Modified redox signalling in vasculature after chronic infusion of the
insulin receptor antagonist, S961
1Kristen J Bubb, 2Rebecca H Ritchie and 1Gemma A Figtree1Cardiovascular and Thoracic Health, Kolling Institute of Medical Research, Sydney Medical School, University of Sydney, St Leonards, NSW, 2065, Australia.2Heart Failure Pharmacology Laboratory, Basic Science Domain, Baker Heart and Diabetes Institute, Melbourne, VIC, Australia.
Correspondence:Dr Kristen BubbLevel 12, Kolling Building
Royal North Shore Hospital
This article has been accepted for publication and undergone full peer review but has not been through the copyediting, typesetting, pagination and proofreading process, which may lead to differences between this version and the Version of Record. Please cite this article as doi: 10.1111/micc.12501
Background: Type 2 diabetes and associated vascular complications cause substantial morbidity and mortality. It is important to investigate mechanisms and test therapies in relevant physiological models, yet few animal models adequately recapitulate all aspects of the human condition. Objective: We sought to determine the potential of using an insulin receptor antagonist, S961, in mice for investigating vascular pathophysiology. Methods: S961 was infused into mice for four weeks. Blood glucose was monitored and insulin measured at the end of the protocol. Blood pressure and pressor responses to vasodilators were measured in cannulated mice and vascular reactive oxygen and nitrogen species were measured in isolated tissue. Results: S961 infusion induced hyperglycaemia and hyperinsulinemia. There was evidence of increased vascular reactive oxygen and nitrogen species and modification of nitric oxide-mediated signalling. Pressor responses to a nitric oxide donor were attenuated, but responses to bradykinin were preserved. Conclusions: Infusion of S961, an insulin receptor antagonist, results in the production of a mouse model of type 2 diabetes that may be useful for investigating redox signalling in the vasculature of insulin-resistant mice over the short term. It is limited by both the transient nature of the hyperglycaemia and incomplete functional analogy to the human condition.
Keywords: diabetes, endothelial nitric oxide synthase, reactive nitrogen species, superoxide, glucose
ANOVA – analysis of variance
eNOS – endothelial nitric oxide synthase
MABP – mean arterial blood pressure
Nox – NADPH (nicotinamide adenine dinucleotide phosphate) oxidase
NO – nitric oxide
SEM – standard error of the mean
SNP – sodium nitroprusside
STZ – Streptozotocin
T2D – type 2 diabetes
Vascular complications are the major factor impacting on morbidity and mortality in diabetics. These complications include atherosclerosis, which pre-disposes to myocardial infarction, stroke and peripheral vascular disease, as well as microvascular diseases and subsequent retinopathy,
nephropathy and neuropathy
. Treatments are tailored towards
the types of vascular
complications, i.e. antihypertensive therapies and statins for hypertension and atherosclerosis and anti-angiogenesis therapies for retinopathy 2. Treatment of the many co-existing conditions tends to require polypharmacy. This is associated with contraindications and morbidity3. The incidence of type 2 diabetes (T2D) has sharply risen on a global scale over the past four decades 1 and the
forecast from the latest projections are >600 million cases by 2040 4. Therefore, it is increasingly important to discover novel and superior means to address and prevent diabetic vascular complications; and pre-clinical research using physiological models is essential in this process.
T2D makes up 90-95% of diagnosed diabetes cases 5, making research into this type of diabetes particularly important; yet many pre-clinical vascular studies historically focussed on type 1 diabetic models which are produced more efficiently. There are both pharmacological and genetic models available to study T2D in large and small animals. All models have both advantages and disadvantages. Pre-clinical diabetic models do not fully recapitulate the human condition 6 and with the multiple etiologies of T2D, simplistic models are particularly troublesome. Additionally, popular models currently in use suffer from specific limitations. For example, caution must be taken when using streptozotocin (STZ) to induce insulin insufficiency due to non-specific toxicity introducing
complexities in interpretation of signalling pathways
, and increased oxidative stress, even in
animals who are not successful in reaching a diabetic stage
. Whilst genetic models, particularly
those utilising mutations in leptin signalling (i.e. ‘obese’ ob/ob and ‘diabetes’ db/db) have proven to be both reliable and to mimic the complications of T2D relatively well 10, they are not as readily available, cheap or easy to use, particularly if cross-breeding to other mutant strains is required; and have received criticism as lacking therapeutically translatable findings 11. In addition, the rapid onset of obesity in these mice, the marked blood glucose increases in db/db mutants and the specific single-locus receptor mutation, which is not akin to T2D patients, are also problematic 12.
