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Obesity is the main factor involved in the onset of many diseases. Threonine supplementation has been demonstrated to reduce fat mass and serum triglycerides in already obese mice. However, it is unclear whether threonine could inhibit the development of obesity in mice without previous high-fat diet induction. In the present study, mice were fed a chow diet (CD) or a high-fat diet (HFD), supplemented or not with threonine (3.0% in drinking water) for 15 weeks. Results showed that mice subjected to chronic threonine supplementation showed decreased body weight, epididymal white adipose tissue weight, serum low-density lipoprotein cholesterol, and total cholesterol in comparison with HFD-fed mice. In the epididymal adipose tissue, gene expressions of sterol regulatory element-binding protein 1c and fatty acid synthase were up-regulated, while hormone sensitive lipase, adiponectin and fibroblast growth factor 21 were down-regulated. In the liver tissue, gene expressions of sirtuin1, adenosine monophosphate-activated protein kinase and peroxisome proliferator activated receptor γ co-activator 1α were up-regulated by threonine supplementation in HFD-fed mice. These results suggest that long-term threonine supplementation inhibited fat mass and improved lipid metabolism, making it a potential agent to prevent the development of diet-induced obesity.

Emerging evidence shows that amino acids can modulate lipid metabolism. Aromatic amino acids (AAAs) serve as important precursors of several neurotransmitters and metabolic regulators that play a vital role in regulating nutrient metabolism. But whether AAAs have a lipid-lowering function remains unknown. Here mice were fed amino acid-defined diets containing AAAs at 1.82% and 3.64% for 3 weeks. We demonstrated that double AAA intake significantly decreased the serum and hepatic triglycerides and serum low-density lipoprotein cholesterol, but increased the high-density lipoprotein cholesterol as well as insulin tolerance. Combined metabolomic and transcriptomic analysis showed that the hepatic acidic pathway of bile acid synthesis was responsible for the improvement in lipid metabolism by AAA treatment. This study suggests that AAAs have the potential to ameliorate steatosis and provides a new alternative to improve lipid metabolism.

Branched chain amino acids (BCAAs), especially leucine (Leu), have been reported to decrease fat deposition. However, extensive studies have not been conducted to determine whether Leu and isoleucine (Ile) have the same effects in mice fed a high fat diet (HFD). Already obese mice induced by HFD were assigned to the following groups: HFD, HFD + Leu, HFD + Ile, and HFD + Leu + Ile. Chow diet was set as a negative control throughout the study. The results showed that adding Leu, Ile and their combination in drinking water significantly decreased relative white adipose tissue (WAT) weight and serum triglycerides, low-density lipoprotein, free fatty acid, and insulin compared with the HFD control. Insulin sensitivity and hepatic steatosis impaired by HFD were alleviated after Leu or Ile supplementation. Furthermore, lipid metabolism-related genes were modified, and the genes involved in the browning of WAT and beige selective markers were upregulated by dietary supplementation with Leu, Ile and their combination. Intestinal microbiota analysis showed that the ratio of Firmicutes-to-Bacteroidetes was decreased after Leu or Ile treatment. This study indicated that dietary supplementation with Leu or Ile reduced body weight and WAT weight by regulating lipid metabolism-related genes and browning of WAT, providing a new tool in the management of obesity and metabolic disorders.

Branched chain amino acids (BCAA), especially leucine (Leu), have been reported to decrease fat deposition. However, opposite effects of BCAA on lipid metabolism have been observed. To determine the role of BCAA in lipid metabolism, an amino acid-defined diet was formulated and C57BL/6J mice were assigned into the following groups: amino acid-defined control diet and control diet supplemented with Leu, isoleucine, or valine. Nitrogen was balanced by proportionally mixed amino acids except BCAA. Results showed that dietary Leu supplementation significantly increased the levels of serum triglycerides, total cholesterol, low-density lipoprotein-cholesterol, high-density lipoprotein-cholesterol and urea nitrogen. Metabolomics showed that biosynthesis of unsaturated fatty acids was altered by Leu supplementation. Leu treatment up-regulated the expression of genes related to fat synthesis and down-regulated the expression of genes related to fatty acid synthesis. Furthermore, the genes and proteins of selective markers involved in browning of white adipose tissue (WAT) were up-regulated by dietary supplementation with Leu. This study indicated that dietary supplementation with Leu, but not isoleucine or valine, significantly affected lipid metabolism by regulating lipid metabolism-related genes and serum fatty acid concentration, providing a new tool in the management of obesity and metabolic disorders.

