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Purpose [18F]-2-Fluoro-2-deoxy-D-glucose PET/CT (FDG PET/CT) is a sensitive and quantitative technic for detecting inflammatory process. Glucose uptake is correlated with an increased anaerobic glycolysis seen in activated inflammatory cells such as monocytes, lymphocytes, and granulocytes. The aim of the study was to assess the inflammatory status at the presumed peak of the inflammatory phase in non-critically ill patients requiring admission for COVID-19. Methods Patients admitted with COVID-19 were prospectively enrolled. FDG PET/CT was performed from day 6 to day 14 of the onset of symptoms. Depending on FDG PET/CT findings, patients’ profiles were classified as “inflammatory” or “low inflammatory.” FDG PET/CT data were compared with chest CT evolution and short-term clinical outcome. All inflammatory sites were reported to screen potential extra-pulmonary tropism. Results Thirteen patients were included. Maximum standardized uptake values ranged from 4.7 to 16.3 in lungs. All patients demonstrated increased mediastinal lymph nodes glucose uptake. Three patients (23%) presented mild nasopharyngeal, two patients (15%) bone marrow, and five patients (38%) splenic mild increase in glucose uptake. No patient had significant digestive focal or segmental glucose uptake. There was no significant physiological myocardial glucose uptake in all patients except one. There was no correlation between PET lung inflammatory status and chest CT evolution or short-term clinical outcome. Conclusion Inflammatory process at the presumed peak of the inflammatory phase in COVID-19 patients is obvious in FDG PET/CT scans. Glucose uptake is heterogeneous and typically focused on lungs. Trial registration NCT04441489. Registered 22 June 2020 (retrospectively registered). Electronic supplementary material The online version of this article (10.1007/s00259-020-04968-8) contains supplementary material, which is available to authorized users.

SummaryMultiple islet autoantibodies (AAb) predict type 1 diabetes (T1D) and hyperglycemia within 10 years. By contrast, T1D develops in just ∼15% of single AAb+ (generally against glutamic acid decarboxylase, GADA+) individuals; hence the single GADA+ state may represent an early stage of T1D amenable to interventions. Here, we functionally, histologically, and molecularly phenotype human islets from non-diabetic, GADA+ and T1D donors. Similar to the few remaining beta cells in T1D islets, GADA+ donor islets demonstrated a preserved insulin secretory response. By contrast, alpha cell glucagon secretion was dysregulated in both T1D and GADA+ islets with impaired glucose suppression of glucagon secretion. Single cell RNA sequencing (scRNASeq) of GADA+ alpha cells revealed distinct abnormalities in glycolysis and oxidative phosphorylation pathways and a marked downregulation of PKIB, providing a molecular basis for the loss of glucose suppression and the increased effect of IBMX observed in GADA+ donor islets. The striking observation of a distinct early defect in alpha cell function that precedes beta cell loss in T1D suggests that not only overt disease, but also the progression to T1D itself, is bihormonal in nature.

Over the past few years, an increase use of low-intensity shockwave therapy (LISWT) for the treatment of erectile dysfunction (ED) has been observed. However, many controversies in the scientific literature about its efficacy still exist. We aimed to investigate changes in the pattern of usage and attitudes toward LISWT for ED among experts over the last years. A dedicated online survey was sent to delegates at the Congress for the EAU Section of Andrological Urology (ESAU) in Prague from 31st October to 1st November 2019. The survey captured demographic data, professional background, and the experience and personal knowledge about LISWT. The results were compared with a previous survey conducted in 2016 and investigating the same topics in order to assess changes in the knowledge and opinion on LISWT over time. Overall, 172 and 192 questionnaires were available from 2019 and 2016, respectively. Participants in the 2019 survey were 80 (45.7%) urologists, 52 (29.7%) uro-andrologists, and 34 (19.4%) residents in urology, from 30 different countries. Compared to the past, we observed an increase of respondents who were familiar with LISWT (75 vs. 95%; p < 0.0001) and who performed LISWT in their routine practice (38% in 2019 vs. 14% in 2016; p < 0.0001). Conversely, no changes in terms of overall perception of treatment efficacy were registered over time (68% in 2019 vs. 72% in 2016 considered LISWT as an effective treatment, respectively (p = 0.5)). An increased proportion of physicians had suggested a wider application of LISWT, and some even advocated its use in nonvasculogenic ED (33% in 2019 vs. 5.9% in 2016; p < 0.0001). The awareness and clinical application of LISWT for ED have increased over time, despite the lack of robust evidence in terms of effectiveness. Further research on the benefit of LISWT is needed to guide physicians in the therapeutic management of ED patients.

Given the global epidemic in type 2 diabetes, novel antidiabetic drugs with increased efficacy and reduced side effects are urgently needed. Previous work has shown that M3 muscarinic acetylcholine (ACh) receptors (M3Rs) expressed by pancreatic β cells play key roles in stimulating insulin secretion and maintaining physiological blood glucose levels. In the present study, we tested the hypothesis that a positive allosteric modulator (PAM) of M3R function can improve glucose homeostasis in mice by promoting insulin release. One major advantage of this approach is that allosteric agents respect the ACh-dependent spatiotemporal control of M3R activity. In this study, we first demonstrated that VU0119498, a drug known to act as a PAM at M3Rs, significantly augmented ACh-induced insulin release from cultured β cells and mouse and human pancreatic islets. This stimulatory effect was absent in islets prepared from mice lacking M3Rs, indicative of the involvement of M3Rs. VU0119498 treatment of wild-type mice caused a significant increase in plasma insulin levels, accompanied by a striking improvement in glucose tolerance. These effects were mediated by β-cell M3Rs, since they were absent in mutant mice selectively lacking M3Rs in β cells. Moreover, acute VU0119498 treatment of obese, glucose-intolerant mice triggered enhanced insulin release and restored normal glucose tolerance. Interestingly, doses of VU0119498 that led to pronounced improvements in glucose homeostasis did not cause any significant side effects due to activation of M3Rs expressed by other peripheral cell types. Taken together, the data from this proof-of-concept study strongly suggest that M3R PAMs may become clinically useful as novel antidiabetic agents.

Background: Experimental and clinical data suggest that reducing inflammation without affecting lipid levels may reduce the risk of cardiovascular disease. Yet, the inflammatory hypothesis of atherothrombosis has remained unproved. Methods: We conducted a randomized, double-blind trial of canakinumab, a therapeutic monoclonal antibody targeting interleukin-1β, involving 10,061 patients with previous myocardial infarction and a high-sensitivity C-reactive protein level of 2 mg or more per liter. The trial compared three doses of canakinumab (50 mg, 150 mg, and 300 mg, administered subcutaneously every 3 months) with placebo. The primary efficacy end point was nonfatal myocardial infarction, nonfatal stroke, or cardiovascular death. RESULTS: At 48 months, the median reduction from baseline in the high-sensitivity C-reactive protein level was 26 percentage points greater in the group that received the 50-mg dose of canakinumab, 37 percentage points greater in the 150-mg group, and 41 percentage points greater in the 300-mg group than in the placebo group. Canakinumab did not reduce lipid levels from baseline. At a median follow-up of 3.7 years, the incidence rate for the primary end point was 4.50 events per 100 person-years in the placebo group, 4.11 events per 100 person-years in the 50-mg group, 3.86 events per 100 person-years in the 150-mg group, and 3.90 events per 100 person-years in the 300-mg group. The hazard ratios as compared with placebo were as follows: in the 50-mg group, 0.93 (95% confidence interval [CI], 0.80 to 1.07; P = 0.30); in the 150-mg group, 0.85 (95% CI, 0.74 to 0.98; P = 0.021); and in the 300-mg group, 0.86 (95% CI, 0.75 to 0.99; P = 0.031). The 150-mg dose, but not the other doses, met the prespecified multiplicity-adjusted threshold for statistical significance for the primary end point and the secondary end point that additionally included hospitalization for unstable angina that led to urgent revascularization (hazard ratio vs. placebo, 0.83; 95% CI, 0.73 to 0.95; P = 0.005). Canakinumab was associated with a higher incidence of fatal infection than was placebo. There was no significant difference in all-cause mortality (hazard ratio for all canakinumab doses vs. placebo, 0.94; 95% CI, 0.83 to 1.06; P = 0.31). Conclusions: Antiinflammatory therapy targeting the interleukin-1β innate immunity pathway with canakinumab at a dose of 150 mg every 3 months led to a significantly lower rate of recurrent cardiovascular events than placebo, independent of lipid-level lowering. (Funded by Novartis; CANTOS ClinicalTrials.gov number, NCT01327846.)

