Effect of high versus low doses of fat and vitamin A dietary supplementation on fatty acid composition of phospholipids in mice

Dietary fat and vitamin A provide important precursors for potent bioactive ligands of nuclear hormone receptors, which regulate various enzymes involved in lipid homeostasis, metabolism and inflammation. We determined the effects of dietary fat and dietary vitamin A on hepatic expression of two fatty acid metabolizing enzymes, elongase 6 (ELOVL6) and stearoyl-coenzyme A desaturase 1 (SCD1) and the concentration of saturated fatty acids (SAFA) and monounsaturated fatty acid (MUFA) of phospholipids in serum and liver. Mice (n = 6) were fed 4 weeks with diets containing 2, 5 and 25 % of fat or vitamin A (0, 2,500 and 326,500 RE/kg as retinyl palmitate). MUFAs and SAFAs were measured using GC and ESI–MS/MS. Hepatic expression of metabolizing enzymes was determined using QRT-PCR. ELOVL6 was significantly down-regulated in response to a high-fat diet (p < 0.001) and significantly up-regulated in response to low-fat diet (p < 0.05). SCD1 expression was significantly lower in high- versus low-fat diet (p < 0.05). The vitamin A content in the diet did not influence the hepatic expression of both enzymes. In plasma, the amounts of MUFAs bound to phospholipids significantly decreased in response to a high-fat diet and increased after a low-fat diet. This tendency was also observed in the liver for various phospholipids sub-classes. In summary, this study shows that fat content in the diet has a stronger impact than the content of vitamin A on hepatic gene expression of SCD1 and ELOVL6 and thereby on MUFA and SAFA concentrations in liver and plasma.     

Isosteviol has beneficial effects on palmitate-induced α-cell dysfunction and gene expression

Background

Long-term exposure to high levels of fatty acids impairs insulin secretion and exaggerates glucagon secretion. The aim of this study was to explore if the antihyperglycemic agent, Isosteviol (ISV), is able to counteract palmitate-induced α-cell dysfunction and to influence α-cell gene expression.

Methodology/Principal Findings

Long-term incubation studies with clonal α-TC1–6 cells were performed in the presence of 0.5 mM palmitate with or without ISV. We investigated effects on glucagon secretion, glucagon content, cellular triglyceride (TG) content, cell proliferation, and expression of genes involved in controlling glucagon synthesis, fatty acid metabolism, and insulin signal transduction. Furthermore, we studied effects of ISV on palmitate-induced glucagon secretion from isolated mouse islets. Culturing α-cells for 72-h with 0.5 mM palmitate in the presence of 18 mM glucose resulted in a 56% (p<0.01) increase in glucagon secretion. Concomitantly, the TG content of α-cells increased by 78% (p<0.01) and cell proliferation decreased by 19% (p<0.05). At 18 mM glucose, ISV (10⁻⁸ and 10⁻⁶ M) reduced palmitate-stimulated glucagon release by 27% (p<0.05) and 27% (p<0.05), respectively. ISV (10⁻⁶ M) also counteracted the palmitate-induced hypersecretion of glucagon in mouse islets. ISV (10⁻⁶ M) reduced α-TC1–6 cell proliferation rate by 25% (p<0.05), but ISV (10⁻⁸ and 10⁻⁶ M) had no effect on TG content in the presence of palmitate. Palmitate (0.5 mM) increased Pcsk2 (p<0.001), Irs2 (p<0.001), Fasn (p<0.001), Srebf2 (p<0.001), Acaca (p<0.01), Pax6 (p<0.05) and Gcg mRNA expression (p<0.05). ISV significantly (p<0.05) up-regulated Insr, Irs1, Irs2, Pik3r1 and Akt1 gene expression in the presence of palmitate.

Conclusions/Significance

ISV counteracts α-cell hypersecretion and apparently contributes to changes in expression of key genes resulting from long-term exposure to palmitate. ISV apparently acts as a glucagonostatic drug with potential as a new anti-diabetic drug for the treatment of type 2 diabetes.     

