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Approximately 30% of patients with epilepsy are drug-refractory. There is an urgent need to elucidate the exact pathophysiology of different types of epilepsies and the mechanisms of action of both antiseizure medication and metabolic therapies to treat patients more effectively and safely. For example, it has been demonstrated that exogenous ketone supplement (EKS)-generated therapeutic ketosis, as a metabolic therapy, may decrease epileptic activity in both animal models and humans, but its exact mechanism of action is unknown. However, it was demonstrated that therapeutic ketosis, among others, can increase adenosine level, which may enhance activity of A1 adenosine receptors (A 1 Rs) in the brain. It has also been demonstrated previously that adenosine has anti-epileptic effect through A 1 Rs in different models of epilepsies. Thus, it is possible that (i) therapeutic ketosis generated by the administration of EKSs may exert its anti-epileptic effect through, among other mechanisms, increased adenosine level and A 1 R activity and that (ii) the enhanced activity of A 1 Rs may be a necessary anti-epileptic mechanism evoked by EKS administration-generated ketosis. Moreover, EKSs can evoke and maintain ketosis without severe side effects. These results also suggest that the therapeutic application of EKS-generated ketosis may be a promising opportunity to treat different types of epilepsies. In this literature review, we specifically focus on the putative role of A 1 Rs in the anti-epileptic effect of EKS-induced ketosis.

Ketosis-a metabolic state characterized by elevated levels of ketone bodies in the blood or urine-reduces the performance and health of dairy cows and causes substantial economic losses for the dairy industry. Currently, beta-hydroxybutyric acid is the gold standard for determining ketosis in cows; however, as this method is only applicable postpartum, it is not conducive to the early intervention of ketosis in dairy cows. In this study, the sera of dry, periparturient, postpartum ketotic, and healthy cows were analyzed by both transcriptomics and metabolomics techniques. Moreover, changes of gene expression and metabolites were observed, and serum physiological and biochemical indexes were detected by ELISA. The purpose was to screen biomarkers that can be used to detect the incidence of dry or periparturient ketosis in cows. The results showed that ketotic cows had increased levels of glycolipid metabolism indexes, oxidizing factors, and inflammatory factors during dry periods and liver damage, which could be used as early biomarkers to predict the onset of ketosis. Transcriptomic results yielded 20 differentially expressed genes (DEGs) between ketotic and healthy cows during dry, peripartum, and postpartum periods. GO and KEGG enrichment analyses indicated that these DEGs were involved in amino acid metabolism, energy metabolism, and disease-related signaling pathways. The metabolomics sequencing results showed that ketotic cows mainly showed enrichment in tricarboxylic acid cycle, butyric acid metabolism, carbon metabolism, lysine degradation, fatty acid degradation, and other signaling pathways. Metabolites differed between ketotic and healthy cows in dry, pre-parturition, and post-parturition periods. Combined transcriptomics and metabolomics analyses identified significant enrichment in the glucagon signaling pathway and the lysine degradation signaling pathway in dry, periparturient, and postpartum ketotic cows. PRKAB2 and SETMAR-key DEGs of the glucagon signaling pathway and lysine degradation signaling pathway, respectively-can be used as key marker genes for determining the early onset of ketosis in dairy cows., Competing Interests: Declaration of competing interest We declare that we have no financial and personal relationships with other people or organizations that could unduly influence our work, and that we have no professional or other personal interests of any nature or type with any product, service and/or company that could be perceived to influence the content of this article., (Copyright © 2024. Published by Elsevier Inc.)

In dairy cows, the occurrence of subclinical ketosis (SCK) is particularly high during early lactation. Previously, we documented alterations in the abundance of adiponectin (ADPN) in anestrus cows with SCK in comparison to cows in estrus. In the present study, 60 cows were divided into two groups: control (C, n = 30) and SCK (n = 30). Based on cow's estrus situation in two group at 55-60 days postpartum, 15 anestrus SCK cows and estrus cows were designated the SCK-A group and C-E group, respectively. The SCK-A group had downregulated serum and follicular fluid ADPN levels compared with the C-E group. The serum ADPN level was positively correlated with the insulin level and follicle growth rate, and there was a positive correlation between ADPN and glucose in the follicular fluid. Primary culture of dairy cow granulosa cells (GCs) was established to observe the effect of low glucose (Glu) and/or ADPN on GCs cyclins and proteins important for steroid synthesis. The results showed that the addition of 1 µg/mL ADPN alleviated the negative effects of low Glu treatment on the proliferation of GCs and the expression of steroid secretion related protein proteins. Treatment with LY294002 (PI3K inhibitor) four experimental GCs groups: control (0 µg/mL ADPN), 1 µg/mL ADPN, LY294002 inhibitor, and 1 µg/mL ADPN+LY294002. The results showed that ADPN promotes the secretion of steroid hormones by GCs through the PI3K-AKT. In summary, ADPN plays a crucial role in ameliorating postpartum anestrus in dairy cows with SCK., Competing Interests: Declaration of Competing Interest There are no conflicts of interest related to this study., (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)

Ketosis in dairy cows is often accompanied by the dysregulation of lipid homeostasis in the liver. Acetyl-coenzyme A acetyltransferase 2 (ACAT2) is specifically expressed in the liver and is important for regulating lipid homeostasis in ketotic cows. Lentinan (LNT) has a wide range of pharmacological activities, and this study investigates the protective effects of LNT on β-hydroxybutyrate (BHBA)-induced lipid metabolism disorder in bovine hepatocytes (BHECs) and elucidates the underlying mechanisms. BHECs were first pretreated with LNT to investigate the effect of LNT on BHBA-induced lipid metabolism disorder in BHECs. ACAT2 was then silenced or overexpressed to investigate whether this mediated the protective action of LNT against BHBA-induced lipid metabolism disorder in BHECs. Finally, BHECs were treated with LNT after silencing ACAT2 to investigate the interaction between LNT and ACAT2. LNT pretreatment effectively enhanced the synthesis and absorption of cholesterol, inhibited the synthesis of triglycerides, increased the expression of ACAT2, and elevated the contents of very low-density lipoprotein and low-density lipoprotein cholesterol, thereby ameliorating BHBA-induced lipid metabolism disorder in BHECs. The overexpression of ACAT2 achieved a comparable effect to LNT pretreatment, whereas the silencing of ACAT2 aggravated the effect of BHBA on inducing disorder in lipid metabolism in BHECs. Moreover, the protective effect of LNT against lipid metabolism disorder in BHBA-induced BHECs was abrogated upon silencing of ACAT2. Thus, LNT, as a natural protective agent, can enhance the regulatory capacity of BHECs in maintaining lipid homeostasis by upregulating ACAT2 expression, thereby ameliorating the BHBA-induced lipid metabolism disorder.

Background: Fluorine-18 fluorodeoxyglucose (FDG) with positron emission tomography (PET) is the standard for detecting myocardial inflammation in cardiac sarcoidosis, requiring preparation with the ketogenic diet (KD) to achieve myocardial glucose suppression. Despite this, incomplete myocardial glucose suppression remains a significant issue, and strategies to reduce myocardial glucose uptake (MGU) and identify incomplete myocardial glucose suppression are required. This study sought to understand the relationship between point-of-care beta-hydroxybutyrate (BHB) and different patterns of MGU and between KD and fasting duration with MGU in patients undergoing evaluation for cardiac sarcoidosis., Methods: We prospectively included 471 outpatients who underwent FDG-PET for cardiac sarcoidosis evaluation, followed the KD for 1 (n=100), 2 (n=29), and ≥3 days (n=342), fasted for at least 12 hours, and had BHB levels measured immediately before FDG injection. Images were classified as (1) no MGU (negative), (2) focal/multifocal (positive), (3) diffuse (nondiagnostic), or (4) nonspecific uptake (NS-MGU)., Results: Cardiac FDG-PET scans were interpreted as the following: 376 (79.83%) negative; 61 (12.95%) positive; 14 (2.97%) diffuse; and 20 (4.25%) NS-MGU. There was a strong negative relationship between BHB levels and MGU ( P <0.0001). BHB levels increased significantly with KD duration ( P <0.0001) and fasting time ( P =0.0067). The combined rate of diffuse, NS-MGU, and positive scans (34%, 28%, 16%) decreased inversely with KD duration (1, 2, and ≥3 days, respectively). However, MGU was not different across different fasting times ( P =0.6). Blood glucose levels were not associated with MGU ( P =0.17) and only weakly associated with BHB levels (R 2 =0.03; P <0.001)., Conclusions: We observed a strong inverse relationship between ketosis and patterns of MGU. Longer KD and fasting durations are associated with higher ketosis. However, only KD duration was associated with lower rates of MGU. Measurement of BHB levels before FDG-PET using point-of-care testing is feasible and may facilitate the management of patients referred for myocardial inflammation., Competing Interests: None.

Ketosis, a commonly observed energy metabolism disorder in dairy cows during the peripartal period, is distinguished by increased concentrations of BHB in the blood. This condition has a negative impact on milk production and quality, causing financial losses. An untargeted metabolomics approach was performed on plasma samples from cows between 5 and 7 DIM diagnosed as controls (CON; BHB <1.2 mM, n = 30), subclinically ketotic (SCK; 1.2 < BHB <3.0 mM, n = 30), or clinically ketotic (CK; BHB >3.0 mM, n = 30). Cows were selected from a commercial farm of 214 Holstein cows (average 305-d yield in the previous lactation of 35.42 ± 7.23 kg/d; parity, 2.41 ± 1.12; BCS, 3.1 ± 0.45). All plasma and milk samples (n = 90) were subjected to liquid chromatography-MS-based metabolomic analysis. Statistical analyses were performed using GraphPad Prism 8.0, MetaboAnalyst 4.0, and R version 4.1.3. Compared with the CON group, both SCK and CK groups had greater milk fat, freezing point, and fat-to-protein ratio, as well as lower milk protein, lactose, solids-not-fat, and milk density. Within 21 d after calving, compared with CON, the SCK group experienced a reduction of 2.65 kg/d in milk yield, while the CK group experienced a decrease of 7.7 kg/d. Untargeted metabolomics analysis facilitated the annotation of a total of 5,259 and 8,423 metabolites in plasma and milk. Differentially affected metabolites were screened in CON versus SCK, CON versus CK, and SCK versus CK (unpaired t-test, false discovery rate <0.05; and absolute value of log(2)-fold change >1.5). A total of 1,544 and 1,888 differentially affected metabolites were detected in plasma and milk. In plasma, glycerophospholipid metabolism, pyrimidine metabolism, tryptophan metabolism, sphingolipid metabolism, amino sugar and nucleotide sugar metabolism, phenylalanine metabolism, and steroid hormone biosynthesis were identified as important pathways. Weighted gene co-expression network analysis (WGCNA) indicated that tryptophan metabolism is a key pathway associated with the occurrence and development of ketosis. Increases in 5-hydroxytryptophan and decreases in kynurenine and 3-indoleacetic acid in SCK and CK were suggestive of an impact at the gut level. The decrease of most glycerophospholipids indicated that ketosis is associated with disordered lipid metabolism. For milk, pyrimidine metabolism, purine metabolism, pantothenate and CoA biosynthesis, amino sugar and nucleotide sugar metabolism, nicotinate and nicotinamide metabolism, sphingolipid metabolism, and fatty acid degradation were identified as important pathways. The WGCNA indicated that purine and pyrimidine metabolism in plasma was highly correlated with milk yield during the peripartal period. Alterations in purine and pyrimidine metabolism characterized ketosis, with lower levels of these metabolites in both milk and blood underscoring reduced efficiency in nitrogen metabolism. Our results may help to establish a foundation for future research investigating mechanisms responsible for the occurrence and development of ketosis in peripartal cows., (© 2024, The Authors. Published by Elsevier Inc. on behalf of the American Dairy Science Association®. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).)

