BackgroundIn non-insulin-dependent, type 2, diabetes mellitus (T2D), glucose metabolism is compromised, and the heart loses its metabolic flexibility. The Zucker Diabetic Fatty rat (ZDF) model, which replicates the pathophysiology of T2D in patients, shows that as T2D progresses so does heart failure. Heart ketone metabolism seems to play a role in mitigating the heart failure process. This study assesses ketone metabolism in a ZDF heart failure model using cardiac PET imaging.MethodsSix lean ZDF rats (CTRL) and six diabetic obese ZDF rats (T2D) were evaluated for coronary flow reserve (CFR) using [13N]ammonia ([13N]NH3) cardiac PET. In addition, rats were evaluated with [11C]acetoacetate ([11C]AcAc) PET during rest and stress conditions to assess ketone metabolism, both at baseline and under an acute exogenous ketone ester oral supplementation. Blood chemistry, cardiac function and hemodynamic parameters were also evaluated under these conditions.ResultsCFR was impaired in the T2D model (CTRL: 1.8 ± 0.5; T2D: 1.4 ± 0.2, p < 0.05) suggesting the development of heart failure in the T2D model. Blood ketones increased more than 2-fold after supplementation. The [11C]AcAc heart ketone uptake values with and without ketone supplementation were similar for the CTRL group, and these values were higher than for T2D rats. For the T2D group, the uptake decreased by 20% at rest under ketone supplementation vs. no supplementation (p < 0.05) and remained unchanged under stress with and without supplementation. Because of this decrease at rest, the stress/rest ratio after supplementation increases to the level observed in CTRL. [11C]AcAc heart ketone metabolism showed a slight decrease under stress for the CTRL group, but not for the T2D. Under ketone supplementation, the metabolism stress/rest ratio increased only in T2D (1.25 ± 0.29, p = 0.03 compared to baseline).ConclusionIn a rat model of T2D and CFR impairment, we were able to measure changes in ketone metabolism using [11C]AcAc PET at rest and under stress with and without acute ketone supplementation. Our findings suggest that the heart ketone metabolism of T2D rats is impaired during the heart failure process. Ketone supplementation may have the potential to restore this cardiac reserve.

Background: Glucagon-like peptide 1 receptor agonists (GLP-1 RAs) and sodium-glucose co-transporter-2 inhibitors (SGLT2i) demonstrated cardiovascular (CV) and kidney outcomes benefit in persons with type 2 diabetes (T2D). The long-acting amylin analogue, cagrilintide, is under investigation for the treatment of obesity and T2D. Endotrophin, a pro-fibrotic fragment derived from collagen type VI, is an independent risk marker of mortality, CV and kidney outcomes, and dulaglutide treatment decreased levels of this biomarker compared with insulin glargine in persons with T2D. We investigated for the first time the effect of semaglutide, empagliflozin, and cagrilintide on endotrophin in type 2 diabetic rats. Method: We measured endotrophin (by the rodent PRO-C6 ELISA) in serum collected at baseline and study end from diabetic ZDF rats treated with either vehicle (n = 10), semaglutide (n = 7), empagliflozin (n = 4), or cagrilintide (n = 10). The dose chosen for semaglutide (50 nmol/kg), empagliflozin (30 mg/kg), and cagrilintide (10 nmol/kg) was based on previous research. Results: Levels of endotrophin increased significantly during the study in vehicle-treated rats, reflecting the disease progression (median [95% CI] for increase: 182.5 [55.87-298.0], P &amp;lt; 0.01). Interestingly, treatment with semaglutide, empagliflozin, and cagrilintide each attenuated this increase, resulting in significantly lower levels of endotrophin compared to vehicle at study end (P &amp;lt; 0.01), (P &amp;lt; 0.05), and (P &amp;lt; 0.01), respectively. Conclusion: We developed a novel, robust assay to detect the clinically relevant endotrophin biomarker in rodents. Levels of serum endotrophin increased over time in vehicle-treated rats, which is in line with endotrophin predicting disease progression in persons with T2D. In addition to the beneficial effects on metabolic factors, semaglutide, empagliflozin, and cagrilintide each reduced levels of endotrophin, suggesting a potential effect to reduce fibrosis. Disclosure A.Møller: None. S.A.Melander: None. A.T.Larsen: Employee; Nordic Bioscience A/S. C.F.G.Laursen: None. F.Genovese: Employee; Nordic Bioscience A/S, Stock/Shareholder; Nordic Bioscience A/S. M.A.Karsdal: Speaker's Bureau; Pfizer Inc. D.Rasmussen: Employee; Nordic Bioscience A/S, Stock/Shareholder; Nordic Bioscience A/S. K.Henriksen: Employee; Nordic Bioscience A/S, Stock/Shareholder; Nordic Bioscience A/S.

