MITF regulates IDH1 and NNT and drives a transcriptional program protecting cutaneous melanoma from reactive oxygen species.

Microphthalmia-associated transcription factor (MITF) plays pivotal roles in melanocyte development, function, and melanoma pathogenesis. MITF amplification occurs in melanoma and has been associated with resistance to targeted therapies. Here, we show that MITF regulates a global antioxidant program that increases survival of melanoma cell lines by protecting the cells from reactive oxygen species (ROS)-induced damage. In addition, this redox program is correlated with MITF expression in human melanoma cell lines and patient-derived melanoma samples. Using a zebrafish melanoma model, we show that MITF decreases ROS-mediated DNA damage in vivo . Some of the MITF target genes involved, such as IDH1 and NNT , are regulated through direct MITF binding to canonical enhancer box (E-BOX) sequences proximal to their promoters. Utilizing functional experiments, we demonstrate the role of MITF and its target genes in reducing cytosolic and mitochondrial ROS. Collectively, our data identify MITF as a significant driver of the cellular antioxidant state. One Sentence Summary: MITF promote melanoma survival via increasing ROS tolerance.

NNT mediates redox-dependent pigmentation via a UVB- and MITF-independent mechanism.

Ultraviolet (UV) light and incompletely understood genetic and epigenetic variations determine skin color. Here we describe an UV- and microphthalmia-associated transcription factor (MITF)-independent mechanism of skin pigmentation. Targeting the mitochondrial redox-regulating enzyme nicotinamide nucleotide transhydrogenase (NNT) resulted in cellular redox changes that affect tyrosinase degradation. These changes regulate melanosome maturation and, consequently, eumelanin levels and pigmentation. Topical application of small-molecule inhibitors yielded skin darkening in human skin, and mice with decreased NNT function displayed increased pigmentation. Additionally, genetic modification of NNT in zebrafish alters melanocytic pigmentation. Analysis of four diverse human cohorts revealed significant associations of skin color, tanning, and sun protection use with various single-nucleotide polymorphisms within NNT. NNT levels were independent of UVB irradiation and redox modulation. Individuals with postinflammatory hyperpigmentation or lentigines displayed decreased skin NNT levels, suggesting an NNT-driven, redox-dependent pigmentation mechanism that can be targeted with NNT-modifying topical drugs for medical and cosmetic purposes.

Vitamin D deficiency exacerbates UV/endorphin and opioid addiction.

The current opioid epidemic warrants a better understanding of genetic and environmental factors that contribute to opioid addiction. Here we report an increased prevalence of vitamin D (VitD) deficiency in patients diagnosed with opioid use disorder and an inverse and dose-dependent association of VitD levels with self-reported opioid use. We used multiple pharmacologic approaches and genetic mouse models and found that deficiencies in VitD signaling amplify exogenous opioid responses that are normalized upon restoration of VitD signaling. Similarly, physiologic endogenous opioid analgesia and reward responses triggered by ultraviolet (UV) radiation are repressed by VitD signaling, suggesting that a feedback loop exists whereby VitD deficiency produces increased UV/endorphin-seeking behavior until VitD levels are restored by cutaneous VitD synthesis. This feedback may carry the evolutionary advantage of maximizing VitD synthesis. However, unlike UV exposure, exogenous opioid use is not followed by VitD synthesis (and its opioid suppressive effects), contributing to maladaptive addictive behavior.

Reduced MC4R signaling alters nociceptive thresholds associated with red hair.

Humans and mice with natural red hair have elevated basal pain thresholds and an increased sensitivity to opioid analgesics. We investigated the mechanisms responsible for higher nociceptive thresholds in red-haired mice resulting from a loss of melanocortin 1 receptor (MC1R) function and found that the increased thresholds are melanocyte dependent but melanin independent. MC1R loss of function decreases melanocytic proopiomelanocortin transcription and systemic melanocyte-stimulating hormone (MSH) levels in the plasma of red-haired (Mc1re/e ) mice. Decreased peripheral α-MSH derepresses the central opioid tone mediated by the opioid receptor OPRM1, resulting in increased nociceptive thresholds. We identified MC4R as the MSH-responsive receptor that opposes OPRM1 signaling and the periaqueductal gray area in the brainstem as a central area of opioid/melanocortin antagonism. This work highlights the physiologic role of melanocytic MC1R and circulating melanocortins in the regulation of nociception and provides a mechanistic framework for altered opioid signaling and pain sensitivity in red-haired individuals.

