Effects of Rapid Weight Loss on Systemic and Adipose Tissue

ISSN 2472-1972

Effects of Rapid Weight Loss on Systemic and

Adipose Tissue Inflammation and

Metabolism in Obese

Postmenopausal Women

Jos´eO.Alem´an,

1,2,8

Neil M. Iyengar,

Jeanne M. Walker,

Ginger L. Milne,

Joel Correa Da Rosa,

Yupu Liang,

Dilip D. Giri,

Xi Kathy Zhou,

Michael N. Pollak,

Clifford A. Hudis,

Jan L. Breslow,

Peter R. Holt,

and Andrew J. Dannenberg

Laboratory of Biochemical Genetics and Metabolism, Rockefeller University, New York, New York

10065;

Department of Medicine, Memorial Sloan-Kettering Cancer Center, New York, New York 10065;

Rockefeller Hospital, Rockefeller University, New York, New York 10065;

Division of Clinical

Pharmacology, Vanderbilt University Medical Center, Nashville, Tennessee 37232;

Department of

Pathology, Memorial Sloan-Kettering Cancer Center, New York, New York 10065;

Department of Health

Care Policy and Research, Weill Cornell Medical College, New York, New York 10065;

Departments of

Medicine and Oncology, McGill University, Montreal, Quebec H3T 1E2, Canada; and

Department of

Medicine, Weill Cornell Medical College, New York, New York 10065

*These authors are co-senior authors.

Context: Obesity is associated with subclinical white adipose tissue inflammation, as defined by the

presence of crown-like structures (CLSs) consisting of dead or dying adipocytes encircled by macro-

phages. In humans, bariatric surgery-induced weight loss leads to a decrease in CLSs, but the effects of

rapid diet-induced weight loss on CLSs and metabolism are unclear.

Objective: To determine the effects of rapid very-low-calorie diet-induced weight loss on CLS density,

systemic biomarkers of inflammation, and metabolism in obese postmenopausal women.

Design: Prospective cohort study.

Setting: Rockefeller University Hospital, New York, NY.

Participants: Ten obese, postmenopausal women with a mean age of 60.6 years (standard deviation, 6

3.6 years).

Main Outcome Measures: Effects on CLS density and gene expression in abdominal subcutaneous

adipose tissue, cardiometabolic risk factors, white blood count, circulating metabolites, and oxidative

stress (urinary isoprostane-M) were measured.

Results: Obese subjects lost approximately 10% body weight over a mean of 46 days. CLS density

increased in subcutaneous adipose tissue without an associated increase in proinflammatory gene

expression. Weight loss was accompanied by decreased fasting blood levels of high-sensitivity C-reactive

protein, glucose, lactate, and kynurenine, and increased circulating levels of free fatty acids, glycerol,

b-hydroxybutyrate, and 25 hydroxyvitamin D. Levels of urinary isoprostane-M declined.

Conclusion: Rapid weight loss stimulated lipolysis and an increase in CLS density in subcutaneous

adipose tissue in association with changes in levels of circulating metabolites, and improved systemic

biomarkers of inflammation and insulin resistance. The observed change in levels of metabolites

Abbreviations: CLS, crown-like structure; CVD, cardiovascular disease; GSEA, Gene Set Enrichment Analysis; GSVA, Gene Set

Variation Analysis; H&E, hematoxylin and eosin; hsCRP, high-sensitivity C-reactive protein; PCR, polymerase chain reaction;

SHBG, steroid hormone-binding globulin; VLCD, very-low-calorie diet.

Received 23 February 2017

Accepted 20 April 2017

First Published Online 25 April 2017

June 2017 | Vol. 1, Iss. 6

doi: 10.1210/js.2017-00020 | Journal of the Endocrine Society | 625–637

(i.e., lactate, b-hydroxybutyrate, 25 hydroxyvitamin D) may contribute to the anti-inflammatory effect

of rapid weight loss.

This article has been published under the terms of the Creative Commons Attribution Non-

Commercial, No-Derivatives License (CC BY-NC-ND; https://creativecommons.org/licenses/by-nc-

nd/4.0/).

