Volume 48 | June 2021

Background

Over the past 20 years, insights from human and mouse genetics have illuminated the central role of the brain leptin-melanocortin pathway in controlling mammalian food intake, with genetic disruption resulting in extreme obesity, and more subtle polymorphic variations influencing the population distribution of body weight. At the end of 2020, the U.S. Food and Drug Administration (FDA) approved setmelanotide, a melanocortin 4 receptor agonist, for use in individuals with severe obesity due to either pro-opiomelanocortin (POMC), proprotein convertase subtilisin/kexin type 1 (PCSK1), or leptin receptor (LEPR) deficiency.

Scope of review

Herein, we chart the melanocortin pathway's history, explore its pharmacology, genetics, and physiology, and describe how a neuropeptidergic circuit became an important druggable obesity target.

Major conclusions

Unravelling the genetics of the subset of severe obesity has revealed the importance of the melanocortin pathway in appetitive control; coupling this with studying the molecular pharmacology of compounds that bind melanocortin receptors has brought a new obesity drug to the market. This process provides a drug discovery template for complex disorders, which for setmelanotide took 25 years to transform from a single gene into an approved drug.

Background

Diabetes always involves variable degrees of β-cell demise and malfunction leading to insufficient insulin secretion. Besides clinical investigations, many research projects used rodent islets to study various facets of β-cell pathophysiology. Their important contributions laid the foundations of steadily increasing numbers of experimental studies resorting to isolated human islets.

Scope of review

This review, based on an analysis of data published over 60 years of clinical investigations and results of more recent studies in isolated islets, addresses a question of translational nature. Does the information obtained in vitro with human islets fit with our knowledge of insulin secretion in man? The aims are not to discuss specificities of pathways controlling secretion but to compare qualitative and quantitative features of glucose-induced insulin secretion in isolated human islets and in living human subjects.

Major conclusions

Much of the information gathered in vitro can reliably be translated to the in vivo situation. There is a fairly good, though not complete, qualitative and quantitative coherence between insulin secretion rates measured in vivo and in vitro during stimulation with physiological glucose concentrations, but the concordance fades out under extreme conditions. Perplexing discrepancies also exist between insulin secretion in subjects with Type 2 diabetes and their islets studied in vitro, in particular concerning the kinetics. Future projects should ascertain that the experimental conditions are close to physiological and do not alter the function of normal and diabetic islets.

Objective

Recent studies suggest that excess dietary fructose contributes to metabolic dysfunction by promoting insulin resistance, de novo lipogenesis (DNL), and hepatic steatosis, thereby increasing the risk of obesity, type 2 diabetes (T2D), non-alcoholic steatohepatitis (NASH), and related comorbidities. Whether this metabolic dysfunction is driven by the excess dietary calories contained in fructose or whether fructose catabolism itself is uniquely pathogenic remains controversial. We sought to test whether a small molecule inhibitor of the primary fructose metabolizing enzyme ketohexokinase (KHK) can ameliorate the metabolic effects of fructose.

Methods

The KHK inhibitor PF-06835919 was used to block fructose metabolism in primary hepatocytes and Sprague Dawley rats fed either a high-fructose diet (30% fructose kcal/g) or a diet reflecting the average macronutrient dietary content of an American diet (AD) (7.5% fructose kcal/g). The effects of fructose consumption and KHK inhibition on hepatic steatosis, insulin resistance, and hyperlipidemia were evaluated, along with the activation of DNL and the enzymes that regulate lipid synthesis. A metabolomic analysis was performed to confirm KHK inhibition and understand metabolite changes in response to fructose metabolism in vitro and in vivo. Additionally, the effects of administering a single ascending dose of PF-06835919 on fructose metabolism markers in healthy human study participants were assessed in a randomized placebo-controlled phase 1 study.

Results

Inhibition of KHK in rats prevented hyperinsulinemia and hypertriglyceridemiafrom fructose feeding. Supraphysiologic levels of dietary fructose were not necessary to cause metabolic dysfunction as rats fed the American diet developed hyperinsulinemia, hypertriglyceridemia, and hepatic steatosis, which were all reversed by KHK inhibition. Reversal of the metabolic effects of fructose coincided with reductions in DNL and inactivation of the lipogenic transcription factor carbohydrate response element-binding protein (ChREBP). We report that administering single oral doses of PF-06835919 was safe and well tolerated in healthy study participants and dose-dependently increased plasma fructose indicative of KHK inhibition.

