Creatine supplementation reduces the cerebral oxidative and metabolic stress responses to acute in utero hypoxia in the late-gestation fetal sheep.

Prophylactic creatine treatment may reduce hypoxic brain injury due to its ability to sustain intracellular ATP levels thereby reducing oxidative and metabolic stress responses during oxygen deprivation. Using microdialysis, we investigated the real-time in vivo effects of fetal creatine supplementation on cerebral metabolism following acute in utero hypoxia caused by umbilical cord occlusion (UCO). Fetal sheep (118 days' gestational age (dGA)) were implanted with an inflatable Silastic cuff around the umbilical cord and a microdialysis probe inserted into the right cerebral hemisphere for interstitial fluid sampling. Creatine (6 mg kg-1  h-1 ) or saline was continuously infused intravenously from 122 dGA. At 131 dGA, a 10 min UCO was induced. Hourly microdialysis samples were obtained from -24 to 72 h post-UCO and analysed for percentage change of hydroxyl radicals (• OH) and interstitial metabolites (lactate, pyruvate, glutamate, glycerol, glycine). Histochemical markers of protein and lipid oxidation were assessed at post-mortem 72 h post-UCO. Prior to UCO, creatine treatment reduced pyruvate and glycerol concentrations in the microdialysate outflow. Creatine treatment reduced interstitial cerebral • OH outflow 0 to 24 h post-UCO. Fetuses with higher arterial creatine concentrations before UCO presented with reduced levels of hypoxaemia ( PO2${P_{{{\rm{O}}_{\rm{2}}}}}$ and SO2${S_{{{\rm{O}}_{\rm{2}}}}}$ ) during UCO which associated with reduced interstitial cerebral pyruvate, lactate and • OH accumulation. No effects of creatine treatment on immunohistochemical markers of oxidative stress were found. In conclusion, fetal creatine treatment decreased cerebral outflow of • OH and was associated with an improvement in cerebral bioenergetics following acute hypoxia. KEY POINTS: Fetal hypoxia can cause persistent metabolic and oxidative stress responses that disturb energy homeostasis in the brain. Creatine in its phosphorylated form is an endogenous phosphagen; therefore, supplementation is a proposed prophylactic treatment for fetal hypoxia. Fetal sheep instrumented with a cerebral microdialysis probe were continuously infused with or without creatine-monohydrate for 10 days before induction of 10 min umbilical cord occlusion (UCO; 131 days' gestation). Cerebral interstitial fluid was collected up to 72 h following UCO. Prior to UCO, fetal creatine supplementation reduced interstitial cerebral pyruvate and glycerol concentrations. Fetal creatine supplementation reduced cerebral hydroxyl radical efflux up to 24 h post-UCO. Fetuses with higher arterial creatine concentrations before UCO and reduced levels of systemic hypoxaemia during UCO were associated with reduced cerebral interstitial pyruvate, lactate and • OH following UCO. Creatine supplementation leads to some improvements in cerebral bioenergetics following in utero acute hypoxia.

Assessing Creatine Supplementation for Neuroprotection against Perinatal Hypoxic-Ischaemic Encephalopathy: A Systematic Review of Perinatal and Adult Pre-Clinical Studies.

There is an important unmet need to develop interventions that improve outcomes of hypoxic-ischaemic encephalopathy (HIE). Creatine has emerged as a promising neuroprotective agent. Our objective was to systematically evaluate the preclinical animal studies that used creatine for perinatal neuroprotection, and to identify knowledge gaps that need to be addressed before creatine can be considered for pragmatic clinical trials for HIE. METHODS: We reviewed preclinical studies up to 20 September 2021 using PubMed, EMBASE and OVID MEDLINE databases. The SYRCLE risk of bias assessment tool was utilized. RESULTS: Seventeen studies were identified. Dietary creatine was the most common administration route. Cerebral creatine loading was age-dependent with near term/term-equivalent studies reporting higher increases in creatine/phosphocreatine compared to adolescent-adult equivalent studies. Most studies did not control for sex, study long-term histological and functional outcomes, or test creatine post-HI. None of the perinatal studies that suggested benefit directly controlled core body temperature (a known confounder) and many did not clearly state controlling for potential study bias. CONCLUSION: Creatine is a promising neuroprotective intervention for HIE. However, this systematic review reveals key knowledge gaps and improvements to preclinical studies that must be addressed before creatine can be trailed for neuroprotection of the human fetus/neonate.

