Systematic review of thyroid function in NKX2-1-related disorders: Screening and diagnosis.

BACKGROUND: NKX2-1-related disorders (NKX2-1-RD) are rare conditions affecting lung, thyroid, and brain development, primarily caused by pathogenic variants or deletions in the NKX2-1 gene. Congenital hypothyroidism (CH) is a common endocrine manifestation, leading to irreversible intellectual disability if left untreated. OBJECTIVES: The aim was to evaluate the current evidence for the use of screening and diagnostic techniques for endocrine alterations in patients with NKX2-1-RD. METHODS: This systematic review was reported following the PRISMA guidelines. Two separate research questions in PICO format were addressed to cover initial screening and diagnosis procedures for endocrine diseases in patients with NKX2-1-RD. Eligibility criteria focused on patients with genetic confirmation of the disease and hypothyroidism. Various databases were searched, and data were extracted and assessed independently by two reviewers. RESULTS: Out of 1012 potentially relevant studies, 46 were included, for a total of 113 patients. CH was the most frequent endocrine alteration (45% of patients). Neonatal screening was reported in only 21% of patients based on blood TSH measurements. TSH thresholds varied widely across studies, making hypothyroidism detection ranges difficult to establish. Diagnostic tests using serum TSH were used to diagnose hypothyroidism or confirm its presence. 35% of patients were diagnosed at neonatal age, and 42% at adult age. Other hormonal dysfunctions identified due to clinical signs, such as anterior pituitary deficiencies, were detected later in life. Thyroid scintigraphy and ultrasonography allowed for the description of the thyroid gland in 30% of cases of hypothyroidism. Phenotypic variability was observed in individuals with the same variants, making genotype-phenotype correlations challenging. CONCLUSION: This review highlights the need for standardized protocols in endocrine screening for NKX2-1-RD, emphasizing the importance of consistent methodology and hormone threshold levels. Variability in NKX2-1 gene variants further complicates diagnostic efforts. Future research should concentrate on optimizing early screening protocols and diagnostic strategies.

Detrimental Effect of Cannabidiol on the Early Onset of Diabetic Nephropathy in Male Mice.

Anti-inflammatory and antidiabetogenic properties have been ascribed to cannabidiol (CBD). CBD-based medicinal drugs have been approved for over a lustrum, and a boom in the commercialization of CBD products started in parallel. Herein, we explored the efficacy of CBD in streptozotocin (STZ)-induced diabetic mice to prevent diabetic nephropathy at onset. Eight-to-ten-week-old C57BL6J male mice were treated daily intraperitoneally with 10 mg/kg of CBD or vehicle for 14 days. After 8 days of treatment, mice were challenged with STZ or vehicle (healthy-control). At the end of the study, non-fasting blood glucose (FBG) level was 276 ± 42 mg/dL in vehicle-STZ-treated compared to 147 ± 9 mg/dL (p ≤ 0.01) in healthy-control mice. FBG was 114 ± 8 mg/dL in vehicle-STZ-treated compared to 89 ± 4 mg/dL in healthy-control mice (p ≤ 0.05). CBD treatment did not prevent STZ-induced hyperglycemia, and non-FBG and FBG levels were 341 ± 40 and 133 ± 26 mg/dL, respectively. Additionally, treatment with CBD did not avert STZ-induced glucose intolerance or pancreatic beta cell mass loss compared to vehicle-STZ-treated mice. Anatomopathological examination showed that kidneys from vehicle-STZ-treated mice had a 35% increase of glomerular size compared to healthy-control mice (p ≤ 0.001) and presented lesions with a 43% increase in fibrosis and T cell infiltration (p ≤ 0.001). Although treatment with CBD prevented glomerular hypertrophy and reduced T cell infiltration, it significantly worsened overall renal damage (p ≤ 0.05 compared to vehicle-STZ mice), leading to a more severe renal dysfunction than STZ alone. In conclusion, we showed that CBD could be detrimental for patients with type 1 diabetes, particularly those undergoing complications such as diabetic nephropathy.

(+)-trans-Cannabidiol-2-hydroxy pentyl is a dual CB1R antagonist/CB2R agonist that prevents diabetic nephropathy in mice.

