Type 2 diabetes is associated with increased activation of brain regions that regulate sympathetic drive to the heart

S Sethi,1,2,3,4 RA Augustine,1,3,4 GT Bouwer,1,3,4 DO Schwenke,1,2 CH Brown,1,3,4 RR Lamberts1,2.

1Department of Physiology, 2HeartOtago, 3Brain Health Research Centre and 4Centre for Neuroendocrinology, University of Otago, Dunedin, NZ.

Aim

Type 2 diabetes mellitus (DM) might cause serious heart complications. The heart is partly controlled by the brain via the parasympathetic and sympathetic nervous systems, but although increased sympathetic drive to the heart is observed in DM, the brain regions responsible are unknown. Beta-blockers and other current therapies for heart complications target the heart instead of the sympathetic drive from the brain, and they are less effective in DM than non-DM individuals. We therefore aimed to determine whether DM is associated with altered neuronal activation patterns in the main brain regions regulating sympathetic drive to the heart, namely, the rostral ventrolateral medulla (RVLM), the hypothalamic paraventricular nucleus (PVN) and the nucleus tractus solitarius (NTS). We hypothesised that there would be higher activation of neurons in each of these regions in DM.

Method

In the brains of 20-week-old male DM and non-DM Zucker Diabetic Fatty rats, double-label immunohistochemistry was performed for ΔFosB (a marker of chronic neuronal activation) and tyrosine hydroxylase (TH, a marker of sympathetic neurons) in the RVLM and NTS, and for ΔFosB and either oxytocin, vasopressin or corticotrophin-releasing hormone in the PVN. Data were analysed using unpaired t-tests and all results are expressed as mean ± SEM.

Result

More activated TH-positive neurons were observed in the RVLM of DM (9±1, N=10) compared to non-DM rats (3±0, N=8; P<0.001), and more activated corticotrophin-releasing hormone-positive neurons were observed in the PVN of DM rats (DM: 74±3, n=8 vs non-DM: 59±5, N=7; P<0.05). Moreover, there was increased labelling of ΔFosB in the NTS of DM (25 ± 4, N = 10) compared to non-DM rats (15±2, N=8; P<0.05).

In conclusion, this is the first study to show that DM is associated with higher activation of neurons in brain regions regulating cardiac sympathetic drive. Understanding the alterations in central neural circuits that are activated in DM may eventually lead to more effective pharmacological therapies for diabetic heart disease.

Improved gene transfer for the treatment of neurological disease using modified viral vector AAV-PHP.eB

SN Mathiesen,1,2 L Schoderboeck,1 SM Ohline,2 HE Wicky,1 WC Abraham,2 SM Hughes.1

Departments of 1Biochemistry and 2Psychology, Brain Health Research Centre, Brain Research New Zealand—Rangahau Roro Aotearoa, University of Otago, Dunedin.

Aim

Methods that effectively deliver therapeutics to the central nervous system (CNS) are crucial for the treatment of neurological diseases. The use of adeno-associated viral (AAV) vectors for gene therapy is appealing, as some vectors can cross the blood-brain barrier, allowing them to be administered via minimally invasive methods that are feasible for translation to humans. A novel AAV vector evolved from AAV9 in-vivo, AAV-PHP.eB, has been reported to produce more effective CNS transduction than AAV9 in some strains of mice, but not in others. The present study compared the efficacy of two AAV vectors, AAV-PHP.eB and AAV9, in targeting mouse CNS and peripheral tissues after administration via various routes, and in two genetically diverse mouse strains.

Method

C57BL/6 mice were administered a combination of AAV-PHP.eB and AAV9 encoding different coloured fluorescent reporter proteins, either by intravenous injection (N=9), intranasal injection (N=6), or intrahippocampal injection (N=5). B6C3 mice (N=4) received the same vectors via intravenous injection. Four weeks after vector administration, tissue sections were examined for reporter protein expression and cell-type transduction.

Result

In C57BL/6 mice, AAV-PHP.eB led to higher brain transduction than AAV9 (P<0.05, t-test), but lower liver transduction. However, both vectors produced only minimal brain transduction in B6C3 mice. The two vectors also showed equal transduction efficiency after intrahippocampal and intranasal injection. AAV-PHP.eB transduced more neuronal than glial cells (P<0.05, t-test).

Modification of AAV vectors to improve CNS transduction may be a viable method for overcoming the challenges of invasive administration. However, there appear to be critical blood brain barrier differences, even between mouse strains, that determine the transport of vectors from the bloodstream to the CNS. These findings highlight the need for vectors modified to suit specific features of the human blood-brain barrier for treatment of human disease.

A crossover comparison of four cardiopulmonary exercise testing modalities in severe lower-limb osteoarthritis patients

BH Roxburgh,1,2 HA Campbell,1 JD Cotter,2 U Reymann,1 D Gwynne-Jones,1 MJA Williams,3 KN Thomas.1

1Department of Surgical Sciences, Dunedin School of Medicine, 2School of Physical Education, Sport and Exercise Sciences, 3Department of Medicine, Dunedin School of Medicine, University of Otago, Dunedin.

