How heatwaves affect short-term emergency hospital admissions due to bacterial foodborne diseases.

The coming decades are likely to see of extreme weather events becoming more intense and frequent across Europe as a whole and around the Mediterranean in particular. The reproduction rate of some microorganisms, including the bacteria that cause foodborne diseases, will also be affected by these events. The aim of this study was thus to ascertain whether there might be a statistically significant relationship between emergency hospital admissions due to the principal bacterial foodborne diseases (BFDs) and the various meteorological variables, including heatwaves. We conducted a time-series study, with daily observations of both the dependent variable (emergency hospital admissions due to BFDs) and the independent variables (meteorological variables and control variables of chemical air pollution) across the period 2013-2018 in the Madrid Region (Spain), using Generalised Linear Models with Poisson regression, in which control and lag variables were included for the purpose of fitting the models. We calculated the threshold value of the maximum daily temperature above which such admissions increased statistically significantly, analysed data for the whole year and for the summer months alone, and estimated the relative and attributable risks. The estimated attributable risk was 3.6 % for every one-degree rise in the maximum daily temperature above 12 °C throughout the year, and 12.21 % for every one degree rise in temperature above the threshold heatwave definition temperature (34 °C) in summer. Furthermore, different meteorological variables displayed a statistically significant association. Whereas hours of sunlight and mean wind speed proved significant in the analyses of both the whole year and summer, the variables "rain" and "relative humidity", only showed a significant relationship in the analysis for the whole year. High ambient temperature is a risk factor that favours the increase in emergency hospitalisations attributable to the principal BFDs, with a greater impact being observed on days coinciding with heatwave periods. The results yielded by this study could serve as a basis for implementing BFD prevention strategies, especially on heatwave days.

Short-term effects of fine particulate matter from biomass combustion and Saharan dust intrusions on emergency hospital admissions due to mental and behavioural disorders, anxiety and depression in Spain.

Climate change is affecting both the frequency and scale of wildfires, as well as the increase in the number of days with Saharan dust intrusions. Traditionally, studies have focused on the extent to which the increase in fine particulate matter (PM) has had an impact on cardio-respiratory diseases, but (apart from PM) not on how the meteorological and pollution conditions in these situations affect other diseases, such as those linked to mental health. This study therefore sought to ascertain how daily mean PM10, PM 2.5, NO2, O3 concentrations and daily maximum temperature in heat waves influence daily emergency hospital admissions in Spain caused by mental and behavioural disorders, depression and anxiety on days with PM from biomass combustion and/or Saharan dust intrusions, as compared to days without such conditions, across the period 2009-2018. Our results indicate that on days on which there is biomass combustion, PM concentrations have a statistically significant effect on emergency admissions due to mental disorders, probably related with the toxicity of these particles. Yet on days with intrusions of Saharan dust rather than PM, it is the other variables considered in the analysis that are most closely linked to these types of admissions. The results of this study thus point to the need to implement public health prevention plans which take into account the joint effect of various environmental risk factors that act synergistically in given situations.

Short-term effects of tropospheric ozone and other environmental factors on emergency admissions due to pregnancy complications: A time-series analysis in the Madrid Region.

Air pollution has been linked to adverse neonatal outcomes, mainly in the case of prolonged exposures. This study focuses on the short-term effects on maternal health. We conducted a retrospective ecological time-series study in the Madrid Region covering the period 2013-2018. The independent variables were mean daily concentrations of tropospheric ozone (O3), particulate matter (PM10/PM2.5) and nitrogen dioxide (NO2), as well as noise levels. The dependent variables were daily emergency hospital admissions due to complications in pregnancy, childbirth and the puerperium. Poisson generalised linear regression models were fitted to quantify the relative and attributable risks, controlling for trend, seasonality, the autoregressive nature of the series, and a number of meteorological factors. There were 318 069 emergency hospital admissions due to obstetric complications across the 2191 days of study. Of this total: 13 164 (95%CI: 9930-16 398) admissions were attributable to exposure to O3, the only pollutant to show a statistically significant (p < 0.05) association with admissions due to hypertensive disorders; and 10 575 (95%CI: 3573-17 566) admissions were attributable to daytime noise levels, while admissions due to hyperemesis gravidarum and vomiting were related to exposure to night noise. Other pollutants which also displayed statistically significant associations were: NO2 concentrations, with admissions due to vomiting and preterm labour; PM10 concentrations, with premature rupture of membranes: and PM2.5 concentrations, with total complications. Exposure to a range of air pollutants, and ozone in particular, is associated with a higher number of emergency hospital admissions due to gestational complications. Hence, surveillance of environmental effects on maternal health should be intensified, and plans and strategies to minimise these should be drawn up.