Objective: In response to bacterial inflammation, anorexia of acute illness is protective and is associated with the induction of fasting metabolic programs such as ketogenesis. Forced feeding during the anorectic period induced by bacterial inflammation is associated with suppressed ketogenesis and increased mortality. As ketogenesis is considered essential in fasting adaptation, we sought to determine the role of ketogenesis in illness-induced anorexia., Methods: A mouse model of inducible hepatic specific deletion of the rate limiting enzyme for ketogenesis (HMG-CoA synthase 2, Hmgcs2) was used to investigate the role of ketogenesis in endotoxemia, a model of bacterial inflammation, and in prolonged starvation., Results: Mice deficient of hepatic Hmgcs2 failed to develop ketosis during endotoxemia and during prolonged fasting. Surprisingly, hepatic HMGCS2 deficiency and the lack of ketosis did not affect survival, glycemia, or body temperature in response to endotoxemia. Mice with hepatic ketogenic deficiency also did not exhibit any defects in starvation adaptation and were able to maintain blood glucose, body temperature, and lean mass compared to littermate wild-type controls. Mice with hepatic HMGCS2 deficiency exhibited higher levels of plasma acetate levels in response to fasting., Conclusions: Circulating hepatic-derived ketones do not provide protection against endotoxemia, suggesting that alternative mechanisms drive the increased mortality from forced feeding during illness-induced anorexia. Hepatic ketones are also dispensable for surviving prolonged starvation in the absence of inflammation. Our study challenges the notion that hepatic ketogenesis is required to maintain blood glucose and preserve lean mass during starvation, raising the possibility of extrahepatic ketogenesis and use of alternative fuels as potential means of metabolic compensation., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Author(s). Published by Elsevier GmbH.. All rights reserved.)

High-yielding dairy cows in early lactation often encounter difficulties in meeting the energy requirements essential for maintaining milk production. This is primarily attributed to insufficient dry matter intake, which consequently leads to sustained lipolysis of adipose tissue. Fatty acids released by lipolysis can disrupt metabolic homeostasis. Autophagy, an adaptive response to intracellular environmental changes, is considered a crucial mechanism for regulating lipid metabolism and maintaining a proper cellular energy status. Despite its close relationship with aberrant lipid metabolism and cytolipotoxicity in animal models of metabolic disorders, the precise function of diacylglycerol o-acyltransferase 1 (DGAT1) in bovine adipose tissue during periods of negative energy balance is not fully understood, particularly regarding its involvement in lipolysis and autophagy. The objective of the present study was to assess the effect of DGAT1 on both lipolysis and autophagy in bovine adipose tissue and isolated adipocytes. Adipose tissue and blood samples were collected from cows diagnosed as clinically ketotic (n = 15) or healthy (n = 15) following a veterinary evaluation based on clinical symptoms and serum concentrations of BHB, which were 3.19 mM (interquartile range = 0.20) and 0.50 mM (interquartile range = 0.06), respectively. Protein abundance of DGAT1 and phosphorylation levels of unc-51-like kinase 1 (ULK1), were greater in adipose tissue from cows with ketosis, whereas phosphorylation levels of phosphoinositide 3-kinase (PI3K), protein kinase B (AKT), and mammalian target of rapamycin (mTOR) were lower. Furthermore, when adipocytes isolated from the harvested adipose tissue of 15 healthy cows were transfected with DGAT1 overexpression adenovirus or DGAT1 small interfering RNA followed by exposure to epinephrine (EPI), it led to greater ratios and protein abundance of phosphorylated hormone-sensitive triglyceride lipase (LIPE) to total LIPE and adipose triglyceride lipase (ATGL), while inhibiting the protein phosphorylation levels of ULK1, PI3K, AKT, and mTOR. Overexpression of DGAT1 in EPI-treated adipocytes reduced lipolysis and autophagy, whereas silencing DGAT1 further exacerbated EPI-induced lipolysis and autophagy. Taken together, these findings indicate that upregulation of DGAT1 may function as an adaptive response to suppress adipocytes lipolysis, highlighting the significance of maintaining metabolic homeostasis in dairy cows during periods of negative energy balance., (The Authors. Published by Elsevier Inc. on behalf of the American Dairy Science Association®. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).)

Background: The interrelationship between cellular metabolism and the epithelial-to-mesenchymal transition (EMT) process has made it an interesting topic to investigate the adjuvant effect of therapeutic diets in the treatment of cancers. However, the findings are controversial. In this study, the effects of glucose limitation along and with the addition of beta-hydroxybutyrate (bHB) were examined on the expression of specific genes and proteins of EMT, Wnt, Hedgehog, and Hippo signaling pathways, and also on cellular behavior of gastric cancer stem-like (MKN-45) and non-stem-like (KATO III) cells., Methods and Results: The expression levels of chosen genes and proteins studied in cancer cells gradually adopted a low-glucose condition of one-fourth, along and with the addition of bHB, and compared to the unconditioned control cells. The long-term switching of the metabolic fuels successfully altered the expression profiles and behaviors of both gastric cancer cells. However, the results for some changes were the opposite. Glucose limitation along and with the addition of bHB reduced the CD44 + population in MKN-45 cells. In KATO III cells, glucose restriction increased the CD44 + population. Glucose deprivation alleviated EMT-related signaling pathways in MKN-45 cells but stimulated EMT in KATO III cells. Interestingly, bHB enrichment reduced the beneficial effect of glucose starvation in MKN-45 cells, but also alleviated the adverse effects of glucose restriction in KATO III cells., Conclusions: The findings of this research clearly showed that some controversial results in clinical trials for ketogenic diet in cancer patients stemmed from the different signaling responses of various cells to the metabolic changes in a heterogeneous cancer mass., (© 2024. The Author(s), under exclusive licence to Springer Nature B.V.)

Background: Genetic mitochondrial diseases impact over 1 in 4000 individuals, most often presenting in infancy or early childhood. Seizures are major clinical sequelae in some mitochondrial diseases including Leigh syndrome, the most common pediatric presentation of mitochondrial disease. Dietary ketosis has been used to manage seizures in mitochondrial disease patients. Mitochondrial disease patients often require surgical interventions, leading to anesthetic exposures. Anesthetics have been shown to be toxic in the setting of mitochondrial disease, but the impact of a ketogenic diet on anesthetic toxicities in this setting has not been studied., Aims: Our aim in this study was to determine whether dietary ketosis impacts volatile anesthetic toxicities in the setting of genetic mitochondrial disease., Methods: The impact of dietary ketosis on toxicities of volatile anesthetic exposure in mitochondrial disease was studied by exposing young Ndufs4(-/-) mice fed ketogenic or control diet to isoflurane anesthesia. Blood metabolites were measured before and at the end of exposures, and survival and weight were monitored., Results: Compared to a regular diet, the ketogenic diet exacerbated hyperlactatemia resulting from isoflurane exposure (control vs. ketogenic diet in anesthesia mean difference 1.96 mM, Tukey's multiple comparison adjusted p = .0271) and was associated with a significant increase in mortality during and immediately after exposures (27% vs. 87.5% mortality in the control and ketogenic diet groups, respectively, during the exposure period, Fisher's exact test p = .0121). Our data indicate that dietary ketosis and volatile anesthesia interact negatively in the setting of mitochondrial disease., Conclusions: Our findings suggest that extra caution should be taken in the anesthetic management of mitochondrial disease patients in dietary ketosis., (© 2024 The Authors. Pediatric Anesthesia published by John Wiley & Sons Ltd.)

It is widely acknowledged that the ketogenic diet (KD) has positive physiological effects as well as therapeutic benefits, particularly in the treatment of chronic diseases. Maintaining nutritional ketosis is of utmost importance in the KD, as it provides numerous health advantages such as an enhanced lipid profile, heightened insulin sensitivity, decreased blood glucose levels, and the modulation of diverse neurotransmitters. Nevertheless, the integration of the KD with pharmacotherapeutic regimens necessitates careful consideration. Due to changes in their absorption, distribution, metabolism, or elimination, the KD can impact the pharmacokinetics of various medications, including anti-diabetic, anti-epileptic, and cardiovascular drugs. Furthermore, the KD, which is characterised by the intake of meals rich in fats, has the potential to impact the pharmacokinetics of specific medications with high lipophilicity, hence enhancing their absorption and bioavailability. However, the pharmacodynamic aspects of the KD, in conjunction with various pharmaceutical interventions, can provide either advantageous or detrimental synergistic outcomes. Therefore, it is important to consider the pharmacokinetic and pharmacodynamic interactions that may arise between the KD and various drugs. This assessment is essential not only for ensuring patients' compliance with treatment but also for optimising the overall therapeutic outcome, particularly by mitigating adverse reactions. This highlights the significance and necessity of tailoring pharmacological and dietetic therapies in order to enhance the effectiveness and safety of this comprehensive approach to managing chronic diseases.

Background: Mastitis is a serious disease which affects animal husbandry, particularly in cow breeding. The etiology of mastitis is complex and its pathological mechanism is not yet fully understood. Our previous research in clinical investigation has revealed that subclinical ketosis can increase the number of somatic cell counts (SCC) in milk, although the underlying mechanism remains unclear. Recent studies have further confirmed the significant role of mastitis., Results: In this study, we aimed to examine the SCC, rumen microbiota, and metabolites in the milkmen of cows with subclinical ketosis. Additionally, we conducted a rumen microbiota transplant into mice to investigate the potential association between rumen microbiota disturbance and mastitis induced by subclinical ketosis in dairy cows. The study has found that cows with subclinical ketosis have a higher SCC in their milk compared to healthy cows. Additionally, there were significant differences in the rumen microbiota and the level of volatile fatty acid (VFA) between cows with subclinical ketosis and healthy cows. Moreover, transplanting the rumen microbiota from subclinical ketosis and mastitis cows into mice can induce mammary inflammation and liver function damage than transplanting the rumen flora from healthy dairy cows., Conclusions: In addition to the infection of mammary gland by pathogenic microorganisms, there is also an endogenous therapeutic pathway mediated by rumen microbiota. Targeted rumen microbiota modulation may be an effective way to prevent and control mastitis in dairy cows., Competing Interests: Declaration of competing interest All authors declare that they have no conflict of interest., (Copyright © 2023. Published by Elsevier Ltd.)