Dual amylin and calcitonin receptor agonist treatment improves insulin sensitivity and increases muscle-specific glucose uptake independent of weight loss

Does receptor balance matter? – Comparing the efficacies of the dual amylin and calcitonin receptor agonists cagrilintide and KBP-336 on metabolic parameters in preclinical models

Abstract EMPA-REG OUTCOME (Empagliflozin, Cardiovascular Outcome Event Trial) trial highlighted the relevance of pharmacological inhibition of sodium-glucose cotransporter 2 (SGLT2) for the treatment of patients with type 2 diabetes mellitus (T2DM) and/or cardiovascular disease. Although the pathways through which SGLT2 inhibitors exert a beneficial effect on the cardiovascular system are still unknown, it has been suggested that energy metabolism regulation and a reduction of systemic inflammation could be some of the mechanisms implicated. Available data also suggests that empagliflozin treatment could be able to exert regulatory effects on adipose tissue (AT) depots. The aim of our study was to evaluate the impact of empagliflozin treatment on the lipidome of visceral (VAT) and subcutaneous adipose tissue (SAT) depots in a rat model of obesity and T2DM. Diabetic obese Zucker Fatty (ZDF) rats were treated with 30 mg/kg/day of empagliflozin p.o for 6 weeks. The lipidomes of VAT and SAT depots were analyzed using ultra-high performance liquid chromatography coupled to mass spectrometry. Empagliflozin's effect on pro-inflammatory markers in AT was analyzed by RT-PCR. In VAT, 18 metabolites were significantly altered in empagliflozin-treated rats vs. controls. Nearly all diglycerides tested (13 of 14) were significantly increased in treated rats, as the most notable altered chemical class. Furthermore, 3 oxidized fatty acids (FA) and FA like gadoleic acid and linoleic acid were also significantly increased. In SAT, a total of 14 metabolites were significantly altered. Most of them (13 of 14) were glycerophospholipids. Significantly lower levels of 4 lysophosphatidylethanolamines, 4 lysophosphatidylcholines, and 3 lysophosphatidylinositols and higher levels of 2 phosphatidylcholines were shown. In contrast to VAT, a significant decrease in most of these metabolites was observed in empagliflozin-treated samples. Several ratios of metabolites were also calculated to infer the potential enzyme activities related to lipid metabolism in both VAT and SAT. However, the ratios studied were only statistically significant in VAT of empagliflozin-treated rats vs. control, where the main potential enzyme activities altered were desaturases and elongases. Empagliflozin treatment also reduces the expression of the pro-inflammatory cytokines interleukin-1 beta (IL-1β), interleukin-6 (IL-6), tumor necrosis factor-alpha (TNFα) and monocyte-chemotactic protein-1 (MCP-1) in VAT, with no changes in SAT, except for interleukin-13 (IL-13), which also decreases compared to untreated diabetic ZDF rats. In conclusion, empagliflozin increased oxidized FA and diglycerides in VAT and decreased glycerophospholipid levels in SAT. The anti-inflammatory effect of empagliflozin in VAT is not observed in SAT. The effect of empagliflozin on the regulation of the adipose tissue lipidome and inflammatory profile is different depending on the localization of the depots. Funding Acknowledgement Type of funding sources: Private company. Main funding source(s): Boehringer Ingelheim Pharma GmbH and Co

Long-acting dual amylin and calcitonin receptor agonists (DACRAs) are novel candidates for treatment of type 2 diabetes (T2D) and obesity due to their beneficial effects on both body weight, glucose control and insulin action. Cagrilintide, which is currently in clinical trials, has shown promising effects on weight loss. In this study we compared a new long-acting DACRA (KBP) to cagrilintide in pre-clinical models of obesity and T2D. In vitro potencies were assessed using receptor assays. In vivo efficacies were investigated head-to-head in high fat diet (HFD) fed obese and T2D (ZDF) rat models. In vitro data showed that both peptides active both the amylin and the calcitonin receptor, with KBP being more potent on both receptors. This was further confirmed in vivo by assessment of acute effects on food intake and CTX suppression. KBP (1.5, 4.5 and 13.5 nmol/kg) and cagrilintide (10, 30 and 100 nmol/kg) induced a potent and dose-dependent weight loss in HFD rats, with the highest dose of KBP being superior to cagrilintide. In diabetic ZDF rats, DACRA treatment improved glucose control and preserved plasma insulin compared to vehicle. Interestingly, despite similar levels of plasma insulin, KBP treatment was superior to cagrilintide in improving glucose control. This was further reflected in the HbA1c levels at study end, where KBP treatment resulted in significantly lower levels compared to cagrilintide. In summary, both DACRAs induced weight loss and improved glucose tolerance, insulin action as well as glucose control. However, KBP treatment results in superior efficacy on both weight loss and glucose control. These findings highlight KBP as a promising once-weekly agent for treatment of obesity and T2D. Disclosure A.T.Larsen: Employee; Nordic Bioscience. N.Sonne: None. K.Mohamed: None. E.Bredtoft: None. F.Andersen: None. M.A.Karsdal: Employee; Nordic Bioscience A/S, Nordic Bioscience A/S, Nordic Bioscience A/S. K.Henriksen: Employee; Nordic Bioscience A/S, Stock/Shareholder; Nordic Bioscience A/S.

