Objectives

In Western society, high-caloric diets rich in fats and sugars have fueled the obesity epidemic and its related disorders. Disruption of the body-brain communication, crucial for maintaining glucose and energy homeostasis, arises from both obesogenic and genetic factors, leading to metabolic disorders. Here, we investigate the role of hypothalamic tanycyte shuttles between the pituitary portal blood and the third ventricle cerebrospinal fluid in regulating energy balance.

Methods

We inhibited vesicle-associated membrane proteins (VAMP1-3)-mediated release in tanycytes by expressing the botulinum neurotoxin type B light chain (BoNT/B) in a Cre-dependent manner in tanycytes. This was achieved by injecting either TAT-Cre in the third ventricle or an AAV1/2 expressing Cre under the control of the tanycyte-specific promoter iodothyronine deiodinase 2 into the lateral ventricle of adult male mice.

Results

In male mice fed a standard diet, targeted expression of BoNT/B in adult tanycytes blocks leptin transport into the mediobasal hypothalamus and results in normal-weight central obesity, including increased food intake, abdominal fat deposition, and elevated leptin levels but no marked change in body weight. Furthermore, BoNT/B expression in adult tanycytes promotes fatty acid storage, leading to glucose intolerance and insulin resistance. Notably, these metabolic disturbances occur despite a compensatory increase in insulin secretion, observed both in response to exogenous glucose boluses in vivo and in isolated pancreatic islets. Intriguingly, these metabolic alterations are associated with impaired spatial memory in BoNT/B-expressing mice.

Conclusions

These findings underscore the central role of tanycytes in brain-periphery communication and highlight their potential implication in the age-related development of type 2 diabetes and cognitive decline. Our tanycytic BoNT/B mouse model provides a robust platform for studying how these conditions progress over time, from prediabetic states to full-blown metabolic and cognitive disorders, and the mechanistic contribution of tanycytes to their development. The recognition of the impact of tanycytic transcytosis on hormone transport opens new avenues for developing targeted therapies that could address both metabolic disorders and their associated cognitive comorbidities, which often emerge or worsen with advancing age.

Background

The advent of liquid biopsies presents a novel, minimally invasive methodology for the detection of disease biomarkers, offering a significant advantage over traditional biopsy techniques. Particularly, the analysis of cell-free RNA (cfRNA) has garnered interest due to its dynamic expression profiles and the capability to study various RNA species, including messenger RNA (mRNA) and long non-coding RNA (lncRNA). These attributes position cfRNA as a versatile biomarker with broad potential applications in clinical research and diagnostics.

Scope of Review

This review delves into the utility of cfRNA biomarkers as prognostic tools for obesity-related comorbidities, such as diabetes, dyslipidemia, and non-alcoholic fatty liver disease.

Major conclusions

We evaluate the efficacy of cfRNA in forecasting metabolic outcomes associated with obesity and in identifying patients likely to experience favorable clinical outcomes following bariatric surgery. Additionally, this review synthesizes evidence from studies examining circulating cfRNA across different physiological and pathological states, with a focus on its role in diabetes, including disease progression monitoring and treatment efficacy assessment. Through this exploration, we underscore the emerging relevance of cfRNA signatures in the context of obesity and its comorbidities, setting the stage for future investigative efforts in this rapidly advancing domain.

Objective

Stable isotope studies have shown that hepatic de novo lipogenesis (DNL) plays an important role in the pathogenesis of intrahepatic lipid (IHL) deposition. Furthermore, previous research has demonstrated that fructose 1-phosphate (F1P) not only serves as a substrate for DNL, but also acts as a signalling metabolite that stimulates DNL from glucose. The aim of this study was to elucidate the mediators of F1P-stimulated DNL, with special focus on two key regulators of intrahepatic glucose metabolism, i.e., glucokinase regulatory protein (GKRP) and carbohydrate response element binding protein (ChREBP).

Methods

Aldolase B deficient mice (Aldob^−/−), characterized by hepatocellular F1P accumulation, enhanced DNL, and hepatic steatosis, were either crossed with GKRP deficient mice (Gckr^−/−) or treated with short hairpin RNAs directed against hepatic ChREBP.

