MSc Barbara Caracciolo
email: barbara.caracciolo@ki.se

MSc L Bäckman

PhD R Monastero

PhD B Winblad

PhD L Fratiglioni

The symptom of low mood in the prodromal stage of mild cognitive impairment and dementia: a cohort study of community-dwelling elderly

Keywords: dementia, depression, MCI (mild cognitive impairment), population-based, early symptoms

Objective: To investigate the symptom of low mood as a predictor of mild cognitive impairment (MCI) and its progression to dementia, taking into account: i) MCI severity, ii) time of assessment, iii) interaction with other factors.

Methods: 764 cognitively healthy elderly living in the community, from the Kungsholmen Project. Participants were assessed by direct interview to detect low mood. Subjects were then followed for six years to identify those who developed MCI. People with incident MCI were followed for a further three years to assess progression to dementia.

Results: People with low mood at baseline had a 2.7-fold (95% Confidence Interval [CI] 1.9-3.7) increased risk of developing MCI at follow-up. The association was stronger for amnestic MCI (aMCI: Hazard Ratio [HR] 5.8; 95% CI 3.1-10.9) compared to global cognitive impairment (other cognitive impairment no dementia, oCIND: HR 2.2; 95% CI 1.5-3.3).

ApoE-ε4 interacted with low mood in a synergistic fashion, increasing the risk of aMCI, while no interaction with psychiatric, vascular, frailty-related, and psychosocial factors was observed. Low mood at baseline, as opposed to low mood co-occurring with MCI, was associated with a 5.3-fold (95% CI 1.2-23.3) increased risk of progression to dementia in aMCI. In contrast, no association was found in oCIND.

Conclusion:

Low mood was more strongly associated with aMCI than with global cognitive impairment. Progression toward dementia was predicted only by low mood manifest in the prodromal stage of MCI. These findings indicate that low mood is particularly prominent in the very early stages of cognitive decline.

INTRODUCTION

Depression is the most studied neuropsychiatric feature of mild cognitive impairment and its association with cognitive deterioration has been shown both cross-sectionally and longitudinally. [START_REF] Monastero | A systematic review of neuropsychiatric symptoms in mild cognitive impairment[END_REF] Prevalence of depression is higher in MCI and dementia than in cognitively healthy subjects. [START_REF] Geda | Prevalence of neuropsychiatric symptoms in mild cognitive impairment and normal cognitive aging: population-based study[END_REF] Subclinical syndromes of depression and isolated depressive symptoms are more common than clinical depression in both MCI and dementia. Interestingly, the most prevalent symptom of depression in MCI is low mood, [START_REF] Monastero | A systematic review of neuropsychiatric symptoms in mild cognitive impairment[END_REF] whereas in dementia apathy is predominant. [START_REF] Monastero | A systematic review of neuropsychiatric symptoms in mild cognitive impairment[END_REF] Longitudinally, depressive symptoms in cognitively healthy persons have been consistently associated with the development of MCI. [START_REF] Rosenberg | Depressive symptoms predict incident cognitive impairment in cognitive healthy older women[END_REF][START_REF] Stepaniuk | Neuropsychiatric impairments as predictors of mild cognitive impairment, dementia, and Alzheimer's disease[END_REF][START_REF] Geda | Depression, apolipoprotein E genotype, and the incidence of mild cognitive impairment: a prospective cohort study[END_REF][START_REF] Barnes | Depressive symptoms, vascular disease, and mild cognitive impairment: findings from the Cardiovascular Health Study[END_REF][START_REF] Lopez | Risk factors for mild cognitive impairment in the Cardiovascular Health Study Cognition Study: part 2[END_REF] In contrast, studies on the progression of MCI toward dementia have produced conflicting evidence. Out of ten studies, [START_REF] Stepaniuk | Neuropsychiatric impairments as predictors of mild cognitive impairment, dementia, and Alzheimer's disease[END_REF][START_REF] Ramakers | Affective symptoms as predictors of Alzheimer's disease in subjects with mild cognitive impairment: a 10-year follow-up study[END_REF][START_REF] Vicini | Differential impact of apathy and depression in the development of dementia in mild cognitive impairment patients[END_REF][START_REF] Wilson | Change in depressive symptoms during the prodromal phase of Alzheimer disease[END_REF][START_REF] Panza | Impact of depressive symptoms on the rate of progression to dementia in patients affected by mild cognitive impairment. The Italian Longitudinal Study on Aging[END_REF][START_REF] Artero | Risk profiles for mild cognitive impairment and progression to dementia are gender specific[END_REF][START_REF] Houde | A predictive depression pattern in mild cognitive impairment[END_REF][START_REF] Gabryelewicz | The rate of conversion of mild cognitive impairment to dementia: predictive role of depression[END_REF][START_REF] Palmer | Predictors of progression from mild cognitive impairment to Alzheimer disease[END_REF][START_REF] Modrego | Depression in patients with mild cognitive impairment increases the risk of developing dementia of Alzheimer type: a prospective cohort study[END_REF] three found that depressive symptoms increased the risk of progressing to dementia; [START_REF] Stepaniuk | Neuropsychiatric impairments as predictors of mild cognitive impairment, dementia, and Alzheimer's disease[END_REF][START_REF] Gabryelewicz | The rate of conversion of mild cognitive impairment to dementia: predictive role of depression[END_REF][START_REF] Modrego | Depression in patients with mild cognitive impairment increases the risk of developing dementia of Alzheimer type: a prospective cohort study[END_REF] two reported an association only in women or for isolated symptoms; [START_REF] Artero | Risk profiles for mild cognitive impairment and progression to dementia are gender specific[END_REF][START_REF] Houde | A predictive depression pattern in mild cognitive impairment[END_REF] three found no association; [START_REF] Wilson | Change in depressive symptoms during the prodromal phase of Alzheimer disease[END_REF][START_REF] Panza | Impact of depressive symptoms on the rate of progression to dementia in patients affected by mild cognitive impairment. The Italian Longitudinal Study on Aging[END_REF][START_REF] Palmer | Predictors of progression from mild cognitive impairment to Alzheimer disease[END_REF] and two recent studies reported that the presence of depressive symptoms decreased the risk of developing dementia. [START_REF] Ramakers | Affective symptoms as predictors of Alzheimer's disease in subjects with mild cognitive impairment: a 10-year follow-up study[END_REF][START_REF] Vicini | Differential impact of apathy and depression in the development of dementia in mild cognitive impairment patients[END_REF] The discrepancy between studies on MCI development and studies on MCI progression may have alternative explanations. First, depressive symptoms may have a different role in different stages of the cognitive decline process. Specifically, symptoms of depression that are the expression of underlying neurodegenerative pathology [START_REF] Sun | Amyloid-associated depression: a prodromal depression of Alzheimer disease?[END_REF] may be prominent during the transition between healthy cognition and initial cognitive impairment, but lose their importance in more advanced stages of cognitive decline. Thus, depressive symptoms may be differentially related to MCI types of different severity that represent different stages of the dementing process. In amnestic MCI (aMCI), [START_REF] Petersen | Mild cognitive impairment: clinical characterization and outcome[END_REF][START_REF] Petersen | Mild cognitive impairment as a clinical entity and treatment target[END_REF] for instance, memory deficits are present in the background of otherwise preserved global cognitive functioning, whereas in other definitional categories, such as other cognitive impairment no dementia (oCIND), [START_REF] Caracciolo | Occurrence of cognitive impairment and dementia in the community: a 9-year-long prospective study[END_REF][START_REF] Larrieu | Incidence and outcome of mild cognitive impairment in a population-based prospective cohort[END_REF] the cognitive functioning is globally compromised, although not yet to a degree sufficient to fulfill the criteria for a diagnosis of dementia. Therefore, oCIND can be interpreted as a more advanced stage of cognitive impairment compared to aMCI.

Second, most studies investigating the progression of MCI to dementia have focused on depressive symptoms co-occurring with MCI. Indeed, if low mood is an early feature of dementia-related neuropathology, its onset should precede the stages when cognitive impairment becomes manifest, first as MCI and later as dementia. When MCI or dementia is detectable, depressive symptoms could also result from a subjective reaction to the cognitive problems, leading to reverse causality. Therefore, as depressive symptoms may be due to different mechanisms, they may have different odds of progressing toward dementia in people with MCI. Heterogeneity of depressive symptoms might also result in different responses to antidepressant drugs. It has been shown that depressive symptoms in MCI are particularly resistant to treatment. [START_REF] Modrego | Depression in patients with mild cognitive impairment increases the risk of developing dementia of Alzheimer type: a prospective cohort study[END_REF] For these reasons, it is important to pay special attention to the time of assessment of the depressive symptoms when studying the association with MCI and the impact on progression from MCI to dementia.

Third, depression as a prodromal feature of cognitive impairment might have characteristics that do not coincide with the typical depressive syndrome, as described by depression interviews, scales and questionnaires. Indeed, isolated depressive symptoms are common in MCI and low mood is the most prevalent symptom. [START_REF] Monastero | A systematic review of neuropsychiatric symptoms in mild cognitive impairment[END_REF] Moreover, in a recent clinically-based study, low mood predicted progression of MCI to AD better than scores from a depression scale. [START_REF] Houde | A predictive depression pattern in mild cognitive impairment[END_REF] Finally, to understand the nature of the relationship between depressive symptoms and MCI, it is important to unfold the pattern of interactions with other factors (e.g., genetic, psychiatric vascular, frailty-related and psychosocial) that are relevant for both depression and MCI and that might modify their association.

The present paper aimed at investigating the symptom of low mood as a predictor of MCI and its progression to dementia in the framework of a large community-based study of the elderly, taking into account: i) MCI severity, ii) time of assessment, iii) interaction with other factors.

METHODS

STUDY POPULATION

Data were gathered from the Kungsholmen Project, a community-based, prospective study of persons aged 75 years and above, who lived in Kungsholmen, Stockholm, Sweden, at the end of 1987. [START_REF] Fratiglioni | Occurrence of dementia in advanced age: the study design of the Kungsholmen Project[END_REF] As illustrated in Figure 1, out of 1700 participants at baseline (1987-1989) 40 persons were excluded due to very low global cognitive status or unknown educational background. To obtain a cognitively healthy cohort, we further excluded: a) prevalent dementia cases (n=225); and b) all prevalent MCI cases (n=337). Of the remaining 1098 persons, 199 (18%) died, and 135 (12%) moved or discontinued their participation before the first follow-up examination. The resulting 764 cognitively healthy cohort members were followed for six years, during which two clinical examinations took place with an interval of three years (first follow-up: 1994-1996; second follow-up: 1997-1998). To ascertain progression toward dementia, incident MCI cases were followed for three further years (third follow-up:1999-2000).

