Beneficial Effects of Bariatric Surgery-Induced by Weight Loss on the Proteome of Abdominal Subcutaneous Adipose Tissue

Abstract

Bariatric surgery (BS) is the most effective treatment for obesity and has a positive impact on cardiometabolic risk and in the remission of type 2 diabetes. Following BS, the majority of fat mass is lost from the subcutaneous adipose tissue depot (SAT). However, the changes in this depot and functions and as well as its relative contribution to the beneficial effects of this surgery are still controversial. With the aim of studying altered proteins and molecular pathways in abdominal SAT (aSAT) after body weight normalization induced by BS, we carried out a proteomic approach sequential window acquisition of all theoretical mass spectra (SWATH-MS) analysis. These results were complemented by Western blot, electron microscopy and RT-qPCR. With all of the working tools mentioned, we confirmed that after BS, up-regulated proteins were associated with metabolism, the citric acid cycle and respiratory electron transport, triglyceride catabolism and metabolism, formation of ATP, pyruvate metabolism, glycolysis/gluconeogenesis and thermogenesis among others. In contrast, proteins with decreased values are part of the biological pathways related to the immune system. We also confirmed that obesity caused a significant decrease in mitochondrial density and coverage, which was corrected by BS. Together, these findings reveal specific molecular mechanisms, genes and proteins that improve adipose tissue function after BS characterized by lower inflammation, increased glucose uptake, higher insulin sensitivity, higher de novo lipogenesis, increased mitochondrial function and decreased adipocyte size.

Keywords: abdominal adipose tissue; bariatric surgery; immune system; lipogenesis; metabolism; mitochondria; proteome.

Figure 1

(A). Effect of obesity and bariatric surgery on protein expression in abdominal subcutaneous adipose tissue depot (aSAT). Venn diagrams showing the qualitative proteomic analysis of aSAT from four obese patients before bariatric surgery (before BS) and the same patients after body weight loss induced by bariatric surgery (after BS). They show the number of unique and overlapping proteins identified individually for each patient before and after bariatric surgery (Patient 1–4), as well as the total of proteins identified. (B). Graphical representation of quantitative proteomics data. Proteins are ranked in a volcano plot according to their statistical p-value (y-axis) and their relative abundance ratio (log2-fold change) between aSAT samples after BS and before BS (x-axis). Off-centered spots are those that vary the most between both groups. The cut-offs for significant changes are fold-changes of 1.5 and p < 0.05.

Figure 2

Heatmap of significantly regulated proteins by weight loss induced by bariatric surgery on aSAT using a sequential window acquisition of all theoretical mass spectra (SWATH-MS) technique. Effects of weight loss induced by bariatric surgery on aSAT protein expression. Three-hundred and sixty-four proteins were significantly regulated, 136 down-regulated and 228 up-regulated. The expression intensity of each protein varies from white to blue. The expression is given in arbitrary units and in order to adequately visualize the changes due to bariatric surgery, the expression value of each protein was normalized taking into account the mean of that protein for the four patients before undergoing bariatric surgery. This value was assigned as 100% and all values represented here were expressed based on that value. The proteins are ordered according to the p-value. In the first column, the p-value varied from 5.25E–12 to 5.47E–05, in the second column from 5.97E–05 to 0.00283, and in the third column from 0.00284 to 0.04071. A value of p < 0.05 was considered statistically significant. Data were plotted using matrix2png version 1.2.2 (http://www.chibi.ubc.ca/matrix2png/). P1–P4 = Patient 1–Patient 4.

Figure 3

Gene ontology (GO) enrichment of 364 significantly regulated proteins according to the FunRich functional annotation. The histograms show for each GO term, cellular component (A; p < 10⁻⁵), biological process (B; p < 0.05) and molecular function (C; p < 0.05), the most significantly enriched categories of up-regulated and down-regulated proteins quantified using the SWATH-MS approach. Extended data of GO enrichment analysis is provided in Table S3.

Figure 4

aSAT hexokinase (HK) activity levels (A) and protein expression levels (B,C) of lipid metabolism and insulin signaling-related enzymes, in control patients and obese patients before BS and the same patients after BS. Values are means ± SEM of six patients per group (HK activity) or 4–5 patients per group (Western blot). Different letters above the bars indicate statistically significant differences, p < 0.05. P.1–P.4 = Patient 1–Patient 4. B = before bariatric surgery, A = after bariatric surgery. Controls = healthy patients with normal BMI. BS = bariatric surgery.

Figure 5

aSAT mRNA expression levels of GLUT4, FAS, S100A8 and ADH1B in control patients and obese patients before BS and after BS and body weight normalization. Values are means ± SEM. Different letters above the bars indicate statistically significant differences, p < 0.05. Control = healthy patients with normal BMI, n = 27. Before BS, n = 145. After BS, n = 6. BS = bariatric surgery.

Figure 6

Mitochondrial morphometrics. Characterization of mitochondrial parameters in aSAT of control patients (healthy patients with normal BMI, n = 5), obese patients before bariatric surgery (before BS, n = 4), and obese patients after bariatric surgery and body weight normalization (after BS, n = 5). Values are means ± SEM. Different letters above the bars indicate statistically significant differences, p < 0.05. The aspect ratio is a measure of mitochondrial length (major axis/minor axis) (A), mitochondria density (B) and coverage (C) were calculated by dividing the number and total area of mitochondria to the cytoplasm area, respectively. Representative electron microscopy images of aSAT sections from control patients (D), obese patients before bariatric surgery (E) and obese patients after bariatric surgery (F), respectively. Bars = 2 µm. The arrows point to the mitochondria.

Figure 7

Adipocyte size distribution. The difference in adipocyte size is evident in representative H&E stained samples of aSAT from lean control patients (A, n = 14), obese patients before bariatric surgery (B, n = 14) and the same patients after bariatric surgery and normalization of body weight (C, n = 6). (D) The mean frequency of adipocyte diameter, indicating that obese patients before BS have greater numbers of adipocytes with diameters > 80 μm and fewer adipocytes with diameters < 80 μm when compared to control or obese patients after BS. Bars = 200 µm. Values are means ± SEM (histogram). Different letters above the bars indicate statistically significant differences, p < 0.05. BS = bariatric surgery.