The skin of mammals serves as their body's first line of protection against external threats, making it one of the most important organs of the human body (36).

Both internal and external factors influence the epidermal ceramide expression profile. For example, downregulating CerS3 can reduce the proportion of unsaturated long-chain ceramides (37). Furthermore, AZGP1, an adipokine, has been shown to improve epidermal barrier function in Alzheimer's disease mice by increasing ceramide 3 (NP), ceramide 5 (AS) and ceramide 1 (EOS) (38).

The composition of ceramides in the stratum corneum is changing with age. Children have more ceramide 8 (NH), and less α-hydroxy and esterified ω-hydroxy ceramides than adults. There is less ceramide 1 in older people compared with that in younger people. Moreover, the degree of fatty acid chain saturation of ceramide 1 is associated with the season, being reduced in autumn and winter, which participates in lamellar and lateral lipid organization. Deficiency of this type of ceramide may be associated with seasonal skin dryness (39). Exposure to ultraviolet irradiation can increase plasma ceramide levels and decrease SM levels in mice (40). Therefore, the level of epidermal ceramides is maintained in dynamic equilibrium by control from a number of factors and plays an important role in skin homeostasis (41).

Although ceramides account for the highest proportion of skin lipids, they are no more important than other lipids. Previous studies found that perturbation of the skin with ceramides alone or mixtures of ceramides and FFAs delayed skin barrier recovery, and normal recovery was only possible with equimolar mixtures of all key lipids, as determined by transepidermal water loss (42,43). These findings demonstrate that FFAs, cholesterol and ceramides are hydrophilic extracellular lipid matrices that are indispensable for skin permeability (44).

Despite the lack of direct evidence, ceramides have been hypothesized to regulate keratinocyte proliferation and differentiation in the skin. Both in vitro and in vivo, keratinocyte differentiation is reported to be accompanied by an increase in ceramide production (45).

Ceramides and their derivatives play a crucial role in controlling inflammation and the immune system. The activation of dendritic cells (DCs) with proinflammatory agents like lipopolysaccharide, TNF or interleukin 1 (IL1A and IL1B) has been shown to increase the intracellular concentrations of ceramides by promoting the stability of Toll-like receptor 4 (TLR4) (46). Changes in the transcriptional levels of ceramide synthetase and ceramidase in the lipid skeleton of TLR9-stimulated wild-type DCs were found to regulate the ceramide ratio (47). Ceramides are intracellular modulators of the NLR family, pyrin domain containing 3 (NLRP3) inflammasome assembly, and studies in microglia have shown that they can promote the release of IL1B (48). A single study has found that in mouse models, the presence of Staphylococcus epidermidis increases the content of ceramides in the epidermis by secreting SMase, which interferes with the growth of other pathogenic microorganisms and affects the immune pathway (49). Ceramides are therefore considered to be bioactive signaling molecules active in a number of inflammatory signaling pathways.

The etiology of DFUs involves peripheral artery occlusive disease, neuropathy and trauma with subsequent infection (54).

DN included motor, peripheral and autonomic neuropathies (55). The manner in which diabetes causes DN is not clear. However, after the occurrence of DN, the muscles will atrophy, resulting in changes in the anatomical structure of the foot, and even the formation of deformities. Due to the abnormal mechanical action, a foot hematoma occurs, which is mainly caused by motor neuropathy. Autonomic neuropathy causes blood vessels to dilate and the temperature of the foot to rise, drying out the skin and making it more vulnerable to damage. A lesion of the peripheral nerve makes the situation worse by depriving the patient of the protective sensation in the foot (56,57). Abnormal glucose metabolism occurs through the polyol and hexosamine pathways, leading to reactive oxygen species (ROS) production and inflammation through mitochondrial damage. In addition, abnormal glucose metabolism, resulting in glycosylation of proteins, the production of advanced glycation end products (AGEs), interaction with age-specific receptors and their own accumulation, can also lead to ROS production and the release of inflammatory factors. Similarly, excess free fat can also generate ROS through β-oxidative catabolism, causing local inflammation. The increase in ROS initiates the oxidative stress mechanism and causes tissue damage (58). Sphingolipid metabolism has been found to be associated with DN (59). The formation of abnormal deoxysphingolipids is harmful to pancreatic β-cells and nerve cells. Elevated levels of deoxysphingolipids have been found in patients with type 1 DM (T1DM) and T2DM with inherited sensory and autonomic neuropathy (60). Furthermore, these studies highlight the potential of plasma ceramide and deoxyceramide species as diagnostic and prognostic biomarkers for DN.

