Laura Braud

Maria Pini

Lucie Muchova

Sylvie Manin

Hiroaki Kitagishi

Daigo Sawaki

Gabor Czibik

Julien Ternacle

Geneviève Derumeaux

Roberta Foresti
email: roberta.foresti@inserm.fr.

Roberto Motterlini
email: roberto.motterlini@inserm.fr

Carbon monoxide-induced metabolic switch in adipocytes improves insulin resistance in obese mice

Introduction

Obesity is one the most important risk factors leading to metabolic dysfunction and type 2 diabetes [START_REF] Stumvoll | Type 2 diabetes: principles of pathogenesis and therapy[END_REF]. Excessive accumulation of visceral fat is accompanied by chronic low grade inflammation contributing to the development of insulin resistance, hyperglycemia and adipose tissue dysfunction [START_REF] Guilherme | Adipocyte dysfunctions linking obesity to insulin resistance and type 2 diabetes[END_REF][START_REF] Barazzoni | Insulin resistance in obesity: an overview of fundamental alterations[END_REF]. Whether malfunction of mitochondria, which play a crucial role in cellular bioenergetics, is implicated in adipose tissue dysfunction is still debated [START_REF] Montgomery | Mitochondrial dysfunction and insulin resistance: an update[END_REF]. Indeed, defective mitochondrial activity can lead to impaired fatty acid oxidation and glycolysis, thereby causing disruption of lipid and glucose metabolism [START_REF] Montgomery | Mitochondrial dysfunction and insulin resistance: an update[END_REF]. It has also been reported that mitochondrial oxidative stress in adipocytes diminishes insulin-stimulated GLUT4 translocation and glucose uptake resulting in insulin resistance [START_REF] Fazakerley | Mitochondrial oxidative stress causes insulin resistance without disrupting oxidative phosphorylation[END_REF]. In contrast, an increased glucose tolerance and insulin sensitivity was observed in mice with genetically altered mitochondrial oxidative phosphorylation [START_REF] Pospisilik | Targeted deletion of AIF decreases mitochondrial oxidative phosphorylation and protects from obesity and diabetes[END_REF] and an enhanced mitochondrial capacity was described in human subjects with insulin resistance [START_REF] Nair | Asian Indians have enhanced skeletal muscle mitochondrial capacity to produce ATP in association with severe insulin resistance[END_REF]. Nevertheless, pharmacological interventions that target mitochondria for the treatment of diabetes have attracted a strong interest over the last decade [START_REF] Green | Prevention of Mitochondrial Oxidative Damage as a Therapeutic Strategy in Diabetes[END_REF][START_REF] Harper | Mitochondrial uncoupling as a target for drug development for the treatment of obesity[END_REF]. For example, mitochondrial uncoupling agents such as 2,4-dinitrophenol or niclosamide ethanolamine have demonstrated promising effects on diabetic symptoms in mice by both promoting substrate oxidation and reducing oxidative stress in mitochondria [START_REF] Tao | Niclosamide ethanolamine-induced mild mitochondrial uncoupling improves diabetic symptoms in mice[END_REF][START_REF] Crunkhorn | Metabolic disease: Mitochondrial uncoupler reverses diabetes [Internet[END_REF].

Emerging evidence indicates that carbon monoxide (CO) modulates metabolism in different cell types [START_REF] Wilson | Carbon monoxide reverses the metabolic adaptation of microglia cells to an inflammatory stimulus[END_REF][START_REF] Lavitrano | Carbon monoxide improves cardiac energetics and safeguards the heart during reperfusion after cardiopulmonary bypass in pigs[END_REF][START_REF] Almeida | Carbon monoxide improves neuronal differentiation and yield by increasing the functioning and number of mitochondria[END_REF][START_REF] Motterlini | Biological signaling by carbon monoxide and carbon monoxide-releasing molecules[END_REF]. CO is a ubiquitous gaseous molecule produced in mammalian cells and tissues through the breakdown of heme catalyzed by heme oxygenase (HO) enzymes [START_REF] Motterlini | Heme Oxygenase-1 As a Target for Drug Discovery[END_REF]. Initially considered as a toxic product, CO is now recognized as a key signaling molecule with vasoactive and cardioprotective effects as well as anti-thrombotic, anti-apoptotic and anti-inflammatory properties [START_REF] Motterlini | The therapeutic potential of carbon monoxide[END_REF][START_REF] Foresti | Signaling and Cellular Functions of Carbon Monoxide (CO)[END_REF]. In relation to cellular metabolism, we have shown that small amounts of CO increase O 2 consumption in cells via a mild uncoupling activity in mitochondria that is accompanied by changes in the production of reactive oxygen species [START_REF] Wilson | Carbon monoxide reverses the metabolic adaptation of microglia cells to an inflammatory stimulus[END_REF]. The use of CO-releasing molecules (CO-RMs), a class of compounds that release controlled quantities of CO into cells and tissues, has helped to unravel this novel mechanism of action and identify the potential therapeutic effects of this gas [START_REF] Wilson | Carbon monoxide reverses the metabolic adaptation of microglia cells to an inflammatory stimulus[END_REF][START_REF] Motterlini | The therapeutic potential of carbon monoxide[END_REF][START_REF] Clark | Cardioprotective actions by a water-soluble carbon monoxidereleasing molecule[END_REF][START_REF] Fayad-Kobeissi | Vascular and angiogenic activities of CORM-401, an oxidant-sensitive CO-releasing molecule[END_REF].

Accordingly, the water soluble CORM-A1 has been shown to improve dietaryinduced obesity, hyperglycemia and insulin resistance in mice [START_REF] Hosick | Chronic carbon monoxide treatment attenuates development of obesity and remodels adipocytes in mice fed a high-fat diet[END_REF][START_REF] Hosick | Chronic treatment with a carbon monoxide releasing molecule reverses dietary induced obesity in mice[END_REF]. However, the mechanisms underlying this beneficial outcome of CO in obesity have been poorly investigated.

In the current study, we examined the effect of CORM-401 on whole body insulin sensitivity and glucose tolerance in C57BL6 mice subjected to an obesogenic high fat diet (HFD). We report that oral administration of CORM-401 reduces the gain in body weight and improves insulin resistance in HFD obese mice. CO delivered by CORM-401 to adipose tissue and cells uncouples mitochondrial respiration leading to an increase in glycolysis to maintain ATP levels. This metabolic switch was associated with higher phosphorylation of the insulin signaling effector Akt and was accompanied by an amelioration of systemic and adipocytes insulin sensitivity. Our data demonstrate that the effect of CO on cellular bioenergetics in adipose tissue serves to counteract insulin resistance in obese mice.

Results

Oral administration of CORM-401 reduces body weight gain and improves insulin resistance in HFD-induced obesity in mice

We have shown recently that CORM-401, a manganese-based compound that releases 3 equivalents of CO/mole (Structure shown in Supplemental Table 1), exerts a stronger vasodilatory effect and angiogenic activity than CORM-A1, a boron compound that liberates only 1 CO/mole [START_REF] Fayad-Kobeissi | Vascular and angiogenic activities of CORM-401, an oxidant-sensitive CO-releasing molecule[END_REF]. Thus, it appears that CORM-401 exhibits enhanced pharmacological activities due to liberation of higher amounts of CO. Since CORM-401 has never been tested in vivo, we first determined the levels of blood COHb in mice receiving CORM-401 at two different doses after oral gavage.

Figure 1A and Supplemental Figure 1A show that COHb significantly increased to 2.5% and 4.5% 30 min after administration of CORM-401 at 15 and 30 mg/kg, respectively, and gradually decreased to control levels over a period of 48 h. These data confirm that CO is released by CORM-401 and delivered into the circulation in a dose-dependent fashion. Based on this kinetic, we decided to assess the metabolic effects of CORM-401 (15 and 30 mg/kg) administered three times a week to mice fed either a standard diet (SD) or a high fat diet (HFD) for 14 weeks. As shown in Supplemental Figure 1B-E, CORM-401 decreased body weight gain and improved both glucose metabolism and insulin resistance in a dose-dependent manner in HFD mice but the effect was significant only at the dose of 30 mg/kg. Because consistent results were obtained with 30 mg/kg CORM-401, we decided to continue our investigation using this dose in two distinct protocols whereby CORM-401 was given in concomitance with the HFD (prevention group, HFD+CORM-P) or 6 weeks after the beginning of HFD ( treatment group, HFD+CORM-T). Body weight gain was significantly increased by the HFD and was reduced by CORM-401 both in the prevention and the treatment group (Figure 1B andC) as reflected by decreased plasma leptin (Figure 1M). Changes in body weight were not due to differences in food intake between the groups (Table 1). In addition, mice fed with SD and SD+CORM-401 displayed similar body weight, suggesting that the compound was well tolerated and the reduced weight gain in HFD-fed mice was not due to a toxic effect of the drug (Supplemental Figure 2A andB). In addition, we did not observe any change in cardiac function parameters by CORM-401 compared to untreated obese mice (Supplemental Table 2). Interestingly, CORM-401 did not affect glucose metabolism in SD mice (Supplemental Figure 2C-F) but improved whole-body glucose tolerance and insulin sensitivity in HFD mice, as shown by a significant decrease in glycemia (Figure 2D andE). In line with this, CORM-401 decreased fasting plasma glycemia and insulin, improving the calculated HOMA-IR in HFD mice (Figure 1 J-L), while plasma cholesterol and triglyceride levels remained unchanged (Figure 1H andI). These results indicate that CORM-401 counteracts body weight gain, impaired glucose tolerance and insulin resistance induced by HFD in mice.