De novo lipogenesis in the differentiating human adipocyte can provide all fatty acids necessary for maturation

The primary products of de novo lipogenesis (DNL) are saturated fatty acids, which confer adverse cellular effects. Human adipocytes differentiated with no exogenous fat accumulated triacylglycerol (TG) in lipid droplets and differentiated normally. TG composition showed the products of DNL (saturated fatty acids from 12:0 to 18:0) together with unsaturated fatty acids (particularly 16:1n-7 and 18:1n-9) produced by elongation/desaturation. There was parallel upregulation of expression of genes involved in DNL and in fatty acid elongation and desaturation, suggesting coordinated control of expression. Enzyme products (desaturation ratios, elongation ratios, and total pathway flux) were also correlated with mRNA levels. We used (13)C-labeled substrates to study the pathway of DNL. Glucose (5 mM or 17.5 mM in the medium) provided less than half the carbon used for DNL (42% and 47%, respectively). Glutamine (2 mM) provided 9-10%, depending upon glucose concentration. In contrast, glucose provided most (72%) of the carbon of TG-glycerol. Pathway analysis using mass isotopomer distribution analysis (MIDA) revealed that the pathway for conversion of glucose to palmitate is complex. DNL in human fat cells is tightly coupled with further modification of fatty acids to produce a range of saturated and unsaturated fatty acids consistent with normal maturation.     

Modulation of palmitate-induced endoplasmic reticulum stress and apoptosis in pancreatic β-cells by stearoyl-CoA desaturase and Elovl6

Induction of endoplasmic reticulum (ER) stress and apoptosis by elevated exogenous saturated fatty acids (FAs) plays a role in the pathogenesis of β-cell dysfunction and loss of islet mass in type 2 diabetes. Regulation of monounsaturated FA (MUFA) synthesis through FA desaturases and elongases may alter the susceptibility of β-cells to saturated FA-induced ER stress and apoptosis. Herein, stearoyl-CoA desaturase (SCD)1 and SCD2 mRNA expression were shown to be induced in islets from prediabetic hyperinsulinemic Zucker diabetic fatty (ZDF) rats, whereas SCD1, SCD2, and fatty acid elongase 6 (Elovl6) mRNA levels were markedly reduced in diabetic ZDF rat islets. Knockdown of SCD in INS-1 β-cells decreased desaturation of palmitate to MUFA, lowered FA partitioning into complex neutral lipids, and increased palmitate-induced ER stress and apoptosis. Overexpression of SCD2 increased desaturation of palmitate to MUFA and attenuated palmitate-induced ER stress and apoptosis. Knockdown of Elovl6 limited palmitate elongation to stearate, increasing palmitoleate production and attenuating palmitate-induced ER stress and apoptosis, whereas overexpression of Elovl6 increased palmitate elongation to stearate and palmitate-induced ER stress and apoptosis. Overall, these data support the hypothesis that enhanced MUFA synthesis via upregulation of SCD2 activity can protect β-cells from elevated saturated FAs, as occurs in prediabetic states. Overt type 2 diabetes is associated with diminished islet expression of SCD and Elovl6, and this can disrupt desaturation of saturated FAs to MUFAs, rendering β-cells more susceptible to saturated FA-induced ER stress and apoptosis.     

