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The value of phenotypic plasticity in energy metabolism and behavioral patterns in response to variations of food availability was handled in adult male voles (Eothenomys miletus) acclimated to random food deprivation (FD) for 4 weeks. During this period, in which voles were fed ad libitum for a randomly assigned 3 days each week, changes in body mass, food intake, resting metabolic rate (RMR), non-shivering thermogenesis (NST) and serum leptin levels were measured. Behavioral observations were made to examine behavioral patterns including activity, feeding, grooming and resting behaviors. The results showed that food intake and gastrointestinal mass increased significantly, but RMR and NST decreased significantly in the FD group compared to the control group. Moreover, the FD group had a significantly higher percentage of feeding behavior and lower activity than those in control group. In addition, serum leptin levels were positively correlated with body fat mass, but showed no significant differ...

To test the hypothesis that mammals that show decrease in body mass under short-day condition should be resistant to high-fat induced obesity, we traced the changes of energy balance in a wild rodent, Apodemus chevrieri, which were acclimated to either long day [16 light (L):8 dark (D), LD] or short day (8 L:16D, SD) and fed either a low-fat diet (LFD) or high-fat diet (HFD) in each photoperiodic manipulation. We found that A. chevrieri was not resistant to high-fat diet-induced obesity, and SD, not HFD, induced the elevation in basal metabolic rate (BMR), nonshivering thermogenesis (NST) and uncoupling protein-1 (UCP1) content in brown adipose tissue (BAT). HFD caused the increase in apparent digestibility and body fat mass, and the decrease in energy intake in both LD and SD A. chevrieri. The enhancement of energy absorption associated with small intestine tissue recruitment can compensate the lower energy intake, which may contribute to the high-fat diet-induced body fat deposition. Thus, a dec...

Environmental factors play an important role in the regulation of a mammal’s physiology and behavior. Consequently, particular species may provide valuable models for understanding the regulation of energy balance. In the present study, tree shrews (Tupaia belangeri) were transferred from a short to a long day photoperiod in cold conditions, in order to test our prediction that short photoperiod may stimulate an increase in thermogenic capacity and energy intake in tree shrews. During the first four weeks of acclimation to short days, T. belangeri increased body mass, whereas during the second four weeks of acclimation to long days, the body mass of tree shrews decreased compared with the short day group. The increase in body mass reflected a significant increase in absolute amounts of body components, such as carcass mass. During long photoperiod associated with cold exposure, livers, kidney, and small intestine mass decreased. T. belangeri decreased resting metabolic rate and energy intake after exposure to long days while during the exposure to short days the shrews started to maintain a stable level after 28 days. Serum leptin levels were positively correlated with body mass, as well as resting metabolic rate and energy intake. The results show that T. belangeri may provide an attractive novel model system for investigation of the regulation of body mass and energy balance at individual levels. Leptin is potentially stimulated by the photoperiod and cold exposure and is responsible for body mass regulation and thermogenesis in T. belangeri.

To investigate the effect of fasting and re-feeding on energy metabolism and thermogenesis in the tree shrew (Tupaia belangeri), the changes in body mass and body fat mass, resting metabolic rate (RMR), non-shivering thermogenesis (NST), body composition, mitochondrial cytochrome c oxidase (COX) activity in liver and brown adipose tissue (BAT), uncoupling protein-1 (UCP1) content of BAT, serum leptin level and post-fasting food intake were monitored and measured. The results showed that fasting induced a significant reduction in body mass and body fat mass. Body mass was restored to the control level in re-feeding tree shrew except for the body fat. RMR and NST decreased significantly in response to fasting, and returned to the control level after re-feeding. Fasting induced significant reduction in total, but not specific, COX activity (nmol O2/min/total tissue) in liver and BAT, and UCP1 content in BAT, which was reversed after re-feeding 48 h. Fasting of 12 h induced a rapid reduction in serum leptin content. There were no post-fasting compensatory increases in food intake. Interestingly, the tree shrew did not recover adipose tissue mass, nor serum leptin levels, upon re-feeding. These results suggest that T. belangeri can adjust the status of its physiology integratively to cope with the lack of food by means of decreasing body mass, adaptive thermogenesis and serum leptin levels, and serum leptin level acted as a starvation signal to mediate predominantly the reduction in body mass and energy expenditure.

Environmental cues, including photoperiod and temperature, play important roles in the adjusting of physiology and behavior in small mammals. In order to determine the contributions of short photoperiod and cold temperatures to seasonal changes in body mass and thermogenesis in Eothenomys miletus, body mass as well as several physiological, hormonal, and biochemical markers (indicative of thermogenic capacity) were examined in seasonally-acclimatized and lab-acclimated animals. Hereby we test our hypothesis that E. miletus can adjust body mass and thermogenesis capacity to survival in winter or short photoperiod and cold temperatures. The results showed that E. miletus adapted to winter by decreasing body mass, and this change was mimicked by exposing animals to cold temperatures and short photoperiod in the lab. E. miletus increased energy intake and thermogenesis and decreased body fat mass and serum leptin levels in winter or under cold temperatures, but not under short photoperiod. Protein contents and uncoupling protein 1 contents of brown adipose tissue increased significantly in winter or at cold temperatures, but not under short photoperiod. Together, these data suggest that the observed physiological regulations from the organismal, hormonal levels to the cellular level of E. miletus are critically important and allow E. miletus to successfully overcome the physiological challenges of a cold environment in winter by increasing thermogenic capacity, energy intake and decreasing body mass and body fat mass. It seems that E. miletus is more sensitive to cold temperatures and leptin may play a potential role in seasonal regulation of body mass and thermogenesis.

To investigate the effect of food restriction on the body mass, thermogenesis and serum leptin level in Apodemus chevrieri, the changes of body mass, body fat mass, resting metabolic rate (RMR), mitochondrial cytochrome coxidase (COX) activity both in liver and brown adipose tissue (BAT), uncoupling protein1 (UCP1) content of BAT, and serum leptin level were measured. The results showed that food restriction induced significant reductions in body mass and body fat mass. RMR decreased significantly in response to food restriction, and food restriction induced significant reduction in total mitochondrial protein content, COX activity both in liver and BAT, and UCP1 content in BAT. Food restriction also induced a reduction in serum leptin level. In contrast, length and mass with content in the small intestine in the food-restricted group was significantly higher relative to the control group. It suggests that the physiological regulations in energy metabolism to adaptively cope with a period of food...

