Previous studies reported that dietary PAD causes deficiency symptoms such as growth retardation, dermatosis, diarrhea, and even death in both mammals [2,3,4,5,6] and poultry [7,8,9,10,11,12,13, 19, 25]. In ducks, growth retardation and excessive exudate from the eyes were exhibited in response to PAD . Consistent with previous studies, the PAD ducks showed growth depression, exudate on eyelids, poor feathering, and a high mortality rate in the present study. Furthermore, poor pantothenic acid status was observed in PAD ducks indicated by reduced plasma pantothenic acid concentration, which is a sensitive biomarker for pantothenic acid status [21, 26, 27]. These results indicate that a severe PAD animal model was successfully established.
It has been shown that pantothenic acid can maintain intestinal function by keeping the structure of intestine integrity of animals [3, 14,15,16]. Insufficient of pantothenic acid would cause intestinal hypofunction in rats, dogs, cats, chicks and fish [4, 5, 17,18,19,20,21]. And a feature common to experimental PAD in different animals is intestinal ulceration [4, 5, 17,18,19,20]. Duodenitis and duodenal ulcers , as well as duodenal changes including eventual atrophy of crypts, diminution in size of villi, were observed in PAD rats . In agreement with the previous studies, we observed small intestine morphological changes indicated by the reduced villus height and villus surface area in PAD ducks in the present study, indicating damage to the intestinal epithelium.
Furthermore, PAD leads to abnormalities in carbohydrate metabolism. Low fasting blood glucose levels and increased sensitivity to insulin were found in PAD rats and dogs [28,29,30,31,32]. In agreement with previous studies, PAD caused fasting hypoglycemia and decreased plasma insulin level in ducks in the present study, indicating abnormal glucose metabolism.
Together, dietary PAD of ducks resulted in growth retardation, alterations of intestinal morphology and function, and abnormal glucose metabolism. However, limited data are available currently concerning the molecular mechanisms behind. Therefore, we used a proteomic approach, for the first time, to investigate the metabolic disorder of small intestine induced by PAD to explain intestinal hypofunction and growth depression. Proteomic analysis revealed 421 differentially expressed proteins in the mucosa of PAD ducks compared to CON birds, indicating an important impact of pantothenic acid on intestinal function. The differentially expressed proteins are mainly enriched in glycolysis and gluconeogenesis, fatty acid beta oxidation, oxidative phosphorylation, TCA cycle, intestinal absorption, regulation of actin cytoskeleton, and oxidative stress. These processes probably underlie the intestinal hypofunction and growth depression induced by PAD. Notably, the CoA-binding proteins were reduced in the intestinal mucosa of PAD ducks, such as ACSL5, ACAD11, ACADM, HADHB, ECI2, ACOX1, and ACOX2. This finding supports the hypothesis that CoA-binding proteins may be depressed as a result of dietary PAD.
Glycolysis and gluconeogenesis
A total of thirteen proteins participating in the glycolysis and gluconeogenesis pathway were altered by PAD. Of these, twelve proteins were involved in glycolysis, two enhanced (ALDOA and HK1) and ten diminished (ALDOB, HKDC1, ENO1, GAPDH, PKM, PFKP, TPI1, PGK1, DLD, and PDHB). HKDC1 and HK1 are two isozymes of hexokinases, which mediate the initial step of glycolysis by catalyzing phosphorylation of D-glucose to D-glucose 6-phosphate . PFKP catalyzes the phosphorylation of fructose 6-phosphate to fructose 1,6-bisphosphate by ATP . ALDOA and ALDOB are two isoforms of aldolase family which are located in skeletal muscle and liver tissue respectively, cleaves fructose-1,6-bisphosphate to triose phosphates . TPI1 catalyzes the interconversion between dihydroxyacetone phosphate and D-glyceraldehyde-3-phosphate . GAPDH catalyzes the conversion of glyceraldehyde 3-phosphate into 1,3-diphosphoglycerate . PGK1 catalyzes one of the two ATP producing reactions in the glycolytic pathway via the reversible conversion of 1,3-diphosphoglycerate to 3-phosphoglycerate . ENO1 catalyzes the formation of phosphoenolpyruvate from 2-phosphoglycerate . PKM catalyzes the second ATP generation reaction in the glycolytic pathway via conversion of phosphoenolpyruvate to pyruvate . PDHB is a subunit of pyruvate dehydrogenase (E1). As the E3 component of pyruvate dehydrogenase complex, DLD oxidizes dihydrolipoic acid to lipoic acid . Pyruvate dehydrogenase complex converts pyruvate to acetyl-CoA [42, 43]. Therefore, ten out of twelve proteins (ALDOB, HKDC1, ENO1, GAPDH, PKM, PFKP, TPI1, PGK1, DLD, and PDHB) were down-regulated in the PAD group suggests that the rate of glycolysis in the intestinal mucosa may be impaired. Besides, one protein (FBP1) involved in gluconeogenesis process was down-regulated by PAD, indicating a depressed gluconeogenesis. FBP1, a rate-limiting enzyme in gluconeogenesis, catalyzes the hydrolysis of fructose 1,6-bisphosphate to fructose 6-phosphate.
