Preclinical exploration of combined glucagon inhibition and liver-preferential insulin for treatment of diabetes using in vitro assays and rat and mouse models.

AIMS/HYPOTHESIS: Normalisation of blood glucose in individuals with diabetes is recommended to reduce development of diabetic complications. However, risk of severe hypoglycaemia with intensive insulin therapy is a major obstacle that prevents many individuals with diabetes from obtaining the recommended reduction in HbA1c. Inhibition of glucagon receptor signalling and liver-preferential insulin action have been shown individually to have beneficial effects in preclinical models and individuals with diabetes (i.e. improved glycaemic control), but also have effects that are potential safety risks (i.e. alpha cell hyperplasia in response to glucagon receptor antagonists and increased levels of liver triacylglycerols and plasma alanine aminotransferase activity in response to glucagon receptor antagonists and liver-preferential insulin). We hypothesised that a combination of glucagon inhibition and liver-preferential insulin action in a dual-acting molecule would widen the therapeutic window. By correcting two pathogenic mechanisms (dysregulated glucagon signalling and non-physiological distribution of conventional insulin administered s.c.), we hypothesised that lower doses of each component would be required to obtain sufficient reduction of hyperglycaemia, and that the undesirable effects that have previously been observed for monotreatment with glucagon antagonists and liver-preferential insulin could be avoided. METHODS: A dual-acting glucagon receptor inhibitor and liver-preferential insulin molecule was designed and tested in rodent models (normal rats, rats with streptozotocin-induced hyperglycaemia, db/db mice and mice with diet-induced obesity and streptozotocin-induced hyperglycaemia), allowing detailed characterisation of the pharmacokinetic and pharmacodynamic properties of the dual-acting molecule and relevant control compounds, as well as exploration of how the dual-acting molecule influenced glucagon-induced recovery and spontaneous recovery from acute hypoglycaemia. RESULTS: This molecule normalised blood glucose in diabetic models, and was markedly less prone to induce hypoglycaemia than conventional insulin treatment (approximately 4.6-fold less potent under hypoglycaemic conditions than under normoglycaemic conditions). However, compared to treatment with conventional long-acting insulin, this dual-acting molecule also increased triacylglycerol levels in the liver (approximately 60%), plasma alanine aminotransferase levels (approximately twofold) and alpha cell mass (approximately twofold). CONCLUSIONS/INTERPRETATION: While the dual-acting glucagon receptor inhibitor and liver-preferential insulin molecule showed markedly improved regulation of blood glucose, effects that are potential safety concerns persisted in the pharmacologically relevant dose range.

The insulin receptor is expressed and functional in cultured blood-brain barrier endothelial cells but does not mediate insulin entry from blood to brain.

Insulin and its receptor are known to be present and functional in the brain. Insulin cerebrospinal fluid concentrations have been shown to correlate with plasma levels of insulin in a nonlinear fashion, indicative of a saturable transport pathway from the blood to the brain interstitial fluid. The aim of the present study was to investigate whether insulin was transported across brain endothelial cells in vitro via an insulin receptor-dependent pathway. The study showed that the insulin receptor was expressed at both the mRNA and protein levels in bovine brain endothelial cells. Luminally applied radiolabeled insulin showed insulin receptor-mediated binding to the endothelial cells. This caused a dose-dependent increase in Akt-phosphorylation, which was inhibited by coapplication of an insulin receptor inhibitor, s961, demonstrating activation of insulin receptor signaling pathways. Transport of insulin across the blood-brain barrier in vitro was low and comparable to that of a similarly sized paracellular marker. Furthermore, insulin transport was not inhibited by coapplication of an excess of unlabeled insulin or an insulin receptor inhibitor. The insulin transport and uptake studies were repeated in mouse brain endothelial cells demonstrating similar results. Although it cannot be ruled out that culture-induced changes in the cell model could have impaired a potential insulin transport mechanism, these in vitro data indicate that peripheral insulin must reach the brain parenchyma through alternative pathways rather than crossing the blood-brain barrier via receptor mediated transcytosis.

Liver X receptor regulates hepatic nuclear O-GlcNAc signaling and carbohydrate responsive element-binding protein activity.

