Emerging Pathophysiological Roles of Ketone Bodies.

The discovery of insulin approximately a century ago greatly improved the management of diabetes, including many of its life-threatening acute complications like ketoacidosis. This breakthrough saved many lives and extended the healthy lifespan of many patients with diabetes. However, there is still a negative perception of ketone bodies stemming from ketoacidosis. Originally, ketone bodies were thought of as a vital source of energy during fasting and exercise. Furthermore, in recent years, research on calorie restriction and its potential impact on extending healthy lifespans, as well as studies on ketone bodies, have gradually led to a reevaluation of the significance of ketone bodies in promoting longevity. Thus, in this review, we discuss the emerging and hidden roles of ketone bodies in various organs, including the heart, kidneys, skeletal muscles, and brain, as well as their potential impact on malignancies and lifespan.

Ketones and the Heart: Metabolic Principles and Therapeutic Implications.

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.

Ketone bodies as chemical signals for the immune system.

Ketone bodies are short-chain fatty acids produced by the liver during periods of limited glucose availability, such as during fasting or low carbohydrate feeding. Recent studies have highlighted important nonmetabolic functions of the most abundant ketone body, ß-hydroxybutyrate (BHB). Notably, many of these functions, including limiting specific sources of inflammation, histone deacetylase inhibition, NFκB inhibition, and GPCR stimulation, are particularly important to consider in immune cells. Likewise, dietary manipulations like caloric restriction or ketogenic diet feeding have been associated with lowered inflammation, improved health outcomes, and improved host defense against infection. However, the underlying mechanisms of the broad benefits of ketosis remain incompletely understood. In this Perspective, we contextualize the current state of the field of nonmetabolic functions of ketone bodies specifically in the immune system and speculate on the molecular explanations and broader physiological significance.

The rumen microbiota contributed to the development of mastitis induced by subclinical ketosis.

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.

One third of cases of new-onset diabetic ketosis in adults are associated with ketosis-prone type 2 diabetes-A systematic review and meta-analysis.

AIMS: Ketosis-prone type 2 diabetes was defined by the World Health Organization in 2019. According to the literature, the diagnosis is based on the presence of ketosis, islet autoantibody negativity and preserved insulin secretion. Our meta-analysis assessed the prevalence and clinical characteristics of ketosis-prone type 2 diabetes among patients hospitalised with diabetic ketoacidosis (DKA) or ketosis. METHODS: The systematic search was performed in five main databases as of 15 October 2021 without restrictions. We calculated the pooled prevalence of ketosis-prone type 2 diabetes (exposed group) within the diabetic population under examination, patients with ketoacidosis or ketosis, to identify the clinical characteristics, and we compared it to type 1 diabetes (the comparator group). The random effects model provided pooled estimates as prevalence, odds ratio and mean difference (MD) with 95% confidence intervals. RESULTS: Eleven articles were eligible for meta-analysis, thus incorporating 2010 patients of various ethnic backgrounds. Among patients presenting with DKA or ketosis at the onset of diabetes, 35% (95% CI: 24%-49%) had ketosis-prone type 2 diabetes. These patients were older (MD = 11.55 years; 95% CI: 5.5-17.6) and had a significantly higher body mass index (BMI) (MD = 5.48 kg/m2 ; 95% CI: 3.25-7.72) than those with type 1 diabetes. CONCLUSIONS: Ketosis-prone type 2 diabetes accounts for one third of DKA or ketosis at the onset of diabetes in adults. These patients are characterised by islet autoantibody negativity and preserved insulin secretion. They are older and have a higher BMI compared with type 1 diabetes. C-peptide and diabetes-related autoantibody measurement is essential to identify this subgroup among patients with ketosis at the onset of diabetes.

Epithelial-mesenchymal transition-related signaling pathways in gastric Cancer cells distinctively respond to long-term experimental ketosis.

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.

Eu- or hypoglycemic ketosis and ketoacidosis in children: a review.

