About

Christoph Buettner, MD, Ph.D, is the Principal Investigator and Professor of Medicine as well as Chancellor Scholar at Rutgers Robert Wood Johnson Medical School. He serves as the Chief of the Division of Endocrinology, Metabolism & Nutrition and holds the position of Vice-Chair for Basic Research in the Department of Medicine. His professional roles indicate a leadership position in both clinical and research domains within the field of endocrinology and metabolism. The Buettner Lab is based at RWJMS in New Brunswick, New Jersey, where he oversees a team including fellows, graduate students, staff, and undergraduate students. The lab's affiliation with the Division of Endocrinology, Metabolism and Nutrition at RWJMS underscores a focus on research and clinical practice in these areas.

Research topics

• Biology
• Cell biology
• Neuroscience
• Medicine
• Internal medicine
• Endocrinology

Selected publications

• eLife Assessment: Time to Eat - A Personalized Circadian Eating Schedule Leads to Weight Loss Without Imposing Calorie Restriction: A Randomized Controlled Pilot Study

  2025-02-26

  peer-reviewOpen access1st authorCorresponding

  Many weight loss strategies are based on the restriction of calories or certain foods. In this pilot study, we preliminary tested a weight loss intervention based solely on increasing the regularity of meals. The assumption is based on the evidence that eating at fixed times allows the circadian system to optimally prepare the food metabolism for these times.In a two-group, single center randomized-controlled single-blind pilot study (pre-registration ) with participants aged 18-65 years and BMI ≥ 22 kg/m2, we used a smartphone application to identify the times at which each participant eats particularly frequently and asked participants of the experimental group to restrict their meals to only these times for six weeks. Control participants received sham treatment. Primary outcome was body weight/BMI and secondary outcome the well-being of participants.Of 148 participants taking part in the pilot study, 121 were randomized, of whom 100 (control: 33, experimental: 67) completed the study. Our results show that the more regular the meals of participants of the experimental group became, the more weight/BMI they lost, averaging 2.62 kg (0.87 kg/m2); p < 0.0001 (BMI: p < 0.0001) compared to an insignificant weight loss of 0.56 kg (0.20 kg/m2) in the control group; p = 0.0918 (BMI: p = 0.0658). Strikingly, weight loss was not related to self-reported changes in calories, food composition, and other food-related factors. Additionally, physical and mental well-being improved significantly.In summary, increasing the regularity of meals may cause participants to lose excess body weight and improve overall well-being. These promising results justify a larger-scale study, albeit with a more rigorous study design.

  PublisherDOI

• eLife Assessment: Three components of glucose dynamics – value, variability, and autocorrelation – are independently associated with coronary plaque vulnerability

  2025-03-04

  peer-reviewOpen access1st authorCorresponding

  Impaired glucose homeostasis leads to numerous complications, with coronary artery disease (CAD) being a major contributor to healthcare costs worldwide. Given the limited efficacy of current CAD screening methods, we investigated the association between glucose dynamics and a predictor of coronary events measured by virtual histology-intravascular ultrasound (%NC), with the aim of predicting CAD using easy-to-measure indices. We found that continuous glucose monitoring (CGM)-derived indices, particularly average daily risk ratio (ADRR) and AC_Var, exhibited stronger predictive capabilities for %NC compared to commonly used indices such as fasting blood glucose (FBG), hemoglobin A1C (HbA1c), and plasma glucose level at 120 min during oral glucose tolerance tests (PG120). Factor analysis identified three distinct components underlying glucose dynamics – value, variability, and autocorrelation – each independently associated with %NC. ADRR was influenced by the first two components and AC_Var by the third. FBG, HbA1c, and PG120 were influenced only by the value component, making them insufficient for %NC prediction. Our results were validated using data sets from Japan (n=64), America (n=53), and China (n=100). CGM-derived indices reflecting the three components of glucose dynamics can serve as more effective screening tools for CAD risk assessment, complementing or possibly replacing traditional diabetes diagnostic methods.

  PublisherDOI

• Overnutrition causes insulin resistance and metabolic disorder through increased sympathetic nervous system activity

  Cell Metabolism · 2024-10-21 · 88 citations

  articleOpen accessSenior author
  PublisherOA PDFDOI

• eLife Assessment: Time to Eat - A Personalized Circadian Eating Schedule Leads to Weight Loss Without Calorie Restriction: A Randomized Controlled Trial