Impact of urban heat islands on morbidity and mortality in heat waves: Observational time series analysis of Spain's five cities.

Urban heat islands (UHIs) have become an especially relevant phenomenon as a consequence of global warming and the growing proportion of people living in cities. The health impacts that are sometimes attributed to the rise in temperature generated in an UHI are not always adequately justified. The objective is to analyse what effect UHIs have on maximum (Tmax) and minimum daily temperatures (Tmin) recorded in urban and non-urban observatories, and quantify the impact on morbidity and mortality during heat waves in Spain's five cities. Data were collected on natural-cause daily mortality and unscheduled emergency hospital admissions (ICD-10: A00-R99) registered in these 5 cities across the period 2014-2018. We analysed daily Tmax and Tmin values at urban and non-urban observatories in these cities, and quantified the impact of Tmax and Tmin values during heat waves in each of these cities, using GLM models that included Tmax only, Tmin only, and both. We controlled for air pollution and other meteorological variables, as well as for seasonalities, trend and the autoregressive nature of the series. The UHI effect was observed in Tmin but not in Tmax, and proved to be greater in coastal cities than in inland and more densely populated cities. The UHI value in relation to the mean Tmin in the summer months ranged from 1.2 °C in Murcia to 4.1 °C in Valencia (difference between urban/non-urban observatories). The modelling process showed that, while a statistically significant association (p < 0.05) was observed in inland cities with Tmax for mortality and hospital admissions in heat waves, in coastal cities the association was obtained with Tmin, and the only impact in this case was the UHI effect on morbidity and mortality. No generalisations can be made about the impact of UHI on morbidity and mortality among the exposed population in cities. Studies on a local scale are called for, since it is local factors that determine whether the UHI effect will have a greater or lesser impact on health during heat-wave events.

Effect of rapamycin on lysosomal accumulation in a CRISPR/Cas9-based cellular model of VPS13A deficiency.

VPS13A is a lipid transfer protein localized at different membrane contact sites between organelles, and mutations in the corresponding gene produce a rare neurodegenerative disease called chorea-acanthocytosis (ChAc). Previous studies showed that VPS13A depletion in HeLa cells results in an accumulation of endosomal and lysosomal markers, suggesting a defect in lysosomal degradation capacity leading to partial autophagic dysfunction. Our goal was to determine whether compounds that modulate the endo-lysosomal pathway could be beneficial in the treatment of ChAc. To test this hypothesis, we first generated a KO model using CRISPR/Cas9 to study the consequences of the absence of VPS13A in HeLa cells. We found that inactivation of VPS13A impairs cell growth, which precludes the use of isolated clones due to the undesirable selection of edited clones with residual protein expression. Therefore, we optimized the use of pool cells obtained shortly after transfection with CRISPR/Cas9 components. These cells are a mixture of wild-type and edited cells that allow a comparative analysis of phenotypes and avoids the selection of clones with residual level of VPS13A expression after long-term growth. Consistent with previous observations by siRNA inactivation, VPS13A inactivation by CRISPR/Cas9 resulted in accumulation of the endo-lysosomal markers RAB7A and LAMP1. Notably, we observed that rapamycin partially suppressed the difference in lysosome accumulation between VPS13A KO and WT cells, suggesting that modulation of the autophagic and lysosomal pathway could be a therapeutic target in the treatment of ChAc.

How heat waves, ozone and sunlight hours affect endocrine and metabolic diseases emergency admissions? A case study in the region of Madrid (Spain).

