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Body mass index and leptin are related to cognitive performance over 10 years in women with and without HIV infection

© The Author(s) 2021. Published by Oxford University Press on behalf of the Endocrine Society. This is an Open Access article distributed under the terms of the Creative Commons Attribution-NonCommercial-NoDerivs licence (https://creativecommons.org/licenses/by-ncnd/4.0/), which permits non-commercial reproduction and distribution of the work, in any medium, provided the original work is not altered or transformed in any way, and that the work is properly cited. For commercial re-use, please contact journals.permissions@oup.com Body mass index and leptin are related to cognitive performance over 10 years in women with


Improved antiretroviral therapies (ART) transformed HIV infection into a treatable chronic condition,
increasing life expectancy among people living with HIV (PLWH). 1,2 Consequently, PLWH may experience concomitant physiological and neuropsychological consequences of aging. These include adverse vascular and metabolic co-morbidities, sometimes due to cumulative ART exposure, and overweight and obesity. In uninfected populations, overweight and obesity during middle-age increase risk for late-life neuropsychological impairment and Alzheimer's Disease and Related Dementias (ADRD). 3,4 Leptin is an adipose tissue hormone, positively correlated with body mass index (BMI), and a proposed modulator of BMI and neuropsychological performance (NP) given peripheral and central nervous system (CNS) effects. [5][6][7] Higher blood leptin levels are associated with lower AD risk within 10 years of clinical onset. [8][9][10] The relationship between peripheral and brain-derived leptin is unknown. However, leptin promotes hippocampal synaptic plasticity 11 affecting learning and memory, [11][12][13][14] aids in satiety regulation, improves insulin sensitivity, 15,16 and acts on the hypothalamus to regulate body weight.
Leptin may be dysregulated in HIV-related lypodystrophies. 17 While several studies examined single BMI measurement associations or BMI trajectories in association with ADRD 3,18 there are fewer reports on adiposity-related metabolic changes. Given the vascular, metabolic, and lipodystrophic effects of both HIV infection and ART, and a relatively unknown HIV-specific aging process, particularly among Black women, we investigated whether baseline and/or 10-year change in BMI and blood leptin levels were associated with NP in middle-aged women with HIV (WWH) and without HIV infection who were participants in the Women's Interagency HIV Study (WIHS) Brooklyn and Chicago sites.

Standard Protocol Approvals, Registrations, and Patient Consents
WIHS was the largest prospective study of HIV infection in women in the United States. 19

Sociodemographic and behavioral measures
All sociodemographic measures were self-reported and included: date of birth, race 19,20 (White, Hispanic any race, African-American, or 'other', i.e., Native American/Alaskan, Asian/Pacific Islander or other); and highest educational level. Participants were asked about their use of marijuana since last visit.

Clinical measures
Body weight and height measures were conducted according to the U.S. National Health and Nutrition Examination Survey (NHANES) III protocol and included body weight (pounds) and body height (inches). 21 Anthropometric measurements were conducted with participants wearing undergarments.
Those who conducted the measurements were recertified every two years. Body weight was recorded to the nearest 1.0 pound, and body height was measured to the nearest 1.00 inch. After software conversion of body weight and height to metric units, BMI was calculated as kilograms per meter squared (kg/m 2 ). Categories of BMI included: underweight, <18.5 kg/m 2 ; 'normal' or healthy weight, 18.5-24.9 kg/m 2 ; overweight, 25.0-29.9 kg/m 2 ; and obese, ≥30 kg/m 2 . 23 Systolic (SBP) and diastolic blood pressures (DBP) were obtained and recorded using a standardized protocol with the mean of multiple resting measurements used for analysis. 23 Hypertension was defined as either average measured SBP >140 mm Hg, or DBP >90 mm Hg, or self-reported hypertension with use of prescribed antihypertensive medications. Diabetes mellitus (DM) was determined by fasting glucose ≥126 mg/dl, HbA1C ≥6.5% or self-reported use of anti-diabetic medication. 19,20,24 Menopause status was self-reported and defined according to the Stages of Reproductive Aging Workshop (STRAW)+10 criteria ). 25 There are four menopausal status categories: premenopause, early perimenopause, late perimenopause, and postmenopause (natural or surgical).