Dual amylin and calcitonin receptor agonists (DACRAs) are known to induce a significant weight loss and improve glucose tolerance and glucose control in rats. However, it is unknown if DACRAs has an insulin sensitizing effect beyond that induced by weight loss and if DACRAs affect tissue specific glucose uptake. Additionally, clamp studies with DACRAs have not previously been performed in diabetic rats. Pre-diabetic ZDSD and diabetic ZDF rats were treated with the DACRA KBP-088 (1.5 nmol//kg, s.c.) for 12 days, and a hyperinsulinemic clamp was conducted on day 13, 24 h post-dosing. In ZDSD rats a hyperinsulinemic euglycemic clamp was carried out, while a hyperinsulinemic isoglycemic clamp was carried out in ZDF rats. In addition, specific glucose uptake was assessed during a hyperinsulinemic isoglycemic clamp in ZDF rats. In ZDSD rats, KBP-088 treatment resulted in a significant reduction in body weight and fasting blood glucose, and improved insulin sensitivity by increasing the glucose infusion rate (GIR) (vehicle: 8.6, KBP-088: 12 mg glucose/kg/min) . In ZDF rats, KBP-088 significantly reduced fasting blood glucose and improved insulin sensitivity (GIR, vehicle: 1.5, KBP-088: 16.5 mg glucose/kg/min) , independent on weight loss. In both studies, KBP-088 increased the rate of glucose clearance, likely be increasing glucose storage, but without altering the endogenous glucose production. Direct assessment of tissue specific glucose uptake showed, that KBP-088 significantly increased glucose uptake in both muscles and adipose tissues when compared to vehicle. In summary, KBP-088 significantly improved insulin sensitivity in both pre-diabetic and diabetic rats and markedly increased tissue specific glucose uptake. Importantly, KBP-088 has an insulin sensitizing effect independent of weight loss, highlighting DACRAs as promising agents for treatment of diabetes. Disclosure A.T.Larsen: Employee; Nordic Bioscience. N.Sonne: None. E.Bredtoft: None. M.A.Karsdal: Employee; Nordic Bioscience A/S, Nordic Bioscience A/S, Nordic Bioscience A/S. K.Henriksen: Employee; Nordic Bioscience A/S, Stock/Shareholder; Nordic Bioscience A/S.

Acidic fibroblast growth factor (aFGF) is a promising regulator of glucose with no adverse effects of hypoglycemia. Previous researches revealed that aFGF mediated adipose tissue remodeling and insulin sensitivity. These findings supported rh-aFGF135 would be used as a new candidate for the treatment of insulin resistance and type 2 diabetes. In this study, we aimed to investigate the hypoglycemic efficacy of recombinant human acidic fibroblast growth factor 135 (rh-aFGF135) with low mitogenic in type 2 diabetic ZDF rats. ZDF rats were treated with rh-aFGF135 at a daily dosage of 0.25 and 0.50 mg/kg by tail intravenous injection for 5 weeks. The blood glucose levels, oral glucose tolerance test, insulin tolerance test, HOMA-IR for insulin resistance, serum biochemical parameters, and the histopathological changes of adipose tissue, liver and other organs were detected at designed time point. The glucose uptake activity and anti-insulin resistance effect of rh-aFGF135 were also detected in HepG2 cells. Results revealed that rh-aFGF135 exhibited a better hypoglycemic effect compared with vehicle group and without the adverse effect of hypoglycemia in ZDF rats. Compared with vehicle group, rh-aFGF135 significantly improved the situation of hyperglycemia and insulin resistance. Rh-aFGF135 decreased ALT, AST, GSP, and FFA levels noticeably compared with vehicle control group (P < 0.01 or P < 0.001). After 5 weeks of treatment, high-dosage rh-aFGF135 could remodel adipose tissue, and has no influence on other organs. H&E staining showed that rh-aFGF135 reduced the size of adipocytes. In addition, rh-aFGF135 may improve insulin resistance partly by increasing the protein expression of p-IRS-1 (human Ser 307). As a hypoglycemic drug for long-term treatment, rh-aFGF135 would be a potentially safe candidate for the therapy of type 2 diabetes.