Fatty acid beta oxidation
In the present study, PAD down-regulated 9 proteins involved in fatty acid beta oxidation, including ACSL5, ACAD11, ACADM, CRAT, DLD, HADHB, ECI2, ACOX1, and ACOX2. Notably, among these, ACSL5, ACAD11, ACADM, HADHB, ECI2, ACOX1, and ACOX2 are belong to CoA-binding proteins, which were all reduced due to PAD. ACSL5 belongs to the acyl-CoA synthetase family, catalyzing free fatty acids into fatty acyl-CoA esters, which play key roles in lipid biosynthesis and fatty acid degradation . ACAD11 and ACADM are two members of fatty acyl-CoA dehydrogenases that catalyze the first step in each cycle of fatty acid beta oxidation . CRAT catalyzes the reversible transfer of an acetyl group from acyl-CoA to carnitine . ECI2 is involved in the beta oxidation of unsaturated fatty acids, catalyzing the formation of 2-trans-enoyl-CoA from 3-cis or trans-enoyl-CoA . HADHB catalyzes the final step of beta-oxidation, in which 3-ketoacyl CoA is cleaved by the thiol group of another molecule of Coenzyme A . ACOX1 and ACOX2 catalyze the first and rate-limiting step of peroxisomal fatty acid beta oxidation, the desaturation of acyl-CoAs to 2-trans-enoyl-CoAs. ACOX1 catalyzes medium to very long straight-chain fatty acids , while ACOX2 catalyze the CoA-esters of very long-chain fatty acids, branched-chain fatty acids and the C27-bile acid intermediates . The down-regulation of all these proteins involved in the fatty acid beta oxidation process in PAD ducks may indicate that this process is impaired. This implication is supported by previous finding in rats that PAD reduced CoA and short-chain acyl-CoA contents , as well as hepatic peroxisomal fatty acid beta oxidation .
PAD down-regulated six proteins involved in the TCA cycle, including DLD, ACO1, ACO2, MDH1, MDH2, and PDHB, and up-regulated one protein, IDH1. PDHB is a subunit of pyruvate dehydrogenase (E1). As the E3 component of pyruvate dehydrogenase complex and α-ketoglutarate dehydrogenase complex, DLD oxidizes dihydrolipoic acid to lipoic acid . Pyruvate dehydrogenase complex converts pyruvate to acetyl-CoA [42, 43]. α-ketoglutarate dehydrogenase catalyzes the oxidation of α-ketoglutarate to form succinyl-CoA, which is a rate-limiting enzyme of the TCA cycle [52, 53]. ACO1 and ACO2 are two isozymes of aconitate hydratase located in cytoplasmic and mitochondrial respectively, which catalyzes the isomerization of citrate to isocitrate via cis-aconitate. MDH1 and MDH2 are two isozymes of malate dehydrogenase located in cytoplasmic and mitochondrial respectively, which catalyzes the reversible interconversion of malate and oxaloacetate . IDH1 is a subunit of isocitrate dehydrogenase, catalyzing the oxidative decarboxylation of isocitrate into α-ketoglutarate . Six out of seven proteins (DLD, ACO1, ACO2, MDH1, MDH2, and PDHB) were down-regulated due to PAD, indicating a decreased TCA cycle.
PAD down-regulated 5 proteins involved in oxidative phosphorylation, including NDUFA5, NDUFA6, ATP5F1, ATP5H, and ATP5PO. NDUFA5 and NDUFA6, the subunits of complex I, are involved in complex I assembly [56, 57]. ATP5F1, ATP5H, and ATP5PO, the subunits of complex V, are involved in complex V assembly [58,59,60]. The down-regulated expression of proteins involved in oxidative phosphorylation, probably indicates that this process is impaired by PAD.
Together, our duodenum mucosal proteomic analysis revealed glycolysis and gluconeogenesis, fatty acid beta oxidation, TCA cycle, and oxidative phosphorylation are probably impaired in response to PAD, consequently resulting in insufficient energy generation in the small intestine and subsequent growth retardation.
Regulation of actin cytoskeleton
Eleven proteins altered by PAD have actin-binding domains and play direct roles in the organization of structure of the cytoskeleton, including two down-regulated proteins (VIL1 and EZR) and nine up-regulated proteins (ACTN1, FN1, MSN, MYL1, MYLK, VCL, TPM3, TPM1, and VIM). VIL1 and EZR (also known as villin 2; VIL2) are microvillar proteins in intestinal epithelial cells . VIL1 is an epithelial cell-specific actin-binding protein that regulates cell migration, cell death, cell morphology, and epithelial-to-mesenchy-mal transition [62, 63]. EZR is critical for the de novo lumen formation and expansion during villus morphogenesis, and EZR absence resulted in abnormal villus morphogenesis [64, 65].