Leptin measures
Baseline and 10-year leptin levels were measured in batch from stored plasma samples. The latter were collected within two visits (12 months before or after) of NP assessment. Paired longitudinal Brooklyn and Chicago samples were run together. Standards and controls were tested in duplicate using commercial ELISAs (Millipore, Billerica, MA). 25,26 The intra-assay coefficient of variation (CV%) was 2% and inter-assay CV% was 10%. Undiluted samples were tested and plates were prepared according to protocol. The 7-point standard curve ranged from 0.5-100 ng/mL, the lower and upper limits of detection of the assay, which was appropriate for all samples. Plates were read using a Molecular Devices Plate reader and Softmax Pro data analysis software (Molecular Devices, Sunnyvale, CA). A 4-point logistic (PL) curve fit was used.

HIV-related variables
Laboratory-confirmed HIV status, AIDS diagnosis, CD4 count, HIV viral load, and ART duration and CNS penetration effectiveness (CPE) 19,20,28 were considered in analyses of WWH only. CPE scoring for each ART was based on published data regarding the differential abilities of ART to cross the bloodbrain barrier. CPE was defined on a 1-4 point scale using the 2010 CPE ranking system 29 , where 4 indicates the highest CNS penetration. The baseline ART regimen CPE sum was subsequently ranked as high (>9), medium (8)(9) or low (<8). 29,30 Neuropsychological performance The NP performance battery included the Letter-Number Sequencing (LNS), Trail Making Test Part B (Trails B), Stroop Test (color word, word reading), Hopkins Verbal Learning Test-Revised (HVLT-R),

Symbol Digit Modalities Test (SDMT), Controlled Oral Word Association Test (COWAT), Category
Fluency Test (Animals), and Grooved Pegboard (GPEG). Performance on these assessments was used to assess seven NP domains: 1) attention/working memory (outcomes: total correct on LNS control and experimental conditions); 2) executive function (outcomes: time to completion on Trails B and Stroop color-word [interference] trial); 3) processing speed (outcomes: total correct on SDMT, time to completion on Stroop word-reading trial); 4) memory (outcome: HVLT-R delayed recall); 5) learning (outcome: total learning across HVLT-R trials); 6) verbal fluency (outcomes: total correct on COWAT and Animals); and 7) fine motor skills (outcomes: total time to completion for each hand on the GPEG).
A c c e p t e d M a n u s c r i p t 6 All timed outcomes were natural log (ln)-transformed to normalize distributions and reverse scored so higher scores represented better performance. Demographically-adjusted T-scores were derived for each outcome based on HIV-seronegative women. 31,32 These demographic factors include age, education, Wide Range Achievement Test reading subtest (WRAT-3) score, race (African American vs not), and ethnicity (Hispanic vs not). T-scores were used to create domain-specific scores and a global NP score as done in previous WIHS and Multicenter AIDS Cohort Study (MACS) analyses. 31,33,34 Global NP performance score was the calculated average of the 7 NP domains.