Fuel stimulated hormone release (HR) and the oxygen consumption rates (OCR) of isolated normal pancreatic islets are positively correlated illustrating that cellular energy state is a critical determinant of stimulus-secretion coupling. We investigated the relationship between hormone release and OCR in islets from donors with T1DM and T2DM, using unique oxygen measurement technology and optimized perifusion protocols. HR and OCR were quantified in 20 human donor islet preparations obtained from the Human Islet Resource Center at the University of Pennsylvania within 3-4 days after isolation (5 each of nondiabetic, T1DM, T2DM and anti-islet AutoAntiBody positive (AAB+). A physiological amino acid mixture (4mM) was used to stimulate glucagon release, then 3 and 16.7mM glucose were added to stimulate insulin release and inhibit glucagon release. Respiration was then uncoupled with FCCP and finally blocked by NaN3. Basal OCR was increased in islets from AAB+, T1DM and T2DM donors compared to controls. Low glucose failed to increase OCR in T1DM and T2DM islets while high glucose-stimulated OCR was both decreased and delayed. Glucose stimulated insulin release much less effectively and failed to suppress glucagon release in both T1DM and T2DM islets. AAB+ islets showed normal insulin release but lacked glucose suppression of glucagon release. The observation of increased basal-OCR of islets in all diabetes related conditions and reduced glucose-stimulated-OCR in T1DM and T2DM islets unrelated to hormone release rates is striking and indicates a profound alteration of bioenergetics regulation in these conditions hitherto unrecognized. Common to this pathophysiology are an increase in relative alpha-cell mass (T1DM and T2DM) or a functional defect of these cells (AAB+) providing a plausible explanation for this phenomenology and implying impaired alpha-cell energy metabolism. Disclosure N.M. Doliba: None. A.V. Rozo: None. W. Qin: None. J. Roman: None. C. Liu: None. A. Naji: None. K.H. Kaestner: None. D.A. Stoffers: None. F. Matschinsky: None. Funding National Institutes of Health (UC4DK112217)

Google Trends (GT) is a free, easily accessible search tool which can be used to analyze worldwide “big data” on the relative popularity of search terms over a specific period of time. To determine worldwide public interest in Peyronie’ disease (PD), erectile dysfunction (ED), premature ejaculation (PE) treatments, their penetrance, variation, and how they compare over time. Worldwide search-engine trends analysis included electronic Google queries from people who searched PD, ED, and PE treatments options from January 2004 to October 2018, worldwide. Join-point regression (JPR) has been performed. Comparison annual relative search volume (ARSV), average annual percentage change (AAPC) and temporal patterns were analyzed to assess loss or gain of interest. Our results showed that for PD it has been a decreased interest for Drug (AAPC: −3.1%, p

Objectives Chronic hyperlipidemia and hyperglycemia are characteristic features of type 2 diabetes (T2DM) that are thought to cause or contribute to β-cell dysfunction by “glucolipotoxicity.” Previously we have shown that acute treatment of pancreatic islets with fatty acids (FA) decreases acetylcholine-potentiated insulin secretion. This acetylcholine response is mediated by M3 muscarinic receptors, which play a key role in regulating β-cell function. Here we examine whether chronic FA exposure also inhibits acetylcholine-potentiated insulin secretion using mouse and human islets. Methods Islets were cultured for 3 or 4 days at different glucose concentration with 0.5 mM palmitic acid (PA) or a 2:1 mixture of PA and oleic acid (OA) at 1% albumin (PA/BSA molar ratio 3.3). Afterwards, the response to glucose and acetylcholine were studied in perifusion experiments. Results FA-induced impairment of insulin secretion and Ca2+ signaling depended strongly on the glucose concentrations of the culture medium. PA and OA in combination reduced acetylcholine potentiation of insulin secretion more than PA alone, both in mouse and human islets, with no evidence of a protective role of OA. In contrast, lipotoxicity was not observed with islets cultured for 3 days in medium containing less than 1 mM glucose and a mixture of glutamine and leucine (7 mM each). High glucose and FAs reduced endoplasmic reticulum (ER) Ca2+ storage capacity; however, preserving ER Ca2+ by blocking the IP3 receptor with xestospongin C did not protect islets from glucolipotoxic effects on insulin secretion. In contrast, an inhibitor of casein kinase 2 (CK2) protected the glucose dependent acetylcholine potentiation of insulin secretion in mouse and human islets against glucolipotoxicity. Conclusions These results show that chronic FA treatment decreases acetylcholine potentiation of insulin secretion and that this effect is strictly glucose dependent and might involve CK2 phosphorylation of β-cell M3 muscarinic receptors., Highlights • Glucolipotoxicity impairs acetylcholine-potentiation of insulin secretion. • Glucose amplification of insulin secretion rather than triggering is damaged by FA. • Inhibitor of casein kinase 2 preserved islet function against glucolipotoxicity.

Loss-of-function mutations of β-cell KATP channels cause the most severe form of congenital hyperinsulinism (KATPHI). KATPHI is characterized by fasting and protein-induced hypoglycemia that is unresponsive to medical therapy. For a better understanding of the pathophysiology of KATPHI, we examined cytosolic calcium ([Ca2+]i), insulin secretion, oxygen consumption, and [U-13C]glucose metabolism in islets isolated from the pancreases of children with KATPHI who required pancreatectomy. Basal [Ca2+]i and insulin secretion were higher in KATPHI islets compared with controls. Unlike controls, insulin secretion in KATPHI islets increased in response to amino acids but not to glucose. KATPHI islets have an increased basal rate of oxygen consumption and mitochondrial mass. [U-13C]glucose metabolism showed a twofold increase in alanine levels and sixfold increase in 13C enrichment of alanine in KATPHI islets, suggesting increased rates of glycolysis. KATPHI islets also exhibited increased serine/glycine and glutamine biosynthesis. In contrast, KATPHI islets had low γ-aminobutyric acid (GABA) levels and lacked 13C incorporation into GABA in response to glucose stimulation. The expression of key genes involved in these metabolic pathways was significantly different in KATPHI β-cells compared with control, providing a mechanism for the observed changes. These findings demonstrate that the pathophysiology of KATPHI is complex, and they provide a framework for the identification of new potential therapeutic targets for this devastating condition.

Cardiovascular complications are the major cause of mortality and morbidity in diabetic patients. The changes in myocardial structure and function associated with diabetes are collectively called diabetic cardiomyopathy. Numerous molecular mechanisms have been proposed that could contribute to the development of diabetic cardiomyopathy and have been studied in various animal models of type 1 or type 2 diabetes. The current review focuses on the role of sodium (Na+) in diabetic cardiomyopathy and provides unique data on the linkage between Na+ flux and energy metabolism, studied with non-invasive 23Na, and 31P-NMR spectroscopy, polarography, and mass spectroscopy. 23Na NMR studies allow determination of the intracellular and extracellular Na+ pools by splitting the total Na+ peak into two resonances after the addition of a shift reagent to the perfusate. Using this technology, we found that intracellular Na+ is approximately two times higher in diabetic cardiomyocytes than in control possibly due to combined changes in the activity of Na+–K+ pump, Na+/H+ exchanger 1 (NHE1) and Na+-glucose cotransporter. We hypothesized that the increase in Na+ activates the mitochondrial membrane Na+/Ca2+ exchanger, which leads to a loss of intramitochondrial Ca2+, with a subsequent alteration in mitochondrial bioenergetics and function. Using isolated mitochondria, we showed that the addition of Na+ (1–10 mM) led to a dose-dependent decrease in oxidative phosphorylation and that this effect was reversed by providing extramitochondrial Ca2+ or by inhibiting the mitochondrial Na+/Ca2+ exchanger with diltiazem. Similar experiments with 31P-NMR in isolated superfused mitochondria embedded in agarose beads showed that Na+ (3–30 mM) led to significantly decreased ATP levels and that this effect was stronger in diabetic rats. These data suggest that in diabetic cardiomyocytes, increased Na+ leads to abnormalities in oxidative phosphorylation and a subsequent decrease in ATP levels. In support of these data, using 31P-NMR, we showed that the baseline β-ATP and phosphocreatine (PCr) were lower in diabetic cardiomyocytes than in control, suggesting that diabetic cardiomyocytes have depressed bioenergetic function. Thus, both altered intracellular Na+ levels and bioenergetics and their interactions may significantly contribute to the pathology of diabetic cardiomyopathy.

Objectives Hyperglycemia and elevated blood lipids are the presumed precipitating causes of β-cell damage in T2DM as the result of a process termed “glucolipotoxicity”. Here, we tested whether glucolipotoxic pathophysiology is caused by defective bioenergetics using islets in culture. Methods Insulin secretion, respiration, ATP generation, fatty acid (FA) metabolite profiles and gene expression were determined in isolated islets treated under glucolipotoxic culture conditions. Results Over time, chronic exposure of mouse islets to FAs with glucose leads to bioenergetic failure and reduced insulin secretion upon stimulation with glucose or amino acids. Islets exposed to glucolipotoxic conditions displayed biphasic changes of the oxygen consumption rate (OCR): an initial increase in baseline and Vmax of OCR after 3 days, followed by decreased baseline and glucose stimulated OCR after 5 days. These changes were associated with lower islet ATP levels, impaired glucose-induced ATP generation, a trend for reduced mitochondrial DNA content and reduced expression of mitochondrial transcription factor A (Tfam). We discovered the accumulation of carnitine esters of hydroxylated long chain FAs, in particular 3-hydroxytetradecenoyl-carnitine. Conclusions As long chain 3-hydroxylated FA metabolites are known to uncouple heart and brain mitochondria [53], [54], [55], we propose that under glucolipotoxic condition, unsaturated hydroxylated long-chain FAs accumulate, uncouple and ultimately inhibit β-cell respiration. This leads to the slow deterioration of mitochondrial function progressing to bioenergetics β-cell failure., Highlights • We found low capacity of islets to generate ATP after glucolipotoxic treatment. • Found biphasic (up/down) respiratory time course as expression of glucolipotoxicity. • We found β-Hydroxylated long FA metabolites as new glucolipotoxicity mediators. • β-Hydroxylated long FAs are known to uncouple Ox/Phos. • We propose defective bioenergetics as main cause of glucolipotoxicity.

