Expression of DNA methyltransferases in the mouse uterus during early pregnancy and susceptibility to dietary folate deficiency.

We have characterized the uterine expression of DNA methyltransferases (DNMTs) during early pregnancy in mice and determined whether a folate-deficient diet (FDD) can affect DNMTs in this context. Within endometrial cells, expressions of DNMT (cytosine-5) 1 (Dnmt1), Dnmt3a, and Dnmt3b were significantly elevated during the prereceptive phase of pregnancy but generally returned to baseline levels during receptive and postimplantation periods. As such, the transcription of DNMT genes is temporally regulated during early pregnancy. When comparisons were made between implantation sites (IS) and inter-IS on day 5 of pregnancy, lower levels of Dnmt3a were detected at IS. Comparisons between IS and inter-IS did not reveal significant expression differences for other DNMT genes. When tissue sections were examined, DNMT3A was specifically lower in the stroma of IS. Reduced DNMT1 and DNMT3B levels were also observed in the luminal and glandular epithelia of IS, whereas no obvious differences in the stroma were detected. In pseudo-pregnant mice subjected to a FDD, levels of Dnmt1 and Dnmt3a (but not Dnmt3b) were significantly upregulated in endometrial tissues, as compared with controls. When tissues from these folate-deficient mice were examined, DNMT1 levels were elevated in both the luminal and glandular epithelia, whereas DNMT3A was upregulated in the luminal epithelium and the stroma. A slight increase in DNMT3B levels was detected in the glandular epithelium. These results indicate that DNMTs may regulate the transcription of endometrial genes associated with embryo implantation and that levels of DNMTs are affected by dietary folate in mice.

Urine-derived stem cells for the therapy of diabetic nephropathy mouse model.

OBJECTIVE: Diabetic nephropathy (DN) is one of the most representative diabetic microangiopathy complications. So far, there have been no satisfactory therapeutic strategies, and the injection of stem cells provides a target for DN therapy. PATIENTS AND METHODS: Urine-derived stem cells (USCs) were obtained from 9 healthy men. 24 mice were randomly and equally divided into control group, DN model group, DN+hUSC group (treated with USCs for 3 times). Hematoxylin-eosin (HE) and Masson staining were used to detect histological changes of kidney injury. Creatinine and blood urea nitrogen (BUN) were measured to assess renal function. Besides, myofibroblast accumulation, macrophage infiltration, cell proliferation, and oxidative stress were detected by immunohistochemical analysis. RESULTS: Compared with DN model group, DN+hUSC group showed lower function loss, cell infiltration, and oxidative stress, as well as less renal fibrosis, histological damage, and cell proliferation. CONCLUSIONS: USC can alleviate inflammation and oxidative stress, reduce renal interstitial fibrosis, improve renal tissue structure and protect renal function through paracrine effect.

Deletion mapping of four loci defined by N-ethyl-N-nitrosourea-induced postimplantation-lethal mutations within the pid-Hbb region of mouse chromosome 7.

As part of a long-term effort to refine the physical and functional maps of the Fes-Hbb region of mouse chromosome 7, four loci [l(7)1Rn, l(7)2Rn, l(7)3Rn, l(7)4Rn] defined by N-ethyl-N-nitrosourea (ENU)-induced, prenatally lethal mutations were mapped by means of trans complementation crosses to mice carrying lethal deletions of the mouse chromosome-7 albino (c) locus. Each locus was assigned to a defined subregion of the deletion map at the distal end of the Fes-Hbb interval. Of particular use for this mapping were preimplantation-lethal deletions having distal breakpoints localized between pid and Omp. Hemizygosity or homozygosity for each of the ENU-induced lethals was found to arrest development after uterine implantation; the specific time of postimplantation death varied, and depended on both the mutation itself and on whether it was hemizygous or homozygous. Based on their map positions outside of and distal to deletions that cause death at preimplantation stages, these ENU-induced mutations identify loci, necessary for postimplantation development, that could not have been discovered by phenotypic analyses of mice homozygous for any albino deletion. The mapping of these loci to specific genetic intervals defined by deletion breakpoints suggests a number of positional-cloning strategies for the molecular isolation of these genes. Phenotypic and genetic analyses of these mutations should provide useful information on the functional composition of the corresponding segment of the human genome (perhaps human 11q13.5).