Between the first and the second follow-up examinations, 28 (30%) cases died and six (6%) refused or moved. Between the second and third follow-up examinations 23 (35%) cases died and four (6%) refused or moved.

All phases of the Kungsholmen Project have been approved by the Ethical Committee of Karolinska Institutet, and informed consent was obtained from all participants.

DATA COLLECTION

All study participants underwent a comprehensive clinical, medical, cognitive, and psychosocial examination both at baseline and at follow-ups, as described in detail elsewhere. [START_REF] Caracciolo | Occurrence of cognitive impairment and dementia in the community: a 9-year-long prospective study[END_REF][START_REF] Fratiglioni | Occurrence of dementia in advanced age: the study design of the Kungsholmen Project[END_REF] Information on medical history was taken from the inpatient register system, which encompasses all hospitals in Stockholm from 1969 onward. The International Classification of Disease, 8th revision (ICD-8) was used in the register system until 1986; [START_REF] Who | International Classification of Disease, Injuries, and Cause of Death, 8th revision (ICD-8)[END_REF] since 1987 the ICD-9th revision (ICD-9) was employed. [START_REF] Who | International Classification of Disease, Injuries, and Cause of Death, 9th revision (ICD-9)[END_REF] Information on drug use (in the preceding two weeks) and blood samples were collected at each examination.

LOW MOOD SYMPTOM

We defined the symptom of low mood as a report of perceived sadness. At baseline, nurses assessed mental health as part of a general health status interview consisting of questions with yes/no answers. The questionnaire investigated different health-related symptoms and included items on depressive symptoms, such as low mood, anxiety, feelings of loneliness, sleeping disturbances, reduced appetite, and tiredness. All of these symptoms except loss of appetite loaded on the same factor, as shown by a factor analysis with varimax rotation performed at baseline. Low mood had the highest loadings on this depression factor (0.81), followed by feeling lonely (0.70) and anxiety (0.64).

At follow-ups, all patients underwent a structured psychiatric interview performed by physicians and based on the Comprehensive Psychopathological Rating Scale (CPRS). [START_REF] Asberg | Construction of a new psychiatric rating instrument, the Comprehensive Psychopathological Rating Scale (CPRS)[END_REF] Report of perceived sadness was drawn from the CPRS interview, and was graded in degree of severity from a scale from 0 (no perceived sadness) to 6 (extreme sadness). [START_REF] Berger | The occurrence of depressive symptoms in the preclinical phase of AD: a population-based study[END_REF] The item was used both as an ordinal variable and as a categorical variable. In the latter case, the item was dichotomized into low mood, for all scores exceeding 0, versus no low mood, for a score equal to 0.

DEFINITION OF MCI

People with MCI included all nondemented participants who fulfilled amnestic MCI (aMCI) [START_REF] Petersen | Mild cognitive impairment: clinical characterization and outcome[END_REF][START_REF] Petersen | Mild cognitive impairment as a clinical entity and treatment target[END_REF] or other cognitive impairment no dementia (oCIND) [START_REF] Caracciolo | Occurrence of cognitive impairment and dementia in the community: a 9-year-long prospective study[END_REF][START_REF] Larrieu | Incidence and outcome of mild cognitive impairment in a population-based prospective cohort[END_REF] criteria. aMCI was defined according to standard criteria [START_REF] Petersen | Mild cognitive impairment: clinical characterization and outcome[END_REF][START_REF] Petersen | Mild cognitive impairment as a clinical entity and treatment target[END_REF] and operationalized according to previous research [START_REF] Caracciolo | Occurrence of cognitive impairment and dementia in the community: a 9-year-long prospective study[END_REF] as follows: 1) Memory complaints were assessed based on memory problems reported by the subjects or informants; 2) Normal general cognitive function was defined as scoring 1 SD above the age-and education-adjusted Mini Mental State Examination mean (MMSE); [START_REF] Folstein | Mini-mental state". A practical method for grading the cognitive state of patients for the clinician[END_REF] 3) Absence of dementia was verified by clinical examination; 4) Normal activities of daily living was characterised by no impairment in Katz' Activities of Daily Living scale; [START_REF] Katz | Assessing self-maintenance: activities of daily living, mobility, and instrumental activities of daily living[END_REF]

and 5)

Objective memory impairment was defined as scoring 1.5 SD below the age-and educationadjusted mean in a verbal memory task (free recall of slowly and rapidly presented words). [START_REF] Wahlin | Free recall and recognition of slowly and rapidly presented words in very old age: a community-based study[END_REF] All cases with global cognitive impairment that did not fulfil criteria for dementia were classified as oCIND [START_REF] Larrieu | Incidence and outcome of mild cognitive impairment in a population-based prospective cohort[END_REF] and operationalized according to previous research [START_REF] Caracciolo | Occurrence of cognitive impairment and dementia in the community: a 9-year-long prospective study[END_REF] as follows: 1) Impaired general cognitive function, defined as scoring 1 SD or more below age-and education-adjusted means on the MMSE derived from the dementia-free population at baseline; and 2) Absence of dementia was verified by clinical examination. aMCI and oCIND were mutually exclusive in the present study.

DIAGNOSIS OF DEMENTIA

Dementia was diagnosed according to the Diagnostic and Statistical Manual of Mental Disorders, Revised Third Edition (DSM-III-R) criteria, [START_REF]APA. Diagnostic and Statistical Manual of Mental DIsorders, Revised Third Edition (DSM-III-R)[END_REF] following a three-step procedure, as described in detail elsewhere. [START_REF] Fratiglioni | Occurrence of dementia in advanced age: the study design of the Kungsholmen Project[END_REF] The dementia diagnoses for dead subjects were made by physicians consulting clinical records and death certificates. [START_REF] Fratiglioni | Very old women at highest risk of dementia and Alzheimer's disease: incidence data from the Kungsholmen Project[END_REF] In the present study, a broad category including all dementia cases, regardless of dementia type was considered. However, Alzheimer's disease (AD) was the most common form of dementia detected, accounting for nearly 80% of all cases. COVARIATES Medical conditions derived from the inpatient register database were: history of psychosis (ICD-8 and ICD-9 codes 291-298), which included episodic mood disorders; history of cerebrovascular disease (ICD-8 and ICD-9 codes 430-438); history of heart disease (ICD-8 or ICD-9 codes 410-414 and 427-428); hip fracture (ICD-8 and ICD-9 codes 820, 821), and diabetes (ICD-8 and ICD-9 code 250). Data on diabetes were integrated with information from other sources, as descried elsewhere (32). High blood pressure was defined as ≥ 180 arterial blood pressure (i.e., systolic Korotkoff phase I and diastolic phase V). Multimorbidity was defined as having two or more chronic conditions. Polypharmacy was defined as the use of more than 5 drugs. [START_REF] Monastero | Heterogeneity in risk factors for cognitive impairment, no dementia: population-based longitudinal study from the Kungsholmen Project[END_REF] Psychotropic drugs use was defined as using one versus none of the following psychoactive drugs: neuroleptics (ATC code: N05A), anxiolytics (ATC code: N05B), other tranquilizers, hypnotics (ATC code: N05C), and antidepressants (ATC code: N06A). APOE genotyping was carried out using a standard polymerase chain reaction (34) and dichotomized as having at least one ApoE-ε4 allele versus having none. Social network was coded according to previous research as a four-level ordinal variable (extensive/moderate/limited/poor social network). [START_REF] Fratiglioni | Influence of social network on occurrence of dementia: a community-based longitudinal study[END_REF]

DATA ANALYSIS

INCIDENCE OF MCI AMONG COGNITIVELY HEALTHY PEOPLE. The cognitively healthy cohort was followed for six years to detect new cases of MCI. Subjects were considered at risk of MCI until they i) were classified for the first time as MCI or demented;

ii) dropped-out of the study due to refusal or moving; or iii) died.

Differences in subject characteristics were tested with Pearson's Chi Square. Independent Cox models were used to estimate the Hazard ratio (HR) and 95% confidence intervals (CI) of developing MCI in relation to baseline low mood (baseline LM). For cases of MCI detected at the first wave of examinations, the baseline for low mood exposure was set at the study baseline. For cases of MCI detected at second wave of examinations, the baseline for low mood exposure was set at the second wave (first follow-up). The relative risks of MCI in relation to baseline low mood were comparable between the two waves, therefore Cox regression analysis was performed using data from both waves. In order to preserve the threeyear follow-up exposure for MCI, baseline LM and all other variables varying with time were entered as time-dependent variables. Low mood co-occurring with incident MCI (follow-up LM) was used as a covariate. Multiplicative interactions were tested within Cox regression.

To test for additive interactions, the attributable proportion (AP) due to interaction was calculated together with the 95% CI. [START_REF] Padyukov | A gene-environment interaction between smoking and shared epitope genes in HLA-DR provides a high risk of seropositive rheumatoid arthritis[END_REF] INCIDENCE OF DEMENTIA AMONG COGNITIVELY HEALTHY PEOPLE. Dementia developed in cognitively healthy people not previously classified as MCI was used as a competitive outcome and the relation with low mood was analyzed with the same methodology described above. EVOLUTION OF MCI. Persons who developed MCI were followed for three further years to assess their progression toward dementia. Independent Cox regression models were run to evaluate the HR and 95% CI of dementia in subjects with MCI in relation to baseline and follow-up low mood. Adjustment and interactions with other covariates were tested as described above.

Missing values were imputed with Multiple Imputation based on available information on other covariates, 37 using Stata 9.0. All other analyses were performed using SPSS, with an alpha level of p < 0.05. 1. Fifty-three percent (n=21) of aMCI and 31 percent (n=37) of oCIND occurred in people with baseline LM. When considering aMCI and oCIND together, the incidence of MCI in people with baseline LM was about 2.5 times higher than that detected among people without baseline LM, and this ratio was constant across the two waves of examinations (Figure 2). Cox regression analysis performed using data from both waves confirmed the increased risk of all outcomes in relation to the presence of baseline LM, although the association was stronger for aMCI. Adjustment for socioeconomic characteristics and other covariates, including follow-up LM, history of psychosis, psychotropic drug use, ApoE-ε4 allele, history of cerebrovascular disease, heart disease, diabetes, high blood pressure, hip fracture, multimorbidity, and polypharmacy, did not substantially change the results and HRs remained stable even after multi-adjustment (Table 2).

