Under conditions of intermittent fasting (IF), caloric restriction (CR), KD and exercise, FFA undergoes extensive β-oxidation in the liver, yielding a significant quantity of ACAC. A portion of this ACAC is converted into β-HB (53). β-HB possesses physicochemical properties such as a small molecular weight and good water solubility (54); it can not only be efficiently transported through the blood circulation but also penetrates the blood-brain barrier (BBB) and the capillary walls of muscle tissue, easily reaching extrahepatic tissues where it is oxidized and decomposed as an energy substrate (Fig. 2). Once there, β-HB can be metabolized and utilized, making it a crucial energy source for the brain, heart, kidneys and skeletal muscle, among other tissues, in times of metabolic stress (55). Consequently, β-HB assumes a pivotal role as a compensatory energy fuel during these periods of increased energy demand (56-58). Furthermore, the contribution of β-HB to energy metabolism is notably amplified during the perinatal lactation period and the neonatal phase (59).

β-HB serves as a mediator for metabolic signaling that influences numerous cellular processes (Fig. 2). β-HB is a ligand for the G protein-coupled receptors (GPCRs), GPR109A (also known as HM74A in humans and PUMA-G in mice) and GPR41. GPR109A is found in adipocytes, retinal tissue and macrophages. Within physiologically relevant concentrations (Ki=0.7 mM), β-HB selectively stimulates GPR109A, leading to the activation or inhibition of various signaling pathways linked to lipid metabolism and cellular growth (60). Moreover, GPR41, also known as free fatty acid receptor 3, is present in sympathetic ganglia; it can inhibit sympathetic activity in mice through the G protein β-γ complex/phospholipase C β/MAPK signaling pathway, thereby suppressing the overall metabolic rate (61,62). Besides GPCRs, β-HB engages directly with ribonucleoproteins; it plays a role in histone acetylation (63), histone lysine β-hydroxybutyrylation (Kbhb) (64) and indirectly promotes protein hyperacetylation (65). Furthermore, β-HB exerts direct regulatory influences on K+ channels and neuronal vesicular glutamate transporters (66) and it inhibits inflammation mediated by the NOD-like receptor family pyrin domain-containing 3 (NLRP3) (67).

The liver is essential for regulating glucose and lipid metabolism and it is a key site for the development of IR. Chronic increase in plasma-free FFA leads to metabolic imbalance in the body and induces IR, which promotes FFA delivery to the liver and hepatic fat deposition. Increased hepatic fat accumulation results in heightened lipotoxicity, adipose tissue inflammation, impaired mitochondrial function and ERS. Excessive deposition of hepatic extracellular matrix forms hepatic fibrosis, which ultimately leads to cirrhosis; T2DM hepatic fibrosis is one of its pathological manifestations (86). It is noteworthy that the abnormal activation of the hepatic gluconeogenesis pathway is a crucial mechanism leading to the hyperglycemic state. This process is regulated by key gluconeogenic enzymes and the increased activity of these enzymes significantly promotes gluconeogenesis, thereby exacerbating hyperglycemia (87).

A persistent, low-grade inflammatory state is recognized as a pivotal element of IR and metabolic disorders. Zhou et al (23) demonstrated that β-HB can inhibit lipid synthesis in the hepatocytes of KKAy mice (T2DM mouse model), thereby reducing hepatic lipid accumulation and decreasing the expression of hepatic inflammatory factors. The mechanism may involve β-HB impairing the binding ability of the transcription factor, C/EBP homologous protein, to the promoters of ribosome biogenesis factor 1 (PES1), thereby leading to downregulated expression of the PES1 protein. Ultimately, this improves liver pathology through two key effects: i) It inhibits the binding of PES1 to the promoters of E1A-binding protein p300 (p300) and cysteine-aspartic acid protease 1; and ii) it reduces p300-mediated acetylation of sterol regulatory element-binding protein 1c (SREBP1c) and the associated inflammatory response pathways. SREBP1c is a core transcription factor for lipid synthesis, and the inhibition of its activity can directly reduce hepatic triglyceride synthesis. Furthermore, the authors observed that KD increased the levels of circulating β-HB, suppressed hepatic PES1 expression and improved hepatic lipid regulation, the inflammatory response, blood glucose levels and IR in KKAy mice (23).

