Treating MS without continuous immunosuppression: an interview with Luciano Rossetti.

Luciano Rossetti, MD, Executive Vice President, Global Head of R&D at Merck KGaA, Darmstadt, Germany speaks to Laura Dormer, Commissioning Editor Luciano Rossetti, MD, is Executive Vice President, Global Head of R&D at Merck KGaA, Darmstadt, Germany, and a member of the Healthcare Executive Committee. As Global Head of R&D, Rossetti leads the strategy for Merck KGaA, Darmstadt, Germany's discovery and development efforts in healthcare. He joined Merck KGaA in July 2014, and has since led the acceleration of several key programs through the pipeline and advanced the innovation of Merck KGaA's discovery teams into development. Under his leadership, R&D has been transformed, with significant potential to deliver the scientific, clinical, regulatory and medical excellence of its teams to patients in need. Before joining Merck KGaA, he served as Senior Vice President, responsible for Global Scientific Strategy and Late Stage Development at Merck Sharp & Dohme, or MSD. In the latter position, he was responsible for clinical development from Phase II to V across all therapeutic areas. Prior to joining MSD in 2006, he spent 18 years in academia during which he had considerable involvement with the pharmaceutical industry in both discovery and development. In his latest assignment, he was Professor of Medicine and led the Diabetes Research & Training Center at the Albert Einstein College of Medicine. He has authored more than 150 peer-reviewed scientific journal articles. He is a Post-Doctoral Fellow of Rome University Medical School and of Yale University Medical School. He holds a Doctorate in Medicine from the Trieste University Medical School, Italy.

Translational Medicine Guide transforms drug development processes: the recent Merck experience.

Merck is implementing a question-based Translational Medicine Guide (TxM Guide) beginning as early as lead optimization into its stage-gate drug development process. Initial experiences with the TxM Guide, which is embedded into an integrated development plan tailored to each development program, demonstrated opportunities to improve target understanding, dose setting (i.e., therapeutic index), and patient subpopulation selection with more robust and relevant early human-based evidence, and increased use of biomarkers and simulations. The TxM Guide is also helping improve organizational learning, costs, and governance. It has also shown the need for stronger external resources for validating biomarkers, demonstrating clinical utility, tracking natural disease history, and biobanking.

Mediobasal hypothalamic SIRT1 is essential for resveratrol's effects on insulin action in rats.

OBJECTIVE: Sirtuin 1 (SIRT1) and its activator resveratrol are emerging as major regulators of metabolic processes. We investigate the site of resveratrol action on glucose metabolism and the contribution of SIRT1 to these effects. Because the arcuate nucleus in the mediobasal hypothalamus (MBH) plays a pivotal role in integrating peripheral metabolic responses to nutrients and hormones, we examined whether the actions of resveratrol are mediated at the MBH. RESEARCH DESIGN AND METHODS: Sprague Dawley (SD) male rats received acute central (MBH) or systemic injections of vehicle, resveratrol, or SIRT1 inhibitor during basal pancreatic insulin clamp studies. To delineate the pathway(s) by which MBH resveratrol modulates hepatic glucose production, we silenced hypothalamic SIRT1 expression using a short hairpin RNA (shRNA) inhibited the hypothalamic ATP-sensitive potassium (K(ATP)) channel with glibenclamide, or selectively transected the hepatic branch of the vagus nerve while infusing resveratrol centrally. RESULTS: Our studies show that marked improvement in insulin sensitivity can be elicited by acute administration of resveratrol to the MBH or during acute systemic administration. Selective inhibition of hypothalamic SIRT1 using a cell-permeable SIRT1 inhibitor or SIRT1-shRNA negated the effect of central and peripheral resveratrol on glucose production. Blockade of the K(ATP) channel and hepatic vagotomy significantly attenuated the effect of central resveratrol on hepatic glucose production. In addition, we found no evidence for hypothalamic AMPK activation after MBH resveratrol administration. CONCLUSIONS: Taken together, these studies demonstrate that resveratrol improves glucose homeostasis mainly through a central SIRT1-dependent pathway and that the MBH is a major site of resveratrol action.

Divergent regulation of energy expenditure and hepatic glucose production by insulin receptor in agouti-related protein and POMC neurons.

