Ectopic Lipid Deposition Is Associated With Insulin Resistance in Postmenopausal Women

Ectopic Lipid Deposition Is Associated With Insulin Resistance in Postmenopausal Women Abstract Context Menopause is associated with an increased incidence of insulin resistance and diabetes. Objective The aim of this study was to explore the lipid deposition in liver and skeletal muscle and investigate the association with insulin sensitivity in postmenopausal and premenopausal women. Design and Setting Single-center cross-sectional study of 55 healthy women between 45 and 60 years of age. We measured lipid deposition in the liver with magnetic resonance spectroscopy, intramuscular and intra-abdominal lipid deposition with MRI, body composition with a dual-energy X-ray absorptiometry scan, and insulin sensitivity with the composite Matsuda Index. Outcome Measures We studied the association between fat distribution, ectopic lipid deposition, and insulin sensitivity in pre- and postmenopausal women. Results Postmenopausal women had an increased lipid deposition in the liver [0.68% (0.44 to 0.99) vs 0.49% (0.38 to 0.64), P = 0.01] and skeletal muscle [3% (2 to 4) vs 2% (1 to 3), P = 0.001] and had a 28% lower Matsuda insulin sensitivity index during an oral glucose tolerance test (6.31 ± 3.48 vs 8.78 ± 4.67, P = 0.05) compared with premenopausal women. Total fat mass and leg fat mass were stronger predictors of ectopic lipid deposition, and visceral fat mass was a stronger predictor of both ectopic lipid deposition and insulin resistance in postmenopausal women compared with premenopausal women. Conclusions For a given subcutaneous and visceral fat depot size, postmenopausal women show increased ectopic lipid deposition and insulin resistance compared with premenopausal women. It is suggested that lipid deposition in liver and skeletal muscle may represent important mechanistic links between the changes in fat depots and the increased incidence of insulin resistance seen after menopause. The prevalence of obesity and metabolic disease has reached epidemic proportions and is a major health concern in the Western world (1). Menopause defines the end of women’s reproductive phase and is associated with an increased occurrence of metabolic disease, including metabolic syndrome (2), diabetes (3), and cardiovascular disease (4). In modern society, women live more than one-third of their lives after menopause. The increased disease burden after the menopausal transition therefore is a major health concern. Through the menopausal transition, the body composition changes from favoring gluteofemoral to truncal fat deposition, particularly visceral fat accumulation (5, 6). In general, increasing gluteofemoral fat mass is associated with improved metabolic health (7), whereas expansion of the visceral fat mass leads to dysfunctional adipose tissue, including adipocyte hypertrophy, macrophage infiltration, and impaired insulin signaling (8). Increased visceral fat mass results in a spillover of excessive amounts of lipids and production of inflammatory cytokines (9) promoting ectopic lipid deposition and lipotoxicity in liver and skeletal muscle and ultimately leading to insulin resistance (10, 11). Young men show increased lipid deposition in liver and skeletal muscle compared with young women (12, 13), and oophorectomized rodents show increased lipid deposition in liver and skeletal muscle compared with sham animals (14, 15). Furthermore, one study found an increased incidence of nonalcoholic fatty liver disease (NAFLD) in older women compared with younger women (16), all in all suggesting a role for female sex hormones in ectopic lipid deposition. Thus, increased ectopic lipid deposition during the menopausal transition could be an important mechanistic link between the fat depot changes and the increased incidence of insulin resistance seen after menopause; however, this link has yet to be investigated. In this cross-sectional study of 55 middle-aged women between 45 and 60 years of age, we hypothesized that postmenopausal women had an increased lipid deposition in liver and skeletal muscle compared with that of premenopausal women and that the increased ectopic lipid deposition in postmenopausal women was associated with increased insulin resistance. Methods Participants Fifty-five women between 45 and 60 years of age were included in the study. Thirty-seven women (18 postmenopausal and 19 premenopausal women) were recruited through advertisement, and 18 women (7 postmenopausal and 11 premenopausal women) were recruited through another study in the same department investigating cellular changes in adipose tissue with menopause. Information on general health and menstrual bleeding history was collected from all women. Exclusion criteria were (1) chronic diseases, (2) infections during the last 4 weeks, (3) smoking, (4) more than 7 alcohol units/wk, (5) premature menopause (before age 40 years), (6) hysterectomy or oophorectomy prior to study inclusion, (7) body mass index (BMI) >35, (8) weight changes >10% within the last year, and (9) dietary changes within the last month. Women with a menstrual period within the last 3 months had their hormonal status analyzed in the follicular phase of the first following menstrual period (between days 1 and 8 of their menstrual cycle) (n = 19). The remaining women were enrolled on a random day (n = 36). The study was approved by the Research Ethics Committees of the Capital Region of Denmark (H-3-2014-096) and performed according to the Declaration of Helsinki. Study design Women were categorized as either premenopausal (menstrual bleeding within the last 12 months) or postmenopausal (no menstrual bleeding within the last 12 months). All women went through (1) MRI of the abdomen and thigh and magnetic resonance spectroscopy (MRS) of the liver, (2) dual-energy X-ray absorptiometry scan, (3) ActivPAL (PALtechnologies, Glasgow, Scotland) measurements of free-living activity (17), and (4) blood samples including hormonal status. Forty-seven of the 55 women further agreed to go through an oral glucose tolerance test (OGTT). All subjects tracked their food intake with a 3-day food diary prior to the MRI/MRS. For every visit, a menstrual bleeding diary was updated and physical activity level was assessed by the Minnesota leisure time physical activity questionnaire (18). Magnetic resonance analyses All women abstained from vigorous exercise and alcohol 48 hours prior to the scans as well as food, liquids, and chewing gum 3 hours prior to the scan. MRI and MRS were performed using a Siemens Magnetom Prisma 3 Tesla matrix magnetic resonance scanner (Erlangen, Germany) at 3-mm intervals. All adipose tissue located from the diaphragm to pelvic floor inside the peritoneum was traced manually as the visceral fat region of interest. Multi-image analysis software (19) (Mango; Research Imaging Institute, Houston, TX) was used to calculate the total volume of visceral fat from the T1-weighted MRI sequence. A single reader, who was blinded to the menopausal status of the subjects, performed all image analyses. Skeletal muscle fat deposition measurements were done as described elsewhere (20). Briefly, skeletal muscle fat deposition was measured in a single MRI slice of the vastus lateralis of the quadriceps muscle, midthigh, as single slice measurements of skeletal muscle fat deposition has shown to be representative of a whole muscle or muscle group (21). The images were analyzed using Siemens Syngo FastView. Fat fraction was calculated as: Fat fraction (%)=(signal fat/signal water +fat ) 100. Magnetic resonance spectroscopy to assess liver fat was performed as described elsewhere (22) but with a few specific adjustments including a 1H magnetic resonance single-voxel PRESS sequence: repetition time/echo time/number of scans/prescan = 3000 ms/33 ms/4/1. Three spectra were sampled and evaluated using the user input independent software LCModel (23), and the volume fraction of fat was calculated in accordance with Hájek et al. (22). The spectral quality was assessed by the half widths full width at half maximum of the water signal. Full width at half maximum was on average 24 Hz, and less than 36 Hz. A single person selected all the volume of interests and performed the MRS examinations blinded to the menopausal status of the subjects. Body composition Fat and fat-free mass for the whole body, trunk, and extremities were measured through dual-energy X-ray absorptiometry scanning (Lunar Prodigy Advance; GE Medical Systems Lunar, Milwaukee, WI). Software (Prodigy, enCORE 2004, version 8.8; GE Lunar Corp, Madison, WI,) was used to estimate regional, total fat, and fat-free tissue masses. ActivPAL activity measurements All participants wore an ActivPAL (PALtechnologies) activity measurement device for four consecutive days to quantify free-living sedentary, upright, and ambulatory activities. By this means, the pattern (sedentary, standing, and stepping) and intensity of a subject’s activities were captured and defined as either sedentary or light- or moderate-intensity activity (17). OGTT All women were instructed not to perform any vigorous exercise 48 hours prior to the experiment and abstain from coffee, tea, or alcohol 24 hours before. They reported to the laboratory after an overnight fast. Blood samples were drawn at time points –10, 0, 15, 30, 60, 90, and 120 minutes after drinking 83 g of glucose monohydrate dissolved in 300 mL of water (24). An estimate of insulin sensitivity was calculated using the composite Matsuda index (25), and area under curve (AUC) was calculated for plasma glucose, serum insulin, and c-peptide. Stumvoll’s first PH index was calculated as a surrogate for β-cell function, as 1283 + 1.829 × Insulin30 – 138.7 × Glucose30 + 3.772 × Insulin0 (26). Laboratory analyses EDTA plasma tubes were immediately spun at 3500g for 15 minutes at 4°C. Serum was stored at room temperature for a minimum of 30 minutes before handling. Estradiol was measured by RIA (Pantex, Santa Monica, CA) with a detection limit of 18 pmol/L. Intra- and interassay coefficient of variations were <8% and <13%, respectively. The estradiol assay was accredited by the Danish Accreditation Fund (DANAK) (27). All additional blood samples were analyzed at the Department of Clinical Biochemistry, Rigshospitalet, Denmark. Statistical analyses All values in tables are presented as means ± SD (28) unless otherwise stated. Variables were log-transformed if appropriate (alanine transaminase, anti-Müllerian hormone, estradiol, FSH, leg fat mass, visceral fat, liver lipid deposition, skeletal muscle lipid deposition, AUC glucose, and Matsuda Index). Differences between groups in lipid deposition in skeletal muscle and liver were analyzed with nonparametric statistics (Mann-Whitney U) and presented as median (interquartile