Altered Extracellular Matrix Dynamics are Associated with Insulin Resistance in Adolescent Children with Obesity

Abstract

Objective:

To examine the hypothesis that abdominal and gluteal adipocyte turnover, lipid dynamics, and fibrogenesis are dysregulated among insulin resistant (IR) compared to insulin sensitive (IS) adolescents with obesity.

Methods:

Seven IS and seven IR adolescent children with obesity participated in a 3-hour OGTT and a multi-section MRI of the abdominal region to examine body fat distribution patterns and liver fat content. An 8-week 70% deuterated water (²H₂O) labeling protocol examined adipocyte turnover, lipid dynamics, and fibrogenesis in vivo from biopsied abdominal and gluteal fat.

Results:

Abdominal and gluteal SAT turnover rates of lipid components were similar among IS and IR adolescent children with obesity. However, the insoluble collagen Iα2 isoform measured from abdominal, but not gluteal, SAT was elevated in IR compared to IS subjects. In addition, abdominal insoluble collagen Iα2 is associated with ratios of visceral-to-total (VAT+SAT) abdominal fat, whole-body and adipose tissue insulin signaling, and trended towards a positive association with liver fat content.

Conclusions:

Altered extracellular matrix dynamics, but not expandability, potentially decreases abdominal SAT lipid storage capacity, contributing to the pathophysiological pathways linking adipose tissue and whole-body insulin resistance with altered ectopic storage of lipids within the liver among IR adolescents with obesity.

INTRODUCTION

Obesity prevalence rates among adolescents in the United States have increased at alarming rates over the past six decades (1), and currently, nearly 1 in 5 children present with obesity (2). Adolescents with obesity are more likely to exhibit insulin resistance and are at an increased risk for the rapid progression of pre-diabetes and Type 2 diabetes (T2D) (3–6). Similarly, insulin resistance during adolescence is closely associated with the accumulation of fat within the liver, resulting in non-alcoholic fatty liver disease (NAFLD) and the increased risk of premature death from more progressive chronic liver diseases and liver cancers during adulthood (7–12).

Independent of the severity of obesity (e.g., body mass index [BMI]), the inability of subcutaneous (SAT) to store lipids has been hypothesized to result in the accumulation of fat within the visceral adipose tissue (VAT) depot and ectopically in other organs, such as the liver (13–15). More recently, Nouws et al. (16) paired SAT cellularity analysis with deuterated water methodologies – the gold standard approach to examine in vivo adipocyte and triglyceride (TG) dynamics (17). This approach demonstrated that adolescent children with obesity and high vs. low VAT/(VAT+SAT) ratios exhibited greater rates of mature adipocyte and TG synthesis rates in abdominal and gluteal SAT. In addition, elevated TG synthesis rates within both SAT depots, independent of visceral adiposity, were associated with increased liver fat accumulation as determined by hepatic fat fraction. These findings, in contrast to their initial hypothesis that decreased SAT TG synthesis would contribute to ectopic fat accumulation within the liver, suggest that the capacity of the SAT to retain and store newly synthesized lipids is decreased (16). As a result, decreased SAT storage appears to have a contributing role in metabolic dysregulation and the increased risk for NAFLD among adolescent children with obesity and adverse patterns of fat partitioning.

More recently, Beals et al. (18) highlighted the critical role of adipose tissue fibrogenesis in adipose tissue expansion capacity in adult persons with obesity and normal or impaired glucose tolerance. The utilization of deuterated water enables researchers to examine collagen synthesis to gain further insight into the relationship between SAT lipid storage and insulin resistance. Beals et al. (18) demonstrated that increased fibrogenesis, not storage capacity, is associated in obese adults with impaired glucose tolerance compared to normal glucose tolerance. However, the potential role of fibrogenesis has yet to be examined among adolescents with obesity. Therefore, this investigation utilized deuterated water methodologies to examine the hypothesis that indices of adipocyte turnover, lipid dynamics, and fibrogenesis in vivo from abdominal and gluteal fat depots would be dysregulated in insulin resistant compared to insulin sensitive adolescents with obesity. Additionally, a 3-hour oral glucose tolerance test (OGTT) and multi-section magnetic resonance imaging (MRI) of the abdominal region were included in the analysis to examine indices of glucose metabolism, abdominal fat distribution patterns, and liver fat content and their relationship with adipocyte turnover, lipid dynamics, and fibrogenesis.