Natural cannabidiol ((-)-CBD) and its derivatives have increased interest for medicinal applications due to their broad biological activity spectrum, including targeting of the cannabinoid receptors type 1 (CB1R) and type 2 (CB2R). Herein, we synthesized the (+)-enantiomer of CBD and its derivative (+)-CBD hydroxypentylester ((+)-CBD-HPE) that showed enhanced CB1R and CB2R binding and functional activities compared to their respective (-) enantiomers. (+)-CBD-HPE Ki values for CB1R and CB2R were 3.1 ± 1.1 and 0.8 ± 0.1 nM respectively acting as CB1R antagonist and CB2R agonist. We further tested the capacity of (+)-CBD-HPE to prevent hyperglycemia and its complications in a mouse model. (+)-CBD-HPE significantly reduced streptozotocin (STZ)-induced hyperglycemia and glucose intolerance by preserving pancreatic beta cell mass. (+)-CBD-HPE significantly reduced activation of NF-κB by phosphorylation by 15% compared to STZ-vehicle mice, and CD3+ T cell infiltration into the islets was avoided. Consequently, (+)-CBD-HPE prevented STZ-induced apoptosis in islets. STZ induced inflammation and kidney damage, visualized by a significant increase in plasma proinflammatory cytokines, creatinine, and BUN. Treatment with (+)-CBD-HPE significantly reduced 2.5-fold plasma IFN-γ and increased 3-fold IL-5 levels compared to STZ-treated mice, without altering IL-18. (+)-CBD-HPE also significantly reduced creatinine and BUN levels to those comparable to healthy controls. At the macroscopy level, (+)-CBD-HPE prevented STZ-induced lesions in the kidney and voided renal fibrosis and CD3+ T cell infiltration. Thus, (+)-enantiomers of CBD, particularly (+)-CBD-HPE, have a promising potential due to their pharmacological profile and synthesis, potentially to be used for metabolic and immune-related disorders.

Δ9-Tetrahydrocannabinolic Acid markedly alleviates liver fibrosis and inflammation in mice.

BACKGROUND: Non-alcoholic fatty liver disease (NAFLD) is the most common liver disease in the Western world, and it is closely associated to obesity, type 2 diabetes mellitus, and dyslipidemia. Medicinal cannabis and some neutral cannabinoids have been suggested as a potential therapy for liver diseases. HYPOTHESIS: Δ9-tetrahydrocannabinolic acid (Δ9-THCA), the non-psychotropic precursor of Δ9-THC, is one of the most abundant cannabinoids presents in Cannabis Sativa. However, its biological activities have been poorly investigated. Herein, we studied the antifibrotic and antiinflammatory activities of Δ9-THCA in two different animal models of liver injury, providing a rationale for additional studies on the medicinal use of this cannabinoid in the treatment of liver fibrosis and the management of NAFLD. STUDY DESIGN: The antifibrotic activity of Δ9-THCA in vitro was investigated in the cell lines LX-2 and NIH-3T3-Col1A2-luc. Non-alcoholic liver fibrosis was induced in mice by CCl4 treatment or, alternatively, by 23-week high fat diet (HFD) feeding. Δ9-THCA was administered daily intraperitoneally during the CCl4 treatment or during the last 3 weeks in HFD-fed mice. METHODS: TGFß-induced profibrotic gene expression was analyzed by luciferase and qPCR assays. Liver fibrosis and inflammation were assessed by immunochemistry and qPCR. Blood glucose, insulin, leptin and triglyceride levels were measured in HFD mice. RESULTS: Δ9-THCA inhibited the expression of Tenascin C (TNC) and Col3A1 induced by TGFß in LX-2 cells and the transcriptional activity of the Col1A2 promoter in fibroblasts. Δ9-THCA significantly attenuated CCl4-induced liver fibrosis and inflammation and reduced T cell and macrophage infiltration. Mice fed HFD for 23 weeks developed severe obesity (DIO), fatty liver and marked liver fibrosis, accompanied by immune cell infiltration. Δ9-THCA, significantly reduced body weight and adiposity, improved glucose tolerance, and drastically attenuated DIO-induced liver fibrosis and immune cell infiltration. CONCLUSIONS: Δ9-THCA prevents TGFß-induced fibrotic markers in vitro and liver inflammation and fibrogenesis in vivo, providing a rationale for additional studies on the medicinal use of this cannabinoid, as well as cannabis preparations containing it, for the treatment of liver fibrosis and the management of NAFLD.

Knockout of PRDX6 induces mitochondrial dysfunction and cell cycle arrest at G2/M in HepG2 hepatocarcinoma cells.

