Rubén Nogueiras’s research while affiliated with Instituto de Investigación Sanitaria de Santiago and other places

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Publications (453)


Modulatory effects of CNNM4 on Protein-L-Isoaspartyl-O-Methyltransferase repair function during Alcohol-Induced hepatic damage
  • Article

December 2024

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32 Reads

Hepatology

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Ma Luz Martínez-Chantar

Background and Aims Alcohol-related liver disease (ALD) is a leading cause of liver-related mortality worldwide, with limited treatment options beyond abstinence and liver transplantation. Chronic alcohol consumption has been linked to magnesium (Mg ²⁺ ) deficiency, which can influence the liver disease progression. The mechanisms underlying Mg ²⁺ homeostasis dysregulation in ALD remain elusive. This study aimed to investigate the role of the Mg ²⁺ transporter Cyclin M4 (CNNM4) in ALD by analyzing its expression patterns in ALD patients and preclinical animal models. Approach and Results In this study, CNNM4 is upregulated in the liver of both ALD patients and animal models. CNNM4 overexpression triggers Mg²⁺ homeostasis dysregulation, linked to ALD progression. We propose a novel therapeutic approach for ALD treatment using N-acetylgalactosamine (GalNAc) silencing RNA (siRNA) technology to specifically modulate Cnnm4 expression in the liver, improving mitochondrial function and alleviating ER stress. Notably, silencing Cnnm4 restores protein isoaspartyl methyltransferase (PCMT1) activity, essential for repairing ethanol-induced protein damage. Enhancing mitochondrial activity through Cnnm4-dependent mechanisms increases SAMe levels, crucial for PCMT1 function, highlighting the interconnected roles of mitochondrial health and protein homeostasis in ALD treatment. Conclusions These findings shed light on the dysregulation of Mg ²⁺ homeostasis in ALD, providing a promising therapeutic approach targeting CNNM4. GalNAc si Cnnm4 therapy boost the repair processes of ethanol damaged proteins through the upregulation of PCMT1 activity.




