Functional differences in visceral and subcutaneous fat pads originate from differences in the adipose stem cell

Abstract

Metabolic pathologies mainly originate from adipose tissue (AT) dysfunctions. AT differences are associated with fat-depot anatomic distribution in subcutaneous (SAT) and visceral omental (VAT) pads. We address the question whether the functional differences between the two compartments may be present early in the adipose stem cell (ASC) instead of being restricted to the mature adipocytes. Using a specific human ASC model, we evaluated proliferation/differentiation of ASC from abdominal SAT-(S-ASC) and VAT-(V-ASC) paired biopsies in parallel as well as the electrophysiological properties and functional activity of ASC and their in vitro-derived adipocytes. A dramatic difference in proliferation and adipogenic potential was observed between the two ASC populations, S-ASC having a growth rate and adipogenic potential significantly higher than V-ASC and giving rise to more functional and better organized adipocytes. To our knowledge, this is the first comprehensive electrophysiological analysis of ASC and derived-adipocytes, showing electrophysiological properties, such as membrane potential, capacitance and K(+)-current parameters which confirm the better functionality of S-ASC and their derived adipocytes. We document the greater ability of S-ASC-derived adipocytes to secrete adiponectin and their reduced susceptibility to lipolysis. These features may account for the metabolic differences observed between the SAT and VAT. Our findings suggest that VAT and SAT functional differences originate at the level of the adult ASC which maintains a memory of its fat pad of origin. Such stem cell differences may account for differential adipose depot susceptibility to the development of metabolic dysfunction and may represent a suitable target for specific therapeutic approaches.

Figure 1. Different proliferation rate in S-ASC and V-ASC.

Growth curves were obtained for each population by haemocytometer cell counting (A), evaluation of BrdU incorporation (B), and MTS assay (C) at each time point. Results are expressed as mean ± SE fold increase of cell counts (A), BrdU (B) and MTS absorbance (C) at each time point over day 1 in at least 4 ASC populations derived from at least 4 independent subjects. *P<0.001 versus respective day 1; §§P<0.01, §§§P<0.001 S- versus V-ASC at the corresponding time point. (D) Ki-67 proliferation index is calculated as mean ± SE percentage of positive cells counted in at least 20 fields for each slide. 4 ASC populations were obtained from 4 different subjects, *P<0.001 versus the corresponding V-ASC population. (E) Population doubling (PD) curves were obtained by cell counting at different passages of S-ASC and V-ASC cultures expanded for about 2 months. Results are expressed as mean ± SE PD numbers obtained by counting cells in triplicates from 3 independent subjects; *P<0.001, versus respective passage 0; §P<0.05, §§§P<0.001 versus the corresponding V-ASC. (F) Immunophenotype of V-ASC obtained at 2 different passages (P3 and 12). Data correspond to mean ± SE FACScan percentage of positive cells for the indicated surface markers previously demonstrated to characterize ASC populations (7); cells were obtained from n = 2 independent subjects. Similar data were obtained for the corresponding S-ASC at the same passage. No statistically significant differences have been observed in the expression of the indicated markers between the two passages. (G) Representative karyotype analysis of V-ASC at passage 12, corresponding to about 3 months of culture. Similar data has been obtained for the corresponding S-ASC at the same passage, in 2 different subjects. Karyotype analysis was performed on ASC obtained from 2 different subjects.

Figure 2. Stemness marker expression in S-ASC and V-ASC.

Quantitative Real Time RT-PCR was performed on mRNA extracted from both S- and V-ASC and from the corresponding in vitro-differentiated adipocytes, to evaluate BMI-1, (A, n = 10 subjects) NANOG (B, n = 5 subjects) and OCT-4 (C, n = 5 subjects) expression. Data are expressed as the mean±SE gene expression versus the housekeeping GAPDH gene. °P<0.001 S- versus V-ASC and *P<0.05 adipocytes versus respective ASC.

Figure 3. Staining of intracellular triglyceride depots in adipocytes obtained from in vitro differentiation of S- and V-ASC.

Dark (A,I; 5× magnification) and bright field (E,M, G,O,H,P; 40× magnification) microscopy, ORO staining of neutral lipids (B,J; 5× magnification; F,N; 40× magnification) and fluorescence microscopy (40× magnification) of in vitro-differentiated S- (C) and V- (K) adipocytes and of the corresponding S- (D) and V- (L) ASC stained with AdipoRed evidentiate a qualitative higher number of lipid droplets and a higher number of differentiated adipocytes in the S- compared to V- populations. No staining was present in ASC, confirming the high specificity of AdipoRed binding to triglycerides. Representative of cell populations obtained at least from n = 4 different subjects.