ObjectivesType 2 diabetes (T2D) is driven by progressive dysfunction and loss of pancreatic β‐cell mass. Imeglimin is a first‐in‐class novel drug candidate that improves glycaemia and glucose‐stimulated insulin secretion in preclinical models and patients. Given evidence that imeglimin can attenuate β‐cell dysfunction and protect β cells in vitro, we postulated that imeglimin could also exert longer term effects to prevent pancreatic β‐cell death and preserve functional β‐cell mass in vivo.MethodsZucker diabetic fatty (ZDF) male rats were treated by oral gavage with imeglimin at a standard dose of 150 mg/kg or vehicle, twice daily for five weeks. At treatment completion, oral glucose tolerance tests were performed in fasted animals before a thorough histomorphometry and immunohistochemical analysis was conducted on pancreas tissue slices to assess cellular composition and disease status.ResultsImeglimin treatment significantly improved glucose‐stimulated insulin secretion (augmentation of the insulinogenic index) and improved glycaemia. Both basal insulinaemia and pancreatic insulin content were also increased by imeglimin. In ZDF control rats, islet structure was disordered with few β‐cells; after imeglimin treatment, islets appeared healthier with more normal morphology in association with a significant increase in insulin‐positive β‐cells. The increase in β‐cell mass was associated with a greater degree of β‐cell proliferation in the presence of reduced apoptosis. Unexpectedly, a decrease in as a α‐cell mass was also documented due to an apparent antiproliferative effect of imeglimin on this cell type.ConclusionIn male ZDF rats, chronic imeglimin treatment corrects a paramount component of type 2 diabetes progression: progressive loss of functional β‐cell mass. In addition, imeglimin may also moderate a‐cell turnover to further ameliorate hyperglycaemia. Cumulatively, these cellular effects suggest that imeglimin may provide for disease modifying effects to preserve functional β‐cell mass.

Liver fructose-1,6-bisphosphatase (FBPase) is a key enzyme in the gluconeogenesis pathway. Inhibiting FBPase activity represents a potential treatment for type 2 diabetes mellitus. A series of novel N-arylsulfonyl-4-arylamino-indole-2-carboxamide derivatives have been disclosed as FBPase inhibitors. Through extensive structure-activity relationship investigations, a promising candidate molecule Cpd118 [sodium (7-chloro-4-((3-methoxyphenyl)amino)-1-methyl-1H-indole-2-carbonyl] [(4-methoxyphenyl)sulfonyl)amide] has been identified with high inhibitory activity against human liver FBPase (IC50, 0.029 ± 0.006 μM) and high selectivity relative to the other six AMP-binding enzymes. Importantly, Cpd118 produced significant glucose-lowering effects on both type 2 diabetic KKAy mice and ZDF rats as demonstrated by substantial reductions in the fasting and postprandial blood glucose levels, as well as the HbA1c level. Furthermore, Cpd118 elicited a favorable pharmacokinetic profile with an oral bioavailability of 99.1%. Moreover, the X-ray crystal structure of the Cpd118-FBPase complex was resolved, which revealed a unique binding mode and provided a structural basis for its high potency and selectivity.

Abstract Background and Aims Type 2 diabetes mellitus is a pathological and chronical condition that induce a rearrangement in carbohydrate metabolism (Kaneto H et al. Antioxid Redox Signal. 2007). Even if the pathological mechanism of DN is not yet clear, NADPH oxidase (NOX) has been demonstrated to be involved in kidney ROS (Reactive Oxygen Species) generation during diabetic state (Palicz A. et al. J Biol Chem. 2001). Therefore, NOX-4, whose p22-phox subunit have shown a pivotal role in diabetes development, has been considered a potential pharmacological target in DN (Gill PS and Wilcox CS. Antioxid Redox Signal. 2006). In previous experiments performed on diabetic rat animal model with DN, we have found a beneficial effect of an extract rich in anthocyanins (ANT), members of flavonoids group (Shih PH. J Agric Food Chem. 2007). The aim of the present work was to investigate the molecular mechanisms involved in the DN and the protective effects of the new extract particularly abundant in ANT, the Red orange and Lemon Extract (RLE). Method Zucker diabetic fatty rats were divided into three groups and treated by gavage daily until 30 weeks of age, as follows: control group (ZF rats) received 1 mL of normal saline; ZDF Group (ZDF rats) received 1mL of normal saline; ZDF + RLE group (ZDF rats) treated with 90 mg/ kg of RLE dissolved in 1mL of normal saline. We examined the gene and protein expression of NOX4 catalytic subunit and its regulatory subunit p22-phox through Real Time PCR and western blot analysis. Results The expression gene levels of NOX4 significantly increased in diabetic rats ZDF, compared to control ZF (***p&amp;lt;0.001). Exposure of ZDF rats with RLE induces a decreased expression of NOX4 (Figure 1) compared to ZDF. On the contrary, there were not significant expression levels variations for regulatory subunit p22-phox. Western blot results are in agreement with Real Time PCR data, in fact an increase in NOX4 protein levels (*p&amp;lt;0.05) is evident, but no significant increase in p22-phox subunit was observed. Conclusion In conclusion, we hypothesize that the protective effect of RLE on the development of DN is related to its ability to inhibit NADPH oxidase activity and opens new perspectives for the treatment of DN.