METHODS

Study participants

Children and adolescents aged 12 to 20 years old with a BMI > 85^th percentile were recruited from the Yale Pediatric Obesity Clinic to participate in the Yale Study of the Pathophysiology of Prediabetes/T2D in Youth, an ongoing investigation aiming to identify the underlying pathophysiology of prediabetes and Type 2 diabetes in youth (NCT01967849). In total, a total of 14 adolescents with obesity volunteered to participate in this research. Subjects with a whole-body insulin sensitivity index (WBISI; determined during the OGTT [described below]) in the top 25% and bottom 25% of the entire Yale Pediatric Obesity Clinic cohort were classified as insulin sensitive (IS) and insulin resistant (IR), respectively. Prior to enrollment, the research team obtained written informed consent and assent and a complete medical history and physical examination. Upon enrollment, total body composition was measured by dual-energy X-ray absorptiometry (Hologic Inc). Of note, data on in vivo TG synthetic fluxes and adipocyte turnover paired with abdominal and gluteal subcutaneous adipose tissue biopsies obtained from adolescent girls with obesity and various VAT/(VAT + SAT) ratios were the focus of an earlier paper (16). Using a subset of this subject population and additionally enrolled participants, the present study focuses on the analysis of fibrogenesis and the relationship with indices of metabolic dysregulation among IS and IR adolescents with obesity. The study was approved by the Yale University Human Investigation Committee in accordance with both the Declarations of Helsinki and Instanbul.

Oral glucose tolerance test

Following an overnight fast, subjects arrived at the Yale Center for Clinical Investigation (YCCI) and height, weight, and waist and hip circumferences were immediately measured. All subjects then underwent an OGTT performed at the YCCI. Whole blood samples were collected from an intravenous (IV) line inserted in the antecubital vein for the analysis of glucose, insulin, C-peptide, and glycerol at baseline and at 10, 20, and 30 min, then every 30 min up to 180 min following oral glucose challenge (1.75 g/kg body weight: 75 g maximum). Homeostatic model assessment for insulin resistance (HOMA-IR; 19) and WBISI (20) were calculated. Finally, adipose tissue insulin resistance (AT-IR) was calculated by multiplying fasting insulin and fasting free fatty acid concentrations (see Table 2 for metabolic phenotype).

Table 2.

Metabolic Profile

Variable	Insulin Sensitive (n = 7)	Insulin Resistant (n = 7)	p- value
Metabolic Measures
Fasting Glucose (mg/dL)	92.64 ± 7.28	94.0 ± 14.07	0.412
2-hour Glucose (mg/dL)	110.71 ± 14.28	108.00 ± 18.98	0.384
Fasting Insulin (μU/mL)	16.0 ± 4.20	42.79 ± 12.78	< 0.001*
2-hour Insulin (μU/mL)	53 (50, 105)	147 (81, 187)	0.009*
Fasting C-peptide (pmol/L)	813.0 ± 114.50	1,480.64 ± 263.13	< 0.001*
2-hour C-peptide (pmol/L)	2,668.14 ± 815.85	4,144.14 ± 1,715.14	0.036*
Adiponectin (ng-mL)	6.13 ± 1.52	4.75 ± 1.57	0.060
Leptin (ng/mL)	60.26 ± 26.97	81.85 ± 51.86	0.174
WBISI	3.21 ± 0.86	1.31 ± 0.47	< 0.001*
HOMA-IR	3.65 ± 0.94	9.85 ± 2.76	< 0.001*
AT-IR	5.88 ± 2.46	15.40 ± 6.37	0.003*
IGI	1.50 (0.87, 4.35)	2.78 (1.19, 6.39)	0.337
Lipids
Total Cholesterol (mg/dL)	161.00 ± 28.21	159.86 ± 42.65	0.954
HDL (mg/dL)	43.57 ± 12.99	37.14 ± 7.06	0.272
LDL (mg/dL)	99.80 ± 27.15	84.67 ± 35.17	0.453
TG (mg/dL)	113.57 ± 52.63	128.29 ± 47.05	0.591
FFA (μmol/L)	0.39 ± 0.14	0.38 ± 0.10	0.838
Liver
PDFF	2.19 ± 1.74	8.59 ± 7.01	0.038*
ALT (units/L)	11 (11, 56)	40 (24, 54)	0.851
AST (units/L)	20 (11, 38)	25 (23, 32)	0.869

Note: Data are presented as mean ± SD or as median (IQR: 25th, 75th percentile).