Freeform/Key Words: crown-like structure, weight loss, metabolism, inflammation

Obesity has reached epidemic proportions, with about two-thirds of US adults classified as

overweight or obese [1]. Complications of obesity include type 2 diabetes, cardiovascular

disease, and increased incidence of and worsened prognosis for several cancers, including

postmenopausal breast cancer [2, 3]. Inflamed adipose tissue is believed to contribute to

obesity-associated complications. This is characterized by immune-cell infiltration, increased

levels of proinflammatory cytokines, as well as adipocyte insulin resistance, mitochondrial

dysfunction, and endoplasmic reticulum stress [4]. Crown-like structures (CLSs) are found in

the white adipose tissue of both obese mice and humans. In cross-sectional studies, the

presence of CLSs is associated with ins ulin resistance, cardiovascular disease (CVD), and

worse prognosis for patients with cancer [ 5–7]. These inflammatory foci represent dead or

dying adipocytes enveloped by macrophages. The macrophages rely on lysosomal exophagy to

phagocytose the dead adipocytes and become foam cells [8, 9]. When white adipose tissue fails

to expand appropriately to store excess energy, ectopic fat deposition occurs in other organs,

leading to insulin resistance—a process known as lipoto xicity. The ability of the CLS mac-

rophages to store free fatty acids may help protect against both local adipocyte dysfunction

and lipotoxicity [10].

In obese rodents, prolonged caloric restriction is associated with a reduction in CLS and

reduced expression of proinflammatory genes [11]. By contrast, rapid weight-loss results in

increased macrophage infiltration in visceral and subcutaneous adipose tissue [12]. In

humans, bariatric surgery-induc ed weight loss leads to a reduction in CLSs, improved

systemic inflammation, and decreased insulin resistance and, possibly, cancer risk [ 13]. The

effects of very-low-calorie diet (VLCD)-mediated rapid weight loss have also been in-

vestigated. Cl´ement and colleagues [14] reported that VLCD-induced rapid weig ht loss leads

to a substantial reductio n in fat mass in association with improved insulin sensitivity, re-

duced circulating triglyceride and cholesterol levels, as well as changes in subcutaneous fat

gene expression. There is considerable evidence that cross-talk between adipose tissue and

distal tissues, including the liver, pancreas, and skeletal muscle, can affect health [15].

Although the effects of rapid weight loss on circulating levels of adipokines and lipids have

been reported [16, 17], much less is known about the effects on systemic levels of metabolites.

In this study, our primary objective was to investigate the effects of VLCD-induced weight

loss on CLS den sity, systemic biomarkers of inflammation, and metabolism in obese post-

menopausal women.

1. Materials and Methods

A. Subjects

Eligible subjects were obese (body mass index, $35 kg/m

) postmenopausal women (defined

by .2 years without menstrual periods). Participants were recruited from the community

through advertis ing or participation in a Rockefeller Hospital registry of subjects previously

screened for research studies. We excluded subjects with clinical CVD; type 2 diabetes and

receiving oral hypoglycemic agents; smokers; regular users of aspirin, fish oils, or vitamin D

supplements; a history of gastrointestinal surgery other than appendectomy; or breast and

other cancers. Fourteen subjects were screened, and 12 of these were evaluated and enrolled.

During the study, two subjects dropped out: one because of acute conjunctivitis and another

626 | Journal of the Endocrine Society | doi: 10.1210/js.2017-00020

for inability to adhere to the diet. Of those who completed the study, three were white, four

were bl ack, and three were Hispanic. This was a single-center study performed at The

Rockefeller University Hospital between September 2012 and August 2013. Subjects

underwent a complete medical examination, standard blood and urine tests, and an elec-

trocardiogram, and all were found to be healthy. A Transparent Reporting of Evaluation with

Nonrandomized Designs flowchart summ arizing subject selection is presented in Figure 1.

B. Sample Size

The primary end point of this study was to determine whether the intervention would sig-

nificantly alter CLS density in subcutan eous adipose tissue. Sample size was primarily

determined by logistics and feasibility considerations. However, with 10 subjects, assuming a

coefficient of variation of 30%, a paired t test applied to this sample provided 80% power to

detect an effect size as small as a 1-unit change in CLS/cm

from before to after weight loss.

This effect size is equivalent to 30% increase from baseline CLS density.

C. Trial Approval and Registration

The study was approved by the institutional review boards at Rockefeller University, Weill

Cornell Medical College, and Memorial Sloan-Kettering Cancer Center, and registered under

ClinicalTrials.gov identifier NCT01699906.