DM is now clearly associated with the development of cardiovascular disease (61). The vascular disease associated with DFUs is peripheral arterial disease of the lower extremity, which refers to an atherosclerotic occlusion from the main iliac artery to the foot artery (62). Hyperglycemia-induced epithelial dysfunction reduces pro-angiogenic signaling and nitric oxide (NO) production. NO prevents smooth muscle relaxation and leads to foot hypoperfusion, which can lead to an impaired skin barrier (63). Patients with diabetes are prone to atherosclerosis, resulting in poor blood perfusion in the foot and neuropathy making patients feel numb, coupled with repeated trauma and unreasonable pressure in daily life, so that the healing of the foot wound is delayed. This is followed by an infection of the foot, which can lead to gangrene and even amputation (64).

In addition, foot deformities, trauma and secondary infections accompanied by weakened resistance further aggravate DFUs, forming a vicious cycle (65).

Regulation of vascular tone requires the production of vasoactive substances, such as NO, by endothelial cells (66). According to the current evidence, ceramides have a detrimental effect on endothelial function. Endothelial dysfunction is caused by decreased NO synthesis or increased NO breakdown due to the generation of ROS (67). Ceramides can activate NADPH oxidase, increasing ROS, leading to increased oxidative stress, which degrades ceramidase (68). Ceramides have been shown to inhibit the de novo pathway of ceramides in diet-induced obese C57Bl/6 mice, which produces normalization of endothelial dysfunction and systemic hypertension (69). However, endothelium-dependent vasodilation is impaired when the endothelium is briefly exposed to exogenous ceramides. This finding indicates that ceramides are crucial in the impairment of the endothelium caused by inflammation or obesity. Exogenous C16 ceramide-mediated endothelial NO synthase (NOS) uncoupling and dysregulated protein phosphatase 2 (PP2A) were found to damage human aortic endothelial cells in a study of atherosclerotic patients (70). Another study in patients with coronary arteries also found that ceramides were associated with coronary endothelial dysfunction (71). However, more mechanistic studies are needed to confirm the findings. Karakashian et al (72) demonstrated that nSMase activity persistently increases with aging, resulting in greater ceramide and endothelial NOS (eNOS) inactivation, as well as a reduction in NO production. In addition, nsMase-derived ceramides participate in the conversion of NO to H₂O₂, which leads to the reduction of NO and the formation of coronary artery disease (73). Ceramides have been shown to decrease eNOS3 (NOS3; also known as eNOS) activity, either under baseline conditions or after stimulation (74). On the other hand, in high-fat diet mice, inhibiting the synthesis of ceramides by the de novo pathway may indirectly improve endothelial dysfunction (75). Moreover, a research study has demonstrated that ceramides can activate NADPH oxidase (76), but other studies contend that ceramides can directly influence the mitochondrial electron transport chain to increase ROS in various cell types, including endothelial cells (77,78). Following a harmful self-amplifying cascade of events, the resultant O₂ can ultimately cause the decoupling of NOS3 and the generation of ROS in endothelial cells. Last but not least, ceramides have the ability to activate the NLPR3 inflammasomes and release inflammatory cytokines, thereby contributing to atherosclerotic lesions and vascular dysfunction (48).

Ceramides have been found to be connected with vasoactivity. Ceramides can not only contract the blood vessels, but also vasodilate them (74). However, the mechanisms by which ceramides induce vasoactivity remain unclear.

A single study has shown that ceramide-induced vasodilation is partially endothelium-dependent via activation of NOS3 and subsequent NO generation, as aforementioned, by impairing endothelium function (79). Angiotensin II (ANGII) may indirectly increase ceramide production, thereby activating SMases in the peripheral vasculature to control vascular function (80).

However, a considerable amount of research has found that ceramides can promote vasoconstriction (74,81,82). Total ceramide levels in the aortic tissue of hypertensive rats and humans were much higher than those in normotensive mice and humans, with the main changes being increases in the levels of ceramides C24:1 and C24:0 (83). However, a correlation between increased blood pressure and increased ceramides in plasma does not indicate a causal relationship (84). Notably, it has been suggested that nSMase-derived ceramides promote the vasoconstriction caused by changes in the levels of thromboxane A2, ANG II and oxygen tension (85,86).

In short, the vascular effects of ceramides appear to be complicated and remain unclear. This vasoactivity may be influenced by a variety of variables. Numerous exogenous and endogenous processes can produce ceramides, and can also transform them into other active molecules. The different vascular effects may result from the different types of vessels and their various diameters, as well as the cell types they act on. Notably, different lengths of ceramides have different pathological functions (87).