Results

Aldob^−/− mice showed higher rates of de novo palmitate synthesis from glucose when compared to wildtype mice (p < 0.001). Gckr knockout reduced de novo palmitate synthesis in Aldob^−/− mice (p = 0.017), without affecting the hepatic mRNA expression of enzymes involved in DNL. In contrast, hepatic ChREBP knockdown normalized the hepatic mRNA expression levels of enzymes involved in DNL and reduced fractional DNL in Aldob^−/− mice (p < 0.05). Of interest, despite downregulation of DNL in response to Gckr and ChREBP attenuation, no reduction in intrahepatic triglyceride levels was observed.

Conclusions

Both GKRP and ChREBP mediate F1P-stimulated DNL in aldolase B deficient mice. Further studies are needed to unravel the role of GKRP and hepatic ChREBP in regulating IHL accumulation in aldolase B deficiency.

Objective

The metabolism of different cells within the same microenvironment can differ and dictate physiological or pathological adaptions. Current single-cell analysis methods of metabolism are not label-free.

Methods

The study introduces a label-free, live-cell analysis method assessing endogenous fluorescence of NAD(P)H and FAD in surface-stained cells by flow cytometry.

Results

OxPhos inhibition, mitochondrial uncoupling, glucose exposure, genetic inactivation of glucose uptake and mitochondrial respiration alter the optical redox ratios of FAD and NAD(P)H as measured by flow cytometry. Those alterations correlate strongly with measurements obtained by extracellular flux analysis. Consequently, metabolically distinct live B-cell populations can be resolved, showing that human memory B-cells from peripheral blood exhibit a higher glycolytic flexibility than naïve B cells. Moreover, the comparison of blood-derived B- and T-lymphocytes from healthy donors and rheumatoid arthritis patients unleashes rheumatoid arthritis-associated metabolic traits in human naïve and memory B-lymphocytes.

Conclusions

Taken together, these data show that the optical redox ratio can depict metabolic differences in distinct cell populations by flow cytometry.

Objectives

This study aimed to evaluate the efficacy of a purification method developed for isolating alpha, beta, and delta cells from pancreatic islets of adult mice, extending its application to islets from newborn and aged mice. Furthermore, it sought to examine transcriptome dynamics in mouse pancreatic endocrine islet cells throughout postnatal development and to validate age-related alterations within these cell populations.

Methods

We leveraged the high surface expression of CD71 on beta cells and CD24 on delta cells to FACS-purify alpha, beta, and delta cells from newborn (1-week-old), adult (12-week-old), and old (18-month-old) mice. Bulk RNA sequencing was conducted on these purified cell populations, and subsequent bioinformatic analyses included differential gene expression, overrepresentation, and intersection analysis.

Results

Alpha, beta, and delta cells from newborn and aged mice were successfully FACS-purified using the same method employed for adult mice. Our analysis of the age-related transcriptional changes in alpha, beta, and delta cell populations revealed a decrease in cell cycling and an increase in neuron-like features processes during the transition from newborn to adult mice. Progressing from adult to old mice, we identified an inflammatory gene signature related to aging (inflammaging) encompassing an increase in β-2 microglobulin and major histocompatibility complex (MHC) Class I expression.

Conclusions

Our study demonstrates the effectiveness of our cell sorting technique in purifying endocrine subsets from mouse islets at different ages. We provide a valuable resource for better understanding endocrine pancreas aging and identified an inflammaging gene signature with increased β-2 microglobulin and MHC Class I expression as a common hallmark of old alpha, beta, and delta cells, with potential implications for immune response regulation and age-related diabetes.

Objective

Hepatic Ca²⁺ signaling has been identified as a crucial key factor in driving gluconeogenesis. The involvement of mitochondria in hormone-induced Ca²⁺ signaling and their contribution to metabolic activity remain, however, poorly understood. Moreover, the molecular mechanism governing the mitochondrial Ca²⁺ efflux signaling remains unresolved. This study investigates the role of the Na⁺/Ca²⁺ exchanger, NCLX, in modulating hepatic mitochondrial Ca²⁺ efflux, and examines its physiological significance in hormonal hepatic Ca²⁺ signaling, gluconeogenesis, and mitochondrial bioenergetics.