The physiological effects of creatine supplementation in fetal sheep before, during, and after umbilical cord occlusion and global hypoxia.

The aim of this study was to investigate the effects of direct creatine infusion on fetal systemic metabolic and cardiovascular responses to mild acute in utero hypoxia. Pregnant ewes (n = 28) were surgically instrumented at 118 days gestation (dGa). A constant intravenous infusion of creatine (6 mg·kg-1·h-1) or isovolumetric saline (1.5 mL·h-1) began at 121 dGa. After 10 days, fetuses were subjected to 10-min umbilical cord occlusion (UCO) to induce mild global hypoxia (saline-UCO, n = 8; creatine-UCO, n = 7) or sham UCO (saline-control, n = 6; creatine-control, n = 7). Cardiovascular, arterial blood gases and metabolites, and plasma creatine were monitored before, during, and then for 72 h following the UCO. Total creatine content in discrete fetal brain regions was also measured. Fetal creatine infusion increased plasma concentrations fivefold but had no significant effects on any measurement pre-UCO. Creatine did not alter fetal physiology during the UCO or in the early recovery stage, up to 24 h after UCO. During the late recovery stage, 24-72 h after UCO, there was a significant reduction in the arterial oxygen pressure and saturation in creatine fetuses (PUCO × TREATMENT = 0.02 and 0.04, respectively). At 72 h after UCO, significant creatine loading was detected in cortical gray matter, hippocampus, thalamus, and striatum (PTREATMENT = 0.01-0.001). In the striatum, the UCO itself increased total creatine content (PUCO = 0.019). Overall, fetal creatine supplementation may alter oxygen flux following an acute hypoxic insult. Increasing total creatine content in the striatum may also be a fetal adaptation to acute oxygen deprivation.NEW & NOTEWORTHY Direct fetal creatine supplementation increased plasma and cerebral creatine concentrations but did not alter fetal body weight, basal cardiovascular output, or blood chemistry. Creatine-treated fetuses displayed changes to arterial oxygenation 24-72 h after acute global hypoxia. An increase in striatum total creatine levels following UCO was also noted and suggests that increasing creatine tissue availability may be an adaptive response against the effects of hypoxia.

Creatine Metabolism in Female Reproduction, Pregnancy and Newborn Health.

Creatine metabolism is an important component of cellular energy homeostasis. Via the creatine kinase circuit, creatine derived from our diet or synthesized endogenously provides spatial and temporal maintenance of intracellular adenosine triphosphate (ATP) production; this is particularly important for cells with high or fluctuating energy demands. The use of this circuit by tissues within the female reproductive system, as well as the placenta and the developing fetus during pregnancy is apparent throughout the literature, with some studies linking perturbations in creatine metabolism to reduced fertility and poor pregnancy outcomes. Maternal dietary creatine supplementation during pregnancy as a safeguard against hypoxia-induced perinatal injury, particularly that of the brain, has also been widely studied in pre-clinical in vitro and small animal models. However, there is still no consensus on whether creatine is essential for successful reproduction. This review consolidates the available literature on creatine metabolism in female reproduction, pregnancy and the early neonatal period. Creatine metabolism is discussed in relation to cellular bioenergetics and de novo synthesis, as well as the potential to use dietary creatine in a reproductive setting. We highlight the apparent knowledge gaps and the research "road forward" to understand, and then utilize, creatine to improve reproductive health and perinatal outcomes.

Renal dysfunction in early adulthood following birth asphyxia in male spiny mice, and its amelioration by maternal creatine supplementation during pregnancy.

