Liver mitochondrial cristae organizing protein MIC19 promotes energy expenditure and pedestrian locomotion by altering nucleotide metabolism.

Liver mitochondria undergo architectural remodeling that maintains energy homeostasis in response to feeding and fasting. However, the specific components and molecular mechanisms driving these changes and their impact on energy metabolism remain unclear. Through comparative mouse proteomics, we found that fasting induces strain-specific mitochondrial cristae formation in the liver by upregulating MIC19, a subunit of the MICOS complex. Enforced MIC19 expression in the liver promotes cristae formation, mitochondrial respiration, and fatty acid oxidation while suppressing gluconeogenesis. Mice overexpressing hepatic MIC19 show resistance to diet-induced obesity and improved glucose homeostasis. Interestingly, MIC19 overexpressing mice exhibit elevated energy expenditure and increased pedestrian locomotion. Metabolite profiling revealed that uracil accumulates in the livers of these mice due to increased uridine phosphorylase UPP2 activity. Furthermore, uracil-supplemented diet increases locomotion in wild-type mice. Thus, MIC19-induced mitochondrial cristae formation in the liver increases uracil as a signal to promote locomotion, with protective effects against diet-induced obesity.

Structure-Activity Relationship and Biological Investigation of SR18292 (16), a Suppressor of Glucagon-Induced Glucose Production.

Despite a myriad of available pharmacotherapies for the treatment of type 2 diabetes (T2D), challenges still exist in achieving glycemic control. Several novel glucose-lowering strategies are currently under clinical investigation, highlighting the need for more robust treatments. Previously, we have shown that suppressing peroxisome proliferator-activated receptor gamma coactivator 1-alpha activity with a small molecule (SR18292, 16) can reduce glucose release from hepatocytes and ameliorate hyperglycemia in diabetic mouse models. Despite structural similarities in 16 to known ß-blockers, detailed structure-activity relationship studies described herein have led to the identification of analogues lacking ß-adrenergic activity that still maintain the ability to suppress glucagon-induced glucose release from hepatocytes and ameliorate hyperglycemia in diabetic mouse models. Hence, these compounds exert their biological effects in a mechanism that does not include adrenergic signaling. These probe molecules may lead to a new therapeutic approach to treat T2D either as a single agent or in combination therapy.

Transcriptome-wide analysis of PGC-1α-binding RNAs identifies genes linked to glucagon metabolic action.

The peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC-1α) is a transcriptional coactivator that controls expression of metabolic/energetic genes, programming cellular responses to nutrient and environmental adaptations such as fasting, cold, or exercise. Unlike other coactivators, PGC-1α contains protein domains involved in RNA regulation such as serine/arginine (SR) and RNA recognition motifs (RRMs). However, the RNA targets of PGC-1α and how they pertain to metabolism are unknown. To address this, we performed enhanced ultraviolet (UV) cross-linking and immunoprecipitation followed by sequencing (eCLIP-seq) in primary hepatocytes induced with glucagon. A large fraction of RNAs bound to PGC-1α were intronic sequences of genes involved in transcriptional, signaling, or metabolic function linked to glucagon and fasting responses, but were not the canonical direct transcriptional PGC-1α targets such as OXPHOS or gluconeogenic genes. Among the top-scoring RNA sequences bound to PGC-1α were Foxo1, Camk1δ, Per1, Klf15, Pln4, Cluh, Trpc5, Gfra1, and Slc25a25 PGC-1α depletion decreased a fraction of these glucagon-induced messenger RNA (mRNA) transcript levels. Importantly, knockdown of several of these genes affected glucagon-dependent glucose production, a PGC-1α-regulated metabolic pathway. These studies show that PGC-1α binds to intronic RNA sequences, some of them controlling transcript levels associated with glucagon action.

Obesity/Type 2 Diabetes-Associated Liver Tumors Are Sensitive to Cyclin D1 Deficiency.