The last decade has been characterized by exciting findings on eu- or hypoglycemic ketosis and ketoacidosis. This review emphasizes the following five key points: 1. Since the traditional nitroprusside-glycine dipstick test for urinary ketones is often falsely negative, the blood determination of ß-hydroxybutyrate, the predominant ketone body, is currently advised for a comprehensive assessment of ketone body status; 2. Fasting and infections predispose to relevant ketosis and ketoacidosis especially in newborns, infants, children 7 years or less of age, and pregnant, parturient, or lactating women; 3. Several forms of carbohydrate restriction (typically less than 20% of the daily caloric intake) are employed to induce ketosis. These ketogenic diets have achieved great interest as antiepileptic treatment, in the management of excessive body weight, diabetes mellitus, and in sport training; 4. Intermittent fasting is more and more popular because it might benefit against cardiovascular diseases, cancers, neurologic disorders, and aging; 5. Gliflozins, a new group of oral antidiabetics inhibiting the renal sodium-glucose transporter 2, are an emerging cause of eu- or hypoglycemic ketosis and ketoacidosis. In conclusion, the role of ketone bodies is increasingly recognized in several clinical conditions. In the context of acid-base balance evaluation, it is advisable to routinely integrate both the assessment of lactic acid and ß-hydroxybutyrate.

Ketogenic diet for epilepsy and obesity: Is it the same?

The term "ketogenic diet" (KD) is used for a wide variety of diets with diverse indications ranging from obesity to neurological diseases, as if it was the same diet. This terminology is confusing for patients and the medical and scientific community. The term "ketogenic" diet implies a dietary regimen characterized by increased levels of circulating ketone bodies that should be measured in blood (beta-hydroxybutyrate), urine (acetoacetate) or breath (acetone) to verify the "ketogenic metabolic condition". Our viewpoint highlights that KDs used for epilepsy and obesity are not the same; the protocols aimed at weight loss characterized by low-fat, low-CHO and moderate/high protein content are not ketogenic by themselves but may become mildly ketogenic when high calorie restriction is applied. In contrast, there are standardized protocols for neurological diseases treatment for which ketosis has been established to be part of the mechanism of action. Therefore, in our opinion, the term ketogenic dietary therapy (KDT) should be reserved to the protocols considered for epilepsy and other neurological diseases, as suggested by the International Study Group in 2018. We propose to adjust the abbreviations in VLCHKD for Very Low CarboHydrate Ketogenic Diet and VLEKD for Very Low Energy Ketogenic Diet, to clarify the differences in dietary composition. We recommend that investigators describe the researchers describing efficacy or side effects of KDs, to clearly specify the dietary protocol used with its unique acronym and level of ketosis, when ketosis is considered as a component of the diet's mechanism of action.

Perioperative urinary ketosis and metabolic acidosis in patients fasted for undergoing gynecologic surgery.

BACKGROUND: Our bodies have adaptive mechanisms to fasting, in which glycogen stored in the liver and muscle protein are broken down, but also lipid mobilisation is triggered. As a result, glycerol and fatty acids are released into the bloodstream, increasing the production of ketone bodies in liver. However, there are limited studies on the incidence of perioperative urinary ketosis, the intraoperative blood glucose changes and metabolic acidosis after fasting for surgery in non-diabetic adult patients. METHODS: We conducted a retrospective cohort study involving 1831 patients undergoing gynecologic surgery under general anesthesia from January to December 2022. Ketosis was assessed using a postoperative urine test, while blood glucose levels and acid-base status were collected from intraoperative arterial blood gas analyses. RESULTS: Of 1535 patients who underwent postoperative urinalysis, 912 (59.4%) patients had ketonuria. Patients with ketonuria were younger, had lower body mass index, and had fewer comorbidities than those without ketonuria. After adjustments, younger age, higher body mass index and surgery starting late afternoon were significant risk factors for postoperative ketonuria. Of the 929 patients assessed with intraoperative arterial blood gas analyses, 29.0% showed metabolic acidosis. Multivariable logistic regression revealed that perioperative ketonuria and prolonged surgery significantly increased the risk for moderate-to-severe metabolic acidosis. CONCLUSION: Perioperative urinary ketosis and intraoperative metabolic acidosis are common in patients undergoing gynecologic surgery, even with short-term preoperative fasting. The risks are notably higher in younger patients with lower body mass index. Optimization of preoperative fasting strategies including implementation of oral carbohydrate loading should be considered for reducing perioperative metabolic derangement due to ketosis.

Short anesthesia without intravenous fluid therapy in children: Results of a prospective non-interventional multicenter observational study.