The roles of microphthalmia-associated transcription factor and pigmentation in melanoma.

MITF and pigmentation play important roles in both normal melanocyte and transformed melanoma cell biology. MITF is regulated by many pathways and it also regulates many targets, some of which are still being discovered and functionally validated. MITF is involved in a wide range of processes in melanocytes, including pigment synthesis and lineage survival. Pigmentation itself plays an important role as the interface between genetic and environmental factors that contribute to melanoma.

cAMP-responsive element-binding protein (CREB)-regulated transcription coactivator 2 (CRTC2) promotes glucagon clearance and hepatic amino acid catabolism to regulate glucose homeostasis.

cAMP-responsive element-binding protein (CREB)-regulated transcription coactivator 2 (CRTC2) regulates transcription of gluconeogenic genes by specifying targets for the transcription factor CREB in response to glucagon. We used an antisense oligonucleotide directed against CRTC2 in both normal rodents and in rodent models of increased gluconeogenesis to better understand the role of CRTC2 in metabolic disease. In the context of severe hyperglycemia and elevated hepatic glucose production, CTRC2 knockdown (KD) improved glucose homeostasis by reducing endogenous glucose production. Interestingly, despite the known role of CRTC2 in coordinating gluconeogenic gene expression, CRTC2 KD in a rodent model of type 2 diabetes resulted in surprisingly little alteration of glucose production. However, CRTC2 KD animals had elevated circulating concentrations of glucagon and a ∼80% reduction in glucagon clearance. When this phenomenon was prevented with somatostatin or a glucagon-neutralizing antibody, endogenous glucose production was reduced by CRTC2 KD. Additionally, CRTC2 inhibition resulted in reduced expression of several glucagon-induced pyridoxal 5'-phosphate-dependent enzymes that convert amino acids to gluconeogenic intermediates, suggesting that it may control substrate availability as well as gluconeogenic gene expression. CRTC2 is an important regulator of gluconeogenesis with tremendous impact in models of elevated hepatic glucose production. Surprisingly, it is also part of a previously unidentified negative feedback loop that degrades glucagon and regulates amino acid metabolism to coordinately control glucose homeostasis in vivo.

The role of the carbohydrate response element-binding protein in male fructose-fed rats.

By 2030, nearly half of Americans will have nonalcoholic fatty liver disease. In part, this epidemic is fueled by the increasing consumption of caloric sweeteners coupled with an innate capacity to convert sugar into fat via hepatic de novo lipogenesis. In addition to serving as substrates, monosaccharides also increase the expression of key enzymes involved in de novo lipogenesis via the carbohydrate response element-binding protein (ChREBP). To determine whether ChREBP is a potential therapeutic target, we decreased hepatic expression of ChREBP with a specific antisense oligonucleotide (ASO) in male Sprague-Dawley rats fed either a high-fructose or high-fat diet. ChREBP ASO treatment decreased plasma triglyceride concentrations compared with control ASO treatment in both diet groups. The reduction was more pronounced in the fructose-fed group and attributed to decreased hepatic expression of ACC2, FAS, SCD1, and MTTP and a decrease in the rate of hepatic triglyceride secretion. This was associated with an increase in insulin-stimulated peripheral glucose uptake, as assessed by the hyperinsulinemic-euglycemic clamp. In contrast, ChREBP ASO did not alter hepatic lipid content or hepatic insulin sensitivity. Interestingly, fructose-fed rats treated with ChREBP ASO had increased plasma uric acid, alanine transaminase, and aspartate aminotransferase concentrations. This was associated with decreased expression of fructose aldolase and fructokinase, reminiscent of inherited disorders of fructose metabolism. In summary, these studies suggest that targeting ChREBP may prevent fructose-induced hypertriglyceridemia but without the improvements in hepatic steatosis and hepatic insulin responsiveness.