Diabetes is a major risk factor for cardiovascular disease, affecting both endothelial and smooth muscle cells. Store-operated Ca2+ channels (SOCCs) have been implicated in many diabetic complications. Vascular dysfunction is common in patients with diabetes, but the role of SOCCs in diabetic vasculopathy is still unclear. Our research aimed to investigate the effects of high glucose (HG) on store-operated Ca2+ entry (SOCE) in small arteries. Small mesenteric arteries from type 2 diabetic Zucker fatty rats (ZDF) versus their non-diabetic controls (Zucker lean, ZL) were examined in a pressurized myograph. Vascular smooth muscle cells (VSMC) were isolated and intracellular Ca2+ was measured (Fura 2-AM). A specific protocol to deplete intracellular Ca2+ stores and thereby open SOCCs, as well as pharmacological SOCE inhibitors (SKF-96365, BTP-2), were used to artificially activate and inhibit SOCE, respectively. High glucose (40mmol/L) relaxed arteries in a SKF-sensitive manner. Diabetic arteries exhibited reduced HG-induced relaxation, as well as reduced contraction after Ca2+ replenishment. Further, the rise in intracellular Ca2+ on account of SOCE is diminished in diabetic versus non-diabetic VSMCs and was insensitive to HG in diabetic VSMCs. The expression of SOCC proteins was measured, detecting a downregulation of Orai1 in diabetes. In conclusion, diabetes leads to a reduction of SOCE and SOCE-induced contraction, which is unresponsive to HG-mediated inhibition. The reduced expression of Orai1 in diabetic arteries could account for the observed reduction in SOCE.

Pharmacological treatment with dual amylin and calcitonin receptor agonists (DACRAs) cause significant weight loss and improvement of glucose homeostasis. In this study, the maximally efficacious dose of the novel DACRA, KeyBiosciencePeptide (KBP)-066, was investigated. Two different rat models were used: high-fat diet (HFD)-fed male Sprague-Dawley rats and male Zucker diabetic fatty (ZDF, fa/fa) rats to determine the maximum weight loss and glucose homeostatic effect, respectively. One acute study and one chronic study was performed in HFD rats. Two chronic studies were performed in ZDF rats: a preventive and an interventive. All studies covered a dose range of 5, 50, and 500 µg/kg KBP-066 delivered by subcutaneous injection. Treatment with KBP-066 resulted in a significant weight reduction of 13%-16% and improved glucose tolerance in HFD rats, which was independent of dose concentration. Dosing with 50 and 500 µg/kg led to a transient but significant increase in blood glucose, both in the acute and the chronic study in HFD rats. All doses of KBP-066 significantly improved glucose homeostasis in ZDF rats, both in the preventive and interventive study. Moreover, dosing with 50 and 500 µg/kg preserved insulin secretion to a greater extent than 5 µg/kg when compared with ZDF vehicle rats. Taken together, these results show that maximum weight loss is achieved with 5 µg/kg, which is within the range of previously reported DACRA dosing, whereas increasing dosing concentration to 50 and 500 µg/kg may further improve preservation of insulin secretion compared with 5 µg/kg in diabetic ZDF rats. SIGNIFICANCE STATEMENT: Here we show that KeyBiosciencePeptide (KBP)-066 induces an equally potent body weight loss across a broad dose range in obese rats. However, higher dosing of KBP-066 may improve insulin action in diabetic rats both as preventive and interventive treatment.