Figure 1: Genetic ablation of AMPKg2 in SF1 neurons. (A) Violin plots depicting expression of the different AMPK gamma isoforms, Prkag1, Prkag2, and Prkag3, across ventromedial nucleus of the hypothalamus (VMH) neuronal clusters identified by Steuernagel et. al [20]. A total of 33694 neurons were divided into 15 clusters and AMPK gamma isoforms expression was compared to the neuronal marker Tubb3 and Nr5a1, a canonical marker of the VMH. The width of the violin plot indicates the frequency of cells given at the different expression levels, where Prkag2 shows continuous, high expression in the different VMH neuronal clusters. (B) Graphical scheme showing AMPKg2 fl/fl mice in which exon 6 (light blue) of Prkag2 is flanked by loxP sites (dark green), were bred with mice carrying a transgene for Cre expression (purple) under the control of SF1 promoter (beige). Excision of the exon 6 leads to a frameshift mutation (red) downstream of exon 6. Cre positive mice (SF1-Cre AMPKg2 fl/fl ) and Cre negative mice (AMPKg2 fl/fl ) were used in the different analyses. (C) Upper: Graphical scheme showing exon 6 region (blue) in Prkag2 and the corresponding region of the CBS1 domain on AMPKg2 (green); red square highlights the part of the exon 6 that codifies for CBS1. Lower: 3D structure of AMPKg2 retrieved from AlphaFold (AF-Q91WG5-F1-v4). Very low probability predictions (Plddt <50) are not displayed for clarity. The approximate resulting area deleted in SF1-Cre AMPKg2 fl/fl animals is highlighted in blue. (D) Left: Graphical scheme of the crossing strategy between SF1-Cre mice, AMPKg2 fl/fl mice, and Ai14-tdTomato mice. Right: Representative confocal image of the hypothalamus depicting tdTomato immunoreactivity (pink) in SF1-positive neurons, mostly located in the VMH, for SF1-Cre-Ai14; AMPKg2 fl/fl . Scale bar 200 mm. (E) Brightfield images of in situ hybridization using BaseScope probe targeting the exon 6 of Prkag2 (red) in the hypothalamus of AMPKg2 fl/fl and SF1-Cre AMPKg2 fl/fl mice. Zoomed images depict areas in ventromedial nucleus (VMH) and arcuate nucleus (ARC) of the hypothalamus. Blue denotes nuclei stained by hematoxylin; black head arrows depict positive cells for the exon 6 of Prkag2. Scale bars 100 mm for lateral images and 50 mm for inner images. (F) Quantification of relative number of positive and negative cells shown as their % in respect to total number of cells, from images in (E), for VMH and ARC in both AMPKg2 fl/fl (n ¼ 2) and SF1-Cre AMPKg2 fl/fl mice (n ¼ 4). Between 3 and 4 brain slices were analyzed and quantified for each animal. (G) Quantification of the total number of positive and negative cells from images in (E), for VMH and ARC in both AMPKg2 fl/fl (n ¼ 2) and SF1-Cre AMPKg2 fl/fl mice (n ¼ 4). Between 3 and 4 brain slices were analyzed and quantified for each animal. Data expressed as mean AE SEM. **P < 0.01, and ***P < 0.001 vs. control. Statistical significance was assessed by Student's t-test. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)
Figure 2: Effect of loss of AMPKg2 in SF1 neurons on energy balance. (AeE) Body weight (A; n ¼ 35 control and 36 KO), daily food intake (B; n ¼ 14 control and 14 KO), food intake normalized by body weight (C; n ¼ 14 control and 14 KO), body mass composition by MRI (D; n ¼ 7 control and 6 KO), and tissue weight (E; n ¼ 31 control and 32 KO; 17e18 weeks old) of control (littermates AMPKg2 fl/fl ) and SF1-Cre AMPKg2 fl/fl (KO) male mice. (FeJ) Body weight (F; n ¼ 41 control and 46 KO), daily food intake (G; n ¼ 18 control and 20 KO), food intake normalized by body weight (H; n ¼ 18 control and 20 KO), body mass composition by MRI (I; n ¼ 18 control and 21 KO), and tissue weight (J; n ¼ 31 control and 36 KO; 23e24 weeks old) of control and SF1-Cre AMPKg2 fl/fl female mice. Data are expressed as mean AE SEM, *P < 0.05, and **P < 0.01 vs. AMPKg2 fl/fl . Statistical significance was assessed by Mixed-effect model (A, F) and Student's t-test (B, C, D, E, G, H, I, J).