BACKGROUND: Studies which analyse the joint effect of acoustic or chemical air pollution variables and different meteorological variables on neuroendocrine disease are practically nonexistent. This study therefore sought to analyse the impact of air pollutants and environmental meteorological variables on daily unscheduled admissions due to endocrine and metabolic diseases in the Madrid Region from January 01, 2013 to December 31, 2018. MATERIAL AND METHODS: We conducted a longitudinal, retrospective, ecological study of daily time series analysed by Poisson regression, with emergency neuroendocrine-disease admissions in the Madrid Region as the dependent variable. The independent variables were: mean daily concentrations of PM10, PM2.5, NO2 and O3; acoustic pollution; maximum and minimum daily temperatures; hours of sunlight; relative humidity; wind speed; and air pressure above sea level. Estimators of the statistically significant variables were used to calculate the relative risks (RRs). RESULTS: A statistically significant association was found between the increase in temperatures in heat waves, RR: 1.123 95% CI (1.001-1.018), and the number of emergency admissions, making it the main risk factor. An association between a decrease in sunlight and an increase in hospital admissions, RR: 1.005 95% CI (1.002 1.008), was likewise observed. Similarly, ozone, in the form of mean daily concentrations in excess of 44 µg/m3, had an impact on admissions due to neuroendocrine disease, RR: 1.010 95% CI (1.007-1.035). The breakdown by sex showed that in the case of women, NO2 was also a risk factor, RR: 1.021 95% CI (1.007-1.035). CONCLUSION: The results obtained in this study serve to identify risk factors for this disease, such as extreme temperatures in heat waves, O3 or NO2. The robust association found between the decrease in sunlight and increase in hospital admissions due to neuroendocrine disease serves to spotlight an environmental factor which has received scant attention in public health until now.

Short-term impact of noise, other air pollutants and meteorological factors on emergency hospital mental health admissions in the Madrid region.

BACKGROUND: A number of environmental factors, such as air pollution, noise in urbanised settings and meteorological-type variables, may give rise to important effects on human health. In recent years, many studies have confirmed the relation between various mental disorders and these factors, with a possible impact on the increase in emergency hospital admissions due to these causes. The aim of this study was to analyse the impact of a range of environmental factors on daily emergency hospital admissions due to mental disorders in the Madrid Autonomous Region (MAR), across the period 2013-2018. METHODOLOGY: Longitudinal ecological time series study analysed by Generalised Linear Models with Poisson regression, with the dependent variable being daily Emergency Hospital Mental Health Admissions (EHMHA) in the MAR, and the independent variable being mean daily concentrations of chemical pollutants, noise levels and meteorological variables. RESULTS: EHMHA were related statistically significantly in the short term with diurnal noise levels. Relative risks (RRs) for total admissions due to mental disorders and self-inflicted injuries, in the case of diurnal noise was RR: 1.008 95%CI (1.003 1.013). Admissions attributable to diurnal noise account for 5.5% of total admissions across the study period. There was no association between hospital admissions and chemical air pollution. CONCLUSION: Noise is a variable that shows a statistically significant short-term association with EHMHA across all age groups in the MAR region. The results of this study may serve as a basis for drawing up public health guidelines and plans, which regard these variables as risk factors for mental disorders, especially in the case of noise, since this fundamentally depends on anthropogenic activities in highly urbanised areas with high levels of traffic density.

Air pollution and meteorological variables' effects on COVID-19 first and second waves in Spain.

The aim of this research is to study the influence of atmospheric pollutants and meteorological variables on the incidence rate of COVID-19 and the rate of hospital admissions due to COVID-19 during the first and second waves in nine Spanish provinces. Numerous studies analyze the effect of environmental and pollution variables separately, but few that include them in the same analysis together, and even fewer that compare their effects between the first and second waves of the virus. This study was conducted in nine of 52 Spanish provinces, using generalized linear models with Poisson link between levels of PM10, NO2 and O3 (independent variables) and maximum temperature and absolute humidity and the rates of incidence and hospital admissions of COVID-19 (dependent variables), establishing a series of significant lags. Using the estimators obtained from the significant multivariate models, the relative risks associated with these variables were calculated for increases of 10 µg/m3 for pollutants, 1 °C for temperature and 1 g/m3 for humidity. The results suggest that NO2 has a greater association than the other air pollution variables and the meteorological variables. There was a greater association with O3 in the first wave and with NO2 in the second. Pollutants showed a homogeneous distribution across the country. We conclude that, compared to other air pollutants and meteorological variables, NO2 is a protagonist that may modulate the incidence and severity of COVID-19, though preventive public health measures such as masking and hand washing are still very important. Supplementary Information: The online version contains supplementary material available at 10.1007/s13762-022-04190-z.

Short-term effects of air pollution and noise on emergency hospital admissions in Madrid and economic assessment.

