The deacetylase Sirt1 is an essential regulator of Aire-mediated induction of central immunological tolerance.

Aire is a transcriptional regulator that induces the promiscuous expression of thousands of tissue-restricted antigens (TRAs) in medullary thymic epithelial cells (mTECs), a step critical for the induction of immunological self-tolerance. Studies have offered molecular insights into how Aire operates, but more comprehensive understanding of this process still remains elusive. Here we found abundant expression of the protein deacetylase Sirtuin-1 (Sirt1) in mature Aire(+) mTECs, wherein it was required for the expression of Aire-dependent TRA-encoding genes and the subsequent induction of immunological self-tolerance. Our study elucidates a previously unknown molecular mechanism for Aire-mediated transcriptional regulation and identifies a unique function for Sirt1 in preventing organ-specific autoimmunity.

Adipose tissue-derived FABP4 mediates glucagon-stimulated hepatic glucose production in gestational diabetes.

AIMS: One of the most common complications of pregnancy is gestational diabetes mellitus (GDM), which may result in significant health threats of the mother, fetus and the newborn. Fatty acid-binding protein 4 (FABP4) is an adipokine that regulates glucose homeostasis by promoting glucose production and liver insulin resistance in mouse models. FABP4 levels are increased in GDM and correlates with maternal indices of insulin resistance, with a rapid decline post-partum. We therefore aimed to determine the tissue origin of elevated circulating FABP4 levels in GDM and to assess its potential contribution in promoting glucagon-induced hepatic glucose production. MATERIALS AND METHODS: FABP4 protein and gene expression was determined in biopsies from placenta, subcutaneous (sWAT) and visceral (vWAT) white adipose tissues from GDM and normoglycaemic pregnant women. FABP4 differential contribution in glucagon-stimulated hepatic glucose production was tested in conditioned media before and after its immune clearance. RESULTS: We showed that FABP4 is expressed in placenta, sWAT and vWAT of pregnant women at term, with a significant increase in its secretion from vWAT of women with GDM compared with normoglycaemic pregnant women. Neutralizing FABP4 from both normoglycaemic pregnant women and GDM vWAT secretome, resulted in a decrease in glucagon-stimulated hepatic glucose production. CONCLUSIONS: This study provides new insights into the role of adipose tissue-derived FABP4 in GDM, highlighting this adipokine, as a potential co-activator of glucagon-stimulated hepatic glucose production during pregnancy.

Fatty acid-binding protein 4: a key regulator of ketoacidosis in new-onset type 1 diabetes.

AIMS/HYPOTHESIS: Fatty acid-binding protein 4 (FABP4) is an adipokine with a key regulatory role in glucose and lipid metabolism. We prospectively evaluated the role of FABP4 in the pathophysiology of diabetic ketoacidosis (DKA) in new-onset type 1 diabetes. METHODS: Clinical and laboratory data were prospectively collected from consecutive children presenting with new-onset type 1 diabetes. In addition to blood chemistry and gases, insulin, C-peptide, serum FABP4 and NEFA were collected upon presentation and 48 h after initiation of insulin treatment. In a mouse model of type 1 diabetes, glucose, insulin, ß-hydroxybutyrate and weight were compared between FABP4 knockout (Fabp4-/-) and wild-type (WT) mice. RESULTS: Included were 33 children (mean age 9.3 ± 3.5 years, 52% male), of whom 14 (42%) presented with DKA. FABP4 levels were higher in the DKA group compared with the non-DKA group (median [IQR] 10.1 [7.9-14.2] ng/ml vs 6.3 [3.9-7] ng/ml, respectively; p = 0.005). The FABP4 level was positively correlated with HbA1c at presentation and inversely correlated with venous blood pH and bicarbonate levels (p < 0.05 for all). Following initiation of insulin therapy, a marked reduction in FABP4 was observed in all children. An FABP4 level of 7.22 ng/ml had a sensitivity of 86% and a specificity of 78% for the diagnosis of DKA, with an area under the receiver operating characteristic curve of 0.78 (95% CI 0.6, 0.95; p = 0.008). In a streptozotocin-induced diabetes mouse model, Fabp4-/- mice exhibited marked hypoinsulinaemia and hyperglycaemia similar to WT mice but displayed no significant increase in ß-hydroxybutyrate and were protected from ketoacidosis. CONCLUSIONS/INTERPRETATION: FABP4 is suggested to be a necessary regulator of ketogenesis in insulin-deficient states.