Mammals, including rodents, are able to maintain a stable body mass, which is a result of balance between energy intake and energy expenditure. Exercise training can increase energy expenditure and is considered one of the most important factors affecting energy balance in mammals. In order to understand the effect of exercise on body mass regulation and energy metabolism, changes of body mass, metabolic rate, energy intake, serum leptin levels and body composition were measured in Apodemus chevrieri during an 8-week forced exercise training with a treadmill. The results showed that exercise training had no significant effects on body mass, but the metabolic rate and energy intake were increased during the 8-week exercise training. Body fat mass and serum leptin levels decreased in the exercise group compared to the control group. There was a positive relationship between serum leptin levels and body fat mass in the control group, but no relationship in the exercise group. Mass of liver and diges...

Ambient conditions, as temperature and photoperiod, play a key role in animals’ physiology and behaviors. To test the hypothesis that the maximum thermal physiological and bioenergetics tolerances are induced by extreme environments in Tupaia belangeri. We integrated the acclimatized and acclimated data in several physiological, hormonal, and biochemical markers of thermogenic capacity and bioenergetics in T. belangeri. Results showed that T. belangeri increased body mass, thermogenesis capacity, protein contents and cytochrome c oxidase (COX) activity of liver and brown adipose tissue in winter-like environments, which indicated that temperature was the primary signal for T. belangeri to regulate several physiological capacities. The associated photoperiod signal also elevated the physiological capacities. The regulations of critical physiological traits play a primary role in meeting the survival challenges of winter-like condition in T. belangeri. Together, to cope with cold, leptin may play a potential role in thermogenesis and body mass regulation, as this hormonal signal is associated with other hormones. The strategies of thermal physiology and bioenergetics differs between typical Palearctic species and the local species. However, the maximum thermal physiology and bioenergetic tolerance maybe is an important strategy to cope with winter-like condition of T. belangeri.

Many small mammals respond to seasonal changes in photoperiod by altering body mass and adiposity. These animals may provide valuable models for understanding the regulation of energy balance. In the present study, we examined the effect on body mass, resting metabolic rate, food intake and body composition in cold-acclimated Apodemus draco by transferring them from a short to long day photoperiod. During the first 4 weeks of exposure to short days, A. draco’s body mass decreased. After the next 4 weeks of exposure to long days, body mass increased in the long day group compared to the short day group. This increase in body mass reflected significant increases in absolute amounts of body components, including wet carcass mass, dry carcass mass and body fat mass. Liver, kidney, and small intestine were enlarged due to longer photoperiod during cold exposure. A. draco increased its resting metabolic rate and energy intake after exposure to long days. Serum leptin levels were positively correlated with body mass, body fat mass, resting metabolic rate as well as energy intake. All of the results indicate that A. draco may provide an attractive novel animal model for investigation of the regulation of body mass and energy balance at the organismal levels. Leptin is potentially involved in the photoperiod-induced body mass regulation and thermogenesis of A. draco during cold exposure.

Environmental cues play important roles in the regulation of an animal’s physiology and behavior. In the present study, we examined the effects of short photoperiod on body weight as well as on several physiological, hormonal, and biochemical measures indicative of thermogenic capacity to test our hypothesis that short photoperiod stimulates increases in thermogenesis without cold stress in Apodemus chevrieri. A. chevrieri were randomly assigned to either a long or short photoperiod for 4 weeks at constant temperature. The short photoperiod group of A. chevrieri showed increases in resting metabolic rate and nonshivering thermogenesis during the 4-week photoperiod acclimation. At the end, A. chevrieri at short photoperiod had lower body weights, higher levels of mitochondrial protein content and cytochrome C oxidase activity in liver and brown adipose tissues, and had higher levels of mitochondrial uncoupling protein-1 contents in brown adipose tissues. No difference in serum leptin levels were found between short and long photoperiod groups, but serum leptin levels were positively correlated with body mass and body fat mass, and negatively correlated with energy intake and uncoupling protein-1 content in brown adipose tissues, respectively. All results suggest that the short photoperiod may induce an increased thermogenesis capacity in A. chevrieri and that leptin is potentially involved in the photoperiod induced body mass regulation and thermogenesis in A. chevrieri.

Pancreatic cancer remains one of the most difficult to treat human cancers despite recent advances in targeted therapy. Inhibition of isoprenylcysteine carboxylmethyltransferase (ICMT), an enzyme that posttranslationally modifies a group of proteins including several small GTPases, suppresses proliferation of some human cancer cells. However, the efficacy of ICMT inhibition on human pancreatic cancer has not been evaluated. In this study, we have evaluated a panel of human pancreatic cancer cell lines and identified those that are sensitive to ICMT inhibition. In these cells, ICMT suppression inhibited proliferation and induced apoptosis. This responsiveness to ICMT inhibition was confirmed in in vivo xenograft tumor mouse models using both a small-molecule inhibitor and shRNA-targeting ICMT. Mechanistically, we found that, in sensitive pancreatic cancer cells, ICMT inhibition induced mitochondrial respiratory deficiency and cellular energy depletion, leading to significant upregulation of p21. Furthermore, we characterized the role of p21 as a regulator and coordinator of cell signaling that responds to cell energy depletion. Apoptosis, but not autophagy, that is induced via p21-activated BNIP3 expression accounts for the efficacy of ICMT inhibition in sensitive pancreatic cancer cells in both in vitro and in vivo models. In contrast, cells resistant to ICMT inhibition demonstrated no mitochondria dysfunction or p21 signaling changes under ICMT suppression. These findings not only identify pancreatic cancers as potential therapeutic targets for ICMT suppression but also provide an avenue for identifying those subtypes that would be most responsive to agents targeting this critical enzyme. Mol Cancer Ther; 16(5); 914–23. ©2017 AACR.

