Brief Report: Divergent low-density lipoprotein receptor (LDLR) linked to low VSV G-dependent viral infectivity and unique serum lipid profile in zebra finches

The low-density lipoprotein receptor (LDLR) is key to cellular cholesterol uptake and is also the main receptor for the vesicular stomatitis virus glycoprotein (VSV G). Here we show that in songbirds LDLR is highly divergent and lacks domains critical for ligand binding and cellular trafficking, inconsistent with universal structure conservation and function across vertebrates. Linked to the LDLR functional domain loss, zebra finches show inefficient infectivity by lentiviruses (LVs) pseudotyped with VSV G, which can be rescued by the expression of human LDLR. Finches also show an atypical plasma lipid distribution that relies largely on high-density lipoprotein (HDL). These findings provide insights into the genetics and evolution of viral infectivity and cholesterol transport mechanisms in vertebrates.     

Insulin secretion and insulin sensitivity in diabetic and non-diabetic subjects with hepatic nuclear factor-1alpha (maturity-onset diabetes of the young-3) mutations

OBJECTIVE: To evaluate insulin secretion and sensitivity in affected (diabetes mellitus or impaired glucose tolerance; n=7) and in unaffected (normal glucose tolerance; n=3) carriers of hepatocyte nuclear factor-1alpha (maturity-onset diabetes of the young-3 (MODY3)) gene mutations. METHODS: Insulin secretion was assessed by an i.v. glucose tolerance test (IVGTT), hyperglycemic clamp and arginine test, and insulin sensitivity by an euglycemic hyperinsulinemic clamp. Results were compared with those of diabetic MODY2 (glucokinase-deficient) and control subjects. RESULTS: The amount of insulin secreted during an IVGTT was decreased in affected MODY3 subjects (46+/-24 (s.d.) pmol/kg body weight (BW)) as compared with values in MODY2 (120+/-49pmol/kg BW) and control (173+/-37pmol/kg BW; P=0.0004) subjects. The amount of insulin secreted during a 10mmol/l glucose clamp was decreased in affected MODY3 subjects (171+/-78pmol/kg BW) and MODY2 subjects (302+/-104pmol/kg BW) as compared with control subjects (770+/-199pmol/kg BW; P=0.0001). Insulin secretion in response to arginine was decreased in affected MODY3 subjects. Milder and heterogeneous defects were observed in the unaffected MODY3 subjects; the amount of insulin secreted during the hyperglycemic clamp was 40-79% of that of controls. The response to arginine was abnormally delayed. Insulin sensitivity was decreased in diabetic but not in non-diabetic MODY3 subjects. CONCLUSIONS: Beta-cell dysfunction in response to glucose and arginine is observed in affected and unaffected MODY3 subjects. The MODY3 and MODY2 subtypes present different insulin secretion profiles. Secondary insulin resistance might contribute to the chronic hyperglycemia of MODY3 patients and modulate their glucose tolerance.     

13C/31P NMR studies of glucose transport in human skeletal muscle

The muscle intracellular (IC) free glucose concentration and the rate of muscle glycogen synthesis were measured by using in vivo ¹³C and ³¹P NMR spectroscopy in normal volunteers under hyperinsulinemic (≈300 pM) clamp conditions at the following three plasma glucose levels: euglycemia (≈6 mM), mild (≈10 mM), and high (≈16 mM) hyperglycemia. In keeping with biopsy studies, muscle IC free glucose concentration at euglycemia (−0.03 ± 0.03 mmol/kg of muscle, mean ± SEM, n = 10) was not statistically different from zero. A small but statistically significant amount of IC free glucose was observed during mild and high hyperglycemia: 0.15 ± 0.08 (n = 5) and 0.43 ± 0.20 mmol/kg of muscle (n = 5), respectively. Muscle glycogen synthesis rate, in mmol per kg of muscle per min, was 111 ± 11 at euglycemia (n = 10), 263 ± 29 during mild hyperglycemia (n = 5), and 338 ± 42 during high hyperglycemia (n = 5), these three rates being significantly different from each other. As previous in vitro and in vivo studies, these rates suggest a K_m (concentration at which unidirectional glucose transport reaches half-maximal rate) of the muscle glucose transport system in the 15–25 mM range under hyperinsulinemic conditions. The low concentrations of muscle IC free glucose observed under hyperinsulinemic conditions were interpreted, with this estimate and in the framework of metabolic control theory, as glucose transport being the predominant step controlling muscle glucose flux not only at euglycemia but also during hyperglycemia.     

