Obesity is characterized by excessive energy intake from Western-type diets, hyperinsulinemia, deposition of triglycerides, accumulation of white adipose tissue (WAT) and hypertrophy of adipocytes in visceral adipose tissue (VAT) and subcutaneous adipose tissue (SAT) ^[1][53]. In animal models and human studies of obesity and insulin resistance, VAT undergoes greater adipocyte hypertrophy than SAT, with a higher degree of macrophage infiltration, more intense levels of inflammation and loss of T regulatory cells (Treg) ^[2][54]. Brown adipose tissue (BAT) only makes up 1–2% of total body fat but is important for non-shivering thermogenesis via uncoupling protein 1 (UCP-1) on cold exposure, or β-adrenergic stimulation. Beigeing or browning of sub-populations of white adipocytes can also contribute to cold-induced thermogenesis via mitochondrial fat oxidation, thereby protecting against obesity ^[3][4][5][55,56,57]. The majority of dietary animal and plant fats are triglycerides, which contain saturated fatty acids (animal fat, hydrogenated vegetable oil, palm oil, coconut oil) or unsaturated fatty acids (fish, vegetable oil).

Triglyceride deposition is an efficient and anhydrous way of storing excess energy substrates compared to glycogen storage. Triglycerides circulate in the blood as apolipoproteins and undergo hydrolysis to produce free fatty acids for β-oxidation and glycerol. As compared to the liver and skeletal muscle, adipocytes lack glycerol kinase and thus have to synthesize glycerol-3-phosphate from glucose or fructose via cytoplasmic glycolysis or by gluconeogenesis from pyruvate in order to manufacture triglycerides. Fatty acids are obtained by adipocytes from insulin-stimulated lipoprotein lipase action on circulating VLDL or by de novo synthesis. Glycerol is combined with three esterified fatty acids to form triacylglycerides and stored as a single fat droplet in the cytoplasm of the adipocyte, squeezing the nucleus to the periphery of the cell. Insulin normally promotes lipogenesis by stimulating glucose and fatty acid uptake by adipocytes, fatty acid synthase (FAS) production of palmitate and WAT triacylglyceride deposition, and inhibits adipocyte hormone-sensitive lipase and lipolysis. Insulin counterregulatory hormones include glucagon, adrenaline, cortisol and growth hormone ^[6][7][8][9][58,59,60,61].

Enlargement of fat storage in adults is achieved mainly by adipocyte hypertrophy in WAT rather than adipocyte hyperplasia. Adipocyte precursor development (adipogenesis) is regulated by CCAAT enhancer binding protein alpha (C/EBPα), PPARγ and sterol regulatory element-binding protein (SREBP)-1c. The diameter of individual human adipocytes ranges from <20 to 300 µm, and thus the stored triglyceride volume can be increased by up to a thousand-fold. Pathological adipocyte hypertrophy is associated with excessive levels of long chain FFAs, impaired β-oxidation of FFA and insulin resistance. Peroxisome proliferator-activated receptor-γ (PPARγ) is inhibited and adipogenesis is suppressed during adult obesity and the loss of adiponectin. This also limits WAT storage capability or ‘fat buffering’ ability and promotes ectopic fat deposition in the liver, pancreas, heart and skeletal muscle and metabolic syndrome. It explains the beneficial effect of the PPARγ agonists thiazolidinediones (pioglitazone, rosiglitazone) on insulin sensitivity in SAT but not in VAT ^{[6][7][8][9][10]}[58,59,60,61,62].