TNF, IL1B and interferon γ are examples of inflammatory agents producing cytotoxicity to pancreatic β-cells by increasing the levels of sphingolipids, particularly ceramides, which are either exogenously delivered or produced endogenously (91). By contrast, several studies have shown that reducing ceramide production, such as inhibition of SPT or inhibition of CerS inhibitors, reduces cytotoxicity to rodent (92) and human (93) β-cells. In fact, there is evidence of a very small increase in ceramides following FFA therapy in trials involving cell apoptosis (94). However, some studies have shown that inhibiting ceramide synthesis reduces β-cell apoptosis (95-98). There may be three reasons to explain this result. First, the increased ceramide level induces apoptosis only at the designated site, namely the mitochondria, without changing the total ceramide mass (93). Second, upon triggering apoptosis, ceramides are changed into another sphingolipid metabolite (glucosylceramide) (99). Third, distinct ceramide isoforms may have different apoptotic potentials. In order to promote apoptosis, hyperglycemia increases the levels of the harmful isoforms of ceramides, namely C22:0, C24:1 and C18:0 (100).

There is increasing evidence that ceramides play an important role in insulin resistance. Insulin resistance is a pathophysiological state characterized by hyperinsulinemia, high blood glucose levels and decreased responsiveness of peripheral tissues to insulin. Numerous studies have shown that excessive consumption of FFAs, the use of glucocorticoids, corpulence and decreased physical activity are some of the major contributors to insulin resistance, in which ceramides serve as a key intermediary (101). Increased ceramide accumulation from palmitate exposure occurs in several cells, including muscle cells (102), adipocytes (103) and cardiomyocytes (104). Simultaneous Akt inhibition results in decreased insulin sensitivity (105). Sphingolipid recycling or the salvage pathway can cause a buildup of ceramides in response to an excessive supply of saturated or unsaturated FAs. Ceramides are an obligatory intermediate in saturated FA-induced insulin resistance (101). Zalewska et al (106) showed that mice administered a high-fat diet expressed higher levels of CerS than mice fed a regular diet. However, Holland et al (107) found that infusion of both highly saturated and highly unsaturated FAs reduced glucose uptake and Akt activation in rats, but infusion of highly saturated FAs alone increased ceramide levels. Both saturated fats and unsaturated fats can promote insulin resistance through different mechanisms, but ceramides only participate in saturated fat-induced insulin resistance (107). However, unsaturated FAs were not associated with increased levels of ceramides, which are not the only lipids responsible for insulin resistance. In line with this, overexpression of acid ceramidase can lower ceramide levels, prevent the buildup of ceramides caused by palmitate and enhance insulin signaling (108).

Glucocorticoids can increase the levels of ceramides, which may be the mechanism for inducing insulin resistance (107,109). By activating enzymes such as SPT, SMase and CerS, the commonly used glucocorticoid dexamethasone increases ceramide levels in a number of cell types and animal species, such as 3T3-L1 cells, wild-type mice and Sprague-Dawley rats (107,110,111). Pretreating C57Bl6/J mice with myriocin, a potent inhibitor of SPT, was shown to avoid some of these effects (112). However, the study involved a low dose of myriocin, so the alterations cannot be ruled out as a result of subsequent changes in the gut microbiota. Peroxisome proliferator-activated receptor α (PPARA) is activated by ceramide accumulation, and thus genetic disruption of PPARA, or other damage causing decreased hepatic PPARA expression, has been reported to inhibit insulin resistance in some conditions (113).

Obesity is known to contribute to T2DM by influencing glucose homeostasis and insulin sensitivity (114). However, the levels of ceramides in the skeletal muscles of rats, mice and diabetic patients have been reduced with long-term aerobic training (115,116). In other studies, for some unknown reason, even after exercise training in rats and people, the level of muscle ceramides does not significantly decrease (115,117,118).

Due to its preservation of the integrity of the internal environment, apoptosis is described as genetically programmed cell death. The mechanism of apoptosis is complex. There are three known apoptotic signaling mechanisms: The intrinsic mitochondrial, intrinsic ER and extrinsic death receptor pathways. A number of studies have shown that ceramides are associated with the induction of β-cell apoptosis (124). Zhang et al (125) found that amyloid peptides induce β-cell apoptosis, partly through the production of ceramides by activating aSMase through activation of K⁺ channels on the cell membrane, which is a marker of cell apoptosis.