Some amino acids (AAs) have been proven to suppress fat mass and improve insulin sensitivity. However, the impact of important essential AAs, threonine, lysine, and methionine, on obesity has not been clarified. In the present study, after an 8 week period of obesity induction, mice were grouped to receive either a high-fat diet (HFD) or HFD supplemented with lysine, threonine, or methionine (3% in drinking water) for another 10 weeks. The results showed that dietary supplementation with threonine significantly decreased body weight, epididymal and perirenal fat pad weights, serum concentrations of glucose, triacylglycerols, total cholesterol, and LDL-cholesterol compared to the HFD group. HOMA-IR and serum leptin and adiponectin were improved by threonine supplementation. In epididymal adipose tissue, threonine treatment significantly down-regulated the expression levels of lipogenesis and up-regulated expressions of lipolysis compared to the HFD group. Threonine addition stimulated the expression of UCP-1 and related genes in brown adipose tissue. However, lysine or methionine supplementation showed little effect on body weight, WAT weight, serum lipid profiles, and lipid-metabolism-related gene expressions of HFD-fed mice. These findings suggest that threonine inhibited fat mass and improved lipid metabolism of already obese mice, providing a potential agent in treating obesity.

The intrinsic physicochemical properties of dissolved organic matter (DOM) may affect the mobility and toxicity of arsenic in aquatic environments. In the present study, the humic acid (HA) was ultra-filtered into five fractions according to molecular weight, and their physicochemical properties were characterized. Complexation of HA fractions with arsenite and arsenate was first determined by differential pulse polarography (DPP). The influences of HA fractions on arsenic toxicity were then examined using Daphnia magna as a model organism. As(V) had a higher affinity with HA than As(III), and their complexation was dependent on the total acidity and fluorescence characteristics of DOM. We demonstrated that the acidity and fluorescence also better explained the As toxicity to daphnids than UV absorbance and hydraulic diameter. Arsenic speciation determined by DPP significantly affected the toxicity of arsenite and arsenate. The results extended the free-ion activity model application to the case of arsenic. The present study clearly indicated that DOM with different molecular weights has distinct physicochemical properties, and could influence the speciation and toxicity of As to different extent.

The C-terminal sequences of porcine thrombin encode a series of peptides with the characteristics of net positive charge and hydrophobicity, suggesting antimicrobial potential. In this study, we synthesized truncated C-terminal peptides to explore their antimicrobial potency and structure-activity relationship. The results showed that some peptides exerted antimicrobial activity against Gram-positive and Gram-negative bacteria, with selectivity for microbial membranes. The antimicrobial potency of the peptides increased with the extension of chain length. Considering toxicity to red blood cells, the 21-mer peptide T-6 displayed the highest therapeutic index of 43.4, suggesting its higher cell selectivity. Typical α-helical conformations were observed upon binding to a bacteria-mimicking environment. The derivatives tended to interact preferentially with negatively charged vesicles compared to zwitterionic vesicles. Flow cytometry and electron microscopy revealed that the peptides targeted bacterial cell membranes and disrupted cytoplasmic membrane integrity, thereby causing the release of cellular contents leading to cell death. Peptide-membrane interaction experiments provided evidence that the peptides killed bacteria via a membrane-mediating mechanism. In summary, the C-terminal sequence of porcine thrombin has antimicrobial functions.