Type 2 diabetes, which is mainly linked to obesity, is associated with increased incidence of liver cancer. We have previously found that in various models of obesity/diabetes, hyperinsulinemia maintains heightened hepatic expression of cyclin D1, suggesting a plausible mechanism linking diabetes and liver cancer progression. Here we show that cyclin D1 is greatly elevated in human livers with diabetes and is among the most significantly upregulated genes in obese/diabetic liver tumors. Liver-specific cyclin D1 deficiency protected obese/diabetic mice against hepatic tumorigenesis, whereas lean/nondiabetic mice developed tumors irrespective of cyclin D1 status. Cyclin D1 dependency positively correlated with liver cancer sensitivity to palbociclib, an FDA-approved CDK4 inhibitor, which was effective in treating orthotopic liver tumors under obese/diabetic conditions. The antidiabetic drug metformin suppressed insulin-induced hepatic cyclin D1 expression and protected against obese/diabetic hepatocarcinogenesis. These results indicate that the cyclin D1-CDK4 complex represents a potential selective therapeutic vulnerability for liver tumors in obese/diabetic patients. SIGNIFICANCE: Obesity/diabetes-associated liver tumors are specifically vulnerable to cyclin D1 deficiency and CDK4 inhibition, suggesting that the obese/diabetic environment confers cancer-selective dependencies that can be therapeutically exploited.

Autophagy mediates hepatic GRK2 degradation to facilitate glucagon-induced metabolic adaptation to fasting.

The liver plays a key role during fasting to maintain energy homeostasis and euglycemia via metabolic processes mainly orchestrated by the insulin/glucagon ratio. We report here that fasting or calorie restriction protocols in C57BL6 mice promote a marked decrease in the hepatic protein levels of G protein-coupled receptor kinase 2 (GRK2), an important negative modulator of both G protein-coupled receptors (GPCRs) and insulin signaling. Such downregulation of GRK2 levels is liver-specific and can be rapidly reversed by refeeding. We find that autophagy, and not the proteasome, represents the main mechanism implicated in fasting-induced GRK2 degradation in the liver in vivo. Reducing GRK2 levels in murine primary hepatocytes facilitates glucagon-induced glucose production and enhances the expression of the key gluconeogenic enzyme Pck1. Conversely, preventing full downregulation of hepatic GRK2 during fasting using adenovirus-driven overexpression of this kinase in the liver leads to glycogen accumulation, decreased glycemia, and hampered glucagon-induced gluconeogenesis, thus preventing a proper and complete adaptation to nutrient deprivation. Overall, our data indicate that physiological fasting-induced downregulation of GRK2 in the liver is key for allowing complete glucagon-mediated responses and efficient metabolic adaptation to fasting in vivo.

Elevated CO2 regulates the Wnt signaling pathway in mammals, Drosophila melanogaster and Caenorhabditis elegans.

Carbon dioxide (CO2) is sensed by cells and can trigger signals to modify gene expression in different tissues leading to changes in organismal functions. Despite accumulating evidence that several pathways in various organisms are responsive to CO2 elevation (hypercapnia), it has yet to be elucidated how hypercapnia activates genes and signaling pathways, or whether they interact, are integrated, or are conserved across species. Here, we performed a large-scale transcriptomic study to explore the interaction/integration/conservation of hypercapnia-induced genomic responses in mammals (mice and humans) as well as invertebrates (Caenorhabditis elegans and Drosophila melanogaster). We found that hypercapnia activated genes that regulate Wnt signaling in mouse lungs and skeletal muscles in vivo and in several cell lines of different tissue origin. Hypercapnia-responsive Wnt pathway homologues were similarly observed in secondary analysis of available transcriptomic datasets of hypercapnia in a human bronchial cell line, flies and nematodes. Our data suggest the evolutionarily conserved role of high CO2 in regulating Wnt pathway genes.

Regulation of Hepatic Metabolism, Recent Advances, and Future Perspectives.

PURPOSE OF REVIEW: The purpose of this review is to provide a brief summary of recent advances in our understanding of liver metabolism. The critical role of the liver in controlling whole-body energy homeostasis makes such understanding crucial to efficiently design new treatments for metabolic syndrome diseases, including type 2 diabetes (T2D). RECENT FINDINGS: Significant advances have been made regarding our understanding of the direct and indirect effects of insulin on hepatic metabolism and the communication between the liver and other tissues. Moreover, the catabolic functions of glucagon, as well as the importance of hepatic redox status for the regulation of glucose production, are emerging as potential targets to reduce hyperglycemia. A resolution to the long-standing question "insulin suppression of hepatic glucose production, direct or indirect effect?" is starting to emerge. New advances in our understanding of important fasting-induced hepatic metabolic fluxes may help design better therapies for T2D.

Insulin regulation of gluconeogenesis.

