Inhibitory Effect Of Butyrate Research Articles

Butyrate prevents the migration and invasion, and aerobic glycolysis in gastric cancer via inhibiting Wnt/β-catenin/c-Myc signaling.

Gastric cancer (GC) remains a common cause of cancer death worldwide. Evidence has found that butyrate exhibited antitumor effects on GC cells. However, the mechanism by which butyrate regulate GC cell proliferation, migration, invasion, and aerobic glycolysis remains largely unknown. The proliferation, migration, and invasion of GC cells were tested by EdU staining, transwell assays. Additionally, protein expressions were determined by western blot assay. Next, glucose uptake, lactate production, and cellular ATP levels in GC cells were detected. Furthermore, the antitumor effects of butyrate in tumor-bearing nude mice were evaluated. We found, butyrate significantly prevented GC cell proliferation, migration, and invasion (p < .01). Additionally, butyrate markedly inhibited GC cell aerobic glycolysis, as shown by the reduced expressions of GLUT1, HK2, and LDHA (p < .01). Moreover, butyrate notably decreased nuclear β-catenin and c-Myc levels in GC cells (p < .01). Remarkably, through activating Wnt/β-catenin signaling with LiCl, the inhibitory effects of butyrate on the growth and aerobic glycolysis of GC cells were diminished (p < .01). Moreover, butyrate notably suppressed tumor volume and weight in GC cell xenograft nude mice in vivo (p < .01). Meanwhile, butyrate obviously reduced nuclear β-catenin, c-Myc, GLUT1, HK2 and LDHA levels in tumor tissues in GC cell xenograft mice (p < .01). Collectively, butyrate could suppress the growth and aerobic glycolysis of GC cells in vitro and in vivo via downregulating wnt/β-catenin/c-Myc signaling. These findings are likely to prove useful in better understanding the role of butyrate in GC.

Abstract P320: The Gut Microbial Metabolite Butyrate Suppresses Cardiac Hypertrophy Via An Epigenetic Mechanism

Introduction: Short-chain fatty acids (SCFA) are one of the gut microbial metabolites that can influence host health and disease. We previously reported that gut dysbiosis is associated with heart failure, and that the proportion of butyrate-producing bacteria is decreased in the gut of patients with heart failure. Purpose: We investigated the molecular mechanism of butyrate in the development of cardiac hypertrophy. Methods and Results: Single-cell transcriptome analysis and co-expression network analysis revealed that G protein-coupled receptors for short-chain fatty acid receptors were not expressed in cardiomyocytes and that Olfr78 was expressed in vascular smooth muscle cells in the heart. On the other hand, treatment with butyrate inhibited ET1-induced and isoproterenol (ISO)-induced hypertrophic growth in cultured neonatal rat cardiomyocytes. Moreover, butyrate increased the acetylation levels of histone H3, suggesting the inhibitory effect of butyrate on HDAC. In addition, butyrate caused the degradation of HDAC2 and up-regulation of Inpp5f, encoding inositol polyphosphate-5-phosphatase f, leading to a significant decrease in the phosphorylation levels of Akt and glycogen synthase kinase 3β (GSK3β). Finally, intraperitoneal injection of butyrate inhibited ISO-induced cardiac hypertrophy in mice. These results suggest that butyrate protects against hypertrophic responses via suppression of the Akt-GSK3β pathway through HDAC inhibition. Conclusion: In the heart, there were no known short-chain fatty acid receptors in cardiomyocytes. However, butyrate was shown to have an epigenetic mechanism in suppressing effect on cardiomyocyte hypertrophy via suppression of HDAC2-Akt-GSK3β axis. Our results uncover a potential link between dysbiosis of intestinal microbiota and the development of cardiac hypertrophy.

IL-33 Is Involved in the Anti-Inflammatory Effects of Butyrate and Propionate on TNFα-Activated Endothelial Cells.