Figure 3: Effect of loss of AMPKg2 in SF1 neurons on energy expenditure and glucose homeostasis. (AeC) ANCOVA analysis of 48h total energy expenditure using body weight as a covariate (A; n ¼ 7 control and 6 KO), daily respiratory exchange ratio (RER, B; n ¼ 7 control and 6 KO), and total locomotor activity (LA, C; n ¼ 7 control and 6 KO) of control (littermates AMPKg2 fl/fl ) and SF1-Cre AMPKg2 fl/fl (KO) male mice. (DeF) ANCOVA analysis of 48h total energy expenditure using body weight as a covariate (D; n ¼ 6 control and 6 KO), RER (E; n ¼ 6 control and 6 KO), and LA (F; n ¼ 6 control and 6 KO) of control and SF1-Cre AMPKg2 fl/fl female mice. (GeJ) Glucose tolerance test and area under the curve (G, H; n ¼ 16 control and 18 KO), insulin tolerance test, and area under the curve (I, J; n ¼ 10 control and 9 KO) of control and SF1-Cre AMPKg2 fl/fl male mice. (KeN) Glucose tolerance test, area under the curve (K, L; n ¼ 16 control and 21 KO), insulin tolerance test, and area under the curve (M, N; n ¼ 10 control and 16 KO) of control and SF1-Cre AMPKg2 fl/fl female mice. Data expressed as mean AE SEM. Statistical significance was assessed by ANCOVA (A, D), two-way ANOVA or Mixed-effect model (G, I, K, M) and Student's t-test (B, C, E, F, H, J, L, N).
Figure 4: Effect of loss of AMPKg2 in SF1 neurons on BAT thermogenesis and WAT browning. (A) Core body temperature of control and SF1-Cre AMPKg2 fl/fl (KO) male mice (n ¼ 25 control and 26 KO). (BeC) Representative BAT infrared thermographic images (B) and BAT temperature (C; n ¼ 8 control and 16 KO) of control and SF1-Cre AMPKg2 fl/fl male mice. (DeE) Representative BAT UCP1 western blot images (D) and BAT UCP1 protein levels (E; n ¼ 6 control and 7 KO) of control (littermates AMPKg2 fl/fl ) and SF1-Cre AMPKg2 fl/fl male mice. a-tubulin was used as control. A black line between the immunoblots depicts samples loaded in the same gel, but not next to each other. (FeG) Representative subcutaneous white adipose tissue (scWAT) immunohistochemistry with anti-UCP1 antibody showing UCP1 staining (F; 20x; scale bar: 20 mm) and UCP1 stained area (G; n ¼ 11 control and 11 KO) of control and SF1-Cre AMPKg2 fl/fl male mice. The mean value of each animal was obtained from 2 to 4 photos of each tissue. (HeI) Representative scWAT hematoxylin-eosin staining (H) and proportion of measured cells corresponding to different cell size (mm 2 ) (I; n ¼ 12 control and 14 KO). The mean value of each animal was obtained from 3 to 4 photos of each tissue, and between 131 (minimum) and 615 (maximum) cells were quantified and classified for each animal. (J) Core body temperature of control and SF1-Cre AMPKg2 fl/fl female mice (n ¼ 19 control and 25 KO). (KeL) Representative BAT infrared thermographic images (K) and BAT temperature (L; n ¼ 19 control and 25 KO) of control and SF1-Cre AMPKg2 fl/fl female mice. (MLN) Representative BAT UCP1 western blot images (M) and BAT UCP1 protein levels (N; n ¼ 7 control and 7 KO) of control and SF1-Cre AMPKg2 fl/fl female mice. a-tubulin was used as control. A black line between the immunoblots depicts samples loaded in the same gel, but not next to each other. (OeP) Representative subcutaneous white adipose tissue (scWAT) immunohistochemistry with anti-UCP1 antibody showing UCP1 staining (O; 20x; scale bar: 20 mm) and UCP1 stained area (P; n ¼ 15 control and 12 KO) of control and SF1-Cre AMPKg2 fl/fl female mice. (QeR) Representative scWAT hematoxylin-eosin staining (Q)
Figure 5: Effect of loss of AMPKg2 in SF1 neurons on temperature challenge. (A) Graphical scheme of the cold exposure procedure. (B) Core body temperature change in cold-exposed control (littermates AMPKg2 fl/fl ) and SF1-Cre AMPKg2 fl/fl (KO) male mice (n ¼ 7 control and 8 KO). (CeE) Representative BAT infrared thermographic images (C; same pair of mice were used for the different time points), BAT temperature (D) and tail base temperature (E) of cold-exposed control and SF1-Cre AMPKg2 fl/fl male mice (n ¼ 7 control and 8 KO). (F) Graphical scheme of the experimental setup. (G) Inner ear temperature of sub-thermoneutral (26 C), thermoneutral (30 C), or supra-thermoneutral (34 C) exposed control and SF1-Cre AMPKg2 fl/fl male mice (n ¼ 6 control and 6 KO). (HeI) representative tail base infrared thermographic images (H) and tail base temperature (I) of sub-thermoneutral (26 C), thermoneutral (30 C) or supra-thermoneutral (34 C) exposed control and SF1-Cre AMPKg2 fl/fl male mice (n ¼ 6 control and 6 KO). Data expressed as mean AE SEM. *P < 0.05, **P < 0.01 vs. AMPKg2 fl/fl . Statistical significance was assessed by two-way ANOVA or Mixed-effect model (B, D, E) and Student's t-test (E, G, I).

