Volume 34 | April 2020

Objective: The most common kidney cancer, clear cell renal cell carcinoma (ccRCC), is closely associated with obesity. The "clearcell" variant of RCC gets its name from the large lipid droplets that accumulate in the tumor cells. Although renal lipid metabolism is altered in ccRCC, the mechanisms and lipids driving this are not well understood.

Methods: We used shotgun lipidomics in human ccRCC tumors and matched normal adjacent renal tissue. To assess MBOAT7s gene expression across tumor severity, we examined histologically graded human ccRCC samples. We then utilized genome editing in ccRCC cell lines to understand the role of MBOAT7 in ccRCC progression.

Results: We identified a lipid signature for ccRCC that includes an increase in arachidonic acid-enriched phosphatidylinositols (AA-PI). In parallel, we found that ccRCC tumors have increased expression of acyltransferase enzyme membrane bound O-acyltransferase domain containing 7 (MBOAT7) that contributes to AA-PI synthesis. In ccRCC patients, MBOAT7 expression increases with tumor grade, and increased MBOAT7 expression correlates with poor survival. Genetic deletion of MBOAT7 in ccRCC cells decreases proliferation and induces cell cycle arrest, and MBOAT7^-/- cells fail to form tumors in vivo. RNAseq of MBOAT7^-/- cells identified alterations in cellmigration and extracellular matrix organization that were functionally validated in migration assays.

Conclusions: This study highlights the accumulation of AA-PI in ccRCC and demonstrates a novel way to decrease the AA-PI pool in ccRCC by limiting MBOAT7. Our data reveal that metastatic ccRCC is associated with altered AA-PI metabolism and identify MBOAT7 as a novel target in advanced ccRCC.

Objective: Adenosine triphosphate (ATP)-binding cassette transporter A1 (ABCA1) influences hepatic cholesterol transportation. Accumulation of hepatic cholesterol leads to fatty liver disease, which is improved by glucagon-like peptide 1 (GLP-1) in diabetes. Therefore, we analyzed the molecular mechanism in the regulation of hepatic ABCA1 by GLP-1 analogue exendin-4.

Methods: Hepatic ABCA1 expression and transcription were checked by western blotting, real-time polymerase chain reaction (PCR), and luciferase assay in HepG2 cells. Chromatin immunoprecipitation (ChIP) and site-directed mutagenesis were employed to determine transcriptional regulation of the ABCA1 gene. Prolactin regulatory element-binding (PREB)-transgenic mice were generated to access the effect of exendin-4 on improving lipid accumulation caused by a high-fat diet (HFD).

Results: Exendin-4 stimulated hepatic ABCA1 expression and transcription via the Ca²⁺/calmodulin (CaM)-dependent protein kinase kinase/CaM-dependent protein kinase IV (CaMKK/CaMKIV) pathway, whereas GLP-1 receptor antagonist exendin9-39 cancelled this effect. Therefore, exendin-4 decreased hepatic lipid content. ChIP showed that PREB could directly bind to the ABCA1 promoter, which was enhanced by exendin-4. Moreover, PREB stimulated ABCA1 promoter activity, and mutation of PREB-binding site in ABCA1 promoter cancelled exendin-4-enhanced ABCA1 promoter activity. Silencing of PREB attenuated the effect of exendin-4 and induced hepatic cholesterol accumulation. Blockade of CaMKK by STO-609 or siRNA cancelled the upregulation of ABCA1 and PREB induced by exendin-4. In vivo, exendin-4 or overexpression of PREB increased hepatic ABCA1 expression and decreased hepatic lipid accumulation and high plasma cholesterol caused by a HFD.

Conclusions: Our data shows that exendin-4 stimulates hepatic ABCA1 expression and decreases lipid accumulation by the CaMKK/CaMKIV/PREB pathway, suggesting that ABCA1 and PREB might be the therapeutic targets in fatty liver disease.

