Mitochondrial uncoupling protein is required for efficient photosynthesis

Uncoupling proteins (UCPs) occur in the inner mitochondrial membrane and dissipate the proton gradient across this membrane that is normally used for ATP synthesis. Although the catalytic function and regulation of plant UCPs have been described, the physiological purpose of UCP in plants has not been established. Here, biochemical and physiological analyses of an insertional knockout of one of the Arabidopsis UCP genes (AtUCP1) are presented that resolve this issue. Absence of UCP1 results in localized oxidative stress but does not impair the ability of the plant to withstand a wide range of abiotic stresses. However, absence of UCP1 results in a photosynthetic phenotype. Specifically there is a restriction in photorespiration with a decrease in the rate of oxidation of photorespiratory glycine in the mitochondrion. This change leads to an associated reduced photosynthetic carbon assimilation rate. Collectively, these results suggest that the main physiological role of UCP1 in Arabidopsis leaves is related to maintaining the redox poise of the mitochondrial electron transport chain to facilitate photosynthetic metabolism.     

The -3826 A-->G variant of the uncoupling protein-1 gene diminishes postprandial thermogenesis after a high fat meal in healthy boys

This study investigated whether the -3826 A-->G nucleotide variant of the uncoupling protein-1 (UCP1) gene is correlated with postprandial thermogenesis after a high fat meal in children. Healthy boys, aged 8-11 yr, were examined for resting energy expenditure and the thermic effect of a meal (TEM), which were measured by indirect calorimetry for 180 min after a high fat (70% fat, 20% carbohydrate, and 10% protein, providing 30% of the daily energy requirement) and a high carbohydrate meal (20% fat, 70% carbohydrate, and 10% protein). The sympatho-vagal activities were assessed by means of spectral analysis of the heart rate variability during the same period. Children were genotyped for UCP1 polymorphism by applying a PCR-restriction fragment length polymorphism using buccal samples. There was no reaction of sympathetic activity to the high carbohydrate meal in either the GG allele or the AA+AG group and no significant difference in TEM. However, after the high fat meal, sympathetic responses were found in both groups; further, the GG allele group showed significantly lower TEM than the AA+AG group. In conclusion, despite fat-induced sympathetic stimulation, GG allele carriers have a lowered capacity of TEM in response to fat intake, suggesting that such impaired UCP1-linked thermogenesis can have adverse effects on the regulation of body weight.     

Kir6.2 is required for adaptation to stress

Reaction to stress requires feedback adaptation of cellular functions to secure a response without distress, but the molecular order of this process is only partially understood. Here, we report a previously unrecognized regulatory element in the general adaptation syndrome. Kir6.2, the ion-conducting subunit of the metabolically responsive ATP-sensitive potassium (K(ATP)) channel, was mandatory for optimal adaptation capacity under stress. Genetic deletion of Kir6.2 disrupted K(ATP) channel-dependent adjustment of membrane excitability and calcium handling, compromising the enhancement of cardiac performance driven by sympathetic stimulation, a key mediator of the adaptation response. In the absence of Kir6.2, vigorous sympathetic challenge caused arrhythmia and sudden death, preventable by calcium-channel blockade. Thus, this vital function identifies a physiological role for K(ATP) channels in the heart.     

Leptin is required for fibrogenic responses induced by thioacetamide in the murine liver

In this study, we investigated hepatic fibrogenesis caused by long-term thioacetamide (TAA) administration in ob/ob mice, a naturally occurring leptin deficient animal. In the lean littermates, prominent hepatic fibrosis, as well as positive staining for alpha smooth muscle actin (alpha-SMA), was induced by treatment with TAA (200 microg/g, IP, 3 times per week) for 4 to 8 weeks as expected. In sharp contrast, almost no hepatic fibrosis developed in ob/ob mice given the equivalent doses of TAA, where specific staining for alpha-SMA barely was detected. Induction of alpha1(I) procollagen mRNA caused by TAA also was prevented in ob/ob mice almost completely. Further, transforming growth factor beta (TGF-beta) mRNA was increased in the liver after TAA treatment for 4 weeks in lean littermates, which also was prevented in ob/ob mice. Interestingly, fibrotic septa in the hepatic lobules, as well as increases in alpha1(I) procollagen mRNA, was observed in ob/ob mice, when they were injected with recombinant murine leptin (1 microg/g daily) in combination with TAA treatment. Leptin per se did not cause any fibrotic changes in the liver in ob/ob mice. These findings clearly indicated that leptin deficiency is responsible for the resistance to TAA-induced profibrogenic responses in ob/ob mice. In conclusion, leptin appears to promote profibrogenic responses in the liver, in part, by up-regulation of TGF-beta.     

