Hepatic p63 regulates glucose metabolism by repressing SIRT1.

OBJECTIVE: p63 is a transcription factor within the p53 protein family that has key roles in development, differentiation and prevention of senescence, but its metabolic actions remain largely unknown. Herein, we investigated the physiological role of p63 in glucose metabolism. DESIGN: We used cell lines and mouse models to genetically manipulate p63 in hepatocytes. We also measured p63 in the liver of patients with obesity with or without type 2 diabetes (T2D). RESULTS: We show that hepatic p63 expression is reduced on fasting. Mice lacking the specific isoform TAp63 in the liver (p63LKO) display higher postprandial and pyruvate-induced glucose excursions. These mice have elevated SIRT1 levels, while SIRT1 knockdown in p63LKO mice normalises glycaemia. Overexpression of TAp63 in wild-type mice reduces postprandial, pyruvate-induced blood glucose and SIRT1 levels. Studies carried out in hepatocyte cell lines show that TAp63 regulates SIRT1 promoter by repressing its transcriptional activation. TAp63 also mediates the inhibitory effect of insulin on hepatic glucose production, as silencing TAp63 impairs insulin sensitivity. Finally, protein levels of TAp63 are reduced in obese persons with T2D and are negatively correlated with fasting glucose and homeostasis model assessment index. CONCLUSIONS: These results demonstrate that p63 physiologically regulates glucose homeostasis.

S100A9 exerts insulin-independent antidiabetic and anti-inflammatory effects.

Type 1 diabetes mellitus (T1DM) is characterized by insulin deficiency leading to hyperglycemia and several metabolic defects. Insulin therapy remains the cornerstone of T1DM management, yet it increases the risk of life-threatening hypoglycemia and the development of major comorbidities. Here, we report an insulin signaling-independent pathway able to improve glycemic control in T1DM rodents. Co-treatment with recombinant S100 calcium-binding protein A9 (S100A9) enabled increased adherence to glycemic targets with half as much insulin and without causing hypoglycemia. Mechanistically, we demonstrate that the hyperglycemia-suppressing action of S100A9 is due to a Toll-like receptor 4-dependent increase in glucose uptake in specific skeletal muscles (i.e., soleus and diaphragm). In addition, we found that T1DM mice have abnormal systemic inflammation, which is resolved by S100A9 therapy alone (or in combination with low insulin), hence uncovering a potent anti-inflammatory action of S100A9 in T1DM. In summary, our findings reveal the S100A9-TLR4 skeletal muscle axis as a promising therapeutic target for improving T1DM treatment.

Glucose sensing by POMC neurons regulates glucose homeostasis and is impaired in obesity.

A subset of neurons in the brain, known as 'glucose-excited' neurons, depolarize and increase their firing rate in response to increases in extracellular glucose. Similar to insulin secretion by pancreatic beta-cells, glucose excitation of neurons is driven by ATP-mediated closure of ATP-sensitive potassium (K(ATP)) channels. Although beta-cell-like glucose sensing in neurons is well established, its physiological relevance and contribution to disease states such as type 2 diabetes remain unknown. To address these issues, we disrupted glucose sensing in glucose-excited pro-opiomelanocortin (POMC) neurons via transgenic expression of a mutant Kir6.2 subunit (encoded by the Kcnj11 gene) that prevents ATP-mediated closure of K(ATP) channels. Here we show that this genetic manipulation impaired the whole-body response to a systemic glucose load, demonstrating a role for glucose sensing by POMC neurons in the overall physiological control of blood glucose. We also found that glucose sensing by POMC neurons became defective in obese mice on a high-fat diet, suggesting that loss of glucose sensing by neurons has a role in the development of type 2 diabetes. The mechanism for obesity-induced loss of glucose sensing in POMC neurons involves uncoupling protein 2 (UCP2), a mitochondrial protein that impairs glucose-stimulated ATP production. UCP2 negatively regulates glucose sensing in POMC neurons. We found that genetic deletion of Ucp2 prevents obesity-induced loss of glucose sensing, and that acute pharmacological inhibition of UCP2 reverses loss of glucose sensing. We conclude that obesity-induced, UCP2-mediated loss of glucose sensing in glucose-excited neurons might have a pathogenic role in the development of type 2 diabetes.