CORM-401 remodels adipose tissue in obese mice

We next investigated the effect of CORM-401 on adipose tissue metabolic and inflammatory profiles. Even though eWAT weight was not affected by CORM-401 (Figure 2A andB), the size of adipocytes was significantly reduced by 25% in the HFD+CORM-P group (Figure 2A andC). Interestingly, conditioned media of eWAT collected from both mice groups administered with CORM-401 displayed lower nonesterified free fatty acid (NEFA) content compared to the HFD group (Figure 2D). Furthermore, the mRNA expression of the key metabolic genes PParg, Adiponectin Fabp4, Hsl, Glut4, Irs1, Hk2 and Vegf was decreased by the HFD (Figure 2E) and restored to basal levels by CORM-401 in the HFD+CORM-P but not in HFD+CORM-T group. In contrast, mRNA expression of PPara, Pgc1a and Cpt1 was decreased by HFD but remained unchanged after CORM-401 treatment (data not shown).

Significant remodeling in other fat pads, particularly in brown adipose tissue (BAT), was evident in the reduction of weight and lipid content in both groups administered with CORM-401 (Supplemental Figure 3A-E). Similarly to eWAT, mRNA expression of the metabolic genes PParg, Vegf, Adiponectin, Pgc1a and Ucp1 were decreased in BAT of HFD-obese mice and fully or partially restored in the HFD+CORM-P group (Supplemental Figure 3G). Conversely, gene expression of metabolic markers in subcutaneous adipose tissue (SubWAT) was not affected by CORM-401 (Supplemental Figure 3F). Despite the fact that HFD did not induce a high-grade inflammation in our model, CORM-401 still modulated local inflammation in obese mice. First, the increased macrophage infiltration induced by HFD was virtually abolished by CORM-401 administration (Figure 2F-G), although the induction of Ccl2 was unchanged by the compound (Figure 2H). Secondly, CORM-401 decreased Hmox-1 expression induced by HFD (Figure 2I) and the levels of IL-6, IL-1β and IL-10 in eWAT conditioned media (Figure 2J). These data demonstrate that the positive effects of CORM-401 on weight gain and metabolism in obese mice correlate with an amelioration of adipose tissue structure, function and inflammation. CORM-401 had no effect on the liver, another crucial organ for the maintenance of glucose homeostasis. In fact, CORM-401 did not modify the increase in liver weight (1.5-fold) and lipid content caused by HFD (Supplemental Figure 4A-C) and only marginally affected the deregulation of the metabolic genes PParg, Hk2, Irs1, Hsl, Fabp4 induced by HFD (Supplemental Figure 4D). We note that the plasmatic indexes of liver injury were not impacted by HFD (Table 2), suggesting nonadvanced hepatic steatosis without major injuries.

CO delivered by CORM-401 increases systemic and adipocytes insulinsensitivity

To elucidate whether the improved insulin resistance promoted by CORM-401 is linked to an effect of CO on adipocytes, we assessed insulin sensitivity in eWAT and 3T3-L1 adipocytes. We first confirmed that CO content in eWAT increased significantly 2 and 6 h after oral gavage with CORM-401 compared to eWAT from mice treated with PBS (CON) or inactive CORM-401 (iCORM) (Figure 3A). We then determined glucose levels in mice treated with PBS, iCORM or CORM-401 1 h prior to intraperitoneal injection of insulin (Figure 3B) and showed that only CORM-401 enhanced systemic insulin sensitivity (Figure 3C andD). In addition, phosphorylation of protein kinase B (Akt) induced by insulin was increased in eWAT from CORM-401-treated mice compared to control (Figure 3E andF). This response was recapitulated in eWAT of obese mice following oral gavage with CORM-401 (Supplemental Figure 5). As observed in eWAT, CO content in 3T3-L1 adipocytes exposed to 50 µM CORM-401 was significantly higher compared to cells treated with PBS or iCORM (Figure 3G). Likewise, only CORM-401-treated adipocytes exhibited increased Akt phosphorylation in basal conditions and after stimulation with insulin (20 nM) for 10 min (Figure 3H, 3I and J). These data demonstrate that CO liberated from CORM-401 accumulates in adipose tissue and regulates Akt signaling to improve insulin sensitivity.

CO uncouples mitochondrial respiration and increases glycolytic rate in adipocytes

We next investigated the effects of CORM-401 on adipocyte bioenergetics based on the emerging evidence that CO is a metabolic regulator in vascular, inflammatory and cancer cells [START_REF] Wilson | Carbon monoxide reverses the metabolic adaptation of microglia cells to an inflammatory stimulus[END_REF][START_REF] Wegiel | Carbon monoxide expedites metabolic exhaustion to inhibit tumor growth[END_REF]. Using MitoStress assays, we showed that CORM-401treated 3T3-L1 adipocytes display a concentration-dependent increase in oxygen consumption rate (OCR) even in the presence of the ATP synthase inhibitor oligomycin (Figure 4A and Supplemental Figure 4), resulting in decreased ATPlinked respiration (or coupled respiration) and higher proton leak (or uncoupled respiration) compared to untreated cells (Figure 4B). This effect was followed by reduced respiration in response to the uncoupling agent FCCP and augmented nonmitochondrial respiration after addition of rotenone/antimycin A. These results indicate an uncoupling activity of CORM-401 in adipocytes, as previously observed in other cell types [START_REF] Wilson | Carbon monoxide reverses the metabolic adaptation of microglia cells to an inflammatory stimulus[END_REF]. Interestingly, in the absence of inhibitors of the electron transport chain, CORM-401 induced a transient increase in OCR lasting for 2 to 3 h depending on the dose used. This effect was concomitant with a rise in glycolysis as assessed by the extracellular acidification rate (ECAR) (Figure 4C andD).

Accordingly, intracellular lactate was increased by CORM-401 (Figure 4E). A glycolysis stress test confirmed that CORM-401, but not iCORM, enhanced ECAR in adipocytes following sequential addition of glucose, oligomycin and 2-DG (Figure 4F and G). Increased glycolysis was not an artifact due to acidification of the medium by CORM-401 since blocking this pathway with 2-DG completely prevented the effects of CORM-401.

Notably, intracellular ATP levels were higher in CORM-401-treated adipocytes compared to iCORM, suggesting that glycolysis is engaged as a compensatory mechanism for maintenance of energy (Figure 4H). ATP content was diminished by 2-DG in iCORM and CORM-401-treated cells; however, this decrease was more pronounced in adipocytes exposed to CORM-401, likely a result of the uncoupling effect of CO and the inhibition of respiration caused by CORM-401 at the end of the experiment (Figure 4H). We wanted to verify if this metabolic switch induced by CORM-401 occurs in vivo by assessing OCR and ECAR in eWAT collected from mice treated with iCORM or CORM-401 (Figure I). Although no significant differences were observed in basal OCR between the two groups, the response to FCCP was lower after treatment with CORM-401 (Figure 4J), as shown in cultured adipocytes. Most importantly, ECAR was also higher in eWAT punches from CORM-401-treated mice (Figure 4K) and lactate levels in conditioned medium from eWAT collected from mice receiving CORM-401 was increased (Figure 4L), confirming stimulation of glycolysis by CO in vivo.

ATP counteracts CO induced-metabolic switch in adipocytes and reverses COinduced increase in insulin sensitivity

Our data indicate that CO induces a metabolic switch, favoring glycolysis, in adipocytes both in vitro and in vivo. If CO decreases mitochondrial ATP production because of transient uncoupling and/or partial inhibition of respiration leading to increased glycolysis to maintain ATP levels, we postulated that ATP per se could serve as the molecular trigger of this metabolic switch. To investigate this hypothesis, we artificially delivered ATP packaged in liposomes to adipocytes and examined the effects of CO on bioenergetics. ATP levels significantly increased in adipocytes after exposure to encapsulated-ATP (Figure 5A). Importantly, exogenous ATP supplementation counteracted the rise in ECAR as well as lactate elicited by CORM-401 without affecting the increase in OCR (Figure 5B-D). Furthermore, ATP delivery to adipocytes reversed the increase in insulin-dependent Akt phosphorylation induced by CO (Figure 5E-F). Thus, the suppression of ECAR and Akt phosphorylation by ATP suggests that the increase in glycolysis by CO is a consequence of the uncoupling action of CO and is directly implicated in the improved insulin sensitivity observed in adipocytes.