^{^}The denotes p-value determined from Pearson χ² analysis.

^*The indicated a significant difference between insulin sensistive and insulin resistant adolescents with obesity (p < 0.05).

Abbreviations: ALT: Alanine Aminotransferase; AST: Aspartate Aminotransferase; AT-IR Adipose Tissue Insulin Resistance; FFA: Free Fatty Acid; HDL: High Density Lipoprotein; HOMA-IR: Homeostatic Model Assessment for Insulin Resistnace; IGI: Insulinogenic Index; LDL: Low Density Lipoprotein; NAFLD: Nonalcoholic Fatty Liver Disease; TG: Triglyceride; PDFF: Proton Density Fat Fraction; WBISI: Whole-Body Insulin Sensitivity Index.

Abdominal fat distribution and intrahepatic fat content by MRI-proton density fat fraction (PDFF)

On the same day and immediately following the OGTT, a multi-section abdominal MRI was performed at the Magnetic Resonance Research Center (Siemens Sonata 3.0 Tesla System; Erlangen, Germany). Abdominal SAT and VAT distribution was determined using a threshold to discriminate fat from soft tissue at the level of the L4/L5 disc space on a single slice. Deep (DeepSAT) and superficial SAT (SupSAT) were determined based on their division by the fascia superficialis (21). The ratios of VAT-to-total (VAT+SAT) and DeepSAT-to-SupSAT were also calculated. Liver fat fraction content was measured by MRI using the proton density fat fraction (PDFF; 22). The advanced MRI-magnitude-based method used to estimate PDFF in children is a noninvasive assessment of liver health moderately associated with histological analysis, and in adults, it is an accurate and reproducible imaging-based biomarker for assessing steatosis and treatment response in nonalcoholic steatohepatitis patients participating in randomized controlled trials (23). For our study, NAFLD was defined as a PDFF ≥ 5.5% (24).

²H₂O labeling protocol

Each participant was next enrolled in 8-week 70% deuterated water (²H₂O; Isotec, Miamisburg, OH) labeling protocol. The ²H₂O labeling protocol was designed to achieve a body water pool enrichment of ~1.0% by providing each patient with two doses of 70 mL of 70% ²H₂O consumed 3–4 hours apart. Participants then drank 40 mL of 70% ²H₂O three times a day for 5 days, followed by a consumption rate of 40 mL twice per day for the remainder of the 8-week labeling protocol. During this time, the protocol was designed to achieve a plateau in body water pool enrichment at ~1.5–2.0%. Compliance with ²H₂O intake was checked weekly by evaluating ²H₂O enrichments in body water were measured from urine by gas chromatography–mass spectrometry (GCMS).

Subcutaneous adipose tissue biopsies

Paired abdominal and gluteal SAT biopsies were performed after having applied EMLA cream and local anesthesia (lidocaine without epinephrine) under sterile conditions at the YCCI Hospital Research Unit. A 2-cm scalpel incision was made for the removal of 2–3 g of abdominal (~5-cm from the umbilicus) and gluteal SAT (outer quadrant of the gluteus; 16).

In vivo adipocyte proliferation and turnover analysis by ²H₂O

A small sample of SAT from each biopsied depot was rinsed in cold saline, digested in collagenase, and immunodepleted for hematopoietic cells as previously described (16). The mature adipocytes were subsequently flash frozen in liquid nitrogen whereas the stromal vascular fraction (SVF; ~60% total) was reconstituted in DMEM.F12 media (1:1) containing 10% FBS and cultured for 16 hours at 37°C. Adherent cells were detached using 0.05% trypsin with EDTA, centrifuged, and the pellet was snap frozen in liquid nitrogen. The frozen mature adipocytes and SVF shipped overnight to the University of California Berkeley laboratory for subsequent analysis of DNA synthesis rates, an index of adipocyte proliferation and turnover expressed as fractional synthesis rates (FSRs). DNA synthesis measured in vivo results from ribose precursors incorporating deuterium from ²H₂O during nucleoside synthesis. Deuterium enrichment into the deoxyribose moiety of adenosine were measured from isolated mature adipocytes and the cells from the SVF by GCMS and application of mass isotopomer distribution analysis (GC-MS-MIDA), thereby representing the production of a new cell exposed to ²H₂O during the S-phase of the cell cycle (17). To adjust for potential differences in labeling times among subjects, measures are expressed as synthesis rates per day (k = -ln [1 – f] / labeling time in days).