Aim

Cardiopulmonary exercise testing (CPET) is the gold standard for assessment of cardiorespiratory fitness (V̇O2peak). Preoperatively, it is used to risk stratify and guide perioperative patient care. Traditionally performed on a cycle, preoperative CPET is difficult for the approximately 10% of New Zealand adults with lower-limb osteoarthritis (OA). The arm ergometer is an alternative modality, utilising the upper body only. However, in healthy individuals, V̇O2peak can be ~30% lower, compared to cycling. The purpose of this study was to compare CPET variables and subjective responses on four different exercise modalities, in patients with OA scheduled for hip or knee arthroplasty (THA/TKA).

Method

In this crossover (within-participants) study, fourteen participants (10 female; age=68±7y; body mass index=31.4±4.3kg.m-2) scheduled for THA (N=5) or TKA (N=9) completed CPET on a cycle, treadmill (TM), cross trainer (XT) and arm ergometer (AE). V̇O2peak, peak heart rate (HRpeak), and pain scores (rated 0–10) were measured, then analysed using a repeated measures analysis of variance.

Result

V̇O2peak was greater on the TM, XT and cycle (21.7±4.7, 21.2±4.3 and 19.6±4.4 ml.min-1.kg-1, respectively) compared to AE (15.7±3.8 ml.min-1.kg-1; P=0.001). HRpeak was higher on the XT than AE (150±17 vs 137±15 beats.min-1; P=0.001), but not different to cycle or TM modalities (145 and 141 beats.min-1 respectively, P>0.48). Peak exercise pain scores were lower on AE (1.7±2.1), compared with TM, XT and cycle (4.5±3, 4.5±2.6 and 4.3±2.9; P=0.01 vs. AE).

Pain scores were higher during CPET using the lower limbs (TM, XT and cycle), compared to AE, in patients scheduled for THA/TKA. Despite this pain, V̇O2peak was higher on these lower-limb modalities, compared to AE.

An old drug for new tricks; metformin as a chemotherapeutic agent for lung cancer

AR Bland, N Shrestha, RL Bower, IA Sammut, RJ Rosengren, JA Ashton.

Department of Pharmacology and Toxicology, School of Biomedical Sciences, University of Otago, Dunedin.

Aim

Crizotinib is an effective first-line therapy for the treatment of EML4-ALK+ lung cancer. However, resistance usually develops after one year. Epidemiological studies have shown that the diabetes drug metformin was associated with a reduced incidence of cancer. Previous in vitro work also found that metformin acts synergistically with low concentrations of crizotinib to reduce cell viability in H3122 ALK+ lung cancer cells. This study aimed to examine if the in vitro synergism translated in vivo in a xenograft lung cancer model. The toxicity of the drug combination was also examined.

Method

Tumor-bearing Nu/J mice received daily administration of vehicle (olive oil), metformin (100 mg/kg), crizotinib (25 mg/kg) or the combination, orally for 14 days (6-7/group). Tumor volume was measured daily. For toxicity testing, Balb/c mice received the same treatment regimen. Full necropsies were performed following the completion of both studies.

Result

Metformin, crizotinib and the combination significantly decreased tumour volume compared to vehicle (612, 424 and 552 vs 943 mm3, respectively). It was also hypothesized that metformin would increase crizotinib concentrations via inhibition of CYP3A4, but no treatments resulted in a CYP3A4 activity change compared to vehicle. This supports the combination efficacy finding, whereby combining the drugs did not enhance tumor suppression as metformin does not increase crizotinib plasma concentrations. All treatments produced no toxicity, as shown by ALT and creatinine plasma levels under the normal threshold.

All drugs were efficacious in reducing the tumour growth rate; however, the combination had no additional therapeutic benefit compared to crizotinib alone. Nevertheless, metformin alone had a significant decrease in tumor volume compared to vehicle, proving efficacy in vivo and should be considered as a potential chemotherapeutic agent. This finding provides justification to further examine the value of metformin in cancer therapy.

Proactive provision of long-acting reversible contraception to New Zealand adolescents

R Duncan,1,2 H Paterson,2 L Anderson,1 N Pickering.1

1Bioethics Centre, 2Department of Women’s and Children’s Health, Dunedin School of Medicine, University of Otago, Dunedin.

Aim

New Zealand adolescents are at risk of unintended pregnancy by not routinely using contraceptives, or using methods with higher failure rates. Adolescents face barriers accessing most forms of contraception, including long-acting reversible contraceptive (LARC) methods. Addressing the barriers could improve uptake of effective methods of contraception, reducing the risk of unintended adolescent pregnancy. On these grounds, we investigated the concept of proactive LARC provision, with tiered contraceptive counselling. Before assessing costs and feasibility of a proactive programme, we must determine the acceptability of such a programme. To gauge acceptability, we consulted with New Zealand adolescents (recipients) and New Zealand general practitioners (GPs) (providers). Our research questions were: (1) Would proactive provision of LARCs be acceptable to adolescents? (2) Would proactive provision of LARCs to adolescents be acceptable to GPs?