Environmental factors, such as photoperiod and temperature, play an important role in the regulation of an animal's physiology and behavior. In the present study, we examined the effects of short photoperiod (SD, 8L:16D) on body mass as well as on several physiological, hormonal, and biochemical measures indicative of thermogenic capacity, to test our hypothesis that short photoperiod stimulates increases thermogenic capacity and energy intake in tree shrews. At the end, these tree shrews (SD) had a significant higher body mass, energy intake, cytochrome C oxidase (COX) activity and uncoupling protein-1 (UCP1) content, serum tri-iodothyronine (T(3)) and thyroxine (T(4)) compared to LD (16L:8D) tree shrews. However, there were no significant differences in serum leptin and melatonin between the two groups. Together, these data suggest tree shrews employ a strategy of maximizing body growth and increasing energy intake in response to cues associated with short photoperiod.

Environmental cues play important roles in the regulation of an animal's physiology and behavior. The purpose of the present study was to test the hypothesis that ambient temperature is a cue to induce adjustments in body mass, energy intake and thermogenic capacity, associated with changes in serum leptin levels in tree shrews (Tupaia belangeri). We found that tree shrews increased basal metabolic rate (BMR), energy intake and subsequently showed a significant decrease in body mass after being returned to warm ambient temperature. Uncoupling protein 1 (UCP1) content in brown adipose tissue (BAT) increased during cold acclimation and reversed after rewarming. The trend of energy intake increased during cold acclimation and decreased after rewarming; the trend of energy intake during cold acclimation was contrary to the trend of energy intake during rewarming. Further, serum leptin levels were negatively correlated with body mass. Together, these data supported our hypothesis that ambient temperature was a cue to induce changes in body mass and metabolic capacity. Serum leptin, as a starvation signal in the cold and satiety signal in rewarming, was involved in the processes of thermogenesis and body mass regulation in tree shrews.

Environmental cues play important roles in the regulation of an animal's physiology and behaviour. The purpose of the present study was to test the hypothesis that ambient temperature was a cue to induce adjustments in body mass, body temperature and thermogenic capacity, associated with changes in serum leptin levels in the tree shrew (Tupaia belangeri chinensis). The results showed that the tree shrew increased resting metabolic rate (RMR), nonshivering thermogenesis (NST) and body mass when exposed to the cold. Uncoupling protein 1 (UCP1) content of brown adipose tissue (BAT) increased in the cold exposure. Also serum leptin levels were positively correlated with body mass. Together, these data supported our hypothesis that ambient temperature was a cue to induce changes in body mass and metabolism. Serum leptin participated in the regulation of energy balance and body weight in the tree shrew.

Environmental factors play an important role in the seasonal adaptation of body mass and thermogenesis in small mammals. The purpose of the present study was to test the hypothesis that ambient temperature triggers adjustments in body mass, body temperature, energy intake, digestible energy intake, metabolic energy intake, and the length and weight of the digestive tract, in Apodemus draco during 42 days of cold exposure. Body mass and body temperature of the cold-acclimated group decreased during the first 28 days and then stabilized at the lower levels. After 14 days of cold acclimation, the body mass of the cold-exposed group was significantly lower and the energy intake, digestible energy intake, and metabolic energy intake were significantly elevated relative to control animals. The differences were maximal after 21 days. The length and weight of the digestive tract (both wet and dry mass of the stomach, small intestine, large intestine, and cecum) changed significantly in response to decreasing temperature. During cold exposure, A. draco was able to maintain physiological functions and reduce its absolute energy demands by reducing the body mass, increasing energy intake, and adjusting the length of the digestive tract.

The present study was designed to examine whether photoperiod alone was effective to induce seasonal changes in physiology in voles ( Eothenomys. ) from the Hengduan Mountain region. Eothenomys miletus were randomly assigned into either long photoperiod (LD; 16L: 8D) or short photoperiod (SD; 8L: 16D) for 4 weeks at constant temperature (25 °C). At the end of acclimation, SD voles showed lower body mass and body fat coupled with higher energy intake than LD voles. SD greatly enhanced the thermogenic capacity of E. miletus , as indicated by an elevated nonshivering thermogenesis (NST), mitochondrial protein in brown adipose tissue (BAT); basal metabolic rate (BMR) was also raised. Although no variations in serum leptin levels were found between SD and LD voles, serum leptin levels were positively correlated with body mass and body fat mass, and negatively correlated with energy intake and UCP1 content in BAT, respectively. To summarize, SD alone is effective in inducing higher thermogenic capacities and energy intake coupled with lower body mass and body fat mass in root voles. Leptin is potentially involved in the photoperiod induced body mass regulation and thermogenesis in E. miletus . Our study shows that SD alone is effective.

Variations in environmental factors instigate significant changes in the physiology and behavior of animals, necessary for their survival. The present study investigated the hypothesis that ambient temperature is a cue capable of inducing changes in body mass, energy intake, and thermogenic capacity. Moreover, the current study determined the potential role of leptin in regulating adaptive thermogenesis in tree shrews (Tupaia belangeri). The tree shrew was chosen as the experimental animal as they inhabit a wide area of Asia and must acclimatize to the cold. Animals were subjected to either 5° C over 28 days to simulate cold acclimation, or maintained under the original climate of room temperature. At 28 days cold-acclimatized shrews had increased body mass by 9.41 g compared to controls. The increase in body mass was found primarily to be due to growth of the digestive organs, combined with significantly increased food intake. Under cold acclimation, uncoupling protein 1 (UCP1) expression in brown adipose tissue (BAT) was significantly elevated, while serum leptin concentration was significantly depressed below control levels. Serum leptin concentration was negatively correlated with body mass, energy intake, and thermogenic capacity during cold acclimation. In summary, these findings indicate that tree shrews adjust energy intake, thermogenic capacity, and body reserves in response to the cold, and further suggest that circulating leptin may act as a key signaling protein to regulate these adaptations.