Energy balance and carbohydrate metabolism in infection and sepsis.

Indirect calorimetry and nitrogen measurements suggest that uncomplicated abdominal surgery produces no significnat change in resting metabolic expenditure and only a slight loss of urinary nitrogen. More severe injury and infections produce larger increases in resting metabolic expenditure and nitrogen loss. Severe injuries can result in a 15 to 30% loss of body weight, but the protein contribution to caloric expenditure does not exceed 20% and is less than expected. The provision of calories and nitrogen can change the course of the septic patient. A continual conversion of alanine carbon to glucose occurs in septic patients, including those who are receiving exogenous glucose at the normal hepatic production rate. In sepsis, the release of glucogenic substrates from peripheral tissues may determine the rate of hepatic gluconeogenesis.

Kinetics of intravenously administered 15N-L-alanine in the evaluation of protein turnover.

After a pulse injection of 15N-L-alanine to a healthy male subject, the distribution of 15N in the various components of blood and urine were determined as function of time. The rapid appearance of the isotope both in the urinary urea and ammonia and in the plasma amide and urea suggests that transamination (and not deamination) may be the key step in the interaction. After 30 to 60 min postinjection, the tracer dynamics represents the overall metabolic pool characteristics and does not reflect the metabolism of alanine only. The nitrogen of alanine is used effectively in the synthesis of body protein.

Metabolism and recycling of urea in man.

The rate of breakdown and reutilization of urea in man has been measured in five normal and two septic patients using 15N and 13C labeled ureas. The labeled molecules of the 15N urea dose were distinguished from the labeled molecules of the recycled urea by analyzing in a mass spectrometer the isotopic nitrogens produced when the recrystalized urine urea was treated with a hypobromite solution. In a normal subject with regular nitrogen intake, it was found that only 4/5 of the produced urea was excreted in urine and the rest was endogenously degraded. Seventy percent of the nitrogen and 63% of the carbon of the degraded urea were returned to the urea pool. On a nitrogen-free diet or after neomycin treatment with regular diet in the normal, the extent of urea splitting is considerably reduced. In the septic patients, breakdown, as well as recycling of urea was almost eliminated. It appears that the reate of endogenous catabolism of urea depends mainly on the activity of the gut flora which may be affected by dietary intake and clinical status of the subject. The method developed here could be applied for the quantitation of urea dynamics under different physiological and pathological conditions.

Parenteral nutrition in the septic patient: nitrogen balance, limiting plasma amino acids, and calorie to nitrogen ratios.

A series of eight septic patients was provided varying levels of beef fibrin protein hydrolysate by central vein in the presence of adequate calories for evaluation of nitrogen retention under septic conditions. The mean nitrogen intake to achieve nitrogen equilibrium was 240 mg/kg of body wt per day. This represents a 40% increase over that required to produce nitrogen equilibrium in normal adults. The mean caloric intake of these patients was 43.3 kcal/kg of body wt per day. The calorie to nitrogen ratio based on the above intake was calculated to be 180:1. In order to utilize effectively calorie to nitrogen ratios in the nutritional care of patients, it is suggested that ratios be standardized using daily total coloric expenditures. Correcting the mean measured resting calorie expenditures of these patients for minimal daily activity, a caloric to nitrogen ratio of 138:1 was obtained. The plasma amino acid ratios in these septic patients confirm the finding that valine and phenylalnine are limiting amino acids in a beef fibrin hydrolysate at infusion levels below 240 mg of N/kg of body wt per day. Analysis of the urinary excretion of total nitrogen, urea, and amino acids in two patients suggests that 30 to 50% of the infused peptides of a beef fibrin hydrolysate are lost in the urine in these septic patients.

Evaluation of whole body nitrogen kinetics in acute metabolic acidosis.

