4A, we generated mice deficient in this transmembrane signaling molecule. METHODS: An embryonic stem (ES) cell clone with a retroviral gene-trap insertion in the sema4A gene was used to generate mice lacking this transmembrane semaphorin. Fundus photography, fluorescein angiography, and electroretinography were used to evaluate retinal anatomy and physiology in mice lacking Sema4A. Electron microscopy and immunohistochemistry with cell-type-specific markers were used to characterize retinal development. In situ hybridization with sema4A-specific riboprobes was used to localize expression of this gene in the developing and adult eye. RESULTS: Fundus photography performed at 14 weeks of age revealed severe retinal degeneration, attenuated retinal vessels, and depigmentation in mice lacking Sema4A. At this age, the outer nuclear layer was reduced to a single row of photoreceptor cells, and the outer plexiform layer was thin and disorganized. Disruption of Sema4A also compromised the physiological function of both rod and cone photoreceptors. Developmental studies in Sema4A-deficient mice revealed abnormal morphology of photoreceptor outer segments during the time at which they establish contacts with apical microvilli of the retinal pigment epithelium (RPE). Sema4A is expressed in the inner retina and RPE during the time at which photoreceptor outer segments elongate. CONCLUSIONS: These findings identify a previously unknown function of Sema4A in the developing visual system and provide a useful model for understanding cell-cell interactions that occur between photoreceptors and the RPE.

Wnk1 kinase deficiency lowers blood pressure in mice: a gene-trap screen to identify potential targets for therapeutic intervention.

The availability of both the mouse and human genome sequences allows for the systematic discovery of human gene function through the use of the mouse as a model system. To accelerate the genetic determination of gene function, we have developed a sequence-tagged gene-trap library of >270,000 mouse embryonic stem cell clones representing mutations in approximately 60% of mammalian genes. Through the generation and phenotypic analysis of knockout mice from this resource, we are undertaking a functional screen to identify genes regulating physiological parameters such as blood pressure. As part of this screen, mice deficient for the Wnk1 kinase gene were generated and analyzed. Genetic studies in humans have shown that large intronic deletions in WNK1 lead to its overexpression and are responsible for pseudohypoaldosteronism type II, an autosomal dominant disorder characterized by hypertension, increased renal salt reabsorption, and impaired K+ and H+ excretion. Consistent with the human genetic studies, Wnk1 heterozygous mice displayed a significant decrease in blood pressure. Mice homozygous for the Wnk1 mutation died during embryonic development before day 13 of gestation. These results demonstrate that Wnk1 is a regulator of blood pressure critical for development and illustrate the utility of a functional screen driven by a sequence-based mutagenesis approach.

Predicting drug efficacy: knockouts model pipeline drugs of the pharmaceutical industry.

One of the major challenges for the pharmaceutical industry is to develop innovative drugs to new targets from the human genome. A systematic approach for target selection could significantly increase the rate of successful new drug development, thereby enhancing industry productivity. It has previously been shown that mouse knockout phenotypes for the targets of the 100 best-selling pharmaceutical drugs correlate well with known drug efficacy. Furthermore, physiological validation of novel pipeline targets of the pharmaceutical industry has been provided using mouse knockout data. These data demonstrate an excellent correlation between knockout phenotype and anticipated drug efficacy, establishing an important marker for superior new drug targets from the genome.

Knockouts model the 100 best-selling drugs--will they model the next 100?

The biopharmaceutical industry is currently faced with a tremendous number of potential drug targets identified through the sequencing of the human genome. The challenge ahead is to delineate those targets with the greatest value for therapeutic intervention. Here, we critically evaluate mouse-knockout technology for target discovery and validation. A retrospective evaluation of the knockout phenotypes for the targets of the 100 best-selling drugs indicates that these phenotypes correlate well with known drug efficacy, illuminating a productive path forward for discovering future drug targets. Prospective mining of the druggable genome is being catalysed by large-scale mouse knockout programs combined with phenotypic screens focused on identifying targets that modulate mammalian physiology in a therapeutically relevant manner.

mdmx is a negative regulator of p53 activity in vivo.

Regulation of p53 protein activity is required for normal embryogenesis, tumor suppression, and cellular response to DNA damage. Here we report that loss of mdmx, a p53-binding protein, results in midgestational embryo lethality, a phenotype that is completely rescued by the absence of p53. Mice homozygous for both mdmx and p53 null mutations are viable and appear developmentally normal. Fibroblasts derived from embryos with reduced mdmx expression demonstrate a decreased growth rate and increased UV-induced apoptosis compared with wild-type cells and contain elevated levels of p53 and several p53 target proteins including the proapoptotic bax protein. These observations demonstrate that mdmx functions as a critical negative regulator of p53 in vivo.

Full-speed mammalian genetics: in vivo target validation in the drug discovery process.

The completion of the Human Genome Project has signaled the beginning of the post-genome era, with a corresponding shift in focus from the sequencing and identification of genes to the exploration of gene function. A rate-limiting step in deriving value from this gene sequence information is determining the potential pharmaceutical applications of genes and their encoded proteins. This validation step is crucial for focusing efforts and resources on only the most promising targets. Strategies using reverse mouse genetics provide excellent methods for validating potential targets and therapeutic proteins in vivo in a mammalian model system.