Excessive WAT hypertrophy is associated with progressive ischemia, endoplasmic reticulum or cellular stress and impaired mitochondrial oxidative capacity in adipocytes. This results in their death or adoption of a senescence-associated secretory phenotype (SASP). Endoplasmic reticulum stress in adipocytes is related to exposure to circulating oxidized low-density lipoprotein (oxLDL) and dyslipidaemia ^{[8][9][11][12][13]}[60,61,63,64,65]. Cellular senescence involves cell cycle arrest, inhibition of autophagy, avoidance of programmed cell death pathways and release of a variety of growth factors, proteases and cytokines via direct secretion or exosomes. This elicits an immune response via damage-associated molecular patterns (DAMPS), Toll-like receptors (TLR-4) and chemokines ^[14][66]. Dead, dying or dysfunctional adipocytes become surrounded by infiltrating macrophages, forming crown-like structures (CLSs) in WAT tissue. A dysfunctional WAT microenvironment is created, characterized by hypoxia, inflammation, oxidative stress and free radical damage of DNA with associated somatic mutations and epigenetic effects. In attempting to phagocytose these large, hypertrophied adipocytes, infiltrating macrophages release the stored triglycerides and free fatty acids (FFA), which can contribute to insulin resistance and hyperinsulinemia. The release of inflammatory cytokines and saturated free fatty acids (FFA) from VAT directly into the portal vein and thence to the liver is particularly important in impaired glucose tolerance and FFA lipotoxicity. This is related to accumulation of long chain FFA metabolites such as diacylglycerol and ceramide, which inhibit insulin-receptor substrate (IRS) interactions with PI3K and promote further insulin resistance ^{[2][6][7][8][9][10]}[54,58,59,60,61,62]. Cellular senescence is designed to prevent the proliferation of cells with a damaged genome; however, the failure of clearance of senescent adipocytes can lead to proliferation or secondary senescence of surrounding cells via paracrine and autocrine signalling. Crown-like structures (CLS) are more often found in VAT than SAT and are closely involved in adipocyte senescense and obesity-related breast carcinogenesis ^[15][16][17][67,68,69]. WAT senescence is commonly associated with ageing, but when diabetes or obesity are present it is independent of chronological age ^[11][63].

One of the main drivers of WAT senescence is increased reactive oxygen species (ROS) generation, oxidative stress and resultant telomere shortening and DNA damage. This activates the DNA damage response (DDR) and p53/p21 pathways, resulting in cell cycle arrest and a WAT SASP, including the secretion of tumour necrosis factor-α (TNF-α) and IL-6, and elevated β-galactosidase activity. DNA damage due to ROS can be reduced by antioxidants such as N-acetyl cysteine or by exercise, which can reduce WAT senescence ^{[14][15][16][17][18][19][20]}[66,67,68,69,70,71,72]. WAT cellular senescence (measured by SA-β-galactosidase activity, CDKN1a, and CDKN2a), adipocyte hypertrophy, adipokine dysregulation (increased leptin secretion, decreased adiponectin), and impaired glucose tolerance and insulin sensitivity began as early as 2 weeks after initiation of a high-fat diet and was attenuated by exercise in murine models of SAT and VAT senescence. Exercise also significantly reduced the high-fat diet-induced expression of profibrotic genes in VAT, including transforming growth factor β1 (TGF-β1), fibronectin (Fn1), and tissue inhibitor of metalloproteinase 1 (TIMP1), compared to sedentary animals fed an HFD ^[19][71]. Furthermore, high-intensity interval training (HIIT) or endurance exercise (END) when administered concurrently with a high-fat diet were able to improve lean mass as a proportion of body weight (Lean mass/BW) by 14%, improve insulin sensitivity by 22% and prevent the increase in body weight (END: 17%, HIIT: 20%) and total body fat mass (END: 46%, HIIT: 50%) compared to sedentary animals in a murine model ^[20][72].

White adipose tissue SASP can be reversed by metformin treatment, which rapidly blocks the release of inflammatory adipokines and inhibits the senescent transcriptional program, leading to adipose cellular quiescence ^[13][65]. Recent clinical research has suggested senolytic agents, such as a combination of the flavonoid quercetin and the Src family tyrosine kinase inhibitor dasatinib, promote apoptosis in senescent cells by interfering with pro-survival networks including ephrin dependence receptor signalling, PI3K–AKT and BCL-2 family members. A combination of oral quercetin and dasatanib in patients with obesity, diabetes and CKD resulted in significantly reduced numbers of SAT β-galactosidase positive senescent adipocytes (−62%), SAT CD68+ macrophages (−28%) and SAT CLS (−86%), as well as circulating plasma SASP factors (IL-1α, IL-2, IL-6, IL-9, MMP-2, MMP-9, MMP-12). Because of their prolonged effect on cellular senescence, short term, intermittent senolytics (‘hit and run’ dosing) can be used, rather than continual dosing. This also helps to prevent serious adverse side effects, particularly of dasatinib and navitoclax ^[14][66].