Background and AimsThe link between gut microbiota and type 2 diabetes (T2D) has been addressed by numerous studies. Streptococcus thermophilus from fermented milk products, has been used as a probiotic in previous research. However, whether heat-killed S. thermophilus can improve the glycemic parameters of diabetic rats remains unanswered. In this study, we evaluated the effect of heat-killed S. thermophilus on T2D model rats and the potential mechanisms of the effect.MethodsZucker diabetic fatty (ZDF) rats were used to generate a diabetic rat model induced by feeding a high-fat diet. Heat-killed S. thermophilus were orally administered to normal and diabetic rats for 12 weeks. Intestinal microbiota analysis, histology analysis, oral glucose tolerance test and measurement of inflammatory factors were performed.ResultsWe found that heat-killed S. thermophilus treatment reduced fasting blood glucose levels and alleviated glucose intolerance and total cholesterol in diabetic ZDF rats. Additionally, heat-killed S. thermophilus increased the interleukin 10 while reducing the levels of lipopolysaccharide, interleukin 6, and tumor necrosis factor-α in diabetic ZDF rats. The heat-killed S. thermophilus treatment can normalize the structure of the intestinal and colon mucosal layer of diabetic rats. The characteristics of the gut microbiota in heat-killed S. thermophilus-treated and control rats were similar. At the genus level, the abundances of beneficial bacteria, including Ruminococcaceae, Veillonella, Coprococcus, and Bamesiella, were all significantly elevated by heat-killed S. thermophilus treatment in ZDF diabetic rats.ConclusionOur study supports the hypothesis that treatment with heat-killed S. thermophilus could effectively improve glycemic parameters in T2D model rats. In addition, the potential mechanisms underlying the protection maybe include changing the composition of gut microbiota, reinforcing the intestinal epithelial barrier and the immunity of the intestinal mucosa, decreasing the level of inflammation, and then reducing the insulin resistance.

Recent data suggest that the individual microbiome influences the pathology of type 2 diabetes (T2D). Linagliptin is a dipeptidyl peptidase 4 (DPP-4) inhibitor used for the treatment of T2D, which is excreted mainly via the bile. We investigated the microbiome of diabetic ZDF vs. normal rats and the effect of linagliptin on the gut bacterial composition over time. Diabetic ZDF and normal rats (two groups each, of six biological replicates) were treated with linagliptin 7.2 mg/kg qd or vehicle, in 5 ml/kg Natrosol 0.5%; faeces were collected at days 0, 3 and 7. The sequencing of microbial 16S rRNA amplicons was performed at CeMeT GmbH in Tübingen, to produce between 50000 and 75000 raw reads per library, at a read length of 2x250 bp. Preprocessing, import QC and quantification of reads assigned to specific operational taxonomic units (OTUs) were carried out using the MEGAN 6 toolbox. Between 14 k and 37 k raw reads were assigned to different bacterial phyla. We then used the Phyloseq package coupled with DESeq2 to determine significant changes in the abundance of OTUs between diabetic ZDF and normal animals, and changes over time in animals treated with linagliptin vs. vehicle. Between 20 and 27 OTUs (quantified at family level) significantly changed in untreated diabetic vs. healthy rats, in agreement with previously published studies. However, we found only weak to moderate changes in the linagliptin treated vs. untreated ZDF rats (i.e., 3 different bacterial families with significant changes in abundance after 7 days). No systematic changes in the biodiversity of the gut phyla could be attributed to linagliptin. This study provides new knowledge on how linagliptin affects the microbiome of diabetic ZDF vs. healthy rats. Some of the changes attributable to linagliptin agree with published studies on the beneficial effect of probiotics in diabetes. However, the observed changes in the microbiota after a short treatment with linagliptin are rather weak compared with the important differences seen in diabetic vs. nondiabetic animals. Disclosure S. Sayols: Employee; Self; Boehringer Ingelheim International GmbH. E. Simon: Employee; Self; Boehringer Ingelheim Pharmaceuticals, Inc. H. Klein: Employee; Self; Boehringer Ingelheim International GmbH. G.G. Leparc: Employee; Self; Boehringer Ingelheim International GmbH. M. Mark: Employee; Self; Boehringer Ingelheim International GmbH. G. Luippold: Employee; Self; Boehringer Ingelheim International GmbH. T. Klein: Employee; Self; Boehringer Ingelheim International GmbH, Boehringer Ingelheim International GmbH. Funding Boehringer Ingelheim; Eli Lilly and Company

