Volume 40 | October 2020

Objectives

Elevated plasma glucagon is an early symptom of diabetes, occurring in subjects with impaired glucose regulation. Here, we explored alpha-cell function in female mice fed a high-fat diet (HFD).

Methods

Female mice expressing the Ca²⁺ indicator GCaMP3 specifically in alpha-cells were fed a high-fat or control (CTL) diet. We then conducted in vivo phenotyping of these mice, as well as experiments on isolated (ex vivo) islets and in the in situperfused pancreas.

Results

In HFD-fed mice, fed plasma glucagon levels were increased and glucagon secretion from isolated islets and in the perfused mouse pancreas was also elevated. In mice fed a CTL diet, increasing glucose reduced intracellular Ca²⁺([Ca²⁺]_i) oscillation frequency and amplitude. This effect was also observed in HFD mice; however, both the frequency and amplitude of the [Ca²⁺]_i oscillations were higher than those in CTL alpha-cells. Given that alpha-cells are under strong paracrine control from neighbouring somatostatin-secreting delta-cells, we hypothesised that this elevation of alpha-cell output was due to a lack of somatostatin (SST) secretion. Indeed, SST secretion in isolated islets from HFD-fed mice was reduced but exogenous SST also failed to suppress glucagon secretion and [Ca²⁺]_i activity from HFD alpha-cells, in contrast to observations in CTL mice.

Conclusions

These findings suggest that reduced delta-cell function, combined with intrinsic changes in alpha-cells including sensitivity to somatostatin, accounts for the hyperglucagonaemia in mice fed a HFD.

Objective

Deletion of Translin (Tsn) from mice induces an unusual metabolic profile characterized by robust adiposity, normal body weight and glucose tolerance. Translin (TN) protein and its partner, trax (TX), form the TN/TX microRNA-degrading enzyme. Since the microRNA system plays a prominent role in regulating metabolism, we reasoned that the metabolic profile displayed by TsnKO mice might reflect dysregulation of microRNA signaling.

Methods

To test this hypothesis, we inserted a mutation, E126A, in Tsnax, the gene encoding TX, that abolishes the microRNA-degrading enzymatic activity of the TN/TX complex. In addition, to help define the cell types that drive the adiposity phenotype, we have also generated mice with floxed alleles of Tsn or Tsnax.

Results

Introduction of the E126A mutation in Tsnax does not impair expression of TN or TX proteins or their co-precipitation. Furthermore, these mice display selective increases in microRNAs that match those induced by Tsn deletion, confirming that this mutation in Tsnax inactivates the microRNA-degrading activity of the TN/TX complex. Mice homozygous for the Tsnax (E126A) mutation display a metabolic profile that closely mimics that of Tsn KO mice.

Selective deletion of Tsn or Tsnax from either adipocytes or hepatocytes, two candidate cell types, does not phenocopy the elevated adiposity displayed by mice with constitutive Tsn deletion or the Tsnax (E126A) mutation. Furthermore, global, conditional deletion of Tsn in adulthood does not elicit increased adiposity.

Conclusion

Taken together, these findings indicate that inactivation of the TN/TX microRNA-degrading enzyme during development is necessary to drive the robust adiposity displayed by Tsn KO mice.

Objectives

Typically, obesity results from an inappropriate balance between energy uptake from nutrient consumption and burning of calories, which leads to a pathological increase in fat mass. Obesity is a major cause of insulin resistance and diabetes. Inhibitory G proteins (Gα_i) form a subfamily that is involved in the regulation of adipose tissue function. Among the three Gα_i members, i.e. Gα_i1, Gα_i2, Gα_i3, the Gα_i2, protein is predominantly expressed in adipose tissue. However, the functions of the Gα_i2 isoform in adipose tissue and its impact on the development of obesity are poorly understood.