INTRODUCTION: The aim of this study was to study the effect of air pollution and noise has on the population in Madrid Community (MAR) in the period 2013-2018, and its economic impact. METHODS: Time series study analysing emergency hospital admissions in the MAR due to all causes (ICD-10: A00-R99), respiratory causes (ICD-10: J00-J99) and circulatory causes (ICD-10: I00-I99) across the period 2013-2018. The main independent variables were mean daily PM2.5, PM10, NO2, 8-h ozone concentrations, and noise. We controlled for meteorological variables, Public Holidays, seasonality, and the trend and autoregressive nature of the series, and fitted generalised linear models with a Poisson regression link to ascertain the relative risks and attributable risks. In addition, we made an economic assessment of these hospitalisations. RESULTS: The following associations were found: NO2 with admissions due to natural (RR: 1.007, 95% CI: 1.004-1.011) and respiratory causes (RR: 1.012, 95% CI: 1.005-1.019); 8-h ozone with admissions due to natural (RR: 1.049, 95% CI: 1.014-1.046) and circulatory causes (RR: 1.088, 95% CI: 1.039-1.140); and diurnal noise (LAeq7-23h) with admissions due to natural (RR: 1.001, 95% CI: 1.001-1.002), respiratory (RR: 1.002, 95% CI: 1.001-1.003) and circulatory causes (RR: 1.003, 95% CI: 1.002-1.005). Every year, a total of 8246 (95% CI: 4580-11,905) natural-cause admissions are attributable to NO2, with an estimated cost of close on €120 million and 5685 (95% CI: 2533-8835) attributed to LAeq7-23h with an estimated cost of close on €82 million. CONCLUSIONS: Nitrogen dioxide, ozone and noise are the main pollutants to which a large number of hospitalisations in the MAR are attributed, and are thus responsible for a marked deterioration in population health and high related economic impact.

Does the meteorological origin of heat waves influence their impact on health? A 6-year morbidity and mortality study in Madrid (Spain).

BACKGROUND: In Spain, two synoptic-scale conditions influence heat wave formation. The first involves advection of warm and dry air masses carrying dust of Saharan origin (North African Dust (NAF) = 1). The second entails anticyclonic stagnation with high insolation and stability (NAF) = 0). Some studies show that the meteorological origin of these heat waves may affect their impact on morbidity and mortality. OBJECTIVE: To determine whether the impact of heat waves on health outcomes in Madrid (Spain) during 2013-2018 varied by synoptic-scale condition. METHODOLOGY: Outcome data consist of daily mortality and daily hospital emergency admissions (morbidity) for natural, circulatory, and respiratory causes. Predictors include daily maximum and minimum temperatures and daily mean concentrations of NO2, PM10, PM2.5, NO2, and O3. Analyses adjust for insolation, relative humidity, and wind speed. Generalized linear models were performed with Poisson link between the variables controlling for trend, seasonality, and auto-regression in the series. Relative Risks (RR) and Attributable Risks (AR) were determined. The RRs for mortality attributable to high temperatures were similar regardless of NAF status. For hospital admissions, however, the RRs for hot days with NAF = 0 are higher than for days with NAF = 1. We also found that atmospheric pollutants worsen morbidity and mortality, especially PM10 concentrations when NAF = 1 and O3 concentrations when NAF = 0. RESULTS: The effect of heat waves on morbidity and mortality depends on the synoptic situation. The impact is greater under anticyclonic stagnation conditions than under Saharan dust advection. Further, the health impact of pollutants such as PM10 and O3 varies according to the synoptic situation. CONCLUSIONS: Based on these findings, we strongly recommend prevention plans to include data on the meteorological situation originating the heat wave, on a synoptic-scale, as well as comprehensive preventive measures against the compounding effect of high temperatures and pollution.

Temporal evolution of threshold temperatures for extremely cold days in Spain.