Methods

Primary mouse hepatocytes from both an AAV-mediated conditional hepatic-specific and a total mitochondrial Na⁺/Ca²⁺ exchanger, NCLX, knockout (KO) mouse models were employed for fluorescent monitoring of purinergic and glucagon/vasopressin-dependent mitochondrial and cytosolic hepatic Ca²⁺ responses in cultured hepatocytes. Isolated liver mitochondria and permeabilized primary hepatocytes were used to analyze the ion-dependence of Ca²⁺ efflux. Utilizing the conditional hepatic-specific NCLX KO model, the rate of gluconeogenesis was assessed by first monitoring glucose levels in fasted mice, and subsequently subjecting the mice to a pyruvate tolerance test while monitoring their blood glucose. Additionally, cultured primary hepatocytes from both genotypes were assessed in vitro for glucagon-dependent glucose production and cellular bioenergetics through glucose oxidase assay and Seahorse respirometry, respectively.

Results

Analysis of Ca²⁺ responses in isolated liver mitochondria and cultured primary hepatocytes from NCLX KO versus WT mice showed that NCLX serves as the principal mechanism for mitochondrial calcium extrusion in hepatocytes. We then determined the role of NCLX in glucagon and vasopressin-induced Ca²⁺ oscillations. Consistent with previous studies, glucagon and vasopressin triggered Ca²⁺ oscillations in WT hepatocytes, however, the deletion of NCLX resulted in selective elimination of mitochondrial, but not cytosolic, Ca²⁺ oscillations, underscoring NCLX’s pivotal role in mitochondrial Ca²⁺ regulation. Subsequent in vivo investigation for hepatic NCLX role in gluconeogenesis revealed that, as opposed to WT mice which maintained normoglycemic blood glucose levels when fasted, conditional hepatic-specific NCLX KO mice exhibited a faster drop in glucose levels, becoming hypoglycemic. Furthermore, KO mice showed deficient conversion of pyruvate to glucose when challenged under fasting conditions. Concurrent in vitro assessments showed impaired glucagon-dependent glucose production and compromised bioenergetics in KO hepatocytes, thereby underscoring NCLX’s significant contribution to hepatic glucose metabolism.

Conclusions

The study findings demonstrate that NCLX acts as the primary Ca²⁺ efflux mechanism in hepatocytes. NCLX is indispensable for regulating hormone-induced mitochondrial Ca²⁺ oscillations, mitochondrial metabolism, and sustenance of hepatic gluconeogenesis.

Objective

Receptor-interacting protein kinase 1 (RIPK1) orchestrates the decision between cell survival and cell death in response to tumor necrosis factor (TNF) and other cytokines. Whereas the scaffolding function of RIPK1 is crucial to prevent TNF-induced apoptosis and necroptosis, its kinase activity is required for necroptosis and partially for apoptosis. Although TNF is a proinflammatory cytokine associated with β-cell loss in diabetes, the mechanism by which TNF induces β-cell demise remains unclear.

Methods

Here, we dissected the contribution of RIPK1 scaffold versus kinase functions to β-cell death regulation using mice lacking RIPK1 specifically in β-cells (Ripk1^β-KO mice) or expressing a kinase-dead version of RIPK1 (Ripk1^D138N mice), respectively. These mice were challenged with streptozotocin, a model of autoimmune diabetes. Moreover, Ripk1^β-KO mice were further challenged with a high-fat diet to induce hyperglycemia. For mechanistic studies, pancreatic islets were subjected to various killing and sensitising agents.

Results

Inhibition of RIPK1 kinase activity (Ripk1^D138N mice) did not affect the onset and progression of hyperglycemia in a type 1 diabetes model. Moreover, the absence of RIPK1 expression in β-cells did not affect normoglycemia under basal conditions or hyperglycemia under diabetic challenges. Ex vivo, primary pancreatic islets are not sensitised to TNF-induced apoptosis and necroptosis in the absence of RIPK1. Intriguingly, we found that pancreatic islets display high levels of the antiapoptotic cellular FLICE-inhibitory protein (cFLIP) and low levels of apoptosis (Caspase-8) and necroptosis (RIPK3) components. Cycloheximide treatment, which led to a reduction in cFLIP levels, rendered primary islets sensitive to TNF-induced cell death which was fully blocked by caspase inhibition.