Ceramides have also been shown to cause apoptosis through their effect on the mitochondria, which controls regional levels of certain lipids like sphingolipids to detect cellular stress (126). BAX and ceramides work together to synergistically induce mitochondrial outer membrane permeabilization (MOMP). According to Ganesan et al (127), when ceramides are present, they are the key molecules in mitochondrial permeabilization, suggesting that the inhibition of ceramides without activating BAX-induced mitochondrial permeabilization, blocks the induction of MOMP, resulting in the death of yeast cells. According to the study by James et al (128), ceramides may be suggested in a new pathway leading to cell apoptosis by inducing the generation of Creola bodies, a marker of apoptotic lung epithelial cells. However, this was found in mouse lung tissues, and the mechanisms in other tissues require more investigation. Meanwhile, ROS also increased as a result of the increased ceramide level, which can contribute to apoptosis. Studies have found that ceramide-mediated insulin resistance-induced apoptosis was associated with the DNA-damage response and mitogen-activated protein kinase pathways, and that ceramides could mediate mitochondrial dysfunction (129,130). ROS are primarily produced by the mitochondria, and impairment of the mitochondria is generally considered to be associated with increased ROS. Apoptosis-inducing factors are generated when ceramides are being synthesized. Ceramides may inhibit the activation of mitochondrial NADPH oxidase, thus blocking electron transport at complex I and complex III of the respiratory chain, and inducing apoptosis by increasing ROS production (131,132).

Ceramides are a mediator of palmitate-induced cell toxicity, according to certain studies (133-135). One hypothesis that has been postulated is that ceramides can induce β-cell death by activating protein kinase C δ-type (PKCD), which is necessary for apoptosis in numerous cell types (136).

Numerous studies have indicated that ER stress is crucial for cell apoptosis (137-139). During diabetes, insulin is produced in large amounts to counteract high blood sugar. In this situation, there is an increased burden on the ER, which serves as the location for the synthesis and folding of released proteins. The unfolded protein response (UPR), which aids in re-establishing normal ER function, is activated by ER stress (140). In addition, miR-204 has been found to activate the apoptotic signaling pathway in β-cells (141).

Another possible way to induce β-cell apoptosis is to inhibit the function of Akt, which is a serine/threonine kinase. First, it was shown that Akt could increase cellular proliferation, but that its inhibition could induce apoptosis (142). Second, Akt upregulates CDKN1B to trigger apoptosis while adversely regulating the transcriptional activity of FOXO1 (143). Third, Akt promotes mTOR/p70S6K signaling and directly phosphorylates and inactivates BCL2 members, including BAD, BAX and BID, to cause apoptosis (144). In addition, AKT induce apoptosis by activating cell signaling such as that of c-Jun N-terminal kinase (JNK) and extracellular signal-regulated kinase (98).

The phosphorylation of insulin receptor substrate-1 (IRS1), a mediator protein that links the binding of insulin and insulin growth factor 1 to related intracellular receptors of the insulin pathways, can activate ceramides to lead to impaired islet signaling (145). The increased IRS1 level induces serine 307 phosphorylation to inhibit insulin signaling (146). Ceramides can also suppress IRS1 expression by triggering the PKR/JNK/Prep1/p160 axis and/or the c-Jun amino-terminal kinase/PBX regulatory protein 1 axis (89). In addition, glucosylceramides have been demonstrated to impair insulin signaling by inhibiting insulin receptors (101,147).

As aforementioned, ceramides can inhibit AKT to cause β-cell apoptosis, and other studies have shown that they can also mediate the secretion of insulin (98,148,149). Ceramides first catalyze the dephosphorylation of AKT by activating PP2A (150). Ceramide secondly prevent the translocation of AKT to the PIP3-PDK1 complex in the plasma membrane. Ceramides can activate PKCZ (151), and then ceramide-induced phosphorylation inhibits this at the serine 473 or threonine 308 residue (24,102). This can form a stable AKT-PKCZ complex to prevent its interactions with PIP3. In further studies, PKC inhibitors were shown to improve insulin sensitivity and block the ceramide-induced loss of AKT activity (152-154). Ceramides were also elevated in other animal models such as Zucker diabetic fatty (ZDF) rats (155) and ob/ob mice (156), as well as in obese humans (157). If ceramides cause insulin resistance by inhibiting AKT, the proximal insulin signaling should be intact; however, there are also abnormalities in this proximal insulin signaling. In addition, the presence of the HIF-2α-neuraminidase 3-ceramide pathway was also observed in mice administered a high-fat diet, and insulin sensitivity was improved in mice ablated with enteric-specific HIF-2α. However, this only occurred in the absence of oxygen (158).

Exosome secretion and/or biogenesis may be impacted by ceramides. The protein adiponectin, which is released by adipocytes, stimulates the synthesis of exosomes in skeletal muscle and endothelial cells, and induces the expression of cadherin 13 (159). Given that research indicates that the microRNA profiles of exosomes were changed in patients with T2DM, Santovito et al (160) reported that exosomes, a metabolic mediator, produced by adipose tissue macrophages, impact insulin sensitivity in mice. Lean mice developed glucose intolerance and insulin resistance after exposure to exosomes from obese mice. By contrast, exosomes from adipose tissue macrophages in lean mice reduced insulin resistance and glucose intolerance in obese animals (161). Exosomes may also convert monocytes into macrophages, lead to inflammation and disrupt insulin signaling to cause T2DM (162).