FNDC5/Irisin Is Not Only a Myokine but Also an Adipokine methods
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Animal Models
Male Sprague Dawley rats (160 g) were housed in 12-h light/12-h dark cycles with free access to a standard chow diet and water. After 5 days of acclimatization, weight-matched animals were assigned to one the following experimental groups (n = 5 per group): a) control ad libitum, b) 36 hours fasting, c) re-feed (36 hours fasting followed by 15 minutes of feeding), d) exercise (ad libitum with free access to the activity wheel for 1 or 3 weeks), e) control activity-based anorexia (ABA) and f) ABA as previously described and listed in,. The body weight, food intake, daily wheel turns, body composition and hormonal determination (ghrelin, leptin and HOMA) of these animals were previously characterized. Diet-induced obesity (DIO) animals (200 g) were fed with a 60% high fat diet D12492 (Research Diets, NJ) over 9 weeks, and age-matched lean rats were used as a control. Male...
Rat muscle/adipose tissues and secretomes processing
The soleus and gastrocnemius muscles, the subcutaneous adipose tissue from the skin of the hips, the visceral fat located inside the peritoneal cavity around the internal organs and the brown (interscapular area) fat locations were processed for secretome collection based on a previously optimized protocol to minimize tissue damage,. Briefly, tissues (n = 5 from each muscle type/fat depot) were carefully excised minimizing rat hair contamination as much as possible and transported from the animal house operating room to the laboratory in a sterile Krebs - Ringer-Hepes (KRH) buffer with penicillin (100 U/ml) and streptomycin (100 µg/ml) at room temperature. The tissues were processed to eliminate any contaminants and washed thoroughly in KRH under sterile conditions in a flow laminar hood. Adipose tissue pieces were centrifuged in a 25 ml tube with 20 ml of KRH...
Immunoblotting
Protein extracts from whole tissue samples and secretomes were processed as previously described,. Briefly, proteins of whole tissue samples were extracted by homogenization using a TissueLyser II (QIAGEN, Tokio, Japan) in cold RIPA buffer [200 mM Tris/HCl (pH 7.4), 130 mM NaCl, 10% (v/v) glycerol, 0.1% (v/v) SDS, 1% (v/v) Triton X-100, 10 mM MgCl2] with anti-proteases and anti-phosphatases (Sigma-Aldrich; St Louis, MO). Homogenates were centrifuged for 30 minutes at 18,000 g in a microfuge at 4°C, and supernatants were frozen at -80°C. Twenty-five µg of adipose tissue secreted proteins, 25 µg of whole tissue and 5 µl of blood plasma from three independent experiments were separated in 12% SDS-PAGE gels and electroblotted onto nitrocellulose membranes as previously described. Equal loading was confirmed by membrane staining with Ponceau S (Sigma-Aldri...
Human adipose tissue acquisition
Human adipose tissue was obtained from an obese patient (body mass index >35) who underwent laparoscopic gastrectomy surgery. The visceral fat was located in the hypogastric region around the internal organs, and the subcutaneous fat was located in the mesogastric region. The tissues were transported from the operating room to the laboratory in sterile KRH buffer with penicillin (100 U/ml) and streptomycin (100 µg/ml) and processed as described above for the rat adipose tissue.
Isolation of mature adipocytes and stromo-vascular fraction (SVF)
Five g of rat adipose tissue from the visceral and subcutaneous locations was washed three to four times with KRH buffer, cut in pieces less than 1 mg and resuspended in an equal volume of 0.2% pre-warmed (37°C) collagenase Type I (Gibco, CA). This tissue was placed in an orbital mixer at 37°C with continuous agitation for 90 minutes. Then, it was centrifuged for 5 minutes at 400 g at room temperature. The supernatant that contained mature adipocytes was recollected. The pellet was identified as the SVF.
Adipose tissue "browning"
Representative western blot images of UCP1 for SAT, VAT and VAT under ad libitum condition and after 3 weeks of exercise are shown (A); UCP1 expression in SAT and VAT among anorexic and control counterparts is shown (B). Histograms of band quantification from at least 3 independent experiments are shown in those cases in which bands exist. Significant protein results are shown as percentages compared with controls in A and with regard to EXER in B (*p<0.05; **p<0.01).
Circulating FNDC5/irisin
FNDC5/Irisin circulating levels were assessed in plasma samples from all the animals studied above ( ). This analysis revealed a significant increase in FNDC5/irisin levels after 1 and 3 weeks of exercise, after 36 hours of fasting and after 15 minutes of re-feeding. Corroborating this finding, we detected a significant increase of FNDC5/irisin levels in ABA control exercised animals (EXER) and CABA. However, DIO and Zucker rats had opposite circulating levels of FNDC5/irisin; whereas DIO animals showed a significant increase, Zucker rats showed significantly diminished levels.
Adipose tissue "browning"
Considering the described potent effect of FNDC5/irisin stimulating white adipose browning and thermogenesis, we tested Ucp1 expression on the subcutaneous and visceral adipose tissues analyzed above ( ). Under normal ad libitum conditions, Ucp1 was expressed exclusively in BAT as expected ( ). However, this expression shows a decreasing trend after 3 weeks of endurance exercise ( ).
Measurement outputs
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Protein expression by western blot
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- Membrane or gel image with visible bands for target and control proteins
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- Band quantification and normalized densitometry values
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- Relative expression values or fold-change comparisons across groups
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Quantify the primary readouts for this experiment: Protein expression by western blot.
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inferred from protocolReporting output
Report representative outputs alongside summary comparisons for Protein expression by western blot.