RESULTS

INCIDENCE

Stratified analyses showed that the increased risk of aMCI and oCIND associated with baseline LM was not substantially modified by follow-up LM nor by the other factors (as listed above). However, an additive interaction was found between baseline LM and ApoE-ε4 allele for the risk of developing aMCI (Attributable Proportion: 0.5, 95% CI: 0.1-0.96) (Figure 3), but not for the risk of developing oCIND. No other interactions were observed. INCIDENCE OF DEMENTIA AMONG COGNITIVELY HEALTHY PEOPLE. During the follow-up, 158 cognitively healthy persons developed dementia by-passing MCI. In these people, the HR for low mood was 1.6 (95% CI 1.1-2.3) after adjustment for age, sex, and, education. Further adjustment for the other covariates did not affect the relationship with baseline LM. However, the association was no longer significant after adjustment for followup LM (HR 1.4, 95% CI 0.9-2.0) and in the multi-adjusted model (HR 1.3, 95% CI 0.9-1.9). EVOLUTION OF MCI. During the 1354.8 person-years (minimum 0.7; maximum 4.5) of follow-up, 50 people with MCI progressed to dementia. Eleven cases of dementia occurred in people with aMCI and 39 cases of dementia occurred in people with oCIND. Among the aMCI persons who progressed to dementia, eight (70%) had baseline LM, detected before the development of aMCI, and three (27%) had follow-up LM, which was detected at the time of aMCI classification. Among the oCIND that progressed to dementia, 14 (33%) had baseline LM and six (15%) had follow-up LM. Cox regression analysis confirmed the association of baseline LM with an increased risk of progression to dementia in people with aMCI (Table 3).

The association was strengthened after further adjustment for ApoE-ε4 allele. Adjustment for other factors did not substantially change the association. On the other hand, follow-up LM was not associated with an increased risk of progression to dementia in people with aMCI (Table 3). Progression to dementia in people with oCIND was linked neither to baseline nor to follow-up LM (Table 3).

DISCUSSION

In our large, prospective, community-based study, low mood reported three years before MCI detection substantially increased the risk of developing MCI. In particular, low mood was associated with a 5.8-fold increased risk of aMCI, a 2.2-fold increased risk of oCIND, and a 2.7-fold increased risk of All-MCI, after adjustment for sociodemographic factors. These findings are in line with previous evidence from other longitudinal studies, which found an association between baseline depressive symptoms and incident MCI, with estimates ranging from a 1.2 to a 16-fold increased risk of MCI. [START_REF] Rosenberg | Depressive symptoms predict incident cognitive impairment in cognitive healthy older women[END_REF][START_REF] Stepaniuk | Neuropsychiatric impairments as predictors of mild cognitive impairment, dementia, and Alzheimer's disease[END_REF][START_REF] Geda | Depression, apolipoprotein E genotype, and the incidence of mild cognitive impairment: a prospective cohort study[END_REF][START_REF] Barnes | Depressive symptoms, vascular disease, and mild cognitive impairment: findings from the Cardiovascular Health Study[END_REF][START_REF] Lopez | Risk factors for mild cognitive impairment in the Cardiovascular Health Study Cognition Study: part 2[END_REF] The excess risk of aMCI associated with low mood was almost triple that for oCIND (HR 5.8 versus 2.2), suggesting the presence of a gradient in the relationship between low mood and cognitive impairment severity. Indeed, in our study the definition of amnestic MCI excluded people with global cognitive impairment (MMSE mean=26). On the other hand, oCIND included more severely impaired cases with global cognitive impairment (MMSE mean=21), who did not fulfill criteria for dementia. In addition, we also observed a group of people that rapidly progressed to dementia by-passing MCI (MMSE mean=8), for whom the excess risk associated with low mood was 60%, lower than that observed in both aMCI and oCIND.

Interestingly, we found a synergistic action of low mood and ApoE-ε4, with increased risk of aMCI in people with both baseline low mood and at least one ApoE-ε4 allele. This is in line with results from a study on primary care patients [START_REF] Geda | Depression, apolipoprotein E genotype, and the incidence of mild cognitive impairment: a prospective cohort study[END_REF] and supports the hypothesis that low mood may be related to AD-type neuropathology. [START_REF] Sun | Amyloid-associated depression: a prodromal depression of Alzheimer disease?[END_REF][START_REF] Kim | The role of apolipoprotein E in Alzheimer's disease[END_REF] Conversely, none of the other factors considered in our investigation interacted with low mood. In particular, no modification of the relationship of baseline low mood with MCI was observed by taking into account history of psychosis, psychotropic drug use, and vascular factors. Indeed, low mood in prodromal MCI can be independent from history of depression. [START_REF] Geda | Depression, apolipoprotein E genotype, and the incidence of mild cognitive impairment: a prospective cohort study[END_REF] It has also been observed that depressive symptoms in MCI are particularly resistant to treatment. [START_REF] Modrego | Depression in patients with mild cognitive impairment increases the risk of developing dementia of Alzheimer type: a prospective cohort study[END_REF][START_REF] Bhalla | Patterns of mild cognitive impairment after treatment of depression in the elderly[END_REF] On the other hand, the lack of interaction of low mood with vascular factors confirms previous findings from the Cardiovascular Health Study and does not support the "vascular depression hypothesis". [START_REF] Barnes | Depressive symptoms, vascular disease, and mild cognitive impairment: findings from the Cardiovascular Health Study[END_REF] Low mood was associated with a 5.3-fold increased risk of progression to dementia in people with aMCI. This finding is in line with some progression studies of MCI [START_REF] Stepaniuk | Neuropsychiatric impairments as predictors of mild cognitive impairment, dementia, and Alzheimer's disease[END_REF][START_REF] Artero | Risk profiles for mild cognitive impairment and progression to dementia are gender specific[END_REF][START_REF] Houde | A predictive depression pattern in mild cognitive impairment[END_REF][START_REF] Gabryelewicz | The rate of conversion of mild cognitive impairment to dementia: predictive role of depression[END_REF][START_REF] Modrego | Depression in patients with mild cognitive impairment increases the risk of developing dementia of Alzheimer type: a prospective cohort study[END_REF] and is at odds with others. [START_REF] Ramakers | Affective symptoms as predictors of Alzheimer's disease in subjects with mild cognitive impairment: a 10-year follow-up study[END_REF][START_REF] Vicini | Differential impact of apathy and depression in the development of dementia in mild cognitive impairment patients[END_REF][START_REF] Wilson | Change in depressive symptoms during the prodromal phase of Alzheimer disease[END_REF][START_REF] Panza | Impact of depressive symptoms on the rate of progression to dementia in patients affected by mild cognitive impairment. The Italian Longitudinal Study on Aging[END_REF][START_REF] Palmer | Predictors of progression from mild cognitive impairment to Alzheimer disease[END_REF] Indeed, most of the studies that found no or inverse association focused on depressive symptoms measured at the same time of MCI detection. [START_REF] Ramakers | Affective symptoms as predictors of Alzheimer's disease in subjects with mild cognitive impairment: a 10-year follow-up study[END_REF][START_REF] Vicini | Differential impact of apathy and depression in the development of dementia in mild cognitive impairment patients[END_REF][START_REF] Panza | Impact of depressive symptoms on the rate of progression to dementia in patients affected by mild cognitive impairment. The Italian Longitudinal Study on Aging[END_REF][START_REF] Palmer | Predictors of progression from mild cognitive impairment to Alzheimer disease[END_REF] We showed that only low mood measured at baseline, three years before the detection of MCI, predicted subsequent progression of MCI to dementia. Conversely, low mood that co-occurred with MCI did not predict further progression to dementia. This results support the hypothesis that the symptom of low mood may be relevant in the prodromal stage of MCI, losing its importance in more advanced stages, when the cognitive deficits are already manifested. Our findings do not confirm previous evidence from a report based on the Religious Order Study, [START_REF] Wilson | Change in depressive symptoms during the prodromal phase of Alzheimer disease[END_REF] which found no increase in depressive symptoms in the prodromal stage of dementia. Indeed, the generalizability of those results has been questioned, as religious order people might have higher resilience against depressive symptoms. [START_REF] Modrego | Depression in the prodromal phase of Alzheimer disease and the reverse causal hypothesis[END_REF] Our study has many strengths, including the community-based design; the longitudinal assessment of the relationship between low mood and cognitive impairment; the comparison of the impact of low mood on different types and degrees of severity of cognitive impairment; the comparison of different times of assessment of low mood in relation with the progression of MCI to dementia; and the thorough assessment of the interaction of low mood with other factors. However, there are some limitations. First, it is difficult to disentangle the directionality of the association between low mood and cognitive impairment even in the framework of a longitudinal study. Reverse causation is always possible. Indeed, people might have performed poorly in the cognitive tests because they were depressed at the time of assessment. This could have generated a spurious association with baseline low mood if those people with low mood at time of MCI detection were already depressed at baseline. However, we controlled for this possibility and found no evidence of a confounding between baseline low mood and follow-up low mood. Second, the assessment of low mood was performed in different settings between the first and the second wave of examinations. Nonetheless, the figures regarding the occurrence of MCI in relation to low mood at the two measuring occasions are very similar, pointing to a substantial stability of the construct of low mood across different measurements. Third, grading of severity of low mood was not available for all participants. However, available information showed that the symptom was mild in most (91%) of the people with low mood who developed MCI. Fourth, the cross-sectional assessment of low mood did not provide any information on duration. Nonetheless, the majority of people with MCI who reported low mood at baseline were euthymic on the subsequent three-year follow-up examination. Fifth, we used dementia as an outcome rather than dementia type. This was done considering the relatively small sample of MCI persons who progressed to dementia. However, in our population nearly 80% of all diagnosis of dementia was classified as probable or possible AD. Future investigations should try to expand our findings using dementia type as an outcome. Finally, in the present cohort there was a group of people who developed dementia without previously being classified as MCI. It is likely that the association between low mood and cognitive decline would have been further strengthened by using a follow-up interval short enough to observe the transitional stage of MCI in all the subjects who developed dementia.

Our results showed that low mood is a predictor of MCI development and its progression towards dementia, and confirmed our working hypothesis that low mood may be more closely related to early, rather than advanced, stages of cognitive decline. There are different possible explanations to this finding. One explanation would be that low mood is a risk factor for cognitive impairment and particularly memory functioning, which would likely be mediated by the interplay between psychosocial stress and the activity of the adreno-cortico-axis, which imbalance has known effects on neurogenesis and hippocampal physiology. [START_REF] Videbech | Hippocampal volume and depression: a meta-analysis of MRI studies[END_REF] Another possible explanation would be that low mood and MCI share a common neuropathogenic substrate. In this case the narrow time frame that we observed for the relationship between low mood and cognitive decline would be explained by the stage of the underlying neurodegenerative process. In earlier stages, when cognitive deterioration is not yet manifest, low mood could be one behavioral sign of neurodegeneration, as in the concept of "amyloidassociated depression". [START_REF] Sun | Amyloid-associated depression: a prodromal depression of Alzheimer disease?[END_REF] In more advanced stages, when the neurodegeneration has become more pervasive, MCI could represent a later manifestation of the process leading to dementia.