BACKGROUND: Acute kidney injury affects ~70% of asphyxiated newborns, and increases their risk of developing chronic kidney disease later in life. Acute kidney injury is driven by renal oxygen deprivation during asphyxia, thus we hypothesized that creatine administered antenatally would protect the kidney from the long-term effects of birth asphyxia. METHODS: Pregnant spiny mice were fed standard chow or chow supplemented with 5% creatine from 20-d gestation (midgestation). One day prior to term (37-d gestation), pups were delivered by caesarean or subjected to intrauterine asphyxia. Litters were allocated to one of two time-points. Kidneys were collected at 1 mo of age to estimate nephron number (stereology). Renal function (excretory profile and glomerular filtration rate) was measured at 3 mo of age, and kidneys then collected for assessment of glomerulosclerosis. RESULTS: Compared with controls, at 1 mo of age male (but not female) birth-asphyxia offspring had 20% fewer nephrons (P < 0.05). At 3 mo of age male birth-asphyxia offspring had 31% lower glomerular filtration rate (P < 0.05) and greater glomerular collagen IV content (P < 0.01). Antenatal creatine prevented these renal injuries arising from birth asphyxia. CONCLUSION: Maternal creatine supplementation during pregnancy may be an effective prophylactic to prevent birth asphyxia induced acute kidney injury and the emergence of chronic kidney disease.

Maternal Creatine Supplementation during Pregnancy Prevents Long-Term Changes in Diaphragm Muscle Structure and Function after Birth Asphyxia.

Using a model of birth asphyxia, we previously reported significant structural and functional deficits in the diaphragm muscle in spiny mice, deficits that are prevented by supplementing the maternal diet with 5% creatine from mid-pregnancy. The long-term effects of this exposure are unknown. Pregnant spiny mice were fed control or 5% creatine-supplemented diet for the second half of pregnancy, and fetuses were delivered by caesarean section with or without 7.5 min of in-utero asphyxia. Surviving pups were raised by a cross-foster dam until 33±2 days of age when they were euthanized to obtain the diaphragm muscle for ex-vivo study of twitch tension and muscle fatigue, and for structural and enzymatic analyses. Functional analysis of the diaphragm revealed no differences in single twitch contractile parameters between any groups. However, muscle fatigue, induced by stimulation of diaphragm strips with a train of pulses (330 ms train/sec, 40 Hz) for 300 sec, was significantly greater for asphyxia pups compared with controls (p<0.05), and this did not occur in diaphragms of creatine + asphyxia pups. Birth asphyxia resulted in a significant increase in the proportion of glycolytic, fast-twitch fibres and a reduction in oxidative capacity of Type I and IIb fibres in male offspring, as well as reduced cross-sectional area of all muscle fibre types (Type I, IIa, IIb/d) in both males and females at 33 days of age. None of these changes were observed in creatine + asphyxia animals. Thus, the changes in diaphragm fatigue and structure induced by birth asphyxia persist long-term but are prevented by maternal creatine supplementation.

Ascorbic acid supplementation improves skeletal muscle oxidative stress and insulin sensitivity in people with type 2 diabetes: Findings of a randomized controlled study.

AIM/HYPOTHESIS: Skeletal muscle insulin resistance and oxidative stress are characteristic metabolic disturbances in people with type 2 diabetes. Studies in insulin resistant rodents show an improvement in skeletal muscle insulin sensitivity and oxidative stress following antioxidant supplementation. We therefore investigated the potential ameliorative effects of antioxidant ascorbic acid (AA) supplementation on skeletal muscle insulin sensitivity and oxidative stress in people with type 2 diabetes. METHODS: Participants with stable glucose control commenced a randomized cross-over study involving four months of AA (2 × 500 mg/day) or placebo supplementation. Insulin sensitivity was assessed using a hyperinsulinaemic, euglycaemic clamp coupled with infusion of 6,6-D2 glucose. Muscle biopsies were measured for AA concentration and oxidative stress markers that included basal measures (2',7'-dichlorofluorescin [DCFH] oxidation, ratio of reduced-to-oxidized glutathione [GSH/GSSG] and F2-Isoprostanes) and insulin-stimulated measures (DCFH oxidation). Antioxidant concentrations, citrate synthase activity and protein abundances of sodium-dependent vitamin C transporter 2 (SVCT2), total Akt and phosphorylated Akt (ser473) were also measured in muscle samples. RESULTS: AA supplementation significantly increased insulin-mediated glucose disposal (delta rate of glucose disappearance; ∆Rd) (p=0.009), peripheral insulin-sensitivity index (p=0.046), skeletal muscle AA concentration (p=0.017) and muscle SVCT2 protein expression (p=0.008); but significantly decreased skeletal muscle DCFH oxidation during hyperinsulinaemia (p=0.007) when compared with placebo. Total superoxide dismutase activity was also lower following AA supplementation when compared with placebo (p=0.006). Basal oxidative stress markers, citrate synthase activity, endogenous glucose production, HbA1C and muscle Akt expression were not significantly altered by AA supplementation. CONCLUSIONS/INTERPRETATION: In summary, oral AA supplementation ameliorates skeletal muscle oxidative stress during hyperinsulinaemia and improves insulin-mediated glucose disposal in people with type 2 diabetes. Findings implicate AA supplementation as a potentially inexpensive, convenient, and effective adjunct therapy in the treatment of insulin resistance in people with type 2 diabetes.