Discussion

We report in this study that treatment with the CO-releasing agent CORM-401 decreases gain in body weight and markedly ameliorates glucose tolerance and insulin resistance in HFD-induced obese mice. We show that this effect is associated with prevention of adipose tissue alterations as indicated by restoration in metabolic gene expression, suppression of macrophage infiltration and reduction in key inflammatory markers (IL-6, IL-1β and HO-1) characteristic of obesity. From a mechanistic point of view, we further show that CORM-401: 1) increases insulin sensitivity in adipose tissue by activation of Akt phosphorylation and 2) triggers a metabolic switch that is dependent on mitochondrial uncoupling by CO and is accompanied by an increase in glycolysis and lactate production. These results reveal an important role of CO in regulating bioenergetic metabolism in adipose tissue thus counteracting the deleterious consequences of obesity.

We observed that administration of CORM-401 for the whole duration of HFD (CORM-P) was more effective in reducing body weight gain than treatment with CORM-401 starting 6 weeks after the beginning of the HDF regime (CORM-T). This effect is most likely due to a longer period of exposure to CO since we noticed that a significant decrease in body weight becomes apparent in both groups 6 weeks after the first administration of CORM-401. Despite these differences, both groups exhibited the same improvement in glucose tolerance and insulin resistance, suggesting that the effect of CORM-401 on body weight is not the only factor explaining a beneficial role of CO on obesity-induced metabolic dysfunction. Indeed, a lower glycemia and increased insulin sensitivity were also found after one single administration of CORM-401 in lean mice, corroborating a direct pharmacological action of CO on glucose metabolism. When we focused on visceral adipose tissue (eWAT), which undergoes significant structural and functional changes during obesity, we also observed some interesting differences. Although CORM-401 did not affect the increased weight of eWAT in HFD-fed mice, a significant reduction in the size of adipocytes and a restoration of metabolic gene expression were evident in the CORM-P group. These effects correlate with the more pronounced decrease in weight gain in this group. However, both CORM-P and CORM-T groups displayed a similar reduction in the secretion of non-esterified fatty acid, infiltration of macrophages and production of the pro-inflammatory cytokine IL-6 in eWAT, supporting the idea that the pharmacological action mediated by CO on these markers is also independent of the decrease in body weight gain. In particular, inhibition of macrophage infiltration and the marked suppression of IL-6, a cytokine strongly implicated in development of metabolic dysfunction during obesity [START_REF] Cai | Local and systemic insulin resistance resulting from hepatic activation of IKK-beta and NF-kappaB[END_REF][START_REF] Kim | Differential effects of interleukin-6 and -10 on skeletal muscle and liver insulin action in vivo[END_REF], indicate another potential mechanism by which CORM-401 exerts its beneficial effect on adipose tissue and insulin resistance. We also observed that treatment with CORM-401 diminishes HO-1 gene induction caused by HFD in adipose tissue. This is in line with previous studies showing that HO-1 is increased during HFD in mice [START_REF] Hosick | Chronic carbon monoxide treatment attenuates development of obesity and remodels adipocytes in mice fed a high-fat diet[END_REF] and humans [START_REF] Jais | Heme Oxygenase-1 Drives Metaflammation and Insulin Resistance in Mouse and Man[END_REF]. HO-1 up-regulation in adipose tissue may be part of the adaptive response mounted by the organism to counteract the inflammatory and metabolic stress elicited by HFD. In fact, systemic induction of HO-1 by cobalt protoporphyrin or genetic over-expression of HO-1 reduces adiposity and improves insulin sensitivity in mice [START_REF] Li | Treatment of obese diabetic mice with a heme oxygenase inducer reduces visceral and subcutaneous adiposity, increases adiponectin levels, and improves insulin sensitivity and glucose tolerance[END_REF][START_REF] Ndisang | Upregulation of the heme oxygenase system ameliorates postprandial and fasting hyperglycemia in type 2 diabetes[END_REF][START_REF] Nicolai | Heme Oxygenase-1 Induction Remodels Adipose Tissue and Improves Insulin Sensitivity in Obesity-Induced Diabetic Rats[END_REF][START_REF] Burgess | Adipocyte Heme Oxygenase-1 Induction Attenuates Metabolic Syndrome in Both Male and Female Obese Mice[END_REF]. However, the role of HO-1 in specific organs involved in metabolic regulation is still controversial since it has also been reported that macrophage and hepatic HO-1 deletion protect against insulin resistance and inflammation [START_REF] Jais | Heme Oxygenase-1 Drives Metaflammation and Insulin Resistance in Mouse and Man[END_REF][START_REF] Huang | Myeloid heme oxygenase-1 haploinsufficiency reduces high fat diet-induced insulin resistance by affecting adipose macrophage infiltration in mice[END_REF]. Notably, in our study we found that CORM-401 does not have any major effect on the liver. Thus, despite the fact that the liver is a crucial organ for the maintenance of glucose, the findings of this study strongly suggest that the beneficial effect of CO on glucose metabolism is mainly driven by its interaction with adipose tissue.

Our results confirm previous findings showing that CORM-A1, another COreleasing agent, significantly diminishes body weight gain and insulin resistance when given intraperitoneally to HFD-fed mice for 30 weeks [START_REF] Motterlini | The therapeutic potential of carbon monoxide[END_REF]. A difference in our study is that CORM-401 was administered orally to mice. In addition, we advance the current knowledge on the pharmacological properties of CO-RMs by reporting that a transient increase in COHb levels is associated with accumulation of CO within the adipose tissue. Thus, we demonstrate that CO reaches the adipose tissue and modulates its function in vivo. This finding is further supported by the complete lack of effects by inactive CORM-401, which is depleted of CO. Importantly, we identified that CO directly impacts insulin sensitivity since phosphorylation of Akt, a major pathway implicated in insulin signaling, is significantly enhanced in adipocytes and adipose tissue after CORM-401 treatment. Despite the fact that increased Akt phosphorylation by CO has been reported in other cell types and tissues [START_REF] Kim | Carbon monoxide protects against hepatic ischemia/reperfusion injury via ROS-dependent Akt signaling and inhibition of glycogen synthase kinase 3β[END_REF][START_REF] Yang | Carbon monoxide releasing molecule induces endothelial nitric oxide synthase activation through a calcium and phosphatidylinositol 3-kinase/Akt mechanism[END_REF][START_REF] Wegiel | Nitric oxide-dependent bone marrow progenitor mobilization by carbon monoxide enhances endothelial repair after vascular injury[END_REF], the specific activation of this signal transduction pathway in adipose tissue likely explains the effect of CO on glucose metabolism independently from the reduction in weight gain.

The additional mechanism that most likely accounts for an improved glucose tolerance is the switch of adipose tissue metabolism towards glycolysis mediated by CO. This effect was confirmed by increased lactate production in adipocytes and in the secretome of adipose tissue after exposure to CORM-401, suggesting augmented glucose utilization. This metabolic switch occurred in concomitance with a rise in oxygen consumption and a decrease in ATP-linked respiration by CORM-401, which we attribute to an uncoupling activity by CO that we have demonstrated previously in isolated mitochondria as well as in endothelial and microglia cells [START_REF] Wilson | Carbon monoxide reverses the metabolic adaptation of microglia cells to an inflammatory stimulus[END_REF][START_REF] Motterlini | Biological signaling by carbon monoxide and carbon monoxide-releasing molecules[END_REF][START_REF] Iacono | A carbon monoxide-releasing molecule (CORM-3) uncouples mitochondrial respiration and modulates the production of reactive oxygen species[END_REF][START_REF] Kaczara | Carbon monoxide released by CORM-401 uncouples mitochondrial respiration and inhibits glycolysis in endothelial cells: A role for mitoBKCa channels[END_REF][START_REF] Sandouka | Carbon monoxide-releasing molecules (CO-RMs) modulate respiration in isolated mitochondria[END_REF]. Thus, a potential defective ATP production in response to CO-mediated uncoupling would trigger glycolysis as a compensatory mechanism to preserve energy levels leading to improved systemic glucose profile. Indeed, ATP levels were maintained after CORM-401 treatment in adipocytes and were significantly decreased only in the presence of the glycolysis inhibitor 2-deoxyglucose. In addition, direct delivery of ATP into adipocytes prevented the metabolic switch (glycolysis and lactate production) and the increase in insulin sensitivity (Akt phosphorylation) without altering the uncoupling effect caused by CO. That is, when ATP is artificially raised in adipocytes, cells sense adequate energy levels and do not engage glycolysis, even though a diminished mitochondrial ATP production by the uncoupling activity of CO is still occurring. The increase in glycolysis exerted by CO in adipocytes is reminiscent of the action of metformin, an antidiabetic drug wellknown to improve insulin resistance in chronic obese patients [START_REF]Fatty acids revert the inhibition of respiration caused by the antidiabetic drug metformin to facilitate their mitochondrial β-oxidation[END_REF] and capable of inhibiting mitochondrial ATP production [START_REF] Zhang | Mitochondrial inhibitor as a new class of insulin sensitizer[END_REF]. Notably, it has been reported that genetic alteration of mitochondrial oxidative phosphorylation improves insulinsensitivity in mice (6) while enhanced mitochondrial capacity in muscle is associated with higher risk of diabetes in humans [START_REF] Nair | Asian Indians have enhanced skeletal muscle mitochondrial capacity to produce ATP in association with severe insulin resistance[END_REF]. It is still debated whether mild increases or decreases in respiratory chain function are beneficial against insulin resistance.