OBJECTIVE: The sites of insulin action in the central nervous system that regulate glucose metabolism and energy expenditure are incompletely characterized. We have shown that mice with hypothalamic deficiency (L1) of insulin receptors (InsRs) fail to regulate hepatic glucose production (HGP) in response to insulin. RESEARCH DESIGN AND METHODS: To distinguish neurons that mediate insulin's effects on HGP from those that regulate energy homeostasis, we used targeted knock-ins to express InsRs in agouti-related protein (AgRP) or proopiomelanocortin (POMC) neurons of L1 mice. RESULTS: Restoration of insulin action in AgRP neurons normalized insulin suppression of HGP. Surprisingly, POMC-specific InsR knock-in increased energy expenditure and locomotor activity, exacerbated insulin resistance and increased HGP, associated with decreased expression of the ATP-sensitive K(+) channel (K(ATP) channel) sulfonylurea receptor 1 subunit, and decreased inhibitory synaptic contacts on POMC neurons. CONCLUSIONS: The contrasting phenotypes of InsR knock-ins in POMC and AgRP neurons suggest a branched-pathway model of hypothalamic insulin signaling in which InsR signaling in AgRP neurons decreases HGP, whereas InsR activation in POMC neurons promotes HGP and activates the melanocortinergic energy expenditure program.

The Irs1 branch of the insulin signaling cascade plays a dominant role in hepatic nutrient homeostasis.

We used a Cre-loxP approach to generate mice with varied expression of hepatic Irs1 and Irs2 to establish the contribution of each protein to hepatic nutrient homeostasis. While nutrient-sensitive transcripts were expressed nearly normally in liver lacking Irs2 (LKO2 mice), these transcripts were significantly dysregulated in liver lacking Irs1 (LKO1 mice) or Irs1 and Irs2 together (DKO mice). Similarly, a set of key gluconeogenic and lipogenic genes was regulated nearly normally by feeding in liver retaining a single Irs1 allele without Irs2 (DKO/1 mice) but was poorly regulated in liver retaining one Irs2 allele without Irs1 (DKO/2 mice). DKO/2 mice, but not DKO/1 mice, also showed impaired glucose tolerance and insulin sensitivity-though both Irs1 and Irs2 were required to suppress hepatic glucose production during hyperinsulinemic-euglycemic clamp. In contrast, either hepatic Irs1 or Irs2 mediated suppression of HGP by intracerebroventricular insulin infusion. After 12 weeks on a high-fat diet, postprandial tyrosine phosphorylation of Irs1 increased in livers of control and LKO2 mice, whereas tyrosine phosphorylation of Irs2 decreased in control and LKO1 mice. Moreover, LKO1 mice -- but not LKO2 mice -- that were fed a high-fat diet developed postprandial hyperglycemia. We conclude that Irs1 is the principal mediator of hepatic insulin action that maintains glucose homeostasis.

Hypothalamic sensing of circulating lactate regulates glucose production.

Emerging studies indicate that hypothalamic hormonal signalling pathways and nutrient metabolism regulate glucose homeostasis in rodents. Although hypothalamic lactate-sensing mechanisms have been described to lower glucose production (GP), it is currently unknown whether the hypothalamus senses lactate in the blood circulation to regulate GP and maintain glucose homeostasis in vivo. To examine whether hypothalamic sensing of circulating lactate is required to regulate GP, we infused intravenous (i.v.) lactate in the absence or presence of inhibition of central/hypothalamic lactate-sensing mechanisms in normal rodents. Inhibition of central/hypothalamic lactate-sensing mechanisms was achieved by three independent approaches. Tracer-dilution methodology in combination with the pancreatic clamp technique was used to assess the effect of i.v. and central/hypothalamic administrations on glucose metabolism in vivo. In the presence of physiologically relevant increases in the levels of plasma lactate, inhibition of central lactate-sensing mechanisms by lactate dehydrogenase inhibitor oxamate (OXA) or ATP-sensitive potassium channels blocker glibenclamide increased GP. Furthermore, direct administration of OXA into the mediobasal hypothalamus increased GP in the presence of similar elevation of circulating lactate. Together, these data indicate that hypothalamic sensing of circulating lactate regulates GP and is required to maintain glucose homeostasis.

SirT1 gain of function increases energy efficiency and prevents diabetes in mice.