Actinin is a component of stress fibers and links the cytoskeleton to adherens-type junctions. As one isoform of actinin, ACTN1 plays a major role in cell migration and adhesion . Besides, ACTN1 can directly binds to VCL, and the two proteins cooperate to organize the cytoskeleton at adhesion junctions . FN1, a glycoprotein component of the extracellular matrix, has a role in cell adhesion and migration . VIM induces changes in cell shape, motility, and adhesion during the epithelial to mesenchymal transition . MYLK induces contraction of the perijunctional actomyosin ring through myosin II regulatory light chain phosphorylation, and thereby increases intestinal epithelial permeability when activated . TPM3 and TPM1 are two isoforms of tropomyosins which play important roles in the regulation of assembly, stability, and motility of the intestinal epithelial cells . Collectively, the over expression of nine proteins (ACTN1, FN1, MSN, MYL1, MYLK, VCL, TPM3, TPM1, and VIM) and down-regulation of two proteins (VIL1 and EZR) in response to PAD probably indicates orchestrated regulation of actin cytoskeleton dynamics and the negative impact on intestinal integrity. Remodeling of the cytoskeleton is fundamental in proliferation, apoptosis, cell invasion and metastasis . Therefore, these altered proteins involved in regulation of actin cytoskeleton in response to PAD probably resulted in morphological changes of small intestine, such as atrophy of their intestinal villus. Furthermore, it is reported that ATP depletion uncouples the gate and fence functions of the tight junction and induces actin network dissolution of epithelial cells . The alterations of intestinal morphology and actin cytoskeleton due to PAD in the present study may be attributed to ATP depletion indicated by impaired glycolysis, fatty acid beta oxidation, TCA cycle, and oxidative phosphorylation processes.
PAD down-regulated three proteins involved in the oxidative stress, including MAOA, CAT, and MGST1, and up-regulated one protein, GCLC. MAOA metabolizes dopamine to dihydroxyphenylacetic acid and H2O2, a potential source of reactive oxygen species . Both CAT and MGST1 take part in the oxidative stress defense as its scavenging of H2O2 [75, 76]. GCLC is a subunit of glutamate cysteine ligase which catalyzes the rate-limiting step in reduced glutathione (GSH) synthesis. And glutamate cysteine ligase is often activated to increase cellular GSH content in response to oxidative stress . Collectively, the reduction of MAOA, CAT, and MGST1 in PAD ducks, as well as the enhanced GCLC, indicates small intestinal oxidative stress was induced. This is supported by the results of increased plasma MDA content and decreased T-SOD activity in the present study and is consistent with previous studies in geese  and fish . It has been shown that pantothenic acid protects cells against oxidative stress by promoting glutathione levels and cellular repair mechanisms [22,23,24]. Furthermore, PAD induced oxidative stress in ducks, which may be associated with intestinal injury and morphological alterations.
A novel and important finding of this study is that four proteins (SLC2A2, VIL1, EZR, and MOGAT2) involved in intestinal absorption were down-regulated in duodenum mucosa as a result of PAD. Intestinal SLC2A2 (GLUT2) is known as a means to transfer glucose and fructose from the lumen to the bloodstream and, thereby, to provide sugar to tissue. And intestinal SLC2A2 deletion in mice induced glucose malabsorption visualized by the delay in the distribution of oral sugar in tissues, as well as decreased microvillus length and body weight gain . It is reported pantothenic acid appears to be part of a glucose carrier system , therefore, PAD may direct reduce SLC2A2 and resulted in abnormal glucose absorption and hypoglycemia in ducks. Villin (VIL1 and EZR) is also involved in the absorptive and secretory function of epithelial cells by modulating F-actin polymerization/depolymerization. Specifically, villin-depleted mice showed a reduction in intestinal glucose absorption . Decreased protein expression of SLC2A2, VIL1, and EZR due to PAD probably impair glucose absorption system, which may provide a possible explanation for fasting hypoglycemia that is seen. MOGAT2 plays a central role in absorption of dietary fat in the small intestine by catalyzing the resynthesis of triacylglycerol in enterocytes . MGAT2 deficient specifically in the small intestine showed a delay in fat absorption in mice . Therefore, a reduction of SLC2A2, VIL1, EZR, and MOGAT2 suggests that glucose and fat malabsorption in the small intestine may be induced by PAD, which probably leads to growth depression observed. This is in line with the previous findings in fish that PAD decreased the digestive and absorptive capacities indicated by the reduced the activities of both intestinal brush border enzymes and digestive enzymes [14, 21].