Figure 1. Transparent Reporting of Evaluation with Nonrandomized Designs flowchart of

the study. Fourteen subjects were screened, of whom 12 were enrolled, and 10 completed the

study. Two subjects completed the weight loss intervention as outpatients, and the remaining

subjects completed as inpatients. WAT, white adipose tissue.

doi: 10.1210/js.2017-00020 | Journal of the Endocrine Society | 627

D. Interventions, Specimen Collect ion, and Analysis

The effect of the VLCD was assessed in subjects after they had lost approximately 10% of body

weight. The diet of each subject before weight loss was evaluated by the Harvard Food

Frequency Questionnaire, which quantifies food intake over a 3-day period [18]. On admis-

sion to the Rockefeller University Hospital, baseline study specimens were collected. Sub-

sequently, participants underwent a 3-day dietary adjustment period, consuming 50% of their

prestudy caloric intake. Of the 10 subjects who completed the VLCD-induced weight loss, two

did so as outpatients with intensive monitoring and eight as inpatients. Blood samples were

drawn biweekly for safety monitoring, which included measurements of electrolytes and liver

function tests. The rate of weight loss did not differ significantly between those studied as

outpatients or inpatients. After losing approximately 10% of their baseline body weight, all

subjects consumed the VLCD for three additional days as inpatients, during which all of the

baseline measurements were repeated. Bloods for end-point analyses were collected under

fasting conditions. Therefore, each study subject acted as her own control.

The VLCD consisted of a commercially available diet (New Direction Program, Robard

Corp., Mount Laurel, NJ) that provided approximately 800 Kcal/d with an estimated mac-

ronutrient energy distribution of 54% protein, 26% carbohydrate, 20% fat (including 4%

saturated fat and 200 mg of cholesterol) and 10 g/d fiber, as previou sly described [19]. This

commercial preparation provided a choice of shakes, soups, bars, and puddings. Subjects had

four choices per day and consumed one item every four waking hours.

Fasting blood samples were analyzed in the Clinical Pathology Laboratory of Memorial

Sloan-Kettering Cancer Center for electrolyte levels, liver function, renal function, lipid

profile, and high-sensitivity C-reactive protein (hsCRP) level. Aliquots of serum for cytokine

measurements, including interleukin (IL)-6, IL-10, and IL-17, were stored at 280°C for

subsequent analysis. Enzyme-linked immunosorbent analyses of leptin, adiponectin, insulin,

steroid hormone-binding globulin (SHBG), estradiol, and IL-6 were performed at the Pollak

Assay Laboratory (McGill University, Quebec , Canada) [6]. Erenna immunoassay analyses

(EMD Millipore, Billerica, MA) of IL-10 and IL-17 were performed by the Rockefeller

Translational Technology Core Laboratory. Body composition was measured by air-

displacement plethysm ography using the BodPod system (COSMED, Italy). Subcutaneous

adipose tissue was obtained by aspiration biopsy using a large-bore, modified liposuction

needle (Anthony Products, Indianapolis, IN) under local anesthesia with lidocaine between

08:00 and 11:00. The initial adipose tissue biopsy specimen was taken in the left lower

quadrant of the abdomen of each subject, whereas the final biopsy specimen was taken in the

right lower quadrant abdomen. The quadrants for the two biopsies were alternated to avoid

the potential of results being confounded by trauma-related to the initial biopsy. Adipose

tissue was formalin fixed and paraffin embedded for immunohistochemistry or frozen in

RNAlater (Ambion Inc, Austin TX) for subsequent RNA extraction and analysis.

E. Urinary F2-isoprostane-M Measurement

Analysis of urinary F2-isoprostane-M was performed on an aliquot of a 24-hour urine col-

lection by liquid chromatography–mass spectrometry in the Vanderbilt University Eicosa-

noid Core Laboratory as described previously [20]. F2-isoprostane-M levels were normalized

to creatinine.

F. CLS Quantification in Adipose Tissue Biopsy Specimens

CLS in abdominal subcutaneous adipose tissue bi opsy specimens were quantified as de-

scribed previously [6]. Biopsy samples were fixed in formalin for 24 hours and then embedded

in a paraffin block. Six sections were cut with a spacing of 50 mm; the first section was stained

with hematoxylin and eosin (H&E) to ensure the sample was representative of adipose tissue.

The remaining five sections were stained for the macrophage marker CD68 (mouse mono-

clonal KP1 antibody; dilution 1:4000; Dako, Glostrup, Denmark) to identify CLS by light

628 | Journal of the Endocrine Society | doi: 10.1210/js.2017-00020

microscopy. The numbers of CLSs per section were recorded by the study pathologist (D.D.G.).

The area of adipose tissue examined in each of the five sections was measured using Image J

software (National Institutes of Health, Bethesda, MD) to calculate CLS density as CLS/cm

G. Adipocyte Diameter

H&E-stained sections were generated from subcutaneous adipose tissue biopsy specimens to

measure adipocyte diameters, as previously described [21, 22]. The H&E-stained sections

were photographed at 320 magnification using an Olympus BX50 microscope (Orangeburg,

NY) and MicroFire digital camera (Optronics Goleta, CA). Mean diamet ers were calcu-

lated using measurements from $30 individual adipocytes for each patient, using the linear

dimensional tool in the Canvas 11 Software (ACD Systems International Victoria, Canada).