Conclusions

Fructose consumption in rats promoted features of metabolic dysfunction seen in metabolic diseases such as T2D and NASH, including insulin resistance, hypertriglyceridemia, and hepatic steatosis, which were reversed by KHK inhibition.

Objective

Mitochondrial aconitase (ACO2) is an essential enzyme that bridges the TCA cycle and lipid metabolism. However, its role in cancer development remains to be elucidated. The metabolic subtype of colorectal cancer (CRC) was recently established. We investigated ACO2's potential role in CRC progression through mediating metabolic alterations.

Methods

We compared the mRNA and protein expression of ACO2 between paired CRC and non-tumor tissues from 353 patients. Correlations between ACO2 levels and clinicopathological features were examined. CRC cell lines with knockdown or overexpression of ACO2 were analyzed for cell proliferation and tumor growth. Metabolomics and stable isotope tracing analyses were used to study the metabolic alterations induced by loss of ACO2.

Results

ACO2 decreased in >50% of CRC samples compared with matched non-tumor tissues. Decreased ACO2 levels correlated with advanced disease stage (P < 0.001) and shorter patient survival (P < 0.001). Knockdown of ACO2 in CRC cells promoted cell proliferation and tumor formation, while ectopic expression of ACO2 restrained tumor growth. Specifically, blockade of ACO2 caused a reduction in TCA cycle intermediates and suppression of mitochondrial oxidative phosphorylation, resulting in an increase in glycolysis and elevated citrate flux for fatty acid and lipid synthesis. Increased citrate flux induced upregulation of stearoyl-CoA desaturase (SCD1), which enhanced lipid desaturation in ACO2-deficent cells to favor colorectal cancer growth. Pharmacological inhibition of SCD selectively reduced tumor formation of CRC with ACO2 deficiency.

Conclusions

Our study demonstrated that the rewiring metabolic pathway maintains CRC survival during compromised TCA cycles and characterized the therapeutic vulnerability of lipid desaturation in a meaningful subset of CRC with mitochondrial dysfunction.

Objectives

The hypothalamic ventromedial nucleus (VMH) plays a major role in metabolic control, but the molecular mechanisms involved remain poorly defined. We analyzed the relevance of the BBSome, a protein complex composed of 8 Bardet–Biedl syndrome (BBS) proteins including BBS1, in VMH steroidogenic factor 1 (SF1) neurons for the control of energy homeostasis and related physiological processes.

Methods

We generated mice bearing selective BBSome disruption, through Bbs1 gene deletion, in SF1 neurons (SF1^Cre/Bbs1^fl/fl). We analyzed the consequence on body weight, glucose homeostasis, and cardiovascular autonomic function of BBSome loss in SF1 neurons.

Results

SF1^Cre/Bbs1^fl/fl mice had increased body weight and adiposity under normal chow conditions. Food intake, energy absorption, and digestive efficiency were not altered by Bbs1 gene deletion in SF1 neurons. SF1^Cre/Bbs1^fl/fl mice exhibited lower energy expenditure, particularly during the dark cycle. Consistent with this finding, SF1^Cre/Bbs1^fl/fl mice displayed reduced sympathetic nerve traffic and expression of markers of thermogenesis in brown adipose tissue. SF1^Cre/Bbs1^fl/fl mice also had lower sympathetic nerve activity to subcutaneous white adipose tissue that was associated with a protein expression profile that promotes lipid accumulation. Notably, despite obesity and hyperinsulinemia, SF1^Cre/Bbs1^fl/fl mice did not exhibit significant changes in glucose metabolism, insulin sensitivity, blood pressure, and baroreflex sensitivity.

Conclusions

Our findings demonstrate that the SF1 neuron BBSome is necessary for the regulation of energy homeostasis through modulation of the activity of the sympathetic nervous system and that the SF1 neuron BBSome is required for the development of obesity-related comorbidities.

Background

Type 2 diabetes (T2D) is a common metabolic disease. Variants in human IGF2 mRNA binding protein 2 (IMP2/IGF2BP2) are associated with increased risk of T2D. IMP2 contributes to T2D susceptibility primarily through effects on insulin secretion. However, the underlying mechanism is not known.