In contrast to research on heat waves, there are no studies in recent years that analyze the temporal evolution of threshold temperatures (Tthreshold) for extremely cold days (ECD). It is unknown whether threshold temperatures have increased more quickly than the minimum daily temperature (Tmin) in recent years. The objective of this study was to analyze the temporal evolution of the minimum daily temperature (Tmin) in a group of Spanish provinces and compare it with the evolution of threshold temperatures. An ecological, retrospective time series study was carried out using daily observations between January 1, 1983 and December 31, 2018 (36 years) in 10 provinces that are representative of the different climate territories in Spain. For each representative observatory in each province, the values of Tmin were obtained for the winter months (November-March). The value of Tthreshold was determined for each province and each year, using dispersion diagrams for the pre-whitened series, with daily mortality due to natural causes displayed on the Y axis (CIEX: A00-R99) and Tmin grouped by 10 degree intervals on the X axis. To determine the temporal evolution of Tmin and Tthreshold for each province, linear models were fitted, with time as the independent variable. During the winter months, Tmin increased at an average rate of 0.2 °C/decade (IC95: 0.1-0.3), while Tthreshold remained practically constant during the period, at 0.1 °C/decade (IC95% -0.1 0.3). These values are much lower than those obtained in the case of heat, both in terms of the evolution of maximum daily temperature and that of Tthreshold. In conclusion, the fact that this trend has been maintained across time in a scenario of climate change, with a slow increase in minimum daily temperatures and constant values of Threshold, suggests a decrease in the number of ECD.

Evolution of the minimum mortality temperature (1983-2018): Is Spain adapting to heat?

The objective of this study was to analyze at the level of Spain's 52 provinces province level the temporal evolution of minimum mortality temperatures (MMT) from 1983 to 2018, in order to determine whether the increase in MMT would be sufficient to compensate for the increase in environmental temperatures in Spain for the period. It also aimed to analyze whether the rate of evolution of MMT would be sufficient, were it to remain constant, to compensate for the predicted increase in temperatures in an unfavorable (RCP 8.5) emissions scenario for the time horizon 2051-2100. The independent variable was made up of maximum daily temperature data (Tmax) for the summer months in the reference observatories of each province for the 1983-2018 period. The dependent variable was daily mortality rate due to natural causes (ICD 10: A00-R99). For each year and province, MMT was determined using a quadratic or cubic fit (p < 0.05). Based on the annual MMT values, a linear fit was carried out that allowed for determining the time evolution of MMT. These values were compared with the evolution of Tmax registered in each observatory during the 1983-2018 analyzed period and with the predicted values of Tmax obtained for an RCP8.5 scenario for the period 2051-2100. The rate of global variance in Tmax in the summer months in Spain during the 1983-2018 period was 0.41 °C/decade, while MMT across the whole country increased at a rate of 0.64 °C/decade. Variations in the provinces were heterogeneous. For the 2051-2100 time horizon, there was predicted increase in Tmax values of 0.66 °C/decade, with marked geographical differences. Although at the global level it is possible to speak of adaptation, the heterogeneities among the provinces suggest that the local level measures are needed in order to facilitate adaptation in those areas where it is not occurring.

Antiphospholipid antibodies, steroid dose, arterial hypertension, relapses, and late-onset predict organ damage in a population of Colombian patients with systemic lupus erythematosus.

Organ damage predicts mortality, increased accrual of detriment, and poor quality of life in systemic lupus erythematosus patients. The objective of this study is to determine the damage-free survival and its predictive factors in a population of Colombian subjects. The method used in this study is the retrospective follow-up of a cohort; damage was measured with SLICC/ACR index. Predictors of impairment were assessed by logistic regression and survival analysis. One hundred sixty-one individuals were included; 28.9% suffered damage, primarily neuropsychiatric, renal, and vascular. Arterial hypertension, antiphospholipid antibodies, prednisone dose, and number of relapses were all predictors of detriment. Onset after age 50 and daily prednisone dose higher than 7.5 mg determined earlier occurrence of damage.

Functional analysis of human glucokinase gene mutations causing MODY2: exploring the regulatory mechanisms of glucokinase activity.

AIMS/HYPOTHESIS: Glucokinase (GCK) acts as a glucose sensor in the pancreatic beta cell and regulates insulin secretion. In the gene encoding GCK the heterozygous mutations that result in enzyme inactivation cause MODY2. Functional studies of naturally occurring GCK mutations associated with hyperglycaemia provide further insight into the biochemical basis of glucose sensor regulation. MATERIALS AND METHODS: Identification of GCK mutations in selected MODY patients was performed by single-strand conformation polymorphism and direct sequencing. The kinetic parameters and thermal stability of recombinant mutant human GCK were determined, and in pull-down assays the effect of these mutations on the association of GCK with glucokinase (hexokinase 4) regulator (GCKR, also known as glucokinase regulatory protein [GKRP]) and 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase (PFKFB1, also known as PFK2) was tested. RESULTS: We identified three novel GCK mutations: the insertion of an asparagine residue at position 161 (inserN161) and two missense mutations (M235V and R308W). We also identified a fourth mutation (R397L) reported in a previous work. Functional characterisation of these mutations revealed that insertion of asparagine residue N161 fully inactivates GCK, whereas the M235V and R308W mutations only partially impair enzymatic activity. In contrast, GCK kinetics was almost unaffected by the R397L mutation. Although none of these mutations affected the interaction of GCK with PFKFB1, we found that the R308W mutation caused protein instability and increased the strength of interaction with GCKR. CONCLUSIONS/INTERPRETATION: Our results show that different MODY2 mutations impair GCK function through different mechanisms such as enzymatic activity, protein stability and increased interaction with GCKR, helping further elucidate the regulation of GCK activity.

