It has been assumed, based largely on morphologic evidence, that human pluripotent stem cells (hPSCs) contain underdeveloped, bioenergetically inactive mitochondria. In contrast, differentiated cells ...harbour a branched mitochondrial network with oxidative phosphorylation as the main energy source. A role for mitochondria in hPSC bioenergetics and in cell differentiation therefore remains uncertain. Here, we show that hPSCs have functional respiratory complexes that are able to consume O2 at maximal capacity. Despite this, ATP generation in hPSCs is mainly by glycolysis and ATP is consumed by the F1F0 ATP synthase to partially maintain hPSC mitochondrial membrane potential and cell viability. Uncoupling protein 2 (UCP2) plays a regulating role in hPSC energy metabolism by preventing mitochondrial glucose oxidation and facilitating glycolysis via a substrate shunting mechanism. With early differentiation, hPSC proliferation slows, energy metabolism decreases, and UCP2 is repressed, resulting in decreased glycolysis and maintained or increased mitochondrial glucose oxidation. Ectopic UCP2 expression perturbs this metabolic transition and impairs hPSC differentiation. Overall, hPSCs contain active mitochondria and require UCP2 repression for full differentiation potential.
While studying metabolic fluxes in human pluripotent stem cells, this paper reveals UCP2 as metabolic switch from glycolysis to OXPHOS, facilitating early differentiation events.
We previously showed that testosterone (T) deficiency enhanced high-fat/low-carbohydrate diet (HFD)-induced hepatic steatosis in rats independent of insulin resistance and that T replacement reduced ...hepatic macrovesicular fat accumulation and inflammation. The present report explores the mechanism of T's protective effects on HFD-induced steatohepatitis. Adult male rats were randomized into four treatment groups for 15 wk: intact rats on regular chow diet or HFD, and castrated rats on HFD with or without T replacement. Fatty acid β-oxidation and de novo synthesis were not changed by castration and T replacement, but expression of lipid export proteins ApoB100 and microsomal triglyceride transfer protein (MTP) was suppressed by HFD in both intact and castrated rats but restored by T replacement. Macrovesicular lipid droplet-related proteins perilipin 1 and fat-specific protein 27 were increased by HFD in castrated rats and suppressed by T replacement. Higher activation/expression of ER stress proteins (PERK, IRE-1α, JNK, NF-κB, and CHOP) was demonstrated in castrated rats fed HFD compared with intact animals, and T replacement suppressed these changes. We conclude that 1) HFD leads to ApoB100/MTP suppression reducing export of lipids; 2) castration promotes progression to steatohepatitis through activation of the ER stress pathway and enhancement of macrovesicular droplet protein expression; and 3) testosterone suppresses ER stress, inhibits the formation of macrovesicular lipid droplets, promotes lipid export, and ameliorates steatohepatitis induced by HFD and castration.
Intrauterine growth restriction (IUGR) with rapid catch-up growth leads to adult obesity and insulin resistance. We have previously shown that IUGR male rats demonstrated increased
de novo
fatty acid ...synthesis in the subcutaneous (SC) fat, but not the visceral fat, during the nursing period prior to the onset of obesity. Young IUGR females do not exhibit the same increase. We further hypothesized that in male IUGR offspring,
de novo
synthesis is a programmed intrinsic effect that persists to adulthood and does not suppress in response to a high fat diet. We measured fatty acid
de novo
synthesis in IUGR adult males (6 months) using deuterium-enriched drinking water as a stable isotope tracer, then further studied the response after consumption of an isocaloric high fat diet. Baseline de novo synthesis in adult females was also studied at age 9 months. Males demonstrated increased baseline
de novo
synthesis in both SC fat and visceral fat. Correspondingly, SC and visceral fat protein expression of lipogenic enzymes acetyl-coA carboxylase-α (ACCα) and fatty acid synthase were upregulated. After the isocaloric high fat diet,
de novo
synthesis was suppressed such that no differences remained between the two groups, although, IUGR SC fat demonstrated persistently increased lipogenic protein expression. In contrast,
de novo
synthesis among adult females is not impacted in IUGR. In conclusion, enhancement of male IUGR SC fat
de novo
synthesis appears to be an early consequence of metabolic programming, whereas enhancement in visceral fat appears to be a later consequence.
Small for gestational age (SGA) leads to increased risk of adult obesity and metabolic syndrome. Offspring exposed to 50% maternal food restriction in utero are born smaller than Controls (FR), ...catch-up in growth by the end of the nursing period, and become obese adults. The objective of the study was to determine stearoyl-CoA desaturase activity (SCD1) and rates of de novo fatty acid synthesis in young FR and Control offspring tissues at the end of the nursing period, as possible contributors to catch-up growth.
From gestational day 10 to term, dams fed ad libitum (Control) or were 50% food-restricted to produce small FR pups. Control dams nursed all pups. At postnatal day 1 (p1) and p21, offspring body tissues were analyzed by GC/MS, and desaturation indices of palmitoleate/palmitate and oleate/stearate were calculated. SCD1 gene expression was determined by real-time PCR on adipose and liver. Offspring were enriched with deuterium that was given to dams in drinking water during lactation and de novo synthesis of offspring body tissues was determined at p21. Primary adipocyte cell cultures were established at p21 and exposed to U(13)C-glucose.