The accumulation of reactive oxygen species (ROS) caused by excessive hepatic lipid deposition is a crucial driving factor for hepatic fibrosis in T2DM (88). Xu et al (89) discovered that upregulation of BDH1 reduces fibrosis, inflammation and apoptosis in the livers of db/db mice, processes that are mediated by ROS. The underlying mechanism may be that β-HB metabolism mediated by BDH1 upregulates the production of fumarate. In turn, fumarate can inhibit the activity of Kelch-like ECH-associated protein 1 (Keap1), thereby activating nuclear factor erythroid 2-related factor 2 (Nrf2). Nrf2 is a core transcription factor for resisting oxidative stress; its activation can induce the expression of antioxidant enzymes, which in turn scavenge excessive ROS (89). BDH1 promotes the interconversion of β-HB with ACAC (37). Therefore, BDH1-mediated hepatic β-HB metabolism opens a new avenue for the treatment of db/db mice.

The kidneys are one of the most susceptible target organs in T2DM. Diabetic kidney disease (DKD) caused by T2DM has become the leading cause of end-stage renal disease and ~40% of T2DM patients will progress to DKD (90). Persistent hyperglycemia can induce functional abnormalities in the glomerular feedback system and mediate cellular damage via glucotoxicity, thereby triggering a state of glomerular hyperfiltration. This early pathological change can further activate a series of cascade reactions, including metabolic disorders, hemodynamic abnormalities, ERS, inflammatory responses and fibrotic processes, leading DKD to progress from early functional impairment to irreversible organic lesions (91). Patients with DKD frequently exhibit early signs of hyperfiltration and albuminuria, as well as glomerular and tubular lesions (92).

SMCT1, as a high-affinity transporter for monocarboxylates, is a key molecule for β-HB uptake in tissues (93). Under the state of hyperinsulinemia in T2DM, the protective effect of β-HB on renal tubular epithelial cells may depend on the 'β-HB uptake and utilization' mediated by SMCT1. The authors (24) observed a decrease in renal SMCT1 expression in patients with DKD and in mice with T2DM. Following overexpression of SMCT1, the levels of β-HB in the serum and kidneys of T2DM mice increased, while the urinary β-HB levels decreased and renal energy metabolism improved. By contrast, a lack of the SLc5α8 gene responsible for encoding SMCT1 led to structural damage and functional impairment of the renal tubules in T2DM. However dietary supplementation with 1,3-butanediol (1,3-BDO), a precursor of β-HB, can ameliorate renal injury in SMCT1-knockout mice (24). Mechanistic studies have shown that hyperinsulinemia inhibits SMCT1, impairs the uptake of β-HB and thereby compromises mitochondrial function and cell survival in renal tubular epithelial cells (94). Therefore, the transport of β-HB into renal tissues mediated by SMCT1 is a necessary process for maintaining the mitochondrial function of renal tubules. In addition, renal β-HB metabolism mediated by BDH1 may also provide a new therapeutic approach for DKD. Both KD intervention and β-HB intervention can increase the serum β-HB levels and upregulate the renal expression of BDH1, thereby ameliorating DKD (95). Mechanistically, β-HB metabolism mediated by BDH1 upregulates the production of fumarate, which in turn activates Nrf2 to inhibit oxidative stress and alleviate renal tubular injury (95). The protective effect of β-HB on the kidney is not limited to renal tubules; it also exerts a protective effect on glomerular cells. Supplementation with β-HB can reduce albuminuria and alleviate renal hypertrophy in mice with DKD (96). In vitro experiments have confirmed that β-HB can protect glomerular podocytes from injury and senescence under stimulation by high glucose combined with transforming growth factor-β (TGF-β). The underlying mechanism may be as follows: β-HB inhibits the activity of glycogen synthase kinase 3β and the phosphorylation of Nrf2, reduces the nuclear export of Nrf2 and increases its nuclear accumulation, thereby enhancing the antioxidant response and delaying podocyte senescence (96). In summary, β-HB protects renal tubules and glomeruli in DKD from oxidative stress-induced injury by inhibiting the nuclear translocation of Nrf2.

It has been indicated that ~80% of individuals with T2DM exhibit signs of overweight or obesity (97). When adipose tissue exceeds its normal storage capacity, it accumulates ectopically (such as in the heart, skeletal muscle, liver and pancreas), leading to increased visceral lipid deposition and the onset of FFA-induced toxicity, commonly referred to as lipotoxicity (98). Lipotoxicity denotes the harmful effects of lipid byproducts on tissues that are not composed of fat, such as the liver, skeletal muscle, heart, kidneys and pancreatic β-cells (99), which are pivotal in the pathogenesis of T2DM IR (100). Furthermore, under healthy conditions, adipokines (such as adiponectin) secreted by adipose tissue can enhance insulin sensitivity. By contrast, the 'endocrine dysfunction' of adipose tissue under pathological states exhibits pathogenicity (101). The increase in adipose tissue mass leads to low-grade inflammation by altering the secretion of adipokines and cytokines, and the production of these adipocytokines is negatively correlated with IR in T2DM (102).