range). Because of collinearity between menopausal status and age, we could not control for age when analyzing the effect of menopausal status on body composition, ectopic lipid deposition, and insulin sensitivity. Predictors in multiple regression models were chosen prior to analyses and based on existing knowledge of associations and outcomes. The size of the study groups limited the number of predictors to a maximum of three (29). Due to collinearity between leg fat mass and total fat mass, a plain subcutaneous fat depot representative was chosen, and total fat mass was left out of the models. Standardized β-values were shown to rank the importance of the predictors in the multiple regression analyses. Regression models were checked for assumptions of the linear model, including normal distribution of the residuals, homogeneity of variance, linearity, and independent observations. All regression analyses were controlled for age. Correlations between serum estradiol and fat masses and ectopic lipid deposition in premenopausal women and all women were performed using a spearman correlation (RS). The dispersion of circulating estradiol levels was limited in postmenopausal women, preventing correlation analyses in this subgroup. Statistical analyses were performed using IBM SPSS Statistics version 22. Results Subject characteristics Subject characteristics are shown in Table 1. Table 1. Subject Characteristics Postmenopausal Women Premenopausal Women n 25 30 Age, ya 55 (49–60)b 48 (45–54) Body composition  Body weight, kg 69.5 ± 12.3 66.1 ± 9.5  Height, cm 166.9 ± 5.3 168.4 ± 6.7  BMI 24.9 ± 4.1 23.3 ± 2.2  Fat-free mass, kg 41.8 ± 4.6 43.0 ± 4.3 Sex hormones  Estradiol, nmol/L 0.08 ± 0.03b 0.33 ± 0.33  FSH, IU/L 75.2 ± 21.6b 27.6 ± 31.3  AMH, pmol/L 0.19 ± 0.05b 2.40 ± 3.88  Testosterone, nmol/L 0.67 ± 0.22 0.84 ± 0.42  Testosterone—free, nmol/L 0.0108 ± 0.0032 0.0154 ± 0.0230 Blood lipids  Total cholesterol, mmol/L 5.5 ± 0.9 5.1 ± 1.1  LDL cholesterol, mmol/L 1.9 ± 0.5 1.8 ± 0.4  HDL cholesterol, mmol/L 3.4 ± 0.7 3.1 ± 1.1  FFA, mmol/L 0.18 ± 0.11 0.16 ± 0.05 Liver parameters  ALT U/L 22 ± 6 24 ± 13  AST U/L 23 ± 4 24 ± 9 Physical activity  Daily METh, total 35.6 ± 1.4 35.4 ± 1.8  Sedentary activity time ≤1.5 METh % of the day 89.8 ± 2.4 90.3 ± 2.6  Light activity time >1.5 and ≤3 METh
% of the day 3.5 ± 1.3 3.3 ± 1.2  Moderate activity time >3 METh % of the day 6.7 ± 2.2 6.4 ± 1.8 Food intake  Daily food intake, kcal 2053 ± 445 1926 ± 341  Daily intake of carbohydrates, g 225 ± 47 207 ± 59  Daily intake of fat, g 84 ± 26 78 ± 23  Daily intake of protein, g 85 ± 26 81 ± 20 Postmenopausal Women Premenopausal Women n 25 30 Age, ya 55 (49–60)b 48 (45–54) Body composition  Body weight, kg 69.5 ± 12.3 66.1 ± 9.5  Height, cm 166.9 ± 5.3 168.4 ± 6.7  BMI 24.9 ± 4.1 23.3 ± 2.2  Fat-free mass, kg 41.8 ± 4.6 43.0 ± 4.3 Sex hormones  Estradiol, nmol/L 0.08 ± 0.03b 0.33 ± 0.33  FSH, IU/L 75.2 ± 21.6b 27.6 ± 31.3  AMH, pmol/L 0.19 ± 0.05b 2.40 ± 3.88  Testosterone, nmol/L 0.67 ± 0.22 0.84 ± 0.42  Testosterone—free, nmol/L 0.0108 ± 0.0032 0.0154 ± 0.0230 Blood lipids  Total cholesterol, mmol/L 5.5 ± 0.9 5.1 ± 1.1  LDL cholesterol, mmol/L 1.9 ± 0.5 1.8 ± 0.4  HDL cholesterol, mmol/L 3.4 ± 0.7 3.1 ± 1.1  FFA, mmol/L 0.18 ± 0.11 0.16 ± 0.05 Liver parameters  ALT U/L 22 ± 6 24 ± 13  AST U/L 23 ± 4 24 ± 9 Physical activity  Daily METh, total 35.6 ± 1.4 35.4 ± 1.8  Sedentary activity time ≤1.5 METh % of the day 89.8 ± 2.4 90.3 ± 2.6  Light activity time >1.5 and ≤3 METh
% of the day 3.5 ± 1.3 3.3 ± 1.2  Moderate activity time >3 METh % of the day 6.7 ± 2.2 6.4 ± 1.8 Food intake  Daily food intake, kcal 2053 ± 445 1926 ± 341  Daily intake of carbohydrates, g 225 ± 47 207 ± 59  Daily intake of fat, g 84 ± 26 78 ± 23  Daily intake of protein, g 85 ± 26 81 ± 20 Data are presented as mean ± SD unless otherwise stated. Abbreviations: ALT, alanine transaminase; AMH, anti-Müllerian hormone; AST, aspartate transaminase; FFA, free fatty acid; HDL, high-density lipoprotein; LDL, low-density lipoprotein. a Data presented as median ± range. b Significantly different from premenopausal (P ≤ 0.05). View Large Table 1. Subject Characteristics Postmenopausal Women Premenopausal Women n 25 30 Age, ya 55 (49–60)b 48 (45–54) Body composition  Body weight, kg 69.5 ± 12.3 66.1 ± 9.5  Height, cm 166.9 ± 5.3 168.4 ± 6.7  BMI 24.9 ± 4.1 23.3 ± 2.2  Fat-free mass, kg 41.8 ± 4.6 43.0 ± 4.3 Sex hormones  Estradiol, nmol/L 0.08 ± 0.03b 0.33 ± 0.33  FSH, IU/L 75.2 ± 21.6b 27.6 ± 31.3  AMH, pmol/L 0.19 ± 0.05b 2.40 ± 3.88  Testosterone, nmol/L 0.67 ± 0.22 0.84 ± 0.42  Testosterone—free, nmol/L 0.0108 ± 0.0032 0.0154 ± 0.0230 Blood lipids  Total cholesterol, mmol/L 5.5 ± 0.9 5.1 ± 1.1  LDL cholesterol, mmol/L 1.9 ± 0.5 1.8 ± 0.4  HDL cholesterol, mmol/L 3.4 ± 0.7 3.1 ± 1.1  FFA, mmol/L 0.18 ± 0.11 0.16 ± 0.05 Liver parameters  ALT U/L 22 ± 6 24 ± 13  AST U/L 23 ± 4 24 ± 9 Physical activity  Daily METh, total 35.6 ± 1.4 35.4 ± 1.8  Sedentary activity time ≤1.5 METh % of the day 89.8 ± 2.4 90.3 ± 2.6  Light activity time >1.5 and ≤3 METh
% of the day 3.5 ± 1.3 3.3 ± 1.2  Moderate activity time >3 METh % of the day 6.7 ± 2.2 6.4 ± 1.8 Food intake  Daily food intake, kcal 2053 ± 445 1926 ± 341  Daily intake of carbohydrates, g 225 ± 47 207 ± 59  Daily intake of fat, g 84 ± 26 78 ± 23  Daily intake of protein, g 85 ± 26 81 ± 20 Postmenopausal Women Premenopausal Women n 25 30 Age, ya 55 (49–60)b 48 (45–54) Body composition  Body weight, kg 69.5 ± 12.3 66.1 ± 9.5  Height, cm 166.9 ± 5.3 168.4 ± 6.7  BMI 24.9 ± 4.1 23.3 ± 2.2  Fat-free mass, kg 41.8 ± 4.6 43.0 ± 4.3 Sex hormones  Estradiol, nmol/L 0.08 ± 0.03b 0.33 ± 0.33  FSH, IU/L 75.2 ± 21.6b 27.6 ± 31.3  AMH, pmol/L 0.19 ± 0.05b 2.40 ± 3.88  Testosterone, nmol/L 0.67 ± 0.22 0.84 ± 0.42  Testosterone—free, nmol/L 0.0108 ± 0.0032 0.0154 ± 0.0230 Blood lipids  Total cholesterol, mmol/L 5.5 ± 0.9 5.1 ± 1.1  LDL cholesterol, mmol/L 1.9 ± 0.5 1.8 ± 0.4  HDL cholesterol, mmol/L 3.4 ± 0.7 3.1 ± 1.1  FFA, mmol/L 0.18 ± 0.11 0.16 ± 0.05 Liver parameters  ALT U/L 22 ± 6 24 ± 13  AST U/L 23 ± 4 24 ± 9 Physical activity  Daily METh, total 35.6 ± 1.4 35.4 ± 1.8  Sedentary activity time ≤1.5 METh % of the day 89.8 ± 2.4 90.3 ± 2.6  Light activity time >1.5 and ≤3 METh
% of the day 3.5 ± 1.3 3.3 ± 1.2  Moderate activity time >3 METh % of the day 6.7 ± 2.2 6.4 ± 1.8 Food intake  Daily food intake, kcal 2053 ± 445 1926 ± 341  Daily intake of carbohydrates, g 225 ± 47 207 ± 59  Daily intake of fat, g 84 ± 26 78 ± 23  Daily intake of protein, g 85 ± 26 81 ± 20 Data are presented as mean ± SD unless otherwise stated. Abbreviations: ALT, alanine transaminase; AMH, anti-Müllerian hormone; AST, aspartate transaminase; FFA, free fatty acid; HDL, high-density lipoprotein; LDL, low-density lipoprotein. a Data presented as median ± range. b Significantly different from premenopausal (P ≤ 0.05). View Large Postmenopausal women were 6.5 (±4.0) years older than premenopausal women [55 years (range: 49 to 60) vs 49 years (range: 45 to 54)] and had on average been postmenopausal for 5.4 (±1.8) years (Table 1). Postmenopausal and premenopausal women showed no significant differences in body weight (69.5 ± 12.3 vs 66.1 ± 9.5 kg, P = 0.26), BMI (24.9 ± 4.1 vs 23.3 ± 2.2, P = 0.08), fat-free mass (41.8 ± 4.6 vs 42.6 ± 4.5, P = 0.53), daily physical activity [35.6 ± 1.4 vs 35.4 ± 1.8 metabolic equivalent hours (METh), P = 0.68], or self-reported daily food intake (2053 ± 444 vs 1926 ± 341 calories, P = 0.24) (Table 1). Fat mass distribution and ectopic lipid deposition Fat mass distribution Postmenopausal women had significantly more visceral fat (0.757 ± 0.507 vs 0.459 ± 0.399 L, P = 0.006) and a trend toward an increased total fat mass (24.7 ± 10.0 vs 20.3 ± 6.7 kg, P = 0.07) and total leg fat mass (9.0 ± 3.6 vs 7.6 ± 2.5 kg, P = 0.08) compared with premenopausal women (Fig. 1a–1c). Figure 1. View largeDownload slide Fat depot sizes in postmenopausal and premenopausal women. (a) Total fat mass (kg), (b) visceral fat mass (L), and (c) leg fat mass (kg). (d) Association between total fat mass and visceral fat mass, (e) association between leg fat mass and visceral fat mass, and (f) association between serum estradiol and visceral fat mass in postmenopausal (white, n = 25) and premenopausal women (black, n = 30). Data are presented as mean ± SEM. *Significantly different from premenopausal women, P < 0.05. Log, logarithm. Figure 1. View largeDownload slide Fat depot sizes in postmenopausal and premenopausal women. (a) Total fat mass (kg), (b) visceral fat mass (L), and (c) leg fat mass (kg). (d) Association between total fat mass and visceral fat mass, (e) association between leg fat mass and visceral fat mass, and (f) association between serum estradiol and visceral fat mass in postmenopausal (white, n = 25) and premenopausal women (black, n = 30). Data are presented as mean ± SEM. *Significantly different from premenopausal women, P < 0.05. Log, logarithm. A 10% increase in total fat mass was associated with a 12% (95% CI: 7% to 18%) increase in visceral fat mass in postmenopausal women and a 13% (95% CI: 7% to 19%) increase in premenopausal women. For a given total fat mass, postmenopausal women stored significantly more visceral fat (P = 0.03) (Fig. 1d). A 10% increase in leg fat mass was associated with an 8% (95% CI: 1% to 14%) increase in visceral fat mass in postmenopausal women and a nonsignificant 1% (95% CI: –6% to 9%) increase in premenopausal women. For a given leg fat mass, postmenopausal women stored significantly more visceral fat (P = 0.02) (Fig. 1e). Visceral fat mass was negatively correlated to serum estradiol in the total group of women (RS = –0.314, P = 0.01) but not in premenopausal women alone (RS = –0.309, P = 0.11) (Fig. 1f). Total fat mass and leg fat mass showed no correlations to serum estradiol (data not shown). Total fat mass, leg fat mass, and visceral fat mass were not significantly associated with age in either postmenopausal or premenopausal women. Lipid deposition in the liver Postmenopausal women had significantly more lipid in the liver compared with premenopausal women [0.68% (0.44 to 0.99) vs 0.49% (0.38 to 0.64), P = 0.01] (Fig. 2a). Figure 2. View largeDownload slide Lipid deposition in the liver and intramuscular lipid deposition in postmenopausal and premenopausal women. (a) Lipid deposition in the liver, (b) association between total fat mass and lipid deposition in the liver, (c) association between visceral fat mass and lipid deposition in the liver, (d) association between leg fat mass and lipid deposition in the liver, (e) association between serum estradiol and lipid deposition in the liver, (f) intramuscular lipid deposition, (g), association between total fat mass and intramuscular lipid deposition, (h) association between visceral fat mass and intramuscular lipid