Our findings argue in favor of reducing respiratory chain function as a promising approach in the treatment of insulin resistance.

Uncoupling agents are renowned to promote weight loss because of their ability to dissipate energy as heat at the expense of ATP production by mitochondria [START_REF] Harper | Mitochondrial uncoupling as a target for drug development for the treatment of obesity[END_REF][START_REF] Speakman | Uncoupled and surviving: individual mice with high metabolism have greater mitochondrial uncoupling and live longer[END_REF]. Therefore, we propose that the transient uncoupling activity of CO elicited by repetitive administration of CORM-401 is a prominent mechanism responsible for the lower weight gain during the HFD regime. Because this uncoupling effect is concomitant to enhanced glycolysis, these two actions of CO appear to be essential in the restoration of metabolic homeostasis in obesity (Figure 6).

In summary, this study provides the first evidence that an orally active COreleasing agent is effective in counteracting glucose intolerance and weight gain in HFD obese mice. The ability of CO to interact with mitochondria and improve adipose tissue function seems to be central to this effect. Thus, our findings support the idea that CORM-401 could be investigated as a prototypic compound for the development of promising insulin-sensitizing agents.

Materials and methods

Chemicals and Reagents. CORM-401 (Mn(CO)4{S2CNMe(CH2CO2H)}) (see chemical structure in Supplemental Figure 1) was synthesized as previously described [START_REF] Crook | Mn(CO)4{S2CNMe(CH2CO2H)}], a new water-soluble COreleasing molecule[END_REF]. Stock solutions (5-10 mM) were prepared by solubilizing CORM-401 in Dulbecco Phosphate Buffer Solution (pH= 7.4) and stored at -20 °C until use. As a negative control, CORM-401 was inactivated (iCORM) by incubation with equimolar concentrations of hydrogen peroxide for 24 h in order to remove CO from CORM-401. The absence of CO release from iCORM was verified using a hemoglobin assay (see below). For the animal experiments, standard diet (A04) and high fat diet (Purified Diet 230 HF) were purchased from SAFE Diet (Augy, France). Dulbecco's modified Eagle's medium (DMEM), penicillin/streptomycin, dexamethasone, new born calf serum and fetal bovine serum (FBS) were purchased from Life Technologies. Pyruvate, isobutylmethylxanthine (IBMX), insulin and all other reagents were obtained from Sigma Aldrich unless otherwise specified.

Animals and experimental design. Eight-week-old male C57BL6 mice weighing approximately 25 g were obtained from Janvier (France). Mice were housed under controlled conditions of temperature (21±1°C), hygrometry (60±10%) and lighting (12 h per day). Animals were acclimatized in the laboratory for one week before the start of the experiments. Mice were fed either a standard diet (SD) or a high fat diet (HFD) for 14 weeks with or without CORM-401 treatment. In preliminary experiments, we tested CORM-401 at 15 mg/kg and 30 mg/kg given orally three times a week to evaluate its dose-dependent effects in our model. Because the effects were more pronounced with 30 mg/kg CORM-401, we continued our study using this dose and randomly divided mice into five groups (n=10 per group): 1) SD mice; 2) HFD mice;

3) SD mice administered with CORM-401 starting at week 6 after the beginning of the diet (SD+CORM); 4) HFD mice administered with CORM-401 starting at week six after the beginning of the diet (treatment group, SD+CORM-T); 5) HFD mice administered with CORM-401 starting at the beginning of the diet (prevention group, SD+CORM-P). Mice were weighed weekly and food consumption was measured at week four and eight. The total amount of food was weighed daily in the afternoon and averaged for each mouse to obtain a daily food consumption measurement per mouse. Mice were sacrificed at the end of 14 weeks after a 6 h fasting and 48 h after the last CORM treatment. Blood was collected for biochemistry analysis and epididymal white adipose tissue (eWAT), inguinal white adipose tissue (iWAT), brown adipose tissue (BAT) and liver were removed and weighed. In an additional set of experiments, 6 h-fasted mice were given PBS, iCORM (30 mg/kg) or CORM-401 (30 mg/kg) by oral gavage. Metabolic assays were performed 1 h after CORM-401 administration for one set of experiment and mice were sacrificed 2 h after CORM-401 administration for seahorse experiment and adipose tissue collection.

Fasting blood glucose, glucose and insulin tolerance tests. After 6 h fasting, wholebody glucose tolerance and insulin sensitivity were assessed in all groups at weeks five/six and 12/13 by intraperitoneal glucose (GTT) and insulin (ITT) tolerance tests, respectively. In addition, GTT and ITT were also evaluated 1 h after oral gavage of CORM-401. First, blood was obtained via tail clip to assess fasting blood glucose (Caresens ® N, DinnoSanteTM). Then, mice received a glucose (1.5 g/kg) or insulin (0.3 UI/kg) solution by intraperitoneal injection and blood glucose was measured at 15, 30, 60, 90 and 120 min after the injection. The HOmeostasis Model Assessment of Insulin Resistance (HOMA-IR) adjusted to rodents was calculated as ([glucose (mg/dl)/18] x [insulin (ng/ml)/0.0347])/108.24 as reported [START_REF] Lee | Comparison between surrogate indexes of insulin sensitivity and resistance and hyperinsulinemic euglycemic clamp estimates in mice[END_REF] The area under the curve (AUC) for the glucose excursion was calculated using Graphpad Prism.

Transthoracic Echocardiography. Transthoracic echocardiography was performed in conscious mice as previously described [START_REF] Sawaki | Visceral Adipose Tissue Drives Cardiac Aging Through Modulation of Fibroblast Senescence by Osteopontin Production[END_REF][START_REF] Ternacle | Short-term high-fat diet compromises myocardial function: a radial strain rate imaging study[END_REF]. Briefly, in hand-gripped mice parasternal images were acquired at the level of papillary muscles using a 13-MHz linear-array transducer with a Vivid 7 ultrasound system (GE Medical System, Chicago, IL). Left ventricular dimensions were serially obtained in M-mode and derived parameters were calculated using standard formulas. Left ventricular mass was determined using the uncorrected cube assumption [START_REF] Sawaki | Visceral Adipose Tissue Drives Cardiac Aging Through Modulation of Fibroblast Senescence by Osteopontin Production[END_REF][START_REF] Ternacle | Short-term high-fat diet compromises myocardial function: a radial strain rate imaging study[END_REF]. Peak systolic values of strain rate in the anterior and posterior wall were obtained using Tissue Doppler Imaging. Strain rate analysis with EchoPac Software (GE Medical System) was performed offline by a blinded observer. Peak systolic strain rate on 8 consecutive cardiac cycles were averaged to reduce the effect of respiratory variations [START_REF] Sawaki | Visceral Adipose Tissue Drives Cardiac Aging Through Modulation of Fibroblast Senescence by Osteopontin Production[END_REF][START_REF] Ternacle | Short-term high-fat diet compromises myocardial function: a radial strain rate imaging study[END_REF].

Preparation of eWAT-conditioned medium (eCM).

Epididymal white adipose tissue (eWAT 0.1 g) was collected and kept at room temperature in a 24-well plate with 1 ml of DMEM/well. The tissue was minced into ∼1 mm 3 pieces and incubated for 1 h at 37°C and 5% CO 2 prior to transferring it into a new plate with fresh DMEM medium containing 4.5 g glucose, 2 mM glutamine, 1% free fatty acid bovine serum albumin, 1% antibiotic and antimycotic solution. eWAT-conditioned medium (eCM) was collected 24 h after incubation and stored at -80°C until analysis.

Plasma and eCM analysis. An enzyme-linked immunosorbent assay (ELISA) kit was used to measure insulin (ALPCO Diagnostics, Salem, NH). Triglycerides (TG), total cholesterol (CHOL), lactate, alanine transaminase (ALAT), aspartate transaminase (ASAT), lactate dehydrogenase, creatinine as well as conjugated and total bilirubin were measured in plasma samples using a Cobas 8000 analyser (Roche, Indianapolis, USA). Cytokines (IL-1β, IL-10 and IL-6) and leptin were measured in plasma and eCM samples using Mesoscale Multiplex and Single-plex plates, respectively (Mesoscale Discovery, Gaithersburg, USA).

Analysis of mRNA expression.