Zhang et al (22) employed a db/db mouse model alongside a T2DM mouse model induced by a high-fat diet (HFD) and treated with streptozocin to investigate how administering a 10% aqueous solution of 1,3-BDO affected these mice. It was discovered that β-HB binds to hydroxycarboxylic acid receptor 2 (HCAR2) and triggers an increase in intracellular Ca²⁺ levels within adipocytes. This process triggers adenylate cyclase, leading to an increase in cyclic adenosine monophosphate (cAMP) levels, which in turn activates protein kinase A (PKA). When activated, PKA restrains the activity of Raf-1 proto-oncogene serine/threonine-protein kinase (Raf1), causing a decrease in the activity of extracellular regulated protein kinases 1/2 (ERK1/2). This reduction ultimately inhibits the phosphorylation of peroxisome proliferator-activated receptor γ (PPARγ) at Ser273 in adipocytes. Such changes in the expression levels of genes regulated by PPARγ contribute to a decrease in IR (22). As a result, β-HB regulates the activity of ERK1/2 through the HCAR2/Ca⁺/cAMP/PKA/Raf1 pathway, ultimately enhancing PPARγ function through post-translational changes and decreasing IR linked to T2DM.

Insufficient secretion of adiponectin is a core feature of adipose tissue dysfunction in T2DM. Nishitani et al (25) found that in KKAy mice, the serum level of β-HB was increased, while in periovarian adipose tissue (visceral adipose tissue), the expression of adiponectin was decreased, the inflammatory response was enhanced, the metabolism of the insulin signaling pathway was weakened, the expression levels of MCT1 and MCT2 were downregulated and the expression of OXCT1 (the enzyme responsible for KB catabolism) was reduced. These findings suggest that abnormal β-HB metabolism is closely associated with adipose tissue dysfunction. In in vitro intervention experiments on 3T3-L1 adipocytes, β-HB can significantly upregulate the mRNA and protein expression levels of adiponectin, while inhibiting inflammatory responses (25). Further mechanistic studies have shown that the regulatory effect of β-HB depends on a novel epigenetic modification, Kbhb. Specifically, β-HB can induce an increase in the Kbhb level of the histone H3 lysine 9 (H3K9) site within the adiponectin gene promoter region in 3T3-L1 adipocytes. This modification serves to open the chromatin structure of the adiponectin gene, facilitate the binding of transcription factors, and thereby activating the expression of the adiponectin gene. Lipocalin expression has also been found to be regulated by two epigenetic modifications (103,104) and promoter activity (105,106), and lipocalin gene expression has been reported to be epigenetically associated with 3T3-L1 adipocytes (104). The research findings of Nishitani et al (25) revealed that β-HB induces an increase in Kbhb at the H3K9 site in 3T3-L1 adipocytes. Therefore, β-HB seems to offer defense in KKAy mice by inducing epigenetic changes in the lipocalin gene within adipocytes. β-HB can directly regulate adiponectin gene expression through epigenetic modifications and this mechanism provides a new perspective for adipose tissue protection.

The positive effects of KD on managing energy metabolism and maintaining glucose homeostasis are still a topic of contention. A notable detail is that KD and the intraperitoneal administration of β-HB produce distinct effects on lipid metabolism. Park et al (107) observed that KD led to an increase in epididymal fat pads and serum leptin levels, while leptin-related signal transducer and activator of transcription 3 (STAT3) signaling was impaired, leading to visceral fat accumulation in diabetic rats with 90% pancreatectomy (Px). By contrast, intraperitoneal administration of β-HB led to decreased testicular fat pads and serum leptin levels, partially restoring hepatic insulin receptor expression and signaling without affecting STAT3 signaling. This discrepancy may be attributed to the fact that a KD inhibits hypothalamic STAT3 phosphorylation, thereby inducing leptin resistance, whereas β-HB injection exerts no effect on STAT3 signal transduction. Additionally, the high-fat content in a KD leads to an increase in FFAs, which promotes fat accumulation. Apart from β-HB, a KD contains substantial amounts of saturated fats and trans fats; these components themselves can disrupt lipid metabolism (such as increasing FFA levels and inducing inflammation) (108). These findings suggest that β-HB exerts a protective effect on lipid metabolism, while a KD is not suitable for lipid metabolism management in non-obese patients with T2DM. Additionally, research has shown that the supraphysiological levels of β-HB (ranging from 15 to 50 mM) triggers the occurrence of lipofuscinosis (109), while it does not promote lipo-browning at physiological concentrations (110).