  2024-05-20

  peer-reviewOpen access1st authorCorresponding

  Many weight loss strategies are based on the restriction of calories or certain foods. Here, we tested a weight loss intervention based solely on increasing the regularity of meals to allow the circadian system to optimally prepare food metabolism for these times.In a two-group, single center randomized-controlled single-blind study (pre-registration DRKS00021419) with participants aged 18-65 years and BMI ≥ 22 kg/m², we used a smartphone application to identify the times at which each participant eats particularly frequently and asked participants of the experimental group to restrict their meals to only these times for six weeks. Control participants received sham treatment. Primary outcome was self-reported body weight/BMI and secondary outcome the well-being of participants.Of 148 participants entering the study, 121 were randomized and of these 100 (control: 33, experimental: 67) finished the study. Our results show that the more regular the meals of participants of the experimental group became, the more weight/BMI they lost, averaging 2.62 kg (0.87 kg/m²); p < 0.0001 (BMI: p < 0.0001) compared to an insignificant weight loss of 0.56 kg (0.20 kg/m²) in the control group; p = 0.0918 (BMI: p = 0.0658). Strikingly, weight loss was not related to changes in self-reported calories, food composition, and other food-related factors. Additionally, physical and mental well-being improved significantly.In summary, increasing the regularity of meals causes participants to lose excess body weight and improves overall well-being.

  PublisherDOI

• Why do some individuals with obesity remain metabolically healthy?

  Trends in Endocrinology and Metabolism · 2024-07-02

  articleOpen access1st authorCorresponding
  PublisherOA PDFDOI

• eLife assessment: Genetic inactivation of zinc transporter SLC39A5 improves liver function and hyperglycemia in obesogenic settings

  2024-12-13

  peer-reviewOpen access1st authorCorresponding
  PublisherDOI

• eLife Assessment: Time to Eat - A Personalized Circadian Eating Schedule Leads to Weight Loss Without Calorie Restriction: A Randomized Controlled Trial

  2024-10-24

  peer-reviewOpen access1st authorCorresponding

  Many weight loss strategies are based on the restriction of calories or certain foods. Here, we tested a weight loss intervention based solely on increasing the regularity of meals, presuming that this allows the circadian system to optimally prepare the food metabolism for these times.In a two-group, single center randomized-controlled single-blind pilot study (pre-registration DRKS00021419) with participants aged 18-65 years and BMI ≥ 22 kg/m², we used a smartphone application to identify the times at which each participant eats particularly frequently and asked participants of the experimental group to restrict their meals to only these times for six weeks. Control participants received sham treatment. Primary outcome was self-reported body weight/BMI and secondary outcome the well-being of participants.Of 148 participants entering the pilot study, 121 were randomized and of these 100 (control: 33, experimental: 67) finished the study. Our results show that the more regular the meals of participants of the experimental group became, the more weight/BMI they lost, averaging 2.62 kg (0.87 kg/m²); p < 0.0001 (BMI: p < 0.0001) compared to an insignificant weight loss of 0.56 kg (0.20 kg/m²) in the control group; p = 0.0918 (BMI: p = 0.0658). Strikingly, weight loss was not related to changes in self-reported calories, food composition, and other food-related factors. Additionally, physical and mental well-being improved significantly.In summary, increasing the regularity of meals causes participants to lose excess body weight and improves overall well-being.

  PublisherDOI

• Brain insulin signaling suppresses lipolysis in the absence of peripheral insulin receptors and requires the MAPK pathway

  Molecular Metabolism · 2023-04-24 · 11 citations

  articleOpen accessCorresponding

  Insulin's ability to counterbalance catecholamine-induced lipolysis defines insulin action in adipose tissue. Insulin suppresses lipolysis directly at the level of the adipocyte and indirectly through signaling in the brain. Here, we further characterized the role of brain insulin signaling in regulating lipolysis and defined the intracellular insulin signaling pathway required for brain insulin to suppress lipolysis. We used hyperinsulinemic clamp studies coupled with tracer dilution techniques to assess insulin's ability to suppress lipolysis in two different mouse models with inducible insulin receptor depletion in all tissues (IRΔWB) or restricted to peripheral tissues excluding the brain (IRΔPER). To identify the underlying signaling pathway required for brain insulin to inhibit lipolysis, we continuously infused insulin +/− a PI3K or MAPK inhibitor into the mediobasal hypothalamus of male Sprague Dawley rats and assessed lipolysis during clamps. Genetic insulin receptor deletion induced marked hyperglycemia and insulin resistance in both IRΔPER and IRΔWB mice. However, the ability of insulin to suppress lipolysis was largely preserved in IRΔPER, but completely obliterated in IRΔWB mice indicating that insulin is still able to suppress lipolysis as long as brain insulin receptors are present. Blocking the MAPK, but not the PI3K pathway impaired the inhibition of lipolysis by brain insulin signaling. Brain insulin is required for insulin to suppress adipose tissue lipolysis and depends on intact hypothalamic MAPK signaling.