The regulation of glyceride synthesis in isolated white-fat cells. The effects of palmitate and lipolytic agents

1. 0.5mm-Palmitate stimulated incorporation of [U-¹⁴C]glucose into glyceride glycerol and fatty acids in normal fat cells in a manner dependent upon the glucose concentration. 2. In the presence of insulin the incorporation of 5mm-glucose into glyceride fatty acids was increased by concentrations of palmitate, adrenaline and 6-N-2′-O-dibutyryladenosine 3′:5′-cyclic monophosphate up to 0.5mm, 0.5μm and 0.5mm respectively. Higher concentrations of these agents produced progressive decreases in the rate of glucose incorporation into fatty acids. 3. The effects of palmitate and lipolytic agents upon the measured parameters of glucose utilization were similar, suggesting that the effects of lipolytic agents are mediated through increased concentrations of free fatty acids. 4. In fat cells from 24h-starved rats, maximal stimulation of glucose incorporation into fatty acids was achieved with 0.25mm-palmitate. Higher concentrations of palmitate were inhibitory. In fat cells from 72h-starved rats, palmitate only stimulated glucose incorporation into fatty acids at high concentrations of palmitate (1mm and above). 5. The ability of fat cells to incorporate glucose into glyceride glycerol in the presence of palmitate decreased with increasing periods of starvation. 6. It is suggested that low concentrations of free fatty acids stimulate fatty acid synthesis from glucose by increasing the utilization of ATP and cytoplasmic NADH for esterification of these free fatty acids. When esterification of free fatty acids does not keep pace with their provision, inhibition of fatty acid synthesis occurs. Provision of free fatty acids far in excess of the esterification capacity of the cells leads to uncoupling of oxidative phosphorylation and a secondary stimulation of fatty acid synthesis from glucose.     

Alternate pathways of linolenic acid biosynthesis in growing cultures of Penicillium chrysogenum

Evidence was obtained that Penicillium chrysogenum can produce linolenate by two biosynthetic pathways, i.e., by elongation of a shorter trienoic acid as well as direct desaturation of 18-C acids. In oxygen deficient cultures, exogenous hexadecatrienoate stimulated [1-¹⁴C]acetate incorporation into labeled octadecatrienoate and [U-¹⁴C]hexadecatrienoate with nonlabeled acetate yielded linolenate that had relatively little label in the 1-C position. With [1-¹⁴C]acetate as the only added substrate, oxygen deficiency inhibited incorporation of label into monoenoic and dienoic acids but not into trienoic acids. Incorporation of the [U-¹⁴C]linoleate into linolenate also was inhibited.In aerated cultures, 1-¹⁴C-label from laurate, palmitate, stearate, oleate, linoleate, and hexadecatrienoate was readily incorporated into linolenate. Decarboxylation and oxidation studies indicated that the longer acids were incorporated largely intact. [U-¹⁴C]Linoleate was incorporated into linolenate in which the fraction of label in 1-C was similar to that of the substrate. These data suggest that this mold has broader synthetic capabilities than do some chloroplast systems for the biosynthesis of linolenate.     

Metabolomics reveals that CAF-derived lipids promote colorectal cancer peritoneal metastasis by enhancing membrane fluidity

Patients with peritoneal metastasis (PM) of colorectal cancer (CRC) have poorer overall survival outcomes than those without PM. Cancer-associated fibroblasts (CAFs) are a major component of the tumor microenvironment and mediate CRC progression and PM. It is imperative to identify and develop novel therapeutic targets for PM-CRC driven by CAFs. Using lipidomics, we reveal that the abundance of phosphatidylcholine (PC) with unsaturated acyl chains was increased in clinical PM-CRC specimens. Additionally, we found that CAFs were present at a higher relative abundance in primary PM-CRC tumors and that membrane fluidity in CRC cells was increased after incubation with CAF-conditioned medium (CM) through three independent methods: lipidomics, fluorescence recovery after photobleaching (FRAP), and generalized polarization. Then, we found that increased membrane fluidity can enhance glucose uptake and metabolism, as supported by real-time bioenergetics analysis and U-¹³C glucose labeling. Interestingly, stearoyl-CoA desaturase 1 (SCD), the rate-limiting enzyme in the biosynthesis of unsaturated fatty acids (uS-FAs), was expressed at low levels in PM and associated with poor prognosis in CRC patients. Importantly, by untargeted metabolomics analysis and fatty acid ([U-¹³C]-stearic acid) tracing analyses, we found that CRC cells take up lipids and lipid-like metabolites secreted from CAFs, which may compensate for low SCD expression. Both in vitro and in vivo experiments demonstrated that sodium palmitate (C16:0) treatment could decrease the CAF-induced change in cell membrane fluidity, limit glucose metabolism, suppress cell invasiveness, and impair tumor growth and intraperitoneal dissemination. An increased C16:0 concentration was shown to induce apoptosis linked to lipotoxicity. Furthermore, C16:0 effectively enhanced the antitumor activity of 5-fluorouracil (5-FU) in vitro and was well tolerated in vivo. Taken together, these findings suggest that adding the saturated fatty acid (S-FA) C16:0 to neoadjuvant chemotherapy may open new opportunities for treating PM-CRC in the future.     