Ketone bodies have a negative image because of ketoacidosis, one of the acute and serious complications in diabetes. The negative image persists despite the fact that ketone bodies are physiologically produced in the liver and serve as an indispensable energy source in extrahepatic organs, particularly during long-term fasting. However, accumulating experimental evidence suggests that ketone bodies exert various health benefits. Particularly in the field of aging research, there is growing interest in the potential organoprotective effects of ketone bodies. In addition, ketone bodies have a potential role in preventing kidney diseases, including diabetic kidney disease (DKD), a diabetic complication caused by prolonged hyperglycemia that leads to a decline in kidney function. Ketone bodies may help alleviate the renal burden from hyperglycemia by being used as an alternative energy source in patients with diabetes. Furthermore, ketone body production may reduce inflammation and delay the progression of several kidney diseases in addition to DKD. Although there is still insufficient research on the use of ketone bodies as a treatment and their effects, their renoprotective effects are being gradually proven. This review outlines the ketone body-mediated renoprotective effects in DKD and other kidney diseases., (Copyright © 2024 The Author(s). Published by Wolters Kluwer Health, Inc. on behalf of the American Society of Nephrology.)

This study is aimed at examining which factors are useful for the diagnosis and distinction of ketoacidosis. We recruited 21 diabetic ketoacidosis (DKA) and alcoholic ketoacidosis (AKA) patients hospitalized in Kawasaki Medical School General Medical Center from April 2015 to March 2021. Almost all patients in this study were brought to the emergency room in a coma and hospitalized. All patients underwent blood gas aspiration and laboratory tests. We evaluated the difference in diagnosis markers in emergencies between DKA and alcoholic ketoacidosis AKA. Compared to AKA patients, DKA patients had statistically higher values of serum acetoacetic acid and lower values of serum lactate, arterial blood pH, and base excess. In contrast, total ketone bodies, β -hydroxybutyric acid, and β -hydroxybutyric acid/acetoacetic acid ratio in serum did not differ between the two patient groups. It was shown that evaluation of each pathology such as low body weight, diabetes, liver dysfunction, and dehydration was important. It is important to perform differential diagnosis for taking medical histories such as insulin deficiency, alcohol abuse, or starvation as the etiology in Japanese subjects with DKA or AKA. Moreover, it is important to precisely comprehend the pathology of dehydration and alcoholic metabolism which would lead to appropriate treatment for DKA and AKA., Competing Interests: HK has received honoraria for lectures and received scholarship grants from Sanofi, Novo Nordisk, Lilly, Boehringer Ingelheim, MSD, Takeda, Ono Pharma, Daiichi Sankyo, Sumitomo Pharma, Mitsubishi Tanabe Pharma, Pfizer, Kissei Pharma, AstraZeneca, Astellas, Novartis, Kowa, Chugai, and Taisho Pharma. KK has been an advisor to, received honoraria for lectures from, and received scholarship grants from Novo Nordisk Pharma, Sanwa Kagaku Kenkyusho, Takeda, Taisho Pharmaceutical Co., Ltd., MSD, Kowa, Sumitomo Pharma, Novartis, Mitsubishi Tanabe Pharma, AstraZeneca, Nippon Boehringer Ingelheim Co., Ltd., Chugai, Daiichi Sankyo, and Sanofi., (Copyright © 2024 Katsumasa Koyama et al.)

Excessive and protracted lipolysis in adipose tissues of dairy cows is a major risk factor for clinical ketosis (CK). This metabolic disease is common in postpartum cows when lipolysis provides fatty acids as an energy substrate to offset negative energy balance. Lipolysis in cows can be induced by the canonical (hormonally induced) and inflammatory pathways. Current treatments for CK focus on improving glucose in blood (i.e., oral propylene glycol [PG], or i.v. dextrose). However, these therapies do not inhibit the canonical and inflammatory lipolytic pathways. Niacin (NIA) can reduce activation of the canonical pathway. Blocking inflammatory responses with cyclooxygenase inhibitors such as flunixin meglumine (FM) can inhibit inflammatory lipolytic activity. The objective of this study was to determine the effects of including NIA and FM in the standard PG treatment for postpartum CK on circulating concentrations of ketone bodies. A 4-group, parallel, individually randomized trial was conducted in multiparous Jersey cows (n = 80) from a commercial dairy in Michigan during a 7-mo period. Eligible cows had CK symptoms (lethargy, depressed appetite, and milk yield) and hyperketonemia (blood β-hydroxybutyrate [BHB] ≥1.2 mmol/L). Cows with CK were randomly assigned to 1 of 3 groups where the first group received 310 g of oral PG once per day for 5 d; the second group received PG for 5 d + 24 g of oral NIA once per day for 3 d (PGNIA); and the third group received PG for 5 d + NIA for 3 d + 1.1 mg/kg i.v. FM once per day for 3 d (PGNIAFM). The control group consisted of cows that were clinically healthy (HC; untreated; BHB <1.2 mmol/L, n = 27) matching for parity and DIM with all 3 groups. Animals were sampled at enrollment (d 0), and d 3, 7, and 14 to evaluate ketone bodies and circulating metabolic and inflammatory biomarkers. Effects of treatment, sampling day, and their interactions were evaluated using mixed effects models. Logistic regression was used to calculate the odds ratio (OR) of returning to normoketonemia (BHB <1.2 mmol/L). Compared with HC, enrolled CK cows exhibited higher blood concentrations of dyslipidemia markers, including nonesterified fatty acids (NEFA) and BHB, and lower glucose and insulin levels. Cows with CK also had increased levels of biomarkers of pain (substance P), inflammation, including lipopolysaccharide-binding protein, haptoglobin, and serum amyloid A, and proinflammatory cytokines IL-4, MCP-1, MIP-1α, and TNFα. Importantly, 72.2% of CK cows presented endotoxemia and had higher circulating bacterial DNA compared with HC. By d 7, the percentage of cows with normoketonemia were higher in PGNIAFM = 87.5%, compared with PG = 58.33%, and PGNIA = 62.5%. At d 7 the OR for normoketonemia in PGNIAFM cows were 1.5 (95% CI, 1.03-2.17) and 1.4 (95% CI, 0.99-1.97) relative to PG and PGNIA, respectively. At d 3, 7, and 14, PGNIAFM cows presented the lowest values of BHB (PG = 1.36; PGNIA = 1.24; PGNIAFM = 0.89 ± 0.13 mmol/L), NEFA (PG = 0.58; PGNIA = 0.59; PGNIAFM = 0.45 ± 0.02 mmol/L), and acute phase proteins. Cows in PGNIAFM also presented the highest blood glucose increment across time points and insulin by d 7. These data provide evidence that bacteremia or endotoxemia, systemic inflammation, and pain may play a crucial role in CK pathogenesis. Additionally, targeting lipolysis and inflammation with NIA and FM during CK effectively reduces dyslipidemia biomarkers, improves glycemia, and improves overall clinical recovery., (© 2023, The Authors. Published by Elsevier Inc. and Fass Inc. on behalf of the American Dairy Science Association®. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).)

The metabolic status of dairy cows directly influences the nutritional quality and flavor of raw milk. A comprehensive comparison of non-volatile metabolites and volatile compounds in raw milk from healthy and subclinical ketosis (SCK) cows was performed using LC-MS, GC-FID, and HS-SPME/GC-MS. SCK can significantly alter the profiles of water-soluble non-volatile metabolites, lipids, and volatile compounds of raw milk. Compared with healthy cows, milk from SCK cows had higher contents of tyrosine, leucine, isoleucine, galactose-1-phosphate, carnitine, citrate, phosphatidylethanolamine species, acetone, 2-butanone, hexanal, dimethyl disulfide and lower content of creatinine, taurine, choline, α-ketoglutaric acid, fumarate, triglyceride species, ethyl butanoate, ethyl acetate, and heptanal. The percentage of polyunsaturated fatty acids in milk was lowered in SCK cows. Our results suggest that SCK can change milk metabolite profiles, disrupt the lipid composition of milk fat globule membrane, decrease the nutritional value, and increase the volatile compounds associated with off-flavors in milk., Competing Interests: Declaration of Competing Interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Yuchao Zhao reports financial support was provided by Beijing Natural Science Foundation. Yuchao Zhao reports financial support was provided by China Postdoctoral Science Foundation. Yuchao Zhao reports financial support was provided by Beijing Postdoctoral Research Foundation., (Copyright © 2023 Elsevier Ltd. All rights reserved.)

In glucose-deprived conditions, ketone bodies are produced by the liver mitochondria, through the catabolism of fatty acids, and are used peripherally, as an alternative energy source. Ketones are produced in the body under normal conditions, including during pregnancy and the neonatal period, when following a ketogenic diet (KD), fasting, or exercising. Additionally, ketone synthesis is also augmented under pathological conditions, including cases of diabetic ketoacidosis (DKA), alcoholism, and several metabolic disorders. Nonetheless, diet is the main regulator of total body ketone concentrations. The KDs are mimicking the fasting state, altering the default metabolism towards the use of ketones as the primary fuel source. Recently, KD has gained recognition as a medical nutrition therapy for a plethora of metabolic conditions, including obesity and diabetes mellitus (DM). The present review aims to discuss the role of ketones, KDs, ketonemia, and ketonuria in DM, presenting all the available new evidence in a comprehensive manner.

Early lactation increases metabolic stress in ketotic dairy cows, leading to mitochondrial damage, apoptosis, and inflammatory response in mammary epithelial cells. The pyrin domain 3 (NLRP3) pathway involving the mitochondrial reactive oxygen species (Mito-ROS)-induced nucleotide-binding oligomerization domain-like receptor has been recognized as a key mechanism in this inflammatory response and cell apoptosis. This study aimed to elucidate the underlying regulatory mechanism of Mito-ROS-NLRP3 pathway-mediated mammary epithelial cell apoptosis in dairy cows with ketosis. Mitochondrial damage and cellular apoptotic program and NLRP3 inflammasome activation were observed in the mammary gland of ketotic cows. Similar damage was detected in MAC-T cells treated with exogenous fatty acids (FFAs). However, NLRP3 inhibitor MCC950 pretreatment or Mito-ROS scavenging by MitoTEMPO attenuated apoptosis in FFA-induced MAC-T cells by inhibiting the NLRP3 inflammasome pathway. These findings reveal that the Mito-ROS-NLRP3 pathway activation is a potent mechanism underlying mammary epithelial cell apoptosis in response to metabolic stress in ketotic dairy cows, which further contributes to reduced milk yield.

