SUMMARY Reduced expression of the Indy (= I am Not Dead, Yet) gene in D. melanogaster and 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) homologue, 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.

Cardiac natriuretic peptides (NP) are major activators of human fat cell lipolysis and have recently been shown to control brown fat thermogenesis. Here, we investigated the physiological role of NP on the oxidative metabolism of human skeletal muscle. NP receptor type A (NPRA) gene expression was positively correlated to mRNA levels of PPARγ coactivator-1α (PGC1A) and several oxidative phosphorylation (OXPHOS) genes in human skeletal muscle. Further, the expression of NPRA, PGC1A, and OXPHOS genes was coordinately upregulated in response to aerobic exercise training in human skeletal muscle. In human myotubes, NP induced PGC-1α and mitochondrial OXPHOS gene expression in a cyclic GMP-dependent manner. NP treatment increased OXPHOS protein expression, fat oxidation, and maximal respiration independent of substantial changes in mitochondrial proliferation and mass. Treatment of myotubes with NP recapitulated the effect of exercise training on muscle fat oxidative capacity in vivo. Collectively, these data show that activation of NP signaling in human skeletal muscle enhances mitochondrial oxidative metabolism and fat oxidation. We propose that NP could contribute to exercise training-induced improvement in skeletal muscle fat oxidative capacity in humans. IntroductionThe cardiac hormones, atrial natriuretic peptide (ANP) and brain natriuretic peptide (BNP), play a major role in the regulation of fluid homeostasis and cardiac physiology (1). Natriuretic peptidemediated (NP-mediated) biological responses are largely mediated through cyclic GMP (cGMP) produced by the guanylyl cyclase domain of NP receptor type A (NPRA) (2). Although classically considered as cardiovascular hormones, we have shown that NP display a potent lipolytic effect in human adipocytes (3). They promote a rapid and sustained rise of intracellular cGMP that activates a cGMP-dependent protein kinase, PRKG1, which then phosphorylates perilipin 1 and hormone-sensitive lipase, necessary steps to initiate lipolysis (4). The potent lipolytic effect of NP is restricted to primates. In contrast, murine adipocytes exhibit a predominance of the clearance receptor NP receptor type C (NPR-C) and a very low expression of the biologically active NPRA (5). Interestingly, the lipolytic effect of NP is fully rescued in adipocytes of NPR-C (also known as Npr3) knockout mice. Moreover, NP induce a "browning" of human white adipocytes (6). This finding may be physiologically relevant considering the presence of functional brown fat in humans (7). Together, these studies suggest that NP plays a potent metabolic role in human adipose tissue. Recent data suggest that mice overexpressing Nppb and Prkg1 are protected from high-fat diet-induced obesity and insulin resistance and show increased energy expenditure (8). This phenotype could be explained by significant changes in skeletal muscle fat oxidative capacity. The physiological relevance and molecular mechanisms of this finding have yet to be addressed in humans. In this study,

Objective: Adipose tissue-derived factors link non-alcoholic fatty liver disease (NAFLD) with obesity, which has also been reported for circulating chemerin. On the other hand, hepatic chemerin and chemokine-like receptor 1 (CMKLR1) mRNA expression has not yet been studied in an extensively characterized patient collective. Design: This study was cross-sectional and experimental in design. Methods: Liver tissue samples were harvested from 47 subjects and histologically examined according to the NAFLD activity score (NAS). The concentrations of chemerin and CMKLR1 were measured using semi-quantitative real-time PCR, and the concentration of serum chemerin was measured using ELISA. To evaluate potential effects of chemerin and CMKLR1, cultured primary human hepatocytes (PHHs) were exposed to selected metabolites known to play a role in NAFLD (insulin, glucagon, palmitoic acid, and interleukin-6 (IL6)). Results: Chemerin and CMKLR1 mRNA levels were elevated in the human liver. Their expression was correlated with the NAS (R 2 Z0.543; P!0.001 and R 2 Z0.355; PZ0.014 respectively) and was significantly elevated in patients with definite non-alcoholic steatohepatitis (NASH) (P!0.05 respectively). Linear regression analysis confirmed an independent association of liver fibrosis, steatosis, inflammation, and hepatocyte ballooning with hepatic chemerin mRNA expression (P!0.05 respectively). The expression of hepatic chemerin and CMKLR1 was correlated with the measures of obesity (P!0.05). The incubation of PHHs with IL6 significantly increased the expression of CMKLR1 mRNA (PZ0.027), while that of chemerin remained unaffected (PO0.05). None of the other metabolites showed an influence (PO0.05). Conclusion: This is the first study to show that chemerin mRNA expression is significantly elevated in the liver of NASH patients and that CMKLR1 expression is upregulated in liver inflammation, whereby IL6 could play a causal role.

In the above article, we unfortunately miscited the findings of Toivonen et al. (2007) in our Discussion, where we wrote: ''In addition, caloric restriction does not increase life span further in flies with reduced Indy expression (Toivonen et al., 2007; Wang et al., 2009), pointing to similar underlying mechanisms in both conditions.'' Toivonen et al. did not investigate caloric restriction and did not see an effect for Indy on Drosophila life span. However, further studies by Wang et al. (2009) supported a role for Indy in Drosophila life span and calorie restriction. We apologize for any confusion our misrepresentation of the Toivonen et al. (2007) paper may have caused.