Short-chain fatty acids (e.g., butyrate and propionate) are able to diminish endothelial cell activation. The aim of this study was to investigate whether intracellular IL-33 mediates the effects of butyrate and propionate on TNFα-induced IL-8 production and vascular cell adhesion molecule-1 (VCAM-1) expression. In addition, it was investigated whether regulating NF-κB and MAPK signaling pathways are involved. Intracellular IL-33 was measured in human endothelial cells (HUVECs) pre-incubated for 24 h with butyrate (0.1 mM or 5 mM), propionate (0.3 mM or 10 mM), or trichostatin A (TSA, 0.5 μM) prior to TNFα (1 ng/mL) stimulation (24 h). The effects of butyrate, propionate, and TSA on TNFα-induced IL-8, vascular cell adhesion molecule-1 (VCAM-1), NF-κB, and MAPK signaling pathways in normal HUVECs and IL-33 siRNA (siIL-33)-transfected HUVECs were compared to study the role of IL-33 in the protective effects of butyrate and propionate. Endogenous IL-33 was highly expressed in the perinuclear in HUVECs, which was significantly reduced by TNFα stimulation. The TNFα-induced reduction in IL-33 was prevented by pre-incubation with butyrate or propionate. Butyrate (0.1 mM), propionate (0.3 mM), and TSA inhibited the IL-8 production and activation of NF-κB. Interestingly, this effect was not observed in siIL-33-transfected HUVECs. The effects of butyrate (5 mM), propionate (10 mM), and TSA (0.5 μM) on VCAM-1 expression and activation of MAPK signaling pathways were not affected by siIL-33 transfection. In conclusion, we showed that the inhibitory effects of butyrate and propionate on TNFα-induced IL-8 production were mediated by the HDACs/IL-33/NF-κB pathway, while their effects on VCAM-1 expression might be associated with the HDACs/MAPK signaling pathway, independently of IL-33.

The role of butyrate in surgical and oncological outcomes in colorectal cancer.

Butyrate is a short-chain fatty acid produced by colonic gut bacteria as a result of fermentation of dietary fibers. In the colon, butyrate is a major energy substrate and contributes to the nutritional support and proliferation of a healthy mucosa. It also promotes the intestinal barrier function by enhancing mucus production and tight junctions. In addition to its pro-proliferative effect in healthy colonocytes, butyrate inhibits the proliferation of cancer cells. The antineoplastic effect of butyrate is associated with the inhibitory effect of butyrate on histone deacetylase (HDAC) enzymes, which promote carcinogenesis. Due to the metabolic shift of cancer cells toward glycolysis, unused butyrate accumulates and inhibits procarcinogenic HDACs. In addition, recent studies suggest that butyrate may improve the healing of colonic tissue after surgery in animal models, specifically at the site of reconnection of colonic ends, anastomosis, after surgical resection. Here, we review current evidence on the impact of butyrate on epithelial integrity and colorectal cancer and present current knowledge on data that support its potential applications in surgical practice.

Community profile governs substrate competition in polyhydroxyalkanoate (PHA)-producing mixed cultures

Polyhydroxyalkanoates (PHAs) are biobased, biodegradable polymers that can be produced efficiently by mixed microbial cultures (MMCs) at low cost. Substrate preferences have been found to vary depending on the microbial composition in the MMCs. In this study, the competition between the uptake of different substrates has been related for the first time to the abundance of certain microbial communities. With a higher abundance of Plasticicumulans enriched in the culture, the inhibitory effect of butyrate and valerate on the uptake of acetate and propionate increased and the hydroxybutyrate:hydroxyvalerate (HB:HV) ratio in the final PHA polymer approached the theoretical value calculated from the volatile fatty acid (VFA) composition. This finding can help understanding of the metabolism of PHA accumulating microorganisms and thus predict better the VFA consumption and PHA composition.

Abstract B13: Immunogenic chemotherapy alters microbiome-induced T-cell activation