Basal insulin analogues have been designed to mimic endogenous insulin secretion for maintaining normoglycaemia. Current insulin therapy for type 2 diabetes (T2D) requires at least daily dosing for patients. However, such daily injections can have poor patient compliance. An insulin analogue with longer duration requiring less frequent administration could improve compliance and maintain better glycaemic control. To achieve extended insulin action pharmacology, a panel of recombinant native single chain (SC) insulin molecules, consisting of B chain to A chain-linker variants, were fused to an antibody fragment crystallizable region (Fc). Characterization of the single chain insulin-Fc (SC-Insulin-Fc) variants were conducted in insulin receptor (IR) and insulin-like growth factor 1 receptor (IGF1R) luminescence-linked reporter assays, and with primary rodent and human hepatocyte assays for phosphorylated AKT. SC-Insulin-Fc-1 and -2 retained potency at the human IR within 6-fold of human insulin, without specificity over IGF1R binding, and induced Akt phosphorylation within a range of endogenous insulin. SC-Insulin-Fc variants lowered blood glucose levels in DIO and db/db mice by more than 50% vs. vehicle controls (p&amp;lt;0.05) for 5 days following a single dose, significantly superior to a standard insulin bolus. Continuous glucose monitoring (CGM) telemetry to profile 24 h control of blood glucose in diabetic ZDF rats was also applied. CGM telemetry revealed enhanced basal and postprandial glucose control in both light and dark cycles (p&amp;lt;0.05 vs. vehicle) up to 10 days following a single administration (90 nmol/kg). Repeat dosing of SC-Insulin-Fc variants (10/30/60 nmol/kg, Q1W) over 5 weeks resulted in dose-dependent improvements of glycaemic control (p≤0.05 vs. vehicle) with a long term readout towards decreasing %HbA1c. This dramatic shift in time spent away from hyperglycaemia toward normoglycaemia suggests once-weekly insulin as a novel viable option to treat human T2D. Disclosure V.G. Howard: Employee; Self; AstraZeneca. Stock/Shareholder; Self; AstraZeneca. C.D. Church: Employee; Self; AstraZeneca, MedImmune. S. Oldham: Employee; Self; AstraZeneca. Stock/Shareholder; Self; AstraZeneca. D.C. Hornigold: Employee; Self; AstraZeneca. Stock/Shareholder; Self; AstraZeneca. J. Trevaskis: Employee; Self; Gilead Sciences, Inc., MedImmune. Stock/Shareholder; Self; AstraZeneca. D. Baker: Stock/Shareholder; Self; AstraZeneca. C.J. Rhodes: Employee; Self; AstraZeneca, MedImmune. A. Rossi: Employee; Self; MedImmune. J. Naylor: Employee; Self; MedImmune. Stock/Shareholder; Self; AstraZeneca. J. Dhillon: Employee; Self; MedImmune. A. Buchanan: Employee; Self; MedImmune. L. Liang: None.

196 - Metabolic stress promotes sustained activation of protein phosphatase 2A (PP2A) in the islet beta-cell: novel regulation by alpha4, a non-canonical subunit of PP2A

IMG-1 is a novel therapeutic compound for the treatment of diabetes. Ten-week-old diabetic ZDF rats were treated with IMG-1 either intravenously (IV) or per oral (PO) once a day and compared to no treatment over a thirty-five-day period. Weights and glucose levels were monitored twice a week. IMG-1 treated animals showed a marked decrease in BG levels within a week, (average BG levels of 179mg/dl for IMG-1 (IV) and 135mg/dl for PO). This finding was enduring for the entire 35-day period. Untreated controls had significantly elevated BG levels throughout the study, with average levels exceeding 400 mg/dl (481 mg/dl). Hemoglobin A1c (Hb1Ac) was measured at days 0 and 35. IMG-1 treated animals had significantly lower Hb1Ac (IV, 9.7%-&amp;gt; 7.4%) and PO (10.8% -&amp;gt;7.5%) while the untreated group maintained increased Hb1Ac levels (10.2%-&amp;gt;12.3%). There was no significant difference in insulin levels between untreated and IMG-1 treated groups at any time point suggesting IMG-1 decreased insulin resistance. Glucagon levels were reduced in both IV and PO IMG-1 treated animals at days 15 (from 115pg/ml and 119pg/ml to 90pg/ml and 92pg/ml, respectively) and 35 (89pg/ml and 92pg/ml). Control animals had no significant difference in glucagon levels throughout the entire study. Based on these results, it appears that IMG-1 is able to normalize both blood sugar in ZDF animals and lower HbA1c and decrease glucagon levels without elevating insulin levels in these animals. Disclosure J.B. Pollett: Employee; Self; Imagine Pharma. N. Thai: Board Member; Self; Imagine Pharma.