Reduced expression of the Indy (‘I am Not Dead, Yet’) gene in lower organisms promotes longevity in a manner akin to caloric restriction. Deletion of the mammalian homolog of Indy (mIndy, Slc13a5) encoding for a plasma membrane associated citrate transporter expressed highly in the liver, protects mice from high-fat diet and aging-induced obesity and hepatic fat accumulation through a mechanism resembling caloric restriction. We aimed to study a possible role of mIndy in human hepatic fat metabolism. In obese, insulin resistant patients with NAFLD, hepatic mIndy expression was increased and mIndy expression was also independently associated with hepatic steatosis. In non-human primates, a two year high fat, high sucrose diet increased hepatic mIndy expression. Liver microarray analysis showed that high mIndy expression was associated with pathways involved in hepatic lipid metabolism and immunological processes. Interleukin-6 (IL-6) was identified as a regulator of mIndy by binding to its cognate receptor. Studies in human primary hepatocytes confirmed that IL-6 markedly induced mIndy transcription via the IL-6-receptor (IL-6R) and activation of the transcription factor Stat3 and a putative start site of the human mIndy promoter was determined. Activation of the IL-6-Stat3 pathway stimulated mIndy expression, enhanced cytoplasmic citrate influx and augmented hepatic lipogenesis in vivo. In contrast, deletion of mIndy completely prevented the stimulating effect of IL-6 on citrate uptake and reduced hepatic lipogenesis. These data show that mIndy is increased in liver of obese humans and non-human primates with NALFD. Moreover, our data identify mIndy as a target gene of IL-6 and determine novel functions of IL-6 via mINDY. Targeting human mINDY may have therapeutic potential in obese patients with NAFLD.

Reduced expression of the INDY (I'm not dead yet) tricarboxylate carrier increased the life span in different species by mechanisms akin to caloric restriction. Mammalian INDY homolog (mIndy, SLC13A5) gene expression seems to be regulated by hormonal and/or nutritional factors. The underlying mechanisms are still unknown. The current study revealed that mIndy expression and [14C]-citrate uptake was induced by physiological concentrations of glucagon via a cAMP-dependent and cAMP-responsive element–binding protein (CREB)–dependent mechanism in primary rat hepatocytes. The promoter sequence of mIndy located upstream of the most frequent transcription start site was determined by 5′-rapid amplification of cDNA ends. In silico analysis identified a CREB-binding site within this promoter fragment of mIndy. Functional relevance for the CREB-binding site was demonstrated with reporter gene constructs that were induced by CREB activation when under the control of a fragment of a wild-type promoter, whereas promoter activity was lost after site-directed mutagenesis of the CREB-binding site. Moreover, CREB binding to this promoter element was confirmed by chromatin immunoprecipitation in rat liver. In vivo studies revealed that mIndy was induced in livers of fasted as well as in high-fat-diet–streptozotocin diabetic rats, in which CREB is constitutively activated. mIndy induction was completely prevented when CREB was depleted in these rats by antisense oligonucleotides. Together, these data suggest that mIndy is a CREB-dependent glucagon target gene that is induced in fasting and in type 2 diabetes. Increased mIndy expression might contribute to the metabolic consequences of diabetes in the liver.

Recent studies have implicated endoplasmic reticulum (ER) stress in insulin resistance associated with caloric excess. In mice placed on a 3-day high fat diet, we find augmented eIF2α signaling, together with hepatic lipid accumulation and insulin resistance. To clarify the role of the liver ER stress-dependent phospho-eIF2α (eIF2α-P) pathway in response to acute caloric excess on liver and muscle glucose and lipid metabolism, we studied transgenic mice in which the hepatic ER stress-dependent eIF2α-P pathway was inhibited by overexpressing a constitutively active C-terminal fragment of GADD34/PPP1R15a, a regulatory subunit of phosphatase that terminates ER stress signaling by phospho-eIF2α. Inhibition of the eIF2α-P signaling in liver led to a decrease in hepatic glucose production in the basal and clamped state, which could be attributed to reduced gluconeogenic gene expression, resulting in reduced basal plasma glucose concentrations. Surprisingly, hepatic eIF2α inhibition also impaired insulin-stimulated muscle and adipose tissue insulin sensitivity. This latter effect could be attributed at least in part by an increase in circulating IGFBP-3 levels in the transgenic animals. In addition, infusion of insulin during a hyperinsulinemic-euglycemic clamp induced conspicuous ER stress in the 3-day high fat diet-fed mice, which was aggravated through continuous dephosphorylation of eIF2α. Together, these data imply that the hepatic ER stress eIF2α signaling pathway affects hepatic glucose production without altering hepatic insulin sensitivity. Moreover, hepatic ER stress-dependent eIF2α-P signaling is implicated in an unanticipated cross-talk between the liver and peripheral organs to influence insulin sensitivity, probably via IGFBP-3. Finally, eIF2α is crucial for proper resolution of insulin-induced ER stress.

Reduced expression of the plasma membrane citrate transporter INDY (acronym I’m Not Dead, Yet ) extends life span in lower organisms. Deletion of the mammalian Indy ( mIndy ) gene in rodents improves metabolism via mechanisms akin to caloric restriction, known to lower blood pressure (BP) by sympathoadrenal inhibition. We hypothesized that mIndy deletion attenuates sympathoadrenal support of BP. Continuous arterial BP and heart rate (HR) were reduced in mINDY-KO mice. Concomitantly, urinary catecholamine content was lower, and the decreases in BP and HR by mIndy deletion were attenuated after autonomic ganglionic blockade. Catecholamine biosynthesis pathways were reduced in mINDY-KO adrenals using unbiased microarray analysis. Citrate, the main mINDY substrate, increased catecholamine content in pheochromocytoma cells, while pharmacological inhibition of citrate uptake blunted the effect. Our data suggest that deletion of mIndy reduces sympathoadrenal support of BP and HR by attenuating catecholamine biosynthesis. Deletion of mIndy recapitulates beneficial cardiovascular and metabolic responses to caloric restriction, making it an attractive therapeutic target.