The extent to which a nutrition-related disorder such as ketosis alters the ruminal microbiota or whether microbiota composition is related to ketosis and potential associations with host metabolism is unknown. We aimed to evaluate variations occurring in the ruminal microbiota of ketotic and nonketotic cows in the early postpartum period, and how those changes may affect the risk of developing the disease. Data on milk yield, dry matter intake (DMI), body condition score, and blood β-hydroxybutyrate (BHB) concentrations at 21 d postpartum were used to select 27 cows, which were assigned (n = 9 per group) to a clinical ketotic (CK, 4.10 ± 0.72 mmol BHB/L, DMI 11.61 ± 0.49 kg/d, ruminal pH 7.55 ± 0.07), subclinical ketotic (SK, 1.36 ± 0.12 mmol BHB/L, DMI 15.24 ± 0.34 kg/d, ruminal pH 7.58 ± 0.08), or control (NK, 0.88 ± 0.14 mmol BHB/L, DMI 16.74 ± 0.67/d, ruminal pH 7.61 ± 0.03) group. Cows averaged 3.6 ± 0.5 lactations and a body condition score of 3.11 ± 0.34 at the time of sampling. After blood serum collection for metabolomics analysis ( 1 H nuclear magnetic resonance spectra), 150 mL of ruminal digesta was collected from each cow using an esophageal tube, paired-end (2 × 300 bp) sequencing of isolated DNA from ruminal digesta was performed via Illumina MiSeq, and sequencing data were analyzed using QIIME2 (v 2020.6) to measure the ruminal microbiota composition and relative abundance. Spearman correlation coefficients were used to evaluate relationships between relative abundance of bacterial genera and concentrations of serum metabolites. There were more than 200 genera, with approximately 30 being significant between NK and CK cows. Succinivibrionaceae UCG 1 taxa decreased in CK compared with NK cows. Christensenellaceae (Spearman correlation coefficient = 0.6), Ruminococcaceae (Spearman correlation coefficient = 0.6), Lachnospiraceae (Spearman correlation coefficient = 0.5), and Prevotellaceae (Spearman correlation coefficient = 0.6) genera were more abundant in the CK group and were highly positively correlated with plasma BHB. Metagenomic analysis indicated a high abundance of predicted functions related to metabolism (37.7%), genetic information processing (33.4%), and Brite hierarchies (16.3%) in the CK group. The 2 most important metabolic pathways for butyrate and propionate production were enriched in CK cows, suggesting increased production of acetyl coenzyme A and butyrate and decreased production of propionate. Overall, the combined data suggested that microbial populations may be related to ketosis by affecting short-chain fatty acid metabolism and BHB accumulation even in cows with adequate feed intake in the early postpartum period., (The Authors. Published by Elsevier Inc. and Fass Inc. on behalf of the American Dairy Science Association®. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).)

De novo capillarization is a primary muscular adaptation to endurance exercise training and is crucial to improving performance. Excess training load, however, impedes such beneficial adaptations, yet we recently demonstrated that such downregulation may be counteracted by ketone ester ingestion (KE) post-exercise. Therefore, we investigated whether KE could increase pro-angiogenic factors and thereby stimulate muscular angiogenesis during a 3-week endurance training-overload period involving 10 training sessions/week in healthy, male volunteers. Subjects received either 25 g of a ketone ester (KE, n = 9) or a control drink (CON, n = 9) immediately after each training session and before sleep. In KE, but not in CON, the training intervention increased the number of capillary contacts and the capillary-to-fibre perimeter exchange index by 44% and 42%, respectively. Furthermore, KE also substantially increased vascular endothelial growth factor (VEGF) and endothelial nitric oxide synthase (eNOS) expression both at the protein and at the mRNA level. Serum erythropoietin concentration was concomitantly increased by 26%. Conversely, in CON the training intervention increased only the protein content of eNOS. These data indicate that intermittent exogenous ketosis during endurance overload training stimulates muscular angiogenesis. This likely resulted from a direct stimulation of muscle angiogenesis, which may be at least partly due to stimulation of erythropoietin secretion and elevated VEGF activity, and/or an inhibition of the suppressive effect of overload training on the normal angiogenic response to training. This study provides novel evidence to support the potential of exogenous ketosis to benefit endurance training-induced muscular adaptation. KEY POINTS: Increased capillarization is a primary muscular adaptation to endurance exercise training. However, excess training load may impede such response. We previously observed that intermittent exogenous ketosis by post-exercise and pre-sleep ketone ester ingestion (KE) counteracted physiological dysregulations induced by endurance overload training. Therefore, we investigated whether KE could increase pro-angiogenic factors thereby stimulating muscular angiogenesis during a 3-week endurance training overload period. We show that the overload training period in the presence, but not in the absence, of KE markedly increased muscle capillarization (+40%). This increase was accompanied by higher circulating erythropoietin concentration and stimulation of the pro-angiogenic factors vascular endothelial growth factor and endothelial nitric oxide synthase in skeletal muscle. Collectively, our data indicate that intermittent exogenous ketosis may evolve as a potent nutritional strategy to facilitate recovery from strenuous endurance exercise, thereby stimulating beneficial muscular adaptations., (© 2023 The Authors. The Journal of Physiology published by John Wiley & Sons Ltd on behalf of The Physiological Society.)

The objective of this observational study was to evaluate the association of transition cow health and estrous expression, detected by an automated activity monitoring system (Smarttag Neck, Nedap Livestock Management), with reproductive performance in lactating Holstein cows. A total of 3,750 lactating Holstein cows (1,563 primiparous cows and 2,187 multiparous cows) from a commercial dairy farm in Slovakia calving from January 2020 until July 2021 were enrolled on an ongoing basis. Activity data were recorded from d 7 until d 60 postpartum. Within this observational period, cows were classified into 3 categories: (1) no estrus event (Estrus0), (2) 1 estrus event (Estrus1), or (3) 2 or more estrus events (Estrus2+). Transition cow health was assessed by farm personnel within the first 30 d in milk (DIM) using standard operating procedures. Generalized linear mixed models were used to analyze continuous and categorical data. Cox proportional hazard models were used for time to event data. The overall prevalence of anestrus was 20.8%. Multiparous cows had a greater risk for anestrus compared with primiparous cows [odds ratio (OR) = 1.4]. Cows with stillbirth (OR = 1.76), retained placenta (OR = 2.19), puerperal metritis (OR = 1.48), or subclinical ketosis (OR = 1.51) had a greater risk for anestrus. In addition, cows calving in summer (OR = 0.82), autumn (OR = 0.38), or winter (OR = 0.56) had a higher incidence of anestrus than cows calving in spring. Estrous expression from d 7 until d 60 postpartum was associated with estrous duration (DU) and estrous intensity at first artificial insemination (AI). Cows in Estrus0 had the shortest DU at first postpartum AI (9.4 ± 0.18 h) compared with cows in Estrus1 (10.5 ± 0.13 h) and Estrus2+ (11.4 ± 0.12 h). Cows in Estrus2+ had a longer DU at first postpartum AI compared with cows in Estrus1. For Estrus0, Estrus1, and Estrus2+ cows, pregnancy per AI at first service was 42.5%, 50.9%, and 55.4%, respectively. Estrous expression from d 7 until d 60 postpartum was associated with time to first AI and time to pregnancy. Compared with Estrus0 cows, Estrus1 [hazard ratio (HR) = 1.43] and Estrus2+ cows (HR = 1.62) had an increased hazard of being inseminated within 100 DIM. Compared with Estrus2+, Estrus1 cows had a reduced hazard of being inseminated within 100 DIM (HR = 0.89). Compared with Estrus0 cows, Estrus1 (HR = 1.24) and Estrus2+ cows (HR = 1.46) had an increased hazard of becoming pregnant within 200 DIM. Median DIM to pregnancy were 121, 96, and 92 for Estrus0, Estrus1, and Estrus2+ cows, respectively. In conclusion, cows with transition cow disorders (i.e., stillbirth, retained placenta, puerperal metritis, or subclinical ketosis) had a greater chance for anestrus compared with healthy cows. Cows in Estrus0 had reduced estrous expression at first AI and inferior reproductive performance compared with cows that displayed estrous activity from d 7 until d 60., (The Authors. Published by Elsevier Inc. and Fass Inc. on behalf of the American Dairy Science Association®. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).)

Type 2 diabetes mellitus (T2DM) shares a common molecular mechanism and underlying pathology with dementia, and studies indicate that dementia is widespread in people with T2DM. Currently, T2DM-induced cognitive impairment is characterized by altered insulin and cerebral glucose metabolism, leading to a shorter life span. Increasing evidence indicates that nutritional and metabolic treatments can possibly alleviate these issues, as there is a lack of efficient preventative and treatment methods. The ketogenic diet (KD) is a very high-fat, low-carbohydrate diet that induces ketosis in the body by producing a fasting-like effect, and neurons in the aged brain are protected from damage by ketone bodies. Moreover, the creation of ketone bodies may improve brain neuronal function, decrease inflammatory expression and reactive oxygen species (ROS) production, and restore neuronal metabolism. As a result, the KD has drawn attention as a potential treatment for neurological diseases, such as T2DM-induced dementia. This review aims to examine the role of the KD in the prevention of dementia risk in T2DM patients and to outline specific aspects of the neuroprotective effects of the KD, providing a rationale for the implementation of dietary interventions as a therapeutic strategy for T2DM-induced dementia in the future.

The ketone bodies beta-hydroxybutyrate and acetoacetate are hepatically produced metabolites catabolized in extrahepatic organs. Ketone bodies are a critical cardiac fuel and have diverse roles in the regulation of cellular processes such as metabolism, inflammation, and cellular crosstalk in multiple organs that mediate disease. This review focuses on the role of cardiac ketone metabolism in health and disease with an emphasis on the therapeutic potential of ketosis as a treatment for heart failure (HF). Cardiac metabolic reprogramming, characterized by diminished mitochondrial oxidative metabolism, contributes to cardiac dysfunction and pathologic remodeling during the development of HF. Growing evidence supports an adaptive role for ketone metabolism in HF to promote normal cardiac function and attenuate disease progression. Enhanced cardiac ketone utilization during HF is mediated by increased availability due to systemic ketosis and a cardiac autonomous upregulation of ketolytic enzymes. Therapeutic strategies designed to restore high-capacity fuel metabolism in the heart show promise to address fuel metabolic deficits that underpin the progression of HF. However, the mechanisms involved in the beneficial effects of ketone bodies in HF have yet to be defined and represent important future lines of inquiry. In addition to use as an energy substrate for cardiac mitochondrial oxidation, ketone bodies modulate myocardial utilization of glucose and fatty acids, two vital energy substrates that regulate cardiac function and hypertrophy. The salutary effects of ketone bodies during HF may also include extra-cardiac roles in modulating immune responses, reducing fibrosis, and promoting angiogenesis and vasodilation. Additional pleotropic signaling properties of beta-hydroxybutyrate and AcAc are discussed including epigenetic regulation and protection against oxidative stress. Evidence for the benefit and feasibility of therapeutic ketosis is examined in preclinical and clinical studies. Finally, ongoing clinical trials are reviewed for perspective on translation of ketone therapeutics for the treatment of HF.