APE2 is required for ATR-Chk1 checkpoint activation in response to oxidative stress

The base excision repair pathway is largely responsible for the repair of oxidative stress-induced DNA damage. However, it remains unclear how the DNA damage checkpoint is activated by oxidative stress at the molecular level. Here, we provide evidence showing that hydrogen peroxide (H₂O₂) triggers checkpoint kinase 1 (Chk1) phosphorylation in an ATR [ataxia-telangiectasia mutated (ATM) and Rad3-related]-dependent but ATM-independent manner in Xenopus egg extracts. A base excision repair protein, Apurinic/apyrimidinic (AP) endonuclease 2 (APE2, APN2, or APEX2), is required for the generation of replication protein A (RPA)-bound single-stranded DNA, the recruitment of a checkpoint protein complex [ATR, ATR-interacting protein (ATRIP), and Rad9] to damage sites, and H₂O₂-induced Chk1 phosphorylation. A conserved proliferating cell nuclear antigen interaction protein box of APE2 is important for the recruitment of APE2 to H₂O₂-damaged chromatin. APE2 3′-phosphodiesterase and 3′-5′ exonuclease activity is essential for single-stranded DNA generation in the 3′–5′ direction from single-stranded breaks, referred to as single-stranded break end resection. In addition, APE2 associates with Chk1, and a serine residue (S86) in the Chk1-binding motif of APE2 is essential for Chk1 phosphorylation, indicating a Claspin-like but distinct role for APE2 in ATR-Chk1 signaling. Our data indicate that APE2 plays a vital and previously unexpected role in ATR-Chk1 checkpoint signaling in response to oxidative stress. Thus, our findings shed light on a distinct mechanism of how an ATR-Chk1–dependent DNA damage checkpoint is mediated by APE2 in the oxidative stress response.Cells are constantly challenged by exogenous and endogenous insults that threaten genomic integrity. Excess accumulation of reactive oxygen species leads to oxidative DNA damage, such as DNA strand breaks with 3′-modified termini, which is often the underlying pathology in a variety of diseases including neurodegenerative diseases and cancer (1–6). Cellular responses to DNA damage are mainly coordinated by two distinct DNA damage checkpoint signaling cascades: ATM (ataxia-telangiectasia mutated)-checkpoint kinase 2 (Chk2) and ATR (ATM and Rad3-related)-checkpoint kinase 1 (Chk1) pathways (7–10). ATM is activated by intermolecular autophosphorylation and dimer dissociation in response to double-stranded beaks (DSBs) (11–13). ATR is activated by primed single-stranded DNA (ssDNA) in response to a variety of DNA damage or replication stresses (14, 15). Oxidative stress has been demonstrated to activate an ATM-dependent DNA damage checkpoint (16–18). However, in previous studies, hyperoxic conditions resulted in the phosphorylation of Chk1 and p53 in an ATR-dependent but ATM-independent fashion (19). Furthermore, it remains unclear which specific DNA structures trigger checkpoint signaling during oxidative stress.To eliminate oxidative DNA damage, base excision repair (BER) has evolved as a major DNA damage repair mechanism (20). In the initial step of BER, oxidatively damaged bases are excised by DNA glycosylases, generating apurinic/apyrimidinic (AP) sites. An incision at the AP site by AP endonuclease or AP lyase generates a single-stranded break (SSB) (21). Subsequently, the SSB is fixed via collaboration between proliferating cell nuclear antigen (PCNA), DNA polymerase β, replication factor C, flap endonuclease 1 (FEN1), and DNA ligase I (22). APE1 (AP endonuclease 1) and APE2 (AP endonuclease 2) are the two characterized AP endonucleases (23, 24). APE2, which has weak AP endonuclease activity and strong 3′-phosphodiesterase and 3′-5′ exonuclease activities, is a key player in PCNA-dependent repair of hydrogen peroxide (H₂O₂)-induced oxidative DNA damage (25–27). However, the biological significance of APE2 in the DNA damage response has not been elucidated. As far as we know, it remains elusive whether the DNA damage checkpoint and BER pathways are coordinated in cellular responses to oxidative stress.We performed a series of studies in checkpoint signaling using Xenopus egg extracts, a well-characterized cell-free model system (28–32). Here, we provide evidence that suggests APE2 is required for ATR-Chk1 checkpoint activation in response to oxidative stress. Molecular characterization of the underlying mechanisms of APE2 has shed light on two distinct roles for APE2 in the ATR-Chk1 checkpoint: ssDNA generation via 3′-5′ SSB end resection and Claspin-like Chk1 binding. These roles of APE2 in the ATR-Chk1 checkpoint will help us better understand the DNA damage response after oxidative stress and provide a clear connection between the BER pathway and DNA damage checkpoint signaling.     