Liver X receptor (LXR)α and LXRß play key roles in hepatic de novo lipogenesis through their regulation of lipogenic genes, including sterol regulatory element-binding protein (SREBP)-1c and carbohydrate responsive element-binding protein (ChREBP). LXRs activate lipogenic gene transcription in response to feeding, which is believed to be mediated by insulin. We have previously shown that LXRs are targets for glucose-hexosamine-derived O-linked ß-N-acetylglucosamine (O-GlcNAc) modification enhancing their ability to regulate SREBP-1c promoter activity in vitro. To elucidate insulin-independent effects of feeding on LXR-mediated lipogenic gene expression in vivo, we subjected control and streptozotocin-treated LXRα/ß(+/+) and LXRα/ß(-/-) mice to a fasting-refeeding regime. We show that under hyperglycemic and hypoinsulinemic conditions, LXRs maintain their ability to upregulate the expression of glycolytic and lipogenic enzymes, including glucokinase (GK), SREBP-1c, ChREBPα, and the newly identified shorter isoform ChREBPß. Furthermore, glucose-dependent increases in LXR/retinoid X receptor-regulated luciferase activity driven by the ChREBPα promoter was mediated, at least in part, by O-GlcNAc transferase (OGT) signaling in Huh7 cells. Moreover, we show that LXR and OGT interact and colocalize in the nucleus and that loss of LXRs profoundly reduced nuclear O-GlcNAc signaling and ChREBPα promoter binding activity in vivo. In summary, our study provides evidence that LXRs act as nutrient and glucose metabolic sensors upstream of ChREBP by modulating GK expression, nuclear O-GlcNAc signaling, and ChREBP expression and activity.

Molecular Engineering of Efficacious Mono-Valent Ultra-Long Acting Two-Chain Insulin-Fc Conjugates.

Here, we describe molecular engineering of monovalent ultra-long acting two-chain insulin-Fc conjugates. Insulin-Fc conjugates were synthesized using trifunctional linkers with one amino reactive group for reaction with a lysine residue of insulin and two thiol reactive groups used for re-bridging of a disulfide bond within the Fc molecule. The ultra-long pharmacokinetic profile of the insulin-Fc conjugates was the result of concertedly slowing insulin receptor-mediated clearance by (1) introduction of amino acid substitutions that lowered the insulin receptor affinity and (2) conjugating insulin to the Fc element. Fc conjugation leads to recycling by the neonatal Fc receptor and increase in the molecular size, both contributing to the ultra-long pharmacokinetic and pharmacodynamic profiles.

Impaired glucocorticoid receptor expression in liver disrupts feeding-induced gene expression, glucose uptake, and glycogen storage.

The transition from a fasted to a fed state is associated with extensive transcriptional remodeling in hepatocytes facilitated by hormonal- and nutritional-regulated transcription factors. Here, we use a liver-specific glucocorticoid receptor (GR) knockout (L-GRKO) model to investigate the temporal hepatic expression of GR target genes in response to feeding. Interestingly, in addition to the well-described fasting-regulated genes, we identify a subset of hepatic feeding-induced genes that requires GR for full expression. This includes Gck, which is important for hepatic glucose uptake, utilization, and storage. We show that insulin and glucocorticoids cooperatively regulate hepatic Gck expression in a direct GR-dependent manner by a 4.6 kb upstream GR binding site operating as a Gck enhancer. L-GRKO blunts preprandial and early postprandial Gck expression, which ultimately affects early postprandial hepatic glucose uptake, phosphorylation, and glycogen storage. Thus, GR is positively involved in feeding-induced gene expression and important for postprandial glucose metabolism in the liver.

Isolation of Adipogenic and Fibro-Inflammatory Stromal Cell Subpopulations from Murine Intra-Abdominal Adipose Depots.

The stromal-vascular fraction (SVF) of white adipose tissue (WAT) is remarkably heterogeneous and consists of numerous cell types that contribute functionally to the expansion and remodeling of WAT in adulthood. A tremendous barrier to studying the implications of this cellular heterogeneity is the inability to readily isolate functionally distinct cell subpopulations from WAT SVF for in vitro and in vivo analyses. Single-cell sequencing technology has recently identified functionally distinct fibro-inflammatory and adipogenic PDGFRß+ perivascular cell subpopulations in intra-abdominal WAT depots of adult mice. Fibro-inflammatory progenitors (termed, "FIPs") are non-adipogenic collagen producing cells that can exert a pro-inflammatory phenotype. PDGFRß+ adipocyte precursor cells (APCs) are highly adipogenic both in vitro and in vivo upon cell transplantation. Here, we describe multiple methods for the isolation of these stromal cell subpopulations from murine intra-abdominal WAT depots. FIPs and APCs can be isolated by fluorescence-activated cell sorting (FACS) or by taking advantage of biotinylated antibody-based immunomagnetic bead technology. Isolated cells can be used for molecular and functional analysis. Studying the functional properties of stromal cell subpopulation in isolation will expand our current knowledge of adipose tissue remodeling under physiological or pathological conditions on the cellular level.

Structure, Aggregation, and Activity of a Covalent Insulin Dimer Formed During Storage of Neutral Formulation of Human Insulin.

