Inflammatory and Metabolic Alterations of Kager's Fat Pad in Chronic Achilles Tendinopathy

<div><p>Background</p><p>Achilles tendinopathy is a painful inflammatory condition characterized by swelling, stiffness and reduced function of the Achilles tendon. Kager’s fat pad is an adipose tissue located in the area anterior to the Achilles tendon. Observations reveal a close physical interplay between Kager’s fat pad and its surrounding structures during movement of the ankle, suggesting that Kager’s fat pad may stabilize and protect the mechanical function of the ankle joint.</p><p>Aim</p><p>The aim of this study was to characterize whether Achilles tendinopathy was accompanied by changes in expression of inflammatory markers and metabolic enzymes in Kager’s fat pad.</p><p>Methods</p><p>A biopsy was taken from Kager’s fat pad from 31 patients with chronic Achilles tendinopathy and from 13 healthy individuals. Gene expression was measured by reverse transcription-quantitative PCR. Focus was on genes related to inflammation and lipid metabolism.</p><p>Results</p><p>Expression of the majority of analyzed inflammatory marker genes was increased in patients with Achilles tendinopathy compared to that in healthy controls. Expression patterns of the patient group were consistent with reduced lipolysis and increased fatty acid β-oxidation. In the fat pad, the pain-signaling neuropeptide substance P was found to be present in one third of the subjects in the Achilles tendinopathy group but in none of the healthy controls.</p><p>Conclusion</p><p>Gene expression changes in Achilles tendinopathy patient samples were consistent with Kager’s fat pad being more inflamed than in the healthy control group. Additionally, the results indicate an altered lipid metabolism in Kager’s fat pad of Achilles tendinopathy patients.</p></div

Inflammatory markers.

<p>mRNA levels of the inflammatory markers <i>TNF-α</i>, <i>IL-1R1</i>, <i>IL-6</i>, <i>IL-10</i> and <i>MCP1</i> and of the macrophage marker <i>CD68</i> were significantly elevated in the Kager’s fat pad of AT patients (n = 30) compared to healthy CON subjects (n = 13). The expression of target gene mRNA was normalized to the expression of <i>TBP</i> mRNA. Normalized mRNA expression for the control subjects was set to 1. * <i>p</i> < 0.05 (compared to CON). <i>TNF-α</i>, tumor necrosis factor-α; <i>IL-1R1</i>, interleukin-1 receptor 1; <i>IL-6</i>, interleukin-6; <i>IL-10</i>, interleukin-10; <i>MCP1</i>, monocyte chemotactant protein-1; <i>CD68</i>, cluster of differentiation 68; CON, control; AT, Achilles tendinopathy; <i>TBP</i>, TATA-binding protein.</p

Energy metabolism, <i>GLUT4</i> and <i>ADIPOQ</i>.

<p>mRNA levels of <i>CS</i>, <i>GLUT4</i> and <i>ADIPOQ</i> were significantly lower, and the mRNA expression of <i>RB1</i> was significantly elevated, in the Kager’s fat pad of AT patients (<i>n</i> = 30) compared to healthy CON subjects (<i>n</i> = 13). The expression of target gene mRNA was normalized to the expression of <i>TBP</i> mRNA. Normalized mRNA expression for the control subjects was set to 1. * <i>p</i> < 0.05 (compared to CON). <i>CS</i>, citrate synthase; <i>GLUT4</i>, glucose transporter 4; <i>ADIPOQ</i>, adiponectin; <i>RB1</i>, retinoblastoma 1; AT, Achilles tendinopathy; CON, control; <i>TBP</i>, TATA-binding protein.</p

Subject characteristics.

<p>Values are presented as mean (min—max).</p><p>F, female; M, male; BMI, body mass index; mo., months.</p><p>Subject characteristics.</p

Lipid metabolism.