  PublisherDOI

• eLife assessment: Hepatic conversion of acetyl-CoA to acetate plays crucial roles in energy stress

  2023-10-30

  peer-reviewOpen access1st authorCorresponding

  Full text Figures and data Side by side Abstract eLife assessment Introduction Results Discussion Materials and methods Appendix 1 Data availability References Peer review Author response Article and author information Metrics Abstract Accumulating evidence indicates that acetate is increased under energy stress conditions such as those that occur in diabetes mellitus and prolonged starvation. However, how and where acetate is produced and the nature of its biological significance are largely unknown. We observed overproduction of acetate to concentrations comparable to those of ketone bodies in patients and mice with diabetes or starvation. Mechanistically, ACOT12 and ACOT8 are dramatically upregulated in the liver to convert free fatty acid-derived acetyl-CoA to acetate and CoA. This conversion not only provides a large amount of acetate, which preferentially fuels the brain rather than muscle, but also recycles CoA, which is required for sustained fatty acid oxidation and ketogenesis. We suggest that acetate is an emerging novel ‘ketone body’ that may be used as a parameter to evaluate the progression of energy stress. eLife assessment This is important work that examines hepatic acetate production via ACOT12/18 in starvation and diabetes. The investigators use solid loss of function strategies in cells, including mouse primary hepatocytes, and in vivo mouse experiments to show that ACOTs are necessary for normal acetate production in the context of fasting and type 1 diabetes. Given that acetate is commonly thought to primarily represent a fermentation product, this study is of interest as it describes hepatic pathways converting fatty acids to acetate. https://doi.org/10.7554/eLife.87419.3.sa0 About eLife assessments Introduction Disordered homeostasis of energy metabolism, which is associated with emergency situations such as untreated diabetes mellitus, prolonged starvation, and ischemic heart/brain disease, is a serious threat to human health (Field et al., 2001; Galgani and Ravussin, 2008; Martinic and von Herrath, 2008; Must et al., 1999). In response to such disorder, the metabolic patterns of multiple organs have to be remodeled to rescue the imbalance and bring whole organism through the crisis (Denechaud et al., 2008; Frühbeck et al., 2001; Goldberg et al., 2018; Hirai et al., 2021; Meier and Gressner, 2004; Nishimoto et al., 2016; Palikaras et al., 2015; Russell and Cook, 1995). Ketone bodies, namely acetoacetate (AcAc), β-hydroxybutyrate (3-hydroxybutyrate, 3-HB), and acetone, are overproduced from fatty acids in the liver under conditions in which carbohydrate availability is reduced, such as diabetes and starvation. These bodies are released into blood and serve as a vital alternative metabolic fuel for extrahepatic tissues including the brain, skeletal muscle, and heart, where they are converted to acetyl-CoA and oxidized in the tricarboxylic cycle (TCA), providing a large amount of energy (Cahill, 2006; D’Acunzo et al., 2021; Dentin et al., 2006; Krishnakumar et al., 2008; Puchalska and Crawford, 2017; Robinson and Williamson, 1980). Previous studies have shown that acetate concentrations are significantly increased in diabetes and prolonged starvation (Akanji et al., 1989; Seufert et al., 1984; Todesco et al., 1993), and acetate is considered to be a nutrient that nourishes the organism by undergoing conversion to acetyl-CoA which is further catabolized in the TCA (Lindsay and Setchell, 1976; Liu et al., 2018; Schug et al., 2015; Schug et al., 2016). Conversely, acetyl-CoA can also be hydrolyzed to acetate by proteins of the acyl-CoA thioesterase (ACOT) family (Swarbrick et al., 2014; Tillander et al., 2017). Unfortunately, it is not clear where, under what conditions and how acetate is produced, nor what is its biological significance. Considering that both acetate and ketone bodies are produced from acetyl-CoA and catabolized back to acetyl-CoA, we thoroughly investigated the production and utilization of acetate, also looking at ketone bodies as a comparison. We suggest that acetate is an emerging novel ‘ketone body’ that plays important roles, similar to those of classic ketone bodies, in energy stress conditions such as diabetes mellitus and prolonged starvation. Note: our description of acetate as an emerging novel ‘ketone body’ does not suggest that it as a real ketone in structure, but emphasizes the high similarity of acetate and classic ketone bodies in terms of both the organ in which they are produced (liver), the substrate from which they are produced (fatty acids-derived acetyl-CoA), the roles they play as important sources of fuel and energy for many extrahepatic peripheral organs, their catabolism back to acetyl-CoA and degradation in the TCA cycle, and the physiological conditions of their production (under energy stresses such as prolonged starvation and untreated diabetes mellitus). Results Acetate is dramatically elevated under energy stress conditions in mammals To investigate whether acetate is produced in a pattern similar to ketone bodies, we measured serum glucose, 3-beta-hydroxybutyrate (3HB), acetoacetate (AcAc), and other metabolites in 17 diabetes mellitus patients and 8 healthy