Systematically testing human HMBS missense variants to reveal mechanism and pathogenic variation.

Defects in hydroxymethylbilane synthase (HMBS) can cause acute intermittent porphyria (AIP), an acute neurological disease. Although sequencing-based diagnosis can be definitive, ∼⅓ of clinical HMBS variants are missense variants, and most clinically reported HMBS missense variants are designated as "variants of uncertain significance" (VUSs). Using saturation mutagenesis, en masse selection, and sequencing, we applied a multiplexed validated assay to both the erythroid-specific and ubiquitous isoforms of HMBS, obtaining confident functional impact scores for >84% of all possible amino acid substitutions. The resulting variant effect maps generally agreed with biochemical expectations and provide further evidence that HMBS can function as a monomer. Additionally, the maps implicated specific residues as having roles in active site dynamics, which was further supported by molecular dynamics simulations. Most importantly, these maps can help discriminate pathogenic from benign HMBS variants, proactively providing evidence even for yet-to-be-observed clinical missense variants.

Short-Term, Combined Fasting and Exercise Improves Body Composition in Healthy Males.

Fasting enhances the beneficial metabolic outcomes of exercise; however, it is unknown whether body composition is favorably modified on the short term. A baseline-follow-up study was carried out to assess the effect of an established protocol involving short-term combined exercise with fasting on body composition. One hundred seven recreationally exercising males underwent a 10-day intervention across 15 fitness centers in the Netherlands involving a 3-day gradual decrease of food intake, a 3-day period with extremely low caloric intake, and a gradual 4-day increase to initial caloric intake, with daily 30-min submaximal cycling. Using dual-energy X-ray absorptiometry analysis, all subjects substantially lost total body mass (-3.9 ± 1.9 kg; p < .001) and fat mass (-3.3 ± 1.3 kg; p < .001). Average lean mass was lost (-0.6 ± 1.5 kg; p < .001), but lean mass as a percentage of total body mass was not reduced. The authors observed a loss of -3.9 ± 1.9% android fat over total fat mass (p < .001), a loss of -2.2 ± 1.9% gynoid over total fat mass (p < .001), and reduced android/gynoid ratios (-0.05 ± 0.1; p < .001). Analyzing 15 preselected single-nucleotide polymorphisms in 13 metabolism-related genes revealed trending associations for thyroid state-related single-nucleotide polymorphisms rs225014 (deiodinase 2) and rs35767 (insulin-like growth factor1), and rs1053049 (PPARD). In conclusion, a short period of combined fasting and exercise leads to a substantial loss of body and fat mass without a loss of lean mass as a percentage of total mass.

International Porphyria Molecular Diagnostic Collaborative: an evidence-based database of verified pathogenic and benign variants for the porphyrias.

With the advent of precision and genomic medicine, a critical issue is whether a disease gene variant is pathogenic or benign. Such is the case for the three autosomal dominant acute hepatic porphyrias (AHPs), including acute intermittent porphyria, hereditary coproporphyria, and variegate porphyria, each resulting from the half-normal enzymatic activities of hydroxymethylbilane synthase, coproporphyrinogen oxidase, and protoporphyrinogen oxidase, respectively. To date, there is no public database that documents the likely pathogenicity of variants causing the porphyrias, and more specifically, the AHPs with biochemically and clinically verified information. Therefore, an international collaborative with the European Porphyria Network and the National Institutes of Health/National Center for Advancing Translational Sciences/National Institute of Diabetes and Digestive and Kidney Diseases (NIH/NCATS/NIDDK)-sponsored Porphyrias Consortium of porphyria diagnostic experts is establishing an online database that will collate biochemical and clinical evidence verifying the pathogenicity of the published and newly identified variants in the AHP-causing genes. The overall goal of the International Porphyria Molecular Diagnostic Collaborative is to determine the pathogenic and benign variants for all eight porphyrias. Here we describe the overall objectives and the initial efforts to validate pathogenic and benign variants in the respective heme biosynthetic genes causing the AHPs.

Vasodilation through levodopa for Parkinson's disease may require high left ventricular assist device flow.

We report implantation of a left ventricular assist device (LVAD) in a patient with Parkinson's disease. Postoperative fluid overload together with insufficient LVAD output in the setting of vasodilation through levodopa likely caused renal hypoperfusion and acute kidney injury. A patient like ours, therefore, requires the highest possible increase of HM3 RPM and LVAD flow early after surgery.

Effect of sitagliptin on energy metabolism and brown adipose tissue in overweight individuals with prediabetes: a randomised placebo-controlled trial.