The coordinated regulation between cellular glucose uptake and endogenous glucose production is indispensable for the maintenance of constant blood glucose concentrations. The liver contributes significantly to this process by altering the levels of hepatic glucose release, through controlling the processes of de novo glucose production (gluconeogenesis) and glycogen breakdown (glycogenolysis). Various nutritional and hormonal stimuli signal to alter hepatic gluconeogenic flux, and suppression of this metabolic pathway during the postprandial state can, to a significant extent, be attributed to insulin. Here, we review some of the molecular mechanisms through which insulin modulates hepatic gluconeogenesis, thus controlling glucose production by the liver to ultimately maintain normoglycemia. Various signaling pathways governed by insulin converge at the level of transcriptional regulation of the key hepatic gluconeogenic genes PCK1 and G6PC, highlighting this as one of the focal mechanisms through which gluconeogenesis is modulated. In individuals with compromised insulin signaling, such as insulin resistance in type 2 diabetes, insulin fails to suppress hepatic gluconeogenesis, even in the fed state; hence, an insight into these insulin-moderated pathways is critical for therapeutic purposes.

Selective Chemical Inhibition of PGC-1α Gluconeogenic Activity Ameliorates Type 2 Diabetes.

Type 2 diabetes (T2D) is a worldwide epidemic with a medical need for additional targeted therapies. Suppression of hepatic glucose production (HGP) effectively ameliorates diabetes and can be exploited for its treatment. We hypothesized that targeting PGC-1α acetylation in the liver, a chemical modification known to inhibit hepatic gluconeogenesis, could be potentially used for treatment of T2D. Thus, we designed a high-throughput chemical screen platform to quantify PGC-1α acetylation in cells and identified small molecules that increase PGC-1α acetylation, suppress gluconeogenic gene expression, and reduce glucose production in hepatocytes. On the basis of potency and bioavailability, we selected a small molecule, SR-18292, that reduces blood glucose, strongly increases hepatic insulin sensitivity, and improves glucose homeostasis in dietary and genetic mouse models of T2D. These studies have important implications for understanding the regulatory mechanisms of glucose metabolism and treatment of T2D.

Adipose Tissue CLK2 Promotes Energy Expenditure during High-Fat Diet Intermittent Fasting.

A promising approach to treating obesity is to increase diet-induced thermogenesis in brown adipose tissue (BAT), but the regulation of this process remains unclear. Here we find that CDC-like kinase 2 (CLK2) is expressed in BAT and upregulated upon refeeding. Mice lacking CLK2 in adipose tissue exhibit exacerbated obesity and decreased energy expenditure during high-fat diet intermittent fasting. Additionally, tissue oxygen consumption and protein levels of UCP1 are reduced in CLK2-deficient BAT. Phosphorylation of CREB, a transcriptional activator of UCP1, is markedly decreased in BAT cells lacking CLK2 due to enhanced CREB dephosphorylation. Mechanistically, CREB dephosphorylation is rescued by the inhibition of PP2A, a phosphatase that targets CREB. Our results suggest that CLK2 is a regulatory component of diet-induced thermogenesis in BAT through increased CREB-dependent expression of UCP1.

Targeting hepatic glucose metabolism in the treatment of type 2 diabetes.

Type 2 diabetes mellitus is characterized by the dysregulation of glucose homeostasis, resulting in hyperglycaemia. Although current diabetes treatments have exhibited some success in lowering blood glucose levels, their effect is not always sustained and their use may be associated with undesirable side effects, such as hypoglycaemia. Novel antidiabetic drugs, which may be used in combination with existing therapies, are therefore needed. The potential of specifically targeting the liver to normalize blood glucose levels has not been fully exploited. Here, we review the molecular mechanisms controlling hepatic gluconeogenesis and glycogen storage, and assess the prospect of therapeutically targeting associated pathways to treat type 2 diabetes.

The Methionine Transamination Pathway Controls Hepatic Glucose Metabolism through Regulation of the GCN5 Acetyltransferase and the PGC-1α Transcriptional Coactivator.

Methionine is an essential sulfur amino acid that is engaged in key cellular functions such as protein synthesis and is a precursor for critical metabolites involved in maintaining cellular homeostasis. In mammals, in response to nutrient conditions, the liver plays a significant role in regulating methionine concentrations by altering its flux through the transmethylation, transsulfuration, and transamination metabolic pathways. A comprehensive understanding of how hepatic methionine metabolism intersects with other regulatory nutrient signaling and transcriptional events is, however, lacking. Here, we show that methionine and derived-sulfur metabolites in the transamination pathway activate the GCN5 acetyltransferase promoting acetylation of the transcriptional coactivator PGC-1α to control hepatic gluconeogenesis. Methionine was the only essential amino acid that rapidly induced PGC-1α acetylation through activating the GCN5 acetyltransferase. Experiments employing metabolic pathway intermediates revealed that methionine transamination, and not the transmethylation or transsulfuration pathways, contributed to methionine-induced PGC-1α acetylation. Moreover, aminooxyacetic acid, a transaminase inhibitor, was able to potently suppress PGC-1α acetylation stimulated by methionine, which was accompanied by predicted alterations in PGC-1α-mediated gluconeogenic gene expression and glucose production in primary murine hepatocytes. Methionine administration in mice likewise induced hepatic PGC-1α acetylation, suppressed the gluconeogenic gene program, and lowered glycemia, indicating that a similar phenomenon occurs in vivo These results highlight a communication between methionine metabolism and PGC-1α-mediated hepatic gluconeogenesis, suggesting that influencing methionine metabolic flux has the potential to be therapeutically exploited for diabetes treatment.