Leptin therapy improves insulin-deficient type 1 diabetes by CNS-dependent mechanisms in mice.

Leptin monotherapy reverses the deadly consequences and improves several of the metabolic imbalances caused by insulin-deficient type 1 diabetes (T1D) in rodents. However, the mechanism(s) underlying these effects is totally unknown. Here, we report that intracerebroventricular (icv) infusion of leptin reverses lethality and greatly improves hyperglycemia, hyperglucagonemia, hyperketonemia, and polyuria caused by insulin deficiency in mice. Notably, icv leptin administration leads to increased body weight while suppressing food intake, thus correcting the catabolic consequences of T1D. Also, icv leptin delivery improves expression of the metabolically relevant hypothalamic neuropeptides proopiomelanocortin, neuropeptide Y, and agouti-related peptide in T1D mice. Furthermore, this treatment normalizes phosphoenolpyruvate carboxykinase 1 contents without affecting glycogen levels in the liver. Pancreatic ß-cell regeneration does not underlie these beneficial effects of leptin, because circulating insulin levels were undetectable at basal levels and following a glucose overload. Also, pancreatic preproinsulin mRNA was completely absent in these icv leptin-treated T1D mice. Furthermore, the antidiabetic effects of icv leptin administration rapidly vanished (i.e., within 48 h) after leptin treatment was interrupted. Collectively, these results unveil a key role for the brain in mediating the antidiabetic actions of leptin in the context of T1D.

SIRT6 promotes metastasis and relapse in HER2-positive breast cancer.

The histone deacetylase sirtuin 6 (SIRT6) has been endowed with anti-cancer capabilities in many tumor types. Here, we investigate the impact of SIRT6-overexpression (SIRT6-OE) in Delta16HER2 mice, which are a bona fide model of HER2-positive breast cancer. After an initial delay in the tumor onset, SIRT6-OE induces a more aggressive phenotype of Delta16HER2 tumors promoting the formation of higher number of tumor foci and metastases than controls. This phenotype of SIRT6-OE tumors is associated with cancer stem cell (CSC)-like features and tumor dormancy, and low senescence and oxidative DNA damage. Accordingly, a sub-set of HER2-positive breast cancer patients with concurrent SIRT6-OE has a significant poorer relapse-free survival (RFS) probability than patients with low expression of SIRT6. ChIP-seq, RNA-seq and RT-PCR experiments indicate that SIRT6-OE represses the expression of the T-box transcription factor 3 (Tbx3) by deacetylation of H3K9ac. Accordingly, loss-of-function mutations of TBX3 or low TBX3 expression levels are predictive of poor prognosis in HER2-positive breast cancer patients. Our work indicates that high levels of SIRT6 are indicative of poor prognosis and high risk of metastasis in HER2-positive breast cancer and suggests further investigation of TBX3 as a downstream target of SIRT6 and co-marker of poor-prognosis. Our results point to a breast cancer subtype-specific effect of SIRT6 and warrant future studies dissecting the mechanisms of SIRT6 regulation in different breast cancer subtypes.

Neddylation of phosphoenolpyruvate carboxykinase 1 controls glucose metabolism.

Neddylation is a post-translational mechanism that adds a ubiquitin-like protein, namely neural precursor cell expressed developmentally downregulated protein 8 (NEDD8). Here, we show that neddylation in mouse liver is modulated by nutrient availability. Inhibition of neddylation in mouse liver reduces gluconeogenic capacity and the hyperglycemic actions of counter-regulatory hormones. Furthermore, people with type 2 diabetes display elevated hepatic neddylation levels. Mechanistically, fasting or caloric restriction of mice leads to neddylation of phosphoenolpyruvate carboxykinase 1 (PCK1) at three lysine residues-K278, K342, and K387. We find that mutating the three PCK1 lysines that are neddylated reduces their gluconeogenic activity rate. Molecular dynamics simulations show that neddylation of PCK1 could re-position two loops surrounding the catalytic center into an open configuration, rendering the catalytic center more accessible. Our study reveals that neddylation of PCK1 provides a finely tuned mechanism of controlling glucose metabolism by linking whole nutrient availability to metabolic homeostasis.