BACKGROUND: The German guidelines recommend that intravenous fluid therapy should not be mandatorily performed in children with short fasting times undergoing short anesthesia, but there is a lack of clinical studies including a large number of pediatric patients. Therefore, we performed a prospective non-interventional multicenter observational study to evaluate the perioperative hemodynamic and metabolic stability of children undergoing short anesthesia without intravenous fluid therapy. AIMS: The primary aim was to assess the incidence of hypotension and the secondary aim was to assess the real preoperative fasting times, the incidence of hypoglycemia and the impact on ketone bodies and acid-base balance. METHODS: Children aged 1 month-18 years undergoing short anesthesia (<1 h) without intravenous fluid therapy were enrolled. Patient demographics, the surgical or diagnostic procedure performed, anesthesia, hemodynamic, laboratory data, and adverse events were documented using a standardized case report form. RESULTS: Four hundred and twenty seven children that were investigated at three pediatric centers from July 2021 to June 2022 (mean age 83.4 ± 58.9 months, body weight 27.9 ± 19.8 kg) were included in the analysis. The real preoperative fasting times were 14.2 ± 3.6 h for solids, 7.2 ± 3.5 h for milk and 5 ± 4.8 h for clear fluids. During the course of anesthesia, hypotension (<2.5th percentile) was detected in 3 of 427 cases (0.7%), hypoglycemia (glucose <3.0 mmol L-1) in 1 of 355 cases (0.3%), and ketosis (ketone bodies ≥0.6 mmol L-1) in 51 of 233 cases (21.9%). The occurrence of ketosis was associated with lower body weight (p <.001) and longer fasting times for solids or milk (p =.021), but not for clear fluids (p =.69). CONCLUSIONS: Our study supported the German guidelines recommendation that perioperative intravenous fluid therapy is not mandatory in children beyond the neonatal period with short pre- and postoperative fasting times undergoing short anesthesia (<1 h).

Impact of dietary ketosis on volatile anesthesia toxicity in a model of Leigh syndrome.

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.

Potential therapeutic effect of a ketogenic diet for the treatment of lymphoedema: Results of an exploratory study.

BACKGROUND: Lymphoedema is a chronic and progressive disease characterised by excessive accumulation of lymph in the interstitial compartment, leading to tissue swelling and fibroadipose deposition. Lymphangiogenesis is partly regulated by ketone body oxidation, and a ketogenic diet (KD) has shown therapeutic efficacy in a preclinical mouse tail lymphoedema model. Therefore, we aimed to investigate the potential therapeutic effect of a KD in patients with secondary lymphoedema. METHODS: Nine patients with unilateral stage 2 lymphoedema secondary to lymphadenectomy were included in this quasi-experimental exploratory study consisting of a short run-in phase to gradually induce ketosis, followed by a classic KD (CKD) and modified Atkins diet (MAD) phase during which patients consumed a CKD and MAD, respectively. Lymphatic function and oedema volume, the primary outcomes, were assessed at baseline and at the end of both the CKD and MAD phase. Secondary outcomes included health-related and lymphedema-specific quality of life (QoL). RESULTS: Seven out of nine patients completed the study protocol. Lymphatic function was improved upon consumption of both a CKD (dermal backflow score [mean ± SD]: 7.29 ± 2.98 vs. 10.86 ± 2.19 at baseline; p = 0.03) and MAD (6.71 ± 2.06; p = 0.02), whereas oedema volume did not decrease during the course of the study (excess limb volume [mean ± SD]: 20.13 ± 10.25% at end of CKD and 24.07 ± 17.77% at end of MAD vs. 20.79 ± 12.96% at baseline; p > 0.99 and p > 0.30, respectively). No changes were observed in health-related, nor lymphoedema-specific QoL at the end of CKD and MAD. CONCLUSIONS: The consumption of a KD improved lymphatic function and was associated with a clinically meaningful reduction in oedema volume in some patients (3/7 at end of CKD, 2/7 at end of MAD) with unilateral stage 2 secondary lymphoedema. These results highlight the potential of a KD to improve lymphatic function in patients with lymphoedema. However, further studies are required to substantiate our findings.

Plasma and milk metabolomics profiles in dairy cows with subclinical and clinical ketosis.

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.

Invited review: Ketone biology-The shifting paradigm of ketones and ketosis in the dairy cow.