After an initial extraction step by mixing Extract-All (Eurobio, France) and chloroform to samples, total RNA purification was performed with a column extraction Kit (RNeasy Mini ® , Qiagen, Germany). Double-strand cDNA was synthesized from total RNA with the High-Capacity cDNA Reverse Transcription Kit (Life Technologies, Carlsbad, CA). Quantitative real-time PCR (qPCR) was performed in a StepOnePlus Real-Time PCR System using commercially available TaqMan primer-probe sets (Life Technologies, Carlsbad, CA). Gene expression was assessed by the comparative CT (ΔΔCT) method with β-actin as the reference gene.

Western Blot Analysis. Snap-frozen eWAT samples (200 mg) were lysed in cell lysis buffer (Cell Signaling, Danvers, MA France) supplemented with 1% phenylmethylsulfonyl fluoride (PMSF). Protein samples were resolved on 12% bis-Tris gels followed by transfer to nitrocellulose membrane. Antibodies for Akt and phospho-Akt (Ser473) were from Cell Signaling (references #4991 and #4060 respectively) and antibodies for β-actin were from Santa Cruz (reference sc-47778).

Bands were visualized by enhanced chemiluminescence and quantified using ImageJ software.

Histology and immunohistochemistry. Fresh tissues were fixed in 10% phosphatebuffered formalin overnight. Paraffin wax sections of 5 µm were processed for haematoxylin-eosin (H&E) and CD68 immunostaining. Images were analyzed using ImageJ software.

3T3-L1 cell culture. 3T3-L1 murine pre-adipocytes (reference 088SP-L1-F) were purchased from the ZenBio company (NC, USA) and cultured in an atmosphere of 5 % CO 2 at 37 °C using DMEM supplemented with 10% newborn calf serum For adipocyte differentiation, cells were stimulated with 3T3-L1 differentiation medium containing IBMX (500 µM), dexamethasone (250 nM) and insulin (175 nM) for 2 days after cells reached confluency. The medium was changed to DMEM containing 10% FBS and insulin (175 nM) after 2 days and adipocytes were then kept into DMEM containing only 10% FBS. Prior to the experiments, adipocytes were subjected to serum deprivation for 16 h with DMEM supplemented with 0.5% FBS.

Cellular Bioenergetic Analysis. Bioenergetic profiles of 3T3-L1 adipocytes were determined using a Seahorse Bioscience XF24 Analyzer (Billerica, MA, USA) that provides real-time measurements of oxygen consumption rate (OCR), indicative of mitochondrial respiration, and extracellular acidification rate (ECAR), an index of glycolysis. An optimal cell density of 20,000 cells/well was determined from preliminary experiments. Bioenergetic measurements were performed in FBS-and bicarbonate-free DMEM (pH 7.4) supplemented with 4.5 g/L glucose, 1% glutamax and 1% pyruvate to match the normal culture conditions of 3T3-L1 cells. In a first set of experiments, the effect of CORM-401 (25-100 μM) on OCR and ECAR was assessed over time. In a second set of experiments, the effect of CORM-401 on mitochondrial function was evaluated by means of a MitoStress test, which allows the measurements of key parameters such as ATP-linked respiration and proton leak after sequential additions of: 1 μg/ml oligomycin (inhibitor of ATP synthesis), 0.7 μM carbonyl cyanide 4-(trifluoromethoxy) phenylhydrazone (FCCP, uncoupling agent) and 1 μM rotenone/antimycin A (inhibitors of complex I and complex III of the respiratory chain, respectively). CORM-401 was added 1 h prior to oligomycin to ensure delivery of CO to cells before assessment of mitochondrial function. In an alternative set of experiments, we investigated the effect of CORM-401 on respiration in the absence of ATP synthesis by adding first oligomycin to cells followed by CORM-401, FCCP and rotenone/antimycin A. In a third set of experiments, the effect of CORM-401 on glycolysis was evaluated by means of a Glycolytic stress test, which allow the measurements of glycolysis and glycolytic reserve capacity after sequential additions of: 10 mM glucose, 1 μg/ml oligomycin and 50 mM of 2-deoxyglucose (inhibitor of glycolysis).The pharmacological action of CORM-401 was compared to that of iCORM-401, which does not release CO.

ATP Assay. Intracellular ATP levels were measured using an ATPLiteTM Bioluminescence Assay Kit (PerkinElmer, France) according to manufacturer's instructions.

Assays for the detection of CO in vivo and in vitro. The levels of carbonmonoxy hemoglobin (COHb) in blood was determined as previously described by our group [START_REF] Nikam | Diverse Nrf2 Activators Coordinated to Cobalt Carbonyls Induce Heme Oxygenase-1 and Release Carbon Monoxide in Vitro and in Vivo[END_REF]. Briefly, blood (5 µl) collected from the mice tail vein at different time points after oral administration with CORM-401 was added to a cuvette containing 4.5 ml of deoxygenated tris(hydroxymethyl) aminomethane solution and spectra recorded as reported above. The percentage of COHb was calculated based on the absorbance at 420 and 432 nm with the reported extinction coefficients for mouse blood [START_REF] Rodkey | Spectrophotometric measurement of carboxyhemoglobin and methemoglobin in blood[END_REF]. The detection of CO content in adipose tissue following oral gavage with CORM-401 was determined by the method of Vreman et al. [START_REF] Vreman | Determination of carbon monoxide (CO) in rodent tissue: effect of heme administration and environmental CO exposure[END_REF]. For this, adipose tissues were initially placed in ice-cold potassium phosphate buffer (1:4 w/v) and stored at -80 C_until analysis. The day of the experiment, tissues were diced and sonicated and forty microliters of this suspension was added to CO-free sealed vials containing 5 mL of 30% (w/v) sulfosalicylic acid and incubated for 30 min on ice. CO released into the vial headspace was quantified by gas chromatography. CO accumulated in cultured 3T3-L1 adipocytes after treatement with CORM-401 was measured spectrophotometrically using a specific scavenger of CO (hemoCD1) as previously described [START_REF] Minegishi | Detection and Removal of Endogenous Carbon Monoxide by Selective and Cell-Permeable Hemoprotein Model Complexes[END_REF].

Statistics.

  Data are expressed as mean values ± standard error of the mean (SEM). Statistical analysis were performed using the unpaired 2-tailed Student's t test or one-way or two-way analysis of variance (ANOVA) with Fisher multiple comparison test. The result were considered significant if the p-value was <0.05. Study approval. All animals received care according to institutional guidelines, and all experiments were approved by the Institutional Ethics committee number 16, Paris, France (licence number 16-090).

FigureFigure 1 .

 1 Figure

Figure 2 .

 2 Figure 2. CORM-401 improves visceral adipose tissue function in HFD-induced obesity. Mice on standard (SD) or high fat diet (HFD) were sacrificed after 14weeks. CORM-401 (30 mg/kg) was given by oral gavage starting either at the beginning (HFD+CORM-T) or after 6 weeks (HFD+CORM-P) HFD. Pictures and representative sections of epididimal white adipose tissue (eWAT) stained with hematoxylin-eosin (A). eWAT was also evaluated for weight (B), adipocytes size (C), free fatty acids (FFA) levels from conditioned media (D), mRNA expression of genes involved in metabolism (E), CD68 expression (F, G), mRNA expression of CCl2 (H) and Hmox-1 (I) as well as interleukins content from conditioned media (J). mRNA

Figure 3 .

 3 Figure 3. CO delivered by CORM-401 increases systemic and adipocytes insulin sensitivity both in vivo and in vitro. CO content was measured in epididimal white adipose tissue (eWAT) after oral gavage with PBS (CON), inactive CORM that does not release CO (iCORM, 30 mg/kg) or CORM-401 (30 mg/kg) (A). An ITT was performed in fasted mice 1 h after oral gavage with PBS (CON), iCORM (30 mg/kg) or CORM-401 (30 mg/kg) (B), while blood glucose levels were measured at the times indicated (C) and represented by area under the curve (ITT AUC) (D). eWAT was evaluated for protein expression of total Akt and phosphorylated Akt assessed by western blot (E, F). Intracellular CO level in 3T3-L1 adipocytes after CORM-401 exposure at 50 µM for 3 h (G). Proteins were extracted from quiescent 3T3-L1 adipocytes after 3 h exposure to PBS (CON), iCORM (50 µM) or CORM-401 (50 µM) (see protocol in H). Expression of total Akt and phosphorylated Akt was assessed by western blot (F, G). Results are expressed as mean ± SEM. A=4-6 mice per group; C-D=10 mice per group; E-F=8 mice per group; G=3 in triplicates, J=4 independent experiments. *p<0.05 vs. control group (CON) and #p<0.05 vs. insulin, values not designated with symbols are not statistically different, one-way ANOVA with Fisher multiple comparison test.

Figure 4 .