Methods

To understand the role of IMP2 in insulin secretion and T2D pathophysiology, we generated Imp2 pancreatic β-cell specific knockout mice (βIMP2KO) by recombining the Imp2^flox allele with Cre recombinase driven by the rat insulin 2 promoter. We further characterized metabolic phenotypes of βIMP2KO mice and assessed their β-cell functions.

Results

The deletion of IMP2 in pancreatic β-cells leads to reduced compensatory β-cell proliferation and function. Mechanically, IMP2 directly binds to Pdx1 mRNA and stimulates its translation in an m6A dependent manner. Moreover, IMP2 orchestrates IGF2-AKT-GSK3β-PDX1 signaling to stable PDX1 polypeptides. In human EndoC-βH1 cells, the over-expression of IMP2 is capable to enhance cell proliferation, PDX1 protein level and insulin secretion.

Conclusion

Our work therefore reveals IMP2 as a critical regulator of pancreatic β-cell proliferation and function; highlights the importance of posttranscriptional gene expression in T2D pathology.

Objective

White adipose tissue (WAT) expansion regulates energy balance and overall metabolic homeostasis. The absence or loss of WAT occurring through lipodystrophy and lipoatrophy contributes to the development of hepatic steatosisand insulin resistance. We previously demonstrated that sole small ubiquitin-like modifier (SUMO) E2-conjugating enzyme Ube2i represses human adipocyte differentiation. The role of Ube2i during WAT development remains unknown.

Methods

To determine how Ube2i impacts body composition and energy balance, we generated adipocyte-specific Ube2i knockout mice (Ube2i^a-KO). CRISPR/Cas9 gene editing inserted loxP sites flanking exons 3 and 4 at the Ube2i locus. Subsequent genetic crosses to Adipoq-Cre transgenic mice allowed deletion of Ube2i in white and brown adipocytes. We measured multiple metabolic endpoints that describe energy balance and carbohydrate metabolism in Ube2i^a-KO and littermate controls during postnatal growth.

Results

Surprisingly, Ube2i^a-KO mice developed hyperinsulinemia and hepatic steatosis. Global energy balance defects emerged from dysfunctional WAT marked by pronounced local inflammation, loss of serum adipokines, hepatomegaly, and near absence of major adipose tissue depots. We observed progressive lipoatrophy that commences in the early adolescent period.

Conclusions

Our results demonstrate that Ube2i expression in mature adipocytes allows WAT expansion during postnatal growth. Deletion of Ube2i in fat cells compromises and diminishes adipocyte function that induces WAT inflammation and ectopic lipid accumulation in the liver. Our findings reveal an indispensable role for Ube2iduring white adipocyte expansion and endocrine control of energy balance.

Objective

Metabolic deregulation is a key hallmark of cancer cells and has been shown to drive cancer growth and metastasis. However, not all metabolic drivers of melanoma are known. Based on our finding that N-acylsphingosine amidohydrolase 1 (ASAH1) is overexpressed in melanoma, the objective of these studies was to establish its role in melanoma tumor growth and metastasis, understand its mechanism of action, and evaluate ASAH1 targeting for melanoma therapy.

Methods

We used publicly available melanoma datasets and patient-derived samples of melanoma and normal skin tissue and analyzed them for ASAH1 mRNA expression and ASAH1 protein expression using immunohistochemistry. ASAH1 was knocked down using short-hairpin RNAs in multiple melanoma cell lines that were tested in a series of cell culture-based assays and mouse-based melanoma xenograft assays to monitor the effect of ASAH1 knockdown on melanoma tumor growth and metastasis. An unbiased metabolomics analysis was performed to identify the mechanism of ASAH1 action. Based on the metabolomics findings, the role of peroxisome-mediated reactive oxygen species (ROS) production was explored in regard to mediating the effect of ASAH1. The ASAH1 inhibitor was used alone or in combination with a BRAFV600E inhibitor to evaluate the therapeutic value of ASAH1 targeting for melanoma therapy.