Figure 4. Differences in adipogenic potential and functional capabilities in ASC and in their in vitro-derived adipocytes.

Expression of the adipogenic genes PPARgamma (A), FABP4 (B) and adiponectin (C) evaluated by quantitative real time RT-PCR is higher in S- compared to derived V-ADIPO. Data are expressed by box charts of gene expression ratio versus GAPDH (C) or fold increase between adipocytes and ASC (A, B). Boxes indicate the 25^th (lower) and 75^th (upper) percentiles. Horizontal lines and dots in the boxes indicate the 50^th percentile value (median) and mean value, respectively. Vertical lines give the 10^th and 90^th percentile limits of the data. Statistical analysis for non-parametric distribution was performed with Wilcoxon text: *P<0.05, ***P<0.001 S- versus V-ADIPO, n = 30 experiments with cells obtained from 14 independent subjects. D: Western Blot analysis of Adiponectin (upper panel) and FABP4 (lower panel) protein expression in ASC compared to the derived adipocytes. Molecular weight (MW) in kDa has been indicated for both standards and proteins of interest. Equal protein loading was verified by probing for the housekeeping protein actin. SAT and VAT samples from the same subject were run as positive controls. Representative of 5 independent experiments performed on cells obtained from 5 subjects. E: Immunofluorescence analysis of FABP4 (left panel) in in vitro-differentiated S-ADIPO (upper panel) and V-ADIPO (lower panel) revealed positivity for the enzyme around the intracellular lipid droplets. Right panels: corresponding bright field microscopy. F: Adipogenic potential of S- and V-ASC has been evaluated as AdipoRed staining of intracellular lipid droplets in the derived adipocytes. Results are expressed as mean ± SE fold increase of AdipoRed absorbance (left axis) in adipocytes versus the corresponding ASC. Lipolytic activity (right axis) of the same adipocytes evaluated as AdipoRed absorbance fold decrease following 12 h treatment with 1 µM isoproterenol. *P<0.001 S- versus V-ADIPO. G: Adiponectin secretion evaluated during in vitro-induced adipogenesis in S- and V-ASC at two different time points (T1 and T2, 14 and 21 days of differentiation respectively) by ELISA adiponectin kit. °P<0.001 T2 versus T1; *P<0.001 S- versus V-ADIPO.

Figure 5. ASC and in vitro-differentiated derived adipocytes express two types of delayed rectifier K⁺ currents.

Light microscopy of a S-ASC (A), V-ASC (B), S-ADIPO (C) and V-ADIPO (D) plated on glass coverslip and impaled by the patch pipette for electrophysiological records (magnification 40X). (E–F) Typical I_K,DR trace currents elicited by a voltage step to 50 mV from a holding potential of −60 mV in Control solution with 4-AP (2 mM), Nifedipine (10 µM) and Ba²⁺(0.1 mM) in S-ASC (Ea), S-ADIPO (Eb), V-ASC (Fa) and V-ADIPO (Fb). In each cell type, I_Ks traces are obtained in the presence of Ibtx (100 nM) and I_BK traces by subtracting I_Ks from I_K,DR. By adding Chr (50 µM) only a very small residual current was recorded (Ibtx+Chr current traces). (G–J) Family of I_BK (G and I) and I_Ks (H and J) family of current traces evaluated as in panels E and F (same cells) from pharmacological dissection with voltage steps of 10 mV increments (from −80 to 50 mV; HP of −60 mV. Note the different ordinate scale in all panels.

Figure 6. Voltage dependence of the two kind of I_K,DR (I_BK and I_Ks) and of I_Kir.

(A) Plots represent the maximum mean value of I_K,DR (a), I_BK and I_Ks (b,c) as a function of the applied voltage step recorded in the presence of Ba²⁺ from all experiments as in Fig. 6 G–J. Note the different ordinate scale for ASC and ADIPO. Panels put in evidence that I_K,DR, I_BK and I_Ks are reduced in size in the ADIPO versus the corresponding ASC. (B) K⁺ currents in representative cells elicited by voltage ramp stimulation in Control external solution without Ba²⁺ recording the total K⁺ currents (I_K,tot) (a, – Ba²⁺) and after adding 0.1 mM Ba²⁺ to block I_Kir and record I_K,DR (b, +Ba²⁺ 0.1 mM). I_Kir have been obtained by detracting current traces recorded in the presence of Ba²⁺ from those in the absence (c). I_K/C_m and V_th mean ± SE values and number of experiments are indicated in Tab 2.