Genetic and clinical characterisation of maturity-onset diabetes of the young in Spanish families

OBJECTIVE: To investigate the frequencies of the major maturity-onset diabetes of the young (MODY) subtypes in a panel of Spanish families and to assess phenotypic differences in patients with the different subtypes of MODY. METHODS: Forty-eight subjects from twenty families with clinical diagnosis of MODY were studied. They underwent a standardised clinical examination and a 75-g oral glucose tolerance test (OGTT) was performed. Estimations of insulin sensitivity (%S) and insulin secretion capacity (%B) were calculated by the computer-solved homeostasis model assessment (HOMA). Mutations in the coding regions of hepatocyte nuclear factor (HNF)-4alpha/MODY1, glucokinase (GCK/MODY2) and HNF-1alpha/MODY3 genes were investigated by single strand comformation polymorphism and sequencing analysis. RESULTS: Mutations in the GCK and HNF-1alpha genes were observed in 5 (25%) and 7 (35%) families respectively. Novel mutations included R385X, M238fsdelT, V226fsdelTinsAA and S418-7del11 in the GCK gene, and S121fsdelC, V133M, R159Q and V259D in the HNF-1alpha gene. No MODY1 families were found. Subjects which were neither MODY2 nor MODY3 (MODY-X) had a higher fasting glucose than subjects in the other groups. Insulin secretion capacity was similar in the three groups and the insulin sensitivity was decreased in MODY-X subjects. Glucose levels were significantly higher and insulin levels significantly lower, throughout the OGTT, in MODY3 compared with MODY2 subjects. CONCLUSIONS: Mutations in the GCK/MODY2 and HNF-1alpha/MODY3 genes account for the majority of cases in a panel of Spanish MODY families, with MODY3 being the most frequent subtype. The relative frequencies and the clinical characteristics of these MODY subtypes are in agreement with data previously reported in other European populations. MODY-X patients seem to present a heterogeneous clinical profile.     

Determination of peritoneal glucose kinetics in rats: implications for the peritoneal implantation of closed-loop insulin delivery systems

Summary Peritoneal glucose kinetics were evaluated in the anaesthetized rat, to assess whether the peritoneal cavity would be a suitable site for the implantation of membrane-protected islets of Langerhans (bioartificial pancreas) or the glucose sensor of an artificial B cell. Glucose was measured in peritoneal fluid samples aspirated by needle puncture. Basal peritoneal and blood glucose concentrations were identical in 16 h fasted (n=4) and non fasted (n=3) animals. After 10 min of an i.v. glucose infusion (n=15) the increment in peritoneal glucose concentration was 63±3% of the increment in blood glucose concentration and both values were significantly correlated (r=0.92; p<0.001). After 10 min of glucose clamping (12.6±0.8 mmol/l), the increment in peritoneal glucose concentration was 69±3% (n=5; p<0.05) of the increment in blood glucose concentration. In three additional experiments it was 93±3% of the increment in blood glucose concentration (NS), after 30 min of glucose clamping (8.0±0.5 mmol/l). Peritoneal glucose concentration monitored by a glucose sensor: (a) followed blood glucose sluggishly during a glucose clamp (n=5), confirming the data shown above, (b) followed blood glucose with a 5 min delay and reached the same plateau after the intravenous injection of 1U insulin (n=3; NS). We conclude that peritoneal glucose reflects blood glucose at basal state and during variations of glycaemia, nevertheless, presenting heterogeneous kinetics. These kinetics might be appropriate for a bioartificial pancreas but not for an in vivo calibration procedure, of a peritoneally implanted glucose sensor.     

Allelic variation in exon 18 of the sulfonylurea receptor 1 (SUR1) gene,insulin secretion and insulin sensitivity in nondiabetic relatives of type 2 diabetic subjects

BACKGROUND: We have previously observed associations of the T-allele of the exon 18 variant (ACC --> ACT; Thr759Thr) of the sulfonylurea receptor 1 (SUR1) gene with type 2 diabetes mellitus (T2DM). Here we assess beta-cell function and insulin sensitivity in carriers of different genotypes at this locus. METHODS: Pre-hepatic insulin secretion rates (ISR) derived by deconvolution of circulating C-peptide levels, and glucose clearance were assessed during graded infusions of intravenous glucose in CC-homozygous (n=6) and CT-heterozygous (n=6) nondiabetic relatives of CT-heterozygous type 2 diabetic subjects. RESULTS: Average ISR over the duration of the study, adjusted for sex, age, BMI and prevailing glucose levels, were lower in CT-heterozygous subjects as compared with CC-homozygous subjects (3.91 +/- 0.40 vs. 4.84 +/- 0.28 pmol/kg x min(-1); p=0.048). The correlation curves relating ISR to glucose levels were significantly different in the two groups (analyses of covariance p=0.029). Glucose clearance was similar in both groups. CONCLUSIONS: Insulin secretion rates, but not insulin sensitivity, assessed during graded infusion of glucose were mildly decreased in nondiabetic relatives of type 2 diabetic subjects, who carry the at risk T-allele of exon 18 variant of the SUR1 gene. These results suggest that the at-risk allele might have a small effect on pancreatic B-cell function and contribute to the development of T2DM in these families.     