<p>mRNA levels of <i>ATGL</i>, <i>HSL</i>, <i>MGL</i>, and <i>ACC2</i> were significantly lower in Kager’s fat pad of AT patients (n = 30) compared to healthy CON subjects (n = 13, except <i>ATGL</i> and <i>HSL</i>: n = 12), while the mRNA expression of <i>CACT</i> and <i>CPT2</i> was significantly elevated. The expression of target gene mRNA was normalized to the expression of <i>TBP</i> mRNA. Normalized mRNA expression for the control subjects was set to 1. * <i>p</i> < 0.05 (compared to CON). <i>ATGL</i>, adipose triglyceride lipase; <i>HSL</i>, hormone-sensitive lipase; <i>MGL</i>, monoacylglycerol lipase; <i>ACC2</i>, acetyl-CoA carboxylase 2; AT, Achilles tendinopathy; CON, control; <i>CACT</i>, carnitine-acylcarnitine translocase; <i>CPT2</i>, carnitine palmitoyltransferase 2; <i>TBP</i>, TATA-binding protein.</p

Characterization of immortalized human brown and white pre-adipocyte cell models from a single donor

<div><p>Brown adipose tissue with its constituent brown adipocytes is a promising therapeutic target in metabolic disorders due to its ability to dissipate energy and improve systemic insulin sensitivity and glucose homeostasis. The molecular control of brown adipocyte differentiation and function has been extensively studied in mice, but relatively little is known about such regulatory mechanisms in humans, which in part is due to lack of human brown adipose tissue derived cell models. Here, we used retrovirus-mediated overexpression to stably integrate human telomerase reverse transcriptase (TERT) into stromal-vascular cell fractions from deep and superficial human neck adipose tissue biopsies from the same donor. The brown and white pre-adipocyte cell models (TERT-hBA and TERT-hWA, respectively) displayed a stable proliferation rate and differentiation until at least passage 20. Mature TERT-hBA adipocytes expressed higher levels of thermogenic marker genes and displayed a higher maximal respiratory capacity than mature TERT-hWA adipocytes. TERT-hBA adipocytes were UCP1-positive and responded to β-adrenergic stimulation by activating the PKA-MKK3/6-p38 MAPK signaling module and increasing thermogenic gene expression and oxygen consumption. Mature TERT-hWA adipocytes underwent efficient rosiglitazone-induced ‘browning’, as demonstrated by strongly increased expression of UCP1 and other brown adipocyte-enriched genes. In summary, the TERT-hBA and TERT-hWA cell models represent useful tools to obtain a better understanding of the molecular control of human brown and white adipocyte differentiation and function as well as of browning of human white adipocytes.</p></div

Subject and biopsy characterization.

<p><b>(A)</b> Patient information. <b>(B)</b> Relative mRNA levels of the thermogenic genes <i>UCP1</i>, <i>PPARGC1A</i> and <i>EBF2</i> in hBAT and hWAT. <b>(C)</b> Relative mRNA levels of the mitochondrial genes <i>CPT1B</i>, <i>CS</i> and <i>COXII</i> in hBAT and hWAT. In (B) and (C), expression levels were normalized to <i>TBP</i> levels. The normalized expression in hWAT was set to 1, except for <i>UCP1</i> in which hBAT was set to 1. Data represent the mean of a technical duplicate without error bars, since only one patient was included. Statistical analyses were not applied.</p

Response to β-adrenergic stimulation.

<p><b>(A)</b> Relative mRNA levels of the β-adrenoceptors <i>ADRB1-3</i> in mature TERT-hBA and TERT-hWA adipocytes at passage 10, 15 and 20. Expression levels were normalized to <i>TBP</i> levels. The normalized expression in TERT-hWA adipocytes was set to 1. RT-qPCR data are presented as mean of means +SEM from 5 independent experiments (two experiments in passage 10 and passage 15 and one experiment in passage 20). Statistical significance was determined by paired two-tailed Student’s t-test. *, p < 0.05 versus TERT-hWA. <b>(B)</b> Relative mRNA levels of thermogenic genes in mature TERT-hBA adipocytes (day 12, passage 12) stimulated with 0.1 μM ISO for 6 h. <b>(C)</b> Relative mRNA levels of thermogenic genes in mature TERT-hBA adipocytes (day 12, passage 13) stimulated with 10 μM FSK for 6 h. In (B) and (C), expression levels were normalized to <i>TBP</i> levels. The normalized expression in vehicle-treated cells was set to 1. Data are presented as mean +SEM of one representative experiment done in technical triplicate. Statistical significance was determined by unpaired two-tailed Student’s t-test. *, p < 0.05 versus vehicle-treated cells. <b>(D)</b> UCP1 protein levels in mature TERT-hBA [day 12, passage 10 (P10) and 15 (P15)] stimulated with 0.1 μM ISO for 24 h. TFIIB was used as a loading control. <b>(E)</b> Western blot analysis for phosphorylated adipocyte mediators in mature TERT-hBA adipocytes (day 12, P9) pretreated with 10 μM propranolol or vehicle for 1 h before being stimulated with 10 μM FSK or 0.1 μM ISO for an additional 1 h. <b>(F-G)</b> Representative time course of oxygen consumption and extracellular acidification rates (OCR and ECAR, respectively) in mature TERT-hBA adipocytes (day 12, passage 9) before and after injection of 10 μM ISO or 10 μM FSK. Data are presented as mean +/- SEM of one representative experiment with 9–12 wells per condition. (H) Western blot analysis for phosphorylated HSL in mature TERT-hWA adipocytes (day 12, passage 9) pretreated with 10 μM propranolol or vehicle for 1 h before being stimulated with 10 μM FSK or 0.1 μM ISO for an additional 1 h.</p