The dysfunctional adipocyte secretome is characterized by the abnormal release of adipokines, which causes detrimental effects on cellular metabolism and proliferation, energy homeostasis and insulin sensitivity. There is an increase in the release of pro-inflammatory adipokines including leptin, insulin-like growth factor-1 (IGF-1), angiopoietin-like-protein-4 (ANGPTL4), MCP-1, IL-8, IL-6, IL-1β, PAI-1, MIP-2 (CXCL2), TIMP and vascular endothelial growth factor (VEGF) in response to adipocyte cellular stress and hyperinsulinemia ^{[20][21][22][23][24][25]}[72,73,74,75,76,77]. These adipokines affect local WAT and distant organs by promoting further adipose hypertrophy, insulin resistance, dyslipidaemia and lipogenesis and inhibiting lipolysis, fatty acid metabolism, browning/beigeing of WAT and UCP-1 expression ^[22][74]. Other adipokines include platelet-derived growth factor-BB (PDGF-BB), granulocyte-colony stimulating factor (G-CSF), hepatocyte growth factor (HGF), resistin, autotaxin and lysophosphatidic acid (LPA) ^[12][23][64,75].

Leptin is primarily produced by SAT and is increased in individuals with higher total body fat ^[25][77]. Leptin is an anorexigenic cytokine due to its activation of pro-opiomelanocortin (POMC)-expressing anorexigenic neurons and suppression of the neuropeptide Y/agouti-related peptide (NPY/AgRP)-expressing orexigenic neurons in the arcuate nucleus of the hypothalamus. Thus, the normal effect of leptin is decreased food intake and increased energy expenditure. Under normal conditions, leptin and insulin both inhibit the effects of orexigenic stimulants (e.g., ghrelin) on the hypothalamic arcuate nucleus. Obese patients develop hypothalamic leptin resistance, reducing their sensitivity to normal satiety signals following a meal and contributing to further weight gain. Mean serum leptin levels are markedly elevated (31.3 ± 24.1 ng/mL) in obese patients compared to levels in normal-weight subjects (7.5 ± 9.3 ng/mL) ^[24][25][76,77]. A chronically elevated leptin level impairs post-prandial GLP-1 release and insulin sensitivity, contributing to hyperinsulinaemia in patients with normal fasting plasma glucose (pre-diabetes) and promoting the development of T2DM and NASH ^[26][27][79,80]. Leptin resistance can be reversed by resveratrol, metformin, GLP-1, GLP-1 analogues (semaglutide) and heat shock protein 90 (HSP-90) inhibitors ^[25][28][77,81].

Leptin is associated with the initiation and promotion of EMT in breast, gastric, lung, ovarian, endometrial and oesophageal adenocarcinoma, particularly in obesity ^{[29][30][31][32][33][34]}[82,83,84,85,86,87]. Many of the signalling pathways that converge on EMT are activated by leptin binding to its receptor Ob-Rb. EMT is a feature of (1) embryogenesis, (2) recovery from tissue trauma or inflammation or (3) carcinogenesis. These three processes each involve stem cell differentiation or de-differentiation of mature cells. EMT is a normal process of healing or fibrosis which is hijacked by cancer cells. Cells acquire immortality due to prolonged or severe cellular stress and inflammation, related to the acquisition of mesenchymal properties, resistance to anoikis, migratory capacity and invasion of the extracellular matrix, chemoresistance, immune evasion, stemness characteristics and metabolic reprogramming, all of which are involved in cancer progression and metastastic disease ^[35][26].

Leptin activation of Ob-R, STAT3 and induction of Yamanaka pluripotency transcription factors OCT4 and SOX2 are thought to be involved in obesity-related tumour growth. Leptin promotes the long stabilization of HIF-1α by inhibiting SIRT-1 and p53, and leptin transcription is reciprocally promoted by the hypoxia response element (HRE) ^[29][30][31][82,83,84]. Leptin helps to suppress the stable anchoring of mature cells and allows the development of an elongated mesenchymal phenotype by promotion of EMT transcription factors Snail, Slug, Zeb1 and Twist. This results in the repression of epithelial E-cadherin and occludin and the promotion of mesenchymal N-cadherin, fibronectin and vimentin. Leptin stimulates RhoA-ROCK pathways and mixed metalloproteases (MMP), and promotes invadopodia, actin cytoskeleton reorganization and focal adhesion formation which are involved in extracellular matrix (ECM) invasion and migration ^[29][32][82,85].