H. Gene Expression

Total RNA was extracted from approximately 0.5 g of frozen adipose tissue using a Qiagen

RNeasy Lipid Tissue Mini Kit (Germantow n, MD). RNA quality was assessed using an

Agilent Bioanalyzer. Approximately, 2 to 3 mg of RNA with RNA integrity number .7 were

submitted for 50-bp paired-end read RNA sequencing of polyA-enriched RNA at the New York

Genome Center. Gene expression was analyzed for pathway enrichment using Gene Set

Enrichment Analysis (GSEA; Broad Institute, Cambridge MA) and Ingenuity Pathway

Analysis (Qiagen).

For heat map generation, genes were annotated using the biomaRt library in R package

Bioconductor (https://www.bioconductor.org/). RNA-seq data were normalized by a regu-

larized logarithmic transformation into a ma trix through DESeq2 [23]. The Gene Set Var-

iation Analysis (GSVA) algorithm [24] was applied to the matrix to quantify pathway activity

for a set of 1454 curated gene sets available in the Molecular Signatures Database. Supervised

heat maps were generated to visually compare pathway activities in before and after weig ht-

loss samples, and to assess subject and pathway similarities. A linear mixed-effects model

was applied to estimate averages for the pathway activity before and after weight loss and to

determine the pathways that differed significantly between before and after VLCD weight

loss. To achieve a more concise visual representation, these averages were displayed in a heat

map (scaled column-wise) based on Euclidean distance and a complete linkage algorithm for

pathway hierarchical cluster ing.

I. Quantitative Real-Time Polymerase Chain Reaction

Transcript expression was assessed after total RNA was isolated from adipose tissue, using

the RNeasy Mini Kit (Qiagen). RNA (100 ng) was reverse transcribed using the qScript cDNA

Synthesis Kit (QuantaBio Beverly, MA) and the resulting cDNA used for real-time poly-

merase chain reaction (PCR) amplif ication using Fast SYBR Green PCR master mix on a 7500

HT real-time PCR system (Applied Biosystems, Foster City, CA). IL-6 was amplified using a

Qiagen QuantiTect primer assay (catalog no. QT00083720). Primers used to amplify tumor

necrosis factor-a were: forward: 5

-CTG CAC TTT GGA GTG AT-3

; reverse: 5

-AGA TGA TCT

GAC TGC CTG GG-3

. Primers used to amplify IL-1b were: forward: 5

-GGA CAA GCT GAG

GAA GAT GC-3

; reverse: 5

-TCG TTA TCC CAT GTG TCG AA-3

. Transcript expression was

normalized to glycerald ehyde 3-phosphate dehydrogenase (GAPDH), which was amplified

using a Qiagen QuantiTect primer assay (catalog no. QT00079247). Expression was de-

termined using the DDCT analysis protocol.

J. Statistical Analyses

Two-tailed paired t tests and Wilcoxon tests were used to compare anthropometric mea-

surements, biochemical variables, serum cytokines, and immunohistochemical results before

and after VLCD-induced weight loss. P , 0.05 was considered significant. For metabolomic

doi: 10.1210/js.2017-00020 | Journal of the Endocrine Society | 629

analyses, matched paired t tests were used to identify compounds that differed sign ificantly

between the before and after weight-loss samples, correcting for multiple measurements.

GraphPad Prism and Excel were used for data visualization.

2. Results

The mean age (6 standard dev iation) of the 10 subjects completing the study was 60.6 6

3.6 years. The mean time to induce ~10% weight loss was 46.2 6 15.3 days (range, 30 to 74 days).

Table 1 shows the mean macronutrient composition of the subjects’ self-selected prestudy

diets and that of the VLCD consumed during weight loss. The daily protein intake of the

VLCD (388 6 45 Kcal protein per day) was similar to the subject’sprestudydiet(3826 28 Kcal

protein per day; P = not significant), whereas fat, carbohydrate, and fiber contents were all

markedly reduced.

A. Effects of the VLCD-Induced Weight Loss on Anthropometric and Clinical Parameters

The before and after weight loss anthropometric and clinical data are shown in Table 2. The

VLCD decreased BMI by 9.6%, including a 3.8% reduction in body fat. Decreases were ob-

served in fasting glucose (13.3%) and insulin (20.3%) levels, and the Homeostatic Model for

Assessment of Insulin Resistance (30.9%), reflecting improved insulin sensitivity. High-

density lipoprotein cholesterol levels decreased (16.0%), with trends towar d decreased

low-density lipoprotein cholesterol (8.2%) and triglyceride (19.3%) levels. Fasting leptin levels

decreased (57.2%), whereas adiponectin was unaffected. Estradiol levels were unaffected by

weight loss, but levels of SHBG increased (56.7%). Weight loss led to a sign ificant increase in

25-hydroxyvitamin D (19.6%) and calcium (2.5%) levels.