Methods

By using AdipoqCreER^T2 mice, we generated adipocyte-specific Gnai2-deficient mice to study Gα_i2 function, specifically in white and brown adipocytes. These mice were fed either a control diet (CD) or a high fat diet (HFD). Mice were examined for obesity development, insulin resistance and glucose intolerance. We examined adipocyte morphology and the development of inflammation in the white adipose tissue. Finally, intracellular cAMP levels as an indicator of Gα_isignaling and glycerol release as an indicator of lipolysis rates were measured to verify the impact of Gα_i2 on the signaling pathway in brown and white adipocytes.

Results

An adipocyte-specific deficiency of Gα_i2 significantly reduced diet-induced obesity, leading to decreased fat masses, smaller adipocytes and decreased inflammation in the white adipose tissue relative to littermate controls. Concurrently, oxygen consumption of brown adipocytes and in vivo measured energy expenditure were significantly enhanced. In addition, glucose tolerance and insulin sensitivity of HFD-fed adipocyte-specific Gnai2-deficient mice were improved compared to the respective controls. In the absence of Gα_i2, adrenergic stimulation of intracellular adipocyte cAMP levels was increased, which correlated with increased lipolysis and energy expenditure.

Conclusion

We conclude that adipocyte Gα_i2 is a major regulator of adipocyte lipid content in diet-induced obesity by inhibiting adipocyte lipolysis in a cAMP-dependent manner resulting in increased energy expenditure.

Objective

Risk alleles for type 2 diabetes at the STARD10 locus are associated with lowered STARD10 expression in the β-cell, impaired glucose-induced insulin secretion, and decreased circulating proinsulin:insulin ratios. Although likely to serve as a mediator of intracellular lipid transfer, the identity of the transported lipids and thus the pathways through which STARD10 regulates β-cell function are not understood. The aim of this study was to identify the lipids transported and affected by STARD10 in the β-cell and the role of the protein in controlling proinsulin processing and insulin granule biogenesis and maturation.

Methods

We used isolated islets from mice deleted selectively in the β-cell for Stard10(βStard10KO) and performed electron microscopy, pulse-chase, RNA sequencing, and lipidomic analyses. Proteomic analysis of STARD10 binding partners was executed in the INS1 (832/13) cell line. X-ray crystallography followed by molecular docking and lipid overlay assay was performed on purified STARD10 protein.

Results

βStard10KO islets had a sharply altered dense core granule appearance, with a dramatic increase in the number of “rod-like” dense cores. Correspondingly, basal secretion of proinsulin was increased versus wild-type islets. The solution of the crystal structure of STARD10 to 2.3 Å resolution revealed a binding pocket capable of accommodating polyphosphoinositides, and STARD10 was shown to bind to inositides phosphorylated at the 3’ position. Lipidomic analysis of βStard10KO islets demonstrated changes in phosphatidylinositol levels, and the inositol lipid kinase PIP4K2C was identified as a STARD10 binding partner. Also consistent with roles for STARD10 in phosphoinositide signalling, the phosphoinositide-binding proteins Pirt and Synaptotagmin 1 were amongst the differentially expressed genes in βStard10KO islets.

Conclusion

Our data indicate that STARD10 binds to, and may transport, phosphatidylinositides, influencing membrane lipid composition, insulin granule biosynthesis, and insulin processing.

Objective

Increasing energy expenditure through activation of brown adipose tissue (BAT) thermogenesis is an attractive approach to counteract obesity. It is therefore essential to understand the molecular mechanisms that control BAT functions. Until now several members of the Janus kinase (JAK) - signal transducer and activator of transcription (STAT) pathway have been implicated as being relevant for BAT physiology. However, whether the STAT family member STAT5 is important for the thermogenic property of adipose tissues is unknown. Therefore, we have investigated the role of STAT5 in thermogenic fat in this paper.

Methods

We performed metabolic and molecular analyses using mice that harbor an adipocyte-specific deletion of Stat5a/b alleles.

