Maternal malnutrition is associated with decreased nutrient transfer to the foetus, which may lead to foetal growth restriction, predisposing children to a variety of diseases. However, regulation of ...placental nutrient transfer during decreased nutrient availability is not fully understood. In the present study, the aim was to investigate changes in levels of placental nutrient transporters accompanying maternal hypoglycaemia following different durations and stages of gestation in rats. Maternal hypoglycaemia was induced by insulin-infusion throughout gestation until gestation day (GD)20 or until end of organogenesis (GD17), with sacrifice on GD17 or GD20. Protein levels of placental glucose transporters GLUT1 (45/55 kDa isotypes) and GLUT3, amino acid transporters SNAT1 and SNAT2, and insulin receptor (InsR) were assessed. On GD17, GLUT1-45, GLUT3, and SNAT1 levels were increased and InsR levels decreased versus controls. On GD20, following hypoglycaemia throughout gestation, GLUT3 levels were increased, GLUT1-55 showed the same trend. After cessation of hypoglycaemia at end of organogenesis, GLUT1-55, GLUT3, and InsR levels were increased versus controls, whereas SNAT1 levels were decreased. The increases in levels of placental nutrient transporters seen during maternal hypoglycaemia and hyperinsulinemia likely reflect an adaptive response to optimise foetal nutrient supply and development during limited availability of glucose.
Oxidative stress is associated with insulin resistance and is thought to contribute to progression toward type 2 diabetes. Oxidation induces cellular damages through increased amounts of reactive ...aldehydes from lipid peroxidation. The aim of our study was to investigate 1) the effect of the major lipid peroxidation end product, 4-hydroxynonenal (HNE), on insulin signaling in 3T3-L1 adipocytes, and 2) whether fatty aldehyde dehydrogenase (FALDH), which detoxifies HNE, protects cells and improves insulin action under oxidative stress conditions.
3T3-L1 adipocytes were exposed to HNE and/or infected with control adenovirus or adenovirus expressing FALDH.
Treatment of 3T3-L1 adipocytes with HNE at nontoxic concentrations leads to a pronounced decrease in insulin receptor substrate (IRS)-1/-2 proteins and in insulin-induced IRS and insulin receptor beta (IR beta) tyrosine phosphorylation. Remarkably, we detect increased binding of HNE to IRS-1/-2-generating HNE-IRS adducts, which likely impair IRS function and favor their degradation. Phosphatidylinositol 3-kinase and protein kinase B activities are also downregulated upon HNE treatment, resulting in blunted metabolic responses. Moreover, FALDH, by reducing adduct formation, partially restores HNE-generated decrease in insulin-induced IRS-1 tyrosine phosphorylation and metabolic responses. Moreover, rosiglitazone could have an antioxidant effect because it blocks the noxious HNE action on IRS-1 by increasing FALDH gene expression. Collectively, our data show that FALDH improves insulin action in HNE-treated 3T3-L1 adipocytes.
Oxidative stress induced by reactive aldehydes, such as HNE, is implicated in the development of insulin resistance in 3T3-L1 adipocytes, which is alleviated by FALDH. Hence, detoxifying enzymes could play a crucial role in blocking progression of insulin resistance to diabetes.
Context:
Insulin resistance can be compensated by increased functional pancreatic β-cell mass; otherwise, diabetes ensues. Such compensation depends not only on environmental and genetic factors but ...also on the baseline β-cell mass from which the expansion originates.
Objective:
Little is known about assembly of a baseline β-cell mass in humans. Here, we examined formation of β-cell populations relative to other pancreatic islet cell types and associated neurons throughout the normal human lifespan.
Design and Methods:
Human pancreatic sections derived from normal cadavers aged 24 wk premature to 72 yr were examined by immunofluorescence. Insulin, glucagon, and somatostatin were used as markers for β-, α-, and δ-cells, respectively. Cytokeratin-19 marked ductal cells, Ki67 cell proliferation, and Tuj1 (neuronal class III β-tubulin) marked neurons.