Ketosis is currently regarded as a major metabolic disorder of dairy cows, reflective of the animal's efforts to adapt to energy deficit while transitioning into lactation. Currently viewed as a pathology by some, ketosis is associatively implicated in milk production losses and peripartal health complications that increase the risk of early removal of cows from the herd, thus carrying economic losses for dairy farmers and jeopardizing the sustainability of the dairy industry. Despite decades of intense research in the mitigation of ketosis and its sequelae, our ability to lessen its purported effects remains limited. Moreover, the association of ketosis to reduced milk production and peripartal disease is often erratic and likely mired by concurrent potential confounders. In this review, we discuss the potential reasons for these apparent paradoxes in the light of currently available evidence, with a focus on the limitations of observational research and the necessary steps to unambiguously identify the effects of ketosis on cow health and performance via controlled randomized experimentation. A nuanced perspective is proposed that considers the dissociation of ketosis-as a disease-from healthy hyperketonemia. Furthermore, in consideration of a growing body of evidence that highlights positive roles of ketones in the mitigation of metabolic dysfunction and chronic diseases, we consider the hypothetical functions of ketones as health-promoting metabolites and ponder on their potential usefulness to enhance dairy cow health and productivity.

Prediction of key milk biomarkers in dairy cows through milk mid-infrared spectra and international collaborations.

At the individual cow level, suboptimum fertility, mastitis, negative energy balance, and ketosis are major issues in dairy farming. These problems are widespread on dairy farms and have an important economic impact. The objectives of this study were (1) to assess the potential of milk mid-infrared (MIR) spectra to predict key biomarkers of energy deficit (citrate, isocitrate, glucose-6 phosphate [glucose-6P], free glucose), ketosis (ß-hydroxybutyrate [BHB] and acetone), mastitis (N-acetyl-ß-d-glucosaminidase activity [NAGase] and lactate dehydrogenase), and fertility (progesterone); (2) to test alternative methodologies to partial least squares (PLS) regression to better account for the specific asymmetric distribution of the biomarkers; and (3) to create robust models by merging large datasets from 5 international or national projects. Benefiting from this international collaboration, the dataset comprised a total of 9,143 milk samples from 3,758 cows located in 589 herds across 10 countries and represented 7 breeds. The samples were analyzed by reference chemistry for biomarker contents, whereas the MIR analyses were performed on 30 instruments from different models and brands, with spectra harmonized into a common format. Four quantitative methodologies were evaluated to address the strongly skewed distribution of some biomarkers. Partial least squares regression was used as the reference basis, and compared with a random modification of distribution associated with PLS (random-downsampling-PLS), an optimized modification of distribution associated with PLS (KennardStone-downsampling-PLS), and support vector machine (SVM). When the ability of MIR to predict biomarkers was too low for quantification, different qualitative methodologies were tested to discriminate low versus high values of biomarkers. For each biomarker, 20% of the herds were randomly removed within all countries to be used as the validation dataset. The remaining 80% of herds were used as the calibration dataset. In calibration, the 3 alternative methodologies outperform the PLS performances for the majority of biomarkers. However, in the external herd validation, PLS provided the best results for isocitrate, glucose-6P, free glucose, and lactate dehydrogenase (coefficient of determination in external herd validation [R2v] = 0.48, 0.58, 0.28, and 0.24, respectively). For other molecules, PLS-random-downsampling and PLS-KennardStone-downsampling outperformed PLS in the majority of cases, but the best results were provided by SVM for citrate, BHB, acetone, NAGase, and progesterone (R2v = 0.94, 0.58, 0.76, 0.68, and 0.15, respectively). Hence, PLS and SVM based on the entire dataset provided the best results for normal and skewed distributions, respectively. Complementary to the quantitative methods, the qualitative discriminant models enabled the discrimination of high and low values for BHB, acetone, and NAGase with a global accuracy around 90%, and glucose-6P with an accuracy of 83%. In conclusion, MIR spectra of milk can enable quantitative screening of citrate as a biomarker of energy deficit and discrimination of low and high values of BHB, acetone, and NAGase, as biomarkers of ketosis and mastitis. Finally, progesterone could not be predicted with sufficient accuracy from milk MIR spectra to be further considered. Consequently, MIR spectrometry can bring valuable information regarding the occurrence of energy deficit, ketosis, and mastitis in dairy cows, which in turn have major influences on their fertility and survival.

Role of diacylglycerol O-acyltransferase 1 (DGAT1) in lipolysis and autophagy of adipose tissue from ketotic dairy cows.

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.

Lipolysis inhibition as a treatment of clinical ketosis in dairy cows: Effects on adipose tissue metabolic and immune responses.