BackgroundActivation of the AMP-activated protein kinase (AMPK) is an attractive approach for the treatment of type 2 diabetes. AMPK activation reduces glucose levels in animal models of type 2 diabetes by increasing glucose uptake in skeletal muscles and reducing hepatic glucose production. Furthermore, AMPK activation ameliorates hepatic steatosis in animal models. For the clinical development of AMPK activators it is essential to have a reliable target engagement marker for appropriate dose finding and to support proof of clinical principle. While the activation of AMPK by quantification of the phosphorylation of AMPK at Thr172 in target tissues can be assessed pre-clinically, this is not feasible in clinical studies. Therefore, we attempted to identify and translate a peripheral target engagement biomarker downstream of AMPK activation for clinical use in blood samples.MethodsFor pharmacological activation of AMPK, two AMPK activators were synthesized (compound 1 and 2). A compound with structural similarities but no pharmacological effect on AMPK phosphorylation was synthesized as negative control (compound 3). Whole blood from healthy volunteers was incubated with an AMPK activator for up to 6 hours and mRNA sequencing was performed. Additionally, human PBMCs were isolated to evaluate Thr172-phosphorylation of AMPK in Western blots. In order to enable identification of translatable biomarker candidates, blood samples from HanWistar rats treated for two weeks with an AMPK activator were also subjected to mRNA sequencing. Furthermore, concentration-response curves for four biomarker candidates were recorded in human blood samples using Nanostring nCounter technology. Finally, ZDF rats were treated with increasing doses of compound 2 for five weeks to investigate the glucose-lowering efficacy. To investigate changes of mRNA expression of two selected biomarker candidates in this ZDF rat study, qRT-PCR was performed.ResultsPharmacological activation of AMPK in human PBMCs revealed an increase in Thr172-phosphorylation of AMPK, confirming target engagement in these blood cells. RNA sequencing of human blood samples identified 608 deregulated genes after AMPK activation. Additionally, AMPK activation led to deregulation of 367 genes in whole blood from HanWistar rats which mapped to the respective human genes. 22 genes out of the intersection of genes deregulated in both species are proposed as potential translatable target engagement biomarker candidates. The most prominent genes were transmembrane glycoprotein NMB (GPNMB, osteoactivin), calcium-binding protein A9 (S100A9), peptidoglycan recognition protein (PGLYRP1) and Ras homolog gene family, member B (RHOB). Specificity for AMPK was shown by testing inactive compound 3 in HanWistar rats. The exposure-effect relationship for GPNMB was investigated in a subchronic study in diabetic ZDF rats. GPNMB showed a dose-dependent up-regulation both acutely and after subchronic dosing. GPNMB up-regulation correlated with an increased Thr172-phosphorylation of AMPK in liver and quadriceps muscle in rats.ConclusionGPNMB has been identified as a translatable target engagement biomarker for use in clinical studies.

BackgroundHypoglycemia is associated with increased mortality rate in patients with diabetes. The underlying mechanisms may involve reduced myocardial tolerance to ischemia and reperfusion (IR) or reduced capacity for ischemic preconditioning (IPC). As IPC is associated with increased myocardial glucose uptake (MGU) during reperfusion, cardioprotection is linked to glucose metabolism possibly by O-linked β-N-acetylglucosamine (O-GlcNAc). We aimed to investigate the impact of hypoglycemia in hearts from animals with diabetes on myocardial IR tolerance, on the efficacy of IPC and whether modulations of MGU and O-GlcNAc levels are involved in the underlying mechanisms.MethodsIn a Langendorff model using diabetic ZDF (fa/fa) and non-diabetic (fa/+) rats (n = 6–7 in each group) infarct size (IS) was evaluated after 40 min of global ischemia and 120 min reperfusion during hypoglycemia [(glucose) = 3 mmol/l] and normoglycemia [(glucose) = 11 mmol/l]. Myocardial glucose uptake and O-GlcNAc levels were evaluated during reperfusion. IPC was induced by 2 × 5 min of global ischemia prior to index ischemia.ResultsIS increased in hearts from animals with (p < 0.01) and without (p < 0.01) diabetes during hypoglycemia compared to normoglycemia. IPC reduced IS during normoglycemia in both animals with (p < 0.01) and without (p < 0.01) diabetes. During hypoglycemia, however, IPC only reduced IS in hearts from animals with diabetes (p < 0.05). IPC increased MGU during reperfusion and O-GlcNAc levels in animals with diabetes during hypo- (MGU: p < 0.05, O-GlcNAc: p < 0.05) and normoglycemia (MGU: p < 0.01, O-GlcNAc: p < 0.05) and in animals without diabetes only during normoglycemia (MGU: p < 0.05, O-GlcNAc: p < 0.01).ConclusionsHypoglycemia increases myocardial susceptibility to IR injury in hearts from animals with and without diabetes. In contrast to hearts from animals without diabetes, the hearts from animals with diabetes are amenable to cardioprotection during hypoglycemia. In parallel with IPC induced cardioprotection, MGU and O-GlcNAc levels increase suggesting that increased MGU and O-GlcNAc levels are involved in the mechanisms of IPC.