Measurement of precursor enrichment for calculating intramuscular triglyceride fractional synthetic rate

Our goal was to assess the validity of the enrichments of plasma free palmitate and intramuscular (IM) fatty acid metabolites as precursors for calculating the IM triglyceride fractional synthetic rate. We infused U-¹³C₁₆-palmitate in anesthetized rabbits for 3 h and sampled adductor muscle of legs using both freeze-cut and cut-freeze approaches. We found that IM free palmitate enrichment (0.70 ± 0.07%) was lower (P < 0.0001) than IM palmitoyl-CoA enrichment (2.13 ± 0.17%) in samples taken by the freeze-cut approach. The latter was close (P = 0.33) to IM palmitoyl-carnitine enrichment (2.42 ± 0.16%). The same results were obtained from the muscle samples taken by the cut-freeze approach, except the enrichment of palmitoyl-CoA (2.21 ± 0.08%) was lower (P = 0.02) than that of palmitoyl-carnitine (2.77 ± 0.17%). Plasma free palmitate enrichment was ∼2-fold that of IM palmitoyl-CoA enrichment and palmitoyl-carnitine enrichment (P < 0.001). These findings indicate that plasma free palmitate overestimated IM precursor enrichment owing to in vivo IM lipid breakdown, whereas IM free palmitate enrichment underestimated the precursor enrichment because of lipid breakdown during muscle sampling and processing. IM palmitoyl-carnitine enrichment was an acceptable surrogate of the precursor enrichment because it was less affected by in vitro lipid breakdown after sampling.     

Dietary conjugated linoleic acid alters long chain polyunsaturated fatty acid metabolism in brain and liver of neonatal pigs

Effects of dietary conjugated linoleic acid (CLA, 1% mixed isomers) on n-6 long-chain polyunsaturated fatty acid (LCPUFA) oxidation and biosynthesis were investigated in liver and brain tissues of neonatal piglets. Fatty acid β-oxidation was measured in tissue homogenates using [1-¹⁴C]linoleic acid (LA) and -arachidonic acid (ARA) substrates, while fatty acid desaturation and elongation were traced using [U-¹³C]LA and GC-MS. Dietary CLA had no effect on fatty acid β-oxidation, but significantly decreased n-6 LCPUFA biosynthesis by inhibition of LA elongation and desaturation. Differences were noted between our ¹³C tracer assessment of desaturation/elongation and simple precursor-product indices computed from fatty acid composition data, indicating that caution should be exercised when employing the later. The inhibitory effects of CLA on elongation/desaturation were more pronounced in pigs fed a low fat diet (3% fat) than a high fat diet (25% fat). Direct elongation of linoleic acid to C20:2n-6 via the alternate elongation pathway might play an important role in n-6 LCPUFA synthesis because more than 40% of the synthetic products of [U-¹³C]LA accumulated in [¹³C]20:2n-6. Overall, the data show that dietary CLA shifted the distribution of the synthetic products of [U-¹³C]LA between elongation and desaturation in liver and decreased the total synthetic products of [U-¹³C]LA in brain by inhibiting LA elongation to C20:2n-6. The impact of CLA on brain LCPUFA metabolism of the developing neonate merits consideration and further investigation.     