Background: The diminished glucose lowering effect of insulin in obesity, called “insulin resistance,” is associated with glucose intolerance, type 2 diabetes, and other serious maladies. Many publications on this topic have suggested numerous hypotheses on the molecular and cellular disruptions that contribute to the syndrome. However, significant uncertainty remains on the mechanisms of its initiation and long-term maintenance.

Scope of review: To simplify insulin resistance analysis, this review focuses on the unifying concept that adipose tissue is a central regulator of systemic glucose homeostasis by controlling liver and skeletal muscle metabolism. Key aspects of adipose function related to insulin resistance reviewed are: 1) the modes by which specific adipose tissues control hepatic glucose output and systemic glucose disposal, 2) recently acquired understanding of the underlying mechanisms of these modes of regulation, and 3) the steps in these pathways adversely affected by obesity that cause insulin resistance.

Major conclusions: Adipocyte heterogeneity is required to mediate the multiple pathways that control systemic glucose tolerance. White adipocytes specialize in sequestering triglycerides away from the liver, muscle, and other tissues to limit toxicity. In contrast, brown/beige adipocytes are very active in directly taking up glucose in response to β adrenergic signaling and insulin and enhancing energy expenditure. Nonetheless, white, beige, and brown adipocytes all share the common feature of secreting factors and possibly exosomes that act on distant tissues to control glucose homeostasis. Obesity exerts deleterious effects on each of these adipocyte functions to cause insulin resistance.

Objective: Regulation of food intake and energy balance depends on a group of hypothalamic neurons that release anorexigenic melanocortins encoded by the Pomc gene. Although the physiological importance of central melanocortins is well appreciated, the genetic program that defines the functional identity of melanocortin neurons and assures high levels of hypothalamic Pomc expression is only beginning to be understood. This study assessed whether the transcriptional regulator PRDM12, identified as a highly expressed gene in adult mouse POMC neurons, plays an important role in the identity and function of melanocortin neurons.

Methods: We first determined the cellular distribution of PRDM12 in the developing hypothalamus. Then we studied mutant mice with constitutively inactivated Prdm12to evaluate possible changes in hypothalamic Pomc expression. In addition, we characterized conditional mutant mice specifically lacking Prdm12 in ISL1-positive or POMC neurons during development. Finally, we measured food intake, body weight progression up to 16 weeks of age, adiposity, and glucose tolerance in adult mice lacking Prdm12 selectively from POMC neurons.

Results: PRDM12 co-expressed with POMC in mouse hypothalamic neurons from early development to adulthood. Mice lacking Prdm12 displayed greatly reduced Pomcexpression in the developing hypothalamus. Selective ablation of Prdm12 from ISL1 neurons prevented hypothalamic Pomc expression. The conditional ablation of Prdm12 limited to POMC neurons greatly reduced Pomc expression in the developing hypothalamus and in adult mice led to increased food intake, adiposity, and obesity.

Conclusions: Altogether, our results demonstrate that PRDM12 plays an essential role in the early establishment of hypothalamic melanocortin neuron identity and the maintenance of high expression levels of Pomc. Its absence in adult mice greatly impairs Pomcexpression and leads to increased food intake, adiposity, and obesity.

Objectives: Nutrient sensing by hypothalamic neurons is critical for the regulation of food intake and energy expenditure. We aimed to identify long- and medium-chain fatty acid species transported into the brain, their effects on energy balance, and the mechanisms by which they regulate activity of hypothalamic neurons.

Methods: Simultaneous blood and cerebrospinal fluid (CSF) sampling was undertaken in rats and metabolic analyses using radiolabeled fatty acid tracers were performed on mice. Electrophysiological recording techniques were used to investigate signaling mechanisms underlying fatty acid-induced changes in activity of pro-opiomelanocortin (POMC) neurons.

Results: Medium-chain fatty acid (MCFA) octanoic acid (C8:0), unlike long-chain fatty acids, was rapidly transported into the hypothalamus of mice and almost exclusively oxidized, causing rapid, transient reductions in food intake and increased energy expenditure. Octanoic acid differentially regulates the excitability of POMC neurons, activating these neurons directly via GPR40 and inducing inhibition via an indirect non-synaptic, purine, and adenosine receptor-dependent mechanism.