Hepatic non-parenchymal S100A9-TLR4-mTORC1 axis normalizes diabetic ketogenesis.

Unrestrained ketogenesis leads to life-threatening ketoacidosis whose incidence is high in patients with diabetes. While insulin therapy reduces ketogenesis this approach is sub-optimal. Here, we report an insulin-independent pathway able to normalize diabetic ketogenesis. By generating insulin deficient male mice lacking or re-expressing Toll-Like Receptor 4 (TLR4) only in liver or hepatocytes, we demonstrate that hepatic TLR4 in non-parenchymal cells mediates the ketogenesis-suppressing action of S100A9. Mechanistically, S100A9 acts extracellularly to activate the mechanistic target of rapamycin complex 1 (mTORC1) in a TLR4-dependent manner. Accordingly, hepatic-restricted but not hepatocyte-restricted loss of Tuberous Sclerosis Complex 1 (TSC1, an mTORC1 inhibitor) corrects insulin-deficiency-induced hyperketonemia. Therapeutically, recombinant S100A9 administration restrains ketogenesis and improves hyperglycemia without causing hypoglycemia in diabetic mice. Also, circulating S100A9 in patients with ketoacidosis is only marginally increased hence unveiling a window of opportunity to pharmacologically augment S100A9 for preventing unrestrained ketogenesis. In summary, our findings reveal the hepatic S100A9-TLR4-mTORC1 axis in non-parenchymal cells as a promising therapeutic target for restraining diabetic ketogenesis.

Hypomorphic mutation of PGC-1beta causes mitochondrial dysfunction and liver insulin resistance.

PGC-1beta is a transcriptional coactivator that potently stimulates mitochondrial biogenesis and respiration of cells. Here, we have generated mice lacking exons 3 to 4 of the Pgc-1beta gene (Pgc-1beta(E3,4-/E3,4-) mice). These mice express a mutant protein that has reduced coactivation activity on a subset of transcription factors, including ERRalpha, a major target of PGC-1beta in the induction of mitochondrial gene expression. The mutant mice have reduced expression of OXPHOS genes and mitochondrial dysfunction in liver and skeletal muscle as well as elevated liver triglycerides. Euglycemic-hyperinsulinemic clamp and insulin signaling studies show that PGC-1beta mutant mice have normal skeletal muscle response to insulin but have hepatic insulin resistance. These results demonstrate that PGC-1beta is required for normal expression of OXPHOS genes and mitochondrial function in liver and skeletal muscle. Importantly, these abnormalities do not cause insulin resistance in skeletal muscle but cause substantially reduced insulin action in the liver.

Enhanced leptin sensitivity and improved glucose homeostasis in mice lacking suppressor of cytokine signaling-3 in POMC-expressing cells.

Suppressor of cytokine signaling-3 (Socs-3) negatively regulates the action of various cytokines, as well as the metabolic hormones leptin and insulin. Mice with haploinsufficiency of Socs-3, or those with neuronal deletion of Socs-3, are lean and more leptin and insulin sensitive. To examine the role of Socs-3 within specific neurons critical to energy balance, we created mice with selective deletion of Socs-3 within pro-opiomelanocortin (POMC)-expressing cells. These mice had enhanced leptin sensitivity, measured by weight loss and food intake after leptin infusion. On chow diet, glucose homeostasis was improved despite normal weight gain. On a high-fat diet, the rate of weight gain was reduced, due to increased energy expenditure rather than decreased food intake; glucose homeostasis and insulin sensitivity were substantially improved. These studies demonstrate that Socs-3 within POMC neurons regulates leptin sensitivity and glucose homeostasis, and plays a key role in linking high-fat diet to disordered metabolism.