B. Effects of the VLCD-Induced Weight Loss on Systemic Markers of Inflammation and

Oxidant Stress

VLCD-induced weight loss was associated with decreased levels of hsCRP (35.3%) and total

white blood cell count (17.4%), largely due to fewer circulating neutrophils (28.3%) (Table 2).

Levels of urinary F2 isoprostane-M, a biomarker of oxidant stress, decreased by 41.1% fol-

lowing VLCD-induced weight loss.

C. Effects of the VLCD-Induced Weight Loss on Subcutaneous Adipose Tissue

Mean adipocyte diameter was 111.1 6 13.0 mm at baseline and 111.7 6 11.3 mm after weight

loss. We also assessed the density of CLSs in abdominal subcu taneous adipose tissue at

baseline and after weight loss [ Fig. 2(a)]. CLSs were detected in all 10 subjects at bas eline.

Interestingly, the density of CLSs increa sed from a mean of 1.78 6 1.04 to 5.33 61.32 CLS/cm

after weight loss [P = 0.01; Fig. 2(b)].

Table 1. Composition of the Diet Before Weight Loss and the VLCD

preWL Diet VLCD

Calories, Kcal/d 2324 6 1041 721 6 83

% Carbohydrate 49 6 8266 1

% Protein 16 6 3546 1

% Fat 36 6 8206 2

% Saturated fat 11 6 446 1

Fiber, g 33 6 18 10 6 4

Values are presented as mean 6 standard deviation.

Abbreviation: preWL, before weight loss.

630 | Journal of the Endocrine Society | doi: 10.1210/js.2017-00020

To further explore the effects of VLCD-induced weight loss on the biology of subcutaneous

adipose tissue, RNA sequencing was carried out on adipose tissue obtained before and after

weight loss. A total of 61 genes were differentially expressed at absolute log fold-change .2

and P , 0.001 (Supplemental Table 1). Of the differentially express ed genes, about two-thirds

decreased and one-third increased after weight loss [Fig. 2(c)]. GSEA identified over-

represented metabolic pathways relating to triacylglycerol and ketone metabolism, and

cellular respiration. As shown in Figure 2(d), analysis by GSVA showe d that VLCD-induced

weight loss was associated with increased expression of genes involved in metabolism of

monocarboxylic acids, fatty acids, the tricarboxylic acid cycle, phospholipids, lipids, organic

acid, and carboxylic acids; and decreas ed expression of genes involved in fatty acid bio-

synthesis, cellular responses to nutrient levels, extracellular signaling, and to stress. Ad-

ditionally, pathway analyses, including GSEA, Ingenuity Pathway Analysis, and GSVA

showed that VLCD-induced weight loss did not affect the expression of genes related to

inflammation. To confirm this finding, quantitative real-time PCR was carried out. Levels of

tumor necrosis factor-a, IL-1b, and IL-6 mRNAs were unaffected by VLCD-induced weight

loss (data not shown).

D. Effects of VLCD-Induced Weight Loss on the Plasma Metabolome

To determine the effects of VLCD-induced weight loss on circulating metabolites, plasma

collected under fasting conditions was subjected to targeted metabolomic analysis. A total of

336 metabolites were detected. Of these, 131 metabolites changed significantly, with 67