In yeast, worms, and flies, an extra copy of the gene encoding the Sirtuin Sir2 increases metabolic efficiency, as does administration of polyphenols like resveratrol, thought to act through Sirtuins. But evidence that Sirtuin gain of function results in increased metabolic efficiency in mammals is limited. We generated transgenic mice with moderate overexpression of SirT1, designed to mimic the Sirtuin gain of function that improves metabolism in C. elegans. These mice exhibit normal insulin sensitivity but decreased food intake and locomotor activity, resulting in decreased energy expenditure. However, in various models of insulin resistance and diabetes, SirT1 transgenics display improved glucose tolerance due to decreased hepatic glucose production and increased adiponectin levels, without changes in body weight or composition. We conclude that SirT1 gain of function primes the organism for metabolic adaptation to insulin resistance, increasing hepatic insulin sensitivity and decreasing whole-body energy requirements. These findings have important implications for Sirtuin-based therapies in humans.

Activation of hypothalamic S6 kinase mediates diet-induced hepatic insulin resistance in rats.

Prolonged activation of p70 S6 kinase (S6K) by insulin and nutrients leads to inhibition of insulin signaling via negative feedback input to the signaling factor IRS-1. Systemic deletion of S6K protects against diet-induced obesity and enhances insulin sensitivity in mice. Herein, we present evidence suggesting that hypothalamic S6K activation is involved in the pathogenesis of diet-induced hepatic insulin resistance. Extending previous findings that insulin suppresses hepatic glucose production (HGP) partly via its effect in the hypothalamus, we report that this effect was blunted by short-term high-fat diet (HFD) feeding, with concomitant suppression of insulin signaling and activation of S6K in the mediobasal hypothalamus (MBH). Constitutive activation of S6K in the MBH mimicked the effect of the HFD in normal chow-fed animals, while suppression of S6K by overexpression of dominant-negative S6K or dominant-negative raptor in the MBH restored the ability of MBH insulin to suppress HGP after HFD feeding. These results suggest that activation of hypothalamic S6K contributes to hepatic insulin resistance in response to short-term nutrient excess.

Leptin controls adipose tissue lipogenesis via central, STAT3-independent mechanisms.

Leptin (encoded by Lep) controls body weight by regulating food intake and fuel partitioning. Obesity is characterized by leptin resistance and increased endocannabinoid tone. Here we show that leptin infused into the mediobasal hypothalamus (MBH) of rats inhibits white adipose tissue (WAT) lipogenesis, which occurs independently of signal transducer and activator of transcription-3 (STAT3) signaling. Correspondingly, transgenic inactivation of STAT3 signaling by mutation of the leptin receptor (s/s mice) leads to reduced adipose mass compared to db/db mice (complete abrogation of leptin receptor signaling). Conversely, the ability of hypothalamic leptin to suppress WAT lipogenesis in rats is lost when hypothalamic phosphoinositide 3-kinase signaling is prevented or when sympathetic denervation of adipose tissue is performed. MBH leptin suppresses the endocannabinoid anandamide in WAT, and, when this suppression of endocannabinoid tone is prevented by systemic CB1 receptor activation, MBH leptin fails to suppress WAT lipogenesis. These data suggest that the increased endocannabinoid tone observed in obesity is linked to a failure of central leptin signaling to restrain peripheral endocannabinoids.

Hypothalamic protein kinase C regulates glucose production.

OBJECTIVE: A selective rise in hypothalamic lipid metabolism and the subsequent activation of SUR1/Kir6.2 ATP-sensitive K(+) (K(ATP)) channels inhibit hepatic glucose production. The mechanisms that link the ability of hypothalamic lipid metabolism to the activation of K(ATP) channels remain unknown. RESEARCH DESIGN AND METHODS: To examine whether hypothalamic protein kinase C (PKC) mediates the ability of central nervous system lipids to activate K(ATP) channels and regulate glucose production in normal rodents, we first activated hypothalamic PKC in the absence or presence of K(ATP) channel inhibition. We then inhibited hypothalamic PKC in the presence of lipids. Tracer-dilution methodology in combination with the pancreatic clamp technique was used to assess the effect of hypothalamic administrations on glucose metabolism in vivo. RESULTS: We first reported that direct activation of hypothalamic PKC via direct hypothalamic delivery of PKC activator 1-oleoyl-2-acetyl-sn-glycerol (OAG) suppressed glucose production. Coadministration of hypothalamic PKC-delta inhibitor rottlerin with OAG prevented the ability of OAG to activate PKC-delta and lower glucose production. Furthermore, hypothalamic dominant-negative Kir6.2 expression or the delivery of the K(ATP) channel blocker glibenclamide abolished the glucose production-lowering effects of OAG. Finally, inhibition of hypothalamic PKC eliminated the ability of lipids to lower glucose production. CONCLUSIONS: These studies indicate that hypothalamic PKC activation is sufficient and necessary for lowering glucose production.