Conclusions

Unlike in many other cell types (e.g., epithelial, and immune), RIPK1 is not required for cell death regulation in β-cells under physiological conditions or diabetic challenges. Moreover, in vivo and in vitro evidence suggest that pancreatic β-cells do not undergo necroptosis but mainly caspase-dependent death in response to TNF. Last, our results show that β-cells have a distinct mode of regulation of TNF-cytotoxicity that is independent of RIPK1 and that may be highly dependent on cFLIP.

Objective

Dietary medium-chain fatty acids (MCFAs), characterized by chain lengths of 8–12 carbon atoms, have been proposed to have beneficial effects on glucose and lipid metabolism, yet the underlying mechanisms remain elusive. We hypothesized that MCFA intake benefits metabolic health by inducing the release of hormone-like factors.

Methods

The effects of chow diet, high-fat diet rich in long-chain fatty acids (LCFA HFD) fed ad libitum or pair-fed to a high-fat diet rich in MCFA (MCFA HFD) on glycemia, hepatic gene expression, circulating fibroblast growth factor 21 (FGF21), and liver fat content in both wildtype and Fgf21 knockout mice were investigated. The impact of a single oral dose of an MCFA-rich oil on circulating FGF21 and hepatic Fgf21 mRNA expression was assessed. In flag-tagged Crebh knockin mice and liver-specific Crebh knockout mice, fed LCFA HFD or MCFA HFD, active hepatic CREBH and hepatic Fgf21 mRNA abundance were determined, respectively.

Results

MCFA HFD improves glucose tolerance, enhances glucose clearance into brown adipose tissue, and prevents high-fat diet-induced hepatic steatosis in wildtype mice. These benefits are associated with increased liver expression of CREBH target genes (Apoa4 and Apoc2), including Fgf21. Both acute and chronic intake of dietary MCFAs elevate circulating FGF21. Augmented hepatic Fgf21 mRNA following MCFA HFD intake is accompanied by higher levels of active hepatic CREBH; and MCFA-induced hepatic Fgf21 expression is blocked in mice lacking Crebh. Notably, while feeding male and female Fgf21 wildtype mice MCFA HFD results in reduced liver triacylglycerol (TG) levels, this liver TG-lowering effect is blunted in Fgf21 knockout mice fed MCFA HFD. The reduction in liver TG levels observed with MCFA HFD was independent of weight loss.

Conclusions

Dietary MCFAs reduce liver fat accumulation via activation of a CREBH-FGF21 signaling axis.

Background and objectives

Fibrosis contributes to 45% of deaths in industrialized nations and is characterized by an abnormal accumulation of extracellular matrix (ECM). There are no specific anti-fibrotic treatments for liver fibrosis, and previous unsuccessful attempts at drug development have focused on preventing ECM deposition. Because liver fibrosis is largely acknowledged to be reversible, regulating fibrosis resolution could offer novel therapeutical options. However, little is known about the mechanisms controlling ECM remodeling during resolution. Changes in proteolytic activity are essential for ECM homeostasis and macrophages are an important source of proteases. Herein, in this study we evaluate the role of macrophage-derived cathepsin D (CtsD) during liver fibrosis.

Methods

CtsD expression and associated pathways were characterized in single-cell RNA sequencing and transcriptomic datasets in human cirrhosis. Liver fibrosis progression, reversion and functional characterization were assessed in novel myeloid-CtsD and hepatocyte-CtsD knock-out mice.