Table 2. Effects of Rapid VLCD-Induced Weight Loss

preWL postWL Difference, % P Value

Weight, kg 101.3 6 9.7 90.9 6 8.2 210.3 ,0.01

BMI, kg/m

38.8 6 3.4 35.1 6 3.0 29.6 0.01

Waist, cm 115.8 6 6.3 107.5 6 7.9 27.2 0.01

% Body fat composition 51.4 6 6.1 47.6 6 5.5 27.4 ,0.01

Metabolic parameters

Glucose, mg/dL 103.4 6 17.6 89.6 6 14.9 213.3 0.01

Insulin, mU/mL 12.3 6 1.8 9.8 6 2.1 220.3 0.07

HOMA-IR 3.30 6 0.59 2.28 6 0.60 230.9 0.04

HDL, mg/dL 54.3 6 10.3 45.6 6 6.2 216.0 0.02

LDL, mg/dL 131.2 6 24.8 120.5 6 27.0 28.2 0.08

TG, mg/dL 96.9 6 28.8 78.2 6 25.0 219.3 0.08

Leptin, ng/mL 61.0 6 21.0 26.1 6 12.2 257.2 ,0.001

Adiponectin, mg/mL 9.8 6 5.8 10.2 6 5.5 4.1 0.20

Estradiol, pg/mL 36.2 6 14.3 33.4 6 11.6 27.7 0.38

SHBG, nmol/L 60.3 6 26.3 94.5 6 33.7 56.7 ,0.001

Calcium, mg/dL 9.06 6 0.27 9.29 6 0.31 2.5 0.01

25-OH vitamin D, ng/mL 22.5 6 10.3 26.9 6 10.3 19.6 0.04

Inflammatory parameters

WBC, 10

/uL 5.96 6 2.00 4.92 6 1.56 217.4 0.01

Neutrophils, 10

/mL 3.22 6 0.60 2.31 6 0.54 228.3 0.01

hsCRP, mg/dL 0.68 6 0.42 0.44 6 0.24 235.3 0.01

IL-6, pg/mL 3.1 6 1.7 2.6 6 1.1 216.1 0.07

IL-10, pg/mL 1.49 6 0.99 1.31 6 0.76 212.1 0.50

IL-17, pg/mL 0.22 6 0.10 0.24 6 0.16 9.1 0.38

MCP-1, pg/mL 312 6 130 292 6 115 26.4 0.32

F2-IsoP-M, ng/mg Cr 1.51 6 0.57 0.89 6 0.29 241.1 0.01

Values are presented as mean 6 standard deviation.

Abbreviations: 25-OH vitamin D, 25 hydroxyvitamin D; BMI, body mass index; F2-IsoP-M, F2-isoprostane-M; HDL,

high-density lipoprotein; HOMA-IR, Homeostatic Model for Assessment of Insulin Resistance; LDL, low-density

lipoprotein; MCP-1, monocyte chemoattractant protein 1; TG, triglyceride; WBC, white blood cell count.

Significance was determined by Wilcoxon signed-rank test.

doi: 10.1210/js.2017-00020 | Journal of the Endocrine Society | 631

increasing (Supplemental Table 2) and 64 decreasing (Supplemental Table 3). The decrease in

plasma levels of creatine and 3-methylhistidine after VLCD-induced weight loss is consistent

with this diet being protein sparing (Table 3). The observed increase in both circulating free

fatty acid and glycerol levels after treatment with the VLCD suggests enhanced lipolysis [Fig.

3(a) and 3(b)]. Treatment with the VLCD also led to elevated ketone levels, including a 16-fold

increase in b-hydroxybutyrate and a 7.6-fold increase in acetoacetate (Table 3). Levels of

lactate, tryptophan, and the tryptophan metabolite kynurenine all decreased after VLCD-

induced weight loss. By contrast, the metabolites p -cresol sulfate and glycolithocholate

sulfate increased after VLCD-induced weight loss, consistent with altered intestinal bac terial

metabolism (Table 3).

3. Discussion

In this study, we determined the effects of VLCD-induced rapid weight loss on both in-

flammation and metabolism in obese postmenopausal women. Consistent with the known

anti-inflammatory effects of weight loss [25], VLCD-induced rapid weight loss resulted in

decreased levels of hsCRP, reduced white blood cell count, and a near-significant reduction in

Figure 2. Effects of VLCD-induced weight loss on subcutaneous adipose tissue. Biopsy

specimens were obtained before and after weight loss and subjected to CD68 immunohistochemistry

and RNA sequencing. (a) Subcutaneous adipose tissue images from the subject with the

largest increase in CLSs after weight loss are shown. (Top panel) preWL. (Bottom panel)

postWL. (b) Line plot of CLS density (CLS/cm

) shown from preWL to postWL in each

individual subject (P = 0.01). (c) Heat map for 61 genes whose expression changed

significantly from preWL to postWL. Subjects (columns) are arranged from preWL to postWL.

Genes (rows) are clustered hierarchically in a supervised manner. (d) Heat map of expression

of 17 lipid metabolism gene pathways altered significantly between preWL and postWL.

postWL, after weight loss; preWL, before weight loss.

632 | Journal of the Endocrine Society | doi: 10.1210/js.2017-00020

circulating IL-6 levels [26]. Obesity has been associated with oxidant stress, which can also

contribute to inflammat ion [27]. We observed decreased levels of urinary F2-isoprostane-M

after weight loss, suggesting decreased oxidative stress [28].