Statistical analysis
BMI and leptin were ln-transformed to meet normality assumptions before consideration as continuous variables. Paired sample t-tests estimated the differences between baseline and follow-up BMI and leptin measures for all women, and separately for WWH and women without HIV. Multivariable linear regression analyses were performed to examine baseline continuous BMI and leptin in association with NP T-scores at 10-year follow-up. Models were run including all participants, and separately for WWH and women without HIV. Multivariable linear regression analyses were again performed to examine 10year change in BMI and leptin in association with follow-up NP T-scores.
Potential covariates included cholesterol level, prevalent diabetes, prevalent hypertension as well as use of hypertensive medication, diabetic medication, menopause status, cocaine, alcohol, marijuana, smoking and/or heroin. 35 Of these, prevalent diabetes, prevalent hypertension, and marijuana use were included since they were deemed statistically relevant (p<0.05) in previous univariate analyses of the relationship between adiposity measures -BMI and leptin-and NP in this group of women. 35 Race, ethnicity, WRAT score, education and age were not included as covariates since they were already included in the demographically-adjusted NP T-scores. Analyses including all women were also adjusted for HIV serostatus (WWH versus women without HIV). In analyses of WWH only, we additionally adjusted for baseline CD4 count, HIV viral load and CPE rank (high >9, medium 8-9, low <8). CD4 count and HIV viral load were analyzed as continuous variables. BMI and leptin were not run in the same regression models, since understanding independent contributions of BMI and leptin is difficult due to their high collinearity. Data analyses were conducted using [ A c c e p t e d M a n u s c r i p t 7

Results
Baseline demographic, BMI, HIV, and NP characteristics of participants are presented in Table 1.
Women were on average 39.8 years old. WWH were approximately 3.1 years older than women without HIV (40.6 years vs 37.5 years, respectively; p=0.005), however educational attainment, a key influencer of NP, did not differ between WWH and women without HIV (Fishers exact test, p=0.85).
Most women (74%) self-described as Black (Caribbean Black or African American), 21% had hypertension, and 14% had diabetes. There were no differences by HIV serostatus. More than half of the women (58%) were pre-menopausal, and menopausal status did not differ by HIV-serostatus (Fishers exact test, p=0.12). Most (62%) women were overweight or obese (≥25.0 kg/m 2 ); <5% had a BMI <18.5 kg/m 2 . WWH had, on average, a lower BMI (28.8 +/-7.5 kg/m 2 ) compared to women without HIV (30.0 +/-8.9 kg/m 2 ), but this was not significant (p=0.14). Compared to women without HIV, WWH   (Table 4). Among WWH only, 10-year increase in BMI was associated with poorer executive function and global performances at follow-up (Table 4). Baseline or change in BMI or leptin measures did not predict any other NP domains (Tables 3 and 4). Changes in BMI or leptin over 10 years were not associated with NP in women without HIV.