Results

We determined that ASAH1 was overexpressed in a large percentage of melanoma cells and regulated by transcription factor E2F1 in a mitogen-activated protein (MAP) kinase pathway-dependent manner. ASAH1 expression was necessary to maintain melanoma tumor growth and metastatic attributes in cell cultures and mouse models of melanoma tumor growth and metastasis. To identify the mechanism by which ASAH1 facilitates melanoma tumor growth and metastasis, we performed a large-scale and unbiased metabolomics analysis of melanoma cells expressing ASAH1 short-hairpin RNAs (shRNAs). We found that ASAH1 inhibition increased peroxisome biogenesis through ceramide-mediated PPARγ activation. ASAH1 loss increased ceramide and peroxisome-derived ROS, which in turn inhibited melanoma growth. Pharmacological inhibition of ASAH1 also attenuated melanoma growth and enhanced the effectiveness of BRAF kinase inhibitor in the cell cultures and mice.

Conclusions

Collectively, these results demonstrate that ASAH1 is a druggable driver of melanoma tumor growth and metastasis that functions by suppressing peroxisome biogenesis, thereby inhibiting peroxisome-derived ROS production. These studies also highlight the therapeutic utility of ASAH1 inhibitors for melanoma therapy.

Objective

Glucose-dependent insulinotropic polypeptide (GIP) and Glucagon-like peptide-1 (GLP-1) are incretin hormones that exert overlapping yet distinct actions on islet β-cells. We recently observed that GIP, but not GLP-1, upregulated islet expression of Transcription Factor 7 (TCF7), a gene expressed in immune cells and associated with the risk of developing type 1 diabetes. TCF7 has also been associated with glucose homeostasis control in the liver. Herein we studied the relative metabolic importance of TCF7 expression in hepatocytes vs. islet β-cells in mice.

Methods

Tcf7 expression was selectively inactivated in adult mouse hepatocytes using adenoviral Cre expression and targeted in β-cells using two different lines of insulin promoter-Cre mice. Glucose homeostasis, plasma insulin and triglycerideresponses, islet histology, hepatic and islet gene expression, and body weight gain were evaluated in mice fed regular chow or high fat diets. Tcf7 expression within pancreatic islets and immune cells was evaluated using published single cell RNA-seq (scRNA-seq) data, and in islet RNA from immunodeficient Rag2^−/−Il2rg^−/− mice.

Results

Reduction of hepatocyte Tcf7 expression did not impair glucose homeostasis, lipid tolerance or hepatic gene expression profiles linked to control of metabolic or immune pathways. Similarly, oral and intraperitoneal glucose tolerance, plasma insulin responses, islet histology, body weight gain, and insulin tolerance were not different in mice with targeted recombination of Tcf7 in insulin-positive β-cells. Surprisingly, islet Tcf7 mRNA transcripts were not reduced in total islet RNA containing endocrine and associated non-endocrine cell types from Tcf7^{βcell−/−} mice, despite Cre-mediated recombination of islet genomic DNA. Furthermore, glucose tolerance was normal in whole body Tcf7^−/− mice. Analysis of scRNA-seq datasets localized pancreatic Tcf7 expression to islet progenitors during development, and immune cells, but not within differentiated islet β-cells or endocrine lineages within mature islets. Moreover, the expression of Tcf7 was extremely low in islet RNA from Rag2^−/−Il2rg^−/− mice and, consistent with expression within immune cells, Tcf7 was highly correlated with levels of Cd3g mRNA transcripts in RNA from wild type mouse islets.

Conclusions

These findings demonstrate that Tcf7 expression is not a critical determinant of glucose homeostasis in mice. Moreover, the detection of Tcf7 expression within islet mRNA is attributable to the expression of Tcf7 RNA in islet-associated murine immune cells, and not in islet β-cells.

Objective

Neuroimmune interactions between the sympathetic nervous system (SNS) and macrophages are required for the homeostasis of multiple tissues, including the adipose tissue. It has been proposed that the SNS maintains adipose tissue macrophages (ATMs) in an anti-inflammatory state via direct norepinephrine (NE) signaling to macrophages. This study aimed to investigate the physiological importance of this paradigm by utilizing a mouse model in which the adrenergic signaling from the SNS to macrophages, but not to other adipose tissue cells, was disrupted.