It is increasingly evident, as with other diseases, that to ensure translation of pre-clinical findings into the clinic, multiple models of the disease should ideally be used 13. In order to investigate therapy in a convenient-to-use model of insulin resistance, we have recently used a selective insulin receptor antagonist, S961, in rabbits to investigate therapeutic options for diabetic vascular and
. This antagonist has an equally high affinity as insulin for the insulin receptor,
and completely inhibits the action of insulin 14. It has been used in rodents by others, and is now well-established as a tool to produce hyperglycemia and insulin resistance 15-17, but cardiovascular parameters have not yet been assessed. We hypothesised that S961 infusion may be a viable and simple alternative murine model for T2D. We aimed to characterise aspects of vascular function and redox signalling in mice treated with S961. We specifically aimed to investigate the impact of S961 on nitric oxide (NO) signalling, nicotinamide adenine dinucleotide phosphate (NADPH) oxidase (Nox)- dependent superoxide production and pressor responses to vasodilators, all of which are particularly relevant to clinical T2D.
Materials and Methods:
The study was approved by the Northern Sydney Local Health District Animal Ethics Committee (approval number RESP/14/278) and conforms to the National Health and Medical Research Council of Australia’s Code of Practice for the Care and Use of Animals for Scientific Purposes. Experiments were performed in male C57BL6 mice aged 8-16 weeks.
Infusion of S961
S961 was infused using Alzet osmotic minipumps (1004) implanted in the upper dorsal subcutaneous region. Initial investigations involved titrating the dose until a stable rise in blood glucose was achieved. During this pilot study, we determined the optimal dose for subsequent studies to be 0.3 mg/kg/day, as there were no significant sustained elevations in blood glucose until this dose was reached. Pumps were implanted for 4 weeks’ continuous infusion of S961 (synthesised by KareBay
Biochem Inc. USA; 96% peptide purity using published sequence and structure 9, 14). Vehicle-treated mice received a solution of sterile saline diluted 1:1 with dimethyl sulfoxide. Dimethyl sulfoxide can have direct antioxidant, anti-nociceptive and inflammatory effects, but these are negligible below 3.3 ml/kg 18. In this study, the total amount infused per day was 2.64 l, equating to 73 l over the 4- week period, which is only ~2.5 ml/kg over the duration of the study.
Blood glucose and insulin measurements
Blood glucose was measured weekly in non-fasted mice at a similar time (1400-1600 hours) chosen to reflect blood glucose levels ~8 hours after last food consumption, using a handheld glucometer and glucose strips that required <10 l of blood (Accu-check Performa, Roche, Australia). Plasma insulin was measured from samples obtained at euthanasia, after 4 weeks of S961 treatment. Venous blood samples were collected into heparinised tubes and centrifuged at 8000 x g for 5 minutes and plasma was extracted. Insulin was measured using a commercially available ELISA assay (Alpco, USA), according to manufacturer’s instructions.
Pressor responses to vasodilators
Mice were maintained under constant anaesthesia (isoflurane, 1.5%, in 100% O2 at 0.6 L/min) throughout the experiment, with respiration rate kept in the range of 80-100 breaths per minute, by minor adjustments of isoflurane level as needed. Body temperature was monitored with a rectal probe and kept constant between 36.5 – 37.0°C by adjusting a heat mat and heat lamp as required. A fluid-filled catheter was prepared using polyvinyl tubing, 0.61 mm outside diameter, tapered to 0.96 mm outside diameter tubing and was attached via 23-gauge needle to a pressure transducer (MLT0670, ADinstruments, Australia). The pressure transducer was connected via a bridge amp to a powerlab system (ADinstruments, Australia). Catheters were filled with heparinised saline (20U/ml)
and pressure transducers were calibrated daily using a manual sphygmomanometer. Catheters were advanced into the left carotid artery for arterial blood pressure measurements and recorded with Labchart 6.0 (AD Instruments, Australia). Mean arterial blood pressure (MABP) was derived from the raw trace using Labchart software. Jugular vein cannulas were implanted for intravenous administration of vasodilators 19. Bradykinin acetate (0.3–10 µg/kg; Sigma Aldrich, Australia) and sodium nitroprusside (SNP, 0.1–10 µg/kg; Sigma Aldrich, Australia) were administered into the jugular vein in ~50 µL bolus doses and MABP was evaluated throughout 20. Changes in MABP were plotted for each dose.