SirT1 regulates adipose tissue inflammation.

OBJECTIVE: Macrophage recruitment to adipose tissue is a reproducible feature of obesity. However, the events that result in chemokine production and macrophage recruitment to adipose tissue during states of energetic excess are not clear. Sirtuin 1 (SirT1) is an essential nutrient-sensing histone deacetylase, which is increased by caloric restriction and reduced by overfeeding. We discovered that SirT1 depletion causes anorexia by stimulating production of inflammatory factors in white adipose tissue and thus posit that decreases in SirT1 link overnutrition and adipose tissue inflammation. RESEARCH DESIGN AND METHODS: We used antisense oligonucleotides to reduce SirT1 to levels similar to those seen during overnutrition and studied SirT1-overexpressing transgenic mice and fat-specific SirT1 knockout animals. Finally, we analyzed subcutaneous adipose tissue biopsies from two independent cohorts of human subjects. RESULTS: We found that inducible or genetic reduction of SirT1 in vivo causes macrophage recruitment to adipose tissue, whereas overexpression of SirT1 prevents adipose tissue macrophage accumulation caused by chronic high-fat feeding. We also found that SirT1 expression in human subcutaneous fat is inversely related to adipose tissue macrophage infiltration. CONCLUSIONS: Reduction of adipose tissue SirT1 expression, which leads to histone hyperacetylation and ectopic inflammatory gene expression, is identified as a key regulatory component of macrophage influx into adipose tissue during overnutrition in rodents and humans. Our results suggest that SirT1 regulates adipose tissue inflammation by controlling the gain of proinflammatory transcription in response to inducers such as fatty acids, hypoxia, and endoplasmic reticulum stress.

Deletion of the mammalian INDY homolog mimics aspects of dietary restriction and protects against adiposity and insulin resistance in mice.

Reduced expression of the Indy (I'm Not Dead, Yet) gene in D. melanogaster and its homolog in C. elegans prolongs life span and in D. melanogaster augments mitochondrial biogenesis in a manner akin to caloric restriction. However, the cellular mechanism by which Indy does this is unknown. Here, we report on the knockout mouse model of the mammalian Indy (mIndy) homolog, SLC13A5. Deletion of mIndy in mice (mINDY(-/-) mice) reduces hepatocellular ATP/ADP ratio, activates hepatic AMPK, induces PGC-1α, inhibits ACC-2, and reduces SREBP-1c levels. This signaling network promotes hepatic mitochondrial biogenesis, lipid oxidation, and energy expenditure and attenuates hepatic de novo lipogenesis. Together, these traits protect mINDY(-/-) mice from the adiposity and insulin resistance that evolve with high-fat feeding and aging. Our studies demonstrate a profound effect of mIndy on mammalian energy metabolism and suggest that mINDY might be a therapeutic target for the treatment of obesity and type 2 diabetes.

Prevention of hepatic steatosis and hepatic insulin resistance by knockdown of cAMP response element-binding protein.

In patients with poorly controlled type 2 diabetes mellitus (T2DM), hepatic insulin resistance and increased gluconeogenesis contribute to fasting and postprandial hyperglycemia. Since cAMP response element-binding protein (CREB) is a key regulator of gluconeogenic gene expression, we hypothesized that decreasing hepatic CREB expression would reduce fasting hyperglycemia in rodent models of T2DM. In order to test this hypothesis, we used a CREB-specific antisense oligonucleotide (ASO) to knock down CREB expression in liver. CREB ASO treatment dramatically reduced fasting plasma glucose concentrations in ZDF rats, ob/ob mice, and an STZ-treated, high-fat-fed rat model of T2DM. Surprisingly, CREB ASO treatment also decreased plasma cholesterol and triglyceride concentrations, as well as hepatic triglyceride content, due to decreases in hepatic lipogenesis. These results suggest that CREB is an attractive therapeutic target for correcting both hepatic insulin resistance and dyslipidemia associated with nonalcoholic fatty liver disease (NAFLD) and T2DM.