Leptin is closely related to obesity, features of T2DM and the progression of breast cancer. Leptin drives breast cancer cell proliferation while increasing GLUT-1 mRNA levels ^[33][86]. Other T2DM-associated molecular changes include elevated insulin, interferon gamma (INF-γ) and oxidative stress (ROS), which contribute to further GLUT-1-mediated progression of both ER-positive and triple-negative breast cancer (TNBC) ^[33][86]. Leptin has greater effects on ER+ breast cancer proliferation and EMT than in ER− breast cancer. This is thought to be due to increased expression of the membrane leptin receptor (ObRl/Rb) in ER+ breast cancer cells and its co-localization with the ER-α, together with leptin effects on breast cancer stromal aromatase activity ^[34][87].

Conversely, there is a decrease in the levels of anti-inflammatory cytokines such as adiponectin, omentin-1 and secreted frizzled-related protein 5 (SFRP5) in obesity ^[36][88]. Adiponectin levels are negatively correlated with increasing BMI, with mean plasma levels of adiponectin in non-obese individuals (8.9 mg/mL) more than double those of obese individuals (3.7 mg/mL, p < 0.0001) ^[24][37][76,89]. Adiponectin stimulates the adiponectin membrane receptors adipoR1 and adipoR2, which increases FFA oxidation in the liver and skeletal muscles. This helps to prevent hepatic steatosis ^[24][76]. Adiponectin has anti-inflammatory properties and regulates peripheral glucose metabolism by sensitizing adipose tissue to insulin. Lowered levels of adiponectin and omentin-1 in obesity and metabolic syndrome contribute to the development of insulin resistance. The remodelling of the extracellular matrix (ECM) by adipose tissue macrophages is associated with insulin resistance secondary to decreased adiponectin levels ^[36][88]. Adiponectin has antioxidant properties, which help to protect against the development of obesity, T2DM and atherogenic cardiovascular diseases ^[38][90]. Omentin-1 enhances the effect of insulin by increasing glucose uptake by VAT, activating insulin receptor substrate (IRS) proteins and inhibiting the mTOR signalling pathway. Omentin-1 also increases adiponectin gene expression and AMPK levels ^[36][88]. SFRP5 binds to Wnt proteins and blocks the action of Wnt5a, which inhibits adipose tissue-related inflammation and insulin resistance ^[36][88].

The abnormal production and secretion of inflammatory adipokines contribute to tumour growth and progression via crosstalk signalling between adipocytes, macrophages and epithelial cells ^[12][64]. Leptin is secreted by adipocytes, fibroblasts and cancer cells, with autocrine, paracrine and endocrine effects in cancers. Leptin activates the Janus kinase 2 (JAK2)/STAT3 (via SH2B1), mitogen-activated protein kinase (MAPK), Nuclear factor kappa B (NF-κB) and phosphoinositide 3-kinase (PI3K)/AKT/mammalian target of rapamycin (mTORC1) intracellular signalling pathways, which regulate EMT, cell proliferation, differentiation and cell apoptosis ^[20][38][72,90]. The aberrant activation of these pathways promotes tumour cell growth, proliferation and survival. Adiponectin has anti-tumour properties, inhibiting the JAK2/STAT3, MAPK, NF-κB, and PI3K/AKT/mTORC1 pathways while activating AMP-activated protein kinase (AMPK) and SIRT-1 ^[12][64]. Whilst adiponectin receptors adipoR1 and adipoR2 are expressed by both ER+ and ER− breast cancers, adiponectin appears to inhibit proliferation more effectively when it activates adiponectin receptors in ER− breast cancers ^[33][86]. A decrease in the circulating levels of adiponectin results in uncontrolled tumour cell proliferation and has been associated with multiple types of cancers ^{[36][38][39][40][41]}[88,90,91,92,93].

WAT-derived exosomes are secretory extracellular vesicles that have recently been implicated in obesity, atherosclerosis and carcinogenesis ^[41][93]. Plasma extracellular vesicles are made up of 80% microvesicles (100–1000 nm diameter), and 20% exosomes (20–100 nm diameter). Exosomes have a phospholipid bi-layer membrane similar to their parent cells, making them stable in plasma but also able to cross the blood–brain barrier or deliver their cargo via endocytosis to recipient cells in distant organs such as the liver and pancreas. Exosomes contain long non-coding RNA (lncRNA), functional mRNAs, miRNAs, small nuclear RNA (snRNA), transfer RNA (tRNA), DNA fragments, growth factors (TGF-1β, TNF-α, TRAIL), enzymes, lipids (25% cholesterol, 25% phosphatidylcholine, 10% sphingomyelin, 10% triglyceride, 6% ceramide), structural proteins (actin, cofilin, tubulin), endosomal sorting complexes required for transport (ESCRT) and secretory signal peptides. Exosome membranes contain tetraspanins (CD9, CD63, CD81), antigen-presenting molecules (major histocompatibility class (MHC) I and II), chaperones (HSP−70 and −90), adhesion molecules (ICAM-1, integrin-α and -β, CD44, P-selectin) and factor receptors/ligands (EGFR, TNFR, TfR, FasL) which are involved in recognition and endocytosis of exosomes by recipient cells, as well as some of their metabolic effects ^{[42][43][44][45][46]}[78,94,95,96,97].