In conclusion, although further studies are needed to disentangle the relative contribution of possible underlying mechanisms, cognitively healthy elderly presenting with the symptom of low mood should be closely monitored as they are at increased risk of MCI and subsequent progression to dementia.

  OF MCI AMONG COGNITIVELY HEALTHY PEOPLE. During the 3711.5 person-years (minimum 1.2; maximum 8.2) of follow-up, 160 persons developed MCI. Of these, 40 were classified as aMCI and 120 as oCIND. Characteristics of the cohort and the cases are shown in Table

Table 3 .*

 3 Hazard ratios (HRs) and 95% confidence intervals (95% CI) for three-year progression of amnestic mild cognitive impairment (aMCI), other cognitive impairment no dementia (oCIND), and overall mild cognitive impairment (All-MCI) to dementia in relation to low mood three years before detection of cognitive impairment (baseline LM) and to low mood reported at the time of detection of cognitive impairment (follow-up LM). Adjusted for: age, sex, education. **Adjusted for: age, sex, education, and ApoE-ε4 allelic status.

Table 1 .

 1 Study population and incident cases of amnestic mild cognitive impairment (aMCI), other cognitive impairment no dementia (oCIND), and overall mild cognitive impairment (All-MCI) by age, gender, and education.

		Study		Incident
		population		cases
			aMCI	oCIND	All-MCI
			n=40	n=120	n=160
	Subjects characteristics
	Age, baseline
	75-79	367 (48)	19 (48)	61 (51)	80 (50)
	80+	397 (52)	21 (52)	59 (49)	80 (50)
	Gender
	Women	576 (75)	29 (73)	92 (77)	121 (76)
	Men	188 (25)	11 (28)	28 (23)	39 (24)
	Education
	High	403 (53)	27 (68)	55 (46)	82 (51)
	Low	361 (47)	13 (32)	65 (54)	78 (49)

Table 2 .

 2 Hazard ratios (HRs) and 95 percent confidence intervals (95% CI) of amnestic mild cognitive impairment (aMCI), other cognitive impairment no dementia (oCIND), and overall mild cognitive impairment (All-MCI) in relation to baseline low mood (three years before MCI detection). **Simultaneous adjustment for age, sex, education, history of psychosis, psychotropic drug use, ApoE-ε4 allele, history of cerebrovascular disease, heart disease, diabetes, high blood pressure, hip fracture, multimorbidity, and polypharmacy.

	aMCI	oCIND	All-MCI
	n=40	n=120	n=160
	HR	HR	HR
	(95% CI)	(95% CI)	(95% CI)

*Adjusted for: age, sex, education. **Adjusted for: age, sex, education, and low mood assessed at the same time of MCI detection (follow-up LM). *

Acknowledgements:

We thank all staff of the Kungsholmen Project for their collaboration in data collection and management.


Aleksandra Vergles Rataj

Andrej Bidovec

Diana Žele

Gorazd

Aleksandra Vergles

Gorazd Vengušt
email: gorazd.vengust@vf.uni-lj.si

F.-J Kaup

Echinococcus multilocularis in the red fox (Vulpes vulpes) in Slovenia

Keywords: Zoonosis, Tapeworm, Echinococcus, Fox

published or not. The documents may come

in the red fox () in Slovenia

Introduction

Human alveolar echinococcosis (AE) represents a zoonotic infection which is usually lethal if left untreated. It is caused by the fox tapeworm, Echinococcus multilocularis (Leuckart 1863) [START_REF] Eckert | Biological, epidemiological, and clinical aspects of echinococcosis, a zoonosis of increasing concern[END_REF]. Its life circle in Europe is predominantly sylvatic, involving the red fox as the main definitive host [START_REF] Romig | The present situation of echinococcosis in Europe[END_REF]). The presence of E. multilocularis in red foxes has been detected in many European countries, including Slovenia's neigh-bours Austria, Hungary, and Italy, but not Croatia [START_REF] Manfredi | Echinococcus multilocularis infection in red foxes in Italy[END_REF][START_REF] Vervaeke | A survey of intestinal helminths of red foxes (Vulpes vulpes) in northern Belgium[END_REF][START_REF] Bagrade | Echinococcus multilocularis is a frequent parasite of red foxes (Vulpes vulpes) in Latvia[END_REF][START_REF] Borecka | Occurrence of Echinococcus multilocularis in red foxes (Vulpes vulpes) in southern Poland[END_REF][START_REF] Romig | Echinococcus multilocularis in Europe-state of the art[END_REF].

Other canids such as wolves, raccoon dogs, coyotes, and wild cats can also be definitive hosts of E. multilocularis [START_REF] Eckert | Biological, epidemiological, and clinical aspects of echinococcosis, a zoonosis of increasing concern[END_REF][START_REF] Kern | Risk factors for alveolar echinococcosis in humans[END_REF]). Several species of small rodents that serve as intermediate hosts become infected by ingesting the tapeworm eggs [START_REF] Smith | Prevalence of zoonotic important parasites in the red fox (Vulpes vulpes) in Great Britain[END_REF][START_REF] Kern | Risk factors for alveolar echinococcosis in humans[END_REF]. Domestic dogs and cats can also act as the definitive hosts of E. multilocularis and may therefore be the most important source of infection for people [START_REF] Deplazes | Echinococcus multilocularis coproantigen detection by enzymelinked immunosorbent assay in fox, dog, and cat populations[END_REF][START_REF] Gottstein | Is high prevalence of Echinococcus multilocularis in wild and domestic animals associated with disease incidence in humans?[END_REF][START_REF] Seimenis | Overview of the epidemiological situation on echinococcosis in the Mediterranean region[END_REF].

Transmission to humans requires ingestion of the parasite eggs in contaminated food, water, soil, or animal fur [START_REF] Kern | Risk factors for alveolar echinococcosis in humans[END_REF][START_REF] Borecka | Occurrence of Echinococcus multilocularis in red foxes (Vulpes vulpes) in southern Poland[END_REF].

Typical histological lesions on liver and lungs caused by E. multilocularis were reported for the first time in Slovenian cattle in 1966 [START_REF] Šenk | Finding of hydatidosis Echinococcus multilocularis veterinarum in cattle[END_REF]. [START_REF] Brglez | Metacestod Echinococcus multilocularis (Leuckart, 1863; Vogel, 1955) in Apodemus flavicollis (Melchior) in Slovenia[END_REF] also confirmed the presence of the larval form of this tapeworm in the abdominal cavity and liver of a rodent Apodemus flavicollis (Melchior 1834), while [START_REF] Logar | Human alveolar echinococcosis in Slovenia[END_REF] in the years 2001-2005 reported a 0.7% prevalence of AE in humans.

Foxes have been present in Slovenia for centuries and inhabit the entire country with an annual catch of approximately 8,000. Despite hunting, the population density of red fox in Slovenia has, due to successful fox vaccination against rabies, increased during the last decade.

The aim of the presented work was to evaluate the prevalence of E. multilocularis in Slovenia, since no systematic studies on the fox have been conducted in this area before.

Material and methods

Locations and sampling

During hunting seasons between 2002 and 2005, the carcasses of 428 foxes were collected as road casualties, or as a part of the oral rabies vaccination control programme throughout Slovenia. Samples were collected from four different regions: Central Slovenia (n=160), South East Slovenia (n=86), East Slovenia (n=109), and West Slovenia (n=73). Carcasses were labelled and stored in individual plastic bags at 4°C with the necropsy performed within 24 h. Before parasitological examination, the intestine was isolated, removed from the abdominal cavity, and stored for at least 4 days at -80°C.

Parasitological investigation

The presence of E. multilocularis was diagnosed using the technique described by [START_REF] Hofer | High prevalence of Echinococcus multilocularis in urban red foxes (Vulpes vulpes) and voles (Arvicola terrestris) in the city of Zürich, Switzerland[END_REF]. The adult parasites were preserved in 70% ethanol. Their identification was based on the microscopic investigation, examining the size and morphology of E. multilocularis [START_REF] Soulsby | Helminths, arthropods and protozoa of domesticated animals, 6th edn[END_REF][START_REF] Boch | Krankheiten des jagdbaren Wildes[END_REF][START_REF] Ballek | Zum Vorkommen von Echinococcus multilocularis und anderen Zestoden und Nematoden beim Rotfuchs (Vulpes vulpes L.) in den Regierungsbezirken Arnsberg[END_REF].

Results

A total of 428 fox intestines were collected from different hunting areas in Slovenia between the years 2002 and 2005. Adult tapeworms were discovered in 11 foxes (2.6%). The highest prevalence 7% (6/86) was detected in South east Slovenia, 1.3% (2/160) in Central Slovenia, 1.8% (2/109) in East Slovenia, and 1.4% (1/73) in West Slovenia. Five animals were infected with more than 1,000 adult worms; in the other six animals, two to 22 adult tapeworms were respectively confirmed. The confidence intervals (CI) were calculated for each region as well as for Slovenia as a whole (Table 1).

Discussion

Red foxes are an important game species in Slovenia, with the hunting bag of around 8,000 foxes per year. According to the statistical data (Statistical Office of the Republic of Slovenia 2008), the hunting-related reduction of foxes in Slovenia has been increasing gradually in the past few years. However, despite that fact fox population is still growing. The increase in the fox population is probably a consequence of initiation of the antirabies vaccination programme [START_REF] Chautan | Role of rabies in recent demographic changes in red fox (Vulpes vulpes) populations in Europe[END_REF][START_REF] Eckert | Echinococcosis: an emerging or re-emerging zoonosis?[END_REF][START_REF] Gloor | The rise of urban fox populations in Switzerland[END_REF]) that has been implemented in Slovenia since 1995 [START_REF] Hostnik | Control of rabies in Slovenia[END_REF]). An increase in the fox population has been reported across the European countries [START_REF] Eckert | Echinococcosis: an emerging or re-emerging zoonosis?[END_REF][START_REF] Manfredi | Echinococcus multilocularis infection in red foxes in Italy[END_REF][START_REF] Sréter | Echinococcus multilocularis in Northern Hungary[END_REF][START_REF] Borecka | Occurrence of Echinococcus multilocularis in red foxes (Vulpes vulpes) in southern Poland[END_REF].