In vivo SAT lipid dynamics by ²H₂O

Abdominal and gluteal triglycerides (TGs) were isolated using the Folch technique to determined TG synthesis and de novo lipogenesis (DNL) based on deuterium incorporation into TG-glycerol and TG-palmitate, respectively, as described previously (17). The TG FSRs were determined using mass isotopomer distribution analysis followed by GC-MS-MIDA (17, 25). Data for FSR were calculated in units d⁻¹ (per day): k = -ln [1 – f] / labeling time in days, which adjusts for potential differences in labeling times among subjects.

In vivo SAT collagen synthesis by ²H₂O

Adipose tissue samples from each depot were subjected to sequential extraction using methods described previously (26), to yield a guanidine-soluble collagen fraction and a guanidine-insoluble collagen fraction. Both these fractions were then digested with trypsin and analyzed by liquid chromatography–mass spectrometry to determine the incorporation of deuterium into collagen type I subunits α1 and α2, using methods described elsewhere (26, 27). Data for FSR were calculated in units d⁻¹ (per day): k = -ln [1 – f] / labeling time in days, which adjusts for potential differences in labeling times among subjects.

Biochemical analyses

Glucose concentrations were examined bedside using a YSI2700-STAT-Analyzer (Yellow Springs Instruments, Yellow Springs, OH). Insulin and C-peptide concentrations from plasma isolated from whole blood by centrifugation were measured using radioimmunoassay (Linco, St. Charles, MO) and ALPCO-Immunoassays (Salem, NH), respectively. Plasma glycerol (Cayman Chemical; Ann Arbor, MI), adiponectin (R&D Systems, Minneapolis, MN), and leptin (Millipore Sigma, Burlington, MA) were measured by enzyme-linked immunosorbent assay according to manufacturer’s instructions. Area-under-the-curves (AUCs) were calculated using the trapezoidal rule.

Statistical analyses

Categorical variables were compared among insulin sensitive (IS) and insulin resistant (IR) adolescent children using Pearson χ² test. Differences in continuous data were determined by independent t-test. For nonparametric data, Mann-Whitney U tests were utilized. Finally, Pearson’s correlations were utilized to examine potential associations among outcome variables. In addition, the Benjamini-Hochberg method (False Discovery Rate) was applied to correct for any limitations related to performing multiple comparisons. Statistical significance was determined by p-value ≤ 0.05.

RESULTS

Subject demographic, anthropometric, and metabolic characteristics

Seven IS and seven IR adolescent children with obesity participated in the study. Although no differences in body composition were observed between to two groups, the ratio of VAT-to-total (VAT+SAT) adipose tissue was greater in IR compared to IS subjects (p = 0.037; Table 1). All other indices of body fat distribution patterns were similar between the two group (Table 1).

Table 1.