Results

We found that STAT5 is necessary for acute cold-induced temperature maintenance and the induction of lipid mobilization in BAT following β₃-adrenergic stimulation. Moreover, mitochondrial respiration of primary differentiated brown adipocytes lacking STAT5 was diminished. Increased sensitivity to cold stress upon STAT5 deficiency was associated with reduced expression of thermogenic markers including uncoupling protein 1 (UCP1), while decreased stimulated lipolysis was linked to decreased protein kinase A (PKA) activity. Additionally, brown remodeling of white adipose tissue was diminished following chronic β₃-adrenergic stimulation, which was accompanied by a decrease in mitochondrial performance.

Conclusion

We conclude that STAT5 is essential for the functionality and the β-adrenergic responsiveness of thermogenic adipose tissue.

Objective

Investigations of autophagy in β-cells have usually focused on its homeostatic function. More dynamic roles in inhibiting glucose-stimulated insulin secretion (GSIS), potentially involving remodelling of cellular lipids, have been suggested from in vitro studies but not evaluated in vivo.

Methods

We employed temporally-regulated deletion of the essential autophagy gene, Atg7, in β-cells. Mice were fed chow or high-fat diets (HFD), in conjunction with deletion of Atg7 for the last 3 weeks (short-term model) or 9 weeks (long-term model). Standard in vivo metabolic phenotyping was undertaken, and 450 lipid species in islets quantified ex vivo using mass spectroscopy (MS). MIN6 cells were also employed for lipidomics and secretory interventions.

Results

β-cell function was impaired by inhibiting autophagy in the longer-term, but conversely improved by 3-week deletion of Atg7, specifically under HFD conditions. This was accompanied by augmented GSIS ex vivo. Surprisingly, the HFD had minimal effect on sphingolipid and neutral lipid species, but modulated >100 phospholipids and ether lipids, and markedly shifted the profile of polyunsaturated fatty acid (PUFA) sidechains from n3 to n6 forms. These changes were partially countered by Atg7 deletion, consistent with an accompanying upregulation of the PUFA elongase enzyme, Elovl5. Loss of Atg7 separately augmented plasmalogens and alkyl lipids, in association with increased expression of Lonp2, a peroxisomal chaperone/protease that facilitates maturation of ether lipid synthetic enzymes. Depletion of PUFAs and ether lipids was also observed in MIN6 cells chronically exposed to oleate (more so than palmitate). GSIS was inhibited by knocking down Dhrs7b, which encodes an enzyme of peroxisomal ether lipid synthesis. Conversely, impaired GSIS due to oleate pre-treatment was selectively reverted by Dhrs7b overexpression.

Conclusions

A detrimental increase in n6:n3 PUFA ratios in ether lipids and phospholipids is revealed as a major response of β-cells to high-fat feeding. This is partially reversed by short-term inhibition of autophagy, which results in compensatory changes in peroxisomal lipid metabolism. The short-term phenotype is linked to improved GSIS, in contrast to the impairment seen with the longer-term inhibition of autophagy. The balance between these positive and negative inputs could help determine whether β-cells adapt or fail in response to obesity.

Objective

Extrahepatic vitamin A is housed within organ-specific stellate cells that support local tissue function. These cells have been reported in the vocal fold mucosa (VFM) of the larynx; however, it is unknown how vitamin A reaches and is disseminated among VFM target cells, how VFM storage and utilization vary as a function of total body stores, and how these parameters change in the context of pathology. Therefore, in this study, we investigated fundamental VFM vitamin A uptake and metabolism.

Methods

Using cadaveric tissue and serum from human donors representing the full continuum of clinical vitamin A status, we established a concentration range and analyzed the impact of biologic and clinical covariates on VFM vitamin A. We additionally conducted immunodetection of vitamin A-associated markers and pharmacokinetic profiling of orally dosed α-retinyl ester (a chylomicron tracer) in rats.