Regulation of energy substrate utilization and hepatic insulin sensitivity by phosphatidylcholine transfer protein/StarD2.

Phosphatidylcholine transfer protein (PC-TP, also known as StarD2) is a highly specific intracellular lipid binding protein with accentuated expression in oxidative tissues. Here we show that decreased plasma concentrations of glucose and free fatty acids in fasting PC-TP-deficient (Pctp(-/-)) mice are attributable to increased hepatic insulin sensitivity. In hyperinsulinemic-euglycemic clamp studies, Pctp(-/-) mice exhibited profound reductions in hepatic glucose production, gluconeogenesis, glycogenolysis, and glucose cycling. These changes were explained in part by the lack of PC-TP expression in liver per se and in part by marked alterations in body fat composition. Reduced respiratory quotients in Pctp(-/-) mice were indicative of preferential fatty acid utilization for energy production in oxidative tissues. In the setting of decreased hepatic fatty acid synthesis, increased clearance rates of dietary triglycerides and increased hepatic triglyceride production rates reflected higher turnover in Pctp(-/-) mice. Collectively, these data support a key biological role for PC-TP in the regulation of energy substrate utilization.

Impaired regulation of hepatic glucose production in mice lacking the forkhead transcription factor Foxo1 in liver.

The hallmark of type 2 diabetes is excessive hepatic glucose production. Several transcription factors and coactivators regulate this process in cultured cells. But gene ablation experiments have yielded few clues as to the physiologic mediators of this process in vivo. We show that inactivation of the gene encoding forkhead protein Foxo1 in mouse liver results in 40% reduction of glucose levels at birth and 30% reduction in adult mice after a 48 hr fast. Gene expression and glucose clamp studies demonstrate that Foxo1 ablation impairs fasting- and cAMP-induced glycogenolysis and gluconeogenesis. Pgc1alpha is unable to induce gluconeogenesis in Foxo1-deficient hepatocytes, while the cAMP response is significantly blunted. Conversely, Foxo1 deletion in liver curtails excessive glucose production caused by generalized ablation of insulin receptors and prevents neonatal diabetes and hepatosteatosis in insulin receptor knockout mice. The data provide a unifying mechanism for regulation of hepatic glucose production by cAMP and insulin.

Identification and characterization of a novel phosphorylation site on the GluR1 subunit of AMPA receptors.

Phosphorylation of various AMPA receptor subunits can alter synaptic transmission and plasticity at excitatory glutamatergic synapses in the central nervous system. Here, we identified threonine-840 (T840) on the GluR1 subunit of AMPA receptors as a novel phosphorylation site. T840 is phosphorylated by protein kinase C (PKC) in vitro and is a highly turned-over phosphorylation site in the hippocampus. Interestingly, the high basal phosphorylation of T840 in the hippocampus is maintained by a persistent activity of a protein kinase, which is counter-balanced by a basal protein phosphatase activity. To study the function of T840, we generated a line of mutant mice lacking this phosphorylation site using a gene knock-in technique. The mice generated lack T840, in addition to two previously identified phosphorylation sites S831 and S845. Using this mouse, we demonstrate that T840 may regulate synaptic plasticity in an age-dependent manner.

Hypothalamic resistin induces hepatic insulin resistance.

Circulating resistin stimulates endogenous glucose production (GP). Here, we report that bi-directional changes in hypothalamic resistin action have dramatic effects on GP and proinflammatory cytokine expression in the liver. The infusion of either resistin or an active cysteine mutant in the third cerebral ventricle (icv) or in the mediobasal hypothalamus stimulated GP independent of changes in circulating levels of glucoregulatory hormones. Conversely, central antagonism of resistin action markedly diminished the ability of circulating resistin to enhance GP. We also report that centrally mediated mechanisms partially control resistin-induced expression of TNF-alpha, IL-6, and SOCS-3 in the liver. These results unveil what we believe to be a novel site of action of resistin on GP and inflammation and suggest that hypothalamic resistin action can contribute to hyperglycemia in type 2 diabetes mellitus.

Adiponectin resistance exacerbates insulin resistance in insulin receptor transgenic/knockout mice.