For analysis of superoxide anion generation, the entire mesenteric vascular bed was isolated at post- mortem and quickly dissected to remove excess fat. The tissue was homogenized in a modified phosphate lysis buffer (250 mmol/L sucrose in PBS (mmol/L: 129 NaCl, 7 Na2HPO4, 3 NaH2PO4.2H2O, pH 7.4, with protease inhibitors (cOmpleteTM EDTA-free, Roche Diagnostics). An ice-cold solution containing lucigenin (20 µmol/L N,N′-Dimethyl-9,9′-biacridinium dinitrate, Sigma Aldrich, Australia) and NADPH (100 μmol/L β-Nicotinamide adenine dinucleotide 2′-phosphate reduced tetrasodium salt hydrate; Sigma Aldrich, Australia) was added to vessel lysate in an opaque 96 well plate and the reaction was conducted at room temperature and tracked using a microplate luminometer (Veritas, Turner Biosystems, USA) with 1 sec integration time and zero delay at each well, repeatedly for 20 cycles. Plate layouts were designed so that equivalent reaction times were allowed for each sample. Average data for superoxide generation for each well was collected over the duration of the plateau phase of responses. Each vascular bed was tested in triplicate and additional replicates of each sample were treated with MnTMPyP (30 µmol/L; a cell-permeable superoxide dismutase mimetic; Merck Millipore, Australia) to determine non-specific signal. Negative control wells included assay buffer with no sample to detect background luminescence. Data were normalized to protein
concentration, which was determined by Pierce BCA assay (Thermofisher Scientific, Australia) and both background luminescence and any non-superoxide generation (signal in presence of MnTMPyP) were subtracted from each value.
Immunoblotting for protein expression
Mesenteric vessels were isolated from the experimental animals (as described above) and were snap-frozen and mechanically homogenized in ice-cold lysis buffer containing 150 mmol/L NaCl, 200 mmol/L Tris-HCl (pH 8.0), 1% Triton X-100, 0.5% deoxycholic acid, 0.1% SDS and, and protease inhibitors (cOmpleteTM EDTA-free, Roche Diagnostics). 50 μg of protein lysate was denatured and run under reducing conditions on SDS-PAGE (Bolt™ pre-cast gels and reagents, Thermofisher Scientific, Australia) and transferred onto Immobilon polyvinylidene fluoride membrane (Merck Millipore, Australia). Membranes were incubated in primary antibodies directed at determining protein expression of the following: endothelial NO synthase (eNOS, anti-eNOS, 610297; 1:1000; BD Biosciences, Australia) and phosphorylation of eNOS (anti-phosphorylated eNOS (serine 1177), 9571; 1:1000; Cell Signaling Technology, USA), which were validated using tissue from eNOS knockout mice as a negative control and recombinant eNOS protein as positive control (data not shown); Nox isoforms (anti-Nox 1, ab55831; 1:1000; Abcam, Australia; anti-Nox 2, ab129068; 1:5000; Abcam, Australia; anti-Nox-4, ab133303; 1:5000; Abcam, Australia); reactive nitrogen species (anti- nitrotyrosine, ab61392; 1:1000; Abcam, Australia); and both expression and phosphorylation of Akt (anti-Akt, 9018S, 1:1000 and anti-phosphorylated Akt [serine 473], 2938S, 1:2000, Cell Signaling Technology, USA). Specific secondary antibodies recognising rabbit or mouse primary antibodies were used (IRDye®, Licor; 1:20,000, USA). Membranes were scanned using an Odyssey imaging platform (Licor, USA), capable of detecting fluorescence of secondary antibodies at two different wavelengths (680 and 800 nm).