Subject Characteristics

Variable	Insulin Sensitive (n = 7)	Insulin Resistant (n = 7)	p- value
Sex (M/W)	1 M; 6 F	1 M; 6 F	^1.000
Age (yr)	16 (15, 17)	17 (14, 17)	0.799
Anthropometrics
Height (m)	1.62 ± 0.08	1.65 ± 0.05	0.314
Weight (kg)	87.89 ± 20.16	101.21 ± 17.87	0.215
BMI (kg/m2)	33.74 ± 7.75	36.90 ± 5.37	0.393
BMI z-score	1.182 ± 0.53	2.23 ± 0.32	0.108
BMI percentile	98.1 (87.7, 98.5)	98.8 (97.5, 99.4)	0.135
Body Surface Area (m2)	1.98 ± 0.24	2.15 ± 0.21	0.179
Body Composition
Body Fat (%)	45.81 ± 6.22	46.9 ± 3.27	0.186
Lean Mass (kg)	44.90 ± 7.76	50.08 ± 7.69	0.265
Fat Mass (kg)	41.39 ± 13.54	46.59 ± 9.92	0.711
Android Fat Mass (kg)	3,500.14 ± 1,353.46	4,079.57 ± 1,102.40	0.397
Android Fat (%)	49.01 ± 5.49	50.24 ± 3.59	0.629
Gynoid Fat Mass (kg)	7,005.43 ± 2,373.50	7,300.0 ± 2,092.54	0.81
Gynoid Fat (%)	47.43 ± 5.59	46.24 ± 3.75	0.65
Android-to-Gynoid Ratio	1.034 ± 0.062	1.090 ± 0.075	0.156
Body Fat Distribution
Waist (cm)	100.64 ± 16.84	109.1 ± 11.69	0.297
Hip (cm)	113.20 ± 15.93	118.29 ± 12.62	0.520
Waist-to-Hip Ratio	0.889 ± 0.074	0.925 ± 0.069	0.364
VAT (cm2)	73.64 ± 46.75	108.66 ± 35.72	0.141
SAT (cm2)	519.59 ± 193.74	520.16 ± 151.66	0.995
Deep SAT (cm2)	191.3 (102.9, 234.5)	122.1 (110.7, 222.6)	0.949
Superficial SAT (cm2)	157.24 ± 56.64	156.86 ± 71.19	0.991
Deep-to- Superficial Ratio	1.146 ± 0.380	1.103 ± 0.393	0.836
VAT-to-(VAT+SAT) Ratio	0.124 ± 0.051	0.176 ± 0.047	0.037*
Cardiovascular Measures
Resting SBP (mmHg)	118.33 ± 8.29	109.86 ± 12.16	0.178
Resting DBP (mmHg)	72.33 ± 5.28	69.86 ± 12.42	0.660
Resting HR (bpm)	75.67 ± 10.19	78.57 ± 16.78	0.720

Note: Data are presented as mean ± SD or as median (IQR: 25th, 75th percentile).

^{^}The denotes p- value determined from Pearson χ² analysis.

^*The indicated a significant difference between insulin sensistive and insulin resistant adolescents with obesity (p < 0.05).

Abbreviations: BMI: Body Mass Index; DBP: Diastolic Blood Pressure; HR: Heart Rate; IS: Insulin Sensitive; IR: Insulin Resistant; SAT: Subcutaneous Adipose Tissue; SBP: Systolic Blood Pressure; VAT: Visceral Adipose Tissue.

Both subject groups contained six (85.7%) subjects with fasting plasma glucose concentration within the normal range (< 100 mg/dL) and one subject within the range for pre-diabetes (100 – 125 mg/dL). Likewise, fasting glucose concentrations were similar among groups (Table 2). To the contrary, fasting insulin and C-peptide concentrations were elevated in IR compared to IS subjects (both p < 0.001). Similarly, adipose tissue insulin resistance (AT-IR) was elevated in IR compared to IS adolescent children with obesity (p = 0.003). In response to the 3-hour OGTT, one subject in the IR group (14.3%) presented with glucose intolerance (140 – 199 mg/dL) and all others exhibited normal glucose tolerance (< 140 mg/dL) as determined by 2-hour plasma glucose concentrations. However, whole-body insulin sensitivity (WBISI) was lower and HOMA-IR was elevated in IR compared to IS subjects (both p < 0.001). Finally, MRI analysis indicated that hepatic fat content (PDFF) was greater in IR compared to IS subjects (p = 0.038).

In vivo adipocyte proliferation and turnover analysis by ²H₂O

The ²H₂O body water enrichment was similar among both groups (IS: 1.46% ± 0.31%; IR: 1.17% ± 0.20%; t _[12] = −2.052, p = 0.063). Incorporation of deuterium into DNA of mature adipocytes and cells from the SVF was measured to examine differences in the in vivo cellular proliferation and turnover rates. As a result, no differences in abdominal or gluteal mature adipose tissue FSRs were observed (t _[6.96] = 1.797, p = 0.116; t _[11] = 0.644, p = 0.533, respectively; Figure 1A), although a non-significant trend toward higher mature adipose cell proliferation within the abdominal depot was observed among IR compared to IS subjects. Additionally, no differences in abdominal or gluteal SVF FSRs (t _[12] = 0.061, p = 0.952; t _[11] = −0.106, p = 0.918, respectively; Figure 1B) were observed between subject groups. Similarly, no differences in the mature adipose tissue and SVF FSRs were observed between the abdominal and gluteal adipose tissue depots examined within IS and IR subjects, respectively.