Dietary creatine supplementation during pregnancy: a study on the effects of creatine supplementation on creatine homeostasis and renal excretory function in spiny mice.

Recent evidence obtained from a rodent model of birth asphyxia shows that supplementation of the maternal diet with creatine during pregnancy protects the neonate from multi-organ damage. However, the effect of increasing creatine intake on creatine homeostasis and biosynthesis in females, particularly during pregnancy, is unknown. This study assessed the impact of creatine supplementation on creatine homeostasis, body composition, capacity for de novo creatine synthesis and renal excretory function in non-pregnant and pregnant spiny mice. Mid-gestation pregnant and virgin spiny mice were fed normal chow or chow supplemented with 5 % w/w creatine for 18 days. Weight gain, urinary creatine and electrolyte excretion were assessed during supplementation. At post mortem, body composition was assessed by Dual-energy X-ray absorptiometry, or tissues were collected to assess creatine content and mRNA expression of the creatine synthesising enzymes arginine:glycine amidinotransferase (AGAT) and guanidinoacetate methyltransferase (GAMT) and the creatine transporter (CrT1). Protein expression of AGAT and GAMT was also assessed by Western blot. Key findings of this study include no changes in body weight or composition with creatine supplementation; increased urinary creatine excretion in supplemented spiny mice, with increased sodium (P < 0.001) and chloride (P < 0.05) excretion in pregnant dams after 3 days of supplementation; lowered renal AGAT mRNA (P < 0.001) and protein (P < 0.001) expressions, and lowered CrT1 mRNA expression in the kidney (P < 0.01) and brain (P < 0.001). Creatine supplementation had minimal impact on creatine homeostasis in either non-pregnant or pregnant spiny mice. Increasing maternal dietary creatine consumption could be a useful treatment for birth asphyxia.

Maternal creatine supplementation from mid-pregnancy protects the diaphragm of the newborn spiny mouse from intrapartum hypoxia-induced damage.

We hypothesized that maternal creatine supplementation from mid-pregnancy would protect the diaphragm of the newborn spiny mouse from the effects of intrapartum hypoxia. Pregnant mice were fed a control or 5% creatine-supplemented diet from mid-gestation. On the day before term, intrapartum hypoxia was induced by isolating the pregnant uterus in a saline bath for 7.5-8 min before releasing and resuscitating the fetuses. Surviving pups were placed with a cross-foster dam, and diaphragm tissue was collected at 24 h postnatal age. Hypoxia caused a significant decrease in the cross-sectional area (∼19%) and contractile function (26.6% decrease in maximum Ca2=-activated force) of diaphragm fibers. The mRNA levels of the muscle mass-regulating genes MuRF1 and myostatin were significantly increased (2-fold). Maternal creatine significantly attenuated hypoxia-induced fiber atrophy, contractile dysfunction, and changes in mRNA levels. This study demonstrates that creatine loading before birth significantly protects the diaphragm from hypoxia-induced damage at birth.