Data are expressed as mean ± standard error of the mean (SEM). Student’s t-test was used for comparison between two groups. For multiple comparisons, 1- or 2-way analysis of variance (ANOVA) was used with Bonferroni post-hoc analysis for multiple comparisons. A P value <0.05 was considered statistically significant.
Mice were weighed before commencement of the study protocol and at regular intervals throughout. There was no effect of S961 compared with vehicle on the body weight throughout the study and all mice maintained their pre-study weights (Table 1). Blood glucose concentrations were similar between the two groups prior to S961 infusion, but were substantially higher in S961-treated mice by the post-infusion measurements at 1 week and continued to be significantly higher in S961 mice over the first 3 weeks of infusion (Figure 1 A). During this plateau phase, S961-treated mice had blood glucose levels in the range predicted based on other models of T2D 21 and near identical to mice undergoing another model of T2D in our laboratory (STZ-high fat diet model 22, 22.81.9 (n=11) vs. citrate buffer normal chow controls of 9.60.4 (n=11); compared to S961 model 22.01.0 (n=8) vs. vehicle 9.80.9 (n=6), P>0.05 between the two diabetic models by 1-way ANOVA and P<0.001 T2D vs. control for both models). However, blood glucose levels tended to peak after 2-3 weeks and begin to drop off and eventually returned to similar levels as in vehicle-treated mice by the fourth week of infusion. Despite this, circulating insulin, which was not measured throughout the study, was substantially increased at the conclusion of the study (4-week time point) in S961 vs. vehicle- treated mice (Figure 1 B), validating this model as one of insulin resistance.
To investigate the effects of S961 on vascular redox status, Nox-dependent lucigenin-enhanced chemiluminescence was used to determine vascular superoxide generation in resistance vessels. Superoxide generation was substantially higher in mice treated with S961 compared with vehicle- treated mice (Figure 2 A) and this correlated with the severity of hyperglycaemia over the course of the treatment period (Figure 2 B). Whilst there are modifications of several enzymatic producers of superoxide in T2D, the imbalance in expression of Nox 1, 2 and 4 isoforms in vascular tissue are commonly reported to be key to the increased superoxide generation in other rodent models of diabetes 23. Therefore, we measured the expression of these Nox isoforms in resistance vasculature following S961 treatment. We found no differences in the expression of Nox 1 (Figure 3 A), Nox 2 (Figure 3 B) or Nox 4 (Figure 3 C) following S961 treatment, compared with vehicle.
Increased vascular superoxide production generally results in decreased activity of eNOS and lower NO bioavailability and this is consistently found in diabetic models 1. We assessed eNOS activity by determining the ratio of phosphorylated (at serine 1177) to total eNOS expression, which is a proven method of estimating eNOS activity 24. We found that phosphorylated eNOS was indeed lower in vessels treated with S961 (Figure 4 A, B), suggestive of reduced eNOS activity. Total eNOS expression was higher (Figure 4 A, C), which may indicate compensatory upregulation in an attempt to maintain NO production. Akt is a key regulator of eNOS phosphorylation at serine 1177 25 and is also commonly altered when insulin signalling is disrupted, and so we assessed Akt phosphorylation and total expression in the same samples. Neither total nor phosphorylated Akt were altered after S961 compared with vehicle infusion in mesenteric vessels (Figure 4 D-F).
Increased production of reactive nitrogen species is a common occurrence in vasculature of diabetics and one of the key modifications in vascular disease occurs when free radicals such as superoxide react with NO to form peroxynitrite. Corresponding with decreased eNOS
phosphorylation, we found an approximate doubling in expression of nitrotyrosine (Figure 5), which is a marker of increased reactive nitrogen species, such as peroxynitrite 26, in vessels from S961- treated mice.