Acute effects of leptin require PI3K signaling in hypothalamic proopiomelanocortin neurons in mice.

Normal food intake and body weight homeostasis require the direct action of leptin on hypothalamic proopiomelanocortin (POMC) neurons. It has been proposed that leptin action requires PI3K activity. We therefore assessed the contribution of PI3K signaling to leptin's effects on POMC neurons and organismal energy balance. Leptin caused a rapid depolarization of POMC neurons and an increase in action potential frequency in patch-clamp recordings of hypothalamic slices. Pharmacologic inhibition of PI3K prevented this depolarization and increased POMC firing rate, indicating a PI3K-dependent mechanism of leptin action. Mice with genetically disrupted PI3K signaling in POMC cells failed to undergo POMC depolarization or increased firing frequency in response to leptin. Insulin's ability to hyperpolarize POMC neurons was also abolished in these mice. Moreover, targeted disruption of PI3K blunted the suppression of feeding elicited by central leptin administration. Despite these differences, mice with impaired PI3K signaling in POMC neurons exhibited normal long-term body weight regulation. Collectively, these results suggest that PI3K signaling in POMC neurons is essential for leptin-induced activation and insulin-induced inhibition of POMC cells and for the acute suppression of food intake elicited by leptin, but is not a major contributor to the regulation of long-term organismal energy homeostasis.

EP3 prostaglandin receptors in the median preoptic nucleus are critical for fever responses.

Fever is a result of the action of prostaglandin E2 (PGE2) on the brain and appears to require EP3 prostaglandin receptors (EP3Rs), but the specific neurons on which PGE2 acts to produce fever have not been definitively established. Here we report that selective genetic deletion of the EP3Rs in the median preoptic nucleus of mice resulted in abrogation of the fever response. These observations demonstrate that the EP3R-bearing neurons in the median preoptic nucleus are required for fever responses.

O-GlcNAcylated p53 in the liver modulates hepatic glucose production.

p53 regulates several signaling pathways to maintain the metabolic homeostasis of cells and modulates the cellular response to stress. Deficiency or excess of nutrients causes cellular metabolic stress, and we hypothesized that p53 could be linked to glucose maintenance. We show here that upon starvation hepatic p53 is stabilized by O-GlcNAcylation and plays an essential role in the physiological regulation of glucose homeostasis. More specifically, p53 binds to PCK1 promoter and regulates its transcriptional activation, thereby controlling hepatic glucose production. Mice lacking p53 in the liver show a reduced gluconeogenic response during calorie restriction. Glucagon, adrenaline and glucocorticoids augment protein levels of p53, and administration of these hormones to p53 deficient human hepatocytes and to liver-specific p53 deficient mice fails to increase glucose levels. Moreover, insulin decreases p53 levels, and over-expression of p53 impairs insulin sensitivity. Finally, protein levels of p53, as well as genes responsible of O-GlcNAcylation are elevated in the liver of type 2 diabetic patients and positively correlate with glucose and HOMA-IR. Overall these results indicate that the O-GlcNAcylation of p53 plays an unsuspected key role regulating in vivo glucose homeostasis.

Leptin directly activates SF1 neurons in the VMH, and this action by leptin is required for normal body-weight homeostasis.

Leptin, an adipocyte-derived hormone, acts directly on the brain to control food intake and energy expenditure. An important question is the identity of first-order neurons initiating leptin's anti-obesity effects. A widely held view is that most, if not all, of leptin's effects are mediated by neurons located in the arcuate nucleus of the hypothalamus. However, leptin receptors (LEPRs) are expressed in other sites as well, including the ventromedial hypothalamus (VMH). The possible role of leptin acting in "nonarcuate" sites has largely been ignored. In the present study, we show that leptin depolarizes and increases the firing rate of steroidogenic factor-1 (SF1)-positive neurons in the VMH. We also show, by generating mice that lack LEPRs on SF1-positive neurons, that leptin action at this site plays an important role in reducing body weight and, of note, in resisting diet-induced obesity. These results reveal a critical role for leptin action on VMH neurons.