Methods

We generated a macrophage-specific B2AR knockout mouse (Adrb2^ΔLyz2) by crossing Adrb2^fl/fl and Lyz2^Cre/+ mice. We have previously shown that macrophages isolated from Adrb2^ΔLyz2 animals do not respond to NE stimulation in vitro. Herein we performed a metabolic phenotyping of Adrb2^ΔLyz2 mice on either chow or high-fat diet (HFD). We also assessed the adipose tissue function of Adrb2^ΔLyz2 animals during fasting and cold exposure. Finally, we transplanted Adrb2^ΔLyz2 bone marrow to low-density lipoprotein receptor (LDLR) knockout mice and investigated the development of atherosclerosis during Western diet feeding.

Results

We demonstrated that SNS-associated ATMs have a transcriptional profile indicative of activated beta-2 adrenergic receptor (B2AR), the main adrenergic receptor isoform in myeloid cells. However, Adrb2^ΔLyz2 mice have unaltered energy balance on a chow or HFD. Furthermore, Adrb2^ΔLyz2 mice show similar levels of adipose tissue inflammation and function during feeding, fasting, or cold exposure, and develop insulin resistance during HFD at the same rate as controls. Finally, macrophage-specific B2AR deletion does not affect the development of atherosclerosis on an LDL receptor-null genetic background.

Conclusions

Overall, our data suggest that the SNS does not directly modulate the phenotype of adipose tissue macrophages in either lean mice or mouse models of cardiometabolic disease. Instead, sympathetic nerve activity exerts an indirect effect on adipose tissue macrophages through the modulation of adipocyte function.

Objective

Carbonyl reductase 1 (Cbr1), a recently discovered contributor to tissue glucocorticoid metabolism converting corticosterone to 20β-dihydrocorticosterone (20β-DHB), is upregulated in adipose tissue of obese humans and mice and may contribute to cardiometabolic complications of obesity. This study tested the hypothesis that Cbr1-mediated glucocorticoid metabolism influences glucocorticoid and mineralocorticoid receptor activation in adipose tissue and impacts glucose homeostasis in lean and obese states.

Methods

The actions of 20β-DHB on corticosteroid receptors in adipose tissue were investigated first using a combination of in silico, in vitro, and transcriptomictechniques and then in vivo administration in combination with receptor antagonists. Mice lacking one Cbr1 allele and mice overexpressing Cbr1 in their adipose tissue underwent metabolic phenotyping before and after induction of obesity with high-fat feeding.

Results

20β-DHB activated both the glucocorticoid and mineralocorticoid receptor in adipose tissue and systemic administration to wild-type mice induced glucose intolerance, an effect that was ameliorated by both glucocorticoid and mineralocorticoid receptor antagonism. Cbr1 haploinsufficient lean male mice had lower fasting glucose and improved glucose tolerance compared with littermate controls, a difference that was abolished by administration of 20β-DHB and absent in female mice with higher baseline adipose 20β-DHB concentrations than male mice. Conversely, overexpression of Cbr1 in adipose tissue resulted in worsened glucose tolerance and higher fasting glucose in lean male and female mice. However, neither Cbr1 haploinsfficiency nor adipose overexpression affected glucose dyshomeostasis induced by high-fat feeding.

Conclusions

Carbonyl reductase 1 is a novel regulator of glucocorticoid and mineralocorticoid receptor activation in adipose tissue that influences glucose homeostasis in lean mice.

Objective

Hypothalamic inflammation and endoplasmic reticulum (ER) stress are extensively linked to leptin resistance and overnutrition-related diseases. Surgical intervention remains the most efficient long-term weight-loss strategy for morbid obesity, but mechanisms underlying sustained feeding suppression remain largely elusive. This study investigated whether Roux-en-Y gastric bypass (RYGB) interacts with obesity-associated hypothalamic inflammation to restore central leptin signaling as a mechanistic account for post-operative appetite suppression.

Methods

RYGB or sham surgery was performed in high-fat diet-induced obese Wistar rats. Sham-operated rats were fed ad libitum or by weight matching to RYGB via calorie restriction (CR) before hypothalamic leptin signaling, microglia reactivity, and the inflammatory pathways were examined to be under the control of gut microbiota-derived circulating signaling.