AIMS/HYPOTHESIS: The aim of this study was to evaluate the effect of sitagliptin on glucose tolerance, plasma lipids, energy expenditure and metabolism of brown adipose tissue (BAT), white adipose tissue (WAT) and skeletal muscle in overweight individuals with prediabetes (impaired glucose tolerance and/or impaired fasting glucose). METHODS: We performed a randomised, double-blinded, placebo-controlled trial in 30 overweight, Europid men (age 45.9 ± 6.2 years; BMI 28.8 ± 2.3 kg/m2) with prediabetes in the Leiden University Medical Center and the Alrijne Hospital between March 2015 and September 2016. Participants were initially randomly allocated to receive sitagliptin (100 mg/day) (n = 15) or placebo (n = 15) for 12 weeks, using a randomisation list that was set up by an unblinded pharmacist. All people involved in the study as well as participants were blinded to group assignment. Two participants withdrew from the study prior to completion (both in the sitagliptin group) and were subsequently replaced with two new participants that were allocated to the same treatment. Before and after treatment, fasting venous blood samples and skeletal muscle biopsies were obtained, OGTT was performed and body composition, resting energy expenditure and [18F] fluorodeoxyglucose ([18F]FDG) uptake by metabolic tissues were assessed. The primary study endpoint was the effect of sitagliptin on BAT volume and activity. RESULTS: One participant from the sitagliptin group was excluded from analysis, due to a distribution error, leaving 29 participants for further analysis. Sitagliptin, but not placebo, lowered glucose excursion (-40%; p < 0.003) during OGTT, accompanied by an improved insulinogenic index (+38%; p < 0.003) and oral disposition index (+44%; p < 0.003). In addition, sitagliptin lowered serum concentrations of triacylglycerol (-29%) and very large (-46%), large (-35%) and medium-sized (-24%) VLDL particles (all p < 0.05). Body weight, body composition and energy expenditure did not change. In skeletal muscle, sitagliptin increased mRNA expression of PGC1ß (also known as PPARGC1B) (+117%; p < 0.05), a main controller of mitochondrial oxidative energy metabolism. Although the primary endpoint of change in BAT volume and activity was not met, sitagliptin increased [18F] FDG uptake in subcutaneous WAT (sWAT; +53%; p < 0.05). Reported side effects were mild and transient and not necessarily related to the treatment. CONCLUSIONS/INTERPRETATION: Twelve weeks of sitagliptin in overweight, Europid men with prediabetes improves glucose tolerance and lipid metabolism, as related to increased [18F] FDG uptake by sWAT, rather than BAT, and upregulation of the mitochondrial gene PGC1ß in skeletal muscle. Studies on the effect of sitagliptin on preventing or delaying the progression of prediabetes into type 2 diabetes are warranted. TRIAL REGISTRATION: ClinicalTrials.gov NCT02294084. FUNDING: This study was funded by Merck Sharp & Dohme Corp, Dutch Heart Foundation, Dutch Diabetes Research Foundation, Ministry of Economic Affairs and the University of Granada.

Exercise, fasting, and mimetics: toward beneficial combinations?

Obesity and type 2 diabetes are associated disorders that involve a multiplicity of tissues. Both fasting and physical exercise are known to counteract dyslipidemia/hyperglycemia. Skeletal muscle plays a key role in the control of blood glucose levels, and the metabolic changes and related signaling pathways in skeletal muscle induced by fasting overlap with those induced by exercise. The reduction of fat disposal has been shown to extend to the liver and to white and brown adipose tissue and to involve an increase in their metabolic activities. In recent years signal transduction pathways related to exercise and fasting/food withdrawal in muscle have been intensively studied, both in animals and in humans. Combining fasting/food withdrawal with exercise in animals as well as in humans causes changes unlike those seen during fasting/food withdrawal or exercise alone, which favor repair of muscle over autophagy. In addition, compounds that mimic exercise have been studied in combination with exercise or fasting/food withdrawal. This review addresses our current knowledge of the mechanisms that underlie the individual and combined effects of fasting/food withdrawal, endurance or resistance exercise, and their mimetics, in muscle vs other organs in rodents and humans, and highlights which combinations may improve metabolic disorders.-Jaspers, R. T., Zillikens, M. C., Friesema, E. C. H., delli Paoli, G., Bloch, W., Uitterlinden, A. G., Goglia, F., Lanni, A., de Lange, P. Exercise, fasting, and mimetics: toward beneficial combinations.

Medical and financial burden of acute intermittent porphyria.