volunteers as controls (Figure 1—figure supplement 1A; Figure 1—source data 1). We observed a significant increase of acetate in parallel with the canonical elevation of ketone bodies (3-HB and AcAc) and serum glucose in diabetes mellitus patients as compared with healthy controls (Figure 1A). We then detected acetate in mouse models and found that the levels of serum acetate and ketone bodies were dramatically elevated to the same extent in streptozotocin (STZ)-induced type I diabetic C57BL/6 (Figure 1B) and BALB/c mice (Figure 1—figure supplement 1B) as in type II diabetic db/db mice (Figure 1—figure supplement 1C). As expected, starvation also leads to a marked decrease in serum glucose concentration and an increase in serum acetate and ketone body levels in normal C57BL/6 (Figure 1C) and BALB/c mice (Figure 1—figure supplement 2). These data demonstrate that serum acetate is boosted to the same extent as canonical ketone bodies under energy stresses including those that occur with diabetes mellitus and starvation. For the sake of simplicity, we will refer to this acetate as 'energy stress-induced acetate (ES-acetate)'. Figure 1 with 2 supplements see all Download asset Open asset Acetate is produced at a levels comparable to those of ketone bodies in energy stress conditions. (A) Enrichment of glucose, 3-HB, AcAc, and acetate in clinical serum samples from healthy volunteers and patients with diabetes mellitus (Healthy, n = 8; Diabetes, n = 17). (B) Enrichment of glucose, 3-HB, AcAc, and acetate in the serum of STZ-induced diabetic mice (C57BL/6, n = 5). (C) The levels of acetate, 3-HB, AcAc, and glucose in the serum of C57BL/6 mice (n = 5) starved for the indicated time course. Abbreviations: 3-HB, 3-hydroxybutyrate; AcAc, acetoacetate; NT, untreated control; STZ, streptozotocin. Values are expressed as mean ± standard deviation (SD) and analyzed statistically by two-tailed unpaired Student’s t-test (*p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, n.s., no significant difference). Figure 1—source data 1 Information describing the patient and healthy volunteers for the clinical data depicted in Figure 1. https://cdn.elifesciences.org/articles/87419/elife-87419-fig1-data1-v1.xlsx Download elife-87419-fig1-data1-v1.xlsx ES-acetate is derived from free fatty acids in mammalian cells Next, we asked which nutrients ES-acetate is derived from. In mammals, serum acetate generally has three sources: dietary acetate, as a metabolic product of gut microbiota, and as the intermediate of intracellular biochemical processes (Schug et al., 2016). As the mice used in this work were fed an acetate-free diet, we focused on acetate formed by gut microbiota and endogenous biochemical reactions. To determine whether gut microbiota contribute to the production of ES-acetate, mice were pre-treated with antibiotics to eliminate gut microbes (saline as control) as reported previously (Sivan et al., 2015). We observed that antibiotic pre-treatment failed to obviously affect the production of acetate that was induced by either starvation (Figure 2—figure supplement 1A, B) or diabetes (Figure 2—figure supplement 1C), demonstrating that ES-acetate is mainly produced endogenously. Next we used nuclear magnetic resonance (NMR) (Figure 2—figure supplement 2A, B) and gas chromatography–mass spectrometry (GC–MS) (Figure 2—figure supplement 2C, D) to detect the acetate that was secreted into the culture medium by several cell lines, and found that these cells showed different ability in producing acetate. Consistently, Liu et al., 2018 reported that acetate is derived from glucose in mammalian cells supplied with abundant nutrients. Indeed, we observed the secretion of different amounts of U-13C-acetate after cells were cultured in medium supplemented with U-13C-glucose (Figure 2—figure supplement 3A). Interestingly, we also observed the production of 36.6% of non-U-13C-acetate, indicating that this proportion of acetate is derived from nutrients other than glucose (Figure 2—figure supplement 3B). We then examined whether the acetate secreted by cultured cells is derived from amino acids (AAs) and free fatty acids (FFAs) upon starvation. After different cells were cultured in Hanks’ balanced salt solutions (HBSS, free of glucose, fatty acids and amino acids) supplemented with FFAs or amino acids for 20 hr, supplementation with FFAs (Figure 2—figure supplement 4D, E) rather than amino acids (Figure 2—figure supplement 4A–C) significantly increased acetate levels, suggesting a major contribution of FFAs to acetate production. To confirm this observation, a series of widely used cell lines and mouse primary hepatocytes (MPHs) were cultured in HBSS supplemented with U-13C-palmitate, before secreted U-13C-acetate was measured (Figure 2A). These cell lines displayed quite different abilities to convert palmitate to acetate, and were accordingly divided into FFA-derived acetate-producing cells (FDAPCs: LO2, MPH, AML12, etc.) and no-FFA-derived acetate-producing cells (NFDAPCs: HEK-293T, Huh7, etc.). All of the FDAPCs secreted U-13C-acetate in a manner that was dependent on the dose of U-13C-palmitate supplementation (Figure 2B–D). We also observed that high-fat diet induced a significant increase in acetate production in both normal and STZ-induced diabetic mice (Figure 2E). Taken together, these findings suggest that acetate can be derived