Iron–sulfur (Fe‐S) clusters are essential for mitochondrial metabolism, but their regulation in pulmonary hypertension (PH) remains enigmatic. We demonstrate that alterations of the miR‐210‐ISCU1/2 axis cause Fe‐S deficiencies in vivo and promote PH. In pulmonary vascular cells and particularly endothelium, hypoxic induction of miR‐210 and repression of the miR‐210 targets ISCU1/2 down‐regulated Fe‐S levels. In mouse and human vascular and endothelial tissue affected by PH, miR‐210 was elevated accompanied by decreased ISCU1/2 and Fe‐S integrity. In mice, miR‐210 repressed ISCU1/2 and promoted PH. Mice deficient in miR‐210, via genetic/pharmacologic means or via an endothelial‐specific manner, displayed increased ISCU1/2 and were resistant to Fe‐S‐dependent pathophenotypes and PH. Similar to hypoxia or miR‐210 overexpression, ISCU1/2 knockdown also promoted PH. Finally, cardiopulmonary exercise testing of a woman with homozygous ISCU mutations revealed exercise‐induced pulmonary vascular dysfunction. Thus, driven by acquired (hypoxia) or genetic causes, the miR‐210‐ISCU1/2 regulatory axis is a pathogenic lynchpin causing Fe‐S deficiency and PH. These findings carry broad translational implications for defining the metabolic origins of PH and potentially other metabolic diseases sharing similar underpinnings., National Institutes of Health (U.S.) (U54‐CA151884), National Institutes of Health (U.S.) (R01‐DE016516‐06), National Institutes of Health (U.S.) (EB000244)

Paracrine signaling between pancreatic islet β-cells and α-cells has been proposed to play a role in regulating glucagon responses to elevated glucose and hypoglycemia. To examine this possibility in human islets, we used a metabolomic approach to trace the responses of amino acids and other potential neurotransmitters to stimulation with [U-(13)C]glucose in both normal individuals and type 2 diabetics. Islets from type 2 diabetics uniformly showed decreased glucose stimulation of insulin secretion and respiratory rate but demonstrated two different patterns of glucagon responses to glucose: one group responded normally to suppression of glucagon by glucose, but the second group was non-responsive. The non-responsive group showed evidence of suppressed islet GABA levels and of GABA shunt activity. In further studies with normal human islets, we found that γ-hydroxybutyrate (GHB), a potent inhibitory neurotransmitter, is generated in β-cells by an extension of the GABA shunt during glucose stimulation and interacts with α-cell GHB receptors, thus mediating the suppressive effect of glucose on glucagon release. We also identified glycine, acting via α-cell glycine receptors, as the predominant amino acid stimulator of glucagon release. The results suggest that glycine and GHB provide a counterbalancing receptor-based mechanism for controlling α-cell secretory responses to metabolic fuels.

Type 2 diabetes is characterized by elevated blood glucose levels resulting from a pancreatic β-cell secretory insufficiency combined with insulin resistance, most significantly manifested in skeletal muscle and liver (1). If untreated, diabetic complications develop that cause loss of vision, peripheral neuropathy, impaired kidney function, heart disease, and stroke. The disease has a polygenic basis because numerous genes (the latest count exceeding 20) participate in its pathogenesis, but modern lifestyle characterized by limited physical activity and excessive caloric intake are critical precipitating factors for the current epidemic of type 2 diabetes worldwide (2). It appears that available treatments, including attempts at lifestyle alterations and drug therapies including insulin, are insufficient to stem the tide. Therefore, new approaches, including the development of therapeutic agents with novel mechanisms of action, are needed. Selection of new drug targets to treat type 2 diabetes has to be guided primarily by consideration of established physiological chemistry of glucose homeostasis and of prevailing views about the pathophysiology of type 2 diabetes because the genetics of the disease that could serve as another guiding principle remain prohibitively perplexing. The glucose-phosphorylating enzyme glucokinase (GK) was identified as an outstanding drug target for developing antidiabetic medicines because it has an exceptionally high impact on glucose homeostasis because of its glucose sensor role in pancreatic β-cells and as a rate-controlling enzyme for hepatic glucose clearance and glycogen synthesis, both processes that are impaired in type 2 diabetes (3). Milestones in the 45-year history of GK research are listed in Supplementary Table 1 (Supplementary References S1–S27). In the mid-1990s, Hoffmann La-Roche scientists conducted a high-throughput screen in search of small molecules that could reverse the inhibition of GK by its regulatory protein (GKRP, see further discussion below) and identified a hit molecule that reversed GKRP inhibition by directly stimulating GK (4 …

209 followed by add-on therapy. The efficacy and safety of initial therapy with the fixed-dose combination tablet of sitagliptin and metformin (SITA/MET) compared with metformin alone (MET) was assessed over 44 weeks in patients with type 2 diabetes mellitus and inadequate glycemic control (hemoglobin A1c [HbA1c] ≥7.5%) on diet and exercise. Methods and Materials: The double-blind treatment period included an 18-week, Phase A and a 26-week Phase B. During Phase A, 1250 patients (mean baseline HbA1c 9.9%) were randomized 1:1 to SITA/ MET 50/500 mg BID or MET 500 mg BID, each uptitrated over 4 weeks to SITA/MET 50/1000 mg BID or MET 1000 mg BID, respectively. In Phase A, additional antihyperglycemic agents (AHA) were allowed for patients not meeting progressively stricter glycemic criteria. In Phase B, patients continued their double-blind study medication but, unlike in Phase A, investigators received unmasked HbA1c and FPG results and were to add AHAs as appropriate to achieve glycemic goal per clinical practice. results: In this study, AHAs were initiated by the investigator half as often in the SITA/MET group compared with the MET group (8.8% vs. 16.7% of patients, respectively). Over the 44-week treatment period, HbA1c reductions from baseline were -2.3% (95% CI: -2.4, -2.1) for SITA/MET administered as initial therapy and -1.8% (95% CI: -1.9, -1.6) for MET alone administered as initial therapy. The betweengroup difference of -0.5% favored the SITA/MET group. Fifty percent more patients who initiated treatment with SITA/MET reached the HbA1c goal of

Neural and endocrine factors (i.e., Ach and GLP-1) restore defective glucose-stimulated insulin release in pancreatic islets lacking sulfonylurea type 1 receptors (SUR1−/−) (Doliba NM, Qin W, Vatamaniuk MZ, Li C, Zelent D, Najafi H, Buettger CW, Collins HW, Carr RD, Magnuson MA, and Matschinsky FM. Am J Physiol Endocrinol Metab 286: E834–E843, 2004). The goal of the present study was to assess fuel-induced respiration in SUR1−/− islets and to correlate it with changes in intracellular Ca2+, insulin, and glucagon secretion. By use of a method based on O2 quenching of phosphorescence, the O2 consumption rate (OCR) of isolated islets was measured online in a perifusion system. Basal insulin release (IR) was 7–10 times higher in SUR1−/− compared with control (CON) islets, but the OCR was comparable. The effect of high glucose (16.7 mM) on IR and OCR was markedly reduced in SUR1−/− islets compared with CON. Ach (0.5 μM) in the presence of 16.7 mM glucose caused a large burst of IR in CON and SUR1−/− islets with minor changes in OCR in both groups of islets. In SUR1−/− islets, high glucose failed to inhibit glucagon secretion during stimulation with amino acids or Ach. We conclude that 1) reduced glucose responsiveness of SUR1−/− islets may be in part due to impaired energetics, as evidenced by significant decrease in glucose-stimulated OCR; 2) elevated intracellular Ca2+ levels may contribute to altered insulin and glucagon secretion in SUR1−/− islets; and 3) The amplitudes of the changes in OCR during glucose and Ach stimulation do not correlate with IR in normal and SUR1−/− islets suggesting that the energy requirements for exocytosis are minor compared with other ATP-consuming reactions.