deposition, (i) association between leg fat mass and intramuscular lipid deposition, and (j) association between serum estradiol and intramuscular lipid deposition. All analyses were done in postmenopausal (white, n = 25) and premenopausal women (black, n = 30). Data are presented as median (interquartile range). *Significantly different from premenopausal women, P < 0.05. Log, logarithm. Figure 2. View largeDownload slide Lipid deposition in the liver and intramuscular lipid deposition in postmenopausal and premenopausal women. (a) Lipid deposition in the liver, (b) association between total fat mass and lipid deposition in the liver, (c) association between visceral fat mass and lipid deposition in the liver, (d) association between leg fat mass and lipid deposition in the liver, (e) association between serum estradiol and lipid deposition in the liver, (f) intramuscular lipid deposition, (g), association between total fat mass and intramuscular lipid deposition, (h) association between visceral fat mass and intramuscular lipid deposition, (i) association between leg fat mass and intramuscular lipid deposition, and (j) association between serum estradiol and intramuscular lipid deposition. All analyses were done in postmenopausal (white, n = 25) and premenopausal women (black, n = 30). Data are presented as median (interquartile range). *Significantly different from premenopausal women, P < 0.05. Log, logarithm. A 10% increase in total fat mass was associated with a 19% (95% CI: 10% to 29%) increase in lipid deposition in the liver in postmenopausal women and a nonsignificant 5% (95% CI: –1% to 12%) increase in premenopausal women (Fig. 2b). The association differed significantly with menopausal status (interaction, P = 0.01). A 10% increase in visceral fat mass was associated with a 12% (95% CI: 8% to 17%) increase in lipid deposition in the liver in postmenopausal women and a 5% (95% CI: 2% to 8%) increase in premenopausal women (Fig. 2c). The association differed significantly with menopausal status (interaction, P = 0.005). A 10% increase in leg fat mass was associated with a 13% (95% CI: 3% to 24%) increase in lipid deposition in the liver in postmenopausal women compared with a nonsignificant 1% (95% CI: –8% to 5%) decrease in premenopausal women. The association differed significantly with menopausal status (interaction, P = 0.02) (Fig. 2d). Lipid deposition in the liver was negatively correlated to serum estradiol in the total group of women (RS = –0.303, P = 0.02) and in premenopausal women alone (RS = –0.370, P = 0.05) (Fig. 2e). In a multiple regression analysis with visceral fat mass and leg fat mass as predictors of lipid deposition in the liver, only the visceral fat mass remained a significant predictor in both postmenopausal (P < 0.0001) and premenopausal women (P = 0.001) (Table 2). Table 2. Multiple Regression Analyses of the Predictors of Lipid Deposition in Liver, Intramuscular Lipid Deposition, and Insulin Sensitivity Postmenopausal Women Premenopausal Women Predictors βU
(% Increase) 95% CI βS βU
(% Increase) 95% CI βS Lipid deposition in the liver  Visceral fat mass (10% increase) 11.4a 6.8 to 16.2 1.132 5.3 2.5 to 8.3 0.602  Leg fat mass (10% increase) 3.8a –3.4 to 11.5 0.392 −2.1 −7.2 to 3.3 −0.217 Intramuscular lipid deposition  Visceral fat mass (10% increase) 4.0b 1.1 to 7.1 0.533 2.1 −0.7 to 5.0 0.277  Leg fat mass (10% increase) 2.1 −2.7 to 7.2 0.166 2.3 −3.2 to 8.0 0.154 Insulin sensitivity  Visceral fat mass (10% increase) −1.1 −5.4 to 3.4 −0.123 −0.2 −4.1 to 3.9 −0.017  Lipid deposition in the liver (10% increase) −4.2 −7.1 to 1.2 −0.731 −4.3 −8.5 to 0.1 −0.478  Intramuscular lipid deposition (10% increase) 0.01 −4.0 to 4.2 0.003 −1.8 −5.7 to 2.3 −0.174 Postmenopausal Women Premenopausal Women Predictors βU
(% Increase) 95% CI βS βU
(% Increase) 95% CI βS Lipid deposition in the liver  Visceral fat mass (10% increase) 11.4a 6.8 to 16.2 1.132 5.3 2.5 to 8.3 0.602  Leg fat mass (10% increase) 3.8a –3.4 to 11.5 0.392 −2.1 −7.2 to 3.3 −0.217 Intramuscular lipid deposition  Visceral fat mass (10% increase) 4.0b 1.1 to 7.1 0.533 2.1 −0.7 to 5.0 0.277  Leg fat mass (10% increase) 2.1 −2.7 to 7.2 0.166 2.3 −3.2 to 8.0 0.154 Insulin sensitivity  Visceral fat mass (10% increase) −1.1 −5.4 to 3.4 −0.123 −0.2 −4.1 to 3.9 −0.017  Lipid deposition in the liver (10% increase) −4.2 −7.1 to 1.2 −0.731 −4.3 −8.5 to 0.1 −0.478  Intramuscular lipid deposition (10% increase) 0.01 −4.0 to 4.2 0.003 −1.8 −5.7 to 2.3 −0.174 Analyses were performed in postmenopausal (n = 25) and premenopausal women (n = 30), corrected for age. All analyses were done on log-transformed data. Abbreviations: βS, standardized β (indicating ranking of importance of predictors on outcome); βU, unstandardized β. a Significant interaction (P ≤ 0.05). b Significantly different from postmenopausal (P ≤ 0.05). View Large Table 2. Multiple Regression Analyses of the Predictors of Lipid Deposition in Liver, Intramuscular Lipid Deposition, and Insulin Sensitivity Postmenopausal Women Premenopausal Women Predictors βU
(% Increase) 95% CI βS βU
(% Increase) 95% CI βS Lipid deposition in the liver  Visceral fat mass (10% increase) 11.4a 6.8 to 16.2 1.132 5.3 2.5 to 8.3 0.602  Leg fat mass (10% increase) 3.8a –3.4 to 11.5 0.392 −2.1 −7.2 to 3.3 −0.217 Intramuscular lipid deposition  Visceral fat mass (10% increase) 4.0b 1.1 to 7.1 0.533 2.1 −0.7 to 5.0 0.277  Leg fat mass (10% increase) 2.1 −2.7 to 7.2 0.166 2.3 −3.2 to 8.0 0.154 Insulin sensitivity  Visceral fat mass (10% increase) −1.1 −5.4 to 3.4 −0.123 −0.2 −4.1 to 3.9 −0.017  Lipid deposition in the liver (10% increase) −4.2 −7.1 to 1.2 −0.731 −4.3 −8.5 to 0.1 −0.478  Intramuscular lipid deposition (10% increase) 0.01 −4.0 to 4.2 0.003 −1.8 −5.7 to 2.3 −0.174 Postmenopausal Women Premenopausal Women Predictors βU
(% Increase) 95% CI βS βU
(% Increase) 95% CI βS Lipid deposition in the liver  Visceral fat mass (10% increase) 11.4a 6.8 to 16.2 1.132 5.3 2.5 to 8.3 0.602  Leg fat mass (10% increase) 3.8a –3.4 to 11.5 0.392 −2.1 −7.2 to 3.3 −0.217 Intramuscular lipid deposition  Visceral fat mass (10% increase) 4.0b 1.1 to 7.1 0.533 2.1 −0.7 to 5.0 0.277  Leg fat mass (10% increase) 2.1 −2.7 to 7.2 0.166 2.3 −3.2 to 8.0 0.154 Insulin sensitivity  Visceral fat mass (10% increase) −1.1 −5.4 to 3.4 −0.123 −0.2 −4.1 to 3.9 −0.017  Lipid deposition in the liver (10% increase) −4.2 −7.1 to 1.2 −0.731 −4.3 −8.5 to 0.1 −0.478  Intramuscular lipid deposition (10% increase) 0.01 −4.0 to 4.2 0.003 −1.8 −5.7 to 2.3 −0.174 Analyses were performed in postmenopausal (n = 25) and premenopausal women (n = 30), corrected for age. All analyses were done on log-transformed data. Abbreviations: βS, standardized β (indicating ranking of importance of predictors on outcome); βU, unstandardized β. a Significant interaction (P ≤ 0.05). b Significantly different from postmenopausal (P ≤ 0.05). View Large Lipid deposition in the liver was not significantly associated with age in either postmenopausal or premenopausal women. Intramuscular lipid deposition Postmenopausal women had significantly more intramuscular lipid in the vastus lateralis of the quadriceps muscle compared with premenopausal women [3% (2% to 4%) vs 2% (1% to 3%), P = 0.001] (Fig. 2f). A 10% increase in total fat mass was significantly associated with increased intramuscular lipid deposition in both postmenopausal [8% (95% CI: 3% to 13%)] and premenopausal women [7% (95% CI: 2% to 12%)]. However, for a fixed amount of total body fat, postmenopausal women stored more lipid in skeletal muscle compared with premenopausal women (P = 0.004) (Fig. 2g). Intramuscular lipid deposition was significantly associated with visceral fat mass and leg fat mass only in the postmenopausal women: A 10% increase in visceral fat mass was associated with a 5% (95% CI: 2% to 7%) increase in intramuscular lipid deposition in postmenopausal women compared with a nonsignificant 2% (95% CI: –1% to 5%) increase in premenopausal women. For a given visceral fat mass, postmenopausal women stored significantly more intramuscular lipids (P = 0.01) (Fig. 2h). A 10% increase in leg fat mass was associated with a 5% (95% CI: 1% to 11%) increase in intramuscular lipid deposition in postmenopausal women and a nonsignificant 3% (95% CI: –3% to 8%) increase in premenopausal women. For a given leg fat mass, postmenopausal women stored significantly more intramuscular lipids (P = 0.002) (Fig. 2i). There was no significant correlation between intramuscular lipid deposition and serum estradiol in the total group of women or in premenopausal women alone (Fig. 2j). In a multiple regression analysis with visceral fat mass and leg fat mass as predictors for intramuscular lipid deposition, only visceral fat mass remained as a significant predictor and only in postmenopausal women (P = 0.009) (Table 2). Intramuscular lipid deposition was not significantly associated with age in either postmenopausal or premenopausal women. Insulin sensitivity Postmenopausal women had 19% higher blood glucose (919 ± 277 vs 775 ± 156, P = 0.03) and a 28% lower Matsuda insulin sensitivity index (6.31 ± 3.48 vs 8.78 ± 4.67, P = 0.05) during an OGTT compared with premenopausal women. Insulin (50,190 ± 35,781 vs 39,050 ± 22,194, P = 0.17) and c-peptide AUC (307,430 ± 123,991 vs 262,932 ± 92,389, P = 0.17) were not significantly different between postmenopausal and premenopausal women. However, c-peptide was significantly higher after 120 minutes in postmenopausal women compared with premenopausal women (Fig. 3a–3f). Stumvoll’s index did not differ between groups (data not shown). Figure 3. View largeDownload slide Blood glucose and insulin during an OGTT in postmenopausal and premenopausal women. (a and b) Blood glucose, (c and d) insulin levels, (e) c-peptide, (f) Matsuda insulin sensitivity, and (g) correlation between estradiol and Matsuda insulin sensitivity during an OGTT in postmenopausal (white, n = 20) and premenopausal women (black, n = 27). Data are presented as mean ± SEM. *Significantly different from premenopausal women, P < 0.05. Figure 3. View largeDownload slide Blood glucose and insulin during an OGTT in postmenopausal and premenopausal women. (a and b) Blood glucose, (c and d) insulin levels, (e) c-peptide, (f) Matsuda insulin sensitivity, and (g) correlation between estradiol and Matsuda insulin sensitivity during an OGTT in postmenopausal (white, n = 20) and premenopausal women (black, n = 27). Data are presented as mean ± SEM. *Significantly different from premenopausal women, P < 0.05. Insulin sensitivity was not directly correlated to serum estradiol in the total group of women or in premenopausal women alone (Fig. 3g). Matsuda insulin sensitivity was not significantly associated with age in either postmenopausal or premenopausal women. Insulin sensitivity and total fat mass