Abstract Introduction: The efficacy of cancer immunotherapy is influenced by the patient’s intestinal microbiome. The local and systemic effects of the microbiome on immune function are thought to depend upon a combination of microbial metabolites produced collectively from the diverse bacterial species present in the intestine and the activation of intestine-resident immunocytes such as antigen-presenting cells and T lymphocytes. The interactions between the microbiome and the immune response are regulated by Intestinal epithelial cells, which together maintain intestinal immune homeostasis. The oral chemotherapeutics lenalidomide (Len) and cyclophosphamide (CP) are currently licensed for the treatment of numerous cancers. These agents are also potent immune modulators and have been studied alongside checkpoint inhibition (CPI) with promising results. Few studies have sought to investigate the effect of these immune-modulatory agents on immune responses activated by the intestinal microbiome. In this study the immunomodulatory properties of low-dose Len and CP on IEC function and T-cell activation were investigated in combination with microbial TLR ligands and short-chain fatty acids (SFCA), immunogenic metabolites produced by intestinal bacteria. Results: Both Len and CP altered cytokine expression from IEC in response to stimulation by TLR ligands including lipopolysaccharide, peptidoglycan, and flagellin. Combinations of SCFA modulated the viability and proinflammatory effects of CP on IEC. SCFA and LEN in combination enhanced the expression of antimicrobial peptides from IEC. LEN enhanced IFN-γ and IL-17A expression by CD8+ and CD4+ T-cells. Combinations of LEN and the SCFA acetate further enhanced IFN-γ and IL-17A expression. Cytokines expressed by IEC predispose towards the development of Th2 immune responses; however, in the presence of IEC expressed Th2 skewing cytokines including IL-33 and TSLP, LEN promoted IFN-γ expressing Th1 responses. CP inhibited the ability of the SCFA butyrate to induce T-regulatory cells while Len mitigated the Th1 inhibitory effect of butyrate. Conclusions: LEN and SCFA synergize to drive Th1 responses while CP mitigate their immune-regulatory properties. Both LEN and CP prime IEC function. Collectively these data provide evidence supporting the ability of low-dose LEN and CP to alter components of intestinal immune network consisting of IEC, Immunocytes, and the microbiome. These findings have implications for the homeostasis of intestinal immunity and the maintenance of the intestinal microbiome during chemotherapeutic treatment. The influence of these agents on the microbiome and CPI immunotherapy is under investigation. Citation Format: Peter L. Smith, A.G. Dalgleish. Immunogenic chemotherapy alters microbiome-induced T-cell activation [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2019 Nov 17-20; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2020;8(3 Suppl):Abstract nr B13.

Vancomycin treatment and butyrate supplementation modulate gut microbe composition and severity of neointimal hyperplasia after arterial injury.

Gut microbial metabolites are increasingly recognized as determinants of health and disease. However, whether host -: microbe crosstalk influences peripheral arteries is not understood. Neointimal hyperplasia, a proliferative and inflammatory response to arterial injury, frequently limits the long-term benefits of cardiovascular interventions such as angioplasty, stenting, and bypass surgery. Our goal is to assess the effect of butyrate, one of the principal short chain fatty acids produced by microbial fermentation of dietary fiber, on neointimal hyperplasia development after angioplasty. Treatment of male Lewis Inbred rats with oral vancomycin for 4weeks changed the composition of gut microbes as assessed by 16S rRNA-based taxonomic profiling and decreased the concentration of circulating butyrate by 69%. In addition, rats treated with oral vancomycin had exacerbated neointimal hyperplasia development after carotid angioplasty. Oral supplementation of butyrate reversed these changes. Butyrate also inhibited vascular smooth muscle cell proliferation, migration, and cell cycle progression in a dose-dependent manner invitro. Our results suggest for the first time that gut microbial composition is associated with the severity of arterial remodeling after injury, potentially through an inhibitory effect of butyrate on VSMC.

Effect of butyrate on the heregulin/ErbB-mediated proliferation of human colorectal cancer cells

The expression of ErbB proteins, together with heregulin, was found to be increased in colon cancer cells compared with normal mucosa, and heregulin-stimulated activation of ErbB signaling was observed to contribute to the proliferation of colon cancer cells. Butyrate produced during the fermentation of fiber by intestinal bacteria is known to exert diverse anticancer effects, including the induction of differentiation, cell cycle arrest and growth suppression in human colon cancer cells. In this study, we investigated whether butyrate affects the heregulin/ErbB-mediated proliferation of colon cancer cells. The growth of human CaCo-2 and SNU-C4 colon cancer cells, which express ErbB1-4 proteins, was stimulated by heregulin in a concentration-dependent manner. Pretreatment with sodium butyrate abolished heregulin-stimulated proliferation in both cell lines. Although butyrate did not alter ErbB protein expression and activation, it did block the prolonged activation of Akt and Erk1/2, which are known to be important ErbB downstream molecules mediating heregulin-stimulated proliferation. Our data suggest that the inhibitory effects of butyrate on the heregulin-stimulated proliferation of colorectal cancer cells are, in part, associated with the suppression of the Akt and Erk signaling pathway. Butyrate may act as a preventive and therapeutic agent in the progression of colorectal cancer through the regulation of heregulin-stimulated mitogenic signaling.