In order to assess whether there were functional consequences related to the altered eNOS expression and increased superoxide generation following S961 infusion, we measured pressor responses in anaesthetised mice. Baseline MABP and heart rate were similar in mice treated with S961 or vehicle (Table 1). We gave bolus doses of a NO donor, SNP, which induced dose-dependent reductions in MABP as expected. Mice treated with S961 had significantly impaired responses to SNP (Figure 6 A-C), suggesting modified signalling downstream of NO generation. We also injected bolus doses of bradykinin acetate, which stimulates release of vasodilating substances from the endothelium of blood vessels, including NO, and results in dose-dependent decreases in MABP. Despite the increased superoxide and modified eNOS expression, we found no differences in pressor responses evoked by bradykinin in S961- compared with vehicle-treated mice (Figure 6 D-F).
The main findings of this study were that 4-week infusion of the insulin antagonist, S961, generates a murine model that mimics some aspects of other T2D models in vascular pathophysiology. Like other T2D models, vascular oxidative stress was present in this model and there was evidence of modified NO signalling and nitrosative stress. Together with substantial increases in circulating insulin this makes it a useful model for dissecting out the changes in redox signalling pathways after a relatively acute period of hyperglycaemia and insulin resistance that may occur in the early stages of T2D. However, a major limitation of the study was the lack of any evidence for a dysfunctional vascular endothelium, including no significant increase in baseline MABP and no impairment in
pressor responses when the endothelium was stimulated to release vasodilators in vivo. Therefore, the model may prove a useful complementary model to investigate vascular molecular modifications in response to altered insulin signalling, but perhaps lacks the robustness of some of the other complex models of T2D that do show substantial functional impairment.
One of the major changes we observed in the vasculature after S961 infusion was increased superoxide generation. This is consistent with other models of diabetes27-29 (Table 2) and also with human type 2 diabetic cohorts 30. Major biological sources of superoxide include the mitochondrial respiratory chain, xanthine oxidase and cytochrome P450, but perhaps the most well-characterised in the vasculature is the Nox complex, which was the focus of this study. Increased endothelial Nox 1
and 2 expression have been found in diabetes, insulin resistance and hyperglycaemia
, and are
implicated in endothelial dysfunction and hypertension34. In particular, Nox 2 has been identified as the driving force behind the excessive Nox-dependent superoxide production in T2D and insulin resistance 35. Contribution of Nox 4 to vascular superoxide production in cardiovascular disease has provided the field with challenges. It has been shown to have a protective role in vascular dysfunction, hypertension34 and atherosclerosis 36, but is the prime source of Nox that causes renal complications of diabetes 37. In diabetic vascular dysfunction, vascular Nox 4 expression is increased in an STZ/Apolipoprotein E knockout model of diabetic-induced atherosclerosis and in the db/db mouse but unchanged in an insulin receptor heterozygote (insulin resistant) model 35. We found no differences in expression of Nox 1, 2 or 4 isoforms after S961 infusion. For Nox 4, this is consistent with studies in other models of diabetes. However, for Nox 1 and 2, increased expression is common to many models. This lack of a difference may relate to the time point that we used to examine expression. Furthermore, it is feasible that Nox2 activity was increased, independently of expression, requiring translocation of the p47phox subunit to its membrane subunit p22phox. We did observe an increase in translocation of p47phox to the membrane in rabbits after S961 9, but did not
investigate it in this model. It is also possible that the increase in superoxide levels that we detected were from another source. Both mitochondrial-and non-mitochondrial sources of superoxide are common in diabetes 38 , and mitochondrial dysfunction leading to ROS generation is a strong possibility. Cytochrome P450 is another likely candidate as the source of superoxide generation. This lucigenin-based assay utilises NADPH as a crucial co-factor and it is generally considered a ‘Nox- activity’ assay. However, it has recently been proposed that it is predominantly reflective of cytochrome P450-derived rather than Nox-derived superoxide production39. We did not investigate non-Nox sources of superoxide in this study.