Foxes have a significant role in spreading the E. multilocularis as the definitive hosts. Due to the unknown situation of E. multilocularis in foxes in Slovenia, the monitoring programme has been implemented over the past 4 years. The diagnostic procedures using scraping, sedimentation, and counting techniques are the most reliable methods, but they can be carried out only post-mortem, which may cause some trouble in collecting the samples. It also demands safety precautions [START_REF] Deplazes | Molecular tools for studies on the transmission biology of Echinococcus multilocularis[END_REF].

Parasitological investigation in our study confirmed the presence of adult E. multilocularis in 11/428 (2.6%) foxes in Slovenia within four regions. The prevalence was lower in comparison to Switzerland (44.3%) [START_REF] Hofer | High prevalence of Echinococcus multilocularis in urban red foxes (Vulpes vulpes) and voles (Arvicola terrestris) in the city of Zürich, Switzerland[END_REF], Poland (20.1%) [START_REF] Borecka | Occurrence of Echinococcus multilocularis in red foxes (Vulpes vulpes) in southern Poland[END_REF], Slovak Republic (10.6%) [START_REF] Letková | The red fox (Vulpes vulpes L.) as a source of zoonoses[END_REF], Belarus (7.5%) [START_REF] Shimalov | Helminth fauna of the red fox (Vulpes vulpes Linnaeus, 1758) in southern Belarus[END_REF], Estonia (29.4%) [START_REF] Moks | Echinococcus multilocularis in Estonia[END_REF], Germany, Southern Bavaria (80%) [START_REF] König | Drastic increase in the prevalence of Echinococcus multilocularis in foxes (Vulpes vulpes) in southern Bavaria, Germany[END_REF], and Northern Germany (average provincial prevalence 19.7%) [START_REF] Berke | Emergence of Echinococcus multilocularis among red foxes in northern Germany, 1991-2005[END_REF], while in Belgium a 1.8% prevalence was confirmed [START_REF] Vervaeke | A survey of intestinal helminths of red foxes (Vulpes vulpes) in northern Belgium[END_REF].

The data from neighbouring countries (Austria, Italy, and Hungary) revealed the presence of E. multilocularis [START_REF] Duscher | Echinococcus multilocularis in Austrian Foxes from 1991 until 2004[END_REF][START_REF] Casulli | Echinococcus multilocularis in red foxes (Vulpes vulpes) of the Italian Alpine region: is there a focus of autochthonous transmission?[END_REF][START_REF] Sréter | Echinococcus multilocularis in Northern Hungary[END_REF], while in Croatia [START_REF] Rajković-Janje | Prevalence and seasonal distribution of helminth parasites in red foxes (Vulpes vulpes) from the Zagreb County (Croatia)[END_REF], the presence of E. multilocularis was not reported. According to the latest records, the known range of this parasite has extended across Europe [START_REF] Romig | Echinococcus multilocularis in Europe-state of the art[END_REF]; therefore, the monitoring programmes in regions of former Yugoslavia could contribute valuable data on the presence of this parasite.

In our research, five foxes were heavily infected with more than 1,000 adult tapeworms, four of them originating from South East Slovenia, while in six foxes the presence of only two to 22 adults was confirmed. The distribution of positive diagnosed cases was expected, as the majority of animals originated from the area with the highest population density. As was previously reported [START_REF] Shimalov | Helminth fauna of the red fox (Vulpes vulpes Linnaeus, 1758) in southern Belarus[END_REF][START_REF] Sréter | Echinococcus multilocularis in Northern Hungary[END_REF][START_REF] Moks | Echinococcus multilocularis in Estonia[END_REF][START_REF] Takumi | Evidence for an increasing presence of Echinococcus multilocularis in foxes in The Netherlands[END_REF], the number of tapeworms in our research varies significantly among the individual animals. This is the first report on E. multilocularis in the territory of former Yugoslavia and even in South Eastern Europe. Up to now, the parasite was not discovered in red foxes in Slovenia, but AE has already been reported in cattle [START_REF] Šenk | Finding of hydatidosis Echinococcus multilocularis veterinarum in cattle[END_REF] and in rodents A. flavicollis [START_REF] Brglez | Metacestod Echinococcus multilocularis (Leuckart, 1863; Vogel, 1955) in Apodemus flavicollis (Melchior) in Slovenia[END_REF]. In the years 2001-2005[START_REF] Logar | Human alveolar echinococcosis in Slovenia[END_REF] reported a 0.7% prevalence of AE in humans in Slovenia and suggested that dogs and cats might be a possible source of this infection [START_REF] Romig | Echinococcus multilocularis in animal hosts: new data from western Europe[END_REF][START_REF] Romig | Epidemiology of echinococcosis[END_REF][START_REF] Thompson | Comparative development of Echinococcus multilocularis in its definitive hosts[END_REF]. The general role of domestic cats and dogs in the epidemiology of the infection has not yet been studied in Slovenia; additional studies on this topic are therefore required. Since other European countries have already tested the presence of E. multilocularis in red fox, similar results were expected in our research. E. multilocularis was recently confirmed also in the Baltic states, in most of Belgium, in the south and west of The Netherlands, northern Italy, eastern Austria, and in northern Hungary [START_REF] Romig | Echinococcus multilocularis in Europe-state of the art[END_REF]. Although with low prevalence, the presence of E. multilocularis might present a potential threat for AE in humans. In all the countries where the parasite shows the tendency of spreading, a continuous monitoring programme should be considered.

Acknowledgements The research was funded with a grant from the Veterinary Administration of the Republic of Slovenia. The authors gratefully acknowledge the hunters whose cooperation has made these studies possible, especially Mr. Janez Hafner.


Jun-Ichi Funada

Louise Dennis

Rachel Roberts

Fredrik Karpe

Keith N Frayn
email: keith.frayn@oxlip.ox.ac.uk

Regulation of subcutaneous adipose tissue blood flow is related to measures of vascular and autonomic function

Keywords: Adipose tissue blood flow, body mass index, endothelial function, flowmediated vasodilatation, heart rate variability, insulin resistance ATBF, adipose tissue blood flow, FMD, flow-mediated vasodilatation, HOMA-IR, homeostatic model assessment

Appropriate blood vessel function is important to cardiovascular health. Adipose tissue plays an important role in metabolic homeostasis. Subcutaneous abdominal ATBF (adipose tissue blood flow) is responsive to nutritional stimuli. This response is impaired in obesity, suggesting parallels with endothelial function. We assessed whether regulation of ATBF is related to the regulation of endothelial function, assessed by FMD (flow-mediated vasodilatation) of the brachial artery. Impaired FMD is a marker of atherosclerotic risk so we also assessed relationships between ATBF and a marker of atherosclerosis, common carotid artery IMT (intima-media thickness). Since ATBF is responsive to sympatho-adrenal stimuli we also investigated relationships to HRV (heart rate variability). Seventy-nine healthy volunteers (44 female) were studied fasting and after ingestion of 75 g glucose. FMD, fasting ATBF and the responsiveness of ATBF to glucose were all negatively related to BMI, confirming the adverse cardiovascular effects of adiposity. FMD was related to fasting ATBF (r s = 0.32, P = 0.008) and, at least in males, this relationship was independent of BMI (P = 0.02). Common carotid artery IMT, measured in a subset of participants, was negatively related to fasting ATBF (r s = -0.51, P = 0.02, n = 20). On the other hand, ATBF responsiveness to glucose showed no relation with either FMD or IMT. In multiple regression models, both fasting and stimulated ATBF showed relationships with HRV. Our data show that the regulation of ATBF has features in common with endothelial function, but also relationships with autonomic cardiovascular control as reflected in HRV.

INTRODUCTION

Over the past few years, it has been appreciated that blood flow through metabolically-active tissues is regulated, and that this may relate to cardiovascular and metabolic health [START_REF] Baron | Insulin resistance and vascular function[END_REF][START_REF] Summers | Impaired postprandial tissue regulation of blood flow in insulin resistance: a determinant of cardiovascular risk?[END_REF]. One such tissue is adipose tissue. Subcutaneous adipose tissue blood flow (ATBF) is highly regulated by nutritional state, increasing markedly after meals in lean, healthy subjects [START_REF] Summers | The effect on adipose tissue blood flow of isoenergetic meals containing different amounts and types of fat[END_REF][START_REF] Karpe | Impaired postprandial adipose tissue blood flow response is related to aspects of insulin sensitivity[END_REF]. However, regulation of ATBF is impaired in obese or otherwise insulin-resistant people [START_REF] Karpe | Impaired postprandial adipose tissue blood flow response is related to aspects of insulin sensitivity[END_REF][START_REF] Jansson | Relationship between blood pressure, metabolic variables and blood flow in obese subjects with or without noninsulin-dependent diabetes mellitus[END_REF][START_REF] Dimitriadis | Impaired postprandial blood flow in adipose tissue may be an early marker of insulin resistance in type 2 diabetes[END_REF]. This observation suggests a parallel with endothelial function, which is an important component of cardiovascular health, and is known to be impaired in the early stages of atherosclerosis [START_REF] Celermajer | Non-invasive detection of endothelial dysfunction in children and adults at risk of atherosclerosis[END_REF].

Endothelium-derived NO is one of main regulators of endothelial function in the human vasculature [START_REF] Joannides | Nitric oxide is responsible for flow-dependent dilatation of human peripheral conduit arteries in vivo[END_REF]. ATBF is also highly dependent upon NO in the fasting state: local microinfusion of NG-monomethyl-L-arginine (L-NMMA), an inhibitor of endothelial NO synthase, reduces subcutaneous abdominal ATBF by 40% [START_REF] Ardilouze | Nitric oxide and beta-adrenergic stimulation are major regulators of pre-and postprandial subcutaneous adipose tissue blood flow in humans[END_REF]. But L-NMMA does not affect the ATBF response to glucose ingestion [START_REF] Ardilouze | Nitric oxide and beta-adrenergic stimulation are major regulators of pre-and postprandial subcutaneous adipose tissue blood flow in humans[END_REF], which appears instead to be driven by sympathoadrenal activation and is largely blocked by local or systemic infusion of the beta-adrenergic antagonist, propranolol [START_REF] Ardilouze | Nitric oxide and beta-adrenergic stimulation are major regulators of pre-and postprandial subcutaneous adipose tissue blood flow in humans[END_REF]10]. It might therefore be speculated that endothelial function, as measured by the index of flow-mediated vasodilatation (FMD) in the brachial artery, would relate more to fasting ATBF than to its regulation in the postprandial state. Measurements relating to the activity of the autonomic nervous system, such as heart rate and heart rate variability (HRV), might, in contrast, be expected to relate to postprandial activation of ATBF.