Type 2 diabetic subjects manifest both disordered insulin action and abnormalities in their pancreatic islet cells. Whether the latter represents a primary defect or is a consequence of the former is unknown. To examine the β-cell mass and function of islets from type 2 diabetic patients directly, we isolated islets from pancreata of type 2 diabetic cadaveric donors (n = 14) and compared them with islets from normal donors (n = 14) matched for age, BMI, and cold ischemia time. The total recovered islet mass from type 2 diabetic pancreata was significantly less than that from nondiabetic control subjects (256,260 islet equivalents [2,588 IEq/g pancreas] versus 597,569 islet equivalents [6,037 IEq/g pancreas]). Type 2 diabetic islets were also noted to be smaller on average, and histologically, islets from diabetic patients contained a higher proportion of glucagon-producing α-cells. In vitro study of islet function from diabetic patients revealed an abnormal glucose-stimulated insulin release response in perifusion assays. In addition, in comparison with normal islets, an equivalent number of type 2 diabetic islets failed to reverse hyperglycemia when transplanted to immunodeficient diabetic mice. These results provide direct evidence for abnormalities in the islets of type 2 diabetic patients that may contribute to the pathogenesis of the disease.

Recent improvements in isolated islet transplantation indicate that this therapy may ultimately prove applicable to patients with type I diabetes. An obstacle preventing widespread application of islet transplantation is an insufficient supply of cadaveric pancreata. Non-heart-beating donors (NHBDs) are generally not deemed suitable for whole-organ pancreas donation and could provide a significant source of pancreata for islet transplantation. Isolated pancreatic islets prepared from 10 NHBDs were compared with those procured from 10 brain-dead donors (BDDs). The success of the isolation for the two groups was analyzed for preparation purity, quality, and recovered islet mass. The function of NHBD and BDD islets was evaluated using in vitro and in vivo assays. On the basis of the results of this analysis, an NHBD isolated islet allograft was performed in a type I diabetic. The recovery of islets from NHBDs was comparable to that of control BDDs. In vitro assessment of NHBD islet function revealed function-equivalent BDD islets, and NHBD islets transplanted to non-obese diabetic-severe combined immunodeficient (NOD-SCID) mice efficiently reversed diabetes. Transplantation of 446,320 islet equivalents (IEq) (8,500 IEq/kg of recipient body weight) from a single NHBD successfully reversed the diabetes of a type I diabetic recipient. Normally functioning pancreatic islets can be isolated successfully from NHBDs. A single donor transplant from an NHBD resulted in a state of stable insulin independence in a type I diabetic recipient. These results indicate that NHBDs may provide an as yet untapped source of pancreatic tissue for preparation of isolated islets for clinical transplantation.

Recent success in islet transplantation renews the hope for the complete cure of patients afflicted with Type 1 diabetes. However, in the Edmonton series, two to four pancreas donors were required to obtain a sufficient islet mass to reverse the diabetes of each patient. In view of the donor shortage, this represents a major obstacle preventing greater application of islet transplantation to diabetic patients. We hypothesised that increasing the expression of the insulin gene in transplanted islets would augment their capacity for insulin production, thereby allowing reversal of diabetes with a reduced islet mass.We used a replication defective adenovirus to deliver the human proinsulin gene (Ad-Ins) to isolated human islets. The function of Ad-Ins-transduced human islets was compared to islets transduced with a control vector (Ad-lacz).Ad-Ins-transduced islets produced two to three times more insulin than normal islets or those infected with Ad-lacz, as assessed by in vitro perifusion tests of glucose stimulated insulin release. When transplanted, Ad-Ins-transduced islets normalised the blood glucose of diabetic immunodeficient NOD-Scid mice, and less than half as many Ad-Ins islets were required for reversal of diabetes than when normal islets were transplanted.Our results suggest a simple and effective approach that could enhance the efficiency of islet transplantation for treatment of diabetes in humans.

In the present study, noninvasive 31 P and 23 Na + –nuclear magnetic resonance (NMR) technology and respirometry were used to compare the effect of high glucose (30 mmol/l) with the effect of the antidiabetic sulfonylurea (SU) compound glyburide (GLY) on energy metabolism, Na + flux, insulin, and cAMP release of continuously superfused β-HC9 cells encapsulated in microscopic agarose beads. Both high glucose and GLY increased oxygen consumption in β-HC9 cells (15–30%) with a maximal effect at 8 mmol/l for glucose and at 250 nmol/l for GLY. At the same time, insulin release from β-cells increased by 15- and 25-fold with high glucose or GLY, respectively. The P-creatine (PCr) level was greatly increased and inorganic phosphate (P i ) was decreased with 30 mmol/l glucose in contrast to the decreased level of PCr and increased P i with GLY. ATP levels remained unchanged during both interventions. Studies on isolated mitochondria of β-HC9 cells showed that GLY added to mitochondria oxidizing glutamine or glutamate abolished the stimulation of respiration by ADP (state 3) meanwhile leaving state 3 respiration unchanged during oxidation of other substrates. Exposure of β-HC9 cells to 5 mmol/l glucose decreased intracellular Na + levels monitored by 23 Na + -NMR spectroscopy and 30 mmol/l glucose resulted in a further decrease in cytosolic Na + . In contrast, Na + increased when 1 μmol/l GLY was added to the perfusate containing 5 mmol/l glucose. These data support the hypothesis that glucose activates the β-cell through a “push mechanism” due to substrate pressure enhancing fuel flux, energy production, and extrusion of Na + from the cells in contrast to SU receptor (SUR)-1 inhibitors, which may modify intermediary and energy metabolism secondarily through a “pull mechanism” due to higher energy demand resulting from increased ion fluxes and the exocytotic work load.

Background. Ischemic preconditioning (IPC) protects the myocardium from ischemia reperfusion injury. The effect of IPC on the mitochondria is not well known. However, one of the mechanisms postulated in IPC (the opening of the mitochondrial K(ATP) channels) is likely to result in changes in mitochondrial function. Therefore, the purpose of this study was to determine the effect of IPC on mitochondrial function during ischemia reperfusion. Methods. Isolated rat hearts (n = 6/group) were subjected to (1) 30 minutes of equilibration, 25 minutes of ischemia, and 30 minutes of reperfusion (RP) (control group) or (2) 10 minutes of equilibration, two-5 minute episodes of IPC (each followed by 5 minutes of re-equilibration), 25 minutes of ischemia, and 30 minutes of RP (IPC group). Left ventricular rate pressure product (RPP) was measured. At end-equilibration (end-EQ) and at end-reperfusion (end-RP) mitochondria were isolated. Mitochondrial respiratory function (state 2, 3, and 4), respiratory control index (RCI), rate of oxidative phosphorylation (ADP/Delta t), and ADP:O ratio were measured by polarography with the use of NADH- or FADH-dependent substrates. Results. IPC improved recovery of RPP at end-RP (72% +/- 5% in IPC vs 30% +/- 4% in control, P.05). Ischemia reperfusion (IR) decreased state 3, ADP/Delta t, and RCI in both groups compared with end-EQ. IPC improved state 3 (47 +/- 3 in IPC vs 37 +/- 2 ng-atoms O/min/mg protein in control), ADP/Delta t (17 +/- 1 in IPC vs 13 +/- 1 nmol/s/mg protein in control), and RCI (3.7 +/- 0.1 in IPC vs 2.1 +/- 0.2 in control) at end-RP compared with control with the use of NADH-dependent substrate (P.05 vs control). IPC also improved state 3 (85 +/- 6 in IPC vs 71 +/- 4 ng-atoms O/min/mg protein in control), ADP/Delta t (18 +/- 2 in IPC vs 12 +/- 1 nmol/s/mg protein in control), RCI (2 +/- 0.1 in IPC vs 1.5 +/- 0.1 in control), and ADP:O ratios (1.4 +/- 0.04 in IPC vs 1.7 +/- 0.09 in control) at end-RP compared with control with the use of FADH-dependent substrate (P.05 vs control). Conclusions. The cardioprotective effects of IPC can be attributed at least in part to the preservation of mitochondrial function during reperfusion.

Coenzyme Q10 (CoQ10) protects myocardium from ischemia-reperfusion (IR) injury as evidenced by improved recovery of mechanical function, ATP, and phosphocreatine during reperfusion. This protection may result from CoQ10's bioenergetic effects on the mitochondria, from its antioxidant properties, or both. The purpose of this study was to elucidate the effects of CoQ10 supplementation on mitochondrial function during myocardial ischemia-reperfusion using an isolated mitochondrial preparation.Isolated hearts (n = 6/group) from rats pretreated with liposomal CoQ10 (10 mg/kg iv, CoQ10), vehicle (liposomal only, Vehicle), or saline (Saline) 30 min before the experiments were subjected to 15 min of equilibration (EQ), 25 min of ischemia (I), and 40 min of reperfusion (RP). Left ventricular-developed pressure (DP) was measured. Mitochondria were isolated at end-equilibration (end-EQ), at end-ischemia (end-I), and at end-reperfusion (end-RP). Mitochondrial respiratory function (State 2, 3, and 4, respiratory control index (RCI, ratio of State 3 to 4), and ADP:O ratio) was measured by polarography using NADH (alpha-ketoglutarate, alpha-KG)- or FADH (succinate, SA)-dependent substrates.CoQ10 improved recovery of DP at end-RP (67 +/- 11% in CoQ10 vs 47 +/- 5% in Vehicle and 50 +/- 11% in Saline, P0.05 vs Vehicle and Saline). CoQ10 did not change preischemic mitochondrial function. IR decreased State 3 and RCI in all groups using either substrate. CoQ10 had no effect in the mitochondrial oxidation of alpha-KG at end-I. CoQ10 improved State 3 at end-I when SA was used (167 +/- 21 in CoQ10 vs 120 +/- 10 in Saline and 111 +/- 10 ng-atoms O/min/mg protein in Vehicle, P0.05). Using alpha-KG as a substrate, CoQ10 improved RCI at end-RP (4.2 +/- 0.2 in CoQ10 vs 3.2 +/- 0.2 in Saline and 3.0 +/- 0.3 in Vehicle, P0.05). Using SA, CoQ10 improved State 3 (181 +/- 10 in CoQ10 vs 142 +/- 9 in Saline and 140 +/- 12 ng-atoms O/min/mg protein in Vehicle, P0.05) and RCI (2.21 +/- 0.06 in CoQ10 vs 1.85 +/- 0.11 in Saline and 1.72 +/- 0.08 in Vehicle, P0.05) at end-RP.The cardioprotective effects of CoQ10 can be attributed to the preservation of mitochondrial function during reperfusion as evidenced by improved FADH-dependent oxidation.