eNOS uncoupling and superoxide production are closely linked
. We observed
reduced eNOS phosphorylation, possibly indicative of reduced eNOS activity 43 and also evidence of insulin resistance in S961-treated mice. Given that phosphorylated eNOS is normalised to the increased eNOS expression, it may be that the activity of eNOS is not reduced as much as indicated by the phosphorylated protein expression. However, the very fact that eNOS expression was higher after S961, alludes to compensatory upregulation to preserve endothelial function, which has also
been found in type 1 diabetes
. The pro-oxidative environment following S961 treatment did not
to impact on pressor responses following endothelium-stimulation, and may be due, at least in part, to upregulated eNOS expression (Figure 7). It could also be due to compensation from other endothelium-derived factors. Upregulation of the prostacyclin pathway can contribute to prevention of endothelial dysfunction in diabetes 45, and this or upregulation of other endothelium-derived factors is likely in our study. This was not tested here but future studies may use inhibitors of NOS, such as L-NAME, to determine the NO contribution to the response. This could also be more effectively investigated in isolated vascular function studies using a series of pharmacological interventions and combinations, such as inhibitors of NOS, cyclooxygenases or purported EDHFs (i.e. TRAM-34, iberiotoxin, apamin). In any case, lack of evidence for a dysfunctional endothelium
phenotype is a key limitation of the model. Most other T2D models exhibit vascular dysfunction 46-49 (Table 2) and it is a well-established, central feature of the human disease and its complications 50-53.
The lack of functional endothelial dysfunction in the S961-treated mice was also surprising given the marked endothelial dysfunction that we saw in our rabbit model 9. The failure to maintain consistent hyperglycaemia with S961 for >3 weeks in our mice may have been a factor. It may be postulated that longer periods of hyperglycaemia in mice might achieve endothelial dysfunction. However, even very acute hyperglycaemia, such as consumption of a high sugar beverage or oral glucose tolerance test can result in impaired endothelial dysfunction in humans. Interestingly, a key outcome of a recent systematic review was that bursts of hyperglycaemia, while impairing measures such as flow-mediated dilatation (macrovascular) caused no functional deficit in the microvasculature 54 . This may partly explain the major difference between our mouse and rabbit model of S961 infusion, as only aortic (macrovascular) function was assessed in the rabbits, and it was impaired after just 7 days of S961-induced hyperglycaemia. Whereas in our mice, the in vivo pressure measurements represent contributions from both the macro and the microcirculation and these were not altered by S961 over the 4-week period where hyperglycaemia occurred for most of the time. This suggests primarily an NO-mediated functional deficit after short term hyperglycaemia as the macrovasculature relies heavily on NO for endothelium-dependent vasodilatation, whereas the microcirculation has substantial contribution from other endothelium-derived factors such as endothelium-dependent hyperpolarisation 55 and prostacyclin 56.
The fact that we found evidence consistent with increased reactive nitrogen species, likely due to peroxynitrite generation, further supports the suggestion that eNOS was upregulated to overcome
lowering of NO bioavailability
due to superoxide-induced
endothelial uncoupling (Figure 7)
Despite no endothelial dysfunction, our S961-treated mice exhibited impaired NO-induced pressor
responses. Dysfunctional NO-mediated relaxation has also been found in T2D patients 50-52, in ob/ob mice 46 and STZ-treated rats 45, the latter corresponding with decreased soluble guanylate cyclase and protein kinase G.
In establishing the S961 mouse model, we discovered that the doses used in the handful of other mouse studies varied widely. Our initial dosing took into account several other papers that obtained
stable increases in blood glucose using doses reported to be 10-20 nmol/L per week
16, 57, 58
calculated the reported doses to equate to ~0.029 µg/kg/day based on the pump model used. However, in our hands this dose was insufficient in producing any rise in glucose. We therefore commenced a pilot trial and determined the appropriate concentration to be 1000-fold higher than reported in other subcutaneous infusion studies. It was equivalent to what we previously used in a rabbit model 8, 9 after accounting for species differences 59. Our dose was also closer to the doses used in several other studies using an intraperitoneal dosing regimen. These studies used around 10- fold less per day 15, but likely achieved more efficient absorption and possibly had a higher purity formulation of S961. The final choice of S961 dose resulted in average peak blood glucose levels almost identical to another model of T2D in our hands (low-dose STZ, high fat diet) and equivalent to the range generally reported in the literature 21. The lowering of blood glucose after week 2 likely reflects beta cell expansion 16 to improve insulin production. Indeed, insulin levels were ~6-fold higher at week 4 in S961-treated mice, similar to previously reported increases after S961 infusion 15, 16. Mini-pump malfunction and drug decay were both ruled-out as contributory factors in the loss of hyperglycaemic response, as re-implantation of freshly prepared mini-pumps at the mid-point of the experiment did not prevent the blood glucose from dropping. In future studies, it may be preferable to use daily intraperitoneal injections rather than mini-pump infusions, as stable hyperglycaemia was maintained beyond 3 weeks in another protocol using twice daily intraperitoneal injections of S961 15. It is interesting to note that ob/ob mice, although being a classic model for insulin resistant
T2D and prone to a plethora of diabetic complications, are not consistently hyperglycaemic 46, 60 and this depends heavily on mouse background strain 12.