Impaired endothelial function is associated with increased atherosclerotic risk [START_REF] Hashimoto | Association of coronary risk factors and endothelium-dependent flow-mediated dilatation of the brachial artery[END_REF][START_REF] Shechter | Long-term association of brachial artery flow-mediated vasodilation and cardiovascular events in middle-aged subjects with no apparent heart disease[END_REF]. Increased intima-media thickness of the common carotid artery (IMT) is a surrogate marker of early atherosclerosis [13], although not always closely associated with impairment of FMD [START_REF] Yan | Relationship between carotid artery intima-media thickness and brachial artery flowmediated dilation in middle-aged healthy men[END_REF]. As noted earlier, ATBF responsiveness to nutrients is impaired in obesity or insulin resistance, but it is not known whether it is also associated with direct markers of atherosclerosic risk.

We have therefore investigated whether there are relationships between measurements of endothelial function and HRV, and ATBF and its responsiveness to glucose ingestion. Our working hypothesis was that, because of the common involvement of endothelial NO generation, there would be stronger relationships in the fasting state than with postprandial stimulation of ATBF. On the other hand, we hypothesised that HRV would relate specifically to the postprandial regulation of ATBF. We also took the opportunity to assess relationships between these measures of vascular function and measurements of early atherosclerosis, as reflected by IMT, in a pilot study in a sub-group of these volunteers.

MATERIALS AND METHODS

Subjects

We recruited 79 healthy volunteers (44 female) by advertisement and from the Oxford BioBank (OBB), a collection of samples and clinical data from the local Oxfordshire healthy population [START_REF] Tan | The in vivo effects of the Pro12Ala PPAR2 polymorphism on adipose tissue NEFA metabolism: the first use of the Oxford Biobank[END_REF]. The OBB is set up such that the database can be screened, and suitable participants invited to attend for further studies. The studies were approved by the Oxfordshire Clinical Research Ethics Committee, and all subjects gave their informed consent.

The study was conducted in two phases. In both phases, ATBF and FMD were measured in the fasting state, together with standard biochemical and cardiovascular measurements (heart rate, blood pressure). In one phase, measurements of ATBF and FMD were combined with measurements of HRV. The participants in this phase were also given an oral glucose tolerance test (oGTT) to assess metabolic and cardiovascular responses to a challenge. In the other phase, ATBF and FMD measurements were combined with ultrasound measurements of carotid intima-media thickness (IMT). Some of these participants were given the oGTT. The disposition of the participants amongst the various procedures is outlined in Fig. 1.

Those who were to have measurements of HRV attended for a screening visit to assess suitability. Some of the participants in this phase went on to take part in a dietary intervention; full details have been published separately [START_REF] Hodson | Does the DASH diet lower blood pressure by altering peripheral vascular function?[END_REF]. In addition, some aspects relating to adipose tissue fatty acid composition, adipocyte size and insulin sensitivity in a sub-set of the volunteers have been published separately [START_REF] Roberts | Markers of de novo lipogenesis in adipose tissue: associations with small adipocytes and insulin sensitivity in humans[END_REF].

All subjects (both phases) were asked to refrain from strenuous exercise or alcohol intake for 24 hours before the experimental day. Investigations started around 8 am following an overnight fast. We did not standardize the stage of the menstrual cycle for pre-menopausal female participants. Details of the procedures are given below.

Measurement of adipose tissue blood flow

ATBF was measured by the 133 Xe washout technique [START_REF] Larsen | Blood flow through human adipose tissue determined with radioactive xenon[END_REF][START_REF] Karpe | Effects of insulin on adipose tissue blood flow in man[END_REF]. In brief, 1 MBq 133 Xe was injected into the para-umbilical area of the subcutaneous adipose tissue to a depth of approximately 10 mm. After an equilibration period of 30 min, ATBF was monitored by collecting continuous 20 s readings from a γ-counter probe placed over the site of injection [START_REF] Samra | Modification and validation of a commercially available portable detector for measurement of adipose tissue blood flow[END_REF]. In 74 of the volunteers, as noted above, following a period of measurement of fasting blood flow, an oral glucose load (75 g) was given, and readings were continued for the next 2 h. Blood flow was calculated from a semilog plot of the disappearance of 133 Xe counts versus time in 10 min intervals. ATBF was then calculated according to the following equation: ATBF = slope of semilog plot of (counts per second) x partition coefficient x 100 [START_REF] Larsen | Blood flow through human adipose tissue determined with radioactive xenon[END_REF]. The partition coefficient between adipose tissue and blood was taken as 10 ml.g -1 . This value was based on a review of appropriate literature and represents a typical value [START_REF] Jansson | Comparison of two methods to assess the tissue/blood partition coefficient for xenon in subcutaneous adipose tissue in man[END_REF]. Peak ATBF was calculated as previously [START_REF] Karpe | Impaired postprandial adipose tissue blood flow response is related to aspects of insulin sensitivity[END_REF], and the ATBF response to glucose as peak minus baseline.

Heart rate variability measurement

An electrocardiograph (ECG) was recorded during the screening visit to assess suitability for inclusion, and for acclimatisation of the volunteers. Subjects with >3% ectopic beats were excluded at the screening visit. HRV was then assessed on the study day, when two fasting baseline measurements of HRV were recorded for 10 min following the same procedure. Recordings were also made following consumption of glucose at 20, 70 and 130 min.

Subjects were semi-supine in a quiet room. Respiration was controlled using an electronic metronome at a rate comfortable for the subject at 9-11 breaths per minute. A chest strain gauge was worn to assess respiratory compliance. A 3-lead continuous ECG signal was recorded using a Powerlab/8Sp, MLS310 HRV Module (AD Instruments Pty Ltd, Castle Hill, NSW, Australia). The R-R intervals for the last consecutive 5 min for each 10 min recording were manually selected and analyzed (minimum of 250 cycles). Time and frequency-domain analyses of HRV and Poincaré plot were performed. All R-R intervals were edited by visual inspection to exclude ectopic beats or artefacts.

The various parameters of HRV tended to be highly-intercorrelated, and for illustration the key parameter of standard deviation of R-R intervals (SDnn) is presented in Results. There was no consistent pattern of change in any of the HRV parameters following glucose ingestion, so average values for SDnn over the whole experimental day are presented.

Ultrasound study FMD measurements were made in a dark, quiet, temperature-controlled laboratory using a high-resolution ultrasound machine (ATL 5000, Philips Ltd., Andover, MA, USA) equipped with a 14 MHz linear artery transducer. The limit of axial resolution was 0.1mm and all subjects rested in the supine position for at least 10 min before the investigation.

FMD, the index of endothelium-dependent vasodilatation, was determined by the maximal change in the diameter of the brachial artery during hyperaemia [START_REF] Celermajer | Non-invasive detection of endothelial dysfunction in children and adults at risk of atherosclerosis[END_REF]22], which was created by placing a cuff on the forearm and inflating it to 200 mmHg for 5 min, thereby occluding blood flow to the forearm. Longitudinal scans of the right brachial artery were obtained proximal to the antecubital fossa with the probe positioned so that the optimum images could be obtained. The peak diameter change during reactive hyperaemia occurred approximately 1 min after cuff deflation. Three consecutive frames at rest and at peak reactive hyperaemia were stored in order to determine FMD. The brachial artery diameter, between mediaadventitia interface on the anterior wall and intima-lumen interface on the posterior wall, was measured manually four times in each frame. A total of 12 measurements in each condition were taken to determine FMD. Arterial flow velocity was measured using a pulsed Doppler signal in the centre of the vessel at baseline and during the first 15 s of reactive hyperaemia after cuff deflation. The degree of hyperaemic response was assessed as the brachial blood flow at peak hyperaemia as a percentage of the resting flow. In 20 of the volunteers, additional ultrasound measurements were made of the common carotid artery. A region of interest for each common carotid artery was selected at least 1cm proximal to the carotid bulb. The region of interest was manually moved to a more proximal position to avoid any plaque formation. Carotid artery intima-media thickness (IMT) was evaluated as the distance between the intima-lumen interface on both anterior and posterior walls of the bilateral common carotid artery [13,[START_REF] O'leary | Carotid-artery intima and media thickness as a risk factor for myocardial infarction and stroke in older adults. Cardiovascular Health Study Collaborative Research Group[END_REF]. The average IMT of the bilateral common carotid arteries was employed in this investigation.

Ultrasound measurements were made by one of two observers (ALD, JF) who were responsible for the two phases of the study. One of them (ALD) performed HRV measurements. The other (JF) performed common carotid artery IMT measurements. Hence, IMT and HRV measurements were not made in the same individuals, as described earlier and shown on Fig. 1. Intra-observer variability was assessed by making repeated measurements on volunteers on separate occasions. For FMD, the correlation coefficient between first and second measurements was 0.81 (n = 28), coefficient of variation of repeated measurements (c.v.) 13%. For IMT (n = 19), the correlation coefficient was 0.90, and the c.v. 5.7%. For SDnn (n = 25), the correlation coefficient was 0.92 and the c.v. 15%.

Analytical methods

Samples of blood were taken in the fasting state and at intervals throughout the glucose tolerance test into heparinised syringes. Plasma concentrations of glucose, insulin, nonesterified fatty acids (NEFA) and triacylglycerols (TG) were measured as described previously [START_REF] Hodson | Does the DASH diet lower blood pressure by altering peripheral vascular function?[END_REF].

Calculations and statistical analysis

Glucoregulatory insulin resistance in the fasting state (HOMA-IR) was assessed from fasting glucose and insulin concentrations [24]. In those participants who underwent the oGTT, indices of insulin sensitivity were calculated from responses of plasma glucose, insulin and NEFA concentrations as described by Belfiore et al. [START_REF] Belfiore | Insulin sensitivity indices calculated from basal and OGTT-induced insulin, glucose, and FFA levels[END_REF]. These refer to post-glucose glucoregulatory insulin sensitivity (ISIgly) and the suppression of NEFA (ISInefa).

All statistical analyses were performed using SPSS Statistics version 17.0 for Windows (SPSS Inc, Chicago, IL). Several of the variables displayed skewed distributions. Spearman's rank correlation method was used to evaluate the relationships between ATBF and other variables. Partial correlation coefficients were calculated on log-transformed data.