A fatty acid elongase ELO with novel activity from Dictyostelium discoideum

Fatty acid elongation was examined in the cellular slime mold, Dictyostelium discoideum. Profiling of the total fatty acid content of D. discoideum indicated that fatty acid elongation is active. Orthologs of the fatty acid elongase ELO family were identified in the D. discoideum genome and the cDNA for one, eloA, was cloned and functionally characterized by expression in yeast. EloA is a highly active ELO with strict substrate specificity for monounsaturated fatty acids, in particular 16:1^Δ9 to produce the unusual 18:1^Δ11 fatty acid. This is the first report on fatty acid elongation in a cellular slime mold.     

A COMPARATIVE STUDY OF THE METABOLISM OF DE NOVO SYNTHESIZED FATTY ACIDS FROM ACETATE AND GLUCOSE, AND EXOGENOUS FATTY ACIDS, IN SLICES OF RABBIT CEREBRAL CORTEX DURING DEVELOPMENT

Slices of rabbit cerebral cortex, from the foetal stage to the adult have been used to compare lipid synthesis from fatty acids synthesized de novo from [U-¹⁴C]glucose and [1-¹⁴C]acetate, with lipid synthesis from exogenous albumin-bound [1-¹⁴C]palmitate. Incorporation into cellular lipid has been determined in terms of DNA, protein, wet wt. of tissue and wet weight of whole brain. On a wet wt. basis, maximum incorporation of glucose carbon into lipid occurred in the foetal brain while lipid synthesis from acetate and palmitate was maximum at 4–14 days after birth. Glucose and acetate were incorporated into a diversity of lipids (with increasing amounts of phosphatidylcholine synthesized during maturation), while palmitate was incorporated into the free fatty acid and triglyceride fractions. A greater proportion of acetate was incorporated into fatty acids of chain-length longer than C16 compared with the incorporation of palmitate. However, on a molar basis de novo synthesized and exogenous palmitate were elongated, desaturated and incorporated into phospholipids at a similar rate, while exogenous palmitate was incorporated to a greater extent than de nova synthesized fatty acid into the triglyceride fraction. This difference in metabolism may be due to the different size of the non-esterified fatty acid pool in the two situations. At the period of their most active formation, the very long-chain fatty acids may be synthesized from a pool of the C18 series of fatty acids (saturated and monoenoic) not in equilibrium with the bulk of C₁₈ acids in cerebral lipids. This could be a pool of acyl groups derived from ethanolamine phospholipids.     

Susceptibility of Pancreatic Beta Cells to Fatty Acids Is Regulated by LXR/PPARα-Dependent Stearoyl-Coenzyme A Desaturase

Chronically elevated levels of fatty acids-FA can cause beta cell death in vitro. Beta cells vary in their individual susceptibility to FA-toxicity. Rat beta cells were previously shown to better resist FA-toxicity in conditions that increased triglyceride formation or mitochondrial and peroxisomal FA-oxidation, possibly reducing cytoplasmic levels of toxic FA-moieties. We now show that stearoyl-CoA desaturase-SCD is involved in this cytoprotective mechanism through its ability to transfer saturated FA into monounsaturated FA that are incorporated in lipids. In purified beta cells, SCD expression was induced by LXR- and PPARα-agonists, which were found to protect rat, mouse and human beta cells against palmitate toxicity. When their SCD was inhibited or silenced, the agonist-induced protection was also suppressed. A correlation between beta cell-SCD expression and susceptibility to palmitate was also found in beta cell preparations isolated from different rodent models. In mice with LXR-deletion (LXRβ^-/- and LXRαβ^-/-), beta cells presented a reduced SCD-expression as well as an increased susceptibility to palmitate-toxicity, which could not be counteracted by LXR or PPARα agonists. In Zucker fatty rats and in rats treated with the LXR-agonist TO1317, beta cells show an increased SCD-expression and lower palmitate-toxicity. In the normal rat beta cell population, the subpopulation with lower metabolic responsiveness to glucose exhibits a lower SCD1 expression and a higher susceptibility to palmitate toxicity. These data demonstrate that the beta cell susceptibility to saturated fatty acids can be reduced by stearoyl-coA desaturase, which upon stimulation by LXR and PPARα agonists favors their desaturation and subsequent incorporation in neutral lipids.     