 4 Figure 4. CO induces a metabolic switch in adipocytes in vitro and in vivo. Oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) were measured in 3T3-L1 cells after addition of PBS (CON) or CORM-401 (25, 50, 100 μM). A MitoStress assay was performed on 3T3-L1 adipocytes after first addition of PBS or CORM-401 followed by sequential addition of oligomycin, FCCP and rotenone/antimycin A (A). ATP-linked respiration rate was calculated by subtracting OCR values after oligomycin addition from basal OCR values and proton leak was calculated by subtracting OCR values after R/AA addition from OCR values after oligomycin addition (B). OCR (C) and ECAR (D) were measured for 6 h after addition of CORM-401. Lactate (E) and intracellular ATP (H) were measured 3 h after exposure to iCORM or CORM-401 with or without 2-deoxyglucose (2-DG). A glycolytic assay was performed after addition of iCORM or CORM-401 (50 μM) followed by sequential addition of glucose, oligomycin and 2-DG (F). Glycolysis rate was calculated by subtracting ECAR values after glucose addition from basal ECAR values and glycolytic reserve was calculated by subtracting ECAR values after oligomycin addition from basal ECAR values (G). Experiments using the Seahorse Analyzer were performed on punches of eWAT collected from mice 2 h after oral

Table 1 . Food intake Food intake (g/mouse/day)

 1 Results are means ± SEM; 2 Means with different superscript letters are significantly different among the same period (before treatment or during treatment), p < 0.05, one-way ANOVA with Fisher multiple comparison test.

	Tables
		1-2 SD	HFD	HFD+CORM-T HFD+CORM-P
	4 th week	3.8 ± 0.2 a 2.8 ± 0.2 b 2.8 ± 0.2 b	2.6 ± 0.3 b
	8 th week	3.6 ± 0.1 a 3.1 ± 0.1 b 3.0 ± 0.2 b	2.7 ± 0.1 b
	1

Table 2 .

 2 Blood analysis Conjugated bilirubin (µmol/L) 0.62 ± 0.2 a 0.47 ± 0.07 a 0.46 ± 0.06 a 0.36 ± 0.03 a Lactate deshydrogenase (U/L) 191.2 ± 56.3 a 248.1 ± 46.5 a 321.3 ± 93.8 a 238 ± 66.3 a ALAT: Aspartate amino transferase, ASAT: Alanine amino transferase. 1 Results are means ± SEM; 2 Means with different superscript letters are significantly different, p < 0.05, one-way ANOVA with Fisher multiple comparison test.

		SD	HFD	HFD+CORM-T HFD+CORM-P
	ASAT/ALAT	3.0 ± 0.5 a	2.5 ± 0.9 a	3.6 ± 0.9 a	2.9 ± 0.7 a
	Total bilirubin (µmol/L)	6.46 ± 2.1 a	4.8 ± 2.1 a	7.4 ± 2.2 a	4.82 ± 1.9 a
	Creatinine (mmol/L)	8 ± 0.8 a	10.13 ± 1.8 a 8.37 ± 1.0 a	7.43 ± 1.5 a

Acknowledgements

This work was supported by a grant from the French National Research Agency (ANR) (ANR-15-RHUS-0003). The authors thank Dr. Jayne-Louise Wilson for her support in performing experiments with the Seahorse Analyzer. The authors also thank Rachid Souktani and Cécile Lecointe for help in the animal facility platform; Xavier Decrouy, Christelle Gandolphe and Wilfried Verbecq-Morlot from the histology platform; and Stéphane Moutereau at Henri Mondor Hospital for blood analysis.

Author Contributions

L.B., R.F. and R.M. designed the study, performed experiments, collected and analyzed data and wrote the manuscript. L.B. and M.P. performed metabolic tests. L.M. performed CO measurements in adipose tissue. S.M. performed histological staining. H.K. provided critical materials and advice on the use of hemoCD. L.B., M.P., D.S. and G.C. collected organs for analysis. J.T. performed the echocardiography. G.D. analyzed the echocardiography and read the manuscript. R.M. is the guarantor of this work and, as such, had full access to all the data in this study and takes responsibility for the integrity of the data and the accuracy of the data analysis.

Conflict of interest:

The authors have declared that no conflict of interest exists.


William Souza Da Cruz

Jean-Max Dutertre

Jean-Baptiste Rigaud
email: rigaud@emse.frguillaume.hubert@onera.fr

Guillaume Hubert

Evidence of a Dynamic Fault Model in the DICE Radiation-Hardened Cell

hardened memory cell, to show the possibility of inducing failures in that same memory cell and to propose a countermeasure to detect and treat such failures. All data obtained and analyses presented were performed through parametric simulations of electronic devices and circuits, taking into account a simple approach based on transient pulses consistent with TCAD or multi-physics tools, allowing observing effects in standard or hardened cells [START_REF] Artola | Seu prediction from set modeling using multi-node collection in bulk transistors and srams down to the 65 nm technology[END_REF].

This article is organized as follows. Section II addresses the effects of radiation in integrated circuits (IC), focusing on SEE, techniques for mitigating these effects, it also introduces the radiation-hardened Dual-Interlock-Storage-Cell (DICE). Section III presents the possibility of injecting a dynamicfault in the DICE cell, showing through simulations that it is possible to prevent the writing of data in a memory cell of this type. Section IV proposes a countermeasure against such dynamic-faults, describing its operation and showing through results by simulation its effectiveness. The conclusions of this study are presented in section V.

II. RADIATION HARDENING

A. Radiation-induced faults and mitigation techniques 1) Radiation-induced errors: When exposed to harsh and radioactive environments ICs may suffer from various types of radioactive effects. In this work, we do not consider the cumulative total ionizing dose effects induced by X or gamma rays, protons and electrons, that can cause IC malfunction mainly due to threshold shifts and increased leakage [START_REF] Barnaby | Total-ionizing-dose effects in modern cmos technologies[END_REF]. We rather focused on SEEs that are induced by ionizing particles passing through a device and that may induce transient errors [START_REF] Baumann | Soft errors in advanced computer systems[END_REF].

As an ionizing particle passes through silicon, it creates electron-hole pairs along its track due to direct ionization by the incident particle itself or ionization by secondary particles created by nuclear reactions between the incident particle and the affected device [START_REF] Dodd | Physics-based simulation of single-event effects[END_REF]. These charge carriers may recombine without any noticeable effect on the circuit's activity. An exception exists when the charge carriers are induced in the vicinity of a transistors reverse biased PN junction (drain/bulk, source/bulk or Nwell/Psubstrate): a place where there exists a strong electric field [START_REF] Dodd | Physics-based simulation of single-event effects[END_REF]. As a consequence, the charge carriers drift in opposite directions and a current pulse is induced (it vanishes as the charges are exhausted). It may last a few Abstract-Hardware redundancy techniques are routinely used for mitigation purposes in radiation-hardened integrated circuits. Duplication indeed has been proved effective to harden the well known DICE memory cell against SEUs. In this work, we nonetheless report, on simulation basis, the possibility of a dynamic fault mechanism that may turn an SET into an error. We also introduce and validate a countermeasure that alleviates this weakness.

Index Terms-Radiation hardening, DICE memory cell, dynamic fault model, SEE

I. INTRODUCTION

Electronic devices are continuously improved in order to reduce their dimensions [START_REF] Colinge | Physics of Semiconductor Devices, ser. Physics of semiconductor devices[END_REF] and still become more robust to failures [START_REF] Duzellier | Radiation effects on electronic devices in space[END_REF]. Among the different causes of failures to which an electronic device is subject, there are environment perturbations (e.g. by intense solar activity) [START_REF] Ziegler | Effect of cosmic rays on computer memories[END_REF] and humaninduced perturbations (e.g. laser attacks aimed at acquiring sensitive information) [START_REF] Kim | Faults, injection methods, and fault attacks[END_REF]. The study of the different effects to which these devices are exposed is important to maintain their proper functioning when operating in extreme environments or under malicious attacks.

Considering space applications, the robustness of electronic devices in the face of ionizing radiation is of great importance. It can cause damage to collected data, operational problems in the systems, and premature wear of electronic components. This study focuses on Single Event Effects (SEE), which are random in nature and can be caused by a single ionizing particle with enough energy to induce disturbances or even a permanent failure in electronic systems [START_REF] Baumann | Soft errors in advanced computer systems[END_REF]. This type of effect can be classified a s d estructive o r t ransient, t he latter being the focus of this study.

There are different approaches to mitigate or counter such effects on electronic devices and systems, including the use of new materials and manufacturing processes [START_REF] Mistry | A 45nm logic technology with high-k+ metal gate transistors, strained silicon, 9 cu interconnect layers, 193nm dry patterning, and 100% pb-free packaging[END_REF], new MOSFET layouts [START_REF] Gimenez | Layout Techniques for MOSFETs[END_REF] and through the design of circuit-level hardening techniques that detect and prevent certain types of failures [START_REF] Dutertre | Integration of robustness in the design of a cell[END_REF].

