Stress increases the risk of type 2 diabetes onset in women: A 12-year longitudinal study using causal modelling

<div><p>Background</p><p>Type 2 diabetes is associated with significant morbidity and mortality. Modifiable risk factors have been found to contribute up to 60% of type 2 diabetes risk. However, type 2 diabetes continues to rise despite implementation of interventions based on traditional risk factors. There is a clear need to identify additional risk factors for chronic disease prevention. The aim of this study was to examine the relationship between perceived stress and type 2 diabetes onset, and partition the estimates into direct and indirect effects.</p><p>Methods and findings</p><p>Women born in 1946–1951 (n = 12,844) completed surveys for the Australian Longitudinal Study on Women’s Health in 1998, 2001, 2004, 2007 and 2010. The total causal effect was estimated using logistic regression and marginal structural modelling. Controlled direct effects were estimated through conditioning in the regression model. A graded association was found between perceived stress and all mediators in the multivariate time lag analyses. A significant association was found between hypertension, as well as physical activity and body mass index, and diabetes, but not smoking or diet quality. Moderate/high stress levels were associated with a 2.3-fold increase in the odds of diabetes three years later, for the total estimated effect. Results were only slightly attenuated when the direct and indirect effects of perceived stress on diabetes were partitioned, with the mediators only explaining 10–20% of the excess variation in diabetes.</p><p>Conclusions</p><p>Perceived stress is a strong risk factor for type 2 diabetes. The majority of the effect estimate of stress on diabetes risk is not mediated by the traditional risk factors of hypertension, physical activity, smoking, diet quality, and body mass index. This gives a new pathway for diabetes prevention trials and clinical practice.</p></div

Comparison of sociodemographic characteristics between women with incident type 2 diabetes compared with women without a diagnosis of type 2 diabetes over the observation period.

<p>Comparison of sociodemographic characteristics between women with incident type 2 diabetes compared with women without a diagnosis of type 2 diabetes over the observation period.</p

Longitudinal associations between the hypothesised mediators and type 2 diabetes, using a time lag approach.

<p>Each analysis is adjusted for the potential confounders of SES (measured by educational attainment) and age, as well as secular trends (time by survey). The aim here is to identify the relationship between each mediator and the outcome of type 2 diabetes. The combined effect of all the mediators is modelled in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0172126#pone.0172126.t004" target="_blank">Table 4</a>.</p

Total causal effects of perceived stress on type 2 diabetes using a time lag approach (assuming physical activity as a time varying mediator).^a^,^b

<p>Total causal effects of perceived stress on type 2 diabetes using a time lag approach (assuming physical activity as a time varying mediator).<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0172126#t004fn001" target="_blank">^a</a>^,<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0172126#t004fn002" target="_blank">^b</a></p

Simplified Directed Acyclic Graph showing hypothesised causal mechanism between perceived stress and type 2 diabetes taking into account potential confounders and mediators.

<p>According to the DAG, perceived stress (measured at Survey 2) may be mediated through physical activity, diet quality (or BMI instead of diet), hypertension and smoking status and confounded by age and socioeconomic status (i.e. highest educational qualification).</p

Longitudinal associations between perceived stress and hypothesised mediators using a time lag approach.

<p>Each analysis is adjusted for the potential confounders of SES (measured by educational attainment) and age, as well as secular trends (time by survey).</p

Densitometric analysis of h-CaD and ERK2 expression in NIL and L myometrium.

<p>Densitometric analysis was performed for 8×NIL and 8×L myometrial samples following immunodetection of phospho-h-CaD, total h-CaD, phospho-ERK 1/2, total ERK 1/2 and α-SMA. (A) Onset of labor was associated with a statistically significant, 2-fold up-regulation in h-CaD phosphorylation, whilst (B) total h-CaD expression remained unchanged. (C) ERK2 phosphorylation remained unchanged following the onset of labor, however (D) total ERK2 expression underwent a statistically significant 1.4-fold up-regulation in association with the onset of labor. *p<0.05, **p<0.02.</p

Protein phosphorylation associated with myometrial contractility.

<p>Myometrial strips were snap frozen at specific stages during the development of contractions. The tissue was then pulverised, protein extracted and separated by 1D SDS-PAGE. (A) Phosphorylated myometrial proteins were labelled in gel with Pro-Q® Diamond Phospho Stain and visualised using a FujiFilm LAS-3000 luminescent image analyser. The gel was then SYPRO® Ruby Protein stained to visualise total protein and ascertain equal loading. Lanes M_P and M_T illustrate the Peppermint™ Stick molecular weight marker labelled with Pro-Q® Diamond Stain and SYPRO Ruby Protein Stain respectively. Dotted line indicates removal on none-relevant gel lanes. (B) Separated proteins were western transferred to nitrocellulose membrane. Total protein loading was visualised using SYPRO Ruby Blot Stain and equal loading across lanes confirmed. Membranes were then probed with mouse anti-phosphotyrosine (1∶2500) and anti-mouse IgG-HRP (1∶2500). The immunoreactive bands were detected using a FujiFilm LAS-3000 luminescent image analyser. Positive and negative controls were protein extracts from capacitated and non-capacitated human spermatozoa respectively. Black arrowheads and white arrowheads indicate phosphorylation events associated with contraction and relaxation respectively. (i), (ii) and (iv) highlight bands exhibiting phasic phosphorylation, whilst phosphorylation remained increased for bands (iii) and (v). Representative images shown from 3 replicates. M_r = relative molecular mass×1000. M_T = molecular weight marker stained for total protein detection, M_P = molecular weight marker stained for phospho protein detection.</p

h-CaD and ERK 1/2 phosphorylation during myometrial contractions <i>in vitro</i>.

<p>(A) Myometrial strips were snap frozen at specific stages during the development of contractions. The tissue was then pulverised and subjected to protein extraction. Protein (20 µg) was separated by 1D PAGE and transferred to a nitrocellulose membrane. (B) Membranes were probed with antibodies against phospho-h-CaD (1∶2000), total h-CaD (1∶2000), phospho-ERK 1/2 (1∶2000) and total ERK 1/2 (1∶2000), as well as α-SMA (1∶10000), representative images shown of 5 replicates. (C) Non-parametric analyses of the non-normalised raw data revealed a statistically significant 2-fold increase in h-CaD phosphorylation during the transition from point 1 to point 2. No significant difference was observed between contraction points 1 and 3 or 2 and 3. *p<0.05. M_r = relative molecular mass×1000.</p

h-CaD and ERK 1/2 expression in NIL and L term human myometrium.

<p>NIL and L myometrial protein extracts were separated by SDS-PAGE and western transferred onto nitrocellulose membrane. Membranes were probed with antibodies against phospho-h-CaD (1∶2000), total h-CaD (1∶2000), phospho-ERK 1/2 (1∶2000), total ERK 1/2 (1∶2000) and α-SMA (1∶10000). A total 8×NIL and 8×L samples were analysed. Representative image demonstrates detection of these proteins on a single blot containing 4×NIL and 4×L myometrial samples, as well as a calibrator sample that was included to allow densitometric comparison across separate blots. M_r = relative molecular mass×1000.</p