from FFAs in energy stress conditions. Figure 2 with 4 supplements see all Download asset Open asset Acetate is derived from free fatty acids (FFAs) in mammalian cells. (A) The amount of U-13C-acetate secreted by the indicated cell lines cultured in U-13C-palmitate-containing Hanks’ balanced salt solution (HBSS) for 20 hr (n = 3). (B–D) The amount of U-13C-acetate secreted by MPH (B), LO2 (C), and AML12 (D) cells cultured in HBSS supplemented with increasing doses of U-13C-palmitate for 20 hr (n = 3). (E) Enrichment of acetate in the serum of untreated or STZ-induced diabetic C57BL/6 mice (n = 10) fed with a high-fat diet (HFD) or a control diet. Abbreviations: MPH, mouse primary hepatocyte; STZ, streptozotocin; UD, undetectable. Values are expressed as mean ± standard deviation (SD) and were analyzed statistically using a two-tailed unpaired Student’s t-test (A, E) or one-way analysis of variance (ANOVA) (B–D) (*p < 0.05, ****p < 0.0001, n.s., no significant difference). ACOT12 and ACOT8 are involved in acetate production in mammalian cells It has been reported that acyl-CoAs with different lengths of carbon chain could be hydrolyzed to FFAs specifically by a corresponding ACOTs family protein (Tillander et al., 2017). Acetyl-CoA, the shortest chain of acyl-CoA and the critical product of β-oxidation, is hydrolyzed to acetate by acyl-CoA thioesterase 12 (ACOT12) (Swarbrick et al., 2014). We next analyzed the GEO database and found out that the expression of Acot1/2/8/12 is upregulated significantly alongside the increase of β-oxidation and ketogenesis in mice liver after 24 hr of fasting (Figure 3A; Figure 3—figure supplement 1A). To determine which ACOT is responsible for ES-acetate production, we overexpressed a series of ACOTs in HEK-293T cells and observed large amount of acetate production when either ACOT8 or ACOT12 was overexpressed (Figure 3B), indicating that these two ACOTs are involved in ES-acetate production. Consistently, the protein levels of both Acot12 and Acot8 are upregulated robustly in the livers of either starved mice or STZ-induced type I diabetic mice (Figure 3—figure supplement 1B, C). Furthermore, when ACOT12 and ACOT8 were separately overexpressed in NFDAPCs HEK-293T and Huh7, FFA-derived acetate was significantly increased (Figure 3—figure supplement 1D, E). Similarly, overexpression of wildtype ACOT12 and ACOT8, rather than their enzyme activity-dead mutants, in HEK-293T (Figure 3C) and Huh7 (Figure 3D) cells drastically increased the production of U-13C-acetate derived from U-13C-palmitate (Ishizuka et al., 2004; Swarbrick et al., 2014). By contrast, knockdown (KD) of ACOT12 or ACOT8 in FDAPCs MPH (Figure 3E, F) and LO2 (Figure 3—figure supplement 1F, G) diminished U-13C-acetate production. These data reveal that ACOT12 and ACOT8 are responsible for ES-acetate production. Figure 3 with 1 supplement see all Download asset Open asset ACOT12 and ACOT8 are involved in acetate production in mammalian cells. (A) Heatmap showing the differential hepatic expression of genes in the fed and fasted groups. RNAseq analysis data from Goldstein et al., 2017. (B) The secretion of acetate (upper panel) by HEK-239T cell lines overexpressing various ACOTs, and the protein levels of expressed ACOTs (lower panel). (C,D) HEK-293T (C) and Huh7 (D) cell lines overexpressing control vector, wildtype (WT) ACOT12 and ACOT8 or enzyme activity-dead mutants (Mut) of these two enzymes were cultured in Hanks’ balanced salt solution (HBSS) containing U-13C-palmitate for 20 hr, before U-13C-acetate was detected. (E, F) U-13C-acetate secreted by Acot12- or Acot8-knockdown mouse primary hepatocytes (MPH) after incubation in U-13C-palmitate-containing HBSS for 20 hr. Abbreviations: ACOT8 Mut, ACOT8 H78A mutant; ACOT12 Mut, ACOT12 R312E mutant; shAcot8, short hairpin RNA targeting mouse Acot8 gene; shAcot12, short hairpin RNA targeting mouse Acot12 gene; UD, undetectable. Values are expressed as mean ± standard deviation (SD) (n = 3) of three independent experiments and analyzed using unpaired Student’s t-tests (**p < 0.01, ***p < 0.001, ****p < 0.0001, n.s., no significant difference). Figure 3—source data 1 Complete, unedited immunoblots, as well as immunoblots including sample and band identification, are provided for the immunoblots presented in Figure 3. https://cdn.elifesciences.org/articles/87419/elife-87419-fig3-data1-v1.zip Download elife-87419-fig3-data1-v1.zip Hepatic ACOT12 and ACOT8 are responsible for ES-acetate production in energy stress conditions Next we were prompted to determine which organ and subcellular structures are mainly involved in the generation of ES-acetate. First, we analyzed the expression of ACOTs individually at mRNA level in various tissues of human and mice by employing the GTEx and GEO databases. ACOT12 is mainly expressed in human liver together with genes encoding ketogenic enzymes (HMGCS2, HMGCSL, ACAT1, and BDH1), whereas ACOT8 is expressed ubiquitously at a relative high level in most tissues (Figure 4—figure supplement 1). Acot12 is also expressed mainly in mouse liver and kidney, whereas Acot8 seems to be expressed at a much lower level in nearly all of the mouse tissues examined (Figure 4—figure supplement 2A). Differing from their mRNA expression patterns in the GEO database, we observed high levels of both Acot12 and Acot8 proteins in mouse liver and kidney (Figure 