Multiple regression analyses were performed using fasting ATBF and peak ATBF as dependent variables. Independent variables were chosen as those hypothesised to affect fasting or peak ATBF. Regression analysis was run as a backward elimination model, with stepwise removal of variables not achieving a significance of P < 0.10 (SPSS default). Logtransformed data were used where appropriate.

So far as possible, analyses have been performed across the whole cohort. However, relationships with HRV and with IMT are necessarily limited to the subgroups in whom they were measured.

RESULTS

The characteristics of the participants are given in Table 1.

Fasting ATBF and univariate relationships with other variables

Fasting ATBF covered a wide range (3.2 -10.2 ml.100g -1 .min -1 ), and was significantly negatively related to glucoregulatory insulin resistance assessed with the HOMA index (Table 2, Fig. 2A). It was less strongly related to measures of post-glucose insulin sensitivity, ISIgly and ISInefa (Table 2).

Amongst indices of cardiovascular function, fasting ATBF was positively related to FMD (Fig. 2C, Table 3) but not to other indices. Fasting ATBF and FMD were each negatively related to both age and BMI. The relationship between fasting ATBF and FMD (Fig. 2C) gave the impression of two groups. Further exploration showed that this was age-dependent. If the group were split at the median age (42 years), then the correlation was strong in the younger group (r s = 0.45, P = 0.01) but absent in the older group (r s = 0.26, P = 0.13).

The relationships between fasting ATBF, HOMA index, and BMI were complex. In a partial correlation controlling for BMI, the relationship between fasting ATBF and HOMA-IR was not significant (P = 0.52), while significance was also largely lost when the relationship between fasting ATBF and BMI was controlled for HOMA-IR (P = 0.07).

In the sub-study of relationships with IMT, fasting ATBF and IMT were negatively related (Table 3).

Peak ATBF and the ATBF response to glucose

Fasting and peak ATBF were related (r s = 0.63, P < 0.001). ATBF response to glucose, the difference between fasting and peak ATBF, was not related to the fasting value (P = 0.90) but was related to the peak value (r s = 0.71, P < 0.001).

Peak ATBF and the ATBF response were strongly related to measures of post-glucose insulin sensitivity, both ISIgly and ISInefa (Table 2, Fig. 2B). Amongst cardiovascular function indices (Table 3), peak ATBF was positively related to FMD and to SDnn, a measure of HRV. Both peak ATBF and the ATBF response were related to the hyperaemic response to release of occlusion, and weakly negatively related to heart rate.

Peak ATBF and the ATBF response to glucose were negatively related to BMI, as expected (Table 3). Relationships with metabolic measurements (not shown) were generally not striking other than negative relationships of ATBF response with fasting NEFA and TG concentrations (r s = -0.30, P = 0.02, n = 61; r s = -0.34, P = 0.01, n = 56 respectively); peak ATBF did not relate to fasting NEFA but did to TG (r s = -0.41, P = 0.002). Fasting NEFA concentrations were also positively related to both systolic blood pressure and to heart rate (r s = 0.24, P = 0.05, n = 65; r s = 0.29, P = 0.02, n = 62 respectively).

Multiple regression analysis

In order to understand relationships between ATBF and other variables jointly, we conducted multiple regression analyses for fasting and for peak ATBF (Table 4). SDnn and fasting insulin contributed to a significant model for fasting ATBF, whereas SDnn and FMD were significant in the peak ATBF model, after allowing for the effect of other variables, which did not contribute substantially to these models. We looked for differences between men and women. In the whole cohort, age was well matched but BMI was significantly lower in women. We therefore truncated the BMI range to include only those with BMI greater than 22 and less than 33 kg/m 2 , which gave wellmatched groups (Table 5). Adipose tissue blood flow (fasting, peak and response to glucose) was identical in men and women, whereas FMD and hyperaemic response were significantly greater in women (Table 5). Insulin sensitivity assessed by HOMA was identical, but NEFA concentrations were higher, and TG concentrations lower in women than in men (Table 5).

Sex-specific features

We then assessed whether the relationships amongst the measured variables differed in men and women. In general they were much stronger in men than in women (e.g. men, fasting ATBF vs FMD, r s = 0.57, P = 0.002, n = 26). This relationship was still significant in a partial correlation controlling for BMI (r = 0.45, P = 0.024), although in a partial correlation controlling for both age and BMI the significance was marginal (r = 0.39, P = 0.060). The ATBF response to glucose was generally less strongly related to other vascular parameters than the fasting ATBF, except that in women, the ATBF response correlated negatively with heart rate (r s = -0.43, P = 0.007, n = 38) and positively with SDnn (r s = 0.46, P = 0.02, n = 26).

DISCUSSION

Adipose tissue is metabolically very active, playing an important role in the minute-to-minute regulation of the supply and removal of circulating lipid fuel [26]. Because TG and NEFA are not soluble in the plasma and require transport mechanisms for delivery to, or removal from, the tissue, it seems reasonable that in adipose tissue, more than in other tissues, there might be important interrelationships between ATBF and metabolism. Thus, there is a need for tissue-specific regulation of blood flow to meet the specific metabolic activities of adipose tissue. We showed previously [START_REF] Samra | Effects of epinephrine infusion on adipose tissue: interactions between blood flow and lipid metabolism[END_REF] that adrenaline infusion causes a rise in ATBF with increased efflux of NEFA from adipose tissue, but also increased TG clearance. Appropriate regulation of ATBF may therefore have importance in metabolic physiology in the extraction and release of lipids by adipose tissue.

In this study, we explored relationships between the regulation of subcutaneous ATBF and a number of aspects of cardiovascular function. We found some relationship between fasting ATBF and FMD of the brachial artery, a widely-accepted assessment of endothelial function. This is perhaps not surprising given that NO is a major regulator of fasting ATBF [START_REF] Ardilouze | Nitric oxide and beta-adrenergic stimulation are major regulators of pre-and postprandial subcutaneous adipose tissue blood flow in humans[END_REF]. Endothelial NO generation is usually assumed to be the major determinant of FMD, although formal assessment should include a test of glyceryl-trinitrate administration [START_REF] Celermajer | Non-invasive detection of endothelial dysfunction in children and adults at risk of atherosclerosis[END_REF], which we did not do. Interestingly, a stronger relationship between fasting ATBF and FMD was seen in men than in women. This might indicate that endothelial function is influenced by other factors, perhaps including menstrual cycle, in women. It should be noted that we did not standardise the stage of the menstrual cycle for female participants, which is likely to have increased variability in some of our measurements: FMD is known to vary during the menstrual cycle [28,[START_REF] Williams | Variations in endothelial function and arterial compliance during the menstrual cycle[END_REF], although there is no information about ATBF. In addition, fasting ATBF was itself reasonably strongly negatively related to BMI and to insulin resistance, as we have found previously [START_REF] Karpe | Impaired postprandial adipose tissue blood flow response is related to aspects of insulin sensitivity[END_REF][START_REF] Summers | Subcutaneous abdominal adipose tissue blood flow: variation within and between subjects and relationship to obesity[END_REF]. Furthermore, fasting ATBF was also moderately strongly related to common carotid artery IMT, a surrogate marker of early atherosclerosis, suggesting that reduced ATBF is associated, directly or indirectly, with adverse functional cardiovascular consequences.

ATBF may increase several-fold in response to glucose (or mixed meal) ingestion [START_REF] Summers | The effect on adipose tissue blood flow of isoenergetic meals containing different amounts and types of fat[END_REF][START_REF] Karpe | Effects of insulin on adipose tissue blood flow in man[END_REF], although it does not respond to a pure fat load [START_REF] Evans | Effects of an oral and intravenous fat load on adipose tissue and forearm lipid metabolism[END_REF]. This response is mediated via sympatho-adrenal activation rather than via NO generation, and is largely blocked by local or systemic propranolol administration [START_REF] Ardilouze | Nitric oxide and beta-adrenergic stimulation are major regulators of pre-and postprandial subcutaneous adipose tissue blood flow in humans[END_REF]10]. We found no relationship between the ATBF response to glucose ingestion and FMD, but ATBF responsiveness was related to other aspects of cardiovascular function in which sympathetic activity may play a role, namely systolic blood pressure and heart rate, and, in women, HRV measured as SDnn. Multivariate analysis in fact suggested that both fasting and stimulated ATBF were related to HRV.

We observed several differences in responses between men and women. The greater FMD in women is expected [32,[START_REF] Nishiyama | Sex and limb-specific ischemic reperfusion and vascular reactivity[END_REF] because of their smaller brachial artery diameter; FMD is highly dependent upon arterial diameter [START_REF] Nishiyama | Sex and limb-specific ischemic reperfusion and vascular reactivity[END_REF][START_REF] Silber | Why is flow-mediated dilation dependent on arterial size? Assessment of the shear stimulus using phase-contrast magnetic resonance imaging[END_REF]. However, this sex difference is not always observed [35] and may disappear after the menopause [32]. Our group, with a mean age of 45 years, were predominantly pre-menopausal. The hyperaemic response was also greater in women than in men. The physiological significance of this response is not clear, and this sex-difference has not been observed in other studies [32]. Post-occlusion hyperaemia was not different between two groups who differed considerably in remnant lipoprotein-cholesterol concentrations, and in FMD, after a fat load [22]. We observed a relatively strong relationship between hyperaemic response to occlusion, and the ATBF response to glucose ingestion. The hyperaemic response is not predominantly endotheliumdependent but is thought to relate to locally-generated factors including metabolites [START_REF] Raitakari | Enhanced peripheral vasodilation in humans after a fatty meal[END_REF]. Possibly our results suggest that similar mediators affect the postprandial ATBF response. The greater plasma NEFA and lower TG concentrations that we found in women compared with men are to be expected from other studies; greater NEFA concentrations may relate to greater fat mass in women [START_REF] Mittendorfer | Relationship between body fat mass and free fatty acid kinetics in men and women[END_REF] and lower TG concentrations to faster removal [START_REF] Saito | Serum lipoprotein lipase in healthy subjects: effects of gender and age, and relationships to lipid parameters[END_REF][START_REF] Matthan | Gender-specific differences in the kinetics of nonfasting TRL, IDL, and LDL apolipoprotein B-100 in men and premenopausal women[END_REF]. We did not find a sex-difference in ATBF, so this is unlikely to have contributed. The weak positive relationship that we noted between plasma NEFA concentrations and systolic blood pressure, which was seen equally in both sexes, confirms earlier findings (reviewed in [START_REF] Sarafidis | Non-esterified fatty acids and blood pressure elevation: a mechanism for hypertension in subjects with obesity/insulin resistance?[END_REF]). It has been suggested recently that this may reflect sympathetic activation by fatty acids [41], although we did not see relationships with HRV.