Abstract Environmental cues play important roles in the regulation of an animal's physiology and behavior. The purpose of the present study was to test the hypothesis that ambient temperature was a cue to induce adjustments in body mass, energy intake and thermogenic capacity, associated with the role of the liver for adaptive thermogenesis in tree shrews (Tupaia belangeri), a unique species of small mammals in the Oriental realm. Many targets were measured, such as the state 3 and the state 4 of mitochondrial respiration, the protein content of mitochondria of the liver in T. belangeri under prolonged cold acclimation (5±1°C, 12L : 12D light cycle) during different times [0 d (control), 7 d, 14 d, 21 d, 28 d]. We found the total protein, mitochondrial protein, and the state 3 and the state 4 of mitochondrial respiration of the liver greatly increased by 39.9%, 39.3%, 84.9% and 181.1% after 28 d in T. belangeri when exposed to cold, as compared with the controls, respectively. Thus, the liver plays a key role in the adaptive thermogenesis during cold acclimation in tree shrews; we believed that evidence from the physiological ecology is equally supportive of the island origin of tree shrews, T. belangeri.

Environmental factors play an important role in the seasonal adaptation of body mass and thermogenesis in small, wild mammals. The purpose of the present study was to test the hypothesis that ambient temperature was a cue to trigger the seasonal adjustments in body mass, energy intake, uncoupling protein 1 (UCP1) in brown adipose tissue (BAT), and other biochemical characteristics of Eothenomys miletus during 49 days of cold exposure. Our data demonstrated that cold acclimation induced a remarkable decrease in body mass, a significant increase in energy intake and metabolic rate, and high expression of UCP1 in BAT of E. miletus. Biochemical characteristics of BAT and liver respiration were also increased following cold acclimation. These data suggest that E. miletus reduced the body mass and increased energy intake and expenditure under cold acclimation. Increased expression of UCP1 was potentially involved in the regulation of energy metabolism and thermogenic capacity following cold acclimation.

Thermogenic characteristics and evaporative water loss were measured at different temperatures in Tupaia belangeri . The thermal neutral zone (TNZ) of T. belangeri was 30–35 °C. Mean body temperature was 39.76±0.27 °C and mean body mass was 100.86±9.09 g. Basal metabolic rate (BMR) was 1.38±0.03 ml O 2 /g h. Average minimum thermal conductance ( C m ) was 0.13±0.01 ml O 2 /g h °C. Evaporative water loss in T. belangeri increased when the temperature rose; the maximal evaporative water loss was 3.88±0.41 mg H 2 O/g h at 37.5 °C. The results may reflect features of small mammals in the sub-tropical plateau region: T. belangeri had high basal metabolic rate and high total thermal conductance, compared with the predicted values based on their body mass whilst their body temperatures are relatively high; T. belangeri has high levels of evaporative water loss and poor water-retention capacity. Evaporative water loss plays an important role in temperature regulation.

The pygmy loris ( Nycticebus pygmaeus ) is a small prosimian living in Vietnam, Laos, eastern Cambodia and the south part of China. In China it is only found in Pingbian, Hekou, Jinping, Luchun of Yunnan. As N. pygmaeus is seriously threatened by hunting, trade and habitat destruction, it is listed in Appendix II of CITES, and in 2006 the IUCN classified it as “vulnerable”. In order to understand the characteristics of energy metabolism and thermoregulation of N. pygmaeus , the resting metabolic rate (RMR) and body temperature ( T b ) at different ambient temperature ( T a ) of pygmy lorises, as well as body mass, energy intake, digestable energy intake, digestability and the thermal conductance were measured in captivity. The results obtained mainly are as follows: (1) Pygmy loris feed dry food averaged 12.90 ± 1.02 g/d. They could gain 214.87 ± 16.65 kJ/d from food intake, and earned 200.15 ± 16.36 kJ digestable energy intake per day with 90.13 ± 1.34% of the digestability. (2) The T b at room temperatures was a little low (35.23 ± 0.16 °C) and varied with T a from 25 °C to 35 °C. There was a positive relationship between T b and T a , which was described as: T b = 27.22 + 0.34 T a ( r = 0.880). (3) The resting metabolic rate (RMR) of the pygmy loris was 0.3844 ± 0.0162 mlO 2 /g/h, which was 51.91 ± 1.90% of the previous predicted rate by Kleiber (1961) [21] . (4) The average thermal conductance of the pygmy loris ( N . pygmaeus ) was 0.0449 ± 0.0031 mlO 2 /g/h/°C. These characteristics of energy metabolism and thermoregulation of N. pygmaeus in Yunnan Daweishan Nature Reserve might be considered as the adaptive characteristics to their environment in tropical semi-evergreen forests and secondary forests.

Eothenomys miletus is an important species inhabiting Hengduan mountains region. In order to study adaptive strategy and the role of serum leptin level in response to a 49 d cold exposure, body mass, energy intake, basal metabolic rate (BMR), nonshivering thermogenesis (NST) in E. miletus were measured. During cold exposure (5±1 oC), body mass decreased; serum leptin levels decreased significantly and were positively correlated with body mass and fat mass; energy intake, BMR and NST were higher at 5 °C than that of controls. These results suggest that E. miletus enhanced thermogenic capacity and increased maintenance cost during cold acclimation, resulting in increased energy intake. Serum leptin participated in the regulation of energy balance and body mass in E. miletus.

Pep-1-K (PK) is a good cell-selective antimicrobial peptide designed from cell-penetrating peptide Pep-1. To develop novel short antimicrobial peptides with higher cell selectivity and shorter length compared with PK, several PK analogs were designed by the deletion, addition and/or substitution of amino acids. Among these analogs, PK-12-KKP (KKPWWKPWWPKWKK) showing the sequence and structure homology with a Trp/Pro-rich natural antimicrobial peptide, indolicidin (IN), displayed a 20-fold higher cell selectivity as compared to IN. Circular dichroism analysis revealed that PK-12-KKP adopts a folded structure combined with some portions of unordered structure. PK-12-KKP selectively binds to negatively charged bacterial membrane-mimetic vesicles, and its high phospholipid selectivity corresponds well with its high cell selectivity. Moreover, it showed very weak potential in depolarization of the cytoplasmic membrane of Staphylococcus aureus at 8 microM (4x minimal inhibitory concentration) and dye leakage from negatively charged liposomes. These results suggest that the ultimate target of our designed PK-12-KKP maybe the intracellular components (e.g. protein, DNA or RNA) rather than the cytoplasmic membranes. Collectively, our designed short Trp/Pro-rich peptide, PK-12-KKP, appears to be an excellent candidate for future development as a novel antimicrobial agent.