A specific covalently linked dimeric species of insulin high molecular weight products (HMWPs), formed during prolonged incubation of a neutral pharmaceutical formulation of human insulin, were characterized in terms of tertiary structure, self-association, biological activity, and fibrillation properties. The dimer was formed by a covalent link between A21Asn and B29Lys. It was analyzed using static and dynamic light scattering and small-angle X-ray scattering to evaluate its self-association behavior. The tertiary structure was obtained using nuclear magnetic resonance and X-ray crystallography. The biological activity of HMWP was determined using 2 in vitro assays, and its influence on fibrillation was investigated using Thioflavin T assays. The dimer's tertiary structure was nearly identical to that of the noncovalent insulin dimer, and it was able to form hexamers in the presence of zinc. The dimer exhibited reduced propensity for self-association in the absence of zinc but significantly postponed the onset of fibrillation in insulin formulations. Consistent with its dimeric state, the tested species of HMWP showed little to no biological activity in the used assays. This study is the first detailed characterization of a specific type of human insulin HMWP formed during storage of a marketed pharmaceutical formulation. These results indicate that this specific type of HMWP is unlikely to antagonize the physical stability of the formulation, as HMWP retained a tertiary structure similar to the noncovalent dimer and participated in hexamer assembly in the presence of zinc. In addition, increasing amounts of HMWP reduce the rate of insulin fibrillation.

Treatment of diabetic rats with insulin or a synthetic insulin receptor agonist peptide leads to divergent metabolic responses.

In addition to lowering of blood glucose, treatment with insulin also induces lipid synthesis and storage. Patients with type 2 diabetes often suffer from lipid-related comorbidities including dyslipidemia, obesity, and fatty liver disease. We examined here in two separate studies changes in lipid dynamics in Zucker diabetic fatty (ZDF) rats, in response to 7 days of treatment with either insulin or the insulin receptor agonist peptide S597. In concert with blood glucose normalization, the treated rats displayed large increases in hepatic de novo lipid synthesis and deposition of newly synthesized lipids in adipose tissue depots, accompanied by weight gain and expansion of adipose depots. In both treatment groups, heavy water labeling revealed that after 2 h (study A), de novo lipogenesis was responsible for 80% of newly stored hepatic triglyceride (TG)-palmitate, and after 5 days (study B), ∼60% of newly deposited TG-palmitate in adipose tissues originated from this pathway. Interestingly, in both studies, treatment with the insulin mimetic peptide resulted in significantly lower blood TG levels, plasma TG production rates, and hepatic de novo synthesized fatty acid in plasma TG compared with insulin. There were no differences in plasma TG turnover (clearance rate) in response to either treatment, consistent with differential actions on the liver. These results show that in ZDF rats, treatment with a synthetic insulin-receptor-activating peptide or with insulin to lower blood glucose is accompanied by different effects on hepatic lipid anabolism and blood TG profiles.

Insulin analog with additional disulfide bond has increased stability and preserved activity.

Insulin is a key hormone controlling glucose homeostasis. All known vertebrate insulin analogs have a classical structure with three 100% conserved disulfide bonds that are essential for structural stability and thus the function of insulin. It might be hypothesized that an additional disulfide bond may enhance insulin structural stability which would be highly desirable in a pharmaceutical use. To address this hypothesis, we designed insulin with an additional interchain disulfide bond in positions A10/B4 based on Cα-Cα distances, solvent exposure, and side-chain orientation in human insulin (HI) structure. This insulin analog had increased affinity for the insulin receptor and apparently augmented glucodynamic potency in a normal rat model compared with HI. Addition of the disulfide bond also resulted in a 34.6°C increase in melting temperature and prevented insulin fibril formation under high physical stress even though the C-terminus of the B-chain thought to be directly involved in fibril formation was not modified. Importantly, this analog was capable of forming hexamer upon Zn addition as typical for wild-type insulin and its crystal structure showed only minor deviations from the classical insulin structure. Furthermore, the additional disulfide bond prevented this insulin analog from adopting the R-state conformation and thus showing that the R-state conformation is not a prerequisite for binding to insulin receptor as previously suggested. In summary, this is the first example of an insulin analog featuring a fourth disulfide bond with increased structural stability and retained function.

Novel covalently linked insulin dimer engineered to investigate the function of insulin dimerization.

An ingenious system evolved to facilitate insulin binding to the insulin receptor as a monomer and at the same time ensure sufficient stability of insulin during storage. Insulin dimer is the cornerstone of this system. Insulin dimer is relatively weak, which ensures dissociation into monomers in the circulation, and it is stabilized by hexamer formation in the presence of zinc ions during storage in the pancreatic ß-cell. Due to the transient nature of insulin dimer, direct investigation of this important form is inherently difficult. To address the relationship between insulin oligomerization and insulin stability and function, we engineered a covalently linked insulin dimer in which two monomers were linked by a disulfide bond. The structure of this covalent dimer was identical to the self-association dimer of human insulin. Importantly, this covalent dimer was capable of further oligomerization to form the structural equivalent of the classical hexamer. The covalently linked dimer neither bound to the insulin receptor, nor induced a metabolic response in vitro. However, it was extremely thermodynamically stable and did not form amyloid fibrils when subjected to mechanical stress, underlining the importance of oligomerization for insulin stability.