Reverse transcription followed by quantitative (real-time) polymerase chain reaction (RT-qPCR) has become the gold standard in mRNA expression analysis. However, it requires an accurate choice of reference genes for adequate normalization. The aim of this study was to validate the reference genes for qPCR experiments in the brain of rats in the model of mild ketosis established through supplementation with medium-chain triglycerides (MCT) and intermittent fasting. This approach allows to reproduce certain neuroprotective effects of the classical ketogenic diet while avoiding its adverse effects. Ketogenic treatment targets multiple metabolic pathways, which may affect the reference gene expression. The standard chow of adult Wistar rats was supplemented with MCT (2 ml/kg orogastrically, during 6 h of fasting) or water (equivolume) for 1 month. The mRNA expression of 9 housekeeping genes (Actb, B2m, Gapdh, Hprt1, Pgk1, Ppia, Rpl13a, Sdha, Ywhaz) in the medial prefrontal cortex, dorsal and ventral hippocampus was measured by RT-qPCR. Using the RefFinder® online tool, we have found that the reference gene stability ranking strongly depended on the analyzed brain region. The most stably expressed reference genes were found to be Ppia, Actb, and Rpl13a in the medial prefrontal cortex; Rpl13a, Ywhaz, and Pgk1 in the dorsal hippocampus; Ywhaz, Sdha, and Ppia in the ventral hippocampus. The B2m was identified as an invalid reference gene in the ventral hippocampus, while Sdha, Actb, and Gapdh were unstable in the dorsal hippocampus. The stabilities of the examined reference genes were lower in the dorsal hippocampus compared to the ventral hippocampus and the medial prefrontal cortex. When normalized to the three most stably expressed reference genes, the Gapdh mRNA was upregulated, while the Sdha mRNA was downregulated in the medial prefrontal cortex of MCT-fed animals. Thus, the expression stability of reference genes strongly depends on the examined brain regions. The dorsal and ventral hippocampal areas differ in reference genes stability rankings, which should be taken into account in the RT-qPCR experimental design., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2023 Schwarz et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Case History: Serum and liver samples from 35, 2-year-old dairy heifers that had fractured one or both humeri post-calving between July and December 2019 were submitted to a diagnostic laboratory for analysis. Serum samples were analysed for albumin, β-hydroxybutyrate (BHB), creatinine, Ca, Mg, phosphate, non-esterified fatty acids (NEFA), and serum Cu concentration. Liver samples were analysed for liver Cu concentration. Data were compared to published reference intervals. Data values for heifers that prior to fracture had grazed fodder beet were also compared to values for those that had grazed pasture., Clinical Findings: Sixty-nine percent of heifers with humeral fracture had serum creatinine concentrations below the lower value of the reference range (55-130 µmol/L). In 3/32 (9%) heifers, serum NEFA concentrations were increased above the reference value indicating body fat mobilisation (≥1.2 mmol/L for peri-partum cows) and in 20/35 (57%) heifers BHB serum concentrations were above the reference value indicating subclinical ketosis (≥1.1 mmol/L for peri-partum cows). In 24/35 (69%) heifers, liver Cu concentration was low (≤ 44 µmol/kg) or marginal (45-94 µmol/kg). The concentration of Cu in serum was low (≤ 4.5 µmol/L) in 2/33 (6%) heifers and marginal (4.6-7.9 µmol/L) in 5/33 (15%) heifers. There was moderate positive correlation between the logged concentrations of Cu in paired liver and serum samples, r (31)  = 0.43; (95% CI = 0.1-0.79; p = 0.014). One heifer had a serum phosphate concentration below the lower limit of the reference range (< 1.10 mmol/L). For all heifers, the concentrations of albumin, Ca, and Mg in serum were within the reference intervals (23-38 g/L, 2.00-2.60 mmol/L, and 0.49-1.15 mmol/L respectively). Over winter, 15/35 (43%) heifers grazed predominantly pasture, 14/35 (40%) grazed fodder beet and 6/35 (17%) had a mixed diet., Clinical Relevance: In some of these heifers with humeral fractures, there was evidence for protein and/or energy malnutrition in the form of elevated NEFA and BHB concentrations and low creatinine concentrations in serum. Liver Cu concentrations were also reduced in most affected heifers. However, the absence of a control group means it is not possible to determine if these are risk factors for fracture or features common to all periparturient heifers. Clinical trials and molecular studies are needed to determine the true contribution of Cu and protein-energy metabolism to the pathogenesis of spontaneous humeral fractures in dairy heifers., Abbreviations: BHB: ß-hydroxybutyrate; NEFA: Non-esterified fatty acids.

Data were obtained from studies in Australia, Canada, and the United States using individual cow data from 28,230 Holstein cows to evaluate associations between parity and disease. Our goal was to develop understanding of disease risks for cows of differing parity. We hypothesized that there would be increased risks of disease and changes in metabolite concentrations with increased parity. Parity ≥5 represented 2,533 cows or 9.0%, parity 4 was 9.8% (2,778), parity 3 as 19.0% (5,355), parity 2 as 28.1% (7,925), and parity 1 was 34.1% (9,639) of the sample. Of these cows, 15.5% were in Australia, 14.7% in Canada, and 69.8% in the United States. Lactational incidence (LI) risk of clinical hypocalcemia increased with parity from 0.1% for parity 1 to 13% for parity ≥5 cows. The marked increase suggests profound differences in metabolism with increased parity. The LI of clinical mastitis was 17.4%. The odds of mastitis increased with parity to 2.5 times greater in parity ≥5 than in parity 1. The LI of lameness increased with parity; specifically, the odds of lameness was 5.6 times greater for parity ≥5 than parity 1. Dystocia incidence was 8.7% and greatest for parity 1 cows. The LI of retained placenta was 7.4% and increased with parity, with the odds for parity ≥5 2.3 times greater than for parity 1. The LI of metritis was 10% and of endometritis 14%, with the greatest odds in parity 1. The LI of clinical ketosis was 3.3% with a marked increase in odds with parity. The prevalence of subclinical ketosis was 26.8% with only cows in parity 1 having lower odds than other parities. Parity ≥5 cows had greater odds (odds ratio = 1.7) of respiratory disease than parity 1 cows, which were lesser than other parities. Metabolite concentrations were evaluated in 5,154 Holstein cows in the precalving, calving, and immediate postcalving data sets. Metabolic measures near peak lactation provided 1,906 observations. Concentrations of β-hydroxybutyrate (BHB) and nonesterified fatty acids increased with parity on d 1 to 3 of lactation and at peak lactation. On d 1 to 3 after calving differences in glucose, nonesterified fatty acids, and BHB indicated a greater reliance on mobilized lipid to export energy to peripheral tissues as BHB for greater parity cows. Differences in concentrations among parity groups were marked at times, for example >0.20 mM in Ca for parity 1 and 2 to parity ≥5 and >0.33 mM for all older parities compared with parity 1 for P on the day of calving. The marked increase suggests profound differences in metabolism with increased parity are probably influenced, in part, by increased production. We found marked differences in concentrations of metabolites with parity that are consistent with reduced reproduction, health, and body condition for higher parity cows. These unfavorable differences in metabolism in Ca, P, glucose, and cholesterol concentrations for higher parity cows also complement the often-substantial differences in disease risk with parity and suggest a need to carefully consider the parity structure in study design. Managers and advisors will need to consider methods to reduce risk of health disorders tailored to cows of different ages., (The Authors. Published by Elsevier Inc. and Fass Inc. on behalf of the American Dairy Science Association®. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).)

Ketosis is a metabolic disease of dairy cows in the perinatal period, β-hydroxybutyrate (β-HB) is the main component of ketosis. High levels of β-HB can trigger oxidative stress and inflammatory response in dairy cows, leading to decreased milk yield and multiple postpartum diseases. Forsythin (FOR), the major constituent of the herbal medicine Forsythia, has anti-inflammatory, anti-oxidant, and antiviral effects. FOR was demonstrated to have an antioxidant effect on PC12 cells. However, the effects of FOR on β-HB-stimulated bovine macrophages (BMs) has not been reported. Thus, the aim of the present study was to investigate the effects of FOR on β-HB-stimulated BMs. Firstly, the CCK8 test confirmed that FOR (50, 100, 200 μg/mL) has no effect on BMs activity, and we selected these concentrations for subsequent experiments. Secondly, through detecting the oxidation indexes ROS, MDA and antioxidant indexes CAT and SOD, we confirmed the antioxidant effect of FOR on BMs. Next, qRT-PCR confirmed that FOR dramatically reduced the mRNA levels of IL-1β and IL-6. Furthermore, the western blotting confirmed that FOR observably down-regulated β-HB-stimulated phosphorylation of p38, ERK and Akt and up-regulated expression of Nrf2, and HO-1. Above results suggested that FOR plays antioxidant effects on β-HB-induced BMs through p38, ERK and PI3K/Akt, Nrf2 and HO-1 signaling pathways. Therefore, we speculated that FOR may be a potential medicine to alleviate β-HB-induced inflammatory response and provide a preliminary reference for the research and development of FOR., Competing Interests: Declaration of Competing Interest We declare that we have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022. Published by Elsevier Ltd.)

Background: Ketosis is a common metabolic disorder during the post-partum transition period of dairy cattle. How the method of reproduction, parturition time, and calf birth weight affect the occurrence of ketosis on dairy herds remains elusive., Objectives: This study investigated factors associated with the severity of ketosis., Methods: We divided 186 Holstein cows into three classifications based on the highest β-hydroxybutyrate (BHBA) concentration during the post-partum transition period, namely non-ketosis (<1.2 mmol/L, n = 94), subclinical ketosis (1.2-2.9 mmol/L, n = 58), and clinical ketosis (≥3.0 mmol/L, n = 34). We evaluated characteristics of cows associated with the severity of ketosis., Results: Ketosis was not associated with the method of reproduction, parturition time, pregnancy wastage, premature delivery, retained placenta, and type of calf. Cows calving in spring and especially summer were at higher risk of severe ketosis (p < 0.01). Cows with increased body condition score (BCS) at parturition, age, lactation number, and calving interval were more likely to develop severe ketosis (p < 0.05). Cows with clinical ketosis produced most milk (29.9 ± 1.0 kg) from days four to six, whereas cows without ketosis produced the least (21.3 ± 0.8 kg) (p < 0.001). Heavier calf birth weight resulted in high risk of severe ketosis (p < 0.01), due to increased milk yield during the early lactation., Conclusions: The severity of ketosis is associated with the calving season, BCS at parturition, age, lactation number, calving interval, milk yield in the early lactation period, and calf birth weight. Nonetheless, it was not associated with the method of reproduction, parturition time, pregnancy wastage, premature delivery, retained placenta, and type of calf. This study is the first to investigate the associations between ketosis and calf birth weight. Our findings could help predict cows at risk of ketosis and take precautions., (© 2022 The Authors. Veterinary Medicine and Science published by John Wiley & Sons Ltd.)

Objectives: Ketogenic diets have reported efficacy for neurological dysfunctions; however, there are limited published human clinical trials elucidating the mechanisms by which nutritional ketosis produces therapeutic effects. The purpose of this present study was to investigate animal models that report variations in nervous system function by changing from a standard animal diet to a ketogenic diet, synthesise these into broad themes, and compare these with mechanisms reported as targets in pain neuroscience to inform human chronic pain trials., Methods: An electronic search of seven databases was conducted in July 2020. Two independent reviewers screened studies for eligibility, and descriptive outcomes relating to nervous system function were extracted for a thematic analysis, then synthesised into broad themes., Results: In total, 170 studies from eighteen different disease models were identified and grouped into fourteen broad themes: alterations in cellular energetics and metabolism, biochemical, cortical excitability, epigenetic regulation, mitochondrial function, neuroinflammation, neuroplasticity, neuroprotection, neurotransmitter function, nociception, redox balance, signalling pathways, synaptic transmission and vascular supply., Discussion: The mechanisms presented centred around the reduction of inflammation and oxidative stress as well as a reduction in nervous system excitability. Given the multiple potential mechanisms presented, it is likely that many of these are involved synergistically and undergo adaptive processes within the human body, and controlled animal models that limit the investigation to a particular pathway in isolation may reach differing conclusions. Attention is required when translating this information to human chronic pain populations owing to the limitations outlined from the animal research.