Biosynthesis of C(20) and C(22) Fatty Acids by Developing Seeds of Limnanthes alba: CHAIN ELONGATION AND Delta5 DESATURATION

The storage triacylglycerols of meadowfoam (Limnanthes alba) seeds are composed essentially of C₂₀ and C₂₂ fatty acids, which contain an unusual Δ5 double bond. When [1-¹⁴C]acetate was incubated with developing seed slices, ¹⁴C-labeled fatty acids were synthesized with a distribution similar to the endogenous fatty acid profile. The major labeled product was cis-5-eicosenoate, with smaller amounts of palmitate, stearate, oleate, cis-5-octadecenoate, eicosanoate, cis-11-eicosenoate, docosanoate, cis-5-docosenoate, cis-13-docosenoate, and cis-5,cis-13-docosadienoate. The label from [¹⁴C]acetate and [¹⁴C]malonate was used preferentially for the elongation of endogenous oleate to produce cis-[¹⁴C]11-eicosenoate, cis-13-[¹⁴C]docosenoate, and cis-5,cis-13-[¹⁴C]docosadienoate and for the elongation of endogenous palmitate to produce the remaining C₂₀ and C₂₂ acyl species. The Δ5 desaturation of the preformed acyl chain and chain elongation of oleate and palmitate were demonstrated in vivo by incubation of the appropriate 1-¹⁴C-labeled free fatty acids. Using [1-¹⁴C]acyl-CoA thioesters as substrates, these enzyme activities were also demonstrated in vitro with a cell-free homogenate.     

Thermodynamic and Kinetic Framework of Selenocysteyl-tRNASec Recognition by Elongation Factor SelB

SelB is a specialized translation elongation factor that delivers selenocysteyl-tRNA^Sec (Sec-tRNA^Sec) to the ribosome. Here we show that Sec-tRNA^Sec binds to SelB·GTP with an extraordinary high affinity (K_d = 0.2 pm). The tight binding is driven enthalpically and involves the net formation of four ion pairs, three of which may involve the Sec residue. The dissociation of tRNA from the ternary complex SelB·GTP·Sec-tRNA^Sec is very slow (0.3 h⁻¹), and GTP hydrolysis accelerates the release of Sec-tRNA^Sec by more than a million-fold (to 240 s⁻¹). The affinities of Sec-tRNA^Sec to SelB in the GDP or apoforms, or Ser-tRNA^Sec and tRNA^Sec to SelB in any form, are similar (K_d = 0.5 μm). Thermodynamic coupling in binding of Sec-tRNA^Sec and GTP to SelB ensures at the same time the specificity of Sec- versus Ser-tRNA^Sec selection and rapid release of Sec-tRNA^Sec from SelB after GTP cleavage on the ribosome. SelB provides an example for the evolution of a highly specialized protein-RNA complex toward recognition of unique set of identity elements. The mode of tRNA recognition by SelB is reminiscent of another specialized factor, eIF2, rather than of EF-Tu, the common delivery factor for all other aminoacyl-tRNAs, in line with a common evolutionary ancestry of SelB and eIF2.     

