Molecular targets of a human HNF1 alpha mutation responsible for pancreatic beta-cell dysfunction

The reverse tetracycline-dependent transactivator system was employed in insulinoma INS-1 cells to achieve controlled inducible expression of hepatocyte nuclear factor-1 alpha (HNF1 alpha)-P291fsinsC, the most common mutation associated with subtype 3 of maturity-onset diabetes of the young (MODY3). Nuclear localized HNF1 alpha-P291fsinsC protein exerts its dominant-negative effects by competing with endogenous HNF1 alpha for the cognate DNA-binding site. HNF1 alpha controls multiple genes implicated in pancreatic beta-cell function and notably in metabolism- secretion coupling. In addition to reduced expression of the genes encoding insulin, glucose transporter-2, L-pyruvate kinase, aldolase B and 3-hydroxy-3-methylglutaryl coenzyme A reductase, induction of HNF1 alpha-P291fsinsC also significantly inhibits expression of mitochondrial 2-oxoglutarate dehydrogenase (OGDH) E1 subunit mRNA and protein. OGDH enzyme activity and [(14)C]pyruvate oxidation were also reduced. In contrast, the mRNA and protein levels of mitochondrial uncoupling protein-2 were dramatically increased by HNF1 alpha-P291fsinsC induction. As predicted from this altered gene expression profile, HNF1 alpha-P291fsinsC also inhibits insulin secretory responses to glucose and leucine, correlated with impaired nutrient-evoked mitochondrial ATP production and mitochondrial membrane hyperpolarization. These unprecedented results suggest the molecular mechanism of HNF1 alpha-P291fsinsC causing beta-cell dysfunction.     

Analysis of protein dimerization and ligand binding of orphan receptor HNF4alpha

Hepatocyte nuclear factor 4alpha (HNF4alpha) (NR2A1), an orphan member of the nuclear receptor superfamily, binds DNA exclusively as a homodimer even though it is very similar in amino acid sequence to retinoid X receptor alpha (RXRalpha), which heterodimerizes readily with other receptors. Here, experimental analysis of residues involved in protein dimerization and studies on a reported ligand for HNF4alpha are combined with a structural model of the HNF4alpha ligand-binding domain (LBD) (residues 137 to 384). When K300 (in helix 9) and E327 (in helix 10) of HNF4alpha1 were converted to the analogous residues in RXRalpha (E390 and K417, respectively) the resulting construct did not heterodimerize with the wild-type HNF4alpha, although it was still able to form homodimers and bind DNA. Furthermore, the double mutant did not heterodimerize with RXR or RAR but was still able to dimerize in solution with an HNF4alpha construct truncated at amino acid residue 268. This suggests that the charge compatibility between helices 9 and 10 is necessary, but not sufficient, to determine dimerization partners, and that additional residues in the HNF4alpha LBD are also important in dimerization. The structural model of the HNF4alpha LBD and an amino acid sequence alignment of helices 9 and 10 in various HNF4 and other receptor genes indicates that a K(X)(26)E motif can be used to identify HNF4 genes from other organisms and that a (E/D(X)(26-29)K/R) motif can be used to predict heterodimerization of many, but not all, receptors with RXR. In vitro analysis of another HNF4alpha mutant construct indicates that helix 10 also plays a structural role in the conformational integrity of HNF4alpha. The structural model and experimental analysis indicate that fatty acyl CoA thioesters, the proposed HNF4alpha ligands, are not good candidates for a traditional ligand for HNF4alpha. Finally, these results provide insight into the mechanism of action of naturally occurring mutations in the human HNF4alpha gene found in patients with maturity onset diabetes of the young 1 (MODY1).     

Structural basis of natural promoter recognition by a unique nuclear receptor, HNF4alpha. Diabetes gene product

HNF4alpha (hepatocyte nuclear factor 4alpha) plays an essential role in the development and function of vertebrate organs, including hepatocytes and pancreatic beta-cells by regulating expression of multiple genes involved in organ development, nutrient transport, and diverse metabolic pathways. As such, HNF4alpha is a culprit gene product for a monogenic and dominantly inherited form of diabetes, known as maturity onset diabetes of the young (MODY). As a unique member of the nuclear receptor superfamily, HNF4alpha recognizes target genes containing two hexanucleotide direct repeat DNA-response elements separated by one base pair (DR1) by exclusively forming a cooperative homodimer. We describe here the 2.0 angstroms crystal structure of human HNF4alpha DNA binding domain in complex with a high affinity promoter element of another MODY gene, HNF1alpha, which reveals the molecular basis of unique target gene selection/recognition, DNA binding cooperativity, and dysfunction caused by diabetes-causing mutations. The predicted effects of MODY mutations have been tested by a set of biochemical and functional studies, which show that, in contrast to other MODY gene products, the subtle disruption of HNF4alpha molecular function can cause significant effects in afflicted MODY patients.     