Evaporative water loss (EWL) and energy metabolism were measured at different temperatures in Eothenomys miletus and Apodemus chevrieri in dry air. The thermal neutral zone (TNZ) of E. miletus was 22.5–30 °C and that of A. chevrieri was 20–27.5 °C. Mean body temperatures of the two species were 35.75±0.5 and 36.54±0.61 °C. Basal metabolic rates (BMR) were 1.92±0.17 and 2.7±0.5 ml O2/g h, respectively. Average minimum thermal conductance (Cm) were 0.23±0.08 and 0.25±0.06 ml O2/g h °C. EWL in E. miletus and A. chevrieri increased with the increase in temperature; the maximal EWL at 35 °C was 4.78±0.6 mg H2O/g h in E. miletus, and 5.92±0.43 mg H2O/g h in A. chevrieri. Percentage of evaporative heat loss to total heat production (EHL/HP) increased with the increase in temperature; the maximal EHL/HP was 22.45% at 30 °C in E. miletus, and in A. chevrieri it was 19.96% at 27.5 °C. The results may reflect features of small rodents in the Hengduan mountains region: both E. miletus and A. chevrieri have high levels of BMR and high levels of total thermal conductance, compared with the predicted values based on their body masses, while their body temperatures are relatively low. EWL plays an important role in temperature regulation.

Melittin (ME), a linear 26-residue non-cell-selective antimicrobial peptide, displays strong lytic activity against bacterial and human red blood cells. To design ME analogue with improved cell selectivity, we synthesized a melittin diastereomer (ME-D) with D-amino acid in the leucine zipper sequence (Leu-6, Lue-13 and Ile-20). Compared to ME, ME-D exhibited the same or 2-fold higher antibacterial activity but 8-fold less hemolytic activity. Circular dichroism analysis revealed that ME-D has much less alpha-helical content in alpha-helical content in the presence of zwitterionic EYPC/cholesterol (10 : 1, w/w) liposomes compared to negatively charged EYPE/EYPG (7 : 3, w/w) liposomes. The blue shift of the fluorescence emission maximum of ME-D in zwitterionic EYPC/ cholesterol (10 : 1, w/w) liposomes was much smaller than in negatively charged EYPE/EYPG (7 : 3, w/w) liposomes. These results suggested that the improvement in therapeutic index/cell selectivity of ME-D is correlated with its less permeability to zwitterionic membranes.

Melittin (ME), a non-cell-selective antimicrobial peptide, contains the leucine zipper motif, wherein every seventh amino acid is leucine or isolucine. Here, we attempted to generate novel cell-selective peptides by substituting amino acids in the leucine zipper sequence of ME with peptoid residues. We generated a series of ME analogues by replacing Leu-6, Lue-13 and Ile-20 with Nala, Nleu, Nphe, or Nlys, and we examined their secondary structure, self-association activity, cell selectivity and mode of action. Circular dichroism spectroscopy indicated that the substitutions disrupt the α-helical structure of ME in micelles of sodium dodecyl sulfate and on negatively charged and zwitterionic phospholipid vesicles. Substitution by Nleu, Nphe, or Nlys but not Nala disturbed the self-association in an aqueous environment, interaction with zwitterionic membranes, and toxicity to mammalian cells of ME but did not affect the interaction with negatively charged membranes or antibacterial activity. Notably, peptides with Nphe or Nlys substitution had the highest therapeutic indices, consistent with their lipid selectivity. In addition, all of peptoid residue-containing ME analogues had little or no ability to induce membrane disruption, membrane depolarization and lipid flip-flop. Taken together, our studies indicate that substitution of the leucine zipper motif in ME with peptoid residues increases its selectivity against bacterial cells by impairing self-association activity and changes its mode of antibacterial action from membrane-targeting mechanism to possible intracellular targeting mechanism. Furthermore, our ME analogues especially those with Nleu, Nphe, or Nlys substitutions, may be therapeutically useful antimicrobial peptides.

Piscidin 1 (Pis-1) is a novel cytotoxic peptide with a cationic alpha-helical structure that was isolated from the mast cells of hybrid striped bass [Silphaduang, U., and Noga, E. J. (2001) Nature 414, 268-269]. Pis-1 is not selective for bacterial versus mammalian cells. In the present study, to develop novel antibiotic peptides with selectivity for bacterial cells, we examined the effect of substituting two glycine residues, Gly8 and Gly13, with Ala or Pro on this peptide's structure and biological activities. The bacterial cell selectivity of the peptides decreased in the following order: Gly-->Pro analogues > Gly-->Pro/Ala analogues > Pis-1 > Gly-->Ala analogues. The antimicrobial and hemolytic activities and abilities to permeabilize the model phospholipid membranes were higher for Pis-1 with Gly or Pro at position 8 than for its counterparts with either Gly or Pro at position 13. We determined the tertiary structure of Pis-1 and its analogues in the presence of SDS micelles by NMR spectroscopy. We found that Pis-1 has an alpha-helical structure from Phe2 to Thr21. Also, Pis-1 AA (Gly8, Gly13-->Ala8, Ala13) with higher antibacterial and hemolytic activity than Pis-1 has a stable alpha-helical structure from Phe2 to Thr21. Pis-1 PG (Gly-->Pro8) with bacterial cell selectivity has a hinge structure at Pro8, which provides flexibility in piscidin, followed by a three-turn helix from Val10 to Gly22 in the C-terminal region. Taken together, our results demonstrate that the conformational flexibility provided by introduction of a Pro at position 8, coupled with the primary anchoring of phenylalanines and histidines in the N-terminus to the cell membrane and the optimal length of the C-terminal amphipathic alpha-helix, are the critical factors that confer antibacterial activity and bacterial cell selectivity to Pis-1 PG. Pis-1 PG may be a good candidate for the development of a new drug with potent antibacterial activity but without cytotoxicity.