The data obtained after a pulse dose of L-[15N] alanine and [13C] urea in control and acidotic conscious dogs were analyzed to compute the whole body nitrogen turnover rates. Acute acidosis was induced and maintained by continuous HCl infusion. On the basis of a four pool model, the mean daily protein synthesis rate in the normal dog was calculated to be 10.8 g/kg compared to 7.6 g/kg in acidosis. Since all dogs were in negative nitrogen balance, the daily catabolic rate of protein was greater than the synthetic rate and the mean daily catabolic rate was 14.4 g protein/kg in normal dogs compared to 10.5 g protein/kg in acidotic dogs. The body urea pool size and excretion rates were decreased by 24 and 27%, respectively, due to acute acidosis, without any change in the fractional turnover rate. Thus the adaptive response to the induced acid challenge appears to be a reduction in the synthesis and breakdown rates of protein and also a decrease in the production and excretion rate of urea.

Whole body protein synthesis and catabolism in septic man.

The protein catabolic response to sepsis has been measured in three patients and in two normal subjects using a pulse injections of L-[15N]alanine. In addition, the urea kinetics were measured using a pulse administration of [15N]urea. Several nitrogen models which simulated the metabolic pathways of nitrogen-labeled compounds were tried. Best curve fits and acceptable confidence limits were obtained with a four-pool model containing two metabolic pools and two urea pools. Using this model, synthesis and catabolism rates were calculated for a fast and slow protein turnover pool. The mean daily total protein synthesis rate in the normal was 3.695 g/kg compared to 4.479 g/kg in sepsis. Because all subjects were in negative nitrogen balance, the mean total protein catabolic rate in the normal was 4.379 g/kg, compared to 5.298 g/kg in sepsis. These data suggest an increase in both protein synthesis and catabolism during sepsis.

Prediction of resting energy expenditure from fat-free mass and fat mass.

On the basis of literature values, the relationship between fat-free mass (FFM), fat mass (FM), and resting energy expenditure [REE (kJ/24 h)] was determined for 213 adults (86 males, 127 females). The objectives were to develop a mathematical model to predict REE based on body composition and to evaluate the contribution of FFM and FM to REE. The following regression equations were derived: 1) REE = 1265 + (93.3 x FFM) (r2 = 0.727, P < 0.001); 2) REE = 1114 + (90.4 x FFM) + (13.2 x FM) (R2 = 0.743, P < 0.001); and 3) REE = (108 x FFM) + (16.9 x FM) (R2 = 0.986, P < 0.001). FM explained only a small part of the variation remaining after FFM was accounted for. The models that include both FFM and FM are useful in examination of the changes in REE that occur with a change in both the FFM and FM. To account for more of the variability in REE, FFM will have to be divided into organ mass and skeletal muscle mass in future analyses.

Effect of weight reduction on resting energy expenditure, substrate utilization, and the thermic effect of food in moderately obese women.

It is not known whether the decrease in the thermic effect of food (TEF) in obesity is a consequence of obesity or a factor contributing to the development of obesity. The resting energy expenditure (REE) of 24 obese, nondiabetic, postmenopausal women was 5481 +/- 110 kJ/24 h (1310 +/- 26.4 kcal/24 h). After weight loss (12.7 +/- 0.45 kg) the REE was significantly decreased (4858 +/- 94 kJ/24 h, or 1161 +/- 22.4 kcal/24 h) and equivalent to the REE of 4866 +/- 119 kJ/24 h (1163 +/- 28.5 kcal/24 h) in 24 never-obese, postmenopausal women. The TEF, expressed as a percentage of the calories ingested, was 8.2 +/- 0.50% for obese subjects, 8.7 +/- 0.57% for postobese subjects, and 9.8 +/- 0.54% for never-obese subjects. Compared with never-obese subjects, the TEF was significantly reduced in obese subjects (P = 0.043) and remained unchanged after weight loss (P = 0.341). These findings indicate that the lower TEF in the obese subjects is uncorrected by weight loss, and thus it is a contributor to obesity rather than a consequence of obesity.

Contribution of skeletal muscle protein in elevated rates of whole body protein catabolism in trauma patients.