Fasting induces basolateral uptake transporters of the SLC family in the liver via HNF4alpha and PGC1alpha

Fasting induces numerous adaptive changes in metabolism by several central signaling pathways, the most important represented by the HNF4alpha/PGC-1alpha-pathway. Because HNF4alpha has been identified as central regulator of basolateral bile acid transporters and a previous study reports increased basolateral bile acid uptake into the liver during fasting, we hypothesized that HNF4alpha is involved in fasting-induced bile acid uptake via upregulation of basolateral bile acid transporters. In rats, mRNA of Ntcp, Oatp1, and Oatp2 were significantly increased after 48 h of fasting. Protein expression as determined by Western blot showed significant increases for all three transporters 72 h after the onset of fasting. Whereas binding activity of HNF1alpha in electrophoretic mobility shift assays remained unchanged, HNF4alpha binding activity to the Ntcp promoter was increased significantly. In line with this result, we found significantly increased mRNA expression of HNF4alpha and PGC-1alpha. Functional studies in HepG2 cells revealed an increased endogenous NTCP mRNA expression upon cotransfection with either HNF4alpha, PGC-1alpha, or a combination of both. We conclude that upregulation of the basolateral bile acid transporters Ntcp, Oatp1, and Oatp2 in fasted rats is mediated via the HNF4alpha/PGC-1alpha pathway.     

In vivo role of the HNF4alpha AF-1 activation domain revealed by exon swapping

The gene encoding the nuclear receptor hepatocyte nuclear factor 4alpha (HNF4alpha) generates isoforms HNF4alpha1 and HNF4alpha7 from usage of alternative promoters. In particular, HNF4alpha7 is expressed in the pancreas whereas HNF4alpha1 is found in liver, and mutations affecting HNF4alpha function cause impaired insulin secretion and/or hepatic defects in humans and in tissue-specific 'knockout' mice. HNF4alpha1 and alpha7 isoforms differ exclusively by amino acids encoded by the first exon which, in HNF4alpha1 but not in HNF4alpha7, includes the activating function (AF)-1 transactivation domain. To investigate the roles of HNF4alpha1 and HNF4alpha7 in vivo, we generated mice expressing only one isoform under control of both promoters, via reciprocal swapping of the isoform-specific first exons. Unlike Hnf4alpha gene disruption which causes embryonic lethality, these 'alpha7-only' and 'alpha1-only' mice are viable, indicating functional redundancy of the isoforms. However, the former show dyslipidemia and preliminary results indicate impaired glucose tolerance for the latter, revealing functional specificities of the isoforms. These 'knock-in' mice provide the first test in vivo of the HNF4alpha AF-1 function and have permitted identification of AF-1-dependent target genes.     

Monocyte and macrophage dysfunction as a cause of HIV-1 induced dysfunction of innate immunity

HIV-1 can establish both long lived and productive infection of macrophages (M?) but circulating monocytes are less permissive to infection. Multiple studies have identified extensive changes to monocyte and M? phenotype, differentiation or function. These include alterations in Toll-like receptor signaling and resultant changes to cytokine responses, specific defects in phagocytosis and microbial killing and modulation of apoptotic responses, all of which may perturb the important role of these cells in innate immunity. Interpretation of contradictory data however, is complicated by the use of different experimental models and many of the reported effects may be an indirect consequence of HIV 1 infection that result from exposure to viral products or from disruption of cellular and cytokine networks in the immune system, rather than the direct consequence of productive HIV 1 infection. Future research should focus on refining experimental models and on elucidating the physiological mechanisms of monocyte/ M? dysfunction during HIV 1 infection.     

Evaluation of sepsis induced cardiac dysfunction as a predictor of mortality

Background

Sepsis is characterized by life threatening organ dysfunction with dysregulated immune response. Cardiac dysfunction seen in sepsis is unique as it is reversible within 7–10?days. Initial study by Parker et al. in 1984, showed, paradoxically lower ejection fraction in survivors of septic shock. Subsequent meta-analysis did not support that survivors had lower ejection fraction. Aim of our study was to assess the sepsis induced cardiac dysfunction by 2D echocardiography and Troponin I.

Methods

After obtaining institutional ethical committee approval (ref 125/2016), a prospective observational study was done in an university medical college from February 2016 to April 2016. Inclusion criteria were patients diagnosed with sepsis by new sepsis definition. Pregnant patients and patients with poor echo window were excluded. Echocardiographic assessment was done within 48?h of diagnosis of sepsis by standard methods. Primary outcome was ICU mortality and secondary outcome was ICU length of stay. Statistical analysis was done using STATA? (Version14, College station TX).

Results

Fifty eight patients were screened, ten were excluded due to poor echo window. Baseline characteristics were similar in survivors and non survivors, except APACHE II, SOFA age and cumulative fluid balance. Echocardiographic parameters, mitral annular plane systolic excursion (MAPSE), E/e’ and LV systolic function assessed by visual gestalt method were found to be statistically significant. Parameters found significant in bivariate analysis were used as a covariate in logistic regression. APACHE II and MAPSE were significant co-variates in logistic regression with ROC (0.95) and calibration was satisfactory (chi2(df8),1.98, p?=?0.98).

Conclusions

Sepsis induced cardiac dysfunction assessed by echocardiography showed measurement of MAPSE when combined with APACHE II was a good predictor of mortality. Among the echocardiographic parameters MAPSE alone was a good predictor of mortality. Results of this study need further validation from larger study.