Matsuda insulin sensitivity index was significantly associated with total fat mass in postmenopausal women [a 10% increase in total fat mass was associated with a 7.3% decrease (95% CI: –10.4% to 1.0%) in insulin sensitivity] but not in premenopausal women [–3.2% (95% CI: –9.2% to 3.2%)] (Fig. 4a). Figure 4. View largeDownload slide Association between lipid deposition and Matsuda insulin sensitivity in postmenopausal and premenopausal women. Association between Matsuda insulin sensitivity and (a) total fat mass, (b) visceral fat mass, (c) leg fat mass, (d) lipid deposition in the liver, and (e) intramuscular lipid deposition in postmenopausal (white, n = 20) and premenopausal women (black, n = 27). Log, logarithm. Figure 4. View largeDownload slide Association between lipid deposition and Matsuda insulin sensitivity in postmenopausal and premenopausal women. Association between Matsuda insulin sensitivity and (a) total fat mass, (b) visceral fat mass, (c) leg fat mass, (d) lipid deposition in the liver, and (e) intramuscular lipid deposition in postmenopausal (white, n = 20) and premenopausal women (black, n = 27). Log, logarithm. Insulin sensitivity and visceral fat mass In postmenopausal women, Matsuda insulin sensitivity index was significantly associated with visceral fat mass, as a 10% increase in visceral fat mass was associated with a 6.4% decrease (95% CI: –8.9% to 3.1%) in insulin sensitivity. Premenopausal women showed a trend to an association between visceral fat mass and insulin sensitivity [a 10% increase in visceral fat mass was associated with a 3.0% decrease (95% CI: –5.8% to 0.1%) in insulin sensitivity]. The association differed significantly with menopausal status (interaction, P = 0.003) (Fig. 4b). Insulin sensitivity and leg fat mass There was no significant association between Matsuda insulin sensitivity and leg fat mass in either postmenopausal or premenopausal women (Fig. 4c). Insulin sensitivity and lipid deposition in the liver Postmenopausal and premenopausal women showed no significant differences in the association between lipid deposition in the liver and insulin sensitivity, as a 10% increase in lipid deposition in the liver was associated with a significant decrease in insulin sensitivity in both postmenopausal [–4.5% (95% CI: –3.1% to 5.9%)] and premenopausal women [–4.9% (95% CI: –1.6% to 8.1%)] (Fig. 4d). Insulin sensitivity and intramuscular lipid deposition A 10% increase in intramuscular lipid deposition was associated with a 6.1% decrease in insulin sensitivity (95% CI: –1.6% to 10.5%) in postmenopausal women, whereas, in premenopausal women, a 10% increase in intramuscular lipid deposition was associated with a nonsignificant 2.8% decrease (95% CI: –6.9% to 1.6%) in insulin sensitivity (Fig. 4e). Predictors of insulin sensitivity In a multiple regression analysis with visceral fat mass, lipid deposition in the liver, and intramuscular lipid deposition as predictors of insulin sensitivity, lipid deposition in the liver was the strongest predictor of insulin sensitivity in both postmenopausal and premenopausal women but was only a statistically significant predictor in postmenopausal women (P = 0.01) (Table 2). Discussion In this cross-sectional study of 55 women between 45 and 60 years of age, we found that postmenopausal women had an increased lipid deposition in the liver and skeletal muscle compared with that of premenopausal women. Total fat mass, visceral fat mass and leg fat mass were stronger predictors of ectopic lipid deposition and decreased insulin sensitivity in postmenopausal compared with premenopausal women. In accordance with our findings, a previous study (16) showed that older postmenopausal Asian women had an increased incidence of NAFLD compared with younger premenopausal Asian women. However, study participants had a broad range of ages (21 to 80 years old) and the researchers used abdominal ultrasonography to diagnose NAFLD, a method unable to provide reliable quantitative information, and with a lower limit of detection at around 15% to 20% lipid in the hepatocyte (30), which greatly exceeds the lipid deposition in the liver of the healthy group of women in this study. Increased visceral fat mass is known to be associated with ectopic lipid deposition in the liver in both rodents (11) and diabetic and nondiabetic humans (31, 32). This is in agreement with our findings as both postmenopausal and premenopausal women showed significant positive associations between visceral fat mass and lipid deposition in the liver. Interestingly, the association between visceral fat mass and lipid deposition in the liver was significantly steeper in the postmenopausal women, and for a given visceral fat mass, postmenopausal women stored more intramuscular lipids. These findings indicate that the health-related consequences linked to expanding the visceral fat depot increase after menopause. In support of this, we showed that increased visceral fat mass was strongly associated with decreased insulin sensitivity only in postmenopausal women. It has been suggested that leg fat does not play the same pathological role in the development of metabolic disease as other fat depots and may instead be protective (7, 33). In accordance with this, we found no significant associations between leg fat mass and lipid deposition in liver and skeletal muscle in premenopausal women. In contrast, postmenopausal women showed significant positive associations between leg fat mass and lipid deposition in liver and skeletal muscle, indicating that the beneficial role of leg fat mass could change with menopausal status. However, leg fat mass was not associated with intramuscular lipid deposition when controlling for visceral fat mass and was not a significant predictor of insulin resistance in either postmenopausal or premenopausal women. Thus, the importance of leg fat mass in whole-body insulin resistance seemed to be limited. Whether ectopic lipid deposition in the liver is related to total body fat is debated (34, 35). In this study, the association between total fat mass and ectopic lipid deposition varied with menopausal status; we showed that total fat mass was a significantly stronger predictor of lipid depositions in liver and skeletal muscle in postmenopausal women compared with premenopausal women. The fact that fat depot size was a stronger predictor of ectopic lipid deposition in postmenopausal women could indicate that postmenopausal women have a more dysfunctional adipose tissue both in the subcutaneous and visceral fat depot, with a lower threshold for lipid spillover compared with premenopausal women. In support of our findings, oophorectomized mice show increased adipocyte size and infiltration of both macrophages and T-cells in both intra-abdominal and subcutaneous adipose tissue (36–38), ectopic lipid deposition in liver, and skeletal muscle (15, 39) and decreased insulin sensitivity (36, 38), indicating that loss of ovarian function leads to a general dysfunction in both subcutaneous and visceral adipose tissue. However, these findings still need further investigation in humans. Postmenopausal women showed increased blood glucose throughout the OGTT, which could be an indication of decreased β-cell function, as insulin secretion has been shown to be the major determinant of glucose disposal after an oral glucose load (40, 41). However, pre- and postmenopausal women showed no significant differences in Stumvoll’s 1st PH index, a surrogate for β-cell function. Furthermore, although not significantly elevated, insulin levels tended to be generally increased throughout the OGTT, leaving a possible role for peripheral insulin resistance in the decreased insulin sensitivity seen with menopause, as reported in oophorectomized rodents (42). Inactivity is known to affect lipid accumulation in adipose tissue and ectopic sites and is associated with decreased insulin sensitivity (43). Studies in mice have shown that loss of ovarian function leads to decreased physical activity (37, 38). In contrast, human studies investigating physical activity level over menopause have failed to identify a decrease in fitness with menopause when controlling for differences in lean body mass (5, 6). In agreement with this, we found no differences in daily physical activity between postmenopausal and premenopausal women. Thus, loss of physical fitness with menopause was hardly responsible for the differences in ectopic lipid deposition and glucose metabolism found in this study. Our study has some limitations. Postmenopausal women were 6.5 years older than premenopausal women, and because of the study design, the effects of menopause and age on ectopic lipid deposition and insulin resistance could not be addressed separately. However, age was not significantly associated with fat depot size, ectopic lipid deposition, or insulin sensitivity in either postmenopausal or premenopausal women. To further address the impact of age on ectopic lipid deposition and insulin resistance, we repeated all analyses in the subset of women who had an age between 49 and 54 years, leaving 11 postmenopausal women (mean age: 52.7) and 11 premenopausal women (mean age: 52.1) in the analyses. As the results of the analyses in this subgroup of women were in accordance with the results from the complete study group, we conclude that age is less likely to have played any major role in fat distribution, ectopic lipid deposition, and insulin sensitivity in the relatively narrow age span investigated in this study. We conclude that postmenopausal women have an increased lipid deposition in liver and skeletal muscle compared with premenopausal women. Total, visceral, and leg fat mass were all stronger predictors of ectopic lipid deposition in postmenopausal women, suggesting a general adipose tissue dysfunction with menopause associated with a decreased whole-body insulin sensitivity These findings bring additional value to the understanding of the increased occurrence of metabolic disease with menopause. Lipid deposition in liver and skeletal muscle may represent important mechanistic links between the changes in fat depots and the increased incidence of insulin resistance seen after menopause. Abbreviations: Abbreviations: AUC area under curve BMI body mass index METh metabolic equivalent hours MRS magnetic resonance spectroscopy NAFLD nonalcoholic fatty liver disease OGTT oral glucose tolerance test Acknowledgments Financial Support: This work was supported by Augustinus Fonden Grant 17-2354 (to J.A.). Disclosure Summary: The authors have nothing to disclose References 1. 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Oxford University Press
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Copyright © 2018 Endocrine Society
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0021-972X