Results:
Most β-cell neogenesis was observed preterm with a burst of β-cell proliferation peaking within the first 2 yr of life. Thereafter, little indication of β-cell growth was observed. Postnatal proliferation of α- and δ-cells was rarely seen, but a wave of ductal cell proliferation was found mostly associated with exocrine cell expansion. The β-cell to α-cell ratio doubled neonatally, reflecting increased growth of β-cells, but during childhood, there was a 7-fold change in the β-cell to δ-cell ratio, reflecting an additional loss of δ-cells. A close association of neurons to pancreatic islets was noted developmentally and retained throughout adulthood. Negligible neuronal association to exocrine pancreas was observed.
Conclusion:
Human baseline β-cell population and appropriate association with other islet cell types is established before 5 yr of age.
Selective insulin resistance, where insulin's ability to lower blood glucose via the Akt signaling pathway gradually decreases while retaining its effectiveness in activating other signaling pathways ...(e.g. MAPK), is a key pathophysiological mechanism underlying the hyperglycemia, dyslipidemia and vascular dysfunction in type 2 diabetes (T2D). Here we hypothesize that a selective signaling insulin analog that activates Akt signaling but has a considerably reduced effect on MAPK should counteract selective insulin resistance and provide effective glucose lowering with improved vascular function, reduced hepatic lipid accumulation and generally alleviate insulin resistance over time compared to human insulin (HI). NNC-965, a basal insulin analog with 4 amino acid modifications and a C18 fatty diacid, was designed as a functionally selective insulin, with greater Akt than MAPK activation in several cellular systems and in vivo. NNC-965 fully activates glucose regulating pathways, including inhibition of glucose output, glycogen accumulation and inhibition of gluconeogenic mRNAs in rat hepatocytes as well as stimulation of glucose uptake and inhibition of lipolysis in rat adipocytes. In contrast, NNC-965 displayed reduced induction of lipogenic mRNA and de novo lipogenesis in rat liver cells compared to HI. In a mouse model of T2D, treatment with NNC-965 for 6 weeks to the same glucose level as insulin 700 (an insulin degludec-like analog) resulted in 35% less liver triglycerides and improved acetylcholine mediated vasodilation in ex vivo studies using mesenteric arterioles. In conclusion, NNC-965 is a novel functionally selective insulin analog that can potentially improve insulin resistance and eventually reduce cardiovascular risk by restoring balanced insulin signaling.
Disclosure
G.S.Olsen: Employee; Novo Nordisk A/S. G.Povlen: Employee; Novo Nordisk. T.A.Pedersen: Employee; Novo Nordisk A/S. R.Slaaby: Employee; Novo Nordisk, Stock/Shareholder; Novo Nordisk. B.F.Hansen: Employee; Novo Nordisk A/S, Stock/Shareholder; Novo Nordisk A/S. T.Glendorf: Employee; Novo Nordisk A/S, Stock/Shareholder; Novo Nordisk A/S. D.Demozay: None. H.Hvid: Employee; Novo Nordisk A/S. E.Nishimura: Employee; Novo Nordisk A/S. J.Brandt: Employee; Novo Nordisk A/S.
Abstract
Mammalian metabolism has evolved to adapt to changes in nutrient status. Insulin, the key anabolic hormone, facilitates intracellular storage of nutrient fuels and plays a pivotal role in ...the transition away from catabolism upon refeeding. Although circulating insulin relative to nutrient levels has been well characterized during fasting and refeeding, how pancreatic β-cell biology caters to acute changes in insulin demand has not been sufficiently addressed. Here, we examined the dynamics of (pro)insulin production and associated changes in β-cell ultrastructure during refeeding after a 72-hour fast in male rats. We found that fasted β-cells had marked degranulation, which inversely coordinated with the upregulation of autophagolysomal and lysosomal organelles. There was also expanded Golgi that correlated with enhanced (pro)insulin biosynthetic capacity but, conversely, blunted in vivo insulin secretion. Within 4 to 6 hours of refeeding, proinsulin biosynthesis, cellular ultrastructure, in vivo insulin secretion, and glucose tolerance normalized to levels near those of fed control animals, indicating a rapid replenishment of normal insulin secretory capacity. Thus, during a prolonged fast, the β-cell protects against hypoglycemia by markedly reducing insulin secretory capacity in vivo but is simultaneously poised to efficiently increase (pro)insulin production upon refeeding to effectively return normal insulin secretory capacity within hours.