INTRODUCTION: A small proportion of patients with acute intermittent porphyria (AIP) suffer from recurrent porphyric attacks, with a severely diminished quality of life. In this retrospective case-control study, the burden of disease is quantified and compared among three AIP patient subgroups: cases with recurrent attacks, cases with one or occasional attacks and asymptomatic carriers. METHODS: Data from patient records and questionnaires were collected in patients between 1960 and 2016 at the Erasmus Medical Center, Rotterdam, the Netherlands. We collected symptoms related to porphyria, porphyria related complications, attack frequency, hospitalisation frequency, hospitalisation days related to acute porphyric attacks, frequency of heme infusions and medical healthcare costs based on hospitalisations and heme therapy. RESULTS: In total 11 recurrent AIP cases, 24 symptomatic AIP cases and 53 AIP carriers as controls were included. All recurrent patients reported porphyria related symptoms, such as pain, neurological and/or psychiatric disorders, and nearly all developed complications, such as hypertension and chronic kidney disease. In the recurrent cases group, the median lifelong number of hospitalisation days related to porphyric attacks was 82 days per patient (range 10-374), and they spent a median of 346 days (range 34-945) at a day-care facility for prophylactic heme therapy; total follow-up time was 243 person-years (PYRS). In the symptomatic non-recurrent group the median lifelong number of hospitalisation days related to porphyric attacks was 7 days per patient (range 1-78), total follow-up time was 528 PYRS. The calculated total medical healthcare cost for recurrent cases group was €5.8 million versus €0.3 million for the symptomatic cases group.

Correction to: Medical and financial burden of acute intermittent porphyria.

Due to a typesetting error the wrong figure 2 was used.

Blood pressure-independent renoprotection in diabetic rats treated with AT1 receptor-neprilysin inhibition compared with AT1 receptor blockade alone.

ARNI [dual AT1 (angiotensin II type 1) receptor-neprilysin inhibition] exerts beneficial effects on blood pressure and kidney function in heart failure, compared with ARB (AT1 receptor blockade) alone. We hypothesized that ARNI improves cardiac and kidney parameters in diabetic TGR(mREN2)27 rats, an angiotensin II-dependent hypertension model. Rats were made diabetic with streptozotocin for 5 or 12 weeks. In the final 3 weeks, rats were treated with vehicle, irbesartan (ARB) or irbesartan+thiorphan (ARNI). Blood pressure, measured by telemetry in the 5-week group, was lowered identically by ARB and ARNI. The heart weight/tibia length ratio in 12-week diabetic animals was lower after ARNI compared with after ARB. Proteinuria and albuminuria were observed from 8 weeks of diabetes onwards. ARNI reduced proteinuria more strongly than ARB, and a similar trend was seen for albuminuria. Kidneys of ARNI-treated animals showed less severe segmental glomerulosclerosis than those of ARB-treated animals. After 12 weeks, no differences between ARNI- and ARB-treated animals were found regarding diuresis, natriuresis, plasma endothelin-1, vascular reactivity (acetylcholine response) or kidney sodium transporters. Only ARNI-treated rats displayed endothelin type B receptor-mediated vasodilation. In conclusion, ARNI reduces proteinuria, glomerulosclerosis and heart weight in diabetic TGR(mREN2)27 rats more strongly than does ARB, and this occurs independently of blood pressure.

Optimum AT1 receptor-neprilysin inhibition has superior cardioprotective effects compared with AT1 receptor blockade alone in hypertensive rats.

Neprilysin inhibitors prevent the breakdown of bradykinin and natriuretic peptides, promoting vasodilation and natriuresis. However, they also increase angiotensin II and endothelin-1. Here we studied the effects of a low and a high dose of the neprilysin inhibitor thiorphan on top of AT1 receptor blockade with irbesartan versus vehicle in TGR(mREN2)27 rats with high renin hypertension. Mean arterial blood pressure was unaffected by vehicle or thiorphan alone. Irbesartan lowered blood pressure, but after 7 days pressure started to increase again. Low- but not high-dose thiorphan prevented this rise. Only during exposure to low-dose thiorphan plus irbesartan did heart weight/body weight ratio, cardiac atrial natriuretic peptide expression, and myocyte size decrease significantly. Circulating endothelin-1 was not affected by low-dose thiorphan with or without irbesartan, but increased after treatment with high-dose thiorphan plus irbesartan. This endothelin-1 rise was accompanied by an increase in renal sodium-hydrogen exchanger 3 protein abundance, and an upregulation of constrictor vascular endothelin type B receptors. Consequently, the endothelin type B receptor antagonist BQ788 no longer enhanced endothelin-1-induced vasoconstriction (indicative of endothelin type B receptor-mediated vasodilation), but prevented it. Thus, optimal neprilysin inhibitor dosing reveals additional cardioprotective effects on top of AT1 receptor blockade in renin-dependent hypertension.