Peroxiredoxin 6 (PRDX6) has been associated with tumor progression and cancer metastasis. Its acting on phospholipid hydroperoxides and its phospholipase-A2 activity are unique among the peroxiredoxin family and add complexity to its action mechanisms. As a first step towards the study of PRDX6 involvement in cancer, we have constructed a human hepatocarcinoma HepG2PRDX6-/- cell line using the CRISPR/Cas9 technique and have characterized the cellular response to lack of PRDX6. Applying quantitative global and redox proteomics, flow cytometry, in vivo extracellular flow analysis, Western blot and electron microscopy, we have detected diminished respiratory capacity, downregulation of mitochondrial proteins and altered mitochondrial morphology. Autophagic vesicles were abundant while the unfolded protein response (UPR), HIF1A and NRF2 transcription factors were not activated, despite increased levels of p62/SQSTM1 and reactive oxygen species (ROS). Insulin receptor (INSR), 3-phosphoinositide-dependent protein kinase 1 (PDPK1), uptake of glucose and hexokinase-2 (HK2) decreased markedly while nucleotide biosynthesis, lipogenesis and synthesis of long chain polyunsaturated fatty acids (LC-PUFA) increased. 254 Cys-peptides belonging to 202 proteins underwent significant redox changes. PRDX6 knockout had an antiproliferative effect due to cell cycle arrest at G2/M transition, without signs of apoptosis. Loss of PLA2 may affect the levels of specific lipids altering lipid signaling pathways, while loss of peroxidase activity could induce redox changes at critical sensitive cysteine residues in key proteins. Oxidation of specific cysteines in Proliferating Cell Nuclear Antigen (PCNA) could interfere with entry into mitosis. The GSH/Glutaredoxin system was downregulated likely contributing to these redox changes. Altogether the data demonstrate that loss of PRDX6 slows down cell division and alters metabolism and mitochondrial function, so that cell survival depends on glycolysis to lactate for ATP production and on AMPK-independent autophagy to obtain building blocks for biosynthesis. PRDX6 is an important link in the chain of elements connecting redox homeostasis and proliferation.

Peroxiredoxin 6 Down-Regulation Induces Metabolic Remodeling and Cell Cycle Arrest in HepG2 Cells.

Peroxiredoxin 6 (Prdx6) is the only member of 1-Cys subfamily of peroxiredoxins in human cells. It is the only Prdx acting on phospholipid hydroperoxides possessing two additional sites with phospholipase A2 (PLA2) and lysophosphatidylcholine-acyl transferase (LPCAT) activities. There are contrasting reports on the roles and mechanisms of multifunctional Prdx6 in several pathologies and on its sensitivity to, and influence on, the redox environment. We have down-regulated Prdx6 with specific siRNA in hepatoblastoma HepG2 cells to study its role in cell proliferation, redox homeostasis, and metabolic programming. Cell proliferation and cell number decreased while cell volume increased; import of glucose and nucleotide biosynthesis also diminished while polyamines, phospholipids, and most glycolipids increased. A proteomic quantitative analysis suggested changes in membrane arrangement and vesicle trafficking as well as redox changes in enzymes of carbon and glutathione metabolism, pentose-phosphate pathway, citrate cycle, fatty acid metabolism, biosynthesis of aminoacids, and Glycolysis/Gluconeogenesis. Specific redox changes in Hexokinase-2 (HK2), Prdx6, intracellular chloride ion channel-1 (CLIC1), PEP-carboxykinase-2 (PCK2), and 3-phosphoglycerate dehydrogenase (PHGDH) are compatible with the metabolic remodeling toward a predominant gluconeogenic flow from aminoacids with diversion at 3-phospohglycerate toward serine and other biosynthetic pathways thereon and with cell cycle arrest at G1/S transition.

The firing frequency of spontaneous action potentials and their corresponding evoked exocytosis are increased in chromaffin cells of CCl4 -induced cirrhotic rats with respect to control rats.

High catecolamine plasma levels because of sympathetic nervous system over-activity contribute to cirrhosis progression. The aim of this study was to investigate whether chromaffin cells of the adrenal gland might potentiate the deleterious effect exerted by this over-activity. Electrophysiological patch-clamp and amperometric experiments with carbon-fibre electrodes were conducted in single chromaffin cells of control and CCl4 -induced cirrhotic rats. The spontaneous action potential firing frequency was increased in chromaffin cells of cirrhotic rats with respect to control rats. The exocytosis evoked by that firing was also increased. However, exocytosis elicited by ACh did not vary between control and cirrhotic rats. Exocytosis triggered by depolarizing pulses was also unchanged. Amperometric recordings confirmed the lack of increased catecholamine charge released in cirrhosis after ACh or depolarization stimuli. However, the amperometric spikes exhibited faster kinetics of release. The overall Ca2+ entry through voltage-dependent Ca2+ channels (VDCC), or in particular through Cav1 channels, did not vary between chromaffin cells of control and cirrhotic rats. The inhibition of VDCC by methionine-enkephaline or ATP was not either altered, but it was increased by adrenaline in cells of cirrhotic rats. When a cocktail composed by the three neurotransmitters was tested in order to approach a situation closer to the physiological condition, the inhibition of VDCC was similar between both types of cells. In summary, chromaffin cells of the adrenal gland might contribute to exacerbate the sympathetic nervous system over-activity in cirrhosis because of an increased exocytosis elicited by an enhanced spontaneous electrical activity.