Combined in vitro and in silico analyses of missense mutations in GNPTAB provide new insights into the molecular bases of mucolipidosis II and III alpha/beta

Mucolipidosis (ML) II and III alpha/beta are inherited lysosomal storage disorders caused by mutations in GNPTAB encoding the α/β‐precursor of GlcNAc‐1‐phosphotransferase. This enzyme catalyzes the initial step in the modification of more than 70 lysosomal enzymes with mannose 6‐phosphate residues to ensure their intracellular targeting to lysosomes. The so‐called stealth domains in the α‐ and β‐subunit of GlcNAc‐1‐phosphotransferase were thought to be involved in substrate recognition and/or catalysis. Here, we performed in silico alignment analysis of stealth domain‐containing phosphotransferases and showed that the amino acid residues Glu³⁸⁹, Asp⁴⁰⁸, His⁹⁵⁶, and Arg⁹⁸⁶ are highly conserved between different phosphotransferases. Interestingly, mutations in these residues were identified in patients with MLII and MLIII alpha/beta. To further support the in silico findings, we also provide experimental data demonstrating that these four amino acid residues are strictly required for GlcNAc‐1‐phosphotransferase activity and thus may be directly involved in the enzymatic catalysis.     

Clinical Features and Therapeutic Outcomes in Men with Advanced Prostate Cancer and DNA Mismatch Repair Gene Mutations

Mismatch repair (MMR) gene mutations are rare in prostate cancer, and their histological and clinical characteristics are largely unknown. We conducted a retrospective study to explore disease characteristics and treatment outcomes of men with metastatic prostate cancer harboring germline and/or somatic MMR mutations detected using clinical-grade genomic assays. Thirteen patients with a deleterious MMR gene mutation were identified. Median age was 64 yr, 75% had grade group 5 (Gleason sum 9 or 10), 23% had intraductal histology, 46% had metastatic disease at initial diagnosis, and 31% had visceral metastases. Most patients (46%) had MSH6 mutations, 73% demonstrated microsatellite instability, and median tumor mutational load was 18/Mb (range, 3–165 mutations/Mb). Surprisingly, responses to standard hormonal therapies were very durable (median progression-free survival [PFS] of 67 mo to initial androgen deprivation and median PFS of 26 mo to abiraterone/enzalutamide). Two of four men receiving PD-1 inhibitors achieved a ≥50% prostate-specific antigen response at 12 wk, with a median PFS duration in these four men of 9 mo. Despite aggressive clinical and pathological features, patients with MMR-mutated advanced prostate cancer appear to have particular sensitivity to hormonal therapies, as well as anecdotal responses to PD-1 inhibitors. Certain histological features (grade group 5, intraductal carcinoma) should prompt evaluation for MMR deficiency. These data are only hypothesis generating.

An extremely high dose of losartan affords superior renoprotection in the remnant model

BACKGROUND: Rats subjected to 5/6 renal ablation (NX) exhibit large renal amounts of angiotensin II (Ang II) and of its main receptor, AT-1R. At previously used doses, AT-1R blockers (ARB) offer only partial renal protection. A possible explanation for this limited effect is that these doses are insufficient to block most of the abnormally expressed AT-1R. We investigated whether extremely high doses of the ARB, losartan (L), offer better protection than conventional doses in the NX model. METHODS: Thirty days after NX, tail-cuff pressure (TCP), albuminuria (U(alb)V, mg/day), glomerulosclerosis index (GSI), fractional interstitial area (%INT), and macrophage infiltration (MO) were evaluated in a separate group (NX(pre)). The remaining rats were then subdivided among 4 groups: NX+V, receiving vehicle; NX+L50, treated with L, at the "conventional" dose of 50 mg/kg/day; NX+L500, receiving L, 500 mg/kg/day; and NX+HH, receiving hydrochlorothiazide and hydralazine to lower blood pressure to a similar extent as in group L500. RESULTS: After a month of treatment, blood pressure and renal vascular resistance were lowest in group L500. Glomerular pressure was lowered by a similar extent by L50 and L500, while GFR was similar among groups. U(alb)V, TCP, and renal injury were only partially reduced by L50 120 days after renal ablation. By contrast, L500 arrested renal inflammation and glomerular/interstitial injury at pretreatment levels, and promoted regression of hypertension and U(alb)V, causing no apparent untoward effects. CONCLUSION: The renal protection afforded by ARB in NX is dose dependent. Maximal protection may require doses several fold higher than those currently employed.