Molecular pathophysiology of hepatic glucose production.

Maintaining blood glucose concentration within a relatively narrow range through periods of fasting or excess nutrient availability is essential to the survival of the organism. This is achieved through an intricate balance between glucose uptake and endogenous glucose production to maintain constant glucose concentrations. The liver plays a major role in maintaining normal whole body glucose levels by regulating the processes of de novo glucose production (gluconeogenesis) and glycogen breakdown (glycogenolysis), thus controlling the levels of hepatic glucose release. Aberrant regulation of hepatic glucose production (HGP) can result in deleterious clinical outcomes, and excessive HGP is a major contributor to the hyperglycemia observed in Type 2 diabetes mellitus (T2DM). Indeed, adjusting glycemia as close as possible to a non-diabetic range is the foremost objective in the medical treatment of patients with T2DM and is currently achieved in the clinic primarily through suppression of HGP. Here, we review the molecular mechanisms controlling HGP in response to nutritional and hormonal signals and discuss how these signals are altered in T2DM.

Pharyngeal pumping inhibition and avoidance by acute exposure to high CO2 levels are both regulated by the BAG neurons via different molecular pathways.

Carbon dioxide (CO2) is a key molecule in many biological processes. Studies in humans, mice, D. melanogaster, C. elegans, unicellular organisms and plants have shed light on the molecular pathways activated by elevated levels of CO2. However, the mechanisms that organisms use to sense and respond to high CO2 levels remain largely unknown. Previous work has shown that C. elegans quickly avoid elevated CO2 levels using mechanisms that involve the BAG, ASE and AFD neurons via cGMP- and calcium- signaling pathways. Here, we discuss our recent finding that exposure of C. elegans to high CO2 levels leads to a very rapid cessation in the contraction of the pharynx muscles. Surprisingly, none of the tested CO2 avoidance mutants affected the rapid pumping inhibition response to elevated CO2 levels. A forward genetic screen identified that the hid-1-mediated pathway of dense core vesicle maturation regulates the pumping inhibition, probably through affecting neuropeptide secretion. Genetic studies and laser ablation experiments showed that the CO2 response of the pharyngeal muscle pumping is regulated by the BAG neurons, the same neurons that mediate CO2 avoidance.

The response to high CO2 levels requires the neuropeptide secretion component HID-1 to promote pumping inhibition.

Carbon dioxide (CO2) is a key molecule in many biological processes; however, mechanisms by which organisms sense and respond to high CO2 levels remain largely unknown. Here we report that acute CO2 exposure leads to a rapid cessation in the contraction of the pharynx muscles in Caenorhabditis elegans. To uncover the molecular mechanisms underlying this response, we performed a forward genetic screen and found that hid-1, a key component in neuropeptide signaling, regulates this inhibition in muscle contraction. Surprisingly, we found that this hid-1-mediated pathway is independent of any previously known pathways controlling CO2 avoidance and oxygen sensing. In addition, animals with mutations in unc-31 and egl-21 (neuropeptide secretion and maturation components) show impaired inhibition of muscle contraction following acute exposure to high CO2 levels, in further support of our findings. Interestingly, the observed response in the pharynx muscle requires the BAG neurons, which also mediate CO2 avoidance. This novel hid-1-mediated pathway sheds new light on the physiological effects of high CO2 levels on animals at the organism-wide level.

Evolutionary conserved role of c-Jun-N-terminal kinase in CO2-induced epithelial dysfunction.