The density of CLS, a histological marker of adipose inflammation, is increased in both

visceral and subcutaneous adipose tissue from obese vs lean subjects and correlates with

elevated levels of circulating proinflammatory mediators [29]. To complement our analysis of

systemic markers of inflammation, we explored the effects of rapid VLCD-induced weight loss

on CLS density in subcutaneous adipose tissue. Interestingly, VLCD-induced weight loss was

associated with a significant increase in CLS density in the absence of a change in adipocyte

size or expression of proinflammatory genes. Weight loss after bariatric surgery has been

associated with a reduction in CLS [13]. By contrast, in obese mice, Kosteli et al. [12] showed

that rapid weight loss stimulated lipolysis, resulting in the accumulation of macrophages in

adipose tissue [12]. Elevated level s of free fatty acids, a consequence of increased lipolysis,

were found to function as chemoattractants, leading to increased macrophage content. We

also observed a significant increase in lipolysis after VLCD-induced weight loss. Taken to-

gether, we speculate that VLCD-mediated rapid weight loss stimulated lipolysis, contributing

to the observed increase in CLS density.

The increase in CLS density in response to VLCD-induced weight loss has potentially

significant implications. These CLS macrophages can store excess free fatty acids [29, 30],

thereby protecting against lipotoxicity and contributing to the observed improvement in

insulin sensitivity and decreased systemic inflammation following rapid weight loss. There

are other possible explanations for how rapid VLCD-induced weight loss could lead to both an

increase in CLS density in abdominal subcutaneous fat and a decrease in systemic in-

flammatory biomarkers. One possibility is that rapid weight loss has different effects on

visceral vs subcutaneous fat. Perhaps a decrease in CLS density occurred in visceral fat,

contributing to reduced systemic inflammation. Another possibility is that the overall de-

crease in fat mass mediated by the VLCD led to a decrease in systemic inflammation. A

limitation of our study is that the effects of rapid weight loss were only measured at a sing le

time point when fat remodeling was occurring. It is possible that a decrease in CLS density

would be observed later, once a new steady state is achieved.

Although weight loss was associated with a small increase in CLS density, we did not

detect a comparable increase in the express ion of proinflammatory genes. There are multipl e

potential explanations for this findin g. Lactate has been observed to stimulate M1 polari-

zation of macrophages [31]. Plasma lactate levels were reduced after rapid weight loss, pre-

sumably reflecting improved adipose tissue oxygenation [32]. Because lactate has been reported

to stimulate M1 polarization of macrophages, a fall in adipose tissue lactate levels in concert with

reduced circulating levels could prevent an increase in proinflammatory gene expression. We

also observed a 16-fold increase in b-hydroxybutyrate after weight loss. Youm et al. [33] found

that b-hydroxybutyrate blocked inflammation mediated by the NLRP3 inflammasome. It is

possible, therefore, that increased b-hydroxybutyrate levels contributed to the lack of a local

inflammatory phenotype in subcutaneous adipose tissue. Consistent with findings in other

weight-loss studies [34], we observed an increase in circulating 25 hydroxyvitamin D levels after

Table 3. VLCD-Induced Weight Loss Was Associated With Changes in Levels of Plasma Metabolites

Increased Plasma

Metabolite

Mean Pairs Ratio

PostWL/PreWL P Value

Decreased Plasma

Metabolite

Mean Pairs Ratio

PostWL/PreWL I Value

b-Hydroxybutyrate 15.98 ,0.001 3-Methylhistidine 0.63 0.01

p-Cresol sulfate 10.05 0.03 Creatine 0.69 ,0.01

Glycolithocholate sulfate 8.76 0.03 Kynurenine 0.74 ,0.001

Acetoacetate 7.64 ,0.001 Lactate 0.74 ,0.01

Tryptophan 0.78 ,0.001

Values are presented as mean pairs ratio of postWL vs preWL levels.

Abbreviations: PostWL, after weight loss; PreWL, before weight loss.

doi: 10.1210/js.2017-00020 | Journal of the Endocrine Society | 633

VLCD-induced weight loss. Because vitamin D possesses anti-inflammatory properties [35], the

increase in 25 hydroxyvitamin D levels might also help to explain the lack of increased

proinflammatory gene expression after VLCD-induced weight loss.