Recently, we designed a novel cell-selective antimicrobial peptide (TPk) with intracellular mode of action from Pro --> Nlys (Lys peptoid residue) substitution in a noncell-selective cathelicidin-derived Trp/Pro-rich antimicrobial peptide, tritrpticin-amide (TP; VRRFPWWWPFLRR-NH(2)) (Biochemistry 2006; 45: 13007-13017). In this study, to elucidate the effect of Pro --> Nlys substitution on therapeutic index and mode of action of other noncell-selective cathelicidin-derived Trp/Pro-rich antimicrobial peptides and develop novel short antimicrobial peptides with high cell selectivity/therapeutic index, we synthesized Nlys-substituted antimicrobial peptides, TPk, STPk and INk, in which all proline residues of TP, symmetric TP-analogue (STP; KKFPWWWPFKK-NH(2)) and indolicidin (IN; ILPWKWPWWPWRR-NH(2)) were replaced by Nlys, respectively. Compared to parent Pro-containing peptides (TP, STP and IN), Nlys substituted peptides (TPk, STPk and Ink) had 4- to 26-fold higher cell selectivity/therapeutic index. Parent Pro-containing peptides induced a significant depolarization of the cytoplasmic membrane of intact Staphylococcus aureus at their MIC, whereas Nlys-substituted antimicrobial peptides did not cause visible membrane depolarization at their MIC. These results suggest that the antibacterial action of Nlys-substituted peptides is probably not due to the disruption of bacterial cytoplasmic membranes but the inhibition of intracellular components. Taken together, our results showed that Pro --> Nlys substitution in other noncell-selective Trp/Pro-rich antimicrobial peptides such as STP and IN as well as TP can improve the cell selectivity/therapeutic index and change the mode of antibacterial action from membrane-disrupting to intracellular targeting. In conclusion, our findings suggested that Pro --> Nlys substitution in noncell-selective Trp/Pro-rich antimicrobial peptides is a promising method to develop cell-selective antimicrobial peptides with intracellular target mechanism.

To investigate the effect of Pro --peptoid residue substitution on cell selectivity and the mechanism of antibacterial action of Pro-containing beta-turn antimicrobial peptides, we synthesized tritrpticin-amide (TP, VRRFPWWWPFLRR-NH(2)) and its peptoid residue-substituted peptides in which two Pro residues at positions 5 and 9 are replaced with Nleu (Leu peptoid residue), Nphe (Phe peptoid residue), or Nlys (Lys peptoid residue). Peptides with Pro --Nphe (TPf) or Pro --Nleu substitution (TPl) retained antibacterial activity but had significantly higher toxicity to mammalian cells. In contrast, Pro --Nlys substitution (TPk) increased the antibacterial activity but decreased the toxicity to mammalian cells. Tryptophan fluorescence studies indicated that the bacterial cell selectivity of TPk is closely correlated with a preferential interaction with negatively charged phospholipids. Interestingly, TPk was much less effective at depolarizing of the membrane potential of Staphylococus aureus and Escherichia coli spheroplasts and causing the leakage of a fluorescent dye entrapped within negatively charged vesicles. Furthermore, confocal laser-scanning microscopy showed that TPk effectively penetrated the membrane of both E. coli and S. aureus and accumulated in the cytoplasm, whereas TP and TPf did not penetrate the cell membrane but remained outside or on the cell membrane. These results suggest that the bactericidal action of TPk is due to inhibition of the intracellular components after penetration of the bacterial cell membrane. In addition, TPK with Lys substitution effectively depolarized the membrane potential of S. aureus and E. coli spheroplasts. TPK induced rapid and effective dye leakage from bacterial membrane-mimicking liposomes and did not penetrate the bacterial cell membranes. These results suggested that the ability of TPk to penetrate the bacterial cell membranes appears to involve the dual effects that are related to the increase in the positive charge and the peptide's backbone change by peptoid residue substitution. Collectively, our results showed that Pro --Nlys substitution in Pro-containing beta-turn antimicrobial peptides is a promising strategy for the design of new short bacterial cell-selective antimicrobial peptides with intracellular mechanisms of action.

Here, we report the successful design of a novel bacteria-selective antimicrobial peptide, Pep-1-K (KKTWWKTWWTKWSQPKKKRKV). Pep-1-K was designed by replacing Glu-2, Glu-6, and Glu-11 in the cell-penetrating peptide Pep-1 with Lys. Pep-1-K showed strong antibacterial activity against reference strains (MIC = 1-2 microM) of Gram-positive and Gram-negative bacteria as well as against clinical isolates (MIC = 1-8 microM) of methicillin-resistant Staphylococcus aureus and multidrug-resistant Pseudomonas aeruginosa. In contrast, Pep-1-K did not cause hemolysis of human erythrocytes even at 200 microM. These results indicate that Pep-1-K may be a good candidate for antimicrobial drug development, especially as a topical agent against antibiotic-resistant microorganisms. Tryptophan fluorescence studies indicated that the lack of hemolytic activity of Pep-1-K correlated with its weak ability to penetrate zwitterionic phosphatidylcholine/cholesterol (10:1, w/w) vesicles, which mimic eukaryotic membranes. Furthermore, Pep-1-K caused little or no dye leakage from negatively charged phosphatidylethanolamine/phosphatidylglycerol (7:3, w/w) vesicles, which mimic bacterial membranes but had a potent ability to cause depolarization of the cytoplasmic membrane potential of intact S. aureus cells. These results suggested that Pep-1-K kills microorganisms by not the membrane-disrupting mode but the formation of small channels that permit transit of ions or protons but not molecules as large as calcein.