SirT1 knockdown in liver decreases basal hepatic glucose production and increases hepatic insulin responsiveness in diabetic rats.

Hepatic gluconeogenesis is a major contributing factor to hyperglycemia in the fasting and postprandial states in type 2 diabetes mellitus (T2DM). Because Sirtuin 1 (SirT1) induces hepatic gluconeogenesis during fasting through the induction of phosphoenolpyruvate carboxylase kinase (PEPCK), fructose-1,6-bisphosphatase (FBPase), and glucose-6-phosphatase (G6Pase) gene transcription, we hypothesized that reducing SirT1, by using an antisense oligonucleotide (ASO), would decrease fasting hyperglycemia in a rat model of T2DM. SirT1 ASO lowered both fasting glucose concentration and hepatic glucose production in the T2DM rat model. Whole body insulin sensitivity was also increased in the SirT1 ASO treated rats as reflected by a 25% increase in the glucose infusion rate required to maintain euglycemia during the hyperinsulinemic-euglycemic clamp and could entirely be attributed to increased suppression of hepatic glucose production by insulin. The reduction in basal and clamped rates of glucose production could in turn be attributed to decreased expression of PEPCK, FBPase, and G6Pase due to increased acetylation of signal transducer and activator of transcription 3 (STAT3), forkhead box O1 (FOXO1), and peroxisome proliferator-activated receptor-gamma coactivator 1alpha (PGC-1alpha), known substrates of SirT1. In addition to the effects on glucose metabolism, SirT1 ASO decreased plasma total cholesterol, which was attributed to increased cholesterol uptake and export from the liver. These results indicate that inhibition of hepatic SirT1 may be an attractive approach for treatment of T2DM.

N-acylphosphatidylethanolamine, a gut- derived circulating factor induced by fat ingestion, inhibits food intake.

N-acylphosphatidylethanolamines (NAPEs) are a relatively abundant group of plasma lipids of unknown physiological significance. Here, we show that NAPEs are secreted into circulation from the small intestine in response to ingested fat and that systemic administration of the most abundant circulating NAPE, at physiologic doses, decreases food intake in rats without causing conditioned taste aversion. Furthermore, (14)C-radiolabeled NAPE enters the brain and is particularly concentrated in the hypothalamus, and intracerebroventricular infusions of nanomolar amounts of NAPE reduce food intake, collectively suggesting that its effects may be mediated through direct interactions with the central nervous system. Finally, chronic NAPE infusion results in a reduction of both food intake and body weight, suggesting that NAPE and long-acting NAPE analogs may be novel therapeutic targets for the treatment of obesity.

Dissecting enzyme regulation by multiple allosteric effectors: nucleotide regulation of aspartate transcarbamoylase.

The enzyme aspartate transcarbamoylase (ATCase, EC 2.1.3.2 of Escherichia coli), which catalyzes the committed step of pyrimidine biosynthesis, is allosterically regulated by all four ribonucleoside triphosphates (NTPs) in a nonlinear manner. Here, we dissect this regulation using the recently developed approach of random sampling-high-dimensional model representation (RS-HDMR). ATCase activity was measured in vitro at 300 random NTP concentration combinations, each involving (consistent with in vivo conditions) all four NTPs being present. These data were then used to derive a RS-HDMR model of ATCase activity over the full four-dimensional NTP space. The model accounted for 90% of the variance in the experimental data. Its main elements were positive ATCase regulation by ATP and negative by CTP, with the negative effects of CTP dominating the positive ones of ATP when both regulators were abundant (i.e., a negative cooperative effect of ATP x CTP). Strong sensitivity to both ATP and CTP concentrations occurred in their physiological concentration ranges. UTP had only a slight effect, and GTP had almost none. These findings support a predominant role of CTP and ATP in ATCase regulation. The general approach provides a new paradigm for dissecting multifactorial regulation of biological molecules and processes.