The human HNF-3 genes: cloning, partial sequence and mutation screening in patients with impaired glucose homeostasis.

Hepatocyte nuclear factors 3 (HNF-3 alpha, -3 beta and -3 gamma) belong to an evolutionarily conserved family of transcription factors that are critical for diverse biological processes such as development, differentiation and metabolism. Gene expression studies have shown that HNF3 proteins are critical regulators of the early-onset type 2 diabetes genes HNF-1 alpha, HNF-4 alpha and IPF-1/PDX-1 (MODY3, 1 and 4, respectively) and of glucagon transcription and pancreatic alpha-cell function. In this study, we investigated whether genetic variation in the genes encoding HNF-3 alpha, HNF-3 beta and HNF-3 gamma predisposes humans to hyperglycemic or hypoglycemic syndromes. In addition, we report the cloning and partial nucleotide sequence of the human HNF-3 alpha, -3 beta and -3 gamma genes. Mutation screening included 96 subjects with type 2 diabetes mellitus, as well as one family with persistent neonatal hypoglycemia. No functional mutations were detected in the coding sequences of the three HNF-3 genes. Our results suggest that mutations in HNF-3 genes are not a common cause of type 2 diabetes mellitus. The data provided will facilitate genetic studies in other populations and will advance our understanding of the role HNF-3 plays in the development of diabetes mellitus and other metabolic disorders of glucose homeostasis.

Collagenous colitis and lymphocytic colitis: clinical and endoscopic findings.

OBJECTIVE: collagenous colitis (CC) and lymphocytic colitis (LC) are two entities of unknown cause, characterized by chronic watery diarrhea, grossly normal-appearing colonic mucosa and abnormal histopathological findings in colonic biopsies. The clinical features of the disease are based mainly on case reports or small uncontrolled series. Although normal colonoscopic findings are, as a rule, part of the diagnosis of CC, several cases of macroscopic colitis associated with CC have been reported, and the spectrum of endoscopic mucosal changes has not been described in large series. METHODS: we present a retrospective study of all patients who underwent total colonoscopy and mucosal biopsy in our Endoscopy Unit between 1991 and 1997. Clinical and endoscopic findings in patients diagnosed as having CC or LC were recorded. RESULTS: of 676 patients studied, 398 suffered from chronic diarrhea. Collagenous colitis was diagnosed in 22 patients and LC in 10. Eleven per cent of the patients with CC and 20% of those with LC did not have diarrhea. Macroscopic colitis was observed in 6 out of 22 patients with CC (27%) and in 4 out of 10 with LC (40%). Macroscopic lesions included edema, erythema, abnormal vascular pattern, superficial erosions or ulcerations and hemorrhagic lacerations. In this series 7.03% of the patients with chronic diarrhea were diagnosed as having CC or LC. CONCLUSIONS: collagenous colitis and LC are two entities that should be considered in the differential diagnosis of chronic diarrhea. Total colonoscopy with multiple biopsies that include the right colon are mandatory. The presence of macroscopic lesions on endoscopy does not rule out a diagnosis of either entity. We identified patients who fulfilled the histopathological criteria for CC or LC but who did not have diarrhea.

Impaired glucose homeostasis and neonatal mortality in hepatocyte nuclear factor 3alpha-deficient mice.