Loss-of-function mutations affecting the cholesterol transporter ATP-binding cassette transporter subfamily A member 1 (ABCA1) impair cellular cholesterol efflux and are associated with reduced HDL-cholesterol (HDL-C) levels. ABCA1 may also be important in regulating β-cell cholesterol homeostasis and insulin secretion. We sought to determine whether loss-of-function ABCA1 mutations affect β-cell secretory capacity in humans by performing glucose-potentiated arginine tests in three subjects homozygous for ABCA1 mutations (age 25 ± 11 years), eight heterozygous subjects (28 ± 7 years), and eight normal control subjects pair-matched to the heterozygous carriers. To account for any effect of low HDL-C on insulin secretion, we studied nine subjects with isolated low HDL-C with no ABCA1 mutations (age 26 ± 6 years) and nine pair-matched control subjects. Homozygotes for ABCA1 mutations exhibited enhanced oral glucose tolerance and dramatically increased β-cell secretory capacity that was also greater in ABCA1 heterozygous subjects than in control subjects, with no differences in insulin sensitivity. Isolated low HDL-C subjects also demonstrated an increase in β-cell secretory capacity but in contrast to those with ABCA1 mutations, exhibited impaired insulin sensitivity, supporting β-cell compensation for increased insulin demand. These data indicate that loss-of-function mutations in ABCA1 in young adults may be associated with enhanced β-cell secretory capacity and normal insulin sensitivity and support the importance of cellular cholesterol homeostasis in regulating β-cell insulin secretion.

Glucokinase activators (GKAs) are being developed and clinically tested for potential antidiabetic therapy. The potential benefits and limitations of this approach continue to be intensively debated. To contribute to the understanding of experimental pharmacology and therapeutics of GKAs, we have tested the efficacy of one of these agents (Piragliatin) in isolated islets from humans with type 2 diabetes mellitus (T2DM), from mice with glucokinase (GK) mutations induced by ethyl-nitroso-urea (ENU) as models of Maturity Onset Diabetes of the Young linked to GK and Permanent Neonatal Diabetes Mellitus linked to GK (PNDM-GK) and finally of islets rendered glucose insensitive by treatment with the sulphonyl urea compound glyburide in organ culture. We found that the GKA repaired the defect in all three instances as manifest in increased glucose-induced insulin release and elevated intracellular calcium responses. The results show the remarkable fact that acute pharmacological activation of GK reverses secretion defects of β-cells caused by molecular mechanism that differ vastly in nature, including the little understood multifactorial lesion of β-cells in T2DM of man, the complex GK mutations in mice resembling GK disease and acute sulphonylurea failure of mouse β-cells in tissue culture. The implications of these results are to be discussed on the theoretical basis underpinning the strategy of developing these drugs and in light of recent results of clinical trials with GKAs that failed for little understood reasons.

Glucokinase (GK ; EC 2.7.1.1.) phosphorylates and regulates glucose metabolism in insulin-producing pancreatic beta-cells, hepatocytes, and certain cells of the endocrine and nervous systems allowing it to play a central role in glucose homeostasis . Most importantly, it serves as glucose sensor in pancreatic beta-cells mediating glucose-stimulated insulin biosynthesis and release and it governs the capacity of the liver to convert glucose to glycogen. Activating and inactivating mutations of the glucokinase gene cause autosomal dominant hyperinsulinemic hypoglycemia and hypoinsulinemic hyperglycemia in humans, respectively, illustrating the preeminent role of glucokinase in the regulation of blood glucose and also identifying the enzyme as a potential target for developing antidiabetic drugs . Small molecules called glucokinase activators (GKAs) which bind to an allosteric activator site of the enzyme have indeed been discovered and hold great promise as new antidiabetic agents. GKAs increase the enzyme’s affinity for glucose and also its maximal catalytic rate. Consequently, they stimulate insulin biosynthesis and secretion, enhance hepatic glucose uptake, and augment glucose metabolism and related processes in other glucokinase-expressing cells. Manifestations of these effects, most prominently a lowering of blood glucose, are observed in normal laboratory animals and man but also in animal models of diabetes and patients with type 2 diabetes mellitus (T2DM ) . These compelling concepts and results sustain a strong R&D effort by many pharmaceutical companies to generate GKAs with characteristics allowing for a novel drug treatment of T2DM.

Fatty acids, acetylcholine, and GLP-1 enhance insulin secretion in a glucose-dependent manner. However, the interplay between glucose, fatty acids, and the neuroendocrine regulators of insulin secretion is not well understood. Therefore, we studied the acute effects of PA (alone or in combination with glucose, acetylcholine, or GLP-1) on isolated cultured mouse islets. Two different sets of experiments were designed. In one, a fixed concentration of 0.5 mM of PA bound to 0.15 mM BSA was used; in the other, a PA ramp from 0 to 0.5 mM was applied at a fixed albumin concentration of 0.15 mM so that the molar PA/BSA ratio changed within the physiological range. At a fixed concentration of 0.5 mM, PA markedly inhibited acetylcholine-stimulated insulin release, the rise of intracellular Ca2+, and enhancement of cAMP production but did not influence the effects of GLP-1 on these parameters of islet cell function. 2-ADB, an IP3receptor inhibitor, reduced the effect of acetylcholine on insulin secretion and reversed the effect of PA on acetylcholine-stimulated insulin release. Islet perfusion for 35–40 min with 0.5 mM PA significantly reduced the calcium storage capacity of ER measured by the thapsigargin-induced Ca2+release. Oxygen consumption due to low but not high glucose was reduced by PA. When a PA ramp from 0 to 0.5 mM was applied in the presence of 8 mM glucose, PA at concentrations as low as 50 μM significantly augmented glucose-stimulated insulin release and markedly reduced acetylcholine's effects on hormone secretion. We thus demonstrate that PA acutely reduces the total oxygen consumption response to glucose, glucose-dependent acetylcholine stimulation of insulin release, Ca2+, and cAMP metabolism, whereas GLP-1's actions on these parameters remain unaffected or potentiated. We speculate that acute emptying of the ER calcium by PA results in decreased glucose stimulation of respiration and acetylcholine potentiation of insulin secretion.

Foxa1 and Foxa2 play both redundant and distinct roles in early pancreas development. We demonstrate here that inducible ablation of both transcription factors in mature mouse beta-cells leads to impaired glucose homeostasis and insulin secretion. The defects in both glucose-stimulated insulin secretion and intracellular calcium oscillation are more pronounced than those in beta-cells lacking only Foxa2. Unexpectedly, in contrast to the severe reduction of beta-cell-enriched factors contributing to metabolic and secretory pathways, expression of a large number of genes that are involved in neural differentiation and function is significantly elevated. We further demonstrate that expression of carbohydrate response element-binding protein (ChREBP or Mlxipl), an important transcriptional regulator of carbohydrate metabolism, is significantly affected in compound Foxa1/a2 mutant beta-cells. ChREBP expression is directly controlled by Foxa1 and Foxa2 in both the fetal endocrine pancreas as well as mature islets. These data demonstrate that Foxa1 and Foxa2 play crucial roles in the development and maintenance of beta-cell-specific secretory and metabolic pathways.