Figure 1.

In vivo Adipocyte Proliferation and Turnover Analysis by ²H₂O. No differences in abdominal or gluteal mature adipose tissue (panel A) or SVF FSRs (panel B) were observed between subject groups. Data are presented as means ± S.D.

In vivo SAT lipid dynamics by ²H₂O

Incorporation of deuterium into newly synthesized TG-glycerol and TG-palmitate were measured to examine differences in the in vivo synthesis of TGs and de novo lipogenesis (DNL). No differences in abdominal or gluteal TG synthesis (t _[12] = 1.444, p = 0.174; t _[12] = 1.006, p = 0.334, respectively; Figure 2A) were observed between subject groups. Additionally, no differences in total or daily DNL synthesis were observed in the abdominal (t _[6.86] = 1.074, p = 0.319; t _[12] = 0.622, p = 0.546, respectively) or gluteal depots (t _[12] = 0.983, p = 0.345; t _[12] = 0.459, p = 0.657, respectively; Figures 2B and C) Likewise, no differences in TG synthesis and DNL were observed between the abdominal and gluteal adipose tissue depots examined within IS and IR subjects, respectively.

Figure 2.

In vivo SAT Lipid Dynamics by ²H₂O. No differences in abdominal or gluteal TG synthesis (panel A) were observed between subject groups. Likewise, no differences in total (panel B) or daily DNL synthesis (panel C) within the abdominal or gluteal depots were observed between subject groups. Data are presented as means ± S.D.

Fibrogenesis (collagen synthesis rates) by ²H₂O

Numerous collagen isoforms were also examined between IS and IR subjects to determine indices of fibrogenesis. Although no differences in the soluble Iα1 (t _[12] = 0.300, p = 0.769; t _[10] = 0.986, p = 0.347, respectively; Figure 3A), soluble Iα2 (t _[12] = 0.536, p = 0.602; t _[10] = 0.576, p = 0.577, respectively; Figure 3B), and insoluble Iα1 FSRs (t _[11] = 1.272, p = 0.230; t _[8] = 1.317, p = 0.224, respectively; Figure 3C) were observed between subject groups, abdominal insoluble Iα2 FSR was greater in IR compared to IS subjects (t _[12] = 0.611, p < 0.001; Figure 3D). No difference in the insoluble Iα2 FSR measured from the gluteal depot was observed between subject groups (t _[9] = 0.805, p = 0.441). Additionally, no differences in collagen isoforms were observed between the abdominal and gluteal adipose tissue depots examined in IS and IR subjects, respectively.

Figure 3.

Fibrogenesis (collagen synthesis rates) by ²H₂O. Although no differences in the soluble Iα1 (panel A), soluble Iα2 (panel B), and insoluble Iα1 (panel C) were observed between subject groups within the abdominal or gluteal depots, abdominal insoluble Iα2 FSR was greater in IR compared to IS subjects (panel D). No difference in the insoluble Iα2 FSR measured from the gluteal depot was observed between subject groups. * indicated a significant difference between IS and IR subjects (p = 0.05). Data are presented as means ± S.D.

Next, we examined the associations of the adipose tissue insoluble collagen Iα2 measured from the abdominal depot with differences in abdominal fat distribution, metabolic function, and liver fat content. Abdominal insoluble collagen Iα2 was positively associated with the ratio of VAT-to-total (VAT + SAT) adipose tissue (r = 0.643, p = 0.007; Figure 4A). In addition, a positive association was observed for fasting insulin (r = 0.579, p = 0.015; Figure 4B). Although no association was observed with the overall insulin response during the OGTT (assess by AUC analysis; r = 0.119, p = 0.343; Figure 4C), increased abdominal insoluble collagen Iα2 was associated with lower WBISI (determined by OGTT; r = −0.540, p = 0.023; Figure 4D), greater AT-IR (r = 0.466, p = 0.047; Figure 4E), and (due to small sample size) trended towards a positive association with PDFF (r = 0.447, p = 0.073; Figure 4F). Interestingly, when controlling for each variable, only the relationship between abdominal insoluble collagen Iα2 and the ratio of VAT-to-total (VAT + SAT) adipose tissue remained.