Within this context, this study was carried out in order to evaluate the functioning of a SEU (Single Event Upset) hundreds of picoseconds and may have an amplitude as large as a few mA (depending on the amount of induced charges and on the dynamic of their collection) [START_REF] Baumann | Soft errors in advanced computer systems[END_REF]. This charge carriers collection phenomenon can be decomposed in two successive parts [START_REF] Wang | Single event upset: An embedded tutorial[END_REF]. At first, the depletion region (hence the electric field) is stretched along the particle track, the charges nearby are collected in a few picoseconds generating a peak current: a phenomenon called funneling [START_REF] Wang | Single event upset: An embedded tutorial[END_REF]. In a second time, the remaining charges are collected in a longer phenomenon, called diffusion [START_REF] Wang | Single event upset: An embedded tutorial[END_REF]. The current decreases slowly until all charges are collected. In turn, this current pulse creates a transient voltage pulse, which may induce an error if induced (1) directly in a memory cell (a SEU) or (2) in a logic gate and if it propagates until being captured by a downstream Flip-Flop (a Single Event Transient, SET).

2) Mitigation techniques: Since the first evidence of SEEs [START_REF] Ziegler | Effect of cosmic rays on computer memories[END_REF], the radiation effect community started studying and developing countermeasures. Several mitigation principles were introduced: Error Detection And Correction techniques (or EDAC, e.g. based on spatial or temporal redundancy), sensors monitoring the currents at the root cause of SEEs (e.g. the bulk current sensors introduced by [START_REF] Neto | Using bulk built-in current sensors to detect soft errors[END_REF]), cells hardening through architecture redesign [START_REF] Dutertre | Integration of robustness in the design of a cell[END_REF], [START_REF] Calin | Upset hardened memory design for submicron CMOS technology[END_REF], or even the use of the Silicon On Insulator (SOI) technology as an alternative to the usual CMOS bulk [START_REF] Roche | Technology downscaling worsening radiation effects in bulk: Soi to the rescue[END_REF]. Spatial or temporal redundancy techniques [START_REF] Pratt | Improving fpga design robustness with partial tmr[END_REF], [START_REF] Nicolaidis | Time redundancy based soft-error tolerance to rescue nanometer technologies[END_REF] are often used to mitigate SEEs, they respectively consist in making several times the same logical operations with redundant hardware blocks or at different moments (using the same hardware). It relies on the assumption that only one set of operations would be erroneous due to the local and nondeterministic nature of SEEs, making it possible to detect errors (in the case of duplication) or even to correct them (using triplication with majority vote [START_REF] Nicolaidis | Time redundancy based soft-error tolerance to rescue nanometer technologies[END_REF]).

In our research, we considered a SEU-hardened memory cell using a duplication technique, it is described hereafter.

B. The radiation-hardened Dual-Interlock-Storage-Cell

The DICE memory cell [START_REF] Calin | Upset hardened memory design for submicron CMOS technology[END_REF] achieves SEU hardening through duplication of the internal nodes storing its logical state. A usual 6T SRAM memory cell has two internal nodes which logical values, 01 or 10, represent either a 1 state or a 0 state. The DICE memory cell, depicted in figure 1, has four internal nodes Q1, Q11, Q2, Q22 to store its logical value: either 1010 to represent the logical state 1, or 0101 to represent state 0. It has four CMOS inverters with an original connection of the transistor gates. It is designed to recover from a single radiation-induced logical inversion of any of its internal nodes (see subsection II-C for a simulation-based illustration of the mechanism at play). Access to the internal nodes for writing and reading operations takes place simultaneously through four access transistors (M8, M9, M10, M11 in figure 1).

C. Simulation of the DICE cell mitigating a SEU

In this subsection, we illustrate on simulation-based, the SEU mitigation mechanism of the DICE memory cell 1 . As a radiation-induced transient current is at the root cause of SEEs, we used a current source to model this phenomenon in our analog simulations. It outputs a tunable double exponential current transient based on the modeling works reported in [START_REF] Douin | Influence of laser pulse duration in single event upset testing[END_REF], [START_REF] Sarafianos | Building the electrical model of the photoelectric laser stimulation of a pmos transistor in 90 nm technology[END_REF]. Orders of magnitude of amplitudes and temporal profiles used in electrical simulations are consistent with transient pulse issued from TCAD or multi-physics tools [START_REF] Artola | Seu prediction from set modeling using multi-node collection in bulk transistors and srams down to the 65 nm technology[END_REF], [START_REF] Rostand | Compact modelling of single event transient in bulk mosfet for spice: Application to elementary circuit[END_REF]. The main parameters we considered were the current peak value (up to a few hundreds of µA) and the pulse duration (up to several hundreds of ps). Simulations were performed on circuits designed at the 65 nm CMOS technology node (VDD = 1.2 V). From the schematic of the DICE cell (and of the other gates used in our tests), we derived a layout and extracted its parasitic elements that we used in our simulations.

We considered an SEE arising when the DICE cell stores a 0 state (Q1 = Q2 = 0 and Q11 = Q22 = 1). It consists in a transient current denoted I rad in figure 2 (top red waveform). It was applied between the drain of transistor M0 (a SEE-sensitive transistor in state 0) and its bulk biasing contact to VDD. It starts at 2.6 ns in the simulation, at this time the DICE cell is in memory mode (i.e. its access transistors are in OFF state with their command signal wr at 0). It has a peak amplitude of 200 µA, a rise time of 0.04 ns and a fall time of 0.38 ns. Figure 2 also displays the effect of the transient current on the cell's four internal nodes Q1, Q11, Q2, and Q22.

The main effect of the transient current is to drive Q1's voltage from 0 V to 1.2 V. As a result, transistor M1 is turned OFF, which puts node Q11 into a high impedance state (HZ) because transistor M5 is also OFF: the HZ state preserves Q11's voltage that remains almost unchanged. Q1's voltage modification also turns transistor M7 ON; while M3 is ON, a direct consequence is that the voltage of node Q22 goes approximately to VDD/2. As the transient current vanishes, Q1 returns to its original 0 V voltage (the change in Q22 voltage was not sufficient to prevent this through the command of transistor M0). Then, Q22 also returns to its initial voltage. The DICE cell was able to withstand the SEU thanks to the duplication of its internal nodes and to the preservation of its state on the duplicated nodes. For illustration purposes, we also ascertained the same SEU mitigation property when increasing the transient current duration to 0.17 ns and 0.37 ns (drawn in light gray in Fig. 2). These parameters are far beyond the threshold needed to induce a SEU in a 6T SRAM cell [START_REF] Gorbunov | Design of 65 nm cmos sram for space applications: A comparative study[END_REF], [START_REF] Hubert | Single-event transient modeling in a 65-nm bulk cmos technology based on multi-physical approach and electrical simulations[END_REF] (note that these shapes of transient currents are no longer consistent with those induced by a SEE, however, it is an easy way to illustrate the mitigation properties of the DICE cell against an increase of the collected charge). Similar behaviors and the same SEU mitigation property are observed when the transient current is applied to the other nodes of the DICE cell (either in state 1 or 0).

This illustrates the strength of the DICE memory cell in mitigating SEUs in memory mode. However, its immunity to SEU may be put at risk during the writing phase as we explain in the next section.

III. INJECTION OF A DYNAMIC FAULT INTO A RADIATION-HARDENED CELL

The previous section acknowledges on simulation basis the (already known) ability of the DICE memory cell to withstand SEUs. However, if a single transient current is unable to induce a flip of the DICE's state, we wondered whether it may prevent a value to be written inside the DICE during the writing phase. Thus, inducing an error through a mechanism that shares similarities with a SET. It may happen if the logical state to be written is the complementary value of the DICE current state (e.g. while trying to write a 1 inside a DICE cell storing a 0). The DICE cells are interconnected in parallel, so that each one can be accessed at a time for writing or reading through the activation of its wr signal. Substituting DICE to SRAM cells doubles the number of bit lines (a DICE cell has four inputs/outputs): denoted bus data 1,2 and bus data 1,2 . We considered a case study of a circuit using duplication as a radiation-hardening technique. Hence, the value to be written is duplicated in signals data i 1 and data i 2 which are fed to the bit lines through four buffers (and two inverters for inversion purposes) commanded by a write signal. The data o 1 and data o 2 output signals are obtained thank to sense amplifier blocks connected to the bit lines (they are not considered in this study). The write phase involved the successive activation of two signals: write and wr to propagate, respectively, the data to be written to the bit lines and then inside the DICE cell (they respectively last 140 ps and 360 ps, settings related to a reliable write in error-free operation). The dynamic fault is caused by a current pulse (denoted I rad in Fig. 4) starting shortly after the activation of the write signal: it has an amplitude of 100 µA and a duration of 200 ps. It is induced on the data i 1 signal and propagates, in the form of SETs, to the bus data 1 and bus data 1 bit lines (depicted in black2 in the third and fourth waveforms of Fig. 4). It prevents them from reaching the normal 1.2 V and 0 V voltages they should attain before the activation of wr (the bus data 2 and bus data 2 bit lines, depicted in red, are not corrupted). As a result, nodes Q1 and Q11 are almost stuck at their initial 0 V and 1.2 V values during the whole write window (depicted in black in the two bottom waveforms). Though, nodes Q2 and Q22 (depicted in red) are at first evolving to follow the write value, they finally return to their initial value when the write window closes (wr goes back to 0 V). Note that this dynamic fault phenomenon resembles the SEU mitigation mechanism described in subsection II-C: the voltages of two nodes (here Q1 and Q11) prevent the DICE cell state from changing.