Results

RYGB, other than CR-induced adiposity reduction, ameliorates hypothalamic gliosis, inflammatory signaling, and ER stress, which are linked to enhanced hypothalamic leptin signaling and responsiveness. Mechanistically, we demonstrate that RYGB interferes with hypothalamic ER stress and toll-like receptor 4 (TLR4) signaling to restore the anorexigenic action of leptin, which most likely results from modulation of a circulating factor derived from the altered gut microbial environment upon RYGB surgery.

Conclusions

Our data demonstrate that RYGB interferes with hypothalamic TLR4 signaling to restore the anorexigenic action of leptin, which most likely results from modulation of a circulating factor derived from the post-surgical altered gut microbial environment.

Objective

Liver glycogen levels are dynamic and highly regulated by nutrient availability as the levels decrease during fasting and are restored during the feeding cycle. However, feeding in the presence of fructose in water suppresses glycogen accumulation in the liver by upregulating the expression of the glucose-6-phosphatase catalytic subunit (G6pc) gene, although the exact mechanism is unknown. We generated liver-specific knockout MED13 mice that lacked the transcriptional Mediator complexkinase module to examine its effect on the transcriptional activation of inducible target gene expression, such as the ChREBP- and FOXO1-dependent control of the G6pc gene promoter.

Methods

The relative changes in liver expression of lipogenic and gluconeogenic genes as well as glycogen levels were examined in response to feeding standard low-fat laboratory chow supplemented with water or water containing sucrose or fructose in control (Med13^fl/fl) and liver-specific MED13 knockout (MED13-LKO) mice.

Results

Although MED13 deficiency had no significant effect on constitutive gene expression, all the dietary inducible gene transcripts were significantly reduced despite the unchanged insulin sensitivity in the MED13-LKO mice compared to that in the control mice. G6pc gene transcription displayed the most significant difference between the Med13 ^fl/fl and MED13-LKO mice, particularly when fed fructose. Following fasting that depleted liver glycogen, feeding induced the restoration of glycogen levels except in the presence of fructose. MED13 deficiency rescued the glycogen accumulation defect in the presence of fructose. This resulted from the suppression of G6pc expression and thus G6PC enzymatic activity.

Among two transcriptional factors that regulate G6pc gene expression, FOXO1binding to the G6pc promoter was not affected, whereas ChREBP binding was dramatically reduced in MED13-LKO hepatocytes. In addition, there was a marked suppression of FOXO1 and ChREBP-β transcriptional activities in MED13-LKO hepatocytes.

Conclusions

Taken together, our data suggest that the kinase module of the Mediator complex is necessary for the transcriptional activation of metabolic genes such as G6pc and has an important role in regulating glycogen levels in the liver through altering transcription factor binding and activity at the G6pc promoter.

Objective

Non-alcoholic fatty liver disease (NAFLD) is a silent pandemic associated with obesity and the metabolic syndrome, and also increases cardiovascular- and cirrhosis-related morbidity and mortality. A complete understanding of adaptive compensatory metabolic programmes that modulate non-alcoholic steatohepatitis (NASH) progression is lacking.

Methods and results

Transcriptomic analysis of liver biopsies in patients with NASH revealed that NASH progression is associated with rewiring of metabolic pathways, including upregulation of de novo lipid/cholesterol synthesis and fatty acid remodelling. The modulation of these metabolic programmes was achieved by activating sterol regulatory element-binding protein (SREBP) transcriptional networks; however, it is still debated whether, in the context of NASH, activation of SREBPs acts as a pathogenic driver of lipotoxicity, or rather promotes the biosynthesis of protective lipids that buffer excessive lipid accumulation, preventing inflammation and fibrosis. To elucidate the pathophysiological role of SCAP/SREBP in NASH and wound-healing response, we used an Insig1 deficient (with hyper-efficient SREBPs) murine model challenged with a NASH-inducing diet. Despite enhanced lipid and cholesterol biosynthesis, Insig1 KO mice had similar systemic metabolism and insulin sensitivity to Het/WT littermates. Moreover, activating SREBPs resulted in remodelling the lipidome, decreased hepatocellular damage, and improved wound-healing responses.

Conclusions

Our study provides actionable knowledge about the pathways and mechanisms involved in NAFLD pathogenesis, which may prove useful for developing new therapeutic strategies. Our results also suggest that the SCAP/SREBP/INSIG1 trio governs transcriptional programmes aimed at protecting the liver from lipotoxic insults in NASH.