OBJECTIVE: Adiponectin increases insulin sensitivity and contributes to insulin's indirect effects on hepatic glucose production. RESEARCH DESIGN AND METHODS: To examine adiponectin's contribution to insulin action, we analyzed adiponectin levels and activation of AMP-activated protein kinase (AMPK) in insulin receptor transgenic/knockout mice (L1), a genetic model of resistance to insulin's indirect effects on hepatic glucose production. RESULTS: In euglycemic, insulin-resistant L1 mice, we detected hyperadiponectinemia with normal levels of adiponectin receptor-1 and -2. Moreover, adiponectin administration is unable to lower glucose levels or induce activation of AMPK, consistent with a state of adiponectin resistance. In a subset of hyperglycemic L1 mice, we observed decreased mRNA expression of AdipoR2 in liver and muscle, as well as decreased peroxisome proliferator-activated receptor (PPAR)alpha target gene expression in liver, raising the possibility that deterioration of adiponectin/AdipoR2 signaling via PPARalpha activation contributes to the progression from compensated insulin resistance to diabetes. In contrast, we failed to detect changes in other markers of the systemic or local inflammatory response. CONCLUSIONS: These data provide evidence for a mechanism of adiponectin resistance and corroborate the notion that adiponectin potentiates hepatic insulin sensitivity.

Brain glucose metabolism controls the hepatic secretion of triglyceride-rich lipoproteins.

Increased production of very low-density lipoprotein (VLDL) is a critical feature of the metabolic syndrome. Here we report that a selective increase in brain glucose lowered circulating triglycerides (TG) through the inhibition of TG-VLDL secretion by the liver. We found that the effect of glucose required its conversion to lactate, leading to activation of ATP-sensitive potassium channels and to decreased hepatic activity of stearoyl-CoA desaturase-1 (SCD1). SCD1 catalyzed the synthesis of oleyl-CoA from stearoyl-CoA. Curtailing the liver activity of SCD1 was sufficient to lower the hepatic levels of oleyl-CoA and to recapitulate the effects of central glucose administration on VLDL secretion. Notably, portal infusion of oleic acid restored hepatic oleyl-CoA to control levels and negated the effects of both central glucose and SCD1 deficiency on TG-VLDL secretion. These central effects of glucose (but not those of lactate) were rapidly lost in diet-induced obesity. These findings indicate that a defect in brain glucose sensing could play a critical role in the etiology of the metabolic syndrome.

Role of transcription factor NFAT in glucose and insulin homeostasis.

Compromised immunoregulation contributes to obesity and complications in metabolic pathogenesis. Here, we demonstrate that the nuclear factor of activated T cell (NFAT) group of transcription factors contributes to glucose and insulin homeostasis. Expression of two members of the NFAT family (NFATc2 and NFATc4) is induced upon adipogenesis and in obese mice. Mice with the Nfatc2-/- Nfatc4-/- compound disruption exhibit defects in fat accumulation and are lean. Nfatc2-/- Nfatc4-/- mice are also protected from diet-induced obesity. Ablation of NFATc2 and NFATc4 increases insulin sensitivity, in part, by sustained activation of the insulin signaling pathway. Nfatc2-/- Nfatc4-/- mice also exhibit an altered adipokine profile, with reduced resistin and leptin levels. Mechanistically, NFAT is recruited to the transcription loci and regulates resistin gene expression upon insulin stimulation. Together, these results establish a role for NFAT in glucose/insulin homeostasis and expand the repertoire of NFAT function to metabolic pathogenesis and adipokine gene transcription.

Critical role of STAT3 in leptin's metabolic actions.

Leptin has pleiotropic effects on glucose homeostasis and feeding behavior. Here, we validate the use of a cell-permeable phosphopeptide that blocks STAT3 activation in vivo. The combination of this biochemical approach with stereotaxic surgical techniques allowed us to pinpoint the contribution of hypothalamic STAT3 to the acute effects of leptin on food intake and glucose homeostasis. Leptin's ability to acutely reduce food intake critically depends on intact STAT3 signaling. Likewise, hypothalamic signaling of leptin through STAT3 is required for the acute effects of leptin on liver glucose fluxes. Lifelong obliteration of STAT3 signaling via the leptin receptor in mice (s/s mice) results in severe hepatic insulin resistance that is comparable to that observed in db/db mice, devoid of leptin receptor signaling. Our results demonstrate that the activation of the hypothalamic STAT3 pathway is an absolute requirement for the effects of leptin on food intake and hepatic glucose metabolism.