Milk is an important food for mammalian neonates, but its insufficient production is a nutritional problem for humans and other animals. Recent studies indicate that dietary supplementation with L-arginine (Arg) increases milk production in mammals, including sows, rabbits, and cows. However, the underlying molecular mechanisms remain largely unknown. The present study was conducted with porcine mammary epithelial cells (PMECs) to test the hypothesis that Arg enhances milk protein synthesis via activation of the mechanistic target of rapamycin (mTOR) cell signaling. PMECs were cultured for 4 days in Arg-free basal medium supplemented with 10, 50, 200, or 500 μmol/L Arg. Rates of protein synthesis and degradation in cells were determined with the use of L-[ring-2,4

In this study, the influence of isoleucine and arginine on the biological activity and peptide-membrane interactions of linear avian β-defensin-4 (RL38) analogs was investigated. Results of biological activities showed that the antimicrobial activities of AvBD-4 analogs were closely related to hydrophobicity and amphipathicity. The peptide GLI19 with high hydrophobicity value and amphipathicity displayed broad spectrum antimicrobial activity against both gram-negative and gram-positive, whereas GLR19 with increasing multiple charges only exhibited activity against gram-negative. The interaction between peptides and the liposome membrane demonstrated that the peptides preferentially bound to negatively charged phospholipids over zwitterionic phospholipids, which supported the antimicrobial activity data. The outer membranes assay further demonstrated that GLI19 had a greater capacity than the other tested peptides to penetrate the cell membrane at a low concentration. Collectively, the peptides derived from the bactericidal domain of linear β- defensins by truncation and hydrophobic amino acid substitution may be effective high-potential antibacterial agents.

Avian β-defensin 6 (AvBD-6) is an antimicrobial peptide that plays significant roles in the innate immunity of chickens. To explore the effects of disulfide bonds on antimicrobial activity of AvBD-6, two peptides with or without Cys residues were designed and expressed in Pichia Pastoris. The peptide AvBD-6-B was obtained by removing six Cys residues of AvBD-6. According to the codon bias of Pichia Pastoris, the genes of AvBD-6 and AvBD-6-B were synthesized. The Bgl II-linearized recombinant plasmids pGAPHαM-AvBD-6 and pGAPHαM-AvBD-6-B were transformed into Pichia Pastoris GS115 by electroporation. The recombinant AvBD-6 and AvBD-6-B were expressed in YPD for 48 h, 72 h and 96 h at 30 �C Tricine-SDS-PAGE analysis demonstrated that both AvBD-6 and AvBD-6-B were expressed in Pichia Pastoris. The concentration of recombinant AvBD-6 and AvBD-6-B reached 114.9 mg/l and 93.8 mg/l, respectively. The expression products exhibited antimicrobial activity against Gram-positive and Gram-negative bacteria. Antimicrobial activity of AvBD-6-B suggests that the removal of six Cys residues had no significant effect on the antimicrobial activity of avian β-defensins. Neither peptide showed hemolytic activity. This study could serve as an impetus for the production of this antimicrobial peptide as a replacement for antibiotics in animal feed.

Antimicrobial peptides are major components of the innate self-defence system and a large number of peptides have been designed to study the mechanism of action. In the present study, a small combinatorial library was designed to study whether the biological activity of Val/Arg-rich peptides is associated with targeted cell membranes. The peptides were produced by segregating hydrophilic residues on the polar side and hydrophobic residues on the opposite side. The peptides displayed strong antimicrobial activity against Gram-negative and Gram-positive bacteria, but weak haemolysis even at a concentration of 256 µM. CD spectra showed that the peptides formed α-helical-rich structure in the presence of negatively charged membranes. The tryptophan fluorescence and quenching experiments indicated that the peptides bound preferentially to negatively charged phospholipids over zwitterionic phospholipids, which corresponds well with the biological activity data. In the in vivo experiment, the peptide G6 decreased the bacterial counts in the mouse peritoneum and increased survival after 7 days. Overall, a high binding affinity with negatively charged phospholipids correlated closely with the cell selectivity of the peptides and some peptides in this study may be likely candidates for the development of antibacterial agents.