From studies in rabbits
, rats 17 and mice
15, 16 it is apparent that S961 recapitulates many aspects of
T2D, including hyperglycaemia, hyperinsulinemia, glucose intolerance and impaired glucose uptake and decreased hepatic glycogen. Interestingly, our model closely mimics the phenotype of a specific tissue insulin-receptor null model (insulin receptor expressed only in brain, liver and pancreas) 61. Like ours, the insulin-receptor null mice were void of interference in insulin signalling-mediated autophosphorylation cascades in vessels. Despite this crucial difference from other insulin resistant models (Table 1), it is important that, consistent with other models, eNOS phosphorylation was reduced after S961 as this is a likely surrogate marker of insulin resistance, and is postulated to be due to a free fatty acid-mediated impairment independent of modified Akt phosphorylation 61.
To summarise, S961-induced hyperglycaemia is a relevant model to study some aspects of redox signalling in vasculature of T2D mice. Whilst it is also useful for testing therapeutic avenues to overcome diabetic vascular dysfunction in rabbits, its usefulness in this respect may be limited in mice, at least over a four-week protocol. There is scope to improve the model by combining it with another perturbation to induce vascular dysfunction, for example high fat diet, or even combined high fat/high fructose diet, which was recently found to induce insulin resistance and T2D-associated hypertension 62. The major usefulness of S961 infusion in mice may be as a secondary model of T2D alongside another model, as it is increasingly apparent that multiple models of disease are needed to ensure greater chance of translation of promising pre-clinical findings into humans.
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The authors acknowledge Ms Charmaine Chu Wen Lo and Mr Michael Tsang for technical assistance and the National Health and Medical Research Council of Australia (GAF, RHR) and Heart Foundation (GAF) for Fellowship funding.
With the rising incidence of type 2 diabetes is it increasingly important to prevent and treat vascular complications that arise. As no animal model is perfect, it is necessary to investigate multiple models. In this study, we showed for the first time that infusion of the insulin receptor antagonist, S961, in mice results in modified vascular redox signalling, with increased levels of reactive oxygen and nitrogen species. However, not all aspects of vascular complications seen in other mouse models of type 2 diabetes were recapitulated. This may be a useful complimentary model in addition to the more robust models of type 2 diabetes, to isolate modifications due to specific insulin signalling changes.
Baseline 4-week endpoint
Treatment Vehicle S961 P Vehicle S961 P
Body weight 29.3 1.5 28.1 0.8 0.88 30.6 1.2 29.3 0.6 0.84
MABP 93 2 97 2 0.14
Heart rate 499 15 484 17 0.54
Data shown as mean SEM. Statistical analysis by Student’s t-test or 1-way ANOVA. Vehicle n = 6, S961 n = 8 mice.
STZ-based T2D models
Ob / Ob Db / Db
Hyperglycaemia ✓* ✓ ✘ & ✓ ✓
Hyperinsulinaemia ✓ ✓ ✓ ✓
Endothelial dysfunction ✘ ✓ & ✘ ✓ ✓
Decreased phosphorylated eNOS ✓ ✓ ✘ & ✓ ✓
Impaired NO-donor mediated relaxation ✓ ✓ ✓ ✘
Modified nitric oxide signalling ✓ ✓ ✓
Increased reactive nitrogen species ✓ ✓
Increased reactive oxygen species ✓ ✓ ✓ ✓
Modified NADPH oxidase signalling ✘ ✓ ✓
* occurs but may be transient; & refers to conflicting findings in the literature, where these complications have been observed in these models but have also frequently been found unchanged.