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Male Sprague Dawley rats (160 g) were housed in 12-h light/12-h dark cycles with free access to a standard chow diet and water. After 5 days of acclimatization, weight-matched animals were assigned to one the following experimental groups (n = 5 per group): a) control ad libitum, b) 36 hours fasting, c) re-feed (36 hours fasting followed by 15 minutes of feeding), d) exercise (ad libitum with free access to the activity wheel for 1 or 3 weeks), e) control activity-based anorexia (ABA) and f) ABA as previously described and listed in,. The body weight, food intake, daily wheel turns, body composition and hormonal determination (ghrelin, leptin and HOMA) of these animals were previously characterized. Diet-induced obesity (DIO) animals (200 g) were fed with a 60% high fat diet D12492 (Research Diets, NJ) over 9 weeks, and age-matched lean rats were used as a control. Male obese Zucker rats (fa/fa, n = 10) and their control counterparts, lean Zucker rats (fa/-, n = 10), were purchased from Charles River laboratories (Barcelona, Spain) at 10 weeks of age and were fed chow ad libitum for 12 weeks. Animals were euthanized by decapitation; their b...
The soleus and gastrocnemius muscles, the subcutaneous adipose tissue from the skin of the hips, the visceral fat located inside the peritoneal cavity around the internal organs and the brown (interscapular area) fat locations were processed for secretome collection based on a previously optimized protocol to minimize tissue damage,. Briefly, tissues (n = 5 from each muscle type/fat depot) were carefully excised minimizing rat hair contamination as much as possible and transported from the animal house operating room to the laboratory in a sterile Krebs - Ringer-Hepes (KRH) buffer with penicillin (100 U/ml) and streptomycin (100 µg/ml) at room temperature. The tissues were processed to eliminate any contaminants and washed thoroughly in KRH under sterile conditions in a flow laminar hood. Adipose tissue pieces were centrifuged in a 25 ml tube with 20 ml of KRH for 5 minutes at room temperature to remove blood cells and cell debris. Fat pieces of approximately 2 g were incubated in 6 well cell culture dishes (Iwaki, Tokio, Japan) in 4 ml of serum-free medium that was changed twice every two hours and again after 16 hours. Finally, fresh 4 ml of serum/phen...
Protein extracts from whole tissue samples and secretomes were processed as previously described,. Briefly, proteins of whole tissue samples were extracted by homogenization using a TissueLyser II (QIAGEN, Tokio, Japan) in cold RIPA buffer [200 mM Tris/HCl (pH 7.4), 130 mM NaCl, 10% (v/v) glycerol, 0.1% (v/v) SDS, 1% (v/v) Triton X-100, 10 mM MgCl2] with anti-proteases and anti-phosphatases (Sigma-Aldrich; St Louis, MO). Homogenates were centrifuged for 30 minutes at 18,000 g in a microfuge at 4°C, and supernatants were frozen at -80°C. Twenty-five µg of adipose tissue secreted proteins, 25 µg of whole tissue and 5 µl of blood plasma from three independent experiments were separated in 12% SDS-PAGE gels and electroblotted onto nitrocellulose membranes as previously described. Equal loading was confirmed by membrane staining with Ponceau S (Sigma-Aldrich; St Louis, MO) in the case of secretome protein extracts, or by measuring the amount of GAPDH in whole tissue protein extracts. Primary anti-FNDC5, anti-UCP-1 and anti-PGC1 were purchased from Abcam (Cambridge, UK); anti-Irisin was purchased from Phoenix Pharmaceuticals Inc. (CA, USA); anti-IL6 a...
Human adipose tissue was obtained from an obese patient (body mass index >35) who underwent laparoscopic gastrectomy surgery. The visceral fat was located in the hypogastric region around the internal organs, and the subcutaneous fat was located in the mesogastric region. The tissues were transported from the operating room to the laboratory in sterile KRH buffer with penicillin (100 U/ml) and streptomycin (100 µg/ml) and processed as described above for the rat adipose tissue.
Five g of rat adipose tissue from the visceral and subcutaneous locations was washed three to four times with KRH buffer, cut in pieces less than 1 mg and resuspended in an equal volume of 0.2% pre-warmed (37°C) collagenase Type I (Gibco, CA). This tissue was placed in an orbital mixer at 37°C with continuous agitation for 90 minutes. Then, it was centrifuged for 5 minutes at 400 g at room temperature. The supernatant that contained mature adipocytes was recollected. The pellet was identified as the SVF.
Representative western blot images of UCP1 for SAT, VAT and VAT under ad libitum condition and after 3 weeks of exercise are shown (A); UCP1 expression in SAT and VAT among anorexic and control counterparts is shown (B). Histograms of band quantification from at least 3 independent experiments are shown in those cases in which bands exist. Significant protein results are shown as percentages compared with controls in A and with regard to EXER in B (*p<0.05; **p<0.01).
FNDC5/Irisin circulating levels were assessed in plasma samples from all the animals studied above ( ). This analysis revealed a significant increase in FNDC5/irisin levels after 1 and 3 weeks of exercise, after 36 hours of fasting and after 15 minutes of re-feeding. Corroborating this finding, we detected a significant increase of FNDC5/irisin levels in ABA control exercised animals (EXER) and CABA. However, DIO and Zucker rats had opposite circulating levels of FNDC5/irisin; whereas DIO animals showed a significant increase, Zucker rats showed significantly diminished levels.
Considering the described potent effect of FNDC5/irisin stimulating white adipose browning and thermogenesis, we tested Ucp1 expression on the subcutaneous and visceral adipose tissues analyzed above ( ). Under normal ad libitum conditions, Ucp1 was expressed exclusively in BAT as expected ( ). However, this expression shows a decreasing trend after 3 weeks of endurance exercise ( ).
Machine-readable layer
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