Conclusions: MCFA octanoic acid is a central signaling nutrient that targets POMC neurons via distinct direct and indirect signal transduction pathways to instigate changes in energy status. These results could explain the beneficial health effects that accompany MCFA consumption.

Objective: The liver is regularly exposed to changing metabolic and inflammatory environments. It must sense and adapt to metabolic need while balancing resources required to protect itself from insult. Peroxisome proliferator activated receptor gamma coactivator-1 alpha (PGC-1α) is a transcriptional coactivator expressed as multiple, alternatively spliced variants transcribed from different promoters that coordinate metabolic adaptation and protect against inflammation. It is not known how PGC-1α integrates extracellular signals to balance metabolic and anti-inflammatory outcomes.

Methods: Primary mouse hepatocytes were used to evaluate the role(s) of different PGC-1α proteins in regulating hepatic metabolism and inflammatory signaling downstream of tumor necrosis factor alpha (TNFα). Gene expression and signaling analysis were combined with biochemical measurement of apoptosis using gain- and loss-of-function in vitro and in vivo.

Results: Hepatocytes expressed multiple isoforms of PGC-1α, including PGC-1α4, which microarray analysis showed had common and isoform-specific functions linked to metabolism and inflammation compared with canonical PGC-1α1. Whereas PGC-1α1 primarily impacted gene programs of nutrient metabolism and mitochondrial biology, TNFα signaling showed several pathways related to innate immunity and cell death downstream of PGC-1α4. Gain- and loss-of-function models illustrated that PGC-1α4 uniquely enhanced expression of anti-apoptotic gene programs and attenuated hepatocyte apoptosis in response to TNFα or lipopolysaccharide (LPS). This was in contrast to PGC-1α1, which decreased the expression of a wide inflammatory gene network but did not prevent hepatocyte death in response to cytokines.

Conclusions: PGC-1α variants have distinct, yet complementary roles in hepatic responses to metabolism and inflammation, and we identify PGC-1α4 as an important mitigator of apoptosis.

Objectives: Lipolysis, hydrolysis of triglycerides to fatty acids in adipocytes, is tightly regulated, poorly understood, and, if perturbed, can lead to metabolic diseases including obesity and type 2 diabetes. The goal of this study was to identify the genetic regulators of lipolysis and elucidate their molecular mechanisms.

Methods: Adipocytes from abdominal subcutaneous adipose tissue biopsies were isolated and were incubated without (spontaneous lipolysis) or with a catecholamine (stimulated lipolysis) to analyze lipolysis. DNA was extracted and genome-wide genotyping and imputation conducted. After quality control, 939 samples with genetic and lipolysis data were available. Genome-wide association studies of spontaneous and stimulated lipolysis were conducted. Subsequent in vitro gene expression analyses were used to identify candidate genes and explore their regulation of adipose tissue biology.

Results: One locus on chromosome 19 demonstrated genome-wide significance with spontaneous lipolysis. 60 loci showed suggestive associations with spontaneous or stimulated lipolysis, of which many influenced both traits. In the chromosome 19 locus, only HIF3A was expressed in the adipocytes and displayed genotype-dependent gene expression. HIF3A knockdown in vitro increased lipolysis and the expression of key lipolysis-regulating genes.

Conclusions: In conclusion, we identified a genetic regulator of spontaneous lipolysis and provided evidence of HIF3Aas a novel key regulator of lipolysis in subcutaneous adipocytes as the mechanism through which the locus influences adipose tissue biology.

Objective: Diabetes is characterized by pancreatic β-cell dedifferentiation. Dedifferentiating β cells inappropriately metabolize lipids over carbohydrates and exhibit impaired mitochondrial oxidative phosphorylation. However, the mechanism linking the β-cell's response to an adverse metabolic environment with impaired mitochondrial function remains unclear.