Whole body protein breakdown using 15N and skeletal muscle protein breakdown from urinary 3-methylhistidine were measured simultaneously in seven skeletal trauma and eight normal subjects on a standard hypocaloric, protein free diet. The trauma group had a 31% greater resting metabolic energy expenditure than controls. The control males lost 3.73 mumol/kg/day of 3-methylhistidine which suggested a protein breakdown rate of 0.89 g P/kg/day. The control females lost 2.46 mumol/kg/day of 3-methylhistidine or a breakdown rate of 0.58 g P/kg/day. These parameters were 187% greater for males and 163% greater for females in the trauma group. The measured whole body protein breakdown rates were 3.64 g P/kg/day for the control males and 2.69 for females. Skeletal trauma increased both by 73%. Skeletal trauma raised the muscle contribution to the whole body breakdown rate from 24.4 to 40.4% for men and from 21.6 to 33.0% for women. This disproportionate increase in muscle protein breakdown is consistent with muscle protein metabolism being most seriously affected by severe injury.

Plasma amino acid concentrations in geriatric control and hip-fracture patients.

Changes in plasma amino acids, 24-h nitrogen balances, and resting metabolic expenditures (RMEs) were measured in 10 geriatric patients (aged 70-92 y) with hip fracture 1 d after surgical fixation during both a 24-h fasting state and while receiving total peripheral parenteral nutrition (TPPN) for 24 h at 1.5 g amino acids.kg-1.d-1 and 29-30 kcal.kg-1.d-1 and compared with 19 healthy volunteer subjects (aged 70-84 y). RME and 24-h urinary nitrogen losses were also elevated in the trauma patients during both fasting and TPPN. Positive nitrogen balances were evident in both groups during TPPN. Plasma total amino acid concentration was significantly lower in the trauma patients because of lower plasma concentrations of the nonessential amino acids. Phenylalanine and methionine concentrations were significantly higher and lysine lower in the trauma group. In addition, evaluation of the essential amino acid ratios after fasting and TPPN reveal that there are no limiting amino acids during TPPN.

Muscle protein catabolism in the septic patient as measured by 3-methylhistidine excretion.

Muscle protein catabolism has been evaluated using the excretion of urinary 3-methylhistidine (3-MEH) is six normal male and six normal female subjects and in four surgical patients, two of whom developed febrile episodes during the course of their study. In addition, their nutritional status was also evaluated using percentage body weight losses before hospital admittance, creatinine-height ratios, and, in two patients, serum alkaline ribonuclease levels. The results indicate that: 1) prolonged starvation may produced decreased 3-MEH excretion because of an adaptive diminution of muscle breakdown in sustained starvation, decreased 3-MEH excretion also may simply reflect diminished lean body mass, 3-MEH excretion may be increased above basal levels because of superimposed stresses such as fever, and the acute phases of starvation produce increased levels of 3-MEH excretion until adaptive mechanisms occur; 2) creatinine-height ratios are low in starvation, and increase not only with improved nutrition but in response to fever and stress of operation, even when these are superimposed on malnutrition; and 3) alkaline RNAase levels are elevated in malnutrition and decrease with improved nutrition but in response to fever and stress of operation, even when these are superimposed on malnutrition; and 3) alkaline RNAase levels are elevated in malnutrition and decrease with improved nutrition. The enzyme may also be elevated by the stress of operations.

Effects of major skeletal trauma on whole body protein turnover in man measured by L-[1,14C]-leucine.

Skeletal trauma induces excessive urinary nitrogen losses and is thought to stimulate the oxidation of the branched chain amino acids. This study was undertaken to quantitate whole body protein turnover rates and leucine metabolism during the peak nitrogen loss period following skeletal trauma. Quantitation was done in eight healthy and six trauma subjects, who received D5W as their only nutrition for 72 hours, using a 10-hour continuous infusion of L-[1,14C]-Leucine. The controls lost an average of 6 gm of nitrogen/day and the trauma patients 25 gm of nitrogen/day on the study day. Trauma was shown to elevate plasma leucine by 76%, increase the leucine flux through the free leucine pool by 86%, and accelerate leucine oxidation by 277% over the values for controls. Trauma also produced a 50% increase in whole body protein synthesis and a 79% increase in protein breakdown. The data clearly define significant increases in both the protein synthetic and catabolic rates in trauma with a greater increase occurring in catabolism. This is similar to findings for protein turnover in sepsis and burn injury, but is different from that found in elective surgery. A striking aspect of our data is the indication that women do not exhibit the same response to injury that men do. This suggestion, however, is based on a small sample.

Contribution of protein to caloric expenditure following injury.