A. DICE-based RAM memory

B. Simulation-based evidence of dynamic faults

Two important parameters to characterize how transient current contributes to the induction of a fault are the critical charge (Q crit ) and the collected charge (Q coll ) [START_REF] Detcheverry | Seu critical charge and sensitive area in a submicron cmos technology[END_REF]. The collected charge is defined by the integral of the transient electric current over time, allowing the calculation of the charge injected in the node of the circuit by the event [START_REF] Detcheverry | Seu critical charge and sensitive area in a submicron cmos technology[END_REF]. The critical charge, in turn, is the minimum collected charge necessary for such a fault to occur [START_REF] Detcheverry | Seu critical charge and sensitive area in a submicron cmos technology[END_REF]. For evaluation purposes and to allow other studies to be carried out from this one, the charge collected in this simulation was calculated, being a value of approximately 28.5 fC. This value is consistent with the collected charge values expected for nano technologies, as can be seen in [START_REF] Hubert | Impact of scaling on the soft error sensitivity of bulk, fdsoi and finfet technologies due to atmospheric radiation[END_REF], and it is also a value close to the critical charge, since the simulations were performed using the smallest amplitude of electric current and pulse duration that generated a fault in the circuit.

This simulation shows that a SEE induced at a single node (data i 1 ) may induce an error in a SEE-hardened DICE cell if it happens during its writing phase, a so-called dynamic fault. Similar dynamic faults may also be induced when the initial SET arises on node data i 2 and for a 1 state to 0 state writing of the DICE cell.

IV. THWARTING DYNAMIC FAULTS

This section aims to present a countermeasure, named Holding Block (HB), which detects and thwarts the effects of such a dynamic error. In a first step, the description of this circuit explains how to detect a disturbance arising during the writing phase. Then, a solution is presented to guaranty the completion of the writing process of the DICE cell. The general overview of this protection circuit inside the memory map is proposed. Finally, simulations demonstrate the positive effect of HB.

A. Countermeasure Description

In the previous section, we demonstrated how a SET induced during the writing phase, may result into creating a dynamic fault that corrupts the DICE memory cell. Here, a countermeasure is presented to fight against this weakness. Its underlying principle is to take advantage of the transient nature of SEEs by extending the write window beyond the perturbation duration (by an amount of time sufficient to ensure the correct completion of the write process). The whole protection circuit is depicted in figure 5. To strengthen the memory, we first need to detect the dynamic fault. To that end, a XOR gate between bus data 1 and bus data 2 is used to raise an alarm signal if there is a difference between the states of the bus wires. HB has one input, the alarm signal and one output, the hold signal. It has to increase the write and wr signals until the attack ends (i.e. alarm returns to zero) and until that the selected memory cell is written to the expected value. So, the hold signal needs to stay at high level even after the alarm signal has been raised and pulled down (its duration is that of the transient perturbation). Simulations of two cases of how the hold signal is built in the HB block are displayed in figure 6. A delay element δ is used to generate a delayed image of the alarm: alarm δ . Two cases have to be considered:

• First, the case of an alarm signal that is longer than δ (as reported in the right part of Fig. 6). Combining alarm and alarm δ with an OR gate, signal or o is obtained which shape is a pulse of a duration equal to the duration of alarm added to δ (passed on to hold through another OR gate). • The second case may arise if the alarm signal is shorter than δ (drawn in the left part of Fig. 6). In this case, or o has a two pulses shape that cannot be used to extend the write window. We used a simple RS OR-AND latch circuit to build the rs o signal and to cover this second case. The obtained signal rs o produced a suitable hold signal when ORed with or o . In both cases, the hold signal keeps a high logical level that may be used to extend the wr and write pulse durations inside the RAM memory. The next subsection describes the insertion of the HB block inside the memory.

B. Countermeasure insertion inside the memory block

Figure 7, describes how to insert two Holding Block circuits in the DICE-based RAM memory. As we have two duplicated data buses, (bus data 1,2 ) and (bus data 1,2 ), an HB circuit is needed for each one. Each bus has its own fault detector, producing signals alarm 1 and alarm 2 . Then, hold 1 and hold 2 signals are looped back inside all the previous wr and write signals. This combinatorial function is done with the two threeinputs OR gates. In normal functioning (i.e. SEE-free), the writing process is guaranteed by the original writing signals write and wr. When a SEE occurs, depending on which bus is targeted, hold 1 or hold 2 will increase the write time window of the memory cell in order to write the correct value inside the DICE cell.

C. Simulation of the dynamic fault countermeasure

Figure 8 reports the simulation results obtained after updating our design with the HB countermeasure (as exposed in Fig. 7). We considered the same settings as in subsection III-B: an SEE that prevented the DICE cell from being written (from state 0 to state 1).

The radiation-induced transient current, I rad , starts at 2.1 ns with a 100 µA peak current and a 200 ps duration (identical to the I rad of Fig. 4). From the onset of this SEE up to 360 ps later (the normal error-free duration of the write signal), the bit line signals and the internal nodes of the DICE evolve in an identical way as in Fig. 4. From that point, the write and wr signals stay activated thanks to the hold 1,2 signals for a duration equal to the SEE duration (200 ps in this instance) added to δ (we set δ equal to the activation duration of wr). As a result, the write window is increased in order to allow for the write process to be completed without any error: the DICE cell goes to state 1. This behavior can be observed on signals bus data 1 , bus data 1 , Q1, Q11, Q2, and Q22. For illustration purposes, the dashed gray waveforms in Fig. 8 reproduce the dynamic fault case.

Note that the alarm 1,2 signals are also used to indicate at higher abstraction level that the write process lasts longer than usual (it may be necessary to delay a subsequent write operation).

V. CONCLUSION

The results presented in this article reveal an original dynamic fault mechanism in the architecture of the DICE radiation-hardened cell: the simulations we performed made it possible to induce a dynamic-fault preventing the correct writing of data in the memory cell. As an improvement alternative, a circuit was proposed to detect and prevent the induction of the faults seen here. The performed simulations indicate an adequate functioning of the proposed mitigation circuit as an additional countermeasure to the system. This first study opens doors for carrying out experimental tests and new studies using multi-physics approach, both in relation to the injection of dynamic-faults in the DICE cell, as well as for a better evaluation of the additional circuit proposed as a countermeasure (a 65 nm CMOS ASIC embedding DICE cells was manufactured). These studies together can validate the data presented here and contribute to the development of electronic systems more robust to failures by SEE.

Fig. 1 .

 1 Fig. 1. Transistor-level view of the DICE cell.

Fig. 2 .

 2 Fig. 2. Simulation of an electric current induced (I rad ) at node Q1 of the DICE cell.

Fig. 3 .

 3 Fig. 3. DICE test bench for dynamic fault testing.

Figure 3

 3 Figure 3 displays the test bench used in our simulations. It represents the architecture of one column of the RAM memory made of DICE memory cells (numbered from 1 to n).The DICE cells are interconnected in parallel, so that each one can be accessed at a time for writing or reading through the activation of its wr signal. Substituting DICE to SRAM cells doubles the number of bit lines (a DICE cell has four inputs/outputs): denoted bus data 1,2 and bus data 1,2 . We considered a case study of a circuit using duplication as a radiation-hardening technique. Hence, the value to be written is duplicated in signals data i 1 and data i 2 which are fed to the bit lines through four buffers (and two inverters for inversion purposes) commanded by a write signal. The data o 1 and data o 2 output signals are obtained thank to sense amplifier blocks connected to the bit lines (they are not considered in this study).

Figure 4

 4 Figure 4 displays the simulation results of a dynamic fault induced into a DICE cell preventing it from passing from a 0 to a 1 state.The write phase involved the successive activation of two signals: write and wr to propagate, respectively, the data to be written to the bit lines and then inside the DICE cell (they

Fig. 4 .

 4 Fig. 4. Simulation of a dynamic fault targeting a DICE cell.

Fig. 5 .

 5 Fig. 5. Architecture of the Holding Block Circuit.

Fig. 6 .

 6 Fig. 6. Simulation of the HB response for different alarm durations.

Fig. 7 .

 7 Fig. 7. HB insertion in the DICE based memory.

Fig. 8 .

 8 Fig. 8. Simulation of the DICE test bench with countermeasures.

Note that we did not consider any charge sharing effect between several sensitive areas in this work.

The simulation results of a SEE-free simulation are also depicted in dashed gray lines in Fig.4for illustration purposes.

ACKNOWLEDGMENT

This research has been supported by a PhD grant from the French région PACA (Emplois Jeunes Doctorants).

This research has been supported by a PhD grant from the French région PACA (Emplois Jeunes Doctorants).