Methods: Here we report that the oxidoreductase cytochrome b5 reductase 3 (Cyb5r3) links FoxO1 signaling to β-cell stimulus/secretion coupling by regulating mitochondrial function, reactive oxygen species generation, and nicotinamide actin dysfunction (NAD)/reduced nicotinamide actin dysfunction (NADH) ratios.

Results: The expression of Cyb5r3 is decreased in FoxO1-deficient β cells. Mice with β-cell-specific deletion of Cyb5r3 have impaired insulin secretion, resulting in glucose intolerance and diet-induced hyperglycemia. Cyb5r3-deficient β cells have a blunted respiratory response to glucose and display extensive mitochondrial and secretory granule abnormalities, consistent with altered differentiation. Moreover, FoxO1 is unable to maintain expression of key differentiation markers in Cyb5r3-deficient β cells, suggesting that Cyb5r3 is required for FoxO1-dependent lineage stability.

Conclusions: The findings highlight a pathway linking FoxO1 to mitochondrial dysfunction that can mediate β-cell failure.

Objective: Chronic inflammation of adipose tissues contributes to obesity-triggered insulin resistance. Unfortunately, the potential molecular mechanisms regarding obesity-associated systemic inflammation and metabolic disorder remain complicated. Here, we report that inactive rhomboid-like protein 2 (iRhom2) was increased in overweight mice with adipose inflammation.

Methods: Mice with deletion of iRhom2 on a C57BL/6J background, mice without deletion of this gene (controls), and mice with deficiency of iRhom2 only in myeloid cells were fed a standard chow diet (SCD) or a high-fat diet (HFD; 60% fat calories). Then the adipose tissues or bone marrow cells were isolated for the further detection.

Results: After 16 weeks on a high-fat diet (HFD), obesity, chronic inflammation in adipose tissues, and insulin resistance were markedly mitigated in iRhom2 knockout (iRhom2 KO) mice, whereas these parameters were exaggerated in iRhom2 overactivated mice. The adverse influences of iRhom2 on adipose inflammation and associated pathologies were determined in db/db mice. We further demonstrated that, in response to an HFD, iRhom2 KO mice and mice with deletion only in the myeloid cells showed less severe adipose inflammation and insulin resistance than control groups. Conversely, transplantation of bone marrow cells from normal mice to iRhom2 KO mice unleashed severe systemic inflammation and metabolic dysfunction after HFD ingestion.

Conclusion: We identified iRhom2 as a key regulator that promotes obesity-associated metabolic disorders. Loss of iRhom2 from macrophages in adipose tissues may indirectly restrain inflammation and insulin resistance via blocking crosslinks between macrophages and adipocytes. Hence, iRhom2 may be a therapeutic target for obesity-induced metabolic dysfunction.

Objective: Considerable uncertainty remains regarding the veracity of measuring myokine irisin more than seven years after its original description. Unresolved issues include the nature of transcription of the irisin precursor fibronectin type III domain containing 5 (FNDC5) gene across species, the reliability of irisin levels measured with commercial enzyme-linked immunosorbent assays (ELISAs), and the overall validity of the recently published reference values for human serum measured with quantitative mass spectrometry. We utilized multiple species and measures to evaluate the robustness of commonly used reagents and methods for reporting irisin.

Methods: Amplification of cDNA was used to assess the FNDC5 transcript patterns in humans and mice. The specificity and sensitivity of different irisin antibodies were examined via western blotting. Quantification of circulating native irisin was conducted with mass spectrometry using an absolute quantification peptide for irisin.

Results: We show that there is a greater transcript diversity of human FNDC5 than currently annotated, but no indication of the expression of transcripts leading to a truncated form of irisin. Available irisin antibodies still bind to patterns of unspecific serum proteins, which compromise reliable measurements of irisin with ELISAs. Absolute quantification of irisin with labeled peptides by mass spectrometry is an advanced method but requires a multi-step sample preparation introducing uncontrollable variations in the measurement.

Conclusion: Our data represent an explicit warning against measuring circulating irisin using available methods. Measuring irisin is akin to chasing shadows.