The relationships that we observed tended to revolve around BMI and insulin sensitivity. The ATBF response to glucose was particularly related to measures of post-glucose insulin sensitivity, as we have shown previously [START_REF] Karpe | Impaired postprandial adipose tissue blood flow response is related to aspects of insulin sensitivity[END_REF]. Fasting ATBF and ATBF responsiveness, as well as FMD, were all negatively related to BMI. This makes it difficult fully to disentangle causal relationships, although the data carry a clear message that increasing BMI is associated with unfavourable cardiovascular changes. These are, in turn, reflected in the strong relationship observed between BMI and carotid artery IMT.

One possible common factor between ATBF regulation and endothelial function might be the presence of perivascular fat. Perivascular adipose tissue accumulates during expansion of fat depots [42] and is now recognized to secrete a variety of factors, including TNF-alpha [42], which may trigger vascular inflammation [START_REF] Eringa | Regulation of vascular function and insulin sensitivity by adipose tissue: focus on perivascular adipose tissue[END_REF][START_REF] Rajsheker | Crosstalk between perivascular adipose tissue and blood vessels[END_REF] and interfere with vascular reactivity. Perivascular adipose tissue is associated with increased NO release at early stages of its accumulation in rodent models, suggesting an adaptive mechanism [START_REF] Gil-Ortega | Adaptative nitric oxide overproduction in perivascular adipose tissue during early diet-Induced obesity[END_REF], and in some human studies has been shown to release vaso-relaxant factors [START_REF] Gao | Perivascular adipose tissue modulates vascular function in the human internal thoracic artery[END_REF]. However, in human vessels perivascular fat is also associated with increased responsiveness to constrictive influences such as that of angiotensin-II [START_REF] Lu | Mechanisms for perivascular adipose tissue-mediated potentiation of vascular contraction to perivascular neuronal stimulation: the role of adipocyte-derived angiotensin II[END_REF]. We did not assess perivascular fat in the present studies, but common relationships between BMI, perivascular adipose tissue accumulation and insulin sensitivity indices could explain some of the common regulation we observed in ATBF and FMD.

In order to elucidate the clinical significance of these peripheral vascular functions, in particular fasting ATBF and ATBF responsiveness, it would be of interest to investigate the effects of dietary intervention on these indices. A recent study showed that the DASH (Dietary Approaches to Stop Hypertension) diet altered neither FMD nor ATBF regulation [START_REF] Hodson | Does the DASH diet lower blood pressure by altering peripheral vascular function?[END_REF] despite highly significant reduction in blood pressure and improvement in lipid parameters. However, the study was of short duration (30 days), and had no significant effect on insulin sensitivity and very little on adiposity (weight change < 1 kg). Therefore, further intervention should be addressed to clarify the clinical significance of these indices on cardiovascular atherosclerotic progression.

AUTHOR CONTRIBUTIONS

Clinical Science Immediate Publication. Published on 03 Jun 2010 as manuscript CS20100066 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/CS20100066 A c c e p t e d M a n u s c r i p t Licenced copy. Copying is not permitted, except with prior permission and as allowed by law.

JF introduced FMD measurements to the other authors, made the first measurements and wrote the first draft of the paper. ALD conducted most of the clinical procedures and commented on the paper. RR helped with clinical studies, collated data and commented on the paper. FK worked on data and contributed to the paper. KNF was responsible for project coordination, data analysis and revising the paper. A c c e p t e d M a n u s c r i p t

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Clinical

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Clinical

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Clinical

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Figure 1 Figure 2

 12 Figure 1 Disposition of the participants amongst the various measurements made

Funada et al. Revision 1. Figure 1

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Table 1 Characteristics of the participants: whole cohort

 1 Values are median (range).

	Women (n = 44)	Men (n = 35)

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Table 2 Univariate inter-relationships between ATBF and insulin sensitivity indices

 2 Data are Spearman rank correlation coefficients for bivariate correlations, shown as r s , P, n. Only relationships significant at P ≤ 0.10 are shown; others are marked NS (non-significant). Note: HOMA-IR is a measure of insulin resistance, whereas ISIgly and ISInefa are measures of insulin sensitivity. ATBF, adipose tissue blood flow; HOMA-IR, homeostatic model assessment of insulin resistance (fasting glucoregulatory insulin resistance); ISIgly, a measure of post-glucose glucoregulatory insulin sensitivity; ISInefa, a measure of post-glucose lipid regulatory insulin sensitivity. Clinical Science Immediate Publication. Published on 03 Jun 2010 as manuscript CS20100066 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/CS20100066 A c c e p t e d M a n u s c r i p tLicenced copy. Copying is not permitted, except with prior permission and as allowed by law.

		HOMA-	ISIgly	ISInefa
		IR
	ATBF fasting (ml.100g -1 .min -1 )	-0.36 0.004	0.33 0.014	0.26 0.056
		63	57	57
	ATBF peak (ml.100g -1 .min -1 )	-0.42 0.001	0.52 <0.001	0.52 <0.001
		61	57	57
	ATBF response (ml.100g -1 .min -1 )	-0.22 0.08	0.46 <0.001	0.50 <0.001
		61	57	57

© 2010 The Authors Journal compilation © 2010 Portland Press Limited

Table 3 Univariate inter-relationships between ATBF, BMI and other indices of cardiovascular function

 3 Data are Spearman rank correlation coefficients for bivariate correlations, shown as r s , P, n. Only relationships significant at P ≤ 0.10 are shown; others are marked NS (non-significant). ATBF, adipose tissue blood flow; FMD, flow-mediated vasodilatation; IMT, intima-media thickness of the common carotid artery; SDnn, standard

	deviation of the R-R intervals.
		BMI	FMD	Hyperaemic	IMT	Systolic BP	Heart rate	SDnn
		(kg/m 2 )	(%)	response (%)	(mm)	(mm Hg)	(min -1 )
	ATBF fasting (ml.100g -1 .min -1 )	-0.41 <0.001	0.32 0.008	NS	-0.51 0.023	NS	NS	0.26 0.062
		77	66		20			53
	ATBF peak (ml.100g -1 .min -1 )	-0.57 <0.001	0.29 0.021	0.29 0.027	NS	NS	-0.20 0.090	0.35 0.010
		74	63	58			71	53
	ATBF response (ml.100g -1 .min -1 )	<0.001 -0.40	NS	0.008 0.35	NS	0.045 -0.24	0.029 -0.26	NS
		74		58		73	71
	BMI (kg/m 2 )	-	-0.33	NS	0.63	NS	NS	-0.26
			0.006		0.003			0.060
			67		20			55

Clinical Science Immediate Publication. Published on 03 Jun 2010 as manuscript CS20100066 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/CS20100066 A c c e p t e d M a n u s c r i p t Licenced copy. Copying is not permitted, except with prior permission and as allowed by law. © 2010 The Authors Journal compilation © 2010 Portland Press Limited

Table 4 Multiple regression analysis of factors associated with fasting and peak ATBF For

 4 the method of analysis, see Calculations and statistical analysis.

		Clinical Science Immediate Publication. Published on 03 Jun 2010 as manuscript CS20100066
		P (overall	P (individual
	THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/CS20100066	model) Dependent variable: Fasting ATBF* Variables included Age Sex BMI* Fasting insulin* sysBP* Heart rate* FMD* SDnn* 0.125 Final model includes: Fasting insulin SDnn 0.013 Dependent variable: Peak ATBF* Variables included Age Sex BMI* Fasting insulin* sysBP* Heart rate* FMD* SDnn* 0.040 Final model includes: FMD SDnn 0.003 A c c e p t e d M a n u s c r i p t variables) 0.079 0.064 0.015 0.013

FMD, flow-mediated vasodilatation; SDnn, standard deviation of the R-R intervals.

* Variables log-transformed to improve normality

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Table 5 Differences between men and women (BMI range truncated to give matched groups

 5 Clinical Science Immediate Publication. Published on 03 Jun 2010 as manuscript CS20100066 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/CS20100066

		Women	Men	Significance
	Age (years)	45 (22 -56; 33)	41 (19 -62; 27)	NS
	BMI (kg/m 2 )	24.8 (22.1 -31.9; 33)	26.4 (22.3 -31.4; 27)	NS
	Fasting ATBF (ml.100g -1 .min -1 )	2.48 (0.85 -10.16; 31)	2.33 (0.87 -7.49; 27)	NS
	Peak ATBF (ml.100g -1 .min -1 )	4.87 (0.88 -13.17; 31)	3.99 (2.02 -11.11; 27)	NS
	ATBF response (ml.100g -1 .min -1 )	2.02 (-1.28 -6.30; 31)	1.62 (-0.24 -5.50; 25)	NS
	FMD (%)	6.11 (0.00 -18.58; 30)	3.64 (0.59 -12.12; 20)	0.027
	Hyperaemic response	1106 (149 -3510; 28)	500 (126 -1244; 17)	0.002
	(%)
	IMT (mm)	0.52 (0.44 -0.61; 6)	0.59 (0.46 -0.76; 6)	NS
	Systolic blood pressure	109 (89 -140; 32)	110 (96 -149; 27)	NS
	(mm Hg) Heart rate (min -1 )	61 (44 -77; 30)	57 (39 -86; 27)	NS
	SDnn	58 (18 -147; 24)	55 (22 -277; 20)	NS
	Plasma glucose	4.9 (4.0 -5.5; 29)	4.9 (4.3 -6.2; 23)	NS
	(mmol/l)
	Plasma insulin (mU/l)	8.8 (0.7 -19.9; 29)	9.7 (6.2 -16.9; 24)	NS
	HOMA-IR	1.8 (0.1 -4.3; 29)	2.0 (1.2 -4.1; 23)	NS
	Plasma NEFA (µmol/l)	576 (224 -1034; 29)	434 (253 -581; 23)	0.001
	Plasma TG (µmol/l)	805 (419 -1664; 27)	1225 (567 -3263; 20)	0.001

)

Values are median (range; n). Significance by Mann-Whitney test. NS, non-significant (all P > 0.15).

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ACKNOWLEDGEMENTS

We thank other members of the research group who helped with the studies, and especially Sandy Humphreys for statistical advice and Marje Gilbert for technical assistance.

FUNDING

This work was supported by the Biotechnology and Biological Sciences Research Council (UK) (grant number BB/D008123/1). Clinical Science Immediate Publication. Published on 03 Jun 2010 as manuscript CS20100066 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/CS20100066

A c c e p t e d M a n u s c r i p t Licenced copy. Copying is not permitted, except with prior permission and as allowed by law.