T2DM is strongly correlated with the development of cardiovascular disease (111). The metabolic environment characterized by hyperglycemia and hyperlipidemia in patients with T2DM increases the risk of developing heart disease (112). Diabetic cardiomyopathy (DCM) refers to heart diseases complicated by or associated with DM, including coronary atherosclerotic heart disease, DCM itself and arrhythmias and cardiac dysfunction caused by autonomic nerve disorders (113). The main pathological features of DCM include cardiomyocyte hypertrophy, myocardial fibrosis and impaired coronary microvascular perfusion. The pathogenesis involves oxidative stress, inflammation and impaired Ca²⁺ handling as well as alterations in substrate metabolism/utilization, insulin signaling, gene regulation, mitochondrial dysfunction, ERS, neurohumoral activation and cardiomyocyte death (114). However, there is currently a lack of effective therapeutic approaches for DCM (115).

β-HB, as a key metabolite of a KD, downregulates the expression of vascular PES1, thereby inhibiting PES1-mediated ubiquitin-dependent degradation of vascular endothelial cadherin (VE-cadherin). Consequently, β-HB upregulates barrier-protective proteins such as VE-cadherin and downregulates pro-leakage proteins such as vascular endothelial growth factor (VEGF), ultimately improving vascular hyperpermeability in T2DM mice; it also assists in reducing fasting blood glucose (116). This finding provides a novel 'PES1/β-HB-targeted' direction for the treatment of vascular complications in T2DM. Ketone esters (KEs) increase the circulating level of β-HB and upregulate the expression of myocardial BDH1, ultimately improving cardiac function in mice with T2DM. The underlying mechanism may be associated with β-HB regulating the expression of mitofusin 2, promoting Parkin-mediated mitophagy and enhancing mitochondrial biogenesis. These processes collectively optimize mitochondrial quality control and reduce the level of oxidative stress (117). Another study also indirectly demonstrated the myocardial antioxidant stress effect of β-HB. Lin et al (118) observed that the expression of BDH1 was decreased in the aorta of T2DM model mice, and systemic overexpression of BDH1 reduced the area of atherosclerotic plaques in T2DM. In their in vitro studies, BDH1 was found to mitigate oxidative stress and inflammatory reactions in Raw264.7 cells (mouse macrophage cell line) by enhancing ferredoxin metabolic flux and stimulating the Nrf2 signaling pathway. The study by Uchihashi et al (119) further supports their conclusion. This study showed that heart-specific overexpression of BDH1 can also improve oxidative stress and cardiac remodeling in heart failure induced by pressure overload. In addition, the reduction in β-HB concentration can be considered a marker of overall FFA oxidation (120). Liepinsh et al (121) found that the plasma β-HB level in Goto-Kakizaki (GK) rats was significantly decreased, while after treatment with light phosphate at a dose of 200 mg/kg for 4 and 8 weeks, the β-HB concentration was further reduced. Therefore, the authors concluded that the cardioprotective effect of light phosphate treatment in GK rats could be explained by partial inhibition of FFA oxidation and increased glucose metabolism.

Individuals with T2DM exhibit an accelerated rate of brain aging at ~26% faster than those without diabetes (122). The effect of diabetes on the central nervous system has garnered considerable attention in recent years. 'Diabetic encephalopathy (DE)' was raised by Reske-Nielsen et al (123) to describe a central nervous system complication associated with diabetes, characterized by cognitive and behavioral deficits. Clinically, DE presents as cognitive dysfunction, decision-making disorders and mood disorders, and pathologically as structural and functional alterations in intracranial tissues. The pathological hallmarks of DE include gray matter, white matter, hippocampal atrophy, compromised synaptic plasticity, glial cell dysfunction and alterations in the structure and function of cerebral blood vessels. The pathological mechanisms of DE encompass an imbalance in pancreatic amyloid polypeptide homeostasis, microRNAs, macrophage autophagy, Lipin1, advanced glycation end products (AGEs), oxidative stress, hyperphosphorylation of Tau proteins and intestinal homeostasis dysregulation (124,125).