The antimicrobial peptide PMAP-36 exists as a homodimer stabilized by an intermolecular disulfide bridge. The dimer of PMAP-36 exhibits a potent and rapid microbicidal activity against a wide spectrum of microorganisms. The gene encoding the antiparallel dimer (PMAP-36)2 was designed and codon-optimized according to bias of Pichia pastoris. The gene was then expressed in the P. pastoris strain GS115. The concentration of the recombinant product reached 106 mg/l. In vitro activity assays indicated that the recombinant peptide showed antimicrobial activities against Gram-positive and Gram-negative bacteria but did not cause hemolysis of chicken erythrocytes. Subsequently, 120 7-day-old male Arbor Acres broilers were used to evaluate the in vivo activities of the peptide. A prophylactic dose of ciprofloxacin lactate was supplemented as the control. The results showed that recombinant (PMAP-36)2 significantly increased the serum IgM content of the birds (p0.05). The recombinant peptide significantly increased the amounts of Bifidobacterium and decreased the amount of Escherichia coli cells in the ceca of the experimental birds (p0.05). The results obtained in the present study indicate that the recombinant (PMAP-36)2 has a potent in vitro and in vivo activity and can be used as an alternative to antibiotic treatment.

We previously reported that GLI13-8, one of cationic antimicrobial peptides from linear avian β-defensin-4 (RL38) analogs, exhibited high antimicrobial activities against both Gram-negative and Gram-positive bacteria. In the present study, we reported the in vitro cytotoxicity of GLI13-8 using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. Results showed that the cytotoxicity of GLI13-8 in three human carcinoma cells (HepG2, SGC7901, and A375) was in a dose-dependent manner. When the concentration of GLI13-8 is

Antimicrobial peptides with amphipathic β-hairpin-like structures have potent antimicrobial properties and low cytotoxicity. The effect of VR or RV motifs on β-hairpin-like antimicrobial peptides has not been investigated. In this study, a series of β-hairpin-like peptides, Ac-C(VR) n D PG (RV) n C-NH 2 ( n = 1, 2, 3, 4, or 5), were synthesized, and the effect of chain length on antimicrobial activity was evaluated. The antimicrobial activity of the peptides initially increased and then decreased with chain length. Longer peptides stimulated the toxicity to mammalian cells. VR3, a 16-mer peptide with seven amino acids in the strand, displayed the highest therapeutic index and represents the optimal chain length. VR3 reduced bacterial counts in the mouse peritoneum and increased the survival rate of mice at 7 days after Salmonella enterica serovar Typhimurium infection in vivo . The circular dichroism (CD) spectra demonstrated that the secondary structure of the peptides was a β-hairpin or β-sheet in the presence of an aqueous and membrane-mimicking environment. VR3 had the same degree of penetration into the outer and inner membranes as melittin. Experiments simulating the membrane environment showed that Trp-containing VRW3 (a VR3 analog) tends to interact preferentially with negatively charged vesicles in comparison to zwitterionic vesicles, which supports the biological activity data. Additionally, VR3 resulted in greater membrane damage than melittin as determined using a flow cytometry-based membrane integrity assay. Collectively, the data for synthetic lipid vesicles and whole bacteria demonstrated that the VR3 peptide killed bacteria via targeting the cell membrane. This assay could be an effective pathway to screen novel candidates for antibiotic development.

A novel α-helical antimicrobial peptide G6 rich in Val/Arg residues has been screened previously. In this study, we further evaluated the biochemical stability, interaction with whole bacteria, and in vivo therapeutic or prophylactic role of the peptide in Salmonella typhimurium-infected mice. The results showed that G6 exhibited strong resistance to pH, heat, and salts. But G6 lost the antimicrobial activity when treated with proteolytic enzymes. G6 had no toxicity on mammalian cell. An intraperitoneal model of sepsis caused by Salmonella typhimurium was established in mice. G6 was administered intraperitoneally 1 h before or after mice were infected with Salmonella typhimurium. For the mice given peptide post-bacterial infection, the mortality of the mice and the peritoneal bacterial counts were significantly lower in the groups that were administered 2.5 mg/kg BW and 5.0 mg/kg BW of G6 (P < 0.05) compared to the PBS-treated group. Similar trend was obtained when G6 was given 1 h prior to Salmonella typhimurium infection. Peptide-membrane experiments showed that G6 was effective in permeabilizing the outer and inner membrane in a dose dependent manner, indicating that the peptide targets the cell membrane. Taken together, the results revealed that the peptide G6 may provide a useful alternative to antibiotic agents to treat or prevent bacterial infections.