Sodium-glucose cotransporter-2 inhibitors (SGLT2is), such as empagliflozin, lower blood glucose in type 2 diabetes mellitus and improve cardiorenal outcomes regardless of diabetes presence. Whether SGLT2is exert any effects on the brain's metabolism has not been studied. We conducted a single-arm clinical trial to investigate the effects of once daily administration of oral empagliflozin (25 mg) for 14 days on systemic and brain metabolism in 21 non-diabetics aged 55 years old or older. Empagliflozin lowered circulating insulin and elevated β-hydroxybutyrate over 34-h periods, both following its first administration and after 14 days of daily administration, with minor alterations in glucose homeostasis. Levels of phosphorylated insulin-like growth factor-1 receptor (pIGF-1R), phosphorylated insulin receptor (pIR), phosphorylated-in-tyrosine insulin receptor substrate-1 (pY-IRS-1), and phosphorylated protein kinase B or AKT (pAKT) were increased in extracellular vesicles enriched for neuronal origin (NEVs) following the first empagliflozin administration, but not after 14 days. Our finding of IGF-1R upregulation in NEVs is promising because several post-mortem and epidemiological studies support the idea that upregulation of IGF signaling may protect against Alzheimer's disease (AD). Moreover, our finding showing activation of insulin signaling and, in particular, the canonical pathway (pIR, pY-IRS-1, pAKT) in NEVs is important because such changes have been repeatedly associated with neuronal survival. Using brain magnetic resonance spectroscopy (MRS), we detected decreased concentrations of the excitatory neurotransmitter glutamate and its precursor glutamine after empagliflozin administration. This finding is also encouraging since glutamatergic excitotoxicity has long been implicated in AD pathology. Overall, our findings may motivate the repurposing of SGLT2is for use in AD and other, related diseases that are characterized by downregulation of IGF-1/insulin signaling in neurons and excitotoxicity.

Ketosis is a common metabolic disorder in peripartal dairy cows that is caused by excessive mobilization of fat and incomplete hepatic metabolism of fatty acids (FFA). Recent data in nonruminant models revealed that sortilin 1 (SORT1) is involved in a variety of lipid metabolism-related diseases. It plays important roles in the regulation of triglyceride (TAG) and total cholesterol (TC) levels. In this study, we first used liver biopsies from healthy cows (serum β-hydroxybutyrate concentration <0.6 mM) and cows diagnosed with clinical ketosis (serum β-hydroxybutyrate concentration >3.0 mM) to assess alterations in cholesterol synthesis, transport, and excretion. Then, to assess mechanistic links between SORT1 and fatty acid-mediated cholesterol metabolism, hepatocytes isolated from 4 healthy female calves (1 d old, 35-45 kg) were challenged with or without a mixture of free fatty acids (FFA; 1.2 mM) to induce metabolic stress. Hepatocytes were then treated with empty adenovirus vectors (with green fluorescent protein; Ad-GFP) or with SORT1-overexpressing adenovirus (Ad-SORT1) for 6 h or with SORT1 inhibitor (SORT1i) for 2 h, followed by a challenge with (Ad-GFP+FFA, Ad-SORT1+FFA, or SORT1i+FFA) or without (Ad-GFP, Ad-SORT1, or SORT1i) 1.2 mM FFA mixture for 12 h. Data analysis of calf hepatocyte treatment comparisons were assessed by 2-way ANOVA, and multiplicity for each experiment was adjusted using the Bonferroni procedure. Expression levels of factors related to cholesterol synthesis, transport, and excretion in liver tissue of cows with ketosis was lower. Hepatocytes challenged with FFA had lower concentrations of TC and mRNA and protein abundances of sterol regulatory element-binding protein 2 (SREBF2), acetyl acyl coenzyme A-cholesterol acyltransferase 2 (ACAT2), ATP-binding cassette transporter A1 (ABCA1), ABC subfamily G member 5 (ABCG5), and ABC subfamily G member 8 (ABCG8). Compared with FFA challenge alone, SORT1i + FFA led to greater protein abundance of 3-hydroxy-3-methyl-glutaryl coenzyme A reductase (HMGCR), ACAT2, and ABCG5, and greater mRNA abundance of ABCG5. Compared with FFA challenge alone, SORT1 overexpression led to lower protein abundance of SREBF2. In contrast, protein abundance of ABCA1 was greater. Overall, our data suggested that exogenous FFA induced abnormal cholesterol metabolism in hepatocytes, whereas a high abundance of SORT1 affected cholesterol esterification and potentially influx into bile. Thus, downregulation of hepatic SORT1 might be a cholesterol-regulated protective mechanism in the presence of a marked increase in FFA., (© 2022, The Authors. Published by Elsevier Inc. and Fass Inc. on behalf of the American Dairy Science Association®. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).)

Ketone bodies are small compounds derived from fatty acids that behave as an alternative mitochondrial energy source when insulin levels are low, such as during fasting or strenuous exercise. In addition to the metabolic function of ketone bodies, they also have several signaling functions separate from energy production. In this perspective, we review the main current data referring to ketone bodies in correlation with nutrition and metabolic pathways as well as to the signaling functions and the potential impact on clinical conditions. Data were selected following eligibility criteria accordingly to the reviewed topic. We used a set of electronic databases (Medline/PubMed, Scopus, Web of Sciences (WOS), Cochrane Library) for a systematic search until July 2022 using MeSH keywords/terms (i.e., ketone bodies, BHB, acetoacetate, inflammation, antioxidant, etc.). The literature data reported in this review need confirmation with consistent clinical trials that might validate the results obtained in in vitro and in vivo in animal models. However, the data on exogenous ketone consumption and the effect on the ketone bodies' brain uptake and metabolism might spur the research to define the acute and chronic effects of ketone bodies in humans and pursue the possible implication in the prevention and treatment of human diseases. Therefore, additional studies are required to examine the potential systemic and metabolic consequences of ketone bodies.

Objective: Aberrant ketogenesis is correlated with the degree of steatosis in non-alcoholic fatty liver disease (NAFLD) patients, and an inborn error of ketogenesis (mitochondrial HMG-CoA synthase deficiency) is commonly associated with the development of the fatty liver. Here we aimed to determine the impact of Hmgcs2-mediated ketogenesis and its modulations on the development and treatment of fatty liver disease., Methods: Loss- and gain-of-ketogenic function models, achieved by Hmgcs2 knockout and overexpression, respectively, were utilized to investigate the role of ketogenesis in the hepatic lipid accumulation during postnatal development and in a high-fat diet-induced NAFLD mouse model., Results: Ketogenic function was decreased in NAFLD mice with a reduction in Hmgcs2 expression. Mice lacking Hmgcs2 developed spontaneous fatty liver phenotype during postnatal development, which was rescued by a shift to a low-fat dietary composition via early weaning. Hmgcs2 heterozygous adult mice, which exhibited lower ketogenic activity, were more susceptible to diet-induced NAFLD development, whereas HMGCS2 overexpression in NAFLD mice improved hepatosteatosis and glucose homeostasis., Conclusions: Our study adds new knowledge to the field of ketone body metabolism and shows that Hmgcs2-mediated ketogenesis modulates hepatic lipid regulation under a fat-enriched nutritional environment. The regulation of hepatic ketogenesis may be a viable therapeutic strategy in the prevention and treatment of hepatosteatosis., (Copyright © 2022 The Author(s). Published by Elsevier GmbH.. All rights reserved.)

Ketosis occurs most frequently in the peripartal period and is associated with liver injury and steatosis. Lysosomes serve as the terminal degradative station and contribute to liver homeostasis through their role in the digestion of dysfunctional organelles and lipid droplets. Transcription factor EB (TFEB) has been identified as a master regulator of lysosomal function. Thus, the objective of the present study was to investigate the status of lysosomal function and TFEB transcriptional activity and potential changes in abundance of upstream effectors of TFEB identified in nonruminants, including mechanistic target of rapamycin kinase complex 1 (mTORC1), protein kinase B (Akt), glycogen synthase kinase β (GSK3β), and extracellular signal-regulated kinase1/2 (ERK1/2), and to explore which factor induces the above changes. Liver and blood samples were collected from healthy cows (n = 10) and ketotic cows (n = 10) that had a similar number of lactations (median = 3, range = 2-4) and days in milk (median = 6 d, range = 3-9 d). Calf hepatocytes were isolated from Holstein calves and treated with 10 ng/mL growth hormone (GH), 3.0 mM β-hydroxybutyrate (BHB), 1.5 ng/mL interleukin-18 (IL-18), 0.15 ng/mL tumor necrosis factor-α (TNF-α), or 1.2 mM free fatty acid (FFA) for 12 h. Serum levels of FFA and activities of alanine aminotransferase and aspartate aminotransferase were greater in ketotic cows, whereas glucose was lower. Additionally, ketotic dairy cows exhibited higher serum concentrations of GH, IL-18, and TNF-α, and lower serum concentration of insulin. The lower protein abundance of lysosome-associated membrane protein 1 (LAMP1) and mRNA abundance of LAMP1 indicated that hepatic lysosomal mass was lower in ketotic cows. Furthermore, lower protein abundance of cathepsin D (CTSD) and mRNA abundance of CTSD and V0 domain of the vacuolar ATPase along with lower activity of β-N-acetylglucosaminidase indicated impairment in hepatic lysosomal function due to ketosis. The lower nuclear abundance, total protein, and mRNA abundance of TFEB and peroxisome proliferator-activated receptor γ coactivator 1 α along with greater phosphorylated (p)-TFEB in the liver of ketotic cows indicated an impairment of hepatic TFEB transcriptional activity. The protein abundances of phosphorylated mTOR (p-mTOR) and its downstream effectors ribosomal protein S6 kinase B (RPS6KB) and eukaryotic factor 4E-binding protein 1 (EIF4EBP1) were greater, whereas p-Akt, p-GSK3β, and p-ERK1/2 were lower in the liver of ketotic cows. Importantly, elevated phosphorylation of mTOR, RPS6KB, and EIF4EBP1 was observed in calf hepatocytes treated with GH, BHB, IL-18, TNF-α, and FFA. Moreover, BHB, TNF-α, and FFA, not GH and IL-18, reduced TFEB transcriptional activity and impaired lysosomal function in calf hepatocytes. Taken together, these data suggest that BHB, TNF-α, and FFA overactivate the hepatic mTORC1 signaling pathway during ketosis and further impaired TFEB transcriptional activity and lysosomal function, which may contribute to liver injury and steatosis., (The Authors. Published by Elsevier Inc. and Fass Inc. on behalf of the American Dairy Science Association®. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).)