Fluorescent n-3 and n-6 Very Long Chain Polyunsaturated Fatty Acids: THREE-PHOTON IMAGING IN LIVING CELLS EXPRESSING LIVER FATTY ACID-BINDING PROTEIN*

Despite the considerable beneficial effects of n-3 and n-6 very long chain polyunsaturated fatty acids (VLC-PUFAs), very little is known about the factors that regulate their uptake and intracellular distribution in living cells. This issue was addressed in cells expressing liver-type fatty acid-binding protein (L-FABP) by real time multiphoton laser scanning microscopy of novel fluorescent VLC-PUFAs containing a conjugated tetraene fluorophore near the carboxyl group and natural methylene-interrupted n-3 or n-6 grouping. The fluorescent VLC-PUFAs mimicked many properties of their native nonfluorescent counterparts, including uptake, distribution, and metabolism in living cells. The unesterified fluorescent VLC-PUFAs distributed either equally in nuclei versus cytoplasm (22-carbon n-3 VLC-PUFA) or preferentially to cytoplasm (20-carbon n-3 and n-6 VLC-PUFAs). L-FABP bound fluorescent VLC-PUFA with affinity and specificity similar to their nonfluorescent natural counterparts. Regarding n-3 and n-6 VLC-PUFA, L-FABP expression enhanced uptake into the cell and cytoplasm, selectively altered the pattern of fluorescent n-6 and n-3 VLC-PUFA distribution in cytoplasm versus nuclei, and preferentially distributed fluorescent VLC-PUFA into nucleoplasm versus nuclear envelope, especially for the 22-carbon n-3 VLC-PUFA, correlating with its high binding by L-FABP. Multiphoton laser scanning microscopy data showed for the first time VLC-PUFA in nuclei of living cells and suggested a model, whereby L-FABP facilitated VLC-PUFA targeting to nuclei by enhancing VLC-PUFA uptake and distribution into the cytoplasm and nucleoplasm.     

A Novel Epimerase That Converts GlcNAc-P-P-undecaprenol to GalNAc-P-P-undecaprenol in Escherichia coli O157

Escherichia coli strain O157 produces an O-antigen with the repeating tetrasaccharide unit α-d-PerNAc-α-l-Fuc-β-d-Glc-α-d-GalNAc, preassembled on undecaprenyl pyrophosphate (Und-P-P). These studies were conducted to determine whether the biosynthesis of the lipid-linked repeating tetrasaccharide was initiated by the formation of GalNAc-P-P-Und by WecA. When membrane fractions from E. coli strains K12, O157, and PR4019, a WecA-overexpressing strain, were incubated with UDP-[³H]GalNAc, neither the enzymatic synthesis of [³H]GlcNAc-P-P-Und nor [³H]GalNAc-P-P-Und was detected. However, when membrane fractions from strain O157 were incubated with UDP-[³H]GlcNAc, two enzymatically labeled products were observed with the chemical and chromatographic properties of [³H]GlcNAc-P-P-Und and [³H]GalNAc-P-P-Und, suggesting that strain O157 contained an epimerase capable of interconverting GlcNAc-P-P-Und and GalNAc-P-P-Und. The presence of a novel epimerase was demonstrated by showing that exogenous [³H]GlcNAc-P-P-Und was converted to [³H]GalNAc-P-P-Und when incubated with membranes from strain O157. When strain O157 was metabolically labeled with [³H]GlcNAc, both [³H]GlcNAc-P-P-Und and [³H]GalNAc-P-P-Und were detected. Transformation of E. coli strain 21546 with the Z3206 gene enabled these cells to synthesize GalNAc-P-P-Und in vivo and in vitro. The reversibility of the epimerase reaction was demonstrated by showing that [³H]GlcNAc-P-P-Und was reformed when membranes from strain O157 were incubated with exogenous [³H]GalNAc-P-P-Und. The inability of Z3206 to complement the loss of the gne gene in the expression of the Campylobacter jejuni N-glycosylation system in E. coli indicated that it does not function as a UDP-GlcNAc/UDP-GalNAc epimerase. Based on these results, GalNAc-P-P-Und is synthesized reversibly by a novel GlcNAc-P-P-Und epimerase after the formation of GlcNAc-P-P-Und by WecA in E. coli O157.