Results

Serum vitamin A was a significant predictor of human VFM concentrations, suggesting that VFM stores may be rapidly metabolized in situ and replenished from the circulatory pool. On a vitamin A-sufficient background, dosed α-vitamin A was detected in rat VFM in both ester and alcohol forms, showing that, in addition to plasma retinol and local stellate cell stores, VFM can access and process postprandial retinyl esters from circulating chylomicra. Both α forms were rapidly depleted, confirming the high metabolic demand for vitamin A within VFM.

Conclusion

This thorough physiological analysis validates VFM as an extrahepatic vitamin A repository and characterizes its unique uptake, storage, and utilization phenotype.

Objectives

Apoptosis-Inducing Factor (AIF) is a protein involved in mitochondrial electron transport chain assembly/stability and programmed cell death. The relevant role of this protein is underlined because mutations altering mitochondrial AIF properties result in acute pediatric mitochondriopathies and tumor metastasis. By generating an original AIF-deficient mouse strain, this study attempted to analyze, in a single paradigm, the cellular and developmental metabolic consequences of AIF loss and the subsequent oxidative phosphorylation (OXPHOS) dysfunction.

Methods

We developed a novel AIF-deficient mouse strain and assessed, using molecular and cell biology approaches, the cellular, embryonic, and adult mice phenotypic alterations. Additionally, we conducted ex vivo assays with primary and immortalized AIF knockout mouse embryonic fibroblasts (MEFs) to establish the cell death characteristics and the metabolic adaptive responses provoked by the mitochondrial electron transport chain (ETC) breakdown.

Results

AIF deficiency destabilized mitochondrial ETC and provoked supercomplex disorganization, mitochondrial transmembrane potential loss, and high generation of mitochondrial reactive oxygen species (ROS). AIF^-/Y MEFs counterbalanced these OXPHOS alterations by mitochondrial network reorganization and a metabolic reprogramming toward anaerobic glycolysis illustrated by the AMPK phosphorylation at Thr172, the overexpression of the glucose transporter GLUT-4, the subsequent enhancement of glucose uptake, and the anaerobic lactate generation. A late phenotype was characterized by the activation of P53/P21-mediated senescence. Notably, approximately 2% of AIF^-/YMEFs diminished both mitochondrial mass and ROS levels and spontaneously proliferated. These cycling AIF^-/Y MEFs were resistant to caspase-independent cell death inducers. The AIF-deficient mouse strain was embryonic lethal between E11.5 and E13.5 with energy loss, proliferation arrest, and increased apoptotic levels. Contrary to AIF^-/Y MEFs, the AIF KO embryos were unable to reprogram their metabolism toward anaerobic glycolysis. Heterozygous AIF^+/-females displayed progressive bone marrow, thymus, and spleen cellular loss. In addition, approximately 10% of AIF^+/- females developed perinatal hydrocephaly characterized by brain development impairment, meningeal fibrosis, and medullar hemorrhages; those mice died 5 weeks after birth. AIF^+/- with hydrocephaly exhibited loss of ciliated epithelium in the ependymal layer. This phenotype was triggered by the ROS excess. Accordingly, it was possible to diminish the occurrence of hydrocephalus AIF^+/- females by supplying dams and newborns with an antioxidant in drinking water.

Conclusions

In a single knockout model and at 3 different levels (cell, embryo, and adult mice) we demonstrated that by controlling the mitochondrial OXPHOS/metabolism, AIF is a key factor regulating cell differentiation and fate. Additionally, by providing new insights into the pathological consequences of mitochondrial OXPHOS dysfunction, our new findings pave the way for novel pharmacological strategies.

Objective

Insulin signalling via phosphoinositide 3-kinase (PI3K) requires PIK3R1-encoded regulatory subunits. C-terminal PIK3R1 mutations cause SHORT syndrome, as well as lipodystrophy and insulin resistance (IR), surprisingly without fatty liver or metabolic dyslipidaemia. We sought to investigate this discordance.