Mitochondrial hydroxymethylglutaryl-CoA synthase 2 (HMGCS2) is the rate-limiting enzyme in ketogenesis. The liver expresses high levels of HMGCS2 constitutively as the main ketogenic organ. It has been suggested that the kidney could be ketogenic as HMGCS2 is expressed in the kidney during fasting and diabetic conditions. However, definitive proof of the capacity for the kidney to produce ketones is lacking. We demonstrated that during fasting, HMGCS2 expression is induced in the proximal tubule of the kidney and is peroxisome proliferator activated receptor-α dependent. Mice with kidney-specific Hmgcs2 deletion showed a minor, likely physiologically insignificant, decrease in circulating ketones during fasting. Conversely, liver-specific Hmgcs2 knockout mice exhibited a complete loss of fasting ketosis. Together, these findings indicate that renal HMGCS2 does not significantly contribute to global ketone production and that during fasting, the increase in circulating ketones is solely dependent on hepatic HMGCS2. Proximal tubule HMGCS2 serves functions other than systemic ketone provision. NEW & NOTEWORTHY The mitochondrial enzyme hydroxymethylglutaryl-CoA synthase 2 (HMGCS2) catalyzes the rate-limiting step of ketogenesis. Although the liver constitutively expresses HMGCS2 and is considered the main ketogenic organ, HMGCS2 is induced in the kidney during fasting, leading to the proposal that the kidney contributes to fasting ketosis. We showed kidney HMGCS2 does not contribute to circulating ketones during fasting and cannot compensate for hepatic ketogenic insufficiency.

Sodium-glucose cotransporter 2 (SGLT2) inhibitors have been shown to increase ketone bodies in patients with type 2 diabetes; however, the underlying mechanisms have not been fully elucidated. Here we examined the effect of the SGLT2 inhibitor dapagliflozin (1 mg/kg/day, formulated in a water, PEG400, ethanol, propylene glycol solution, 4 weeks) on lipid metabolism in obese Zucker rats. Fasting FFA metabolism was assessed in the anesthetized state using a [9,10- 3 H(N)]-palmitic acid tracer by estimating rates of plasma FFA appearance (R a ), whole-body FFA oxidation (R ox ), and nonoxidative disposal (R st ). In the liver, clearance (K β-ox ) and flux (R β-ox ) of FFA into β-oxidation were estimated using [9,10- 3 H]-(R)-bromopalmitate/[U- 14 C]palmitate tracers. As expected, dapagliflozin induced glycosuria and a robust antidiabetic effect; treatment reduced fasting plasma glucose and insulin, lowered glycated hemoglobin, and increased pancreatic insulin content compared with vehicle controls. Dapagliflozin also increased plasma FFA, R a , R ox , and R st with enhanced channeling toward oxidation versus storage. In the liver, there was also enhanced channeling of FFA to β-oxidation, with increased K β-ox , R β-ox and tissue acetyl-CoA, compared with controls. Finally, dapagliflozin increased hepatic HMG-CoA and plasma β-hydroxybutyrate, consistent with a specific enhancement of ketogenesis. Since ketogenesis has not been directly measured, we cannot exclude an additional contribution of impaired ketone body clearance to the ketosis. In conclusion, this study provides evidence that the dapagliflozin-induced increase in plasma ketone bodies is driven by the combined action of FFA mobilization from adipose tissue and diversion of hepatic FFA toward β-oxidation., Competing Interests: Conflict of interest All authors are or were employed by AstraZeneca and have shares in the company., (Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.)

Subclinical ketosis (SCK) in dairy cows, a common metabolic disorder during the peripartal period, is accompanied by systemic inflammation. Excessive release of azurophil granule (AG) contents during degranulation of polymorphonuclear neutrophils (PMN) could contribute to systemic inflammation in SCK cows. Although the increase in blood free fatty acids (FFA) in SCK cows may promote AG degranulation from PMN, the underlying mechanisms are unclear. Thirty multiparous cows (within 3 wk postpartum) with similar lactation numbers (median = 3, range = 2-4) and days in milk (median = 6, range = 3-15) were classified based on serum β-hydroxybutyrate (BHB) level as control (n = 15, BHB < 0.6 mM) or SCK (n = 15, 1.2 mM < BHB < 3.0 mM). Cows with SCK had greater levels of serum haptoglobin, serum amyloid A, IL-1β, IL-6, IL-8 and tumor necrosis factor-α. These proinflammatory factors had strong positive correlations with myeloperoxidase (MPO), a marker protein of PMN AG, whose content was greater in the serum of SCK cows. Both the number of AG and the protein abundance of MPO were lower in PMN isolated from SCK cows. Additionally, we found a greater ratio of blood CH138A + /CD63 high cells and greater mean fluorescence intensity of CD63 on the PMN membrane, further confirming the greater degree of AG degranulation in cows with SCK. In vitro FFA dose response (0, 0.3, 0.6, 1.2, and 2.4 mM for 4 h) and time course (0, 0.5, 1, 2, and 4 h with 0.6 mM) experiments were performed on PMN isolated from control cows. The increase in MPO content in extracellular supernatant resulting from those experiments led to the selection of 0.6 mM FFA for 1 h duration as conditions for subsequent studies. After FFA treatment, release of intracellular MPO was increased along with increased levels of CD63 mean fluorescence intensity on the PMN membrane, confirming that FFA promoted degranulation of AG. In addition, FFA treatment increased reactive oxygen species (ROS) production by PMN, an effect that was attenuated by incubation with diphenyleneiodonium chloride (DPI), a NADPH oxidase-derived ROS inhibitor. The mitochondrial-derived ROS inhibitor carbonyl cyanide 4-(trifluoromethoxy) phenylhydrazone (FCCP) did not affect ROS in response to FFA treatment. Treatment with FFA increased p47 phosphorylation and mRNA abundance of NCF1, NCF2, and CYBB in PMN. Furthermore, DPI, but not FCCP, dampened the degranulation of PMN AG induced by FFA in vitro. These data suggested that the degranulation of AG in PMN induced by FFA was mediated by NADPH oxidase-derived ROS. As verified ex vivo, PMN from SCK cows had greater levels of ROS, phosphorylation of p47, and mRNA abundance of NCF1, NCF2, and CYBB. Overall, the present study revealed that high blood concentrations of FFA in SCK cows induce the production of NADPH oxidase-derived ROS, thereby promoting degranulation of AG in PMN. The stimulatory effect of FFA on the release of AG content during degranulation, especially MPO, provides a new insight into the systemic inflammation experienced by peripartal cows with SCK., (The Authors. Published by Elsevier Inc. and Fass Inc. on behalf of the American Dairy Science Association®. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).)

Optimal cognitive functions are necessary for activities of daily living and self-independence. Cognitive abilities are acquired during early childhood as part of progressive neurodevelopmental milestones; unfortunately, regressive changes can occur as part of physiological aging, or more ominously, pathological diseases, such as Alzheimer's disease (AD). Cases of AD and its milder subset, mild cognitive impairment (MCI), are rising and would impose a burdensome impact beyond the individual level. Various dietary and nutritional approaches have potential for promising results in managing cognitive deterioration. Glucose is the core source of bioenergy in the body; however, glucose brain metabolism could be affected in aging cells or due to disease development. Ketone bodies are an efficient alternate fuel source that could compensate for the deficient glycolytic metabolism upon their supra-physiologic availability in the blood (ketosis), which, in turn, could promote cognitive benefits and tackle disease progression. In this review, we describe the potential of ketogenic approaches to produce cognitive benefits in healthy individuals, as well as those with MCI and AD. Neurophysiological changes of the cognitive brain in response to ketosis through neuroimaging modalities are also described in this review to provide insight into the ketogenic effect on the brain outside the framework of purely molecular explanations.

Background: Ketone bodies have been proposed as an "energy rescue" for the Alzheimer's disease (AD) brain, which underutilizes glucose. Prior research has shown that oral ketone monoester (KME) safely induces robust ketosis in humans and has demonstrated cognitive-enhancing and pathology-reducing properties in animal models of AD. However, human evidence that KME may enhance brain ketone metabolism, improve cognitive performance and engage AD pathogenic cascades is scarce., Objectives: To investigate the effects of ketone monoester (KME) on brain metabolism, cognitive performance and AD pathogenic cascades in cognitively normal older adults with metabolic syndrome and therefore at higher risk for AD., Design: Double-blinded randomized placebo-controlled clinical trial., Setting: Clinical Unit of the National Institute on Aging, Baltimore, US., Participants: Fifty cognitively intact adults ≥ 55 years old, with metabolic syndrome., Intervention: Drinks containing 25 g of KME or isocaloric placebo consumed three times daily for 28 days., Outcomes: Primary: concentration of beta-hydroxybutyrate (BHB) in precuneus measured with Magnetic Resonance Spectroscopy (MRS). Exploratory: plasma and urine BHB, multiple brain and muscle metabolites detected with MRS, cognition assessed with the PACC and NIH toolbox, biomarkers of AD and metabolic mediators in plasma extracellular vesicles, and stool microbiome., Discussion: This is the first study to investigate the AD-biomarker and cognitive effects of KME in humans. Ketone monoester is safe, tolerable, induces robust ketosis, and animal studies indicate that it can modify AD pathology. By conducting a study of KME in a population at risk for AD, we hope to bridge the existing gap between pre-clinical evidence and the potential for brain-metabolic, pro-cognitive, and anti-Alzheimer's effects in humans., Competing Interests: The authors declare no conflicts of interest.

Hypoxia inducible factor alpha (HIF1α) is associated with neuroprotection conferred by diet-induced ketosis but the underlying mechanism remains unclear. In this study we use a ketogenic diet in rodents to induce a metabolic state of chronic ketosis, as measured by elevated blood ketone bodies. Chronic ketosis correlates with neuroprotection in both aged and following focal cerebral ischaemia and reperfusion (via middle cerebral artery occlusion, MCAO) in mouse and rat models. Ketone bodies are known to be used efficiently by the brain and metabolism of ketone bodies is associated with increased cytosolic succinate levels that inhibits prolyl hydroxylases allowing HIF1α to accumulate. Ketosis also regulates inflammatory pathways, and HIF1α is reported to be essential for gene expression of interleukin10 (IL10). Therefore we hypothesised that ketosis-stabilised HIF1α modulates the expression of inflammatory cytokines orchestrating neuroprotection. To test changes in cytokine levels in rodent brain, eight-week-old rats were fed either the standard chow diet (SD) or the ketogenic (KG) diet for 4 weeks before ischaemia experiments (MCAO) were performed and the brain tissues were collected. Consistent with our hypothesis, immunoblotting analysis shows IL10 levels were significantly higher in KG diet rat brain compared to SD, whereas the TNFα and IL6 levels were significantly lower in the brains of KG diet fed group., (© 2022. Springer Nature Switzerland AG.)

Background: Mild cognitive impairment (MCI) is characterized by a decline in cognition and mainly affects older individuals above the age of 60. The global incidence of MCI varies, but it is often underdiagnosed and untreated. There is a distinct lack of approved pharmacologic options to treat MCI. There is, however, evidence to support the efficacy of nutritional interventions, such as ketogenic supplements/diets, which offer ketones as an alternative energy source to brain cells. This article explores the effect of ketones on metabolic activity in the brain and the mechanisms by which ketogenic medium-chain triglycerides (kMCTs) induce ketosis in patients with MCI., Key Takeaways: This article reviews the effect of ketogenic supplements/diets on brain metabolism, including evidence supporting the efficacy of ketones as an efficient fuel for the brain. It discusses the use of oral nutritional ketogenic supplements, with particular reference to the 6-month randomized controlled BENEFIC trial, which showed that consumption of a kMCT drink, BrainXpert Energy Complex, improved cognitive performance in individuals with MCI compared with placebo., Conclusion: While there is a need for more long-term studies, results from the BENEFIC trial revealed the benefits of a brain-specific ketogenic supplement, as a nutritional intervention, on cognitive performance in individuals with MCI., (© 2021. The Author(s).)