Elevated CO(2) levels (hypercapnia) occur in patients with respiratory diseases and impair alveolar epithelial integrity, in part, by inhibiting Na,K-ATPase function. Here, we examined the role of c-Jun N-terminal kinase (JNK) in CO(2) signaling in mammalian alveolar epithelial cells as well as in diptera, nematodes and rodent lungs. In alveolar epithelial cells, elevated CO(2) levels rapidly induced activation of JNK leading to downregulation of Na,K-ATPase and alveolar epithelial dysfunction. Hypercapnia-induced activation of JNK required AMP-activated protein kinase (AMPK) and protein kinase C-ζ leading to subsequent phosphorylation of JNK at Ser-129. Importantly, elevated CO(2) levels also caused a rapid and prominent activation of JNK in Drosophila S2 cells and in C. elegans. Paralleling the results with mammalian epithelial cells, RNAi against Drosophila JNK fully prevented CO(2)-induced downregulation of Na,K-ATPase in Drosophila S2 cells. The importance and specificity of JNK CO(2) signaling was additionally demonstrated by the ability of mutations in the C. elegans JNK homologs, jnk-1 and kgb-2 to partially rescue the hypercapnia-induced fertility defects but not the pharyngeal pumping defects. Together, these data provide evidence that deleterious effects of hypercapnia are mediated by JNK which plays an evolutionary conserved, specific role in CO(2) signaling in mammals, diptera and nematodes.

Ce-emerin and LEM-2: essential roles in Caenorhabditis elegans development, muscle function, and mitosis.

Emerin and LEM2 are ubiquitous inner nuclear membrane proteins conserved from humans to Caenorhabditis elegans. Loss of human emerin causes Emery-Dreifuss muscular dystrophy (EDMD). To test the roles of emerin and LEM2 in somatic cells, we used null alleles of both genes to generate C. elegans animals that were either hypomorphic (LEM-2-null and heterozygous for Ce-emerin) or null for both proteins. Single-null and hypomorphic animals were viable and fertile. Double-null animals used the maternal pool of Ce-emerin to develop to the larval L2 stage, then arrested. Nondividing somatic cell nuclei appeared normal, whereas dividing cells had abnormal nuclear envelope and chromatin organization and severe defects in postembryonic cell divisions, including the mesodermal lineage. Life span was unaffected by loss of Ce-emerin alone but was significantly reduced in LEM-2-null animals, and double-null animals had an even shorter life span. In addition to striated muscle defects, double-null animals and LEM-2-null animals showed unexpected defects in smooth muscle activity. These findings implicate human LEM2 mutations as a potential cause of EDMD and further suggest human LEM2 mutations might cause distinct disorders of greater severity, since C. elegans lacking only LEM-2 had significantly reduced life span and smooth muscle activity.

The physiological and molecular effects of elevated CO2 levels.

Carbon dioxide (CO2) is an end product of cellular respiration, a process by which organisms including all plants, animals, many fungi and some bacteria obtain energy. CO2 has several physiologic roles in respiration, pH buffering, autoregulation of the blood supply and others. Here we review recent findings from studies in mammalian lung cells, Caenorhabditis elegans and Drosophila melanogaster that help shed light on the molecular sensing and response to hypercapnia.

Sensing, physiological effects and molecular response to elevated CO2 levels in eukaryotes.

Carbon dioxide (CO(2)) is an important gaseous molecule that maintains biosphere homeostasis and is an important cellular signalling molecule in all organisms. The transport of CO(2) through membranes has fundamental roles in most basic aspects of life in both plants and animals. There is a growing interest in understanding how CO(2) is transported into cells, how it is sensed by neurons and other cell types and in understanding the physiological and molecular consequences of elevated CO(2) levels (hypercapnia) at the cell and organism levels. Human pulmonary diseases and model organisms such as fungi, C. elegans, Drosophila and mice have been proven to be important in understanding of the mechanisms of CO(2) sensing and response.

Elevated CO2 levels affect development, motility, and fertility and extend life span in Caenorhabditis elegans.

Hypercapnia (high CO(2) levels) occurs in a number of lung diseases and it is associated with worse outcomes in patients with chronic obstructive lung disease (COPD). However, it is largely unknown how hypercapnia is sensed and responds in nonneuronal cells. Here, we used C. elegans to study the response to nonanesthetic CO(2) levels and show that levels exceeding 9% induce aberrant motility that is accompanied by age-dependent deterioration of body muscle organization, slowed development, reduced fertility and increased life span. These effects occur independently of the IGF-R, dietary restriction, egg laying or mitochondrial-induced aging pathways. Transcriptional profiling analysis shows specific and dynamic changes in gene expression after 1, 6, or 72 h of exposure to 19% CO(2) including increased transcription of several 7-transmembrane domain and innate immunity genes and a reduction in transcription of many of the MSP genes. Together, these results suggest specific physiological and molecular responses to hypercapnia, which appear to be independent of early heat shock and HIF mediated pathways.