Figure 4.

Associations of adipose tissue insoluble collage Iα2 (collagen synthesis rates) measured from the abdominal depot were examined with differences in abdominal fat distribution, metabolic function, and liver fat content across both subject groups. Adipose tissue insoluble collage Iα2 is positively associated with the ratio of abdominal visceral-to-total (visceral + subcutaneous) adipose tissue (panel A) and fasting plasma insulin concentrations (panel B). Although no association was observed with the overall insulin response during the OGTT (panel C), increased abdominal insoluble collagen Iα2 was associated with lower WBISI (panel D), greater AT-IR (panel E), and trended towards a positive association with ectopic lipid accumulation within the liver, as indicated by PDFF (panel F).

DISCUSSION

This is the first investigation to examine the in vivo adipocyte turnover, lipid flux, and fibrogenesis in two adipose tissue samples known to differentially regulate insulin sensitivity among a population of adolescent children with obesity. Results from this investigation suggest that abdominal and gluteal SAT turnover rates of lipid components (triglyceride synthesis and breakdown; de novo lipogenesis contribution) of mature adipocytes are similar among IS and IR adolescent children with obesity. However, the insoluble collagen type I, subunit α2 isoform measured from abdominal, but not gluteal, SAT is elevated in IR compared to IS subjects and is associated with indices of abdominal TG synthesis, abdominal fat distribution, and adipose tissue and whole-body insulin signaling.

Previous investigations on adipose tissue expandability and fibrogenesis are limited to adults with obesity. For example, adults with obesity and impaired glucose tolerance have been shown to exhibit greater collage Iα1 FSR (% ˑ wk⁻¹) and expression of collagen encoding genes compared to adults with obesity and normal glucose tolerance (18). Likewise, indices of collagen formation were associated with reduced hepatic and whole-body insulin resistance. In the present study, abdominal insoluble collagen Iα2 synthesis rates are greater in IR compared to IS adolescent children with obesity. Insoluble collagen represents the more cross-linked, stable, and long-lived component of tissue collagen with slower turnover rates compared to guanidine-soluble collagen (26). Although the lower fractional synthesis of insoluble compared to guanidine-soluble collagen observed in the present study is consistent with findings previously demonstrated in rodent pulmonary tissue (26), these results demonstrate that the formation of new collagen within both the abdominal and gluteal SAT depots occurs at a similar rate in both IR and IS adolescents with obesity. In contrast, the formation of stable, cross-linked insoluble collagen, which is characteristic of tissue fibrosis, is elevated and may occur more rapidly within the abdominal, but not gluteal, SAT depot of IR compared to IS subjects.

Moreover, greater abdominal insoluble collagen Iα2 synthesis rates are independently associated with the redistribution of abdominal adipose tissue from the SAT to the VAT depot (41.3% of the total variance). These and previous data suggest that increased formation of stable, cross-linked, insoluble collagen within SAT may reflect the limited capacity for additional SAT expansion required to accommodate excess lipid storage (18). Although results from the present study limit the ability of determining causality, they support the hypothesis that the increased formation of insoluble collagen observed in IR compared to IS subjects contributes to lipid spillover from SAT to VAT, and in turn, serves as a critically important mechanism involved in the complex sequelae of obesity-related metabolic and liver disease pathology.

Increased abdominal insoluble collagen Iα2 was also associated with elevated plasma insulin concentrations, as well as indices of whole body and adipose tissue insulin resistance. Given that chronic hyperinsulinemia has recently been shown to promote collagen formation within rodent adipocytes (28), elevated plasma insulin levels at rest and in response to oral glucose consumption highlight a potential factor contributing to the increased insoluble collagen Iα2 synthesis rates observed among IR subjects in the present study. However, results from the present investigation suggest that chronic exposure to elevated insulin concentrations under fasting condition may be a larger contributor to insoluble collagen formation as opposed to post-prandial increases in insulin. Furthermore, when controlling for abdominal VAT-to-total ratios, the observed relationship with fasting plasma insulin concentrations was no longer observed (note: the relationship of VAT-to-total is independent of insulin concentrations). Although these findings do not eliminate the role of hyperinsulinemia in insoluble collagen formation, and consequently, indices of insulin resistance and liver fat accumulation, more detailed analysis is warranted to closely examine this potential mechanism.