Ketone body production, an alternative fuel upon low glucose availability, reduces hepatic fat accumulation. However, its clinical implications have not been established in patients with nonalcoholic fatty liver disease (NAFLD). We investigated the association between spontaneous fasting ketonuria and liver fibrosis in patients with NAFLD without prediabetes and diabetes mellitus (DM). A total of 6202 patients with ultrasound confirmed NAFLD without prediabetes and DM were enrolled in the study. Using low cut off values of NAFLD fibrosis score (NFS) and fibrosis-4, liver fibrosis was defined as an intermediate-high probability of advanced liver fibrosis. Of the 6202 NAFLD patients, 360 (5.8%) had ketonuria. Compared to the patients without ketonuria, patients with ketonuria were younger (41.1 vs. 44.6 years, p < 0.001), had lower levels of glucose (87.2 vs. 91.0 mg/dL, p < 0.001), and homeostatic model assessment for insulin resistance (1.0 vs. 1.5, p < 0.001). The presence of ketonuria had an inverse association with liver fibrosis, assessed using both NFS (final adjusted odds ratio [aOR], 0.67; 95% confidence interval [CI], 0.45-1.01) and fibrosis-4 (aOR, 0.58; 95% CI, 0.40-0.84). The presence of ketonuria in NAFLD patients without prediabetes and DM may have favorable metabolic effects compared to the absence of ketonuria, independent of traditional metabolic factors.

Objective: To examine the feasibility, safety, systemic biological activity, and cerebral activity of a ketogenic dietary intervention in patients with glioma., Methods: Twenty-five patients with biopsy-confirmed World Health Organization grade 2 to 4 astrocytoma with stable disease after adjuvant chemotherapy were enrolled in an 8-week Glioma Atkins-Based Diet (GLAD). GLAD consisted of 2 fasting days (calories <20% calculated estimated needs) interleaved between 5 modified Atkins diet days (net carbohydrates ≤20 g/d) each week. The primary outcome was dietary adherence by food records. Markers of systemic and cerebral activity included weekly urine ketones, serum insulin, glucose, hemoglobin A1c, insulin-like growth factor-1, and magnetic resonance spectroscopy at baseline and week 8., Results: Twenty-one patients (84%) completed the study. Eighty percent of patients reached ≥40 mg/dL urine acetoacetate during the study. Forty-eight percent of patients were adherent by food record. The diet was well tolerated, with two grade 3 adverse events (neutropenia, seizure). Measures of systemic activity, including hemoglobin A1c, insulin, and fat body mass, decreased significantly, while lean body mass increased. Magnetic resonance spectroscopy demonstrated increased ketone concentrations (β-hydroxybutyrate [bHB] and acetone) in both lesional and contralateral brain compared to baseline. Average ketonuria correlated with cerebral ketones in lesional (tumor) and contralateral brain (bHB R s = 0.52, p = 0.05). Subgroup analysis of isocitrate dehydrogenase-mutant glioma showed no differences in cerebral metabolites after controlling for ketonuria., Conclusion: The GLAD dietary intervention, while demanding, produced meaningful ketonuria and significant systemic and cerebral metabolic changes in participants. Ketonuria in participants correlated with cerebral ketone concentration and appears to be a better indicator of systemic activity than patient-reported food records., Trial Registration Information: ClinicalTrials.gov Identifier: NCT02286167., (© 2021 American Academy of Neurology.)

Background: A better comprehension of the redox status during the periparturient period may facilitate the development of management and nutritional solutions to prevent subclinical hyperketonemia (SCHK) and subclinical hypocalcemia (SCHC) in dairy goats. We aimed to evaluate the variation in the redox status of dairy goats with SCHK and SCHC during their periparturient periods. Guanzhong dairy goats (n = 30) were assigned to SCHK (n = 10), SCHC (n = 10), and healthy (HEAL, n = 10) groups based on their blood β-hydroxybutyrate (BHBA) and calcium (Ca) concentrations. Blood were withdrawn from goats every week from 3 weeks before the expected parturition date to 3 weeks post-kidding. On the same day, the body condition scores (BCS) were evaluated, and the milk yield was recorded for each goat. The metabolic profile parameters and the indicators of oxidative status were determined by using the standard biochemical techniques., Results: In comparison with the HEAL goats, SCHK and SCHC goats presented with a more dramatic decline of BCS post-kidding and a significant decrease in the milk yield at 2- and 3-weeks postpartum, ignoring the obvious increase at 1-week postpartum. The levels of non-esterified fatty acids (NEFA) peaked at parturition, exhibiting significantly higher levels from 1-week prepartum to the parturition day in the SCHK and SCHC groups. The malondialdehyde (MDA) concentration was increased in the SCHK goats from 1-week antepartum until 3-weeks postpartum, with its concentration being significantly higher in the SCHC goats at parturition. The hydrogen peroxide (H 2 O 2 ) concentration was significantly lower in the SCHK and SCHC goats from 2-weeks antepartum to 1-week post-kidding. The total antioxidant capacity (T-AOC) and the superoxide dismutase (SOD) level were decreased at 1-week antepartum in the SCHK and SCHC goats, respectively. The glutathione peroxidase (GSH-Px) level was increased in the SCHK and SCHC goats during the early lactation period., Conclusions: The SCHK and SCHC goats exerted more efforts to maintain their redox homeostasis and to ensure the production performance than the HEAL goats during their periparturient period, probably owing to more intense fat mobilization and lipid peroxidation in the former.

Objectives: A prolonged vasoconstriction/hypoperfusion/hypoxic event follows self-terminating focal seizures. The ketogenic diet (KD) has demonstrated efficacy as a metabolic treatment for intractable epilepsy and other disorders but its effect on local brain oxygen levels is completely unknown. This study investigated the effects of the KD on tissue oxygenation in the hippocampus before and after electrically elicited (kindled) seizures and whether it could protect against a seizure-induced learning impairment. We also examined the effects of the ketone β-hydroxybutyrate (BHB) as a potential underlying mechanism., Methods: Male and female rats were given access to one of three diet protocols 2 weeks prior to the initiation of seizures: KD, caloric restricted standard chow, and ad libitum standard chow. Dorsal hippocampal oxygen levels were measured prior to initiation of diets as well as before and after a 10-day kindling paradigm. Male rats were then tested on a novel object recognition task to assess postictal learning impairments. In a separate cohort, BHB was administered 30 min prior to seizure elicitation to determine whether it influenced oxygen dynamics., Results: The KD increased dorsal hippocampal oxygen levels, ameliorated postictal hypoxia, and prevented postictal learning impairments. Acute BHB administration did not alter oxygen levels before or after seizures., Interpretation: The ketogenic diet raised brain oxygen levels and attenuated severe postictal hypoxia likely through a mechanism independent of ketosis and shows promise as a non-pharmacological treatment to prevent the postictal state., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)

Nutritional ketosis has promise for treating Parkinson's disease. Three previous studies explored the use of a ketogenic diet in cohorts with Parkinson's disease, and, while not conclusive, the data suggest non-motor symptom benefit. Before the ketogenic diet can be considered as a therapeutic option, it is important to establish with greater certainty that there is a reliable symptomatic benefit: which symptoms or groups of symptoms are impacted (if non-motor symptoms, which ones, and by which mechanism), what timescale is needed to obtain benefit, and how large an effect size can be achieved? To accomplish this, further investigation into the disease mechanisms based on pre-clinical data and hints from the clinical outcomes to date is useful to understand target engagement and gauge which mechanism could lead to a testable hypothesis. We review research pertaining to ketogenic diet, exogenous ketones, fasting, clinical studies, and theoretical review papers regarding therapeutic mechanisms from direct ketone body signaling and indirect metabolic effects. Through discussion of these findings and consideration of whether the ketogenic diet can be regarded as therapeutically useful for adjunctive therapy for Parkinson's disease, we identify remaining questions for the clinician to consider prior to recommending this diet., (© 2021. This is a U.S. government work and not under copyright protection in the U.S.; foreign copyright protection may apply.)

Many of the metabolic effects evoked by the ketogenic diet mimic the actions of fasting and the benefits of the ketogenic diet are often attributed to these similarities. Since fasting is a potent autophagy inductor in vivo and in vitro it has been hypothesized that the ketogenic diet may upregulate autophagy. The aim of the present study was to provide a comprehensive evaluation of the influence of the ketogenic diet on the hepatic autophagy. C57BL/6N male mice were fed with two different ketogenic chows composed of fat of either animal or plant origin for 4 weeks. To gain some insight into the time frame for the induction of autophagy on the ketogenic diet, we performed a short-term experiment in which animals were fed with ketogenic diets for only 24 or 48 h. The results showed that autophagy is upregulated in the livers of animals fed with the ketogenic diet. Moreover, the size of the observed effect was likely dependent on the diet composition. Subsequently, the markers of regulatory pathways that may link ketogenic diet action to autophagy were measured, i.e., the activity of mTORC1, activation of AMPK, and the levels of SIRT1, p53, and FOXO3. Overall, observed treatment-specific effects including the upregulation of SIRT1 and downregulation of FOXO3 and p53. Finally, a GC/MS analysis of the fatty acid composition of animals' livers and the chows was performed in order to obtain an idea about the presence of specific compounds that may shape the effects of ketogenic diets on autophagy., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Diabetic retinopathy (DR) is a well-recognized microvascular complication of diabetes. Growing evidence suggests that, in addition to retinal vascular damage, there is significant damage to retinal neural tissue in DR. Studies reveal neuronal damage before clinically evident vascular lesions and DR is now classified as a neurovascular complication. Hyperglycemia causes retinal damage through complex metabolic pathways leading to oxidative stress, inflammation, vascular damage, capillary ischemia, and retinal tissue hypoxia. Retinal hypoxia is further worsened by high oxygen consumption in the rods. Persistent hypoxia results in increases in vascular endothelial growth factor (VEGF) and other pro-angiogenic factors leading to proliferative DR/macular edema and progressive visual impairment. Optimal glucose control has favorable effects in DR. Other treatments for DR include laser photocoagulation, which improves retinal oxygenation by destroying the high oxygen consuming rods and their replacement by low oxygen consuming glial tissue. Hypoxia is a potent stimulator of VEGF, and intravitreal anti-VEGF antibodies are effective in regressing macular edema and in some studies, retinal neovascularization. In this review, we highlight the complex pathophysiology of DR with a focus on retinal oxygen/fuel consumption and hypoxic damage to retinal neurons. We discuss potential mechanisms through which sodium-glucose cotransporter 2 (SGLT2) inhibitors improve retinal hypoxia-through ketone bodies, which are energetically as efficient as glucose and yield more ATP per molecule of oxygen consumed than fat, with less oxidative stress. Retinal benefits would occur through improved fuel energetics, less hypoxia and through the anti-inflammatory/oxidative stress effects of ketone bodies. Well-designed studies are needed to explore this hypothesis., (Published by Oxford University Press on behalf of the Endocrine Society 2021.)