Dairy cows with clinical ketosis (CK) exhibit excessive adipose tissue (AT) lipolysis and systemic inflammation. Lipolysis in cows can be induced by the canonical (hormonally induced) and inflammatory lipolytic pathways. Currently, the most common treatment for CK is oral propylene glycol (PG); however, PG does not reduce lipolysis or inflammation. Niacin (NIA) can reduce the activation of canonical lipolysis, whereas cyclooxygenase inhibitors such as flunixin meglumine (FM) can limit inflammation and inhibit the inflammatory lipolytic pathway. The objective of this study was to determine the effects of including NIA and FM in the standard PG treatment for postpartum CK on AT function. Multiparous Jersey cows (n = 18; 7.1 ± 3.8 DIM) were selected from a commercial dairy. Inclusion criteria were CK symptoms (lethargy, depressed appetite, and drop in milk yield) and high blood levels of BHB (≥1.2 mmol/L). Cows with CK were randomly assigned to one of 3 treatments: (1) PG: 310 g administered orally once per day for 5 d, (2) PG+NIA: 24 g administered orally once per day for 3 d, and (3) PG+NIA+FM: 1.1 mg/kg administered IV once per day for 3 d. Healthy control cows (HC; n = 6) matched by lactation and DIM (±2 d) were sampled. Subcutaneous AT explants were collected at d 0 and d 7 relative to enrollment. To assess AT insulin sensitivity, explants were treated with insulin (1 µL/L) during lipolysis stimulation with a ß-adrenergic receptor agonist (isoproterenol, 1 µM). Lipolysis was quantified by glycerol release in the media. Lipid mobilization and inflammatory gene networks were evaluated using quantitative PCR. Protein biomarkers of lipolysis, insulin signaling, and AT inflammation, including hormone-sensitive lipase, protein kinase B (Akt), and ERK1/2, were quantified by capillary immunoassays. Flow cytometry of AT cellular components was used to characterize macrophage inflammatory phenotypes. Statistical significance was determined by a nonparametric t-test when 2 groups (HC vs. CK) were analyzed and an ANOVA test with Tukey adjustment when 3 treatment groups (PG vs. PG+NIA vs. PG+NIA+FM) were evaluated. At d 0, AT from CK cows showed higher mRNA expression of lipolytic enzymes ABHD5, LIPE, and LPL, as well as increased phosphorylation of hormone-sensitive lipase compared with HC. At d 0, insulin reduced lipolysis by 41% ± 8% in AT from HC, but CK cows were unresponsive (-2.9 ± 4%). Adipose tissue from CK cows exhibited reduced Akt phosphorylation compared with HC. Cows with CK had increased AT expression of inflammatory gene markers, including CCL2, IL8, IL10, TLR4, and TNF, along with ERK1/2 phosphorylation. Adipose tissue from CK cows showed increased macrophage infiltration compared with HC. By d 7, AT from PG+NIA+FM cows had a more robust response to insulin, as evidenced by reduced glycerol release (36.5% ± 8% compared with PG at 26.9% ± 7% and PG+NIA at 7.4% ± 8%) and enhanced phosphorylation of Akt. By d 7, PG+NIA+FM cows presented lower inflammatory markers, including ERK1/2 phosphorylation, and reduced macrophage infiltration, compared with PG and PG+NIA. These data suggest that including NIA and FM in CK treatment improves AT insulin sensitivity and reduces AT inflammation and macrophage infiltration.

Global losses due to dairy cattle diseases: A comorbidity-adjusted economic analysis.

An economic simulation was carried out over 183 milk-producing countries to estimate the global economic impacts of 12 dairy cattle diseases and health conditions: mastitis (subclinical and clinical), lameness, paratuberculosis (Johne's disease), displaced abomasum, dystocia, metritis, milk fever, ovarian cysts, retained placenta, and ketosis (subclinical and clinical). Estimates of disease impacts on milk yield, fertility, and culling were collected from the literature, standardized, meta-analyzed using a variety of methods ranging from simple averaging to random-effects models, and adjusted for comorbidities to prevent overestimation. These comorbidity-adjusted disease impacts were then combined with a set of country-level estimates for lactational incidence or prevalence or both, herd characteristics, and price estimates within a series of Monte Carlo simulations that estimated and valued the economic losses due to these diseases. It was estimated that total annual global losses are US$65 billion (B). Subclinical ketosis, clinical mastitis, and subclinical mastitis were the costliest diseases modeled, resulting in mean annual global losses of approximately US$18B, US$13B, and US$9B, respectively. Estimated global annual losses due to clinical ketosis, displaced abomasum, dystocia, lameness, metritis, milk fever, ovarian cysts, paratuberculosis, and retained placenta were estimated to be US$0.2B, US$0.6B, US$0.6B, US$6B, US$5B, US$0.6B, US$4B, US$4B, and US$3B, respectively. Without adjustment for comorbidities, when statistical associations between diseases were disregarded, mean aggregate global losses would have been overestimated by 45%. Although annual losses were greatest in India (US$12B), the United States (US$8B), and China (US$5B), depending on the measure of losses used (losses as a percentage of gross domestic product, losses per capita, losses as a percentage of gross milk revenue), the relative economic burden of these dairy cattle diseases across countries varied markedly.