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1945-7197
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10.1210/jc.2018-00554
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Abstract

Abstract Context Menopause is associated with an increased incidence of insulin resistance and diabetes. Objective The aim of this study was to explore the lipid deposition in liver and skeletal muscle and investigate the association with insulin sensitivity in postmenopausal and premenopausal women. Design and Setting Single-center cross-sectional study of 55 healthy women between 45 and 60 years of age. We measured lipid deposition in the liver with magnetic resonance spectroscopy, intramuscular and intra-abdominal lipid deposition with MRI, body composition with a dual-energy X-ray absorptiometry scan, and insulin sensitivity with the composite Matsuda Index. Outcome Measures We studied the association between fat distribution, ectopic lipid deposition, and insulin sensitivity in pre- and postmenopausal women. Results Postmenopausal women had an increased lipid deposition in the liver [0.68% (0.44 to 0.99) vs 0.49% (0.38 to 0.64), P = 0.01] and skeletal muscle [3% (2 to 4) vs 2% (1 to 3), P = 0.001] and had a 28% lower Matsuda insulin sensitivity index during an oral glucose tolerance test (6.31 ± 3.48 vs 8.78 ± 4.67, P = 0.05) compared with premenopausal women. Total fat mass and leg fat mass were stronger predictors of ectopic lipid deposition, and visceral fat mass was a stronger predictor of both ectopic lipid deposition and insulin resistance in postmenopausal women compared with premenopausal women. Conclusions For a given subcutaneous and visceral fat depot size, postmenopausal women show increased ectopic lipid deposition and insulin resistance compared with premenopausal women. It is suggested that lipid deposition in liver and skeletal muscle may represent important mechanistic links between the changes in fat depots and the increased incidence of insulin resistance seen after menopause. The prevalence of obesity and metabolic disease has reached epidemic proportions and is a major health concern in the Western world (1). Menopause defines the end of women’s reproductive phase and is associated with an increased occurrence of metabolic disease, including metabolic syndrome (2), diabetes (3), and cardiovascular disease (4). In modern society, women live more than one-third of their lives after menopause. The increased disease burden after the menopausal transition therefore is a major health concern. Through the menopausal transition, the body composition changes from favoring gluteofemoral to truncal fat deposition, particularly visceral fat accumulation (5, 6). In general, increasing gluteofemoral fat mass is associated with improved metabolic health (7), whereas expansion of the visceral fat mass leads to dysfunctional adipose tissue, including adipocyte hypertrophy, macrophage infiltration, and impaired insulin signaling (8). Increased visceral fat mass results in a spillover of excessive amounts of lipids and production of inflammatory cytokines (9) promoting ectopic lipid deposition and lipotoxicity in liver and skeletal muscle and ultimately leading to insulin resistance (10, 11). Young men show increased lipid deposition in liver and skeletal muscle compared with young women (12, 13), and oophorectomized rodents show increased lipid deposition in liver and skeletal muscle compared with sham animals (14, 15). Furthermore, one study found an increased incidence of nonalcoholic fatty liver disease (NAFLD) in older women compared with younger women (16), all in all suggesting a role for female sex hormones in ectopic lipid deposition. Thus, increased ectopic lipid deposition during the menopausal transition could be an important mechanistic link between the fat depot changes and the increased incidence of insulin resistance seen after menopause; however, this link has yet to be investigated. In this cross-sectional study of 55 middle-aged women between 45 and 60 years of age, we hypothesized that postmenopausal women had an increased lipid deposition in liver and skeletal muscle compared with that of premenopausal women and that the increased ectopic lipid deposition in postmenopausal women was associated with increased insulin resistance. Methods Participants Fifty-five women between 45 and 60 years of age were included in the study. Thirty-seven women (18 postmenopausal and 19 premenopausal women) were recruited through advertisement, and 18 women (7 postmenopausal and 11 premenopausal women) were recruited through another study in the same department investigating cellular changes in adipose tissue with menopause. Information on general health and menstrual bleeding history was collected from all women. Exclusion criteria were (1) chronic diseases, (2) infections during the last 4 weeks, (3) smoking, (4) more than 7 alcohol units/wk, (5) premature menopause (before age 40 years), (6) hysterectomy or oophorectomy prior to study inclusion, (7) body mass index (BMI) >35, (8) weight changes >10% within the last year, and (9) dietary changes within the last month. Women with a menstrual period within the last 3 months had their hormonal status analyzed in the follicular phase of the first following menstrual period (between days 1 and 8 of their menstrual cycle) (n = 19). The remaining women were enrolled on a random day (n = 36). The study was approved by the Research Ethics Committees of the Capital Region of Denmark (H-3-2014-096) and performed according to the Declaration of Helsinki. Study design Women were categorized as either premenopausal (menstrual bleeding within the last 12 months) or postmenopausal (no menstrual bleeding within the last 12 months). All women went through (1) MRI of the abdomen and thigh and magnetic resonance spectroscopy (MRS) of the liver, (2) dual-energy X-ray absorptiometry scan, (3) ActivPAL (PALtechnologies, Glasgow, Scotland) measurements of free-living activity (17), and (4) blood samples including hormonal status. Forty-seven of the 55 women further agreed to go through an oral glucose tolerance test (OGTT). All subjects tracked their food intake with a 3-day food diary prior to the MRI/MRS. For every visit, a menstrual bleeding diary was updated and physical activity level was assessed by the Minnesota leisure time physical activity questionnaire (18). Magnetic resonance analyses All women abstained from vigorous exercise and alcohol 48 hours prior to the scans as well as food, liquids, and chewing gum 3 hours prior to the scan. MRI and MRS were performed using a Siemens Magnetom Prisma 3 Tesla matrix magnetic resonance scanner (Erlangen, Germany) at 3-mm intervals. All adipose tissue located from the diaphragm to pelvic floor inside the peritoneum was traced manually as the visceral fat region of interest. Multi-image analysis software (19) (Mango; Research Imaging Institute, Houston, TX) was used to calculate the total volume of visceral fat from the T1-weighted MRI sequence. A single reader, who was blinded to the menopausal status of the subjects, performed all image analyses. Skeletal muscle fat deposition measurements were done as described elsewhere (20). Briefly, skeletal muscle fat deposition was measured in a single MRI slice of the vastus lateralis of the quadriceps muscle, midthigh, as single slice measurements of skeletal muscle fat deposition has shown to be representative of a whole muscle or muscle group (21). The images were analyzed using Siemens Syngo FastView. Fat fraction was calculated as: Fat fraction (%)=(signal fat/signal water +fat ) 100. Magnetic resonance spectroscopy to assess liver fat was performed as described elsewhere (22) but with a few specific adjustments including a 1H magnetic resonance single-voxel PRESS sequence: repetition time/echo time/number of scans/prescan = 3000 ms/33 ms/4/1. Three spectra were sampled and evaluated using the user input independent software LCModel (23), and the volume fraction of fat was calculated in accordance with Hájek et al. (22). The spectral quality was assessed by the half widths full width at half maximum of the water signal. Full width at half maximum was on average 24 Hz, and less than 36 Hz. A single person selected all the volume of interests and performed the MRS examinations blinded to the menopausal status of the subjects. Body composition Fat and fat-free mass for the whole body, trunk, and extremities were measured through dual-energy X-ray absorptiometry scanning (Lunar Prodigy Advance; GE Medical Systems Lunar, Milwaukee, WI). Software (Prodigy, enCORE 2004, version 8.8; GE Lunar Corp, Madison, WI,) was used to estimate regional, total fat, and fat-free tissue masses. ActivPAL activity measurements All participants wore an ActivPAL (PALtechnologies) activity measurement device for four consecutive days to quantify free-living sedentary, upright, and ambulatory activities. By this means, the pattern (sedentary, standing, and stepping) and intensity of a subject’s activities were captured and defined as either sedentary or light- or moderate-intensity activity (17). OGTT All women were instructed not to perform any vigorous exercise 48 hours prior to the experiment and abstain from coffee, tea, or alcohol 24 hours before. They reported to the laboratory after an overnight fast. Blood samples were drawn at time points –10, 0, 15, 30, 60, 90, and 120 minutes after drinking 83 g of glucose monohydrate dissolved in 300 mL of water (24). An estimate of insulin sensitivity was calculated using the composite Matsuda index (25), and area under curve (AUC) was calculated for plasma glucose, serum insulin, and c-peptide. Stumvoll’s first PH index was calculated as a surrogate for β-cell function, as 1283 + 1.829 × Insulin30 – 138.7 × Glucose30 + 3.772 × Insulin0 (26). Laboratory analyses EDTA plasma tubes were immediately spun at 3500g for 15 minutes at 4°C. Serum was stored at room temperature for a minimum of 30 minutes before handling. Estradiol was measured by RIA (Pantex, Santa Monica, CA) with a detection limit of 18 pmol/L. Intra- and interassay coefficient of variations were <8% and <13%, respectively. The estradiol assay was accredited by the Danish Accreditation Fund (DANAK) (27). All additional blood samples were analyzed at the Department of Clinical Biochemistry, Rigshospitalet, Denmark. Statistical analyses All values in tables are presented as means ± SD (28) unless otherwise stated. Variables were log-transformed if appropriate (alanine transaminase, anti-Müllerian hormone, estradiol, FSH, leg fat mass, visceral fat, liver lipid deposition, skeletal muscle lipid deposition, AUC glucose, and Matsuda Index). Differences between groups in lipid deposition in skeletal muscle and liver were analyzed with nonparametric statistics (Mann-Whitney U) and presented