Rapid β-cell ultrastructural changes parallel increased (pro)insulin biosynthesis to meet glycemic demand after fasting-refeeding in rats.
Insulin receptor substrate-2 (IRS-2) plays an essential role in pancreatic islet β-cells by promoting growth and survival. IRS-2 turnover is rapid in primary β-cells, but its expression is highly ...regulated at the transcriptional level, especially by glucose. The aim was to investigate the molecular mechanism on how glucose regulates IRS-2 gene expression in β-cells.
Rat islets were exposed to inhibitors or subjected to adenoviral vector-mediated gene manipulations and then to glucose-induced IRS-2 expression analyzed by real-time PCR and immunoblotting. Transcription factor nuclear factor of activated T cells (NFAT) interaction with IRS-2 promoter was analyzed by chromatin immunoprecipitation assay and glucose-induced NFAT translocation by immunohistochemistry.
Glucose-induced IRS-2 expression occurred in pancreatic islet β-cells in vivo but not in liver. Modulating rat islet β-cell Ca(2+) influx with nifedipine or depolarization demonstrated that glucose-induced IRS-2 gene expression was dependent on a rise in intracellular calcium concentration derived from extracellular sources. Calcineurin inhibitors (FK506, cyclosporin A, and a peptide calcineurin inhibitor CAIN) abolished glucose-induced IRS-2 mRNA and protein levels, whereas expression of a constitutively active calcineurin increased them. Specific inhibition of NFAT with the peptide inhibitor VIVIT prevented a glucose-induced IRS-2 transcription. NFATc1 translocation to the nucleus in response to glucose and association of NFATc1 to conserved NFAT binding sites in the IRS-2 promoter were demonstrated.
The mechanism behind glucose-induced transcriptional control of IRS-2 gene expression specific to the islet β-cell is mediated by the Ca(2+)/calcineurin/NFAT pathway. This insight into the IRS-2 regulation could provide novel therapeutic means in type 2 diabetes to maintain an adequate functional mass.
Insulin resistant individuals display elevated fasting and post-prandial glucose levels which are mainly driven by inadequate inhibition of gluconeogenesis (GNG) and are rarely normalized. The aim of ...this study was to investigate whether direct inhibition of GNG could be a new therapeutical approach to improve glucose homeostasis using in vitro and in vivo models of insulin resistance. Fructose 1,6-bisphosphatase (FBP1) is key in controlling GNG in liver and kidney and loss of function patients display hypoglycaemia episodes and lactate acidosis. Systemic FBP1 inhibition using small molecules has shown to improve glucose homeostasis. To assess the clinical efficacy and safety potential of silencing FBP1 selectively in the liver we dosed insulin resistant DIO rats with a hepatocyte specific GalXC-FBP1 siRNA entity. GalXC-FBP1 siRNA markedly reduced FBP1 mRNA level in the liver by over 90%. This was associated with a complete lack of glucose excursion following a pyruvate challenge indicating inhibition of GNG. Blood glucose level decreased similarly in rats treated with GalXC-FBP1 siRNA or vehicle during an insulin challenge or a prolonged fast suggesting that hepatic FBP1 silencing is not inducing hypoglycaemia. Insulin sensitivity and hyperinsulinemia were improved. Plasma lactate and liver enzymes were not elevated, but a significant 2-fold increase in liver triglycerides was observed in the GalXC-FBP1 siRNA group. Using human hepatocytes in a Liver-on-Chip in vitro model FBP1 silencing reduced glucose production by 30% supporting the relevance of this approach in humans. Collectively these data suggest that liver specific FBP1 silencing has the potential to improve insulin sensitivity in DIO rats without inducing hypoglycaemia but may be associated with a risk of liver steatosis overtime. This concept highlights the difficulty of blocking liver GNG without re-directing the metabolic intermediates fluxes toward triglycerides accumulation in liver.