Results

Analysis of single-cell RNA sequencing datasets demonstrated CtsD was expressed in macrophages and hepatocytes in human cirrhosis. Liver fibrosis progression, reversion and functional characterization were assessed in novel myeloid-CtsD (CtsD^ΔMyel) and hepatocyte-CtsD knock-out mice. CtsD deletion in macrophages, but not in hepatocytes, resulted in enhanced liver fibrosis. Both inflammatory and matrisome proteomic signatures were enriched in fibrotic CtsD^ΔMyel livers. Besides, CtsD^ΔMyel liver macrophages displayed functional, phenotypical and secretomic changes, which resulted in a degradomic phenotypical shift, responsible for the defective proteolytic processing of collagen I in vitro and impaired collagen remodeling during fibrosis resolution in vivo. Finally, CtsD-expressing mononuclear phagocytes of cirrhotic human livers were enriched in lysosomal and ECM degradative signaling pathways.

Conclusions

Our work describes for the first-time CtsD-driven lysosomal activity as a central hub for restorative macrophage function during fibrosis resolution and opens new avenues to explore their degradome landscape to inform drug development.

Objective

During infection, metabolism and immunity react dynamically to promote survival through mechanisms that remain unclear. Pro-opiomelanocortin (POMC) cleavage products are produced and released in the brain and in the pituitary gland. One POMC cleavage product, alpha-melanocyte-stimulating hormone (α-MSH), is known to regulate food intake and energy expenditure and has anti-inflammatory effects. However, it is not known whether α-MSH is required to regulate physiological anti-inflammatory responses. We recently developed a novel mouse model with a targeted mutation in Pomc (Pomc^tm1/tm1 mice) to block production of all α-MSH forms which are required to regulate metabolism. To test whether endogenous α-MSH is required to regulate immune responses, we compared acute bacterial lipopolysaccharide (LPS)-induced inflammation between Pomc^tm1/tm1 and wild-type Pomc^wt/wt mice.

Methods

We challenged 10- to 14-week-old male Pomc^tm1/tm1 and Pomc^wt/wt mice with single i.p. injections of either saline or low-dose LPS (100 μg/kg) and monitored immune and metabolic responses. We used telemetry to measure core body temperature (T_b), ELISA to measure circulating cytokines, corticosterone and α-MSH, and metabolic chambers to measure body weight, food intake, activity, and respiration. We also developed a mass spectrometry method to measure three forms of α-MSH produced in the mouse hypothalamus and pituitary gland.

Results

LPS induced an exaggerated immune response in Pomc^tm1/tm1 compared to Pomc^wt/wt mice. Both groups of mice were hypoactive and hypothermic following LPS administration, but Pomc^tm1/tm1 mice were significantly more hypothermic compared to control mice injected with LPS. Pomc^tm1/tm1 mice also had reduced oxygen consumption and impaired metabolic responses to LPS compared to controls. Pomc^tm1/tm1 mice had increased levels of key proinflammatory cytokines at 2 h and 4 h post LPS injection compared to Pomc^wt/wt mice. Lastly, Pomc^wt/wt mice injected with LPS compared to saline had increased total α-MSH in circulation 2 h post injection.

Conclusions

Our data indicate endogenous α-MSH contributes to the inflammatory immune responses triggered by low-dose LPS administration and suggest that targeting the melanocortin system could be a potential therapeutic for the treatment of sepsis or inflammatory disease.

Objective

Currently, little is known about the mechanism(s) regulating global and specific protein translation during metabolic dysfunction-associated steatohepatitis (MASH; previously known as non-alcoholic steatohepatitis, NASH).

Methods

Unbiased label-free quantitative proteome, puromycin-labelling and polysome profiling were used to understand protein translation activity in vitro and in vivo.

Results

We observed a global decrease in protein translation during lipotoxicity in human primary hepatocytes, mouse hepatic AML12 cells, and livers from a dietary mouse model of MASH. Interestingly, proteomic analysis showed that Rplp1, which regulates ribosome and translation pathways, was one of the most downregulated proteins. Moreover, decreased Esrra expression and binding to the Rplp1 promoter, diminished Rplp1 gene expression during lipotoxicity. This, in turn, reduced global protein translation and Esrra/Rplp1-dependent translation of lysosome (Lamp2, Ctsd) and autophagy (sqstm1, Map1lc3b) proteins. Of note, Esrra did not increase its binding to these gene promoters or their gene transcription, confirming its regulation of their translation during lipotoxicity. Notably, hepatic Esrra-Rplp1-dependent translation of lysosomal and autophagy proteins also was impaired in MASH patients and liver-specific Esrra knockout mice. Remarkably, alternate day fasting induced Esrra-Rplp1-dependent expression of lysosomal proteins, restored autophagy, and reduced lipotoxicity, inflammation, and fibrosis in hepatic cell culture and in vivo models of MASH.