Conclusion
Among WWH or women without HIV infection, who transitioned from being overweight to obese over a decade, associations between baseline BMI and leptin and NP 10-years later, did not differ by HIV status. Higher baseline BMI and leptin levels were associated with poorer EF at follow-up, but higher baseline BMI was associated with better memory performance. In contrast, increasing BMI and leptin levels over 10 years were associated with poorer NP at follow-up across several neuropsychological domains among all women, and among WWH, specifically poorer EF, processing speed, verbal fluency and global performance. Changes in leptin predominated these associations; changes in BMI were associated with fewer neuropsychological domains. NP assessments are useful in the detection, differential etiologic diagnosis, and management of ADRD syndromes. Neuropsychological deficits associated with AD can be differentiated from age-associated NP decline by quantitative and qualitative differences in episodic memory, semantic knowledge, and some aspects of executive functions. 37 Of note, the WIHS NP battery was created to assess the seven domains (EF, memory, attention, processing speed, verbal fluency, motor, and learning) most influenced by HIV infection, as well as aging.
There are few published repeated anthropometric and metabolic adiposity data among WWH or other at-risk populations to compare our results. A previous Brooklyn WIHS cross-sectional study using a different NP battery at the year 2006 baseline described here, 36 reported that higher leptin levels were associated with poorer EF performance, whereas cross-sectional analyses of anthropometric measures across all WIHS sites, showed that higher BMI was associated with better NP across multiple domains, 38 and lower BMI (<18.5 kg/m 2 ) with worse NP. 39 Studies among middle-aged, predominantly White men with HIV have shown no association between cognition and leptin. 39,40 Our sample is comprised of primarily Black women. Among uninfected individuals, leptin differences have been observed by race and sex, with serum leptin being higher in women and Blacks. 41 Studies of older adults with Vascular Cognitive Impairment, have reported inverse associations of BMI, but not leptin, with executive function and verbal memory. 42,43 A c c e p t e d M a n u s c r i p t 9 Since its discovery in 1994, 44 leptin has been explored in relation to its neuroprotective influence on the brain. [8][9][10] Leptin was initially described for its role in feeding, and the homeostatic regulation of energy metabolism. Leptin acts on the arcuate nucleus, a key aggregate of hypothalamic neurons often affected in ADRD. 41 Other mechanisms have been described 44,46 for the association between leptin and NP but most are cross-sectional and observed in uninfected adults >65 years. 4,[8][9][10]47 Observations in older adults compared to middle age life are mixed. 38,42,47,48 Longitudinally, the Framingham Heart Study reported that leptin was neuroprotective when measured within 8 years of late-onset dementia among adults age >65 years. 9 However, over a 24 year follow-up, a single measure of leptin at mid-life (age 38-60 years) was not associated with late-onset dementia among Swedish elderly. 36 The Framingham Heart Study Third Generation Cohort comprised of over 2200 adults, average age, 40 years, reported a cross-sectional neuroprotective effect of leptin among cognitively intact, nonoverweight or obese participants (BMI 18.5-24.9 kg/m 2 ), suggesting that BMI is an effect modifier. 47 Conversely, studies also report inverse cross-sectional associations between leptin and cognition among non-obese adults >65 years. 42,48 These observations may reflect variations in age, sex, adiposity measures and levels, severity and/or domain of cognitive performance, the presence of preclinical or overt dementia, as well as the temporality of association being explored. There are also differential associations of adiposity measures with brain imaging outcomes, which are correlates of NP. 49,50 Among adults with cerebral small vessel disease (without dementia), associations vary by study design (cross-sectional versus longitudinal), anthropometric versus metabolic adiposity measures, and neurodegenerative versus vascular brain outcome. 50 Observed increases in leptin and/or BMI over time in association with worse NP among WWH may be influenced by ART, the chronic HIV aging process, or a combination of both. Adjustment for ART CPE did not alter our findings. While increases in adiposity during the middle age transition may not be positive for brain health, higher adiposity may still be protective for the brain among surviving WWH to later life. It will be interesting to observe whether these associations change as WWH age. This is a sizable study of longitudinal, repeated BMI and leptin measures in association with NP in A c c e p t e d M a n u s c r i p t 10 receptors, leptin resistance); one fasting leptin measure that does not allow consideration of diurnal variation in blood leptin levels; not including both leptin and BMI in the same statistical models due to high collinearity; and lack of comparable baseline NP assessments. Due to multiple comparisons, one must also consider risk for false discoveries. Our analyses were not adjusted for multiple comparisons due to their exploratory nature, however, p-values were <0.005 for some linear regression analysis results. A potential bias is that this was a healthier longitudinal cohort of WWH and at-risk women without HIV. These WWH were highly adherent to their HIV medications, and had low HIV viral loads and high CD4 counts. As with all longitudinal cohort studies, routine follow-up over time may limit the generalizability our results to those who are not enrolled in these rigorous protocols. Finally, our global NP score is study specific, and we do not know how this score relates to clinical ADRD since there are no cohorts today who can address this question given the younger average age of people surviving with HIV infection.  Feinkohl I, Janke J, Slooter AJC, Winterer G, Spies C, Pischon T. Plasma leptin, but not adiponectin, is associated with cognitive impairment in older adults. Psychoneuroendocrinology 2020;120:104783. 49.
Arnoldussen IAC, Gustafson DR, Leijsen EMC, de Leeuw FE, Kiliaan AJ. Adiposity is related to cerebrovascular and brain volumetry outcomes in the RUN  A c c e p t e d M a n u s c r i p t 17