Neuromedin B and gastrin-releasing peptide excite arcuate nucleus neuropeptide Y neurons in a novel transgenic mouse expressing strong Renilla green fluorescent protein in NPY neurons.

Neuropeptide Y (NPY) is one of the most widespread neuropeptides in the brain. Transgenic mice were generated that expressed bright Renilla green fluorescent protein (GFP) in most or all of the known NPY cells in the brain, which otherwise were not identifiable. GFP expression in NPY cells was confirmed with immunocytochemistry and single-cell reverse transcription-PCR. NPY neurons in the hypothalamic arcuate nucleus play an important role in energy homeostasis and endocrine control. Whole-cell patch clamp recording was used to study identified arcuate NPY cells. Primary agents that regulate energy balance include melanocortin receptor agonists, AgRP, and cannabinoids; none of these substances substantially influenced electrical properties of NPY neurons. In striking contrast, neuropeptides of the bombesin family, including gastrin-releasing peptide and neuromedin B, which are found in axons in the mediobasal hypothalamus and may also be released from the gut to signal the brain, showed strong direct excitatory actions at nanomolar levels on the NPY neurons, stronger than the actions of ghrelin and hypocretin/orexin. Bombesin-related peptides reduced input resistance and depolarized the membrane potential. The depolarization was attenuated by several factors: substitution of choline for sodium, extracellular Ni(2+), inclusion of BAPTA in the pipette, KB-R7943, and SKF96365. Reduced extracellular calcium enhanced the current, which reversed around -20 mV. Together, these data suggest two mechanisms, activation of nonselective cation channels and the sodium/calcium exchanger. Since both NPY and POMC neurons, which we also studied, are similarly directly excited by bombesin-like peptides, the peptides may function to initiate broad activation, rather than the cell-type selective activation or inhibition reported for many other compounds that modulate energy homeostasis.

The hypothalamic arcuate nucleus: a key site for mediating leptin's effects on glucose homeostasis and locomotor activity.

Leptin is required for normal energy and glucose homeostasis. The hypothalamic arcuate nucleus (ARH) has been proposed as an important site of leptin action. To assess the physiological significance of leptin signaling in the ARH, we used mice homozygous for a FLPe-reactivatable, leptin receptor null allele (Lepr(neo/neo) mice). Similar to Lepr(db/db) mice, these mice are obese, hyperglycemic, hyperinsulinemic, infertile, and hypoactive. To selectively restore leptin signaling in the ARH, we generated an adeno-associated virus expressing FLPe-recombinase, which was delivered unilaterally into the hypothalamus using stereotaxic injections. We found that unilateral restoration of leptin signaling in the ARH of Lepr(neo/neo) mice leads to a modest decrease in body weight and food intake. In contrast, unilateral reactivation markedly improved hyperinsulinemia and normalized blood glucose levels and locomotor activity. These data demonstrate that leptin signaling in the ARH is sufficient for mediating leptin's effects on glucose homeostasis and locomotor activity.

"AMPing up" our understanding of the hypothalamic control of energy balance.

AMP-activated protein kinase (AMPK) has emerged as a metabolic "fuel gauge," which oscillates between anabolic and catabolic processes that ultimately influence energy balance. A study in this issue of the JCI by Claret et al. now extends the role of AMPK in medial basal hypothalamic neurons (see the related article beginning on page 2325). These findings maintain AMPK signaling as a common cellular mechanism in proopiomelanocortin and neuropeptide Y/agouti-related protein neurons and links hypothalamic AMPK to coordinated energy and glucose homeostasis.

Pharmacological manipulations of CNS sirtuins: potential effects on metabolic homeostasis.