Conclusions

Esrra regulation of Rplp1-mediated translation of lysosome/autolysosome proteins was downregulated during MASH. Alternate day fasting activated this novel pathway and improved MASH, suggesting that Esrra and Rplp1 may serve as therapeutic targets for MASH. Our findings also provided the first example of a nuclear hormone receptor, Esrra, to not only regulate transcription but also protein translation, via induction of Rplp1.

Objective

Proopiomelanocortin (POMC) neurons release potent anorexigenic neuropeptides, which suppress food intake and enhance energy expenditure via melanocortin receptors. Although the importance of central melanocortin in physiological regulation is well established, the underlying genetic mechanisms that define the functional identity of melanocortin neurons and maintain hypothalamic Pomc expression remain to be fully determined. In this study, we investigate the functional significance of Six3, a transcriptional regulator notably expressed in embryonic and adult mouse POMC neurons, in the regulation of hypothalamic Pomc expression and downstream physiological consequences.

Methods

We first evaluated the expression of Six3 in the developing and adult hypothalamus by double fluorescence in situ hybridization. Next, we assessed POMC immunoreactivity in mutant mice selectively lacking Six3 from Pomc-expressing neurons and quantified Pomc mRNA levels in a tamoxifen-inducible Six3 knockout mouse model activated at embryonic E9.5 days. We also determined glucose and insulin sensitivity, daily food intake, body composition and body weight in adult male and female mice lacking Six3 specifically from POMC neurons. Lastly, we assessed the physiological consequences of ablating Six3 from POMC neurons in adult mice.

Results

Six3 and Pomc were co-expressed in mouse hypothalamic neurons during development and adulthood. Mouse embryos deficient in Six3 showed reduced Pomc expression in the developing hypothalamus. Targeted deletion of Six3 specifically from POMC neurons resulted in decreased hypothalamic Pomc expression, increased daily food intake, enhanced glucose sensitivity and mild obesity in male but not in female mice. Finally, conditional removal of Six3 from POMC neurons in adult mice led to a reduction in hypothalamic POMC immunoreactivity with no significant effects in body weight or food intake.

Conclusions

Altogether, our results demonstrate that Six3 plays an essential role in the early establishment of POMC neuron identity and the maintenance of physiological levels of hypothalamic Pomc expression. In addition, our study demonstrates that the functional significance of Six3 expression in POMC neurons is sexually dimorphic and age-dependent.

Objectives

We here assessed whether typical pathogens of laboratory mice affect the development of diet-induced obesity and glucose intolerance, and whether colonization affects the efficacy of the GLP-1R agonist liraglutide and of the GLP-1/GIP co-agonist MAR709 to treat obesity and diabetes.

Methods

Male C57BL/6J mice were experimentally infected with Helicobacter hepaticus, Rodentibacter pneumotropicus and Staphylococcus aureus and compared to a group of uninfected specific and opportunistic pathogen free (SOPF) mice. The development of diet-induced obesity and glucose intolerance was monitored over a period of 26 weeks. To study the influence of pathogens on drug treatment, mice were then subjected for 6 days daily treatment with either the GLP-1 receptor agonist liraglutide or the GLP-1/GIP co-agonist MAR709.

Results

Colonized mice did not differ from SOPF controls regarding HFD-induced body weight gain, food intake, body composition, glycemic control, or responsiveness to treatment with liraglutide or the GLP-1/GIP co-agonist MAR709.

Conclusions

We conclude that the occurrence of H. hepaticus, R. pneumotropicus and S. aureus does neither affect the development of diet-induced obesity or type 2 diabetes, nor the efficacy of GLP-1-based drugs to decrease body weight and to improve glucose control in mice.