4—figure supplement 2B). Consistently, adenovirus-mediated liver-targeted knockdown of either Acot12 or Acot8 dramatically abolished acetate production by starved or diabetic C57BL/6 mice (Figure 4A–F), and conditional deletion of Acot12 or Acot8 in liver dramatically decreased acetate production in starved mice (Figure 4G–J), demonstrating that the liver is the main organ responsible for ES-acetate production. Moreover, U-13C-acetate derived from U-13C-palmitate in glucose-free HBSS was diminished by replenishment of glucose (Figure 4—figure supplement 3), in accordance with the concept that glucose is preferable to fatty acids as an energy source. These observations demonstrate that hepatic ACOT12 and ACOT8 are induced and responsible for ES-acetate production in diabetes mellitus and during starvation. Figure 4 with 3 supplements see all Download asset Open asset ACOT12 and ACOT8 are responsible for acetate production in energy stress conditions. (A, C) Acot12 in mice (C57BL/6) liver was knocked down by adenovirus-based shRNA, followed by detection of Acot12 protein with Western Blot (A) and evaluation of knockdown efficiency by calculating Acot12 level relative to β-actin (C). (B, D) The knockdown efficiency of Acot8 was determined in the same way as that of Acot12. (E) Enrichment of serum acetate in normal diet and 16 hr fasted mice (C57BL/6) with adenovirus-mediated knockdown of Acot12 or Acot8 in the liver. (F) Enrichment of serum acetate in streptozotocin (STZ)-induced diabetic mice (C57BL/6) with adenovirus-mediated knockdown of Acot12 or Acot8 in the liver. (G, H) Acot12 (G) or Acot8 (H) was conditionally deleted in the liver of mice (C57BL/6) by Cre-Loxp in liver, followed by detection of Acot12 and Acot8 protein with Western Blot. (I, J) Enrichment of serum acetate in normal diet and 16 hr fasted mice (C57BL/6) with Cre-Loxp-mediated conditional deletion of Acot12 (I) or Acot8 (J) in liver. Results are expressed as mean ± standard deviation (SD) of three independent experiments in (C, D), n = 10 mice per group in (E, F) and n = 6 mice per group in (I, J). Results were analyzed by unpaired Student’s t-tests (**p < 0.01, ***p < 0.001, ****p < 0.0001, n.s., no significant difference). Figure 4—source data 1 Complete, unedited immunoblots, as well as immunoblots including sample and band identification, are provided for the immunoblots presented in Figure 4. https://cdn.elifesciences.org/articles/87419/elife-87419-fig4-data1-v1.zip Download elife-87419-fig4-data1-v1.zip ACOT12- and ACOT8-catalyzed acetate production is dependent on the oxidation of FFA in both mitochondria and peroxisomes When next made efforts to identify the subcellular domains in which acetate is produced. Immunofluorescence staining and cell fractionation showed that ACOT12 was largely localized in cytosol and ACOT8 mainly in peroxisome (Figure 5A, B). It is well known that fatty acids of different chain lengths can be oxidized to yield acetyl-CoA in either the mitochondria or peroxisomes of hepatocytes, and that mitochondrial acetyl-CoA produced in fatty acid oxidation (FAO) is often exported to the cytosol in the form of citrate, which is further cleaved back to acetyl-CoA by ATP citrate lyase (ACLY) (Figure 5H; Lazarow, 1978; Leighton et al., 1989; Lodhi and Semenkovich, 2014). Thus, we examined acetate production after mitochondria- or peroxisome-yielded acetyl-CoA had been blocked. Knockdown or etomoxir inhibition of carnitine palmitoyltransferase 1 (CPT1), the main mitochondrial fatty acids transporter, decreased more than one-half of U-13C-palmitate-derived U-13C-acetate production in LO2 cell lines, despite mitochondrial β-oxidation being almost completely abolished (Figure 5C–E). Similarly, knockdown of ACLY diminished palmitate-derived acetate production to the same extent as CPT1 KD (Figure 5F). We then knocked down ATP-binding cassette subfamily D member 1 (ABCD1), a peroxisome fatty acids transporter, and observed a less than one-half decline in the production of 13C-palmitate-derived U-13C-acetate (Figure 5G). These results, together with the localization of ACOT12 and ACOT8, suggest that acetyl-CoA produced in FAO in the mitochondria and peroxisome is converted to acetate in the cytosol by ACOT12 and in peroxisomes by ACOT8 (Figure 5H). Figure 5 Download asset Open asset Acetate production is dependent on the oxidation of free fatty acids (FFAs) in both mitochondria and peroxisomes. (A) Co-immunostaining of Flag-ACOT8 with the peroxisome marker catalase and of Flag-ACOT12 with the cytosol marker GAPDH in LO2 cells. Nuclei were stained with DAPI. Scale bars represent 10 μm. (B) The protein levels of Acot12 and Acot8 in the subcellular fractions of mouse primary hepatocyte (MPH) cells. Abbreviations: ER, endoplasmic reticulum; Lyso, lysosome; Mito, mitochondria; Perox, peroxisome. (C, D) U-13C-acetate production (C) and the relative β-oxidation rate (D) in carnitine palmitoyltransferase 1A (CPT1A)-knockdown LO2 cells cultured in Hanks’ balanced salt solution (HBSS) containing U-13C-palmitate for 20 hr. (E) U-13C-acetate production (left) and the relative β-oxidation rate (right) of LO2 cells cultured in U-13C-palmitate-containing HBSS with or without the CPT1 inhibitor etomoxir (20 μM) for 20 hr. (F) U-13C-acetate production in ATP citrate lyase (ACLY)-knockdown LO2 cells cultured in HBSS supplemented with U-13C-palmitate