Methods

The human pathogenic Pik3r1 Y657^∗ mutation was knocked into mice by homologous recombination. Growth, body composition, bioenergetic and metabolic profiles were investigated on chow and high-fat diet (HFD). We examined adipose and liver histology, and assessed liver responses to fasting and refeeding transcriptomically.

Results

Like humans with SHORT syndrome, Pik3r1^WT/Y657∗ mice were small with severe IR, and adipose expansion on HFD was markedly reduced. Also as in humans, plasma lipid concentrations were low, and insulin-stimulated hepatic lipogenesis was not increased despite hyperinsulinemia. At odds with lipodystrophy, however, no adipocyte hypertrophy nor adipose inflammation was found. Liver lipogenic gene expression was not significantly altered, and unbiased transcriptomics showed only minor changes, including evidence of reduced endoplasmic reticulum stress in the fed state and diminished Rictor-dependent transcription on fasting. Increased energy expenditure, which was not explained by hyperglycaemia nor intestinal malabsorption, provided an alternative explanation for the uncoupling of IR from dyslipidaemia.

Conclusions

Pik3r1 dysfunction in mice phenocopies the IR and reduced adiposity without lipotoxicity of human SHORT syndrome. Decreased adiposity may not reflect bona fide lipodystrophy, but rather, increased energy expenditure, and we suggest that further study of brown adipose tissue in both humans and mice is warranted.

Objective

Fasting regimens can promote health, mitigate chronic immunological disorders, and improve age-related pathophysiological parameters in animals and humans. Several ongoing clinical trials are using fasting as a potential therapy for various conditions. Fasting alters metabolism by acting as a reset for energy homeostasis, but the molecular mechanisms underlying the beneficial effects of short-term fasting (STF) are not well understood, particularly at the systems or multiorgan level.

Methods

We performed RNA-sequencing in nine organs from mice fed ad libitum (0 h) or subjected to fasting five times (2–22 h). We applied a combination of multivariate analysis, differential expression analysis, gene ontology, and network analysis for an in-depth understanding of the multiorgan transcriptome. We used literature mining solutions, LitLab™ and Gene Retriever™, to identify the biological and biochemical terms significantly associated with our experimental gene set, which provided additional support and meaning to the experimentally derived gene and inferred protein data.

Results

We cataloged the transcriptional dynamics within and between organs during STF and discovered differential temporal effects of STF among organs. Using gene ontology enrichment analysis, we identified an organ network sharing 37 common biological pathways perturbed by STF. This network incorporates the brain, liver, interscapular brown adipose tissue, and posterior-subcutaneous white adipose tissue; hence, we named it the brain-liver-fats organ network. Using Reactome pathways analysis, we identified the immune system, dominated by T cell regulation processes, as a central and prominent target of systemic modulations during STF in this organ network. The changes we identified in specific immune components point to the priming of adaptive immunity and parallel the fine-tuning of innate immune signaling.

Conclusions

Our study provides a comprehensive multiorgan transcriptomic profiling of mice subjected to multiple periods of STF and provides new insights into the molecular modulators involved in the systemic immunotranscriptomic changes that occur during short-term energy loss.

Objective

Studies in mice have shown that the decrease in lipoprotein lipase (LPL) activity in adipose tissue upon fasting is mediated by induction of the inhibitor ANGPTL4. Here, we aimed to validate this concept in humans by determining the effect of a prolonged fast on ANGPTL4 and LPL gene and protein expression in human subcutaneous adipose tissue.

Methods

Twenty-three volunteers ate a standardized meal at 18.00 h and fasted until 20.00 h the next day. Blood was drawn and periumbilical adipose tissue biopsies were collected 2 h and 26 h after the meal.