Metabolic regulatory mechanisms are designed to maintain stable myocardial function during extremes in physiological insult; they can now be studied in vivo and may provide insight into mechanisms of altered myocardial functional decompensation during disease processes. To determine mechanisms of myocardial stability during hypoxia and acute pressure loading, creatine kinase (CK) kinetics (forward rate constant, Kf, and flux of phosphocreatine, PCr, to adenosine triphosphate, ATP), and nicotinamide adenine dinucleotide (NADH) redox state were determined with 31P nuclear magnetic resonance (NMR) and NADH fluorometry, respectively, and correlated with heart work (heart rate x systolic blood pressure, HR x SBP), cardiac output (CO) and O2 consumption (MVO2) in 15 anesthetized open chest dogs. Hypoxia (PaO2 of 30-35 mm Hg) was produced in 6 dogs with an inspired O2/N2 of 200/3,000. Cardiac loading was produced in 9 dogs by administration of norepinephrine (NE, 1 micrograms/kg/min). Each dog acted as its own control. Baseline NADH fluorometry, 31P-NMR saturation transfer and cardiac function measurements were performed simultaneously in each dog, after which the experimental interventions were made. Similar increases in HR x SBP, CO, and MVO2 which occurred during both interventions were associated with different bioenergetic responses. During NE infusion, the Kf of CK increased from control; during hypoxia, the Kf decreased from control (p less than 0.05). Flux of PCr----ATP was significantly lower during hypoxia than during NE infusion (p less than 0.05). PCr was decreased significantly during NE infusion (p less than 0.05). In addition, NADH redox state increased (from baseline of 100%) during hypoxia (140 +/- 10%) and decreased during NE infusion (78 +/- 6%).(ABSTRACT TRUNCATED AT 250 WORDS)

In insulin-resistant status such as obesity, failure of pancreatic islets to increase insulin secretion leads to diabetes. We sought to screen for the islet genes that facilitate islet adaptation to obesity by comparing gene expression profiles between two strains of obesity-prone inbred mice with different propensities for hyperglycemia. C57BL/6J and AKR/J were fed regular rodent chow or high-fat diet, after which islet morphology, secretory function, and gene expression were assessed. AKR/J had lower blood glucose and higher insulin levels compared with C57BL/6J mice on regular rodent chow or high-fat diet. Insulin secretion was 3.2-fold higher in AKR/J than C57BL/6J mice following intraperitoneal glucose injection. Likewise, glucose-stimulated insulin secretion from isolated islets was higher in AKR/J. Additionally, islet mass was 1.4-fold greater in AKR/J compared with C57BL/6J. To elucidate the factors associated with the differences in islet function, we analyzed the gene expression profiles in islets in AKR/J and C57BL/6J mice. Of 14,000 genes examined, 202 were upregulated and 270 were downregulated in islets from diet-induced obese AKR/J mice compared with C57BL/6J mice. Key genes involved in islet signaling and metabolism, e.g., glucagon-like peptide-1 receptor, sterol Co-A desaturase 1 and 2, and fatty acid desaturase 2 were upregulated in obese AKR/J mice. The expression of multiple extracellular matrix proteins was also increased in AKR/J mice, suggesting a role in modulation of islet mass. Functional analyses of differentially regulated genes hold promise for elucidating factors linking obesity to alterations in islet function.

Neuropeptide Y (NPY), whose role in appetite regulation is well known, is also expressed in pancreatic islets. Although previous studies indicated that application of NPY to pancreatic islets inhibits insulin secretion, its physiological role in the regulation of insulin secretion is not fully understood. We hypothesized that NPY in islets tonically suppresses insulin secretion and the reduction of islet NPY increases insulin secretion. To address the hypothesis, islet function of NPYdeficient mice was analyzed. Although there was little change in glucose homeostasis in vivo, pancreatic islets from NPYdeficient mice had higher basal insulin secretion (1.5 times), glucose-stimulatedinsulinsecretion(1.5times),andisletmass (1.7times),comparedwithwild-typemouse.Nextwesoughtto determine whether the expression of NPY and Y1 receptor in islets was altered in hyperinsulinemia associated with obesity. Islets from C57BL/6J mice on a high-fat diet had 1.9 times higher basal insulin secretion and 2.4 times higher glucosestimulated insulin secretion than control mice, indicating islet adaptation to obesity. Expression of NPY and Y1 receptor mRNA levels was decreased by 70 and 64%, respectively, in high-fat diet islets, compared with controls. NPY and Y1 receptor in islets were also reduced by 91 and 80%, respectively, in leptin-deficient ob/ob mice that showed marked hyperinsulinemia. Together these results suggest that endogenous NPY tonically inhibits insulin secretion from islets and a reduction of islet NPY may serve as one of the mechanisms to increase insulin secretion when islets compensate for insulin resistance associated with obesity. (Endocrinology 148: 5716–5723, 2007)

Fuel stimulation of insulin secretion from pancreatic β-cells is thought to be mediated by metabolic coupling factors that are generated by energized mitochondria, including protons, adenine nucleotides, and perhaps certain amino acids (AA), as for instance aspartate, glutamate, or glutamine (Q). The goal of the present study was to evaluate the role of such factors when insulin release (IR) is stimulated by glucose or AA, alone or combined, using31P,23Na and1H NMR technology, respirometry, and biochemical analysis to study the metabolic events that occur in continuously superfused mouse β-HC9 cells contained in agarose beads and enhanced by the phosphodiesterase inhibitor IBMX. Exposing β-HC9 cells to high glucose or 3.5 mM of a physiological mixture of 18 AA (AAM) plus 2 mM glutamine caused a marked stimulation of insulin secretion associated with increased oxygen consumption, cAMP release, and phosphorylation potential as evidenced by higher phosphocreatine and lower Pipeak areas of31P NMR spectra. Diazoxide blocked stimulation of IR completely, suggesting involvement of ATP-dependent potassium (KATP) channels in this process. However, levels of MgATP and MgADP concentrations, which regulate channel activity, changed only slowly and little, whereas the rate of insulin release increased fast and very markedly. The involvement of other candidate coupling factors was therefore considered. High glucose or AAM + Q increased pHi. The availability of temporal pH profiles allowed the precise computation of the phosphate potential (ATP/Pi× ADP) in fuel-stimulated IR. Intracellular Na+levels were greatly elevated by AAM + Q. However, glutamine alone or together with 2-amino-2-norbornanecarboxylic acid (which activates glutamate dehydrogenase) decreased β-cell Na levels. Stimulation of β-cells by glucose in the presence of AAM + Q (0.5 mM) was associated with rising cellular concentrations of glutamate and glutamine and strikingly lower aspartate levels. Methionine sulfoximine, an inhibitor of glutamine synthetase, blocked the glucose enhancement of AMM + Q-induced IR and associated changes in glutamine and aspartate but did not prevent the accumulation of glutamate. The results of this study demonstrate again that an increased phosphate potential and a functional KATPchannel are essential for metabolic coupling during fuel-stimulated insulin release but illustrate that determining the identity and relative importance of all participating coupling factors and second messengers remains a challenge largely unmet.

Recent advances in functional genomics afford the opportunity to interrogate the expression profiles of thousands of genes simultaneously and examine the function of these genes in a high-throughput manner. In this study, we describe a rational and efficient approach to identifying novel regulators of insulin secretion by the pancreatic beta-cell. Computational analysis of expression profiles of several mouse and cellular models of impaired insulin secretion identified 373 candidate genes involved in regulation of insulin secretion. Using RNA interference, we assessed the requirements of 10 of these candidates and identified four genes (40%) as being essential for normal insulin secretion. Among the genes identified was Hadhsc, which encodes short-chain 3-hydroxyacyl-coenzyme A dehydrogenase (SCHAD), an enzyme of mitochondrial beta-oxidation of fatty acids whose mutation results in congenital hyperinsulinism. RNA interference-mediated gene suppression of Hadhsc in insulinoma cells and primary rodent islets revealed enhanced basal but normal glucose-stimulated insulin secretion. This increase in basal insulin secretion was not attenuated by the opening of the KATP channel with diazoxide, suggesting that SCHAD regulates insulin secretion through a KATP channel-independent mechanism. Our results suggest a molecular explanation for the hyperinsulinemia hypoglycemic seen in patients with SCHAD deficiency.

Foxa1 (formerly hepatic nuclear factor 3α) belongs to the family of Foxa genes that are expressed in early development and takes part in the differentiation of endoderm-derived organs and the regulation of glucose homeostasis. Foxa1−/− pups are growth retarded and hypoglycemic but glucose intolerant in response to an intraperitoneal glucose challenge. However, the mechanism of glucose intolerance in this model has not been investigated. Here, we show that Foxa1−/− islets exhibit decreased glucose-stimulated insulin release in islet perifusion experiments and have significantly reduced pancreatic insulin and glucagon content. Moreover, Foxa1−/− β-cells exhibit attenuated calcium influx in response to glucose and glyburide, suggesting an insulin secretion defect either at the level or upstream of the ATP-sensitive K+ channel. Intracellular ATP levels after incubation with 10 mmol/l glucose were about 2.5 times lower in Foxa1−/− islets compared with controls. This diminished ATP synthesis could be explained by increased expression of the mitochondrial uncoupling protein uncoupling protein 2 (UCP2) in Foxa1-deficient islets, resulting in partially uncoupled mitochondria. Chromatin immunoprecipitation assays indicate that UCP2 is a direct transcriptional target of Foxa1 in vivo. Thus, we have identified a novel function for Foxa1 in the regulation of oxidative phosphorylation in pancreatic β-cells.