for 20 hr. (G) U-13C-acetate production in ATP-binding cassette subfamily D member 1 (ABCD1)-knockdown LO2 cells cultured in HBSS containing U-13C-palmitate for 20 hr. (H) A schematic diagram depicting the mitochondrial and peroxisome pathways of acetate production via the oxidation of FFAs in hepatocytes. Very long- and long-chain fatty acids (VL/LCFAs) are transported through ABCD1 into a peroxisome, where they are further degraded into medium-chain fatty acids (MCFAs) via the fatty acid oxidation (FAO) process. This process involves the production of acetyl-CoA, which is further converted to acetate by peroxisome-localized ACOT8. MCFAs generated in peroxisomes are exported into the cytosol and absorbed directly by mitochondria. Cytosolic acyl-CoA derived from medium- and long-chain fatty acids (M/LCFAs) is transferred into mitochondria through All of the fatty acids and acyl-CoA in mitochondria FAO to be degraded to acetyl-CoA together with is to form citrate in the tricarboxylic cycle is exported into the where it is to acetyl-CoA by is converted to acetate by Values in are expressed as the mean ± standard deviation (SD) (n = 3) of three independent **p < 0.01, ***p < 0.001, ****p < by two-tailed unpaired Student’s Figure data 1 Complete, immunoblots sample and band are presented in Figure with the corresponding data for Download ACOT12- and ACOT8-catalyzed of from acetyl-CoA is for FAO we to the biological significance of ES-acetate production in response to energy stress by a series of serum metabolic Knockdown of Acot12 or Acot8 failed to the levels of blood glucose as well as in fasted and that these two may not be involved in glucose (Figure supplement However, knockdown of these enzymes significant of FFAs and various or fatty whereas levels were not (Figure supplement Considering that in diabetes and after prolonged starvation is mainly transported in the form of fatty rather than these suggest that ACOT12 and ACOT8 be required for degradation of fatty acids in these Indeed, we detected FAO in ACOT12 and ACOT8 knockdown MPH and LO2 cells (Figure Figure supplement We were then prompted to identify the such of A is the that free A is a for many metabolic including those involved in degradation of fatty of the the and the oxidized is important for those et al., We to whether ACOT12- and ACOT8-catalyzed conversion of acetyl-CoA to free plays a in both free level and the and oxidized CoA. To our in Acot12 or Acot8 KD the level of was decreased by and acetyl-CoA increased by and and the of to acetyl-CoA from to and (Figure In accordance with such concentrations of other oxidized and generation levels of as were diminished (Figure supplement By contrast, metabolites that have acetyl-CoA as the substrate for their were increased (Figure supplement It is important to out that all of the oxidized the level of acetyl-CoA is than than that of the other and the acetyl-CoA and plays a significant in the of (Figure Figure supplement This ACOT12- and ACOT8-catalyzed of acetyl-CoA to free and acetate is the in the of level and sustained Figure 6 with 2 supplements see all Download asset Open asset ACOT12 and ACOT8 serve to the for sustained fatty acid oxidation (A, B) primary hepatocytes (MPHs) knocked down for Acot12 (A) or Acot8 (B) were cultured in glucose-free containing acid for 20 hr, before the relative β-oxidation rate was (C) of in knocked down for Acot12 or (D) of acetyl-CoA in knocked down for Acot12 or (E) The of to acetyl-CoA in knocked down for Acot12 or (F) of and various oxidized in Abbreviations: Values are expressed as mean ± standard deviation (SD) (n = 3) of three independent experiments and were analyzed using unpaired Student’s t-tests (*p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, n.s., no significant difference). of acetyl-CoA by ACOT12 and ACOT8 is to ketogenesis from other oxidized a intermediate for the of ketone bodies using acetyl-CoA as a was dramatically in Acot12 and Acot8 KD demonstrating that Acot12 and Acot8 may be of level (Figure the main ketone bodies and were decreased significantly in STZ-induced diabetic mice with knockdown of Acot12 or Acot8 (Figure C). To the that of we detected the protein level of 2 the enzyme for Interestingly, was in KD (Figure indicating that Acot12 and Acot8 are of protein A study showed that is by et al., Thus, we examined the of and observed a clear increase in in KD (Figure corresponding to the increase in acetyl-CoA level (Figure the substrate of This that ACOT12 and ACOT8 are also of by acetyl-CoA to of acetyl-CoA and of Taken together, these suggest that ACOT12 and ACOT8 are upregulated upon energy and in the function of by increasing not only its concentration but also its the production of ketone bodies to fuel the extrahepatic Figure Download asset Open asset ACOT12 and ACOT8 are required for the production of ketone bodies in streptozotocin (STZ)-induced diabetes. (A) of in mouse primary hepatocytes (MPHs) knocked down for Acot12 or Acot8 (n = 3). (B, C) levels of acetoacetate (B) and (C) in STZ-induced diabetic C57BL/6 mice with adenovirus-mediated knockdown of Acot12 or Acot8 in the liver. E) The protein levels of in knocked down for Acot12 (D) and Acot8 G) Acot12 (F) and Acot8 (G) in mice (C57BL/6) liver were knocked down by adenovirus-based shRNA, before protein was detected by Western Blot. (H) Western Blot (upper panel) and