Results

Consistent with previous mouse data, LPL activity in human adipose tissue was significantly decreased by fasting (−60%), concurrent with increased ANGPTL4 mRNA (+90%) and decreased ANGPTL8 mRNA (−94%). ANGPTL4 protein levels in adipose tissue were also significantly increased by fasting (+46%), whereas LPL mRNA and protein levels remained unchanged. In agreement with the adipose tissue data, plasma ANGPTL4 levels increased upon fasting (+100%), whereas plasma ANGPTL8 decreased (−79%). Insulin, levels of which significantly decreased upon fasting, downregulated ANGPTL4 mRNA and protein in primary human adipocytes. By contrast, cortisol, levels of which significantly increased upon fasting, upregulated ANGPTL4 mRNA and protein in primary human adipocytes as did fatty acids.

Conclusion

ANGPTL4 levels in human adipose tissue are increased by fasting, likely via increased plasma cortisol and free fatty acids and decreased plasma insulin, resulting in decreased LPL activity.

This clinical trial was registered with identifier NCT03757767.

Objective

Evidence for AMP-activated protein kinase (AMPK)-mediated regulation of skeletal muscle metabolism during exercise is mainly based on transgenic mouse models with chronic (lifelong) disruption of AMPK function. Findings based on such models are potentially biased by secondary effects related to a chronic lack of AMPK function. To study the direct effect(s) of AMPK on muscle metabolism during exercise, we generated a new mouse model with inducible muscle-specific deletion of AMPKα catalytic subunits in adult mice.

Methods

Tamoxifen-inducible and muscle-specific AMPKα1/α2 double KO mice (AMPKα imdKO) were generated by using the Cre/loxP system, with the Cre under the control of the human skeletal muscle actin (HSA) promoter.

Results

During treadmill running at the same relative exercise intensity, AMPKα imdKO mice showed greater depletion of muscle ATP, which was associated with accumulation of the deamination product IMP. Muscle-specific deletion of AMPKα in adult mice promptly reduced maximal running speed and muscle glycogen content and was associated with reduced expression of UGP2, a key component of the glycogen synthesis pathway. Muscle mitochondrial respiration, whole-body substrate utilization, and muscle glucose uptake and fatty acid (FA) oxidation during muscle contractile activity remained unaffected by muscle-specific deletion of AMPKα subunits in adult mice.

Conclusions

Inducible deletion of AMPKα subunits in adult mice reveals that AMPK is required for maintaining muscle ATP levels and nucleotide balance during exercise but is dispensable for regulating muscle glucose uptake, FA oxidation, and substrate utilization during exercise.

Background

Emerging evidence demonstrates that bone is an endocrine organ capable of influencing multiple physiological and pathological processes through the secretion of hormones. Recent research suggests complex crosstalk between the bone and other metabolic and cardiovascular tissues. It was uncovered that three of these bone-derived hormones—osteocalcin, lipocalin 2, and sclerostin—are involved in the endocrine regulations of cardiometabolic health and play vital roles in the pathophysiological process of developing cardiometabolic syndromes such as type 2 diabetes and cardiovascular disease. Chronic low-grade inflammation is one of the hallmarks of cardiometabolic diseases and a major contributor to disease progression. Novel evidence also implicates important roles of bone-derived hormones in the regulation of chronic inflammation.

Scope of review

In this review, we provide a detailed overview of the physiological and pathological roles of osteocalcin, lipocalin 2, and sclerostin in cardiometabolic health regulation and disease development, with a focus on the modulation of chronic inflammation.

Major conclusions

Evidence supports that osteocalcin has a protective role in cardiometabolic health, and an increase of lipocalin 2 contributes to the development of cardiometabolic diseases partly via pro-inflammatory effects. The roles of sclerostin appear to be complicated: It exerts pro-adiposity and pro-insulin resistance effects in type 2 diabetes and has an anti-calcification effect during cardiovascular disease. A better understanding of the actions of these bone-derived hormones in the pathophysiology of cardiometabolic diseases will provide crucial insights to help further research develop new therapeutic strategies to treat these diseases.