Fatty acids promote M1 polarization of monocyte-derived macrophages in healthy or ketotic dairy cows and a bovine macrophage cell line by impairing mTOR-mediated autophagy.

Excessive concentrations of free fatty acids (FFA) are the main factors causing immune dysfunction and inflammation in dairy cows with ketosis. Polarization of macrophages (the process of macrophages freely switching from one phenotype to another) into M1 or M2 phenotypes is an important event during inflammation induced by environmental stimuli. In nonruminants, mammalian target of rapamycin (mTOR)-mediated autophagy (a major waste degradation process) regulates macrophage polarization. Thus, our objective was to unravel the role of mTOR-mediated autophagy on macrophage polarization in ketotic dairy cows. We performed 4 experiments: (1) In vitro differentiated monocyte-derived macrophages from healthy dairy cows or dairy cows with clinical ketosis (CK) were treated for 24 h with 100 ng/mL LPS and 100 ng/mL IFN-γ or with 10 ng/mL IL4 and 10 ng/mL IL10; (2) Immortalized bovine macrophages were treated for 24 h with 0, 0.3, 0.6, or 1.2 mM FFA, LPS, and IFN-γ, or with IL4 and IL10; (3) Macrophages were pretreated with 2 µM 4,6-dimorpholino-N-(4-nitrophenyl)-1,3,5-triazin-2-amine (MHY1485) for 30 min before treatment with LPS and IFN-γ or IL4 and IL10; (4) Macrophages were pretreated with 100 nM rapamycin (RAPA) for 2 h before treatment with LPS and IFN-γ or IL4 and IL10. Compared with healthy cows, cows with CK had a greater mean fluorescence intensity (MFI) of CD86+, but lower MFI of CD206+ and lower number of autophagosomes and autolysosomes in macrophages. Exogenous FFA treatment upregulated protein abundance of inducible nitric oxide synthase (iNOS) and the MFI of CD86, whereas it downregulated the protein abundance of arginase 1 and the MFI of CD206. In addition, FFA increased the p-p65/p65 protein abundance and tumor necrosis factor α, IL1B, and IL6 mRNA abundance, but decreased LC3-phosphatidylethanolamine conjugate protein abundance and the number of autophagosomes and autolysosomes number. Pretreatment with MHY1485 promoted macrophage M1 polarization and inhibited macrophage M2 polarization via decreased mTOR-mediated autophagy. Activation of mTOR-mediated autophagy by pretreatment with RAPA attenuated the upregulation of inflammation in M1 macrophages that was induced by FFA. These data revealed that high concentrations of FFA promote macrophage M1 polarization in ketotic dairy cows by impairing mTOR-mediated autophagy.

Metabolic shift toward ketosis in asocial cavefish increases social-like affinity.

BACKGROUND: Social affinity and collective behavior are nearly ubiquitous in the animal kingdom, but many lineages feature evolutionarily asocial species. These solitary species may have evolved to conserve energy in food-sparse environments. However, the mechanism by which metabolic shifts regulate social affinity is not well investigated. RESULTS: In this study, we used the Mexican tetra (Astyanax mexicanus), which features riverine sighted surface (surface fish) and cave-dwelling populations (cavefish), to address the impact of metabolic shifts on asociality and other cave-associated behaviors in cavefish, including repetitive turning, sleeplessness, swimming longer distances, and enhanced foraging behavior. After 1 month of ketosis-inducing ketogenic diet feeding, asocial cavefish exhibited significantly higher social affinity, whereas social affinity regressed in cavefish fed the standard diet. The ketogenic diet also reduced repetitive turning and swimming in cavefish. No major behavioral shifts were found regarding sleeplessness and foraging behavior, suggesting that other evolved behaviors are not largely regulated by ketosis. We further examined the effects of the ketogenic diet via supplementation with exogenous ketone bodies, revealing that ketone bodies are pivotal molecules positively associated with social affinity. CONCLUSIONS: Our study indicated that fish that evolved to be asocial remain capable of exhibiting social affinity under ketosis, possibly linking the seasonal food availability and sociality.