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SF1-specific deletion of the energy sensor AMPKγ2 induces obesity
  • Article
  • Full-text available

December 2024

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24 Reads

Molecular Metabolism

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Plasma membrane remodeling determines adipocyte expansion and mechanical adaptability

November 2024

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46 Reads

Adipocytes expand massively to accommodate excess energy stores and protect the organism from lipotoxicity. Adipose tissue expandability is at the center of disorders such as obesity and lipodystrophy; however, little is known about the relevance of adipocyte biomechanics on the etiology of these conditions. Here, we show in male mice in vivo that the adipocyte plasma membrane undergoes caveolar domain reorganization upon lipid droplet expansion. As the lipid droplet grows, caveolae disassemble to release their membrane reservoir and increase cell surface area, and transfer specific caveolar components to the LD surface. Adipose tissue null for caveolae is stiffer, shows compromised deformability, and is prone to rupture under mechanical compression. Mechanistically, phosphoacceptor Cav1 Tyr14 is required for caveolae disassembly: adipocytes bearing a Tyr14Phe mutation at this residue are stiffer and smaller, leading to decreased adiposity in vivo; exhibit deficient transfer of Cav1 and EHD2 to the LD surface, and show distinct Cav1 molecular dynamics and tension adaptation. These results indicate that Cav1 phosphoregulation modulates caveolar dynamics as a relevant component of the homeostatic mechanoadaptation of the differentiated adipocyte.


GLP-1 and GIP agonism has no direct actions in human hepatocytes or hepatic stellate cells

November 2024

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58 Reads

Cellular and Molecular Life Sciences

The use of incretin agonists for managing metabolic dysfunction-associated steatohepatitis (MASH) is currently experiencing considerable interest. However, whether these compounds have a direct action on MASH is still under debate. This study aims to investigate whether GLP-1R/GIPR agonists act directly in hepatocytes and hepatic stellate cells (HSCs). For this, human hepatocyte and HSCs lines, as well as primary human hepatocytes and HSCs treated with Liraglutide, Acyl-GIP or the GLP-1/GIP dual agonist (MAR709) were used. We show that the concentrations of each compound, which were effective in insulin release, did not induce discernible alterations in either hepatocytes or HSCs. In hepatocytes displaying elevated fatty acid content after the treatment with oleic acid and palmitic acid, none of the three compounds reduced lipid concentration. Similarly, in HSCs activated with transforming growth factor-β (TGFb), Liraglutide, Acyl-GIP and MAR709 failed to ameliorate the elevated expression of fibrotic markers. The three compounds were also ineffective in phosphorylating CREB, which mediates insulinotropic actions, in both hepatocytes and HSCs. These findings indicate that incretin agonists have no direct actions in human hepatocytes or hepatic stellate cells, suggesting that their beneficial effects in patients with MASH are likely mediated indirectly, potentially through improvements in body weight, insulin resistance and glycemic control.


Metabolic State Determines Central and Peripheral Mechanisms of Liraglutide-Enhanced Insulin Secretion

November 2024

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37 Reads

While liraglutide effectively treats type 2 diabetes (T2D) and obesity, its mechanism of action across disease progression remains poorly understood. Liraglutide selectively enhances GSIS in islets from glucose-intolerant donors and in islets exposed to prediabetic conditions, but not in normoglycemic or T2D islets. In healthy mice, liraglutide's insulinotropic effect requires tanycyte-mediated central transport, whereas in glucose intolerance it acts directly on islets. Additionally, liraglutide reduces blood glucose in normoglycemic mice through insulin-independent mechanisms involving decreased gluconeogenesis and enhanced peripheral glucose uptake. These findings demonstrate that the therapeutic window for liraglutide's pancreatic effects may be optimal during prediabetes, while its central and insulin-independent actions predominate in other metabolic states.