  PublisherDOI

• eLife Assessment: Hepatic conversion of acetyl-CoA to acetate plays crucial roles in energy stresses

  2023-06-20

  peer-reviewOpen access1st authorCorresponding

  Accumulating evidences indicate that acetate is increased in energy stresses such as diabetes mellitus and prolonged starvation. However, it is largely unknown how and where acetate is produced and what is its biological significance. We observed overproduction of acetate in an amount comparable to ketone bodies in patients and mice with diabetes or starvation. Mechanistically, ACOT 12&8 are dramatically upregulated in liver to convert FFA-derived acetyl-CoA to acetate and CoA. This conversion not only provides large amount of acetate which fuels brain preferentially rather than muscle, but also recycles CoA which is required for sustained fatty acid oxidation and ketogenesis. Taken together, we suggest that acetate is an emerging novel ketone body and may be used as a parameter to evaluate the progression of energy stress in the future.

  PublisherDOI

Recent grants

• NIH Grant R03DK082724

  NIH · $169k · 2010

• NIH Grant R56DK083658

  NIH · $127k · 2017

• NIH Grant K08DK074873

  NIH · $510k · 2010

• NIH Grant R01DK083658

  NIH · $1.9M · 2014

• Core D - Neuropathology

  NIH · $85.6M · 2020

Frequent coauthors

• Kenichi Sakamoto

  Johnson University

  50 shared

• Mary A. Butera

  Icahn School of Medicine at Mount Sinai

  44 shared

• Thomas Scherer

  27 shared

• Claudia Lindtner

  Icahn School of Medicine at Mount Sinai

  23 shared

• Henry H. Ruiz

  18 shared

• Giulia Maurizi

  Icahn School of Medicine at Mount Sinai

  16 shared

• Chun‐Xue Zhou

  Shandong University

  15 shared

• Ling Li

  Sun Yat-sen University

  14 shared

Labs

• Buettner LabPI

Education

• CLINICAL FELLOWSHIP , MEDICINE

  Montefiore Medical Center