as median (interquartile range). Because of collinearity between menopausal status and age, we could not control for age when analyzing the effect of menopausal status on body composition, ectopic lipid deposition, and insulin sensitivity. Predictors in multiple regression models were chosen prior to analyses and based on existing knowledge of associations and outcomes. The size of the study groups limited the number of predictors to a maximum of three (29). Due to collinearity between leg fat mass and total fat mass, a plain subcutaneous fat depot representative was chosen, and total fat mass was left out of the models. Standardized β-values were shown to rank the importance of the predictors in the multiple regression analyses. Regression models were checked for assumptions of the linear model, including normal distribution of the residuals, homogeneity of variance, linearity, and independent observations. All regression analyses were controlled for age. Correlations between serum estradiol and fat masses and ectopic lipid deposition in premenopausal women and all women were performed using a spearman correlation (RS). The dispersion of circulating estradiol levels was limited in postmenopausal women, preventing correlation analyses in this subgroup. Statistical analyses were performed using IBM SPSS Statistics version 22. Results Subject characteristics Subject characteristics are shown in Table 1. Table 1. Subject Characteristics Postmenopausal Women Premenopausal Women n 25 30 Age, ya 55 (49–60)b 48 (45–54) Body composition  Body weight, kg 69.5 ± 12.3 66.1 ± 9.5  Height, cm 166.9 ± 5.3 168.4 ± 6.7  BMI 24.9 ± 4.1 23.3 ± 2.2  Fat-free mass, kg 41.8 ± 4.6 43.0 ± 4.3 Sex hormones  Estradiol, nmol/L 0.08 ± 0.03b 0.33 ± 0.33  FSH, IU/L 75.2 ± 21.6b 27.6 ± 31.3  AMH, pmol/L 0.19 ± 0.05b 2.40 ± 3.88  Testosterone, nmol/L 0.67 ± 0.22 0.84 ± 0.42  Testosterone—free, nmol/L 0.0108 ± 0.0032 0.0154 ± 0.0230 Blood lipids  Total cholesterol, mmol/L 5.5 ± 0.9 5.1 ± 1.1  LDL cholesterol, mmol/L 1.9 ± 0.5 1.8 ± 0.4  HDL cholesterol, mmol/L 3.4 ± 0.7 3.1 ± 1.1  FFA, mmol/L 0.18 ± 0.11 0.16 ± 0.05 Liver parameters  ALT U/L 22 ± 6 24 ± 13  AST U/L 23 ± 4 24 ± 9 Physical activity  Daily METh, total 35.6 ± 1.4 35.4 ± 1.8  Sedentary activity time ≤1.5 METh % of the day 89.8 ± 2.4 90.3 ± 2.6  Light activity time >1.5 and ≤3 METh
% of the day 3.5 ± 1.3 3.3 ± 1.2  Moderate activity time >3 METh % of the day 6.7 ± 2.2 6.4 ± 1.8 Food intake  Daily food intake, kcal 2053 ± 445 1926 ± 341  Daily intake of carbohydrates, g 225 ± 47 207 ± 59  Daily intake of fat, g 84 ± 26 78 ± 23  Daily intake of protein, g 85 ± 26 81 ± 20 Postmenopausal Women Premenopausal Women n 25 30 Age, ya 55 (49–60)b 48 (45–54) Body composition  Body weight, kg 69.5 ± 12.3 66.1 ± 9.5  Height, cm 166.9 ± 5.3 168.4 ± 6.7  BMI 24.9 ± 4.1 23.3 ± 2.2  Fat-free mass, kg 41.8 ± 4.6 43.0 ± 4.3 Sex hormones  Estradiol, nmol/L 0.08 ± 0.03b 0.33 ± 0.33  FSH, IU/L 75.2 ± 21.6b 27.6 ± 31.3  AMH, pmol/L 0.19 ± 0.05b 2.40 ± 3.88  Testosterone, nmol/L 0.67 ± 0.22 0.84 ± 0.42  Testosterone—free, nmol/L 0.0108 ± 0.0032 0.0154 ± 0.0230 Blood lipids  Total cholesterol, mmol/L 5.5 ± 0.9 5.1 ± 1.1  LDL cholesterol, mmol/L 1.9 ± 0.5 1.8 ± 0.4  HDL cholesterol, mmol/L 3.4 ± 0.7 3.1 ± 1.1  FFA, mmol/L 0.18 ± 0.11 0.16 ± 0.05 Liver parameters  ALT U/L 22 ± 6 24 ± 13  AST U/L 23 ± 4 24 ± 9 Physical activity  Daily METh, total 35.6 ± 1.4 35.4 ± 1.8  Sedentary activity time ≤1.5 METh % of the day 89.8 ± 2.4 90.3 ± 2.6  Light activity time >1.5 and ≤3 METh
% of the day 3.5 ± 1.3 3.3 ± 1.2  Moderate activity time >3 METh % of the day 6.7 ± 2.2 6.4 ± 1.8 Food intake  Daily food intake, kcal 2053 ± 445 1926 ± 341  Daily intake of carbohydrates, g 225 ± 47 207 ± 59  Daily intake of fat, g 84 ± 26 78 ± 23  Daily intake of protein, g 85 ± 26 81 ± 20 Data are presented as mean ± SD unless otherwise stated. Abbreviations: ALT, alanine transaminase; AMH, anti-Müllerian hormone; AST, aspartate transaminase; FFA, free fatty acid; HDL, high-density lipoprotein; LDL, low-density lipoprotein. a Data presented as median ± range. b Significantly different from premenopausal (P ≤ 0.05). View Large Table 1. Subject Characteristics Postmenopausal Women Premenopausal Women n 25 30 Age, ya 55 (49–60)b 48 (45–54) Body composition  Body weight, kg 69.5 ± 12.3 66.1 ± 9.5  Height, cm 166.9 ± 5.3 168.4 ± 6.7  BMI 24.9 ± 4.1 23.3 ± 2.2  Fat-free mass, kg 41.8 ± 4.6 43.0 ± 4.3 Sex hormones  Estradiol, nmol/L 0.08 ± 0.03b 0.33 ± 0.33  FSH, IU/L 75.2 ± 21.6b 27.6 ± 31.3  AMH, pmol/L 0.19 ± 0.05b 2.40 ± 3.88  Testosterone, nmol/L 0.67 ± 0.22 0.84 ± 0.42  Testosterone—free, nmol/L 0.0108 ± 0.0032 0.0154 ± 0.0230 Blood lipids  Total cholesterol, mmol/L 5.5 ± 0.9 5.1 ± 1.1  LDL cholesterol, mmol/L 1.9 ± 0.5 1.8 ± 0.4  HDL cholesterol, mmol/L 3.4 ± 0.7 3.1 ± 1.1  FFA, mmol/L 0.18 ± 0.11 0.16 ± 0.05 Liver parameters  ALT U/L 22 ± 6 24 ± 13  AST U/L 23 ± 4 24 ± 9 Physical activity  Daily METh, total 35.6 ± 1.4 35.4 ± 1.8  Sedentary activity time ≤1.5 METh % of the day 89.8 ± 2.4 90.3 ± 2.6  Light activity time >1.5 and ≤3 METh
% of the day 3.5 ± 1.3 3.3 ± 1.2  Moderate activity time >3 METh % of the day 6.7 ± 2.2 6.4 ± 1.8 Food intake  Daily food intake, kcal 2053 ± 445 1926 ± 341  Daily intake of carbohydrates, g 225 ± 47 207 ± 59  Daily intake of fat, g 84 ± 26 78 ± 23  Daily intake of protein, g 85 ± 26 81 ± 20 Postmenopausal Women Premenopausal Women n 25 30 Age, ya 55 (49–60)b 48 (45–54) Body composition  Body weight, kg 69.5 ± 12.3 66.1 ± 9.5  Height, cm 166.9 ± 5.3 168.4 ± 6.7  BMI 24.9 ± 4.1 23.3 ± 2.2  Fat-free mass, kg 41.8 ± 4.6 43.0 ± 4.3 Sex hormones  Estradiol, nmol/L 0.08 ± 0.03b 0.33 ± 0.33  FSH, IU/L 75.2 ± 21.6b 27.6 ± 31.3  AMH, pmol/L 0.19 ± 0.05b 2.40 ± 3.88  Testosterone, nmol/L 0.67 ± 0.22 0.84 ± 0.42  Testosterone—free, nmol/L 0.0108 ± 0.0032 0.0154 ± 0.0230 Blood lipids  Total cholesterol, mmol/L 5.5 ± 0.9 5.1 ± 1.1  LDL cholesterol, mmol/L 1.9 ± 0.5 1.8 ± 0.4  HDL cholesterol, mmol/L 3.4 ± 0.7 3.1 ± 1.1  FFA, mmol/L 0.18 ± 0.11 0.16 ± 0.05 Liver parameters  ALT U/L 22 ± 6 24 ± 13  AST U/L 23 ± 4 24 ± 9 Physical activity  Daily METh, total 35.6 ± 1.4 35.4 ± 1.8  Sedentary activity time ≤1.5 METh % of the day 89.8 ± 2.4 90.3 ± 2.6  Light activity time >1.5 and ≤3 METh
% of the day 3.5 ± 1.3 3.3 ± 1.2  Moderate activity time >3 METh % of the day 6.7 ± 2.2 6.4 ± 1.8 Food intake  Daily food intake, kcal 2053 ± 445 1926 ± 341  Daily intake of carbohydrates, g 225 ± 47 207 ± 59  Daily intake of fat, g 84 ± 26 78 ± 23  Daily intake of protein, g 85 ± 26 81 ± 20 Data are presented as mean ± SD unless otherwise stated. Abbreviations: ALT, alanine transaminase; AMH, anti-Müllerian hormone; AST, aspartate transaminase; FFA, free fatty acid; HDL, high-density lipoprotein; LDL, low-density lipoprotein. a Data presented as median ± range. b Significantly different from premenopausal (P ≤ 0.05). View Large Postmenopausal women were 6.5 (±4.0) years older than premenopausal women [55 years (range: 49 to 60) vs 49 years (range: 45 to 54)] and had on average been postmenopausal for 5.4 (±1.8) years (Table 1). Postmenopausal and premenopausal women showed no significant differences in body weight (69.5 ± 12.3 vs 66.1 ± 9.5 kg, P = 0.26), BMI (24.9 ± 4.1 vs 23.3 ± 2.2, P = 0.08), fat-free mass (41.8 ± 4.6 vs 42.6 ± 4.5, P = 0.53), daily physical activity [35.6 ± 1.4 vs 35.4 ± 1.8 metabolic equivalent hours (METh), P = 0.68], or self-reported daily food intake (2053 ± 444 vs 1926 ± 341 calories, P = 0.24) (Table 1). Fat mass distribution and ectopic lipid deposition Fat mass distribution Postmenopausal women had significantly more visceral fat (0.757 ± 0.507 vs 0.459 ± 0.399 L, P = 0.006) and a trend toward an increased total fat mass (24.7 ± 10.0 vs 20.3 ± 6.7 kg, P = 0.07) and total leg fat mass (9.0 ± 3.6 vs 7.6 ± 2.5 kg, P = 0.08) compared with premenopausal women (Fig. 1a–1c). Figure 1. View largeDownload slide Fat depot sizes in postmenopausal and premenopausal women. (a) Total fat mass (kg), (b) visceral fat mass (L), and (c) leg fat mass (kg). (d) Association between total fat mass and visceral fat mass, (e) association between leg fat mass and visceral fat mass, and (f) association between serum estradiol and visceral fat mass in postmenopausal (white, n = 25) and premenopausal women (black, n = 30). Data are presented as mean ± SEM. *Significantly different from premenopausal women, P < 0.05. Log, logarithm. Figure 1. View largeDownload slide Fat depot sizes in postmenopausal and premenopausal women. (a) Total fat mass (kg), (b) visceral fat mass (L), and (c) leg fat mass (kg). (d) Association between total fat mass and visceral fat mass, (e) association between leg fat mass and visceral fat mass, and (f) association between serum estradiol and visceral fat mass in postmenopausal (white, n = 25) and premenopausal women (black, n = 30). Data are presented as mean ± SEM. *Significantly different from premenopausal women, P < 0.05. Log, logarithm. A 10% increase in total fat mass was associated with a 12% (95% CI: 7% to 18%) increase in visceral fat mass in postmenopausal women and a 13% (95% CI: 7% to 19%) increase in premenopausal women. For a given total fat mass, postmenopausal women stored significantly more visceral fat (P = 0.03) (Fig. 1d). A 10% increase in leg fat mass was associated with an 8% (95% CI: 1% to 14%) increase in visceral fat mass in postmenopausal women and a nonsignificant 1% (95% CI: –6% to 9%) increase in premenopausal women. For a given leg fat mass, postmenopausal women stored significantly more visceral fat (P = 0.02) (Fig. 1e). Visceral fat mass was negatively correlated to serum estradiol in the total group of women (RS = –0.314, P = 0.01) but not in premenopausal women alone (RS = –0.309, P = 0.11) (Fig. 1f). Total fat mass and leg fat mass showed no correlations to serum estradiol (data not shown). Total fat mass, leg fat mass, and visceral fat mass were not significantly associated with age in either postmenopausal or premenopausal women. Lipid deposition in the liver Postmenopausal women had significantly more lipid in the liver compared with premenopausal women [0.68% (0.44 to 0.99) vs 0.49% (0.38 to 0.64), P = 0.01] (Fig. 2a). Figure 2. View largeDownload slide Lipid deposition in the liver and intramuscular lipid deposition in postmenopausal and premenopausal women. (a) Lipid deposition in the liver, (b) association between total fat mass and lipid deposition in the liver, (c) association between visceral fat mass and lipid deposition in the liver, (d) association between leg fat mass and lipid deposition in the liver, (e) association between serum estradiol and lipid deposition in the liver, (f) intramuscular lipid deposition, (g), association between total fat mass and intramuscular lipid deposition, (h) association between visceral fat mass and