Colony stimulating factor 1 is required for mammary gland development during pregnancy.

The study of colony stimulating factor 1 (CSF-1), a homodimeric serum growth factor that regulates mononuclear phagocytes and is involved in maternal-fetal interactions during pregnancy, was dramatically enhanced by the observation that the recessive mutation osteopetrosis, op, is an inactivating mutation in the CSF-1-encoding gene. Homozygous mutant (op/op) mice completely lack CSF-1, are osteopetrotic consequent to a deficiency in osteoclasts, have severely reduced numbers of macrophages, and have reduced fertility evident at the pre- and postimplantation stages of pregnancy. We show here that op/op females have a lactational defect, and consequently, although some are able to produce offspring, few nurture any pups and none feeds a full litter. This lactational defect is due to incomplete mammary gland ductal growth during pregnancy, a precocious development of the lobulo-alveolar system, and despite expression of milk proteins, a failure to switch to a lactational state. These data show that CSF-1 has a role in the development of the mammary gland during pregnancy.     

Gtdap-1 promotes autophagy and is required for planarian remodeling during regeneration and starvation

Remodeling is an integral component of tissue homeostasis and regeneration. In planarians, these processes occur constantly in a simple tractable model organism as part of the animal's normal life history. Here, we have studied the gene Gtdap-1, the planarian ortholog of human death-associated protein-1 or DAP-1. DAP-1, together with DAP-kinase, has been identified as a positive mediator of programmed cell death induced by gamma-IFN in HeLa cells. Although the function of DAP-kinase is well characterized, the role of DAP-1 has not been studied in detail. Our findings suggest that Gtdap-1 is involved in autophagy in planarians, and that autophagy plays an essential role in the remodeling of the organism that occurs during regeneration and starvation, providing the necessary energy and building blocks to the neoblasts for cell proliferation and differentiation. The gene functions at the interface between survival and cell death during stress-inducing processes like regeneration and starvation in sexual and asexual races of planarians. Our findings provide insights into the complex interconnections among cell proliferation, homeostasis, and cell death in planarians and perspectives for the understanding of neoblast stem cell dynamics.     

Leptin receptor signaling is required for intact hypoglycemic counterregulation: A study in male Zucker rats

Hypoglycemia is a major barrier to clinical management of persons with diabetes. Emerging evidence supports a role for leptin in gating hypoglycemic counterregulation. This work demonstrates that male leptin receptor null, Zucker (fa/fa), rats display severe impairments in hypoglycemic counterregulation. Thus, augmenting leptin levels may have clinical utility for preventing hypoglycemia.     

