Intermittent Cold Exposure Induces Distinct Proteomic Signatures in White Adipose Tissue of Mice

Abstract

Adipose tissue exhibits dynamic metabolic and structural changes in response to environmental stimuli, including temperature fluctuations. While continuous cold exposure has been extensively studied, the molecular effects of prolonged intermittent cold exposure (ICE) remain poorly characterized. Here, we present a proteomic analysis of inguinal white adipose tissue (IWAT) from mice subjected to a 16-week regimen of short-term daily ICE (6 °C for 6 h, 5 days per week) without compensatory caloric intake. Mass spectrometry identified 1108 proteins, with 140 differentially expressed between experimental and control groups. ICE significantly upregulated mitochondrial proteins associated with lipid and carbohydrate catabolism, the tricarboxylic acid (TCA) cycle, oxidative phosphorylation, and lipogenesis, including LETM1, AIFM1, PHB, PHB2, ACOT2, NDUA9, and ATP5J. These changes reflect enhanced metabolic activity and mitochondrial remodeling. In contrast, proteins linked to oxidative stress, insulin resistance, inflammation, and extracellular matrix remodeling were downregulated, such as HMGB1, FETUA, SERPH1, RPN1, and AOC3. Notably, gamma-synuclein (SYUG), which inhibits lipolysis, was undetectable in ICE-treated samples. Our findings support the hypothesis that ICE promotes thermogenic reprogramming and metabolic rejuvenation in subcutaneous fat through activation of futile cycles and mitochondrial restructuring. This study offers molecular insights into adaptive thermogenesis and presents intermittent cold exposure as a potential strategy to mitigate adipose tissue aging.

Keywords: adipose tissue; futile cycles; intermittent cold exposure; mitochondrial remodeling; proteomics; thermogenesis.

Figure 1

The animal study design. IBAT—interscapular brown adipose tissue, EWAT—epididymal white adipose tissue, IWAT—inguinal white adipose tissue, TAG—triacylglycerol, LC-ESI-MS—liquid chromatography–electrospray ionization–mass spectrometry.

Figure 2

The effect of intermittent cold exposures on body weight; blood glucose and triglyceride levels; adipose tissues mass; and protein content. (a) Body weight; (b) blood glucose; (c) blood triglycerides; (d) relative adipose tissue mass; (e) total protein content in adipose tissue. * p < 0.05, ** p < 0.01, Mann–Whitney U test.

Figure 3

The effect of intermittent cold exposures on proteome of inguinal adipose tissue. (a) Principal component analysis diagram, where red and blue circles represent control and experimental samples, respectively. Overrepresentation test for differentially expressed proteins and total gene products in the Cellular Components (b) and Biological Processes (c) Gen Ontology (GO) categories.

Figure 3

The effect of intermittent cold exposures on proteome of inguinal adipose tissue. (a) Principal component analysis diagram, where red and blue circles represent control and experimental samples, respectively. Overrepresentation test for differentially expressed proteins and total gene products in the Cellular Components (b) and Biological Processes (c) Gen Ontology (GO) categories.

Figure 4

Schematic representation of an adipocyte of mouse subcutaneous adipose tissue under standard and experimental conditions. (a) Under standard conditions, the use of exogenous fatty acids for lipogenesis is increased, while insulin-dependent glucose transport, de novo fatty acid synthesis, and mitochondriogenesis are suppressed. There is a fusion of lipid droplets into one drop and the growth of it and the whole cell. Compensatory enlargement in the size and stiffness of the extracellular matrix and increased expression of proteins involved in danger signaling favor the accumulation of inflammatory macrophages; in contrast, IgM synthesis suppresses inflammation. Oxidative stress slows down due to the removal of Fe²⁺ from tissues by a system of ceruloplasmin and transferrin. (b) Under experimental conditions, the adipocyte synthesizes fatty acids and triglycerides de novo. For the uninterrupted supply of glucose and ATP to these processes, glycogen deposition and expression of HXK2, which intercepts ATP on the outer membrane of mitochondria, are enhanced. The fatty acids released during lipolysis are directed to the beta oxidation pathway. A part of the acyl-CoA is hydrolyzed by the mitochondrial enzyme ACOT2 to free fatty acids (1). This and the other futile cycles—fatty acid synthesis-beta oxidation (2), lipogenesis–lipolysis (3), as well as, probably, the mitochondrial uncoupling due to carnitine-independent transport of medium-chain fatty acids—intensify energy metabolism. To maintain the high biological quality of mitochondria, mitochondriogenesis and autophagy–mitophagy processes are enhanced.