Phosphorylation of serine 248 of C/EBPα is dispensable for myelopoiesis but its disruption leads to a low penetrant myeloid disorder with long latency.

BACKGROUND: Transcription factors play a key role in lineage commitment and differentiation of stem cells into distinct mature cells. In hematopoiesis, they regulate lineage-specific gene expression in a stage-specific manner through various physical and functional interactions with regulatory proteins that are simultanously recruited and activated to ensure timely gene expression. The transcription factor CCAAT/enhancer binding protein α (C/EBPα) is such a factor and is essential for the development of granulocytic/monocytic cells. The activity of C/EBPα is regulated on several levels including gene expression, alternative translation, protein interactions and posttranslational modifications, such as phosphorylation. In particular, the phosphorylation of serine 248 of the transactivation domain has been shown to be of crucial importance for granulocytic differentiation of 32Dcl3 cells in vitro. METHODOLOGY/PRINCIPAL FINDINGS: Here, we use mouse genetics to investigate the significance of C/EBPα serine 248 in vivo through the construction and analysis of Cebpa(S248A/S248A) knock-in mice. Surprisingly, 8-week old Cebpa(S248A/S248A) mice display normal steady-state hematopoiesis including unaltered development of mature myeloid cells. However, over time some of the animals develop a hematopoietic disorder with accumulation of multipotent, megakaryocytic and erythroid progenitor cells and a mild impairment of differentiation along the granulocytic-monocytic lineage. Furthermore, BM cells from Cebpa(S248A/S248A) animals display a competitive advantage compared to wild type cells in a transplantation assay. CONCLUSIONS/SIGNIFICANCE: Taken together, our data shows that the substitution of C/EBPα serine 248 to alanine favors the selection of the megakaryocytic/erythroid lineage over the monocytic/granulocytic compartment in old mice and suggests that S248 phosphorylation may be required to maintain proper hematopoietic homeostasis in response to changes in the wiring of cellular signalling networks. More broadly, the marked differences between the phenotype of the S248A variant in vivo and in vitro highlight the need to exert caution when extending in vitro phenotypes to the more appropriate in vivo context.

Modeling of C/EBPalpha mutant acute myeloid leukemia reveals a common expression signature of committed myeloid leukemia-initiating cells.

Mutations in the CEBPA gene are present in 7%-10% of human patients with acute myeloid leukemia (AML). However, no genetic models exist that demonstrate their etiological relevance. To mimic the most common mutations affecting CEBPA-that is, those leading to loss of the 42 kDa C/EBPalpha isoform (p42) while retaining the 30kDa isoform (p30)-we modified the mouse Cebpa locus to express only p30. p30 supported the formation of granulocyte-macrophage progenitors. However, p42 was required for control of myeloid progenitor proliferation, and p42-deficient mice developed AML with complete penetrance. p42-deficient leukemia could be transferred by a Mac1+c-Kit+ population that gave rise only to myeloid cells in recipient mice. Expression profiling of this population against normal Mac1+c-Kit+ progenitors revealed a signature shared with MLL-AF9-transformed AML.

Distinct C/EBPalpha motifs regulate lipogenic and gluconeogenic gene expression in vivo.

The C/EBPalpha transcription factor regulates hepatic nitrogen, glucose, lipid and iron metabolism. However, how it is able to independently control these processes is not known. Here, we use mouse knock-in mutagenesis to identify C/EBPalpha domains that specifically regulate hepatic gluconeogenesis and lipogenesis. In vivo deletion of a proline-histidine rich domain (PHR), dephosphorylated at S193 by insulin signaling, dysregulated genes involved in the generation of acetyl-CoA and NADPH for triglyceride synthesis and led to increased hepatic lipogenesis. These promoters bound SREBP-1 as well as C/EBPalpha, and the PHR was required for C/EBPalpha-SREBP transcriptional synergy. In contrast, the highly conserved C/EBPalpha CR4 domain was found to undergo liver-specific dephosphorylation of residues T222 and T226 upon fasting, and alanine mutation of these residues upregulated the hepatic expression of the gluconeogenic G6Pase and PEPCK mRNAs, but not PGC-1alpha, leading to glucose intolerance. Our results show that pathway-specific metabolic regulation can be achieved through a single transcription factor containing context-sensitive regulatory domains, and indicate C/EBPalpha phosphorylation as a PGC-1alpha-independent mechanism for regulating hepatic gluconeogenesis.