Regulation of the proapoptotic factor Bax by Ku70-dependent deubiquitylation.

The DNA end-joining protein Ku70 is one of several proteins that inhibit apoptosis by sequestering the proapoptotic factor Bax from the mitochondria. However, the molecular mechanism underlying Ku70-dependent inhibition of Bax is not fully understood. Here, we show that the absence of Ku70 results in the accumulation of ubiquitylated Bax. Under normal growth conditions, Bax ubiquitylation promotes its degradation. Upon induction of apoptosis in wild-type cells, a significant reduction in the levels of ubiquitylated Bax was observed, whereas in Ku70(-/-) cells, the ubiquitylated Bax was robustly accumulated. Addition of recombinant Ku70 into a protein extract of Ku70(-/-) cells resulted in a decrease in the levels of ubiquitylated Bax, even in the presence of proteasome inhibitors. Moreover, an in vitro deubiquitylation assay demonstrated that recombinant Ku70 hydrolyzed polyubiquitin chains into monoubiquitin units. Thus, Ku70 regulates apoptosis by sequestering Bax from the mitochondria and mediating Bax deubiquitylation. These results shed light on the role of proteasome inhibitors as tumor suppressors.

Intercellular Transmission of Hepatic ER Stress in Obesity Disrupts Systemic Metabolism.

Endoplasmic reticulum stress (ERS) has a pathophysiological role in obesity-associated insulin resistance. Yet, the coordinated tissue response to ERS remains unclear. Increased connexin 43 (Cx43)-mediated intercellular communication has been implicated in tissue-adaptive and -maladaptive response to various chronic stresses. Here, we demonstrate that in hepatocytes, ERS results in increased Cx43 expression and cell-cell coupling. Co-culture of ER-stressed "donor" cells resulted in intercellular transmission of ERS and dysfunction to ERS-naive "recipient" cells ("bystander response"), which could be prevented by genetic or pharmacologic suppression of Cx43. Hepatocytes from obese mice were able to transmit ERS to hepatocytes from lean mice, and mice lacking liver Cx43 were protected from diet-induced ERS, insulin resistance, and hepatosteatosis. Taken together, our results indicate that in obesity, the increased Cx43-mediated cell-cell coupling allows intercellular propagation of ERS. This novel maladaptive response to over-nutrition exacerbates the tissue ERS burden, promoting hepatosteatosis and impairing whole-body glucose metabolism.

The adipokine FABP4 is a key regulator of neonatal glucose homeostasis.

During pregnancy, fetal glucose production is suppressed, with rapid activation immediately postpartum. Fatty acid-binding protein 4 (FABP4) was recently demonstrated as a regulator of hepatic glucose production and systemic metabolism in animal models. Here, we studied the role of FABP4 in regulating neonatal glucose hemostasis. Serum samples were collected from pregnant women with normoglycemia or gestational diabetes at term, from the umbilical circulation, and from the newborns within 6 hours of life. The level of FABP4 was higher in the fetal versus maternal circulation, with a further rise in neonates after birth of approximately 3-fold. Neonatal FABP4 inversely correlated with blood glucose, with an approximately 10-fold increase of FABP4 in hypoglycemic neonates. When studied in mice, blood glucose of 12-hour-old WT, Fabp4-/+, and Fabp4-/- littermate mice was 59 ± 13 mg/dL, 50 ± 11 mg/dL, and 43 ± 11 mg/dL, respectively. Similar to our observations in humans, FABP4 levels in WT mouse neonates were approximately 8-fold higher compared with those in adult mice. RNA sequencing of the neonatal liver suggested altered expression of multiple glucagon-regulated pathways in Fabp4-/- mice. Indeed, Fabp4-/- liver glycogen was inappropriately intact, despite a marked hypoglycemia, with rapid restoration of normoglycemia upon injection of recombinant FABP4. Our data suggest an important biological role for the adipokine FABP4 in the orchestrated regulation of postnatal glucose metabolism.

The short-chain fatty acid propionate increases glucagon and FABP4 production, impairing insulin action in mice and humans.