β-HB is produced in the liver and it can traverse the BBB to provide energy to the brain when glucose levels are low (126). Andersen et al (127) found that cerebral glucose metabolism was reduced in db/db mice, whereas hippocampal β-HB metabolism was increased. Furthermore, an enhancement in mitochondrial oxygen consumption and the rate of ATP synthesis was observed. This suggests that β-HB can partially compensate for insufficient glucose metabolism by enhancing mitochondrial oxidation, thereby maintaining the energy homeostasis of hippocampal neurons. The entry of β-HB into nerve cells depends on MCTs. Pierre et al (128) reported that, at 6 weeks of age, the hippocampus of db/db mice exhibit increased levels of MCT1 and MCT2, and that this upregulated expression of these transporter proteins may support the utilization of KBs. MCT1 is expressed by endothelial cells, astrocytes, oligodendrocytes and microglial cells in the brain, whereas MCT2 is primarily expressed by neurons (129). Thus, the augmented KB metabolism in db/db mice may be related to the expression levels of neuronal transporter proteins.

The hypothalamus, a pivotal regulator of energy homeostasis, has been a subject of debate in terms of its potential influence on energy and glucose homeostasis through central KBs. Park et al (130) administered β-HB at a dose of 12 μg/h into the lateral ventricle of diabetic rats with 90% Px. After 28 days, they observed increased β-HB levels in the hypothalamus and liver, enhanced leptin and insulin signaling in the hypothalamus and elevated STAT3 phosphorylation. By contrast, intraperitoneal injection of β-HB has no effect on hypothalamic signaling in Px rats (107). This discrepancy may be caused by the BBB: β-HB administered via intraperitoneal injection needs to cross the BBB through MCTs. At the cellular and molecular levels, β-HB exhibits multifaceted neuroprotective potential. Majrashi et al (131) demonstrated that supplementation of β-HB at doses of 250 and 500 μM exert a neuroprotective effect on HT22 cells (mouse hippocampal neuronal cell line). The proliferative effect of hippocampal neurons can reduce oxidative stress, maintain energy metabolism, improve mitochondrial function and regulate cell apoptosis. Combined with computational pharmacokinetic and molecular modeling analyses. This study further confirmed the neuroprotective potential of β-HB in cognition-related neurodegenerative diseases. The brain is one of the organs with the highest lipid content and lipids account for ~50% of its dry weight. Dabke et al (132) simulated the effect of endogenous β-HB production induced by a KD on the lipids of neuronal cells. Their research showed that when HT22 cells incubated under low-glucose conditions were treated with β-HB at 5 mM, the levels of cholesterol and phosphatidylserine decreased, while the ratio of phospholipids to cholesterol increased.

DM is linked to various eye-related issues, such as diabetic retinopathy (DR), cataracts, diabetic papillopathy, glaucoma and diseases affecting the ocular surface (133). DR is a major complication of DM that manifests as retinal microangiopathy and stands as the leading cause of vision impairment in middle-aged individuals (134). The condition of diabetes enhances the permeability of the blood-retinal barrier, promoting angiogenesis in the retina (135). The development of DR is complex, and it involves increased production of free radicals, the stimulation of AMP-activated protein kinase/mammalian target of rapamycin signaling pathways, the activation of the renin-angiotensin system, engagement of TGF-β/Smad signaling, the kinin system involving kinin-releasing enzymes, the accumulation of AGEs and various inflammatory agents such as VEGF (136-138). The abnormal signaling pathway of TGF-β plays a role in the development of DR. Systemic inhibition of TGF-β signaling offers protection against obesity, diabetes and liver fat accumulation in mice (139). However, TGF-β has been proposed to safeguard retinal ganglion cells against oxidative harm induced by hyperglycemia by enhancing cellular antioxidant and neuroprotective mechanisms, such as the Nrf2/Keap1 pathway (140).

Trotta et al (141) demonstrated that diabetic mice with heightened ERS markers [phosphorylated (p)ERK, phosphorylated inositol requiring enzyme 1 and activating transcription factor 6α], increased NLRP3 inflammasome activity (NLRP3, apoptosis associated speck-like protein containing a CARD and caspase-1) and increased levels of pro-inflammatory cytokines (IL-1β and IL-18) experienced significant reductions in these parameters following intraperitoneal injections of 50 and 100 mg/kg β-HB. The injections led to increased plasma and retinal β-HB levels as well as enhanced expression of the β-HB receptor, GPR109A. Consequently, ERS markers, NLRP3 inflammasome activation markers and pro-inflammatory cytokine levels were significantly reduced. Moreover, retinal outer nuclear layer cell death was diminished, effectively safeguarding the retina from diabetic-induced damage. Therefore, β-HB may offer protection to the retinas of diabetic mice by mitigating inflammation and ERS through the GPR109A receptor.