WAT exosomes in lean patients are different to those in obese patients in both cargo and number, which is related to adipocyte hypertrophy and the WAT microenvironment in obesity. Lean patients who regularly exercise produce AMPK, which suppresses exosome release from WAT by inhibiting tumour susceptibility gene 101 (TSG101) and promoting Sirtuin 1 (SIRT-1). TSG101 interacts with ESCRT machinery and scavenger receptor class B (CD36) in facilitating endosomal sorting. SIRT-1 is an NAD+-dependent protein deacetylase which normally coordinates autophagy ^[12][64]. WAT cellular stress caused by a high-saturated-fat diet (palmitate), adipocyte hypertrophy, peroxides, inflammation, hypoxia and glycolysis ^[47][98] stimulates the production of WAT exosomes (10-fold increase in number), the trafficking and release of which are controlled by the Ras-associated binding proteins (Rab) and c-Src tyrosine kinases. This is particularly associated with the lipid laden, CD9+ ATM population which accumulates in CLSs ^[45][96]. The protein levels of matrix metalloproteinase-2 (MMP-2), caveolae-associated protein, TGF-β-induced protein ig-h3, thrombospondin-1, fatty acid binding protein-4 (FABP-4), Mimecan and ceruloplasmin are elevated, and septin-11 and leptin levels are reduced, in the VAT-derived exosomes from obese subjects compared to lean subjects ^[24][41][76,93]. Alterations in the contents of WAT-derived exosomes such as miRNA mediate obesity via multiple pathways. For instance, adipose tissue-derived exosomes from obese patients contribute to the development of insulin resistance through the stimulation of peripheral monocytes, resulting in the release of inflammatory cytokines such as TNF-α and IL-6 ^[43][94]. Exosomes derived from dysfunctional large adipocytes have a paracrine effect on smaller recipient adipocytes to promote further lipogenesis and adipocyte hypertrophy. This is mediated by the transfer of lipogenic enzymes (acetyl-CoA carboxylase, glucose-6-phosphate dehydrogenase, fatty acid synthase) and miRNA-33/miRNA-34a in exosomal cargo. Hypoxia increases the exosome levels of these lipogenic enzymes by 3–4 times compared to normoxic conditions. Exosomes derived from obese murine and human adipocytes contain elevated levels of fatty acids, which can be transported to cancer cells together with fatty acid oxidase enzyme (FAO). These are induced by exosomal crosstalk with cancer cells, which enables fatty acid oxidation to proceed, maintaining mitochondrial activity and enhancing proliferation in cancer cells ^[12][42][48][18,64,78].

During obesity, the miRNA profile of WAT-derived exosomes can be completely altered ^[12][64]. miRNA contained in exosomes released from dysfunctional adipocytes are involved in:

• Activation of TGF-β and Wnt/β-catenin pathways (miRNA-23b, miRNA-4429).
• Macrophage M1 polarisation (miRNA-155) and the suppression of M2 macrophage polarization (miRNA-34a).
• Hepatic steatosis, glucose intolerance, insulin resistance (miRNA-34a).
• Vascular remodelling and vascular smooth muscle proliferation (miRNA-221-3p).
• Hypothalamic POMC neuronal regulation of appetite, energy intake and weight gain (miRNA-181b, miRNA-144).
• Cancer growth/EMT (miRNA-23a) ^[21][42][44][73,78,95].

The persistent release of such exosomes could not only explain the increased incidence of cancers in obese patients with metabolic syndrome, but also their poorer outcomes (more advanced cancer, shorter disease-free survival and greater risk of recurrence of cancers) than metabolically normal patients with cancer ^[46][97]. Recidivism of obesity after weight loss may also be related to permanent alterations in the WAT secretome, including exosome release ^[22][74].