WNK1 is required for mitosis and abscission

WNK [with no lysine (K)] protein kinases are found in all sequenced multicellular and many unicellular organisms. WNKs influence ion balance. Two WNK family members are associated with a single gene form of hypertension. RNA interference screens have implicated WNKs in survival and growth, and WNK1 is essential for viability of mice. We found that the majority of WNK1 is localized on cytoplasmic puncta in resting cells. During cell division, WNK1 localizes to mitotic spindles. Therefore, we analyzed mitotic phenotypes in WNK1 knockdown cells. A large percentage of WNK1 knockdown cells fail to complete cell division, displaying defects in mitotic spindles and also in abscission and cell survival. One of the best-characterized WNK1 targets is the protein kinase OSR1 (oxidative stress responsive 1). OSR1 regulates ion cotransporters, is activated in response to osmotic stress by WNK family members, and is largely associated with WNK1. In resting cells, the majority of OSR1, like WNK1, is on cytoplasmic puncta. OSR1 is also in nuclei. In contrast to WNK1, however, OSR1 does not concentrate around spindles during mitosis and does not show a WNK1-like localization pattern in mitotic cells. Knockdown of OSR1 has only a modest effect on cell survival and does not lead to spindle defects. We conclude that decreased cell survival associated with loss of WNK1 is attributable to defects in chromosome segregation and abscission and is independent of the effector kinase OSR1.     

Mrc1 phosphorylation in response to DNA replication stress is required for Mec1 accumulation at the stalled fork

DNA replication stress activates a response pathway that stabilizes stalled forks and promotes the completion of replication. The budding yeast Mec1 sensor kinase, Mrc1 mediator, and Rad53 effector kinase are central to this signal transduction cascade in S phase. We report that Mec1-dependent, Rad53-independent phosphorylation of Mrc1 is required to establish a positive feedback loop that stabilizes Mec1 and the replisome at stalled forks. A structure–function analysis of Mrc1 also uncovered a central region required for proper mediator function and association with replisome components. Together these results reveal new insight into how Mrc1 facilitates checkpoint signal amplification at stalled replication forks.     

Leptin response to endogenous acute stress is independent of pituitary function

There are close interactions between the adipocyte-derived hormone, leptin, and the anterior pituitary, especially the hypothalamic-pituitary-adrenal (HPA) axis. We investigated the relationship between the sympathetic adrenergic system and serum leptin levels, dependent on the function of anterior pituitary hormone axes, in 27 patients without a history of a hormone-secreting pituitary adenoma or other underlying endocrine disease. Based on responses in a routine insulin hypoglycemia test (ITT), the patients were classified as hypopituitary (HP; n=15), growth hormone deficient (GHD; n=6) or controls (CTR; 6 patients with normal responses). Nadir plasma glucose was 1.5+/-0.1 mmol/l at the time of maximum hypoglycemia. Each group had a significant increase in plasma epinephrine; however the magnitude of change was significantly higher in GHD (6.066+/-1.633 nmol/l) compared with HP patients (1.781+/-0.492 nmol/l) (P<0.01). The rise in norepinephrine was delayed (60 min) in the HP and CTR groups. However, in GHD patients there was a considerable increase at the time of hypoglycemia which was significantly different from HP (P<0.001) and CTR (P<0.05) patients. The increase in catecholamines was followed by a quick and significant decrease in serum leptin levels 45 min after an i.v. bolus injection of insulin in HP patients (-4.7+/-2.5%, P<0.05), which was significantly sustained after 60 min (-5.6+/-2.5%, P<0.05). In CTR patients there was a significant decrease in serum leptin levels 60 min after i.v. insulin (-14.4+/-6.9%, P<0.05), while no significant response was observed in the GHD group, although 5 of 6 patients had decreased levels at 45 and 60 min. No differences between the groups were found by ANOVA. In conclusion, an acute increase in endogenous circulating catecholamines is associated with a quick decrease in serum leptin levels. Intact anterior pituitary function seems not to be essential for this hitherto poorly understood mechanism.