FR offspring exhibited higher desaturation index in p1 and p21 adipose tissue, but decreased desaturation index in liver at p21. SCD1 gene expression at p21 was correspondingly increased in adipose and decreased in liver. FR subcutaneous fat demonstrated increased de novo synthesis at p21. Primary cell cultures exhibited increased de novo synthesis in FR.
Adipose tissue is the first site to exhibit increased de novo synthesis and desaturase activity in FR. Therefore, abnormal lipogenesis is already present prior to onset of obesity during the period of catch-up growth. These abnormalities may contribute to future obesity development.
Cyclin-dependent kinases CDK4 and CDK6 are essential for the control of the cell cycle through the G
1
phase. Aberrant expression of CDK4 and CDK6 is a hallmark of cancer, which would suggest that ...CDK4 and CDK6 are attractive targets for cancer therapy. Herein, we report that calcein AM (the calcein acetoxymethyl-ester) is a potent specific inhibitor of CDK4 and CDK6 in HCT116 human colon adenocarcinoma cells, inhibiting retinoblastoma protein (pRb) phosphorylation and inducing cell cycle arrest in the G
1
phase. The metabolic effects of calcein AM on HCT116 cells were also evaluated and the flux between the oxidative and non-oxidative branches of the pentose phosphate pathway was significantly altered. To elucidate whether these metabolic changes were due to the inhibition of CDK4 and CDK6, we also characterized the metabolic profile of a CDK4, CDK6 and CDK2 triple knockout of mouse embryonic fibroblasts. The results show that the metabolic profile associated with the depletion of CDK4, CDK6 and CDK2 coincides with the metabolic changes induced by calcein AM on HCT116 cells, thus confirming that the inhibition of CDK4 and CDK6 disrupts the balance between the oxidative and non-oxidative branches of the pentose phosphate pathway. Taken together, these results indicate that low doses of calcein can halt cell division and kill tumor cells. Thus, selective inhibition of CDK4 and CDK6 may be of greater pharmacological interest, since inhibitors of these kinases affect both cell cycle progression and the robust metabolic profile of tumors.
Understanding nutrient-gene interaction requires tools for both the study of nutrigenomics and the characterization of phenotype. Metabolomics or metabolite profiling is a powerful tool for ...characterizing metabolic phenotype, and tracer-based metabolomics is a subset of metabolomics that focuses on metabolite distribution and flux determination using tracers. In this review, the characterizations of metabolic phenotype by metabolite profiling and by metabolic flux measurements are compared. The rationale and methodologies of tracer-based metabolomics are explained. Tracer-based metabolomics provides a relational database of metabolites linked by the relationship of shared metabolic pathways, common substrates, and cofactors. Such a collection of flux measurements provides precise and accurate information on the operation of the cellular metabolic network and its response to genetic and nutrient environment changes. Nutrient-gene interaction can be studied using the concept of constraint-based modeling, which states that the observed metabolic phenotype is a consequence of constraints from genetic factors and the nutrient environment. Thus, genetic inheritance (genomic constraints) confers a wide range of possible phenotypes whereas selection by metabolic (structural and pathway relationship) and environmental (physical environment and nutrient availability) constraints determines the final observed phenotype. The study of the contribution from nutrient and genetic factors to the survival advantage of cancer cells using flux measurements is a critical first step in our understanding of the relationship between nutrient intake and cancer risk.
Stearoyl-CoA desaturase enzyme 1 (SCD1) is a lipogenic enzyme that is upregulated in obesity, insulin resistance, and cancer. Since glucose is a substrate for both de novo fatty acid synthesis and ...deoxyribose synthesis, we hypothesized that SCD1 affects these multiple synthetic pathways through changes in glucose utilization. This study determined glucose utilization for fatty acid synthesis and cell proliferation in 3T3-L1 preadipocytes during SCD1 inhibition. The effects of SCD1 on cellular metabolism as mediated by its monounstaurated fatty acid products (palmitoleate and oleate) were also observed. 3T3-L1 preadipocytes underwent differentiation induction in conjunction with one of the following treatments for 4 days: (A) no treatment, (B) SCD1 inhibitor CGX0290, (C) CGX0290 + palmitoleate, or (D) CGX0290 + oleate. All cells received medium with 50 % U
13
C-glucose. Cells were harvested on day 7 for studies of fatty acid metabolism, tricarboxylic acid (TCA) cycle activities, and deoxyribose synthesis. CGX0290 decreased fatty acid desaturation, glucose utilization for fatty acid synthesis (acetyl-CoA enrichment), and de novo synthesis. CGX0290 treatment also led to decreased cell density through increased cell death. Further analysis showed that deoxyribose new synthesis and oxidative pentose phosphate pathway activity were unchanged, while non-oxidative transketolase pathway activity was stimulated. Palmitoleate and oleate supplementation each partially ameliorated the effects of CGX0290. In 3T3-L1 cells, SCD1 promotes glucose utilization for fatty acid synthesis. In cell proliferation, SCD1 may promote cell survival, but does not impact the oxidative pathway of deoxyribose production. These effects may be mediated through the production of palmitoleate and oleate.
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