The short-chain fatty acid propionate is a potent inhibitor of molds that is widely used as a food preservative and endogenously produced by gut microbiota. Although generally recognized as safe by the U.S. Food and Drug Administration, the metabolic effects of propionate consumption in humans are unclear. Here, we report that propionate stimulates glycogenolysis and hyperglycemia in mice by increasing plasma concentrations of glucagon and fatty acid-binding protein 4 (FABP4). Fabp4-deficient mice and mice lacking liver glucagon receptor were protected from the effects of propionate. Although propionate did not directly promote glucagon or FABP4 secretion in ex vivo rodent pancreatic islets and adipose tissue models, respectively, it activated the sympathetic nervous system in mice, leading to secretion of these hormones in vivo. This effect could be blocked by the pharmacological inhibition of norepinephrine, which prevented propionate-induced hyperglycemia in mice. In a randomized, double-blind, placebo-controlled study in humans, consumption of a propionate-containing mixed meal resulted in a postprandial increase in plasma glucagon, FABP4, and norepinephrine, leading to insulin resistance and compensatory hyperinsulinemia. Chronic exposure of mice to a propionate dose equivalent to that used for food preservation resulted in gradual weight gain. In humans, plasma propionate decreased with weight loss in the Dietary Intervention Randomized Controlled Trial (DIRECT) and served as an independent predictor of improved insulin sensitivity. Thus, propionate may activate a catecholamine-mediated increase in insulin counter-regulatory signals, leading to insulin resistance and hyperinsulinemia, which, over time, may promote adiposity and metabolic abnormalities. Further evaluation of the metabolic consequences of propionate consumption is warranted.

Regulation of SIRT1 protein levels by nutrient availability.

The mammalian NAD+ dependent deacetylase, SIRT1, was shown to be a key protein in regulating glucose homeostasis, and was implicated in the response to calorie restriction. We show here that levels of SIRT1 increased in response to nutrient deprivation in cultured cells, and in multiple tissues of mice after fasting. The increase in SIRT1 levels was due to stabilization of SIRT1 protein, and not an increase in SIRT1 mRNA. In addition, p53 negatively regulated SIRT1 levels under normal growth conditions and is also required for the elevation of SIRT1 under limited nutrient conditions. These results have important implications on the relationship between sirtuins, nutrient availability and aging.

Characterization of physiological defects in adult SIRT6-/- mice.

The NAD+-dependent SIRT6 deacetylase was shown to be a major regulator of lifespan and healthspan. Mice deficient for SIRT6 develop a premature aging phenotype and metabolic defects, and die before four weeks of age. Thus, the effect of SIRT6 deficiency in adult mice is unknown. Here we show that SIRT6-/- mice in mixed 129/SvJ/BALB/c background reach adulthood, allowing examination of SIRT6-related metabolic and developmental phenotypes in adult mice. In this mixed background, at 200 days of age, more than 80% of the female knock-out mice were alive whereas only 10% of male knock-out mice survived. In comparison to their wild-type littermates, SIRT6 deficient mice have reduced body weight, increased glucose uptake and exhibit an age-dependent progressive impairment of retinal function accompanied by thinning of retinal layers. Together, these results demonstrate a role for SIRT6 in metabolism and age-related ocular changes in adult mice and suggest a gender specific regulation of lifespan by SIRT6.

DeubiKuitylation: a novel DUB enzymatic activity for the DNA repair protein, Ku70.

The Ku70 protein was shown to be involved in multiple cellular pathways including DNA repair, telomere maintenance, V(D)J recombination and Bax mediated apoptosis. Yet, despite this wide spectrum of pathways, until recently the enzymatic activity of Ku70 was elusive. Recent findings demonstrate that Ku70 is associated with the proapoptotic protein Bax and possesses a deubiquitin enzyme (DUB) activity on it. These data suggest a dual role for Ku70 in apoptotic regulation; on one hand the association with Ku70 sequestered Bax away from the mitochondria and plays an antiapoptotic function. On the other hand, this association mediates and promotes Bax deubiquitylation which will block its labeling for proteasomal degradation and in this manner Ku70 will have a proapoptotic role. The exciting finding of Ku70's DUB activity opens numerous avenues for future research. Here we suggest candidate substrate proteins and indicate how the DUB activity of Ku70 on these, might affect the known Ku70's related pathways.