intramuscular lipid deposition, (i) association between leg fat mass and intramuscular lipid deposition, and (j) association between serum estradiol and intramuscular lipid deposition. All analyses were done in postmenopausal (white, n = 25) and premenopausal women (black, n = 30). Data are presented as median (interquartile range). *Significantly different from premenopausal women, P < 0.05. Log, logarithm. Figure 2. View largeDownload slide Lipid deposition in the liver and intramuscular lipid deposition in postmenopausal and premenopausal women. (a) Lipid deposition in the liver, (b) association between total fat mass and lipid deposition in the liver, (c) association between visceral fat mass and lipid deposition in the liver, (d) association between leg fat mass and lipid deposition in the liver, (e) association between serum estradiol and lipid deposition in the liver, (f) intramuscular lipid deposition, (g), association between total fat mass and intramuscular lipid deposition, (h) association between visceral fat mass and intramuscular lipid deposition, (i) association between leg fat mass and intramuscular lipid deposition, and (j) association between serum estradiol and intramuscular lipid deposition. All analyses were done in postmenopausal (white, n = 25) and premenopausal women (black, n = 30). Data are presented as median (interquartile range). *Significantly different from premenopausal women, P < 0.05. Log, logarithm. A 10% increase in total fat mass was associated with a 19% (95% CI: 10% to 29%) increase in lipid deposition in the liver in postmenopausal women and a nonsignificant 5% (95% CI: –1% to 12%) increase in premenopausal women (Fig. 2b). The association differed significantly with menopausal status (interaction, P = 0.01). A 10% increase in visceral fat mass was associated with a 12% (95% CI: 8% to 17%) increase in lipid deposition in the liver in postmenopausal women and a 5% (95% CI: 2% to 8%) increase in premenopausal women (Fig. 2c). The association differed significantly with menopausal status (interaction, P = 0.005). A 10% increase in leg fat mass was associated with a 13% (95% CI: 3% to 24%) increase in lipid deposition in the liver in postmenopausal women compared with a nonsignificant 1% (95% CI: –8% to 5%) decrease in premenopausal women. The association differed significantly with menopausal status (interaction, P = 0.02) (Fig. 2d). Lipid deposition in the liver was negatively correlated to serum estradiol in the total group of women (RS = –0.303, P = 0.02) and in premenopausal women alone (RS = –0.370, P = 0.05) (Fig. 2e). In a multiple regression analysis with visceral fat mass and leg fat mass as predictors of lipid deposition in the liver, only the visceral fat mass remained a significant predictor in both postmenopausal (P < 0.0001) and premenopausal women (P = 0.001) (Table 2). Table 2. Multiple Regression Analyses of the Predictors of Lipid Deposition in Liver, Intramuscular Lipid Deposition, and Insulin Sensitivity Postmenopausal Women Premenopausal Women Predictors βU
(% Increase) 95% CI βS βU
(% Increase) 95% CI βS Lipid deposition in the liver  Visceral fat mass (10% increase) 11.4a 6.8 to 16.2 1.132 5.3 2.5 to 8.3 0.602  Leg fat mass (10% increase) 3.8a –3.4 to 11.5 0.392 −2.1 −7.2 to 3.3 −0.217 Intramuscular lipid deposition  Visceral fat mass (10% increase) 4.0b 1.1 to 7.1 0.533 2.1 −0.7 to 5.0 0.277  Leg fat mass (10% increase) 2.1 −2.7 to 7.2 0.166 2.3 −3.2 to 8.0 0.154 Insulin sensitivity  Visceral fat mass (10% increase) −1.1 −5.4 to 3.4 −0.123 −0.2 −4.1 to 3.9 −0.017  Lipid deposition in the liver (10% increase) −4.2 −7.1 to 1.2 −0.731 −4.3 −8.5 to 0.1 −0.478  Intramuscular lipid deposition (10% increase) 0.01 −4.0 to 4.2 0.003 −1.8 −5.7 to 2.3 −0.174 Postmenopausal Women Premenopausal Women Predictors βU
(% Increase) 95% CI βS βU
(% Increase) 95% CI βS Lipid deposition in the liver  Visceral fat mass (10% increase) 11.4a 6.8 to 16.2 1.132 5.3 2.5 to 8.3 0.602  Leg fat mass (10% increase) 3.8a –3.4 to 11.5 0.392 −2.1 −7.2 to 3.3 −0.217 Intramuscular lipid deposition  Visceral fat mass (10% increase) 4.0b 1.1 to 7.1 0.533 2.1 −0.7 to 5.0 0.277  Leg fat mass (10% increase) 2.1 −2.7 to 7.2 0.166 2.3 −3.2 to 8.0 0.154 Insulin sensitivity  Visceral fat mass (10% increase) −1.1 −5.4 to 3.4 −0.123 −0.2 −4.1 to 3.9 −0.017  Lipid deposition in the liver (10% increase) −4.2 −7.1 to 1.2 −0.731 −4.3 −8.5 to 0.1 −0.478  Intramuscular lipid deposition (10% increase) 0.01 −4.0 to 4.2 0.003 −1.8 −5.7 to 2.3 −0.174 Analyses were performed in postmenopausal (n = 25) and premenopausal women (n = 30), corrected for age. All analyses were done on log-transformed data. Abbreviations: βS, standardized β (indicating ranking of importance of predictors on outcome); βU, unstandardized β. a Significant interaction (P ≤ 0.05). b Significantly different from postmenopausal (P ≤ 0.05). View Large Table 2. Multiple Regression Analyses of the Predictors of Lipid Deposition in Liver, Intramuscular Lipid Deposition, and Insulin Sensitivity Postmenopausal Women Premenopausal Women Predictors βU
(% Increase) 95% CI βS βU
(% Increase) 95% CI βS Lipid deposition in the liver  Visceral fat mass (10% increase) 11.4a 6.8 to 16.2 1.132 5.3 2.5 to 8.3 0.602  Leg fat mass (10% increase) 3.8a –3.4 to 11.5 0.392 −2.1 −7.2 to 3.3 −0.217 Intramuscular lipid deposition  Visceral fat mass (10% increase) 4.0b 1.1 to 7.1 0.533 2.1 −0.7 to 5.0 0.277  Leg fat mass (10% increase) 2.1 −2.7 to 7.2 0.166 2.3 −3.2 to 8.0 0.154 Insulin sensitivity  Visceral fat mass (10% increase) −1.1 −5.4 to 3.4 −0.123 −0.2 −4.1 to 3.9 −0.017  Lipid deposition in the liver (10% increase) −4.2 −7.1 to 1.2 −0.731 −4.3 −8.5 to 0.1 −0.478  Intramuscular lipid deposition (10% increase) 0.01 −4.0 to 4.2 0.003 −1.8 −5.7 to 2.3 −0.174 Postmenopausal Women Premenopausal Women Predictors βU
(% Increase) 95% CI βS βU
(% Increase) 95% CI βS Lipid deposition in the liver  Visceral fat mass (10% increase) 11.4a 6.8 to 16.2 1.132 5.3 2.5 to 8.3 0.602  Leg fat mass (10% increase) 3.8a –3.4 to 11.5 0.392 −2.1 −7.2 to 3.3 −0.217 Intramuscular lipid deposition  Visceral fat mass (10% increase) 4.0b 1.1 to 7.1 0.533 2.1 −0.7 to 5.0 0.277  Leg fat mass (10% increase) 2.1 −2.7 to 7.2 0.166 2.3 −3.2 to 8.0 0.154 Insulin sensitivity  Visceral fat mass (10% increase) −1.1 −5.4 to 3.4 −0.123 −0.2 −4.1 to 3.9 −0.017  Lipid deposition in the liver (10% increase) −4.2 −7.1 to 1.2 −0.731 −4.3 −8.5 to 0.1 −0.478  Intramuscular lipid deposition (10% increase) 0.01 −4.0 to 4.2 0.003 −1.8 −5.7 to 2.3 −0.174 Analyses were performed in postmenopausal (n = 25) and premenopausal women (n = 30), corrected for age. All analyses were done on log-transformed data. Abbreviations: βS, standardized β (indicating ranking of importance of predictors on outcome); βU, unstandardized β. a Significant interaction (P ≤ 0.05). b Significantly different from postmenopausal (P ≤ 0.05). View Large Lipid deposition in the liver was not significantly associated with age in either postmenopausal or premenopausal women. Intramuscular lipid deposition Postmenopausal women had significantly more intramuscular lipid in the vastus lateralis of the quadriceps muscle compared with premenopausal women [3% (2% to 4%) vs 2% (1% to 3%), P = 0.001] (Fig. 2f). A 10% increase in total fat mass was significantly associated with increased intramuscular lipid deposition in both postmenopausal [8% (95% CI: 3% to 13%)] and premenopausal women [7% (95% CI: 2% to 12%)]. However, for a fixed amount of total body fat, postmenopausal women stored more lipid in skeletal muscle compared with premenopausal women (P = 0.004) (Fig. 2g). Intramuscular lipid deposition was significantly associated with visceral fat mass and leg fat mass only in the postmenopausal women: A 10% increase in visceral fat mass was associated with a 5% (95% CI: 2% to 7%) increase in intramuscular lipid deposition in postmenopausal women compared with a nonsignificant 2% (95% CI: –1% to 5%) increase in premenopausal women. For a given visceral fat mass, postmenopausal women stored significantly more intramuscular lipids (P = 0.01) (Fig. 2h). A 10% increase in leg fat mass was associated with a 5% (95% CI: 1% to 11%) increase in intramuscular lipid deposition in postmenopausal women and a nonsignificant 3% (95% CI: –3% to 8%) increase in premenopausal women. For a given leg fat mass, postmenopausal women stored significantly more intramuscular lipids (P = 0.002) (Fig. 2i). There was no significant correlation between intramuscular lipid deposition and serum estradiol in the total group of women or in premenopausal women alone (Fig. 2j). In a multiple regression analysis with visceral fat mass and leg fat mass as predictors for intramuscular lipid deposition, only visceral fat mass remained as a significant predictor and only in postmenopausal women (P = 0.009) (Table 2). Intramuscular lipid deposition was not significantly associated with age in either postmenopausal or premenopausal women. Insulin sensitivity Postmenopausal women had 19% higher blood glucose (919 ± 277 vs 775 ± 156, P = 0.03) and a 28% lower Matsuda insulin sensitivity index (6.31 ± 3.48 vs 8.78 ± 4.67, P = 0.05) during an OGTT compared with premenopausal women. Insulin (50,190 ± 35,781 vs 39,050 ± 22,194, P = 0.17) and c-peptide AUC (307,430 ± 123,991 vs 262,932 ± 92,389, P = 0.17) were not significantly different between postmenopausal and premenopausal women. However, c-peptide was significantly higher after 120 minutes in postmenopausal women compared with premenopausal women (Fig. 3a–3f). Stumvoll’s index did not differ between groups (data not shown). Figure 3. View largeDownload slide Blood glucose and insulin during an OGTT in postmenopausal and premenopausal women. (a and b) Blood glucose, (c and d) insulin levels, (e) c-peptide, (f) Matsuda insulin sensitivity, and (g) correlation between estradiol and Matsuda insulin sensitivity during an OGTT in postmenopausal (white, n = 20) and premenopausal women (black, n = 27). Data are presented as mean ± SEM. *Significantly different from premenopausal women, P < 0.05. Figure 3. View largeDownload slide Blood glucose and insulin during an OGTT in postmenopausal and premenopausal women. (a and b) Blood glucose, (c and d) insulin levels, (e) c-peptide, (f) Matsuda insulin sensitivity, and (g) correlation between estradiol and Matsuda insulin sensitivity during an OGTT in postmenopausal (white, n = 20) and premenopausal women (black, n = 27). Data are presented as mean ± SEM. *Significantly different from premenopausal women, P < 0.05. Insulin sensitivity was not directly correlated to serum estradiol in the total group of women or in premenopausal women alone (Fig. 3g). Matsuda insulin sensitivity was not significantly associated with age in either postmenopausal or premenopausal women. Insulin sensitivity