Hepatocyte nuclear factors 3 (HNF-3) belong to an evolutionarily conserved family of transcription factors that are critical for diverse biological processes such as development, differentiation, and metabolism. To study the physiological role of HNF-3alpha, we generated mice that lack HNF-3alpha by homologous recombination in embryonic stem cells. Mice homozygous for a null mutation in the HNF-3alpha gene develop a complex phenotype that is characterized by abnormal feeding behavior, progressive starvation, persistent hypoglycemia, hypotriglyceridemia, wasting, and neonatal mortality between days 2 and 14. Hypoglycemia in HNF-3alpha-null mice leads to physiological counter-regulatory responses in glucocorticoid and growth hormone production and an inhibition of insulin secretion but fails to stimulate glucagon secretion. Glucagon-producing pancreatic alpha cells develop normally in HNF-3alpha-/- mice, but proglucagon mRNA levels are reduced 50%. Furthermore, the transcriptional levels of neuropeptide Y are also significantly reduced shortly after birth, implying a direct role of HNF-3alpha in the expression of these genes. In contrast, mRNA levels were increased in HNF-3 target genes phosphofructo-2-kinase/fructose-2,6-bisphophatase, insulin growth factor binding protein-1, and hexokinase I of HNF-3alpha-null mice. Mice lacking one or both HNF-3alpha alleles also show impaired insulin secretion and glucose intolerance after an intraperitoneal glucose challenge, indicating that pancreatic beta-cell function is also compromised. Our results indicate that HNF-3alpha plays a critical role in the regulation of glucose homeostasis and in pancreatic islet function.

Functional characterization of the MODY1 gene mutations HNF4(R127W), HNF4(V255M), and HNF4(E276Q).

Genetic studies have shown that mutations in the gene encoding hepatocyte nuclear factor (HNF)-4alpha, a member of the steroid/thyroid hormone receptor superfamily, give rise to early-onset type 2 diabetes (MODY1). The functional properties of mutant HNF-4alpha proteins and the molecular mechanisms by which they impair insulin secretion are largely unknown. In the present study, we have investigated transcriptional activation, DNA binding properties, and protein dimerization activity of three HNF-4alpha missense mutations--HNF4(R127W), HNF4(V255M), and HNF4(E276Q)--that have been associated with type 2 diabetes. We demonstrate that HNF4(E276Q) has lost its ability to bind to HNF-4 consensus binding sites and activate transcription. HNF4(E276Q) had no effect on the functional activity of wild-type HNF-4alpha in the pancreatic beta-cell line HIT-T15, but it exhibited weak dominant-negative activity in other cell types. Analysis of HNF4(E276Q) protein showed that it exists in two forms: a full length 54-kDa protein and a 40-kDa COOH-terminal protein lacking the NH2-terminal transactivation domain and the DNA binding domain. Immunoprecipitation experiments indicate that this truncated protein can bind to wild-type HNF-4alpha and may be responsible for the weak dominant-negative effects seen in these cells. In addition, we show that the transcriptional transactivation of HNF4(R127W) and HNF4(V255M) is indistinguishable from that of wild-type HNF-4alpha, suggesting that they are sequence polymorphisms. Our results demonstrate that HNF4(E276Q) is a loss-of-function mutation and that it identifies glutamic acid 276 in alpha-helix 8 of the ligand-binding domain of HNF-4alpha protein as a critical residue for DNA binding, transcriptional activation, and protein stability in vivo.

Regulation of a transcription factor network required for differentiation and metabolism.

Hepatocyte nuclear factors (HNFs) are a heterogeneous class of evolutionarily conserved transcription factors that are required for cellular differentiation and metabolism. Mutations in HNF-1alphaand HNF-4alpha genes impair insulin secretion and cause type 2 diabetes. Regulation of HNF-4/HNF-1 expression by HNF-3alpha and HNF-3beta was studied in embryoid bodies in which one or both HNF-3alpha or HNF-3beta alleles were inactivated. HNF-3beta positively regulated the expression of HNF-4alpha/HNF-1alpha and their downstream targets, implicating a role in diabetes. HNF-3beta was also necessary for expression of HNF-3alpha. In contrast, HNF-3alpha acts as a negative regulator of HNF-4alpha/HNF-1alpha demonstrating that HNF-3alpha and HNF-3beta have antagonistic transcriptional regulatory functions in vivo. HNF-3alpha does not appear to act as a classic biochemical repressor but rather exerts its negative effect by competing for HNF-3 binding sites with the more efficient activator HNF-3beta. In addition, the HNF-3alpha/HNF-3beta ratio is modulated by the presence of insulin, providing evidence that the HNF network may have important roles in mediating the action of insulin.