Immortalized human neck pre-adipocytes differentiate into mature adipocytes.

<p><b>(A)</b> Population doubling time for TERT-hWA and TERT-hBA pre-adipocytes in passage 8, 13 and 17. <b>(B)</b> The general differentiation protocol for TERT-hBA and TERT-hWA. <b>(C)</b> Representative overview photos of Oil red O-stained TERT-hBA and TERT-hWA adipocytes at day 12 at passage 10 (P10), 15 (P15) and 20 (P20). <b>(D)</b> Relative mRNA levels of the differentiation markers <i>GLUT4</i>, <i>CEBPA</i> and <i>FABP4</i> in pre-adipocytes (day 0) and mature adipocytes (day 12) at P10, P15 and P20. Expression levels were normalized to <i>TBP</i> levels. RT-qPCR data are represented as mean of means +SEM from 4 independent experiments (two experiments in P10 and one experiment in P15 and P20). Statistical significance was determined by paired two-tailed Student’s t-test. *, p < 0.05 versus pre-adipocytes (day 0). <b>(E)</b> Protein levels of FABP4 in TERT-WA and TERT-hBA pre-adipocytes (day 0) and adipocytes (day 12) at P9. AKT was used as a loading control.</p

Conversion of mature TERT-hWA adipocytes into brown-like adipocytes upon exposure to rosiglitazone.

<p><b>(A)</b> The rosiglitazone-induced browning protocol. <b>(B)</b> Relative mRNA levels of <i>UCP1</i> in mature TERT-hWA adipocytes (day 15, passage 7–14), SGBS and hMADS adipocytes exposed to vehicle or 1 μM rosiglitazone from day 12 to 15. <b>(C)</b> Protein levels of UCP1 in TERT-hWA adipocytes (day 15, passage 13) exposed to vehicle or 1 μM rosiglitazone from day 12 to 15. TFIIB was used as a loading control. <b>(D)</b> Relative mRNA levels of the thermogenesis-related genes <i>EBF2</i>, <i>DIO2</i> and <i>PDK4</i> in mature TERT-hWA adipocytes (day 15, passage 12) exposed to vehicle or 1 μM rosiglitazone from day 12 to 15. <b>(E)</b> Relative mRNA levels of the β-adrenoceptors <i>ADRB1-3</i> in mature TERT-hWA adipocytes (day 15, passage 12) exposed to vehicle or 1 μM rosiglitazone from day 12 to 15. <b>(F)</b> Relative mRNA levels of the mitochondrial markers <i>CPT1B</i>, <i>CS</i> and <i>COXII</i> in mature TERT-hWA adipocytes (day 15, passage 12) exposed to vehicle or 1 μM rosiglitazone from day 12 to 15. In (B) and (D-F), expression levels were normalized to <i>TBP</i> levels. The normalized expression in vehicle-treated cells was set to 1. Data are presented as mean +SEM of one representative experiment done in technical triplicate. Statistical significance was determined by unpaired two-tailed Student’s t-test. *, p < 0.05 versus vehicle-treated cells.</p