Butyrate blocks interferon-γ-inducible protein-10 release in human intestinal subepithelial myofibroblasts

Interferon (IFN)-gamma-inducible protein (IP)-10 is a chemoattractant for CXCR 3-expressing T lymphocytes and monocytes. IP-10 has been reported to mediate chronic inflammation such as that in inflammatory bowel disease (IBD). However, the local secretion of IP-10 in the intestine remains unclear. In this study, we investigated IP-10 secretion in human colonic subepithelial myofibroblasts (SEMFs). IP-10 secretion was determined by enzyme-linked immunosorbent assay (ELISA), and IP-10 mRNA expression was evaluated by Northern blotting. Interleukin (IL)-10 mRNA was not detected in unstimulated SEMFs. Interferon (IFN)-gamma strongly induced IP-10 mRNA expression. Tumor necrosis factor (TNF)-alpha also stimulated IP-10 mRNA expression, but this was much weaker than that induced by IFN-gamma. The effects of IFN-gamma and TNF-alpha were detected in a dose- and time-dependent manner. These responses were also observed at the protein levels. The IFN-gamma-induced IP-10 secretion was not affected by acetate or propionate, but was significantly reduced by butyrate. Trichostatin A, a specific inhibitor of histone deacetylase, also blocked the IFN-gamma- and TNF-alpha-induced IP-10 mRNA expression, but the effects of trichostatin A were weaker than those of butyrate. The inhibitory effect of butyrate on IFN-gamma-induced IP-10 release was not associated with STAT (signaling transducer and activator of transcription)-1alpha activation. We demonstrated that human colonic SEMFs are the local site for the secretion IP-10. The regulation of IP-10 release by IFN-gamma and butyrate may play an important role in controlling chronic mucosal inflammation in pathological entities such as IBD.

Butyrate reduces liver metastasis of rat colon carcinoma cells in vivo and resistance to oxidative stress in vitro

Injection of the rat colon carcinoma cell line CC531 into spleen of syngeneic rats results in considerable amounts of liver metastases within 14 days. We investigated whether preincubation of the cells with butyrate reduced their metastatic ability in vivo and whether this was accompanied by reduction in related properties such as secretion of metalloproteinases and their ability to withstand oxidative stress. Butyrate incubation reduced cell growth rate and initiated apoptosis in a dose- and time-related manner, but proliferation was retrieved when cultivation was continued in medium without butyrate. Splenic injection of butyrate treated, proliferating cells resulted in significantly reduced amounts of tumor mass compared to untreated cells. The butyrate treated cells were more susceptible to oxidative stress than control cells, as demonstrated by increased number of apoptotic cells and reduced cell growth after exposure to menadione. A reduction in cellular glutathione was found after prolonged incubation with butyrate. Butyrate appeared not to alter the secretion of active metalloproteinases from the cells although an apparent increase in proforms was demonstrated. Neither did butyrate alter the synthesis of metalloproteinase inhibitors. Lastly, a reduced adhesion of the tumor cells to collagen coated matrix was found after butyrate treatment. Thus, the inhibitory effects of butyrate on tumor malignancy are caused by a diversity of mechanisms.

Sodium butyrate enhances complement-mediated cell injury via down-regulation of decay-accelerating factor expression in colonic cancer cells.

Decay-accelerating factor (DAF) expressed on the surface of colonic cancer cells presents a barrier to complement-mediated clearance by contributing to the ineffectiveness of the humoral immune response. In this study, to investigate the mechanisms responsible for the anti-tumor effects of butyrate, we evaluated how butyrate modulates DAF expression in colonic cancer cells. Three colonic cancer cell lines (HT-29, Caco-2, and T84 cells) were studied. DAF protein expression was assessed by western blot, and DAF mRNA expression was evaluated by northern blot. Complement C3 deposition on the surface of colonic cancer cells was determined by enzyme-linked immunosorbent assay (ELISA). The promoter activity of the DAF gene was assessed by a reporter gene-luciferase assay. Butyrate reduced the basal and interleukin-4 (IL-4)- and tumor necrosis factor-alpha (TNF-alpha)-induced expression of DAF protein and mRNA in HT-29 cells. It increased the susceptibility to complement attack and enhanced C3 deposition on HT-29 cells. The inhibitory effect of butyrate on DAF mRNA expression was also observed in T84 and Caco-2 cells. Butyrate decreased basal DAF expression at both transcriptional and post-transcriptional levels. The inhibitory effect of butyrate on IL-4-induced DAF expression was closely associated with a blockade of IL-4-induced DAF mRNA stability. TNF-alpha-induced transcriptional activation and the increased stability of the DAF gene were also blocked by butyrate. Similar but weak effects were induced by trichostatin A, a potent histone deacetylase inhibitor, suggesting that histone acetylation might participate in butyrate activity. These observations indicate that both a down-regulation of DAF expression and the induction of susceptibility to complement attack contribute to the anti-tumor effects of butyrate in colonic cancer.