The enzyme GK (glucokinase), which phosphorylates glucose to form glucose 6-phosphate, serves as the glucose sensor of insulin-producing β-cells. GK has thermodynamic, kinetic, regulatory and molecular genetic characteristics that are ideal for its glucose sensor function and allow it to control glycolytic flux of the β-cells as indicated by control-, elasticity- and response-coefficients close to or larger than 1.0. GK operates in tandem with the K+ and Ca2+ channels of the β-cell membrane, resulting in a threshold for glucose-stimulated insulin release of approx. 5 mM, which is the set point of glucose homoeostasis for most laboratory animals and humans. Point mutations of GK cause ‘glucokinase disease’ in humans, which includes hypo- and hyper-glycaemia syndromes resulting from activating or inactivating mutations respectively. GK is allosterically activated by pharmacological agents (called GK activators), which lower blood glucose in normal animals and animal models of T2DM. On the basis of crystallographic studies that identified a ligand-free ‘super-open’ and a liganded closed structure of GK [Grimsby, Sarabu, Corbett and others (2003) Science 301 , 370–373; Kamata, Mitsuya, Nishimura, Eiki and Nagata (2004) Structure 12 , 429–438], on thermostability studies using glucose or mannoheptulose as ligands and studies showing that mannoheptulose alone or combined with GK activators induces expression of GK in pancreatic islets and partially preserves insulin secretory competency, a new hypothesis was developed that GK may function as a metabolic switch per se without involvement of enhanced glucose metabolism. Current research has the goal to find molecular targets of this putative ‘GK-switch’. The case of GK research illustrates how basic science may culminate in therapeutic advances of human medicine. Abbreviations: G, glucose; GK, glucokinase; GKA, GK activators; GKRP, GK regulatory protein; GK-AI, GK activity index; GSIR, glucose-stimulated insulin release; MH, mannoheptulose; MODY-2, diabetes mellitus in young people; PNDM, permanent neonatal diabetes mellitus; PHHI, persistent hyperinsulinaemic hypoglycaemia in infancy

Inhibition of ATP-sensitive K+(KATP) channels by an increase in the ATP/ADP ratio and the resultant membrane depolarization are considered essential in the process leading to insulin release (IR) from pancreatic β-cells stimulated by glucose. It is therefore surprising that mice lacking the sulfonylurea type 1 receptor (SUR1−/−) in β-cells remain euglycemic even though the knockout is expected to cause hypoglycemia. To complicate matters, isolated islets of SUR1−/−mice secrete little insulin in response to high glucose, which extrapolates to hyperglycemia in the intact animal. It remains thus unexplained how euglycemia is maintained. In recognition of the essential role of neural and endocrine regulation of IR, we evaluated the effects of acetylcholine (ACh) and glucagon-like peptide-1 (GLP-1) on IR and free intracellular Ca2+concentration ([Ca2+]i) of freshly isolated or cultured islets of SUR1−/−mice and B6D2F1 controls (SUR1+/+). IBMX, a phosphodiesterase inhibitor, was also used to explore cAMP-dependent signaling in IR. Most striking, and in contrast to controls, SUR1−/−islets are hypersensitive to ACh and IBMX, as demonstrated by a marked increase of IR even in the absence of glucose. The hypersensitivity to ACh was reproduced in control islets by depolarization with the SUR1 inhibitor glyburide. Pretreatment of perifused SUR1−/−islets with ACh or IBMX restored glucose stimulation of IR, an effect expectedly insensitive to diazoxide. The calcium channel blocker verapamil reduced but did not abolish ACh-stimulated IR, supporting a role for intracellular Ca2+stores in stimulus-secretion coupling. The effect of ACh on IR was greatly potentiated by GLP-1 (10 nM). ACh caused a dose-dependent increase in [Ca2+]iat 0.1–1 μM or biphasic changes (an initial sharp increase in [Ca2+]ifollowed by a sustained phase of low [Ca2+]i) at 1–100 μM. The latter effects were observed in substrate-free medium or in the presence of 16.7 mM glucose. We conclude that SUR1 deletion depolarizes the β-cells and markedly elevates basal [Ca2+]i. Elevated [Ca2+]iin turn sensitizes the β-cells to the secretory effects of ACh and IBMX. Priming by the combination of high [Ca2+]i, ACh, and GLP-1 restores the defective glucose responsiveness, precluding the development of diabetes but not effectively enough to cause hyperinsulinemic hypoglycemia.

ObjectiveTo restore islet function in patients whose labile diabetes subjected them to frequent dangerous episodes of hypoglycemic unawareness, and to determine whether multiple transplants are always required to achieve insulin independence.Summary Background DataThe recent report by the Edmonton g

Although the role of acetylcholine (Ach) in hepatic glucose metabolism is well elucidated, it is still unclear if it influences gluconeogenesis, glycogenolysis and high-energy phosphate metabolism, and if it does what the mechanisms of this influence are. Therefore, using isolated perfused rat liver as a model, we have studied the effect of Ach on oxygen consumption, synthesis of glucose from lactate and pyruvate, glycogen formation, mitochondrial oxidative phosphorylation and ATP-synthesis. We have established that effects of Ach on oxygen consumption depend on its concentration. When used at a concentration of 10(-7) M, Ach exerts maximum stimulatory effect, while its infusion at 10(-6) M causes a decrease of oxygen consumption by the liver. Moreover, when used at a concentration of 10(-6) M or 10(-7) M, Ach increases rates of glucose production from the gluconeogenic substrates lactate and pyruvate, leading to enhanced glycogen content in perfused liver. It was also shown that Ach possesses a stimulating effect on alanine and aspartate aminotransferases. As detected by 31P NMR spectroscopy, continuous liver perfusion with pyruvate and lactate in the presence of Ach leads to a significant decrease of ATP level, implying enhanced energy requirements for gluconeogenesis under these conditions. Elimination of the described effects of Ach by atropine, the antagonist of muscarinic receptors, and identification of the type 3 muscarinic receptors (m3) in isolated hepatocytes as well as in whole liver, imply that Ach may exert its effect on liver metabolism through m3 receptors.

The possible relationships between intracellular Na+ (Na1+), bioenergetic status and intracellular pH (pH1) in the mechanism for ischemic preconditioning were studied using 23Na and 31P magnetic resonance spectroscopy in isolated Langendorff perfused rat heart. The ischemic preconditioning (three 5-min ischemic episodes followed by two 5-min and one 10-min period of reperfusion) prior to prolonged ischemia (20 min stop-flow) resulted in a decrease in ischemic acidosis and faster and complete recovery of cardiac function (ventricular developed pressure and heart rate) after 30 min of reperfusion. The response of Na1 during ischemia in the preconditioned hearts was characterized by an increase in Na+1 at the end of preconditioning and an accelerated decrease during the first few minutes of reperfusion. During post-ischemic reperfusion, bioenergetic parameters (PCr/P1 and βATP/P, ratios) were partly recovered without any significant difference between control and preconditioned hearts. The reduced acidosis during prolonged ischemia and the accelerated decrease in Na1+ during reperfusion in the preconditioned hearts suggest activation of Na+/H+ exchanger and other ion transport systems during preconditioning, which may protect the heart from intracellular acidosis during prolonged ischemia, and result in better recovery of mechanical function (LVDP and heart rate) during post-ischemic reperfusion.

Background. Ca 2+ overload leads to mitochondrial uncoupling, decreased ATP synthesis, and myocardial dysfunction. Pharmacologically opening of mitochondrial K ATP channels decreases mitochondrial Ca 2+ uptake, improving mitochondrial function during Ca 2+ overload. Ischemic preconditioning (IPC), by activating mitochondrial K ATP channels, may attenuate mitochondrial Ca 2+ overload and improve mitochondrial function during reperfusion. The purpose of these experiments was to study the effect of IPC (1) on mitochondrial function and (2) on mitochondrial tolerance to experimental Ca 2+ overload. Methods. Rat hearts (n = 6/group) were subjected to (a) 30 min of equilibration, 25 min of ischemia, and 30 min of reperfusion (Control) or (b) two 5-min episodes of ischemic preconditioning, 25 min of ischemia, and 30 min of reperfusion (IPC). Developed pressure (DP) was measured. Heart mitochondria were isolated at end-Equilibration (end-EQ) and at end-Reperfusion (end-RP). Mitochondrial respiratory function (state 2, oxygen consumption with substrate only; state 3, oxygen consumption stimulated by ADP; state 4, oxygen consumption after cessation of ADP phosphorylation; respiratory control index (RCI, state 3/state 4); rate of oxidative phosphorylation (ADP/Δt), and ADP:O ratio) was measured with polarography using α-ketoglutarate as a substrate in the presence of different Ca 2+ concentrations (0 to 5 x 10 -7 M) to simulate Ca 2+ overload. Results. IPC improved DP at end-RP. IPC did not improve preischemic mitochondrial respiratory function or preischemic mitochondrial response to Ca 2+ loading. IPC improved state 3, ADP/Δt, and RCI during RP. Low Ca 2+ levels (0.5 and 1 x 10 -7 M) stimulated mitochondrial function in both groups predominantly in IPC. The Control group showed evidence of mitochondrial uncoupling at lower Ca 2+ concentrations (1 x 10 -7 M). IPC preserved state 3 at high Ca 2+ concentrations. Conclusions. The cardioprotective effect of IPC results, in part, from preserving mitochondrial function during reperfusion and increasing mitochondrial tolerance to Ca 2+ loading at end-RP. Activation of mitochondrial K ATP channels by IPC and their improvement in Ca 2+ homeostasis during RP may be the mechanism underlying this protection.