A novel bacterial cell-selective antimicrobial peptide, IsCT-P (ILKKIWKPIKKLF-NH(2)), was designed based on the scorpion-derived alpha-helical antimicrobial peptide, IsCT. Here, we investigated the effect of substituting Pro(8) of IsCT-P with the Ala-peptoid residue (N-methylglycine) on the peptide's structure and mechanism of action. Circular dichroism analysis revealed that the modified peptide, IsCT-a, has a much lower alpha-helicity than IsCT-P in membrane mimicking conditions, suggesting the peptoid residue provides much more structural flexibility than the proline residue. IsCT-a was also much less effective than IsCT-P at causing leakage of fluorescent dye entrapped within negatively charged vesicles and at dissipating the membrane potential of Staphylococcus aureus. Collectively, our results suggest that the antibacterial action of IsCT-a is due to the inhibition of intracellular targets rather than the disruption and depolarization of bacterial cell membranes.

To design melittin (ME) analogues that are not cytotoxic against mammalian cells but which possessing potent antimicrobial activity, we synthesized a ME analogue (ME-w) in which the Trp-19 residue of ME was replaced by a Trp-peptoid residue (Nhtrp). ME-w exhibited similar antimicrobial activity compared to ME against the tested six bacteria and C. albicans. However, it was much less cytotoxic against the hRBCs and HeLa and NIH-3T3 cells than ME. Tryptophan fluorescence and CD spectra revealed that the Trp-19 --> Nhtrp substitution in ME contributed to a much lower helical assembly in an aqueous environment and structural flexibility and exterior localization to zwitterionic membrane which modulates its selectivity toward bacterial cells.

Isoprenylcysteine carboxylmethyltransferase (Icmt) catalyzes the last of the three-step posttranslational protein prenylation process for the so-called CaaX proteins, which includes many signaling proteins, such as most small GTPases. Despite extensive studies on Icmt and its regulation of cell functions, the mechanisms of much of the impact of Icmt on cellular functions remain unclear. Our recent studies demonstrated that suppression of Icmt results in induction of autophagy, inhibition of cell growth and inhibition of proliferation in various cancer cell types, prompting this investigation of potential metabolic regulation by Icmt. We report here the findings that Icmt inhibition reduces the function of mitochondrial oxidative phosphorylation in multiple cancer cell lines. In-depth oximetry analysis demonstrated that functions of mitochondrial complex I, II and III are subject to Icmt regulation. Consistently, Icmt inhibition decreased cellular ATP and depleted critical tricarboxylic acid cycle metabolites, leading to suppression of cell anabolism and growth, and marked autophagy. Several different approaches demonstrated that the impact of Icmt inhibition on cell proliferation and viability was largely mediated by its effect on mitochondrial respiration. This previously unappreciated function of Icmt, which can be therapeutically exploited, likely has a significant role in the impact of Icmt on tumorigenic processes.

FoxO proteins are important regulators in cellular metabolism and are recognized to be nodes in multiple signaling pathways, most notably those involving PI3K/AKT and mTOR. FoxO proteins primarily function as transcription factors, but recent study suggests that cytosolic FoxO1 participates in the regulation of autophagy. In the current study, we find that cytosolic FoxO1 indeed stimulates cellular autophagy in multiple cancer cell lines, and that it regulates not only basal autophagy but also that induced by rapamycin and that in response to nutrient deprivation. These findings illustrate the importance of FoxO1 in cell metabolism regulation independent of its transcription factor function. In contrast to FoxO1, we find the closely related FoxO3a is a negative regulator of autophagy in multiple cancer cell lines, a previously unrecognized function for this protein, different from its function in benign fibroblast and muscle cells. The induction of autophagy by the knockdown of FoxO3a was found not to be mediated through the suppression of mTORC1 signaling; rather, the regulatory role of FoxO3a on autophagy was determined to be through its ability to transcriptionally suppress FoxO1. This complicated interplay of FoxO1 and FoxO3a suggests a complex checks- and balances-relationship between FoxO3a and FoxO1 in regulating autophagy and cell metabolism.

Environmental cues play important roles in the regulation of physiology and behavior in small mammals. In the present study, we performed a factorial experiment (temperature × photoperiod) in which the South China field mouse Apodemus draco (Rodentia: Muridae) was acclimated to different photoperiods (a long photoperiod of 16 h light/8 h dark and a short photoperiod of 8 h light/16 h dark) and temperatures (warm at 30°C and cold at 5°C) to test the hypothesis that photoperiod, temperature, or both together can trigger changes in serum leptin levels, body mass, thermogenesis, and energy intake. Body mass, the resting metabolic rate (RMR), nonshivering thermogenesis (NST), and energy intake significantly decreased in the cold condition. Cold exposure induced increases in mitochondrial protein contents, cytochrome C oxidase (COX) activity, and α-glycerophosphate oxidase (α-GPO) in the liver and brown adipose tissue (BAT). There were no significant differences in mitochondrial protein contents, COX, or α-GPO under different photoperiods. Cold also induced an increase in uncoupling protein 1 in the BAT but showed no significant differences with photoperiod. All of the results indicated that A. draco was more sensitive to temperature. Further, serum leptin levels were involved in the processes of thermogenesis and body mass regulation in A. draco.

The process of autophagy is situated at the intersection of multiple cell signaling pathways, including cell metabolism, growth, and death, and hence is subject to multiple forms of regulation. We previously reported that inhibition of isoprenylcysteine carboxylmethyltransferase (Icmt), which catalyzes the final step in the post-translational prenylation of so-called CAAX proteins, results in the induction of autophagy which enhances cell death in some cancer cells. In this study, using siRNA-mediated knockdown of a group of small GTPases that are predicted Icmt substrates, we identify Rac3 GTPase as a negative regulator of the process of autophagy. Knockdown of Rac3, but not the closely related isoforms Rac1 and Rac2, results in induction of autophagy. Ectopic expression of Rac3, significantly rescues cells from autophagy and cell death induced by Icmt inhibition, strengthening the notion of an isoform-specific autophagy regulatory function of Rac3. This role of Rac3 was observed in multiple cell lines with varying Rac subtype expression profiles, suggesting its broad involvement in the process. The identification of this less-studied Rac member as a novel regulator provides new insight into autophagy and opens opportunities in identifying additional regulatory inputs of the process.