Human native Cav1 channels in chromaffin cells: contribution to exocytosis and firing of spontaneous action potentials.

The present study was performed to evaluate the Cav1 channel subtypes expressed in human chromaffin cells and the role that these channels play in exocytosis and cell excitability. Here we show that human chromaffin cells obtained from organ donors express Cav1.2 and Cav1.3 subtypes using molecular and pharmacological techniques. Immunocytochemical data demonstrated the presence of Cav1.2 and Cav1.3 subtypes, but not Cav1.1 or Cav1.4. Electrophysiological experiments were conducted to investigate the contribution of Cav1 channels to the exocytotic process and cell excitability. Cav1 channels contribute to the exocytosis of secretory vesicles, evidenced by the block of 3µM nifedipine (36.5±2%) of membrane capacitance increment elicited by 200ms depolarizing pulses. These channels show a minor contribution to the initiation of spontaneous action potential firing, as shown by the 2.5 pA of current at the threshold potential (-34mV), which elicits 10.4mV of potential increment. In addition, we found that only 8% of human chromaffin cells exhibit spontaneous action potentials. These data offer novel information regarding human chromaffin cells and the role of human native Cav1 channels in exocytosis and cell excitability.

Monkey adrenal chromaffin cells express α6ß4* nicotinic acetylcholine receptors.

Nicotinic acetylcholine receptors (nAChRs) that contain α6 and ß4 subunits have been demonstrated functionally in human adrenal chromaffin cells, rat dorsal root ganglion neurons, and on noradrenergic terminals in the hippocampus of adolescent mice. In human adrenal chromaffin cells, α6ß4* nAChRs (the asterisk denotes the possible presence of additional subunits) are the predominant subtype whereas in rodents, the predominant nAChR is the α3ß4* subtype. Here we present molecular and pharmacological evidence that chromaffin cells from monkey (Macaca mulatta) also express α6ß4* receptors. PCR was used to show the presence of transcripts for α6 and ß4 subunits and pharmacological characterization was performed using patch-clamp electrophysiology in combination with α-conotoxins that target the α6ß4* subtype. Acetylcholine-evoked currents were sensitive to inhibition by BuIA[T5A,P6O] and MII[H9A,L15A]; α-conotoxins that inhibit α6-containing nAChRs. Two additional agonists were used to probe for the expression of α7 and ß2-containing nAChRs. Cells with currents evoked by acetylcholine were relatively unresponsive to the α7-selctive agonist choline but responded to the agonist 5-I-A-85380. These studies provide further insights into the properties of natively expressed α6ß4* nAChRs.

Selectivity of action of pregabalin on Ca(2+) channels but not on fusion pore, exocytotic machinery, or mitochondria in chromaffin cells of the adrenal gland.

The present study was planned to investigate the action of pregabalin on voltage-dependent Ca(2+) channels (VDCCs) and novel targets (fusion pore formed between the secretory vesicle and the plasma membrane, exocytotic machinery, and mitochondria) that would further explain its inhibitory action on neurotransmitter release. Electrophysiological recordings in the perforated-patch configuration of the patch-clamp technique revealed that pregabalin inhibits by 33.4 ± 2.4 and 39 ± 4%, respectively, the Ca(2+) current charge density and exocytosis evoked by depolarizing pulses in mouse chromaffin cells. Approximately half of the inhibitory action of pregabalin was rescued by l-isoleucine, showing the involvement of α2δ-dependent and -independent mechanisms. Ca(2+) channel blockers were used to inhibit Cav1, Cav2.1, and Cav2.2 channels in mouse chromaffin cells, which were unselectively blocked by the drug. Similar values of Ca(2+) current charge blockade were obtained when pregabalin was tested in human or bovine chromaffin cells, which express very different percentages of VDCC types with respect to mouse chromaffin cells. These results demonstrate that the inhibitory action of pregabalin on VDCCs and exocytosis does not depend on α1 Ca(2+) channel subunit types. Carbon fiber amperometric recordings of digitonin-permeabilized cells showed that neither the fusion pore nor the exocytotic machinery were targeted by pregabalin. Mitochondrial Ca(2+) measurements performed with mitochondrial ratiometric pericam demonstrated that Ca(2+) uptake or release from mitochondria were not affected by the drug. The selectivity of action of pregabalin might explain its safety, good tolerability, and reduced adverse effects. In addition, the inhibition of the exocytotic process in chromaffin cells might have relevant clinical consequences.