The reduction of functional β cell mass is a key feature of type 2 diabetes. Here, we studied metabolic functions and islet gene expression profiles of C57BL/6J mice with naturally occurring nicotinamide nucleotide transhydrogenase (NNT) deletion mutation, a widely used model of diet-induced obesity and diabetes. On high fat diet (HF), the mice developed obesity and hyperinsulinemia, while blood glucose levels were only mildly elevated indicating a substantial capacity to compensate for insulin resistance. The basal serum insulin levels were elevated in HF mice, but insulin secretion in response to glucose load was significantly blunted. Hyperinsulinemia in HF fed mice was associated with an increase in islet mass and size along with higher BrdU incorporation to β cells. The temporal profiles of glucose-stimulated insulin secretion (GSIS) of isolated islets were comparable in HF and normal chow fed mice. Islets isolated from HF fed mice had elevated basal oxygen consumption per islet but failed to increase oxygen consumption further in response to glucose or carbonyl cyanide-4-trifluoromethoxyphenylhydrazone (FCCP). To obtain an unbiased assessment of metabolic pathways in islets, we performed microarray analysis comparing gene expression in islets from HF to normal chow-fed mice. A few genes, for example, those genes involved in the protection against oxidative stress (hypoxia upregulated protein 1) and Pgc1α were up-regulated in HF islets. In contrast, several genes in extracellular matrix and other pathways were suppressed in HF islets. These results indicate that islets from C57BL/6J mice with NNT deletion mutation develop structural, metabolic and gene expression features consistent with compensation and decompensation in response to HF diet.

Intracellular Na+ is approximately two times higher in diabetic cardiomyocytes than in control. We hypothesized that the increase in Na+1 activates the mitochondrial membrane Na+/Ca2+ exchanger, which leads to loss of intramitochondrial Ca2+, with a subsequent alteration (generally depression) in bioenergetic function. To further evaluate this hypothesis, mitochondria were isolated from hearts of control and streptozotocin-induced (4 weeks) diabetic rats. Respiratory function and ATP synthesis were studied using routine polarography and 31P-NMR methods, respectively. While addition of Na+ (1–10 mM) decreased State 3 respiration and rate of oxidative phosphorylation in both diabetic and control mitochondria, the decreases were significantly greater for diabetic than for control. The Na+ effect was reversed by providing different levels of extramitochondrial Ca2+ (larger Ca2+ levels were needed to reverse the Na+ depressant effect in diabetes mellitus than in control) and by inhibiting the Na+/Ca2+exchanger function with diltiazem (a specific blocker of Na+/Ca2+ exchange that prevents Ca2+ from leaving the mitochondrial matrix). On the other hand, the Na+ depressant effect was enhanced by Ruthenium Red (RR, a blocker of mitochondrial Ca2+ uptake, which decreases intramitochondrial Ca2+). The RR effect on Na+ depression of mitochondrial bioenergetic function was larger in diabetic than control. These findings suggest that intramitochondrial Ca2+ levels could be lower in diabetic than control and that the Na+ depressant effect has some relation to lowered intramitochondrial Ca2+. Conjoint experiments with 31P-NMR in isolated superfused mitochondria embedded in agarose beads showed that Na+ (3–30 mM) led to significantly decreased ATP levels in diabetic rats, but produced smaller changes in control. These data support our hypothesis that in diabetic cardiomyocytes, increased Na+ leads to abnormalities of oxidative processes and subsequent decrease in ATP levels, and that these changes are related to Na+ induced depletion of intramitochondrial Ca2+.

Ischemic preconditioning (IPC) protects myocardium from ischemia reperfusion injury by activating mitochondrial K(ATP) channels. However, the mechanism underlying the protective effect of K(ATP) channel activation has not been elucidated. It has been suggested that activation of mitochondrial K(ATP) channels may prevent mitochondrial dysfunction associated with Ca(2+) overload during reperfusion. The purpose of this experiment was to study, in an isolated mitochondrial preparation, the effects of mitochondrial K(ATP) channel opening on mitochondrial function and to determine whether it protects mitochondria form Ca(2+) overload. Mitochondria (mito) were isolated from rat hearts by differential centrifugation (n = 5/group). Mito respiratory function was measured by polarography without (CONTROL) or with a potassium channel opener (PINACIDIL, 100 microM). Different Ca(2+) concentrations (0 to 5 x 10(-7) M) were used to simulate the effect of Ca(2+) overload; state 2, mito oxygen consumption with substrate only; state 3, oxygen consumption stimulated by ADP; state 4, oxygen consumption after cessation of ADP phosphorylation; respiratory control index (RCI: ratio of state 3 to state 4); rate of oxidative phosphorylation (ADP/Deltat); and ADP:O ratio were measured. PINACIDIL increased state 2 respiration and decreased RCI compared to CONTROL. Low Ca(2+) concentrations stimulated state 2 and state 4 respiration and decreased RCI and ADP:O ratios. High Ca(2+) concentrations increased state 2 and state 4 respiration and further decreased RCI, state 3, and ADP/Deltat. PINACIDIL improved state 3, ADP/Deltat, and RCI at high Ca(2+) concentrations compared to CONTROL. Pinacidil depolarized inner mitochondrial membrane, as evidenced by decreased RCI and increased state 2 at baseline. Depolarization may decrease Ca(2+) influx into mito, protecting mito from Ca(2+) overload, as evidenced by improved state 3 and RCI at high Ca(2+) concentrations. The myocardial protective effects resulting from activating K(ATP) channels either pharmacologically or by IPC may be the result of protecting mito from Ca(2+) overload.

Objectives: Mitochondrial abnormalities have been described in cardiac tissue of patients with heart failure. These changes may result from chronic hypoxia. Our goal was to determine whether mitochondrial functional capacity can be improved in patients with heart failure by means of long-term left ventricular assist device therapy, which improves myocardial oxygen supply by decreasing myocardial work. Methods: Mitochondria were isolated from myocardial tissue obtained from 13 patients with heart failure without a left ventricular assist device (HF group) and seven patients with heart failure treated with a left ventricular assist device (LVAD-HF group). Mitochondrial respiratory rates (State 2, State 3, and State 4) were measured by means of polarographic techniques with reduced nicotinamide adenine dinucleotide–dependent (pyruvate/malate, α-ketoglutarate, glutamate) and –independent (succinate) substrates. The respiratory control index of Chance (State 3/State 4) and Lardy (State 3/State 2) and phosphorus to oxygen ratios were determined. Results: The respiratory control index of Chance was higher in LVAD-HF than in HF when using NADH-dependent substrates pyruvate/malate and α-ketoglutarate (pyruvate/malate HF: 4.9 ± 1.0; LVAD-HF: 6.5 ± 1.5; α-ketoglutarate HF: 8.5 ± 2.4; LVAD-HF: 11.8 ± 2.9; both p = 0.04). Similarly, the respiratory control index of Lardy was greater in the LVAD-HF than the HF group when α-ketoglutarate and glutamate were used as substrates (α-ketoglutarate HF: 7.8 ± 1.7; LVAD-HF: 9.9 ± 1.5; glutamate HF: 7.6 ± 2.2; LVAD-HF: 10.7 ± 2.1; both p = 0.04). The phosphorus to oxygen ratio was comparable for both groups using all substrates. No change in mitochondrial respiration was observed after left ventricular assist device therapy with the NADH-independent substrate, succinate. Conclusion: Cardiomyocyte mitochondrial function is improved by long-term therapy with a left ventricular assist device. This improvement suggests that cardiomyocyte metabolic dysfunction in heart failure may be reversed with left ventricular assist device support. (J Thorac Cardiovasc Surg 1998;116:344-9)

Several investigators have shown defects in energy production in hearts from animal models of diabetes mellitus (DM). These defects generally involve changes in oxidative phosphorylation (ox-phos) or in Krebs cycle functions. Abnormalities in ox-phos have been observed in heart mitochondria isolated from streptozotocin-diabetic (Pierce and Dhalla, 1985) and alloxan-diabetic (Puckett and Reddy, 1979) models as well as in genetic diabetic models (Kuo et al., 1983). In DM mitochondria, State 3 is decreased when a variety of substrates are used: Pyr plus malate and palmitylcarnitine plus malate (Kuo et al., 1983), a-ketoglutarate (Taegtmeyer et al, 1987), glutamate (Mokhtar et al., 1993), 3-hy-droxybutyrate and malate, and acetoacetate plus malate (Grinblat et al., 1986). The RCI also decreased in mitochondria from DM animals. The