1. There is a common clinical impression that because the nitrogen excretion after injury roughly parallels the increased resting metabolic expenditure and weight loss, the primary source of extra fuel is derived from protein. This study has been undertaken to evaluate the caloric contribution of protein to the daily resting metabolic expenditure (RME). 2. Under the most extreme circumstances of nitrogen excretion, massive soft tissue injury, the caloric contribution of protein is only 20 percent of the RME. Body fat stores are the only other major source of fuel from which the semistarved patient can meet the increased demands for energy. 3. Any form of severe injury impairs the mechanisms by which protein breakdown and nitrogen excretion are reduced. 4. Patients undergoing elective operation in this study did not have a significant rise in RME or nitrogen excretion.

Net metabolic changes of zinc, copper, nitrogen, and potassium balances in skeletal trauma patients.

Zinc, copper, nitrogen, and potassium balances of 10 male skeletal trauma patients were determined over 5-6 days each. Nutrition consisted of electrolyte/glucose and/or blood/blood product infusions. Patients were started on balance studies within 24 hr following injury. Zinc and copper were analyzed by atomic absorption while nitrogen was measured by the microKjeldahl technique and potassium by flame photometry. The mean daily balances for these patients were -1563 micrograms zinc, -266 micrograms copper, -20.0 g nitrogen and -29 mEq potassium for 5 patients receiving electrolyte/glucose infusions and +1273 micrograms zinc, +322 micrograms copper, -12.9 g nitrogen, and -26 mEq potassium for 5 patients receiving blood/blood products in addition to electrolyte/glucose. Routine daily maintenance supplementation of 2 mg zinc and 2 mg copper is recommended for skeletal trauma patients on electrolyte/glucose and those on electrolyte/glucose with blood/blood products intravenous infusions. Further balance studies are necessary to ascertain the level of zinc and copper supplementation needed by skeletal trauma patients receiving different nutritional support.

Nonsuppressability of gluconeogenesis by glucose in septic patients.

The contribution of alanine to the synthesis of glucose and the oxidation of alanine was evaluated in normal and septic patients using (14C)L-alanine. The data indicate that there is a twofold increase in the conversion of alanine into glucose in sepsis and, further, this increase was observed while the patients were receiving a constant glucose infusion (100 mg/min) prior to and during the single injection of (14C)L-alanine. Failure of glucose to decrease this gluconeogenic response in these septic patients clearly indicates that the controlling mechanism for glucose synthesis is modified following injury and undoubtedly plays a role in the abnormal carbohydrate metabolism observed in injury. The contribution of alanine carbon to oxidation was the same in the control and septic group as measured by the per cent of the (14C)L-alanine dose expired in 3 h. Since the control subjects received glucose continuously during the study with and without amino acids, it is clear that nutritional intake and injury has minimal effect on the oxidation of alanine. This suggests that transamination is not affected by sepsis nor is there an inhibition of pyruvate oxidation following sepsis.

Validity of 3-methylhistidine excretion as an indicator of skeletal muscle protein breakdown in humans.

The urinary excretion of 3-methylhistidine (3MEH) in humans and animals has been used as a biologic marker for skeletal muscle protein breakdown. In rats, it has been recently suggested that there is a significant contribution of 3MEH in urine from the gastrointestinal tract due to the rapid turnover of protein in that tissue. To evaluate this point in humans, six patients with short bowel were evaluated. They were placed on three-day meat-free diets while 24-hour urine collections were obtained. The mean +/- SEM 3MEH in the short-bowel group was 3.27 +/- 0.34 mumol/kg/d and the mean +/- SEM molar ratio of 3MEH to creatinine was 0.0212 +/- 0.0012. These data were not significantly different from the control group at 95% confidence level. The results suggest that the contribution of the small intestine appears to be negligible, therefore urinary 3MEH should continue to be a valid index of skeletal muscle breakdown in man.

Protein and energy sparing of glucose added in hypocaloric amounts to peripheral infusions of amino acids.

After abdominal surgery, patients were given peripheral infusions of amino acids alone for 4 days followed by amino acids plus glucose for 4 days, or the same solutions in the reverse order. Although there was a wide variation in the response of individual subjects, the typical effect of glucose under these conditions was to reduce both nitrogen excretion (average of 2.8 g N/day) and resting metabolic expenditure (average of 110 kcal/day).