Effects of butyrate and propionate on the adhesion, growth, cell cycle kinetics, and protein synthesis of cultured human gingival fibroblasts.

Various periodontal and root canal pathogens, such as the Bacteroides species, can produce significant amounts of short chain fatty acids (SCFA). The roles of SCFA in the pathogenesis of periodontal disease are still not fully understood. We therefore investigated 2 main SCFA, butyrate and propionate, on the functional behavior of cultured human gingival fibroblasts (GF) such as cell growth, protein synthesis, cell adhesion capacity, and cell cycle progression. Butyrate and propionate inhibited the growth of healthy (HGF) and inflamed gingival fibroblasts (IGF) in a dose dependent manner. At concentrations of 4, 8, and 16 mM, butyrate suppressed the cell growth by 11 to 58%, 16 to 60%, and 50 to 71%, respectively. The response of cultured gingival fibroblasts to SCFA showed individual differences. Morphologically, GF became larger and more flattened in appearance following exposure to butyrate (>8 mM) and propionate (>24 mM) for 5 days. Inhibitory effects of butyrate (>2 mM) and propionate (>8 mM) on the growth of GF were due possibly to their inhibition of cell-cycle progression. At concentrations of 2 and 8 mM, butyrate led to G0/G1 arrest. Elevation of the exposure concentration to 8 to 24 mM further result in G2/M phase arrest of GF. On the other hand, propionate, at concentrations ranging from 4 to 24 mM, led to G0/G1 arrest. Butyrate (>2 mM) inhibited the proline-rich protein synthesis of GF. At concentrations of 4, 8, 16, and 24 mM, butyrate inhibited the protein synthesis of HGF-1 by 42%, 43%, 51%, and 54%, respectively. In all strains of cultured GF, the suppressive effect of propionate is less than that of butyrate. At concentration range of 4 to 24 mM, propionate suppressed the protein synthesis of HGF-1 by 23 to 43%. However, both butyrate and propionate (4 to 48 mM) exerted little effects on the adhesion of GF to type I collagen within 3 hours of incubation. These results suggested that SCFA released by pathogenic microorganisms can contribute to the gingival tissue dysfunction and breakdown through their actions on specific biological functions of GF.

Short-chain fatty acids inhibit intestinal trefoil factor gene expression in colon cancer cells.

Intestinal trefoil factor (ITF) gene expression was detected in five colon cancer cell lines. ITF was synthesized by mucous cells of LIM 1215 and LIM 1863 lines, from which it is secreted constitutively. The ITF mRNA transcript was estimated to be 0.6 kb. In LIM 1215 cells, the expression of ITF was potently and dose-dependently inhibited by short-chain fatty acids (butyrate > propionate > acetate) within 8 h of application. The inhibitory effect of butyrate was ablated by actinomycin D and preceded its effects on differentiation of LIM 1215 cells as indicated by induction of alkaline phosphatase activity and counting of periodic acid-Schiff-positive cells. The human ITF promoter contained an 11-residue consensus sequence with high homology to the butyrate response element of the cyclin D1 gene. Mobility shift assays show specific binding of this response element to nuclear protein extracts of LIM 1215 cells. We conclude that butyrate inhibits ITF expression in colon cancer cells and that this effect may be mediated transcriptionally and independently of its effects on differentiation.

Inhibition of thyroxine 5′-deiodination type II in cultured human placental cells by cortisol, insulin, 3′,5′-cyclic adenosine monophosphate, and butyrate