Objective: Enteroendocrine cells (EECs) survey the gut luminal environment and coordinate hormonal, immune and neuronal responses to it. They exhibit well-characterised physiological roles ranging from the control of local gut function to whole body metabolism, but little is known regarding the regulatory networks controlling their differentiation, especially in the human gut. The small molecule isoxazole-9 (ISX-9) has been shown to stimulate neuronal and pancreatic beta-cell differentiation, both closely related to EEC differentiation. Our aim was to use ISX-9 as a tool to explore EEC differentiation.

Methods: We investigated the effects of ISX-9 on EEC differentiation in mouse and human intestinal organoids, using real-time quantitative polymerase chain reaction (RT-qPCR), fluorescent-activated cell sorting, immunostaining and single-cell RNA sequencing.

Results: ISX-9 increased the number of neurogenin3-RFP (Ngn3)-positive endocrine progenitor cells and upregulated NeuroD1 and Pax4, transcription factors that play roles in mouse EEC specification. Single-cell analysis showed induction of Pax4 expression in a developmentally late Ngn3+ population of cells and potentiation of genes associated with progenitors biased toward serotonin-producing enterochromaffin (EC) cells. Further, we observed enrichment of organoids with functional EC cells that was partly dependent on stimulation of calcium signalling in a population of cells residing outside the crypt base. Inducible Pax4 overexpression, in ileal organoids, uncovered its importance as a component of early human endocrine specification and highlighted the potential existence of two major endocrine lineages, the early appearing enterochromaffin lineage and the later developing peptidergic lineage which contains classical gut hormone cell types.

Conclusion: Our data provide proof-of-concept for the controlled manipulation of specific endocrine lineages with small molecules, whilst also shedding new light on human EEC differentiation and its similarity to the mouse. Given their diverse roles, understanding endocrine lineage plasticity and its control could have multiple therapeutic implications.

Objective: In mouse models, deficiency of TTC39B (T39) decreases hepatic lipogenic gene expression and protects against diet-induced steatohepatitis. While assessing the therapeutic potential of antisense oligonucleotides (ASOs) targeting T39, we discovered an unexpected weight loss phenotype. The objective of this study was to determine the mechanism of the resistance to diet-inducedobesity.

Methods: To assess therapeutic potential, we used antisense oligonucleotides (ASO) to knock down T39 expression in a Western or high-fat, high-cholesterol, high-sucrose-diet-fed Ldlr^-/- or wild-type mice.

Results: T39 ASO treatment led to decreased hepatic lipogenic gene expression and decreased hepatic triglycerides. Unexpectedly, T39 ASO treatment protected against diet-induced obesity. The reduced weight gain was seen with two different ASOs that decreased T39 mRNA in adipose tissue macrophages (ATMs), but not with a liver-targeted GalNac-ASO. Mice treated with the T39 ASO displayed increased browning of gonadal white adipose tissue (gWAT) and evidence of increased lipolysis. However, T39 knockout mice displayed a similar weight loss response when treated with T39 ASO, indicating an off-target effect. RNA-seq analysis of gWAT showed a widespread increase in type I interferon (IFN)-responsive genes, and knockout of the IFN receptor abolished the weight loss phenotype induced by the T39 ASO. Some human T39 ASOs and ASOs with different modifications targeting LDLR also induced a type I IFN response in THP1 macrophages.

Conclusion: Our data suggest that extrahepatic targeting of T39 by ASOs in ATMs produced an off-target type 1 IFN response, leading to activation of lipolysis, browning of WAT, and weight loss. While our findings suggest that ASOs may induce off-target type 1 IFN response more commonly than previously thought, they also suggest that therapeutic induction of type 1 IFN selectively in ATMs could potentially represent a novel approach to the treatment of obesity.

Objective: Estrogen receptor-α (ERα) is a nuclear receptor family member thought to substantially contribute to the metabolic regulation of skeletal muscle. However, previous mouse models utilized to assess the necessity of ERα signaling in skeletal muscle were confounded by altered developmental programming and/or influenced by secondary effects, making it difficult to assign a causal role for ERα. The objective of this study was to determine the role of skeletal muscle ERα in regulating metabolism in the absence of confounding factors of development.