It is important to note that the present study is limited by not measuring hepatic collagen synthesis rates. Previous studies have shown that hepatic collagen synthesis rates are strongly associated with progressive liver fibrosis determined by histological assessments among adults with liver disease (29, 30). Future investigations should include additional analysis to further elucidate the relationship between abdominal adipose tissue fibrogenesis and the synthesis of liver collagen turnover and fat. Such investigations will help to identify whether adipose tissue-specific collagen formation is a facilitator in the pathogenesis of impaired insulin signaling and liver fat accumulation among adolescents with obesity. Similarly, results from such investigations will identify therapeutic targets and support clinicians in establishing earlier intervention strategies to prevent and reverse disease progression. Additional limitations to the analysis include the small sample size, lack of documented Tanner stage, lack of adequate control (lean) population, and inability to assess for potential sex differences. However, given the novelty of utilizing deuterated water and the difficulty associated with subject adherence to the prescribed protocol over several weeks, the present results nonetheless provide invaluable insights into examining indices of adipocyte turnover, lipid dynamics, and fibrogenesis in vivo from two metabolically distinct SAT depots. As such, these results help further elucidate an important metabolic mechanism associated with the pathology of a disease that appears to be exhibiting an increased incidence rates among adolescents with obesity in the United States (31).

In summary, the higher synthesis rate of adipose insoluble type I, subunit α2 in the IR compared to IS adolescents with obesity are consistent with observations by Beals et al. (18) in metabolically abnormal adults with obesity. In contrast, adipose tissue TG synthesis rates were similar among both groups investigated, suggesting that abdominal fat distribution patterns have a large role in the relationship between fibrogenesis and metabolic dysregulation. Moreover, similar formation rates of mature adipocytes and cells within the SVF as well as TG synthesis rates and the contribution from DNL all show that the capacity to synthesize and store TG and endogenously synthesize fatty acids were not different between the groups. Taken together, these data suggest that adipose tissue expandability is not impaired in IR compared to IS adolescents with obesity, but that altered extracellular matrix dynamics potentially decrease the capacity of abdominal SAT to store lipids and contribute to the pathophysiological pathways associated with excess VAT accumulation relative to total abdominal adipose tissue. In light of these findings, additional research is necessary to better understand the impact of extracellular matrix dynamics on whole-body and adipose tissue insulin resistance, and subsequently, the ectopic storage of lipids within the liver.

What is already known about the subject?

• Increased adipose tissue fibrogenesis, not storage capacity, is associated in obese adults with impaired glucose tolerance compared to normal glucose tolerance.

• Abdominal and gluteal subcutaneous adipose tissue (SAT) fibrogenesis has yet to be examined among adolescents with obesity.

What are the new findings in your manuscript?

• Abdominal and gluteal SAT turnover rates of lipid components (triglyceride synthesis and breakdown; de novo lipogenesis contribution) were similar among insulin sensitive and insulin resistant adolescent children with obesity.

• The insoluble collagen type I, subunit α2 (Iα2) isoform measured from abdominal, but not gluteal, SAT was elevated in insulin resistant compared to insulin sensitive subjects.

• Increased insoluble Iα2 collagen is associated with indices of abdominal TG synthesis, ratios of visceral-to-total (VAT + SAT) abdominal fat, adipose tissue and whole-body insulin signaling, and trended towards a positive association with liver fat content.

How might these results change the direction of research or focus of clinical practice?

• Altered extracellular matrix dynamics, but not expandability, potentially decreases the capacity of abdominal SAT to store lipids.

• Increased fibrogenesis potentially contributes to the pathophysiological pathways linking adipose tissue and whole-body insulin resistance with altered ectopic storage of lipids within the liver among insulin resistant adolescents with obesity.