The reactive pyruvate metabolite dimethylglyoxal mediates neurological consequences of diabetes

July 2024

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122 Reads

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2 Citations

Complications of diabetes are often attributed to glucose and reactive dicarbonyl metabolites derived from glycolysis or gluconeogenesis, such as methylglyoxal. However, in the CNS, neurons and endothelial cells use lactate as energy source in addition to glucose, which does not lead to the formation of methylglyoxal and has previously been considered a safer route of energy consumption than glycolysis. Nevertheless, neurons and endothelial cells are hotspots for the cellular pathology underlying neurological complications in diabetes, suggesting a cause that is distinct from other diabetes complications and independent of methylglyoxal. Here, we show that in clinical and experimental diabetes plasma concentrations of dimethylglyoxal are increased. In a mouse model of diabetes, ilvb acetolactate-synthase-like (ILVBL, HACL2) is the enzyme involved in formation of increased amounts of dimethylglyoxal from lactate-derived pyruvate. Dimethylglyoxal reacts with lysine residues, forms Nε−3-hydroxy-2-butanonelysine (HBL) as an adduct, induces oxidative stress more strongly than other dicarbonyls, causes blood-brain barrier disruption, and can mimic mild cognitive impairment in experimental diabetes. These data suggest dimethylglyoxal formation as a pathway leading to neurological complications in diabetes that is distinct from other complications. Importantly, dimethylglyoxal formation can be reduced using genetic, pharmacological and dietary interventions, offering new strategies for preventing CNS dysfunction in diabetes.


Prolonged lactation: Preventing obesity since infancy

July 2024

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37 Reads

Open Access Government

Prolonged lactation: Preventing obesity since infancy Dr Vincent Prevot and Professors Markus Schwaninger and Ruben Nogueiras explain the significance of the perinatal maternal lifestyle, specifically prolonged lactation, in shielding offspring from chronic disease. In recent years, the escalating prevalence of obesity and its associated diseases has prompted numerous studies covering broad aspects of metabolism, from the genetic inheritance of disorders to the early emergence of non-genetic metabolic traits. Pregnancy and lactation are now identified as the pivotal periods for establishing these traits, which can develop from infancy into adulthood. Therefore, maternal influences are crucial in the development of pathologies arising from a combination of genetic and non-genetic factors, as well as lifestyle.


Citations (60)


... In the brain, receptors for adipokines are abundantly presented. Evidence has demonstrated that adipokines such as adiponectin cross the blood-brain barrier, and it is known that the neurotrophic and anti-inflammatory properties of adipokines drive their neuroprotective effects against neurodegeneration [8][9][10][11][12][13]. Investigations reported that dysregulated adipokine secretion underlines the co-occurrence of comorbidities such as metabolic syndromes and neurodegenerative diseases [14][15][16][17][18][19][20][21][22]. ...

Reference:

AdipoRon's Impact on Alzheimer's Disease-A Systematic Review and Meta-Analysis
Tanycytic Transcytosis Inhibition Disrupts Energy Balance, Glucose Homeostasis and Cognitive Function in Male Mice
  • Citing Article
  • July 2024

Molecular Metabolism

... Tanycytes are specialized glial cells that provide structural support and regulatory functions within the hypothalamus [11,12]. The processes of tanycytes are closely intertwined with the axonal terminals of GnRH neurons [13], constituting the GnRH-tanycyte unit. ...

Estrogen receptor-α signaling in tanycytes lies at the crossroads of fertility and metabolism
  • Citing Article
  • July 2024

Metabolism

... Hypomethylation of TNXB gene and differential expression of EPPK1 protein in the placenta has been reported to be associated with GDM [45,46]. In a mouse model of diabetes, ILVBL has been reported to be involved in the formation of increased dimethylglyoxal, which induces oxidative stress and disrupts the blood-brain barrier, potentially leading to neurological complications in diabetes [47]. B3GNTL1 was identified as part of a trans-omics biomarker for diabetic kidney disease in diabetic patients [48]. ...