The regulation of conversion of thyroxine (T 4) to 3,5,3′-triiodothyronine (T 3) by the type II iodothyronine deiodinating pathway was studied in normal human placental cells cultured from the chorionic membrane. T 4 5′-deiodination was measured in cell sonicates after intact cells were incubated with test agents for 24 to 48 hours. Stimulation of T 4 5′-deiodination occurred to a similar degree after depriving cells of thyroid hormone in serum-free medium and in medium containing 10% calf serum. Cortisol at 10 to 100 nmol/L in serum-free medium inhibited T 4 5′-deiodination up to 36%, and 1 to 100 nmol/L of insulin inhibited deiodination up to 50%. Dibutyryl-cyclic AMP (dbcAMP) inhibited deiodination, but this appeared to result from the inhibitory effects of butyrate. Addition to the culture media of 8-bromo-cAMP, cholera toxin, and theophylline each caused partial inhibition of T 4 5′-deiodination, strongly suggesting an inhibitory effect of raised intracellular cAMP. Neither α- nor β-adrenergic agonists had any effect when added to the culture medium, nor did glucagon or cysteamine. These results demonstrate a complex, multihormonal control of human placental type II iodothyronine deiodination, and suggest that changes in the activity of this pathway may result in altered intracellular, and conceivably circulating, T 3 concentrations in states of cortisol excess and marked hyperinsulinism. The factor that regulates type II deiodination via cAMP remains to be identified.

Inhibitory effect of short-chain aliphatic acids on deoxythymidine incorporation into DNA in mitogen-stimulated and leukemic lymphocytes.

The effect of short-chain aliphatic acids on [3H]deoxythymidine incorporation into DNA was studied in human mitogen-stimulated lymphocytes. Butyric acid at 1-2 mM level was strongly inhibitory; however, its hydroxy or amino derivatives were ineffective. Valeric and propionic acids were less inhibitory. Formic, acetic, and hexanoic acids did not have any inhibitory effect. The effect of butyrate persisted in washed lymphocytes for 24-48 hr depending on the concentration used and the time of its addition into the incubation medium. The inhibitory effects of butyrate, valerate, and propionate on deoxythymidine and amino acid incorporation into DNA and protein were also found in human leukocytes in myelocytic and lymphocytic leukemias.

Butyrate and propionate: important components of toxic dental plaque extracts.

Extracts of in vitro-cultured human dental plaque contain factors toxic to mammalian cells. Previous studies demonstrated that those toxic factors most readily released from cultured plaque had very low molecular weights and were heat stable. Studies reported here demonstrate that metabolic end products including short-chain fatty acids were present in fractions containing the low-molecular-weight, heat-stable factors. The salts of two of these acids, butyrate and propionate, inhibited proliferation of both mouse L929 cells and human gingival fibroblasts. Furthermore, when tested at concentrations present in plaque extracts, the inhibitory effects of butyrate and propionate accounted for essentially all the inhibitory potential of the extracts. These findings, taken together with those of other groups, suggest that butyrate and propionate, end products of dental plaque metabolism, may have an etiological role in periodontal disease.

Fatty Acid Inhibition of Sulfation Factor-stimulated 35SO4 Incorporation into Embryonic Chicken Cartilage

Abstract The effect of fatty acids and ketones on 35SO4 incorporation into the chondromucoproteins of embryonic chicken cartilage was studied in vitro. Both the nonstimulated incorporation (that occurring in media without added serum) and sulfation factor stimulated incorporation (that occurring in media with 5.0% rat serum added) were investigated. Butyrate and octanoate, from 0.01 mm to 1.0 mm, had minimal effects on nonstimulated 35SO4 incorporation. At 5.0 mm both caused moderate inhibition of 35SO4 incorporation into the cartilage. In the serum stimulated state, butyrate and octanoate, at 0.5 mm, 1.0 mm, and 5.0 mm, resulted in striking dose-related inhibition of 35SO4 incorporation. Palmitate and oleate from 0.1 mm to 1.0 mm had no effect on nonstimulated 35SO4 incorporation, but did cause marked inhibition of the serum-stimulated component of 35SO4 incorporation. β-Hydroxybutyrate, acetoacetate, and acetate, from 0.1 mm to 5.0 mm, had no effect on either nonstimulated or serum-stimulated 35SO4 incorporation. 35SO4 incorporation into cartilage chondromucoproteins was independent of glucose over a concentration range of 2.5 mm to 15.0 mm. In the absence of glucose in the medium, nonstimulated 35SO4 incorporation was markedly inhibited, but the incremental increase stimulated by rat serum was unaffected. The inhibitory effect of butyrate on serum-stimulated 35SO4 incorporation is reversible and this indicates that the fatty acid effect is not a nonspecific toxic action. The data suggest that fatty acids may regulate the synthesis of chondromucoproteins in cartilage and in this way dissociate the lipolytic and glucose mobilizing effects of growth hormone from its stimulation of growth. Though the locus of action of fatty acids on 35SO4 incorporation is unknown, it may be at the site of the action of sulfation factor.