Pep-1-K (PK) is a good cell-selective antimicrobial peptide designed from cell-penetrating peptide Pep-1. To develop novel short antimicrobial peptides with higher cell selectivity and shorter length compared with PK, several PK analogs were designed by the deletion, addition and/or substitution of amino acids. Among these analogs, PK-12-KKP (KKPWWKPWWPKWKK) showing the sequence and structure homology with a Trp/Pro-rich natural antimicrobial peptide, indolicidin (IN), displayed a 20-fold higher cell selectivity as compared to IN. Circular dichroism analysis revealed that PK-12-KKP adopts a folded structure combined with some portions of unordered structure. PK-12-KKP selectively binds to negatively charged bacterial membrane-mimetic vesicles, and its high phospholipid selectivity corresponds well with its high cell selectivity. Moreover, it showed very weak potential in depolarization of the cytoplasmic membrane of Staphylococcus aureus at 8 microM (4x minimal inhibitory concentration) and dye leakage from negatively charged liposomes. These results suggest that the ultimate target of our designed PK-12-KKP maybe the intracellular components (e.g. protein, DNA or RNA) rather than the cytoplasmic membranes. Collectively, our designed short Trp/Pro-rich peptide, PK-12-KKP, appears to be an excellent candidate for future development as a novel antimicrobial agent.

The cell-penetrating peptide Tat (48–60) (GRKKRRQRRRPPQ) derived from HIV-1 Tat protein showed potent antibacterial activity (MIC: 2–8 µM). To investigate the effect of dimerization of Tat (48–60) analog, [Tat(W): GRKKRRQRRRPWQ-NH2], on antimicrobial activity and mechanism of bactericidal action, its dimeric peptides, di-Tat(W)-C and di-Tat(W)-K, were synthesized by a disulfide bond linkage and lysine linkage of monomeric Tat(W), respectively. From the viewpoint of a weight basis and the monomer concentration, these dimeric peptides displayed almost similar antimicrobial activity against six bacterial strains tested but acted more rapidly against Staphylococcus aureus on kinetics of bactericidal activity, compared with monomeric Tat(W). Unlike monomeric Tat(W), these dimeric peptides significantly depolarized the cytoplasmic membrane of intact S. aureus cells at MIC and induced dye leakage from bacterial-membrane-mimicking egg yolk L-α-phosphatidylethanolamine/egg yolk L-α-phosphatidyl-DL-glycerol (7:3, w/w) vesicles. Furthermore, these dimeric peptides were less effective to translocate across lipid bilayers than monomeric Tat(W). These results indicated that the dimerization of Tat analog induces a partial change in the mode of its bactericidal action from intracellular target mechanism to membrane-targeting mechanism. Collectively, our designed dimeric Tat peptides with high antimicrobial activity and rapid bactericidal activity appear to be excellent candidates for future development as novel antimicrobial agents. Copyright © 2009 European Peptide Society and John Wiley & Sons, Ltd.

The cell penetrating peptide, penetratin (RQIKIWFQNRRMKWKK-NH2) showed potent antimicrobial activity (MIC: 0.5-4 microM) without any cytotoxicity against mammalian cells. This study investigated the effect of linking together two peptide chains of penetratin on antimicrobial and cytolytic activities and plausible mode of bactericidal action. Two-stranded penetratin was prepared by a simultaneous solid-phase synthesis of the two strands of a single lysine residue attached to the solid support. Two-stranded penetratin markedly increased cytolytic activity against human erythrocytes and NIH-3T3 mouse fibroblast cells without a significant effect on antimicrobial activity. This finding suggested that penetratin is active as a monomer to bacterial cells but as an oligomer to mammalian cells. Circular dichroism analysis revealed that the alpha-helical content of the membrane-bound penetratin was unaffected by two-stranded Lys-linkage. Penetratin had very weak ability in the depolarization of membrane potential of intact Staphylococcus aureus cells and the fluorescent dye leakage of calcein-entrapped negatively charged bacterial membrane-mimicking vesicles. In contrast, two-stranded penetratin significantly caused membrane depolarization and dye leakage. These results suggest that the two-stranded penetratin induces a significant change in its mode of bactericidal action from the intracellular-target mechanism to the membrane-targeting mechanism.

Arenicin-1 (AR-1) is a novel antimicrobial peptide that was isolated from coelomocytes of the marine polychaeta lugworm Arenicola marina and shown to contain a single disulfide bond between Cys3 and Cys20, forming an 18-residue ring [Ovchinnikova, T. V. et al., FEBS Lett 2004, 577, 209–214]. To determine the role of this disulfide bond, we synthesized AR-1 (RWCVYAYVRVRGVLVRYRRCW) and its linear derivative, arenicin-1-S (AR-1-S: RWSVYAYVRVRGVLVRYRRSW). Activity assays revealed that AR-1-S is somewhat less active against bacterial cells than AR-1. Both peptides were very hydrophobic, and displayed cytotoxicity against human red blood cells. Analysis of the tertiary structures of AR-1 and AR-1-S by NMR spectroscopy disclosed that AR-1 has two-stranded antiparallel β-sheet structures with amphipathicity, while AR-1-S displays a random coil structure in DMSO. Our biological data for AR-1 and AR-1-S indicate that the hydrophobic-hydrophilic balance, disulfide bridge, and the amphipathic β-sheet structure of the peptides play important roles in their biological activities. Elucidation of the structure of AR-1 and its derivative should facilitate the design of novel non-cytotoxic peptide antibiotics with potent antibacterial activities. © 2007 Wiley Periodicals, Inc. Biopolymers (Pept Sci) 88: 208–216, 2007. This article was originally published online as an accepted preprint. The ‘Published Online’ date corresponds to the preprint version. You can request a copy of the preprint by emailing the Biopolymers editorial office at biopolymers@wiley.com