The reactive pyruvate metabolite dimethylglyoxal mediates neurological consequences of diabetes

... These SCFAs are essential for preserving the intestinal barrier and regulating immune functions. As a result, the weakened intestinal barrier allows more bacterial components, such as LPS, to enter the bloodstream, further exacerbating inflammation and liver damage in a self-perpetuating cycle [76][77][78][79]. These mechanisms indeed explain why MASLD appears more common in conditions typically characterized by the disruption of the intestinal barrier, notably inflammatory bowel diseases. ...

The lipopolysaccharide-TLR4 axis regulates hepatic glutaminase 1 expression promoting liver ammonia build-up as steatotic liver disease progresses to steatohepatitis
  • Citing Article
  • June 2024

Metabolism

... Among these, PIAS2 is highly expressed in differentiated papillary thyroid carcinomas but significantly reduced in anaplastic thyroid carcinomas (ATC), a highly lethal, undifferentiated cancer. Recent research [68] identified PIAS2b as essential for mitosis in ATC cells. Silencing PIAS2b with dsRNAi selectively induces cell death in these aggressive cancer cells by disrupting spindle assembly, impairing chromosome-microtubule attachment, and enhancing proteasome activity ( Figure 2). ...

dsRNAi-mediated silencing of PIAS2beta specifically kills anaplastic carcinomas by mitotic catastrophe

... Originally identified as effective treatments for T2D 3,4 , GLP-1R agonists have proven effective in recent years in reducing both body weight and hyperglycaemia 5 . Dual and triple receptor agonists are proving even more effective 6,7 , with weight loss of 20% or more now achievable with the FDA-approved GLP1R-GIPR co-agonist, tirzepatide 8 . These agents bind to receptors in the endocrine pancreas, notably the pancreatic beta cell, potentiating the effects of glucose to stimulate insulin secretion, as well as to receptors in the brain, heart, kidney, adipose tissue and immune cells [9][10][11] to promote energy expenditure and reduce appetite, amongst other beneficial effects. ...

Dual and Triple Incretin-Based Co-agonists: Novel Therapeutics for Obesity and Diabetes

Diabetes Therapy

... This dual role allows LEAP-2 to play a pivotal part in regulating energy metabolism through the ghrelin-GHSR1a signaling pathway. Furthermore, LEAP-2 may offer therapeutic potential by modulating ghrelinrelated responses under both normal and pathological conditions [43,47]. The regulation and effects of LEAP-2 antagonism against GHSR1a on human organ metabolism or disease are shown in Figure 2. In the hypothalamus, LEAP-2 inhibits the cAMP-PKA pathway on NPY/AgRP neurons and phosphorylates AMPK and ACC on NPY neurons, respectively, which reduces food intake and suppresses appetite, resulting in decreased GH secretion. ...

The effects of ghrelin and LEAP-2 in energy homeostasis are modulated by thermoneutrality, high-fat diet and aging

Journal of Endocrinological Investigation

... Western blot analyses were performed on whole hypothalamus or adipose tissue samples as previously described [35]. Briefly, whole cell extracts from tissues were lysed in standard lysis buffer at 4 C. Aliquots of 30 mg proteins were separated by 9%-SDS polyacrylamide gel electrophoresis and transferred to PVDF Immobilon membranes (Millipore). ...

Antiobesity effects of intestinal gluconeogenesis are mediated by the brown adipose tissue sympathetic nervous system
  • Citing Article
  • February 2024

Obesity

... It appears that patients receiving corticosteroids during therapy ultimately underwent a milder infection and recovered more quickly and effectively (Klein et al., 2023), and those with long-COVID symptoms had overall lower serum cortisol levels. Some work has also described reduced levels of gonadotropins within the CNS, which, in the long term, may contribute to accelerated aging of brain tissue and a higher risk of the onset of dementia-like disorders (Davis et al., 2023;Rasika et al., 2024). ...

Seeing through the fog: a neuroendocrine explanation for post-COVID cognitive deficits
  • Citing Article
  • January 2024

Nature Reviews Endocrinology