Disclosure
C. Fledelius: Employee; Novo Nordisk A/S. D. Demozay: Employee; Novo Nordisk A/S. H. Iversen: None. R. S. Ingvorsen: None. L. B. Eriksen: None. A. Blois: Employee; Novo Nordisk. Y. Montauban: None. R. Rijnbrand: Employee; Novo Nordisk. W. Han: None. J. F. Jeppesen: Employee; Novo Nordisk A/S.
Appropriate regulation of insulin receptor substrate 2 (IRS-2) expression in pancreatic β-cells is essential to adequately compensate for insulin resistance. In liver, basal IRS-2 expression is ...controlled via a temporal negative feedback of sterol regulatory element-binding protein 1 (SREBP-1) to antagonize transcription factors forkhead box class O (FoxO)1/FoxO3a at an insulin response element (IRE) on the IRS-2 promoter. The purpose of the study was to examine if a similar mechanism controlled IRS-2 expression in β-cells.
IRS-2 mRNA and protein expression, as well as IRS-2 gene promoter activity, were examined in isolated rat islets. Specific transcription factor association with the IRE on the IRS-2 promoter was examined by chromatin immunoprecipitation (ChIP) assay, and their nuclear translocation was examined by immunofluorescence. A direct in vivo effect of insulin on control of IRS-2 expression in liver and pancreatic islets was also investigated.
In IRS-2 promoter-reporter assays conducted in isolated islets, removal of the IRE decreased basal IRS-2 promoter activity in β-cells up to 80%. Activation of IRS signaling in isolated rat islets by insulin/IGF-I (used as an experimental in vitro tool) or downstream constitutive activation of protein kinase B (PKB) significantly decreased IRS-2 expression. In contrast, inhibition of phosphatidylinositol 3-kinase (PI3K) or PKB significantly increased IRS-2 levels in β-cells. ChIP assays indicated that transcription factors FoxO1 and FoxO3a associated with the IRE on the IRS-2 promoter in β-cells in a PI3K/PKB-dependent manner, whereas others, such as SREBP-1, the transcription factor binding to immunoglobulin heavy chain enhancer 3', and the aryl hydrocarbon receptor nuclear translocator (ARNT), did not. However, only FoxO3a, not FoxO1, was capable of driving IRS-2 promoter activity via the IRE in β-cells. In vivo studies showed insulin was able to suppress IRS-2 expression via activation of SREBP-1 in the liver, but this mechanism was not apparent in pancreatic islets from the same animal.
The molecular mechanism for feedback control of IRS signaling to decrease IRS-2 expression in liver and β-cells is quite distinct, with a predominant role played by FoxO3a in β-cells.
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 HbA
1c
. 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
/
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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.
Graphical abstract
Pharmacokinetic properties and duration of therapeutic action of a pharmaceutical agent can be significantly extended through the combination of two distinct strategies aimed at increasing plasma ...half-life: fatty acid acylation and Fc-conjugation. Using insulin as a case study, we demonstrate that a doubly protracted insulin analog produces a substantial prolongation of pharmacodynamic effect to lower blood glucose in STZ-treated mice when compared to the Fc-only counterparts. This enhancement is further corroborated by direct pharmacokinetic measurements in rat and dog models, demonstrating the potential for once-monthly insulin therapy. The results suggest that this approach might have broad application across a diverse spectrum of peptide- and protein-based therapeutics.
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