Sirtuins are deacetylases and/or mono-ADP-ribosyltransferases found in organisms ranging from bacteria to humans. These enzymes use oxidized nicotinamide adenine dinucleotide (NAD(+)) and a long array of different proteins (e.g.: histones, transcription factors, cofactors, members of the electron transport chain, etc.) as substrates. Sirtuins-mediated reactions yield deacetylated proteins, nicotinamide (NAM) and 2'-O-acetyl-ADP-ribose (O-AADPr) or mono-ADP-ribosylated proteins and NAM. As these post-translational modifications change the activity of their targets and sirtuins depend on NAD(+) to function, these enzymes are thought to link metabolic statuses with cellular gene expression, activity and fate; as such sirtuins are thought to be bona fide metabolic-sensor proteins. Due to their diverse targets, sirtuins affect metabolism, senescence, longevity, circadian rhythms and many other biological and physiological programs. In this review we focus on their known roles on metabolic homeostasis with particular emphasis on their functions in neurons within the central nervous system (CNS). We also touch upon the possible metabolic outcomes of pharmacological manipulations of CNS sirtuins.

Insulin under the influence of light.

The discovery and administration of exogenous insulin has revolutionised diabetes treatment and continues, almost 100 years on, to be the basis for the management of insulin deficiency. However, insulin therapy still has potentially life-threatening side effects such as hypoglycaemia and increased risk of cardiovascular disease. So far, improvements in insulin therapy have focused mainly on modulating its pharmacokinetic and pharmacodynamic properties and improving delivery methods, while variations in the insulin sensitivity of peripheral tissues has received relatively little attention. Notably, tissue insulin sensitivity has been shown to vary considerably around the clock, which could contribute greatly to the effect (and risk of side effects) of a given dose of insulin. Recent evidence suggests that photic inputs regulate diurnal variations in the insulin sensitivity of metabolically relevant tissues via a previously unrecognised mechanism involving the ventromedial hypothalamic nucleus. Therefore, understanding the mechanisms underlying photic control of insulin action is of paramount medical importance. In addition, considering “when” (i.e., the time of day) could assist in deciding “how much” insulin should be administered and hence could aid the fine-tuning of insulin dosage, lowering the risk of side effects, and improving the quality of life of patients with insulin deficiency.

Vav2 catalysis-dependent pathways contribute to skeletal muscle growth and metabolic homeostasis.

Skeletal muscle promotes metabolic balance by regulating glucose uptake and the stimulation of multiple interorgan crosstalk. We show here that the catalytic activity of Vav2, a Rho GTPase activator, modulates the signaling output of the IGF1- and insulin-stimulated phosphatidylinositol 3-kinase pathway in that tissue. Consistent with this, mice bearing a Vav2 protein with decreased catalytic activity exhibit reduced muscle mass, lack of proper insulin responsiveness and, at much later times, a metabolic syndrome-like condition. Conversely, mice expressing a catalytically hyperactive Vav2 develop muscle hypertrophy and increased insulin responsiveness. Of note, while hypoactive Vav2 predisposes to, hyperactive Vav2 protects against high fat diet-induced metabolic imbalance. These data unveil a regulatory layer affecting the signaling output of insulin family factors in muscle.

FKBP10 Regulates Protein Translation to Sustain Lung Cancer Growth.

Cancer therapy is limited, in part, by lack of specificity. Thus, identifying molecules that are selectively expressed by, and relevant for, cancer cells is of paramount medical importance. Here, we show that peptidyl-prolyl-cis-trans-isomerase (PPIase) FK506-binding protein 10 (FKBP10)-positive cells are present in cancer lesions but absent in the healthy parenchyma of human lung. FKBP10 expression negatively correlates with survival of lung cancer patients, and its downregulation causes a dramatic diminution of lung tumor burden in mice. Mechanistically, our results from gain- and loss-of-function assays show that FKBP10 boosts cancer growth and stemness via its PPIase activity. Also, FKBP10 interacts with ribosomes, and its downregulation leads to reduction of translation elongation at the beginning of open reading frames (ORFs), particularly upon insertion of proline residues. Thus, our data unveil FKBP10 as a cancer-selective molecule with a key role in translational reprogramming, stem-like traits, and growth of lung cancer.