Methods: A novel mouse model was developed allowing for induced deletion of ERα in adult female skeletal muscle (ERαKO^ism). ERαshRNA was also used to knockdown ERα (ERαKD) in human myotubes cultured from primary human skeletal muscle cells isolated from muscle biopsies from healthy and obese insulin-resistant women.

Results: Twelve weeks of HFD exposure had no differential effects on body composition, VO₂, VCO₂, RER, energy expenditure, and activity counts across genotypes. Although ERαKO^ism mice exhibited greater glucose intolerance than wild-type (WT) mice after chronic HFD, ex vivo skeletal muscle glucose uptake was not impaired in the ERαKO^ism mice. Expression of pro-inflammatory genes was altered in the skeletal muscle of the ERαKO^ism, but the concentrations of these inflammatory markers in the systemic circulation were either lower or remained similar to the WT mice. Finally, skeletal muscle mitochondrial respiratory capacity, oxidative phosphorylation efficiency, and H₂O₂ emission potential was not affected in the ERαKO^ism mice. ERαKD in human skeletal muscle cells neither altered differentiation capacity nor caused severe deficits in mitochondrial respiratory capacity.

Conclusions: Collectively, these results suggest that ERα function is superfluous in protecting against HFD-induced skeletal musclemetabolic derangements after postnatal development is complete.

Objective: It is well established that the liver-specific miR-122, a bona fide tumor suppressor, plays a critical role in lipid homeostasis. However, its role, if any, in amino acid metabolism has not been explored. Since glutamine (Gln) is a critical energy and anaplerotic source for mammalian cells, we assessed Gln metabolism in control wild type (WT) mice and miR-122 knockout (KO) mice by stable isotope resolved metabolomics (SIRM) studies.

Methods: Six-to eight-week-old WT and KO mice and 12- to 15-month-old liver tumor-bearing mice were injected with [U-¹³C₅,¹⁵N₂]-L-Gln, and polar metabolites from the liver tissues were analyzed by nuclear magnetic resonance (NMR) imaging and ion chromatography-mass spectrometry (IC-MS). Gln-metabolism was also assessed in a Gln-dependent hepatocellular carcinoma (HCC) cell line (EC4). Expressions of glutaminases (Gls and Gls2) were analyzed in mouse livers and human primary HCC samples.

Results: The results showed that loss of miR-122 promoted glutaminolysis but suppressed gluconeogenesis in mouse livers as evident from the buildup of ¹³C- and/or ¹⁵N-Glu and decrease in glucose-6-phosphate (G6P) levels, respectively, in KO livers. Enhanced glutaminolysis is consistent with the upregulation of expressions of Gls (kidney-type glutaminase) and Slc1a5, a neutral amino acid transporter in KO livers. Both Gls and Slc1a5 were confirmed as direct miR-122 targets by the respective 3'-UTR-driven luciferase assays. Importantly, expressions of Gls and Slc1a5 as well as glutaminase activity were suppressed in a Gln-dependent HCC (EC4) cell line transfected with miR-122 mimic that resulted in decreased ¹³C-Gln, ¹³C-á-ketoglutarate, ¹³C-isocitrate, and ¹³C-citrate levels. In contrast, ¹³C-phosphoenolpyruvate and ¹³C-G6P levels were elevated in cells expressing ectopic miR-122, suggesting enhanced gluconeogenesis. Finally, The Cancer Genome Atlas-Liver Hepatocellular Carcinoma (TCGA-LIHC) database analysis showed that expression of GLS is negatively correlated with miR-122 in primary human HCCs, and the upregulation of GLS RNA is associated with higher tumor grade. More importantly, patients with higher expressions of GLS or SLC1A5 in tumors exhibited poor survival compared with those expressing lower levels of these proteins.

Conclusions: Collectively, these results show that miR-122 modulates Gln metabolism both in vitro and in vivo, implicating the therapeutic potential of miR-122 in HCCs that exhibit relatively high GLS levels.