Method

We consulted female adolescents in four focus groups (FGs). We discussed topics of sexual health, contraception, and specifically discussed proactive LARC provision. We performed nine GP semi-structured interviews—these began with discussion of adolescent sexual health and contraception and then we discussed proactive LARC provision. We used a general inductive thematic analysis approach to analyse transcripts.

Result

The FG and GP participants were generally positive about the concept. Emergent FG themes were reproductive health fear, sex and body shame, adolescents’ requirements for sexual health provision, barriers to contraception and sexual health knowledge. Emergent GP themes were contraceptive decision making, the GP role, sexual activity, social context, gauging adolescent understanding, and youth.

Our FGs and interviews indicate that the concept of proactive LARC provision is acceptable to both adolescents and to GPs. Proactive contraception provision to increase adolescent uptake of effective contraceptive methods is therefore a concept worth pursuing. Such an initiative would improve adolescents’ contraceptive knowledge, and could decrease unintended teenage pregnancy by empowering adolescents to control their fertility in a way that suits them.

Rheumatoid arthritis patients’ perspectives on tapering biologic therapy: a qualitative study

SJ Chan,1 LK Stamp,2 N Liebergreen,3 H Ndukwe,1 CA Marra,1 G Treharne.4

1School of Pharmacy, University of Otago, Dunedin, 2Department of Medicine, University of Otago, Christchurch, 3Children’s Issue Centre, Faculty of Law, University of Otago, Dunedin, 4Department of Psychology, University of Otago, Dunedin.

Aim

Implementation of 'treat-to-target' strategies aimed at achieving and maintaining tight disease control, and the development of biologic therapy targeting specific inflammatory mediators has made remission possible for many people with rheumatoid arthritis (RA). Based on the success of preliminary clinical trials, tapering of biologics has been increasingly advocated for the long-term management of RA patients on biologics in sustained remission. However, insights into the possibility of tapering biologics from the perspective of RA patients is lacking. This study sought to better understand RA patients’ perceptions of tapering biologics.

Method

Participants diagnosed with RA and currently taking biologics participated in six focus groups (N=43) or individual interviews (N=2). Interviews and focus group sessions were audio-recorded and transcribed verbatim. Qualitative analysis of the transcripts was carried out using general inductive thematic analysis.

Result

Five themes emerged: fear of the uncertainty of outcome, trade-offs between quality of life and the risk of adverse effects, relief from medication burden, healthcare system support and preference for involvement in decision-making. Participants acknowledged tapering their biologics would provide some relief and freedom in their daily life but were fearful of disease relapse. They were willing to accept the risk of adverse effects associated with long-term biologic treatment in exchange for a better quality of life. When considering tapering, participants wanted assurance of access to treatment and consultation if a flare occurs. Furthermore, participants would prefer to be engaged in shared decision-making with their rheumatologist when making the decision to taper biologics.

Concerns of uncontrolled disease and prompt access to treatment when disease flares are among the key issues that need to be addressed. Decision aids to support shared decision-making approach may facilitate a greater patients' understanding of the trade-offs between risks and benefits involved in tapering their biologic, leading to greater acceptance and adherence to treatment.

The effects of leptin mutations and diet-induced obesity on glucose homeostasis in the zebrafish.

K Kamstra,1 JA Horsfield,2 A Tups.1

1Department of Physiology, School of Biomedical Sciences, 2Department of Pathology, Dunedin School of Medicine, University of Otago, Dunedin, New Zealand

Aim

Leptin is classically thought to be an adipostatic signal communicating information from the adipose tissue to the brain, thereby regulating energy intake and expenditure. Recently, it has been suggested that in the zebrafish (Danio rerio), contrary to humans and other mammals, leptin does not regulate adipostasis but glucose homeostasis. From an evolutionary perspective, this suggests that leptin originated as a glucoregulatory hormone. The aim of this study was to elucidate the effects of leptin mutations and diet-induced weight gain on glucose homeostasis in the zebrafish

Method

We utilized the clustered regularly interspaced short palindromic repeats (CRISPR) system to generate knockout mutant zebrafish for both leptin-a and leptin-b, and for the leptin receptor. Six-month old male mutant fish and wild type control fish (N=11 per group) were exposed to either a six-week long overfeeding regime or normal feeding conditions. Body weights and standard length were measured weekly. Glucose tolerance tests were performed to assess changes in glucose homeostasis at the end of the feeding paradigm.

Result

Under normal feeding conditions, no effect of genotype was found on bodyweight (285±10.14mg vs 289±11.06mg) or standard length (28.79±0.97mm vs 28.94±1.06mm). Glucose tolerance on the other hand was significantly reduced in leptin receptor and leptin-a knockout zebrafish, determined by a two-way ANOVA (P<0.05). The overfeeding paradigm revealed an effect of leptin on bodyweight and somatic growth, as leptin receptor and leptin-a knockout zebrafish displayed aggravated bodyweight gain and increased standard length compared to leptin-b and wild type fish (two-way ANOVA, P<0.05).

These results show that in zebrafish, under normal feeding conditions, leptin regulates glucose homeostasis but not body weight. Only in times of nutrient excess, leptin appears to regulate body weight, somatic growth and glucose homeostasis.