and total fat mass Matsuda insulin sensitivity index was significantly associated with total fat mass in postmenopausal women [a 10% increase in total fat mass was associated with a 7.3% decrease (95% CI: –10.4% to 1.0%) in insulin sensitivity] but not in premenopausal women [–3.2% (95% CI: –9.2% to 3.2%)] (Fig. 4a). Figure 4. View largeDownload slide Association between lipid deposition and Matsuda insulin sensitivity in postmenopausal and premenopausal women. Association between Matsuda insulin sensitivity and (a) total fat mass, (b) visceral fat mass, (c) leg fat mass, (d) lipid deposition in the liver, and (e) intramuscular lipid deposition in postmenopausal (white, n = 20) and premenopausal women (black, n = 27). Log, logarithm. Figure 4. View largeDownload slide Association between lipid deposition and Matsuda insulin sensitivity in postmenopausal and premenopausal women. Association between Matsuda insulin sensitivity and (a) total fat mass, (b) visceral fat mass, (c) leg fat mass, (d) lipid deposition in the liver, and (e) intramuscular lipid deposition in postmenopausal (white, n = 20) and premenopausal women (black, n = 27). Log, logarithm. Insulin sensitivity and visceral fat mass In postmenopausal women, Matsuda insulin sensitivity index was significantly associated with visceral fat mass, as a 10% increase in visceral fat mass was associated with a 6.4% decrease (95% CI: –8.9% to 3.1%) in insulin sensitivity. Premenopausal women showed a trend to an association between visceral fat mass and insulin sensitivity [a 10% increase in visceral fat mass was associated with a 3.0% decrease (95% CI: –5.8% to 0.1%) in insulin sensitivity]. The association differed significantly with menopausal status (interaction, P = 0.003) (Fig. 4b). Insulin sensitivity and leg fat mass There was no significant association between Matsuda insulin sensitivity and leg fat mass in either postmenopausal or premenopausal women (Fig. 4c). Insulin sensitivity and lipid deposition in the liver Postmenopausal and premenopausal women showed no significant differences in the association between lipid deposition in the liver and insulin sensitivity, as a 10% increase in lipid deposition in the liver was associated with a significant decrease in insulin sensitivity in both postmenopausal [–4.5% (95% CI: –3.1% to 5.9%)] and premenopausal women [–4.9% (95% CI: –1.6% to 8.1%)] (Fig. 4d). Insulin sensitivity and intramuscular lipid deposition A 10% increase in intramuscular lipid deposition was associated with a 6.1% decrease in insulin sensitivity (95% CI: –1.6% to 10.5%) in postmenopausal women, whereas, in premenopausal women, a 10% increase in intramuscular lipid deposition was associated with a nonsignificant 2.8% decrease (95% CI: –6.9% to 1.6%) in insulin sensitivity (Fig. 4e). Predictors of insulin sensitivity In a multiple regression analysis with visceral fat mass, lipid deposition in the liver, and intramuscular lipid deposition as predictors of insulin sensitivity, lipid deposition in the liver was the strongest predictor of insulin sensitivity in both postmenopausal and premenopausal women but was only a statistically significant predictor in postmenopausal women (P = 0.01) (Table 2). Discussion In this cross-sectional study of 55 women between 45 and 60 years of age, we found that postmenopausal women had an increased lipid deposition in the liver and skeletal muscle compared with that of premenopausal women. Total fat mass, visceral fat mass and leg fat mass were stronger predictors of ectopic lipid deposition and decreased insulin sensitivity in postmenopausal compared with premenopausal women. In accordance with our findings, a previous study (16) showed that older postmenopausal Asian women had an increased incidence of NAFLD compared with younger premenopausal Asian women. However, study participants had a broad range of ages (21 to 80 years old) and the researchers used abdominal ultrasonography to diagnose NAFLD, a method unable to provide reliable quantitative information, and with a lower limit of detection at around 15% to 20% lipid in the hepatocyte (30), which greatly exceeds the lipid deposition in the liver of the healthy group of women in this study. Increased visceral fat mass is known to be associated with ectopic lipid deposition in the liver in both rodents (11) and diabetic and nondiabetic humans (31, 32). This is in agreement with our findings as both postmenopausal and premenopausal women showed significant positive associations between visceral fat mass and lipid deposition in the liver. Interestingly, the association between visceral fat mass and lipid deposition in the liver was significantly steeper in the postmenopausal women, and for a given visceral fat mass, postmenopausal women stored more intramuscular lipids. These findings indicate that the health-related consequences linked to expanding the visceral fat depot increase after menopause. In support of this, we showed that increased visceral fat mass was strongly associated with decreased insulin sensitivity only in postmenopausal women. It has been suggested that leg fat does not play the same pathological role in the development of metabolic disease as other fat depots and may instead be protective (7, 33). In accordance with this, we found no significant associations between leg fat mass and lipid deposition in liver and skeletal muscle in premenopausal women. In contrast, postmenopausal women showed significant positive associations between leg fat mass and lipid deposition in liver and skeletal muscle, indicating that the beneficial role of leg fat mass could change with menopausal status. However, leg fat mass was not associated with intramuscular lipid deposition when controlling for visceral fat mass and was not a significant predictor of insulin resistance in either postmenopausal or premenopausal women. Thus, the importance of leg fat mass in whole-body insulin resistance seemed to be limited. Whether ectopic lipid deposition in the liver is related to total body fat is debated (34, 35). In this study, the association between total fat mass and ectopic lipid deposition varied with menopausal status; we showed that total fat mass was a significantly stronger predictor of lipid depositions in liver and skeletal muscle in postmenopausal women compared with premenopausal women. The fact that fat depot size was a stronger predictor of ectopic lipid deposition in postmenopausal women could indicate that postmenopausal women have a more dysfunctional adipose tissue both in the subcutaneous and visceral fat depot, with a lower threshold for lipid spillover compared with premenopausal women. In support of our findings, oophorectomized mice show increased adipocyte size and infiltration of both macrophages and T-cells in both intra-abdominal and subcutaneous adipose tissue (36–38), ectopic lipid deposition in liver, and skeletal muscle (15, 39) and decreased insulin sensitivity (36, 38), indicating that loss of ovarian function leads to a general dysfunction in both subcutaneous and visceral adipose tissue. However, these findings still need further investigation in humans. Postmenopausal women showed increased blood glucose throughout the OGTT, which could be an indication of decreased β-cell function, as insulin secretion has been shown to be the major determinant of glucose disposal after an oral glucose load (40, 41). However, pre- and postmenopausal women showed no significant differences in Stumvoll’s 1st PH index, a surrogate for β-cell function. Furthermore, although not significantly elevated, insulin levels tended to be generally increased throughout the OGTT, leaving a possible role for peripheral insulin resistance in the decreased insulin sensitivity seen with menopause, as reported in oophorectomized rodents (42). Inactivity is known to affect lipid accumulation in adipose tissue and ectopic sites and is associated with decreased insulin sensitivity (43). Studies in mice have shown that loss of ovarian function leads to decreased physical activity (37, 38). In contrast, human studies investigating physical activity level over menopause have failed to identify a decrease in fitness with menopause when controlling for differences in lean body mass (5, 6). In agreement with this, we found no differences in daily physical activity between postmenopausal and premenopausal women. Thus, loss of physical fitness with menopause was hardly responsible for the differences in ectopic lipid deposition and glucose metabolism found in this study. Our study has some limitations. Postmenopausal women were 6.5 years older than premenopausal women, and because of the study design, the effects of menopause and age on ectopic lipid deposition and insulin resistance could not be addressed separately. However, age was not significantly associated with fat depot size, ectopic lipid deposition, or insulin sensitivity in either postmenopausal or premenopausal women. To further address the impact of age on ectopic lipid deposition and insulin resistance, we repeated all analyses in the subset of women who had an age between 49 and 54 years, leaving 11 postmenopausal women (mean age: 52.7) and 11 premenopausal women (mean age: 52.1) in the analyses. As the results of the analyses in this subgroup of women were in accordance with the results from the complete study group, we conclude that age is less likely to have played any major role in fat distribution, ectopic lipid deposition, and insulin sensitivity in the relatively narrow age span investigated in this study. We conclude that postmenopausal women have an increased lipid deposition in liver and skeletal muscle compared with premenopausal women. Total, visceral, and leg fat mass were all stronger predictors of ectopic lipid deposition in postmenopausal women, suggesting a general adipose tissue dysfunction with menopause associated with a decreased whole-body insulin sensitivity These findings bring additional value to the understanding of the increased occurrence of metabolic disease with menopause. Lipid deposition in liver and skeletal muscle may represent important mechanistic links between the changes in fat depots and the increased incidence of insulin resistance seen after menopause. Abbreviations: Abbreviations: AUC area under curve BMI body mass index METh metabolic equivalent hours MRS magnetic resonance spectroscopy NAFLD nonalcoholic fatty liver disease OGTT oral glucose tolerance test Acknowledgments Financial Support: This work was supported by Augustinus Fonden Grant 17-2354 (to J.A.). Disclosure Summary: The authors have nothing to disclose References 1. 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Journal

Journal of Clinical Endocrinology and MetabolismOxford University Press

Published: Sep 1, 2018

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