We also determined the effects of VLCD-induced weight loss on a variety of metabolic end

points. Insulin sensitivity improved, as shown by a reduced Homeostatic Model for As-

sessment of Insulin Resistance index. The VLCD diet was associated with reduced levels of

both high-density lipoprotein cholesterol and triglycerides, which, together with a trend

toward decreased low-density lipoprotein cholesterol levels, probably makes the changes

neutral with respect to risk of CVD [36]. Estradiol levels were low, reflecting the post-

menopausal state, and did not change after VLCD-induced weight loss. However, SHBG

levels increased after treatment with the VLCD, which would be predicted to decrease free

estradiol. In all likelihood, the increase in SHBG levels after VLCD-induced weight loss

reflects improved insulin sensitivity [37]. Consistent with the observed decrease in fat mass,

levels of leptin, a proinflammatory adipok ine, declin ed by .50% after VLCD-induced weight

loss. This finding is consistent with the recent report of Magkos et al. [38] who showed that

5.1% to 16.4% weight loss led to a progressive decline in circula ting leptin levels. Increased

circulating levels of estradiol, insulin resistance, and elevated levels of leptin have been

suggested to contribute to the increased risk of breast cancer in obese postmenopausal women

[39]. The changes in free estrogens, insulin resistance, and leptin levels after rapid VLCD-

induced weight loss collectively may lower the risk of breast cancer in this population [40].

Figure 3. Effects of VLCD-induced weight loss on levels of free fatty acids and glycerol in

plasma. (a) Heat map comparing the ratios of plasma free fatty acid levels between postWL

and preWL samples. White indicates unity, with yellow to red indicating increased levels. All

free fatty acid species shown were significantly increased (P , 0.05). (b) Box plot of preWL

and postWL abundance of plasma glycerol relative to total ion counts, and scaled by setting

the preWL loss median to one (*P = 0.01). postWL, after weight loss; preWL, before weight

loss.

634 | Journal of the Endocrine Society | doi: 10.1210/js.2017-00020

Obesity is also associated with numerous immunosuppressive effects [41]. Kynurenine, an

endogenous ligand of the aryl hydrocarbon receptor, has well-documented immunomodu-

latory effects, including stim ulating na

ıve T-cell polarization to the regulatory T-cell phe-

notype [42]. VLCD-induced weight loss was accompanied by reductions in circulating level s of

tryptophan and kynurenine, a tryptophan metabolite. Based on these metabolic changes,

future studies are warranted to evaluate the effects of rapid weight loss on immune function.

In summary, we showed in obese postmenopausal women that rapid diet-induced weight

loss was associated with inc reased CLS density with concomitant improved insulin sensitivity

and decreased circulating biomarkers of inflammation. The increase in CLS density is likely

to reflect adipose remod eling. In cross-sectional studies, we and others have shown that

obesity-associated CLSs are associated with diabetes, CVD, and worse cancer prognosis. The

current study illustrates the dynamic nat ure of CLS and highlights the potential importance

of the rate of weight loss on adipose tissue biology.

Acknowledgments

We thank the clinical research staff and research volunteers who made this work possible. We ac-

knowledge Juana Gonzalez and the Translational Technology Core Laboratory at Rockefeller Univer-

sity Hospital for IL-10 and IL-17 measurements.

Current affiliation: J.O. Alem ´an’s current affiliation is Division of Endocrinology, Diabetes and

Metabolism, Department of Medicine, NYU Langone Medical Center, 522 First Ave, New York, NY

10016. C.A. Huddis’ current affiliation is American Society of Clinical Oncology, 2318 Mill Rd, Suite 800,

Alexandria, VA 22314.

Address all correspondence to: Jos´e Orlando Alem ´an, Rockefeller University, 1230 York Ave, Box 179,

New York, New York 10065. E-mail: [email protected].

This work was supported by the National Center for Advancing Translational Sciences Grant UL1

TR000043, National Institutes of Health Clinical and Translational Science Award program to

Rockefeller University, the Rockefeller University Center for Basic and Translational Research on

Disorders of the Digestive System, from the Leona M. and Harry B. Helmsley Charitable Trust, The

Rockefeller University Sackler Center for Biomedicine and Nutrition from La Fondation Sackler, the

Breast Cancer Research Foundation, the Botwinick-Wolfensohn Foundation (in memory of Mr. and Mrs.

Benjamin Botwinick), and the Memorial Sloan-Kettering Cancer Center Core Grant (P30 CA008748).

Author contributions: J.O. A., N.M.I., C.A.H., J.L.B., P.R.H., and A.J.D. designed the clinical study.

J.O.A. and J.M.W. collected clinical study end points. J.O.A., N.M.I., J.M.W., G.L.M., J.C.R., Y.L., D.D.G.,

X.K.Z., and M.N.P. analyzed the data. J.O.A., J.L.B., P.R.H., and A.J.D. wrote the manuscript. J.L.B. and

A.J.D. edited the manuscript.

Clinical trial registry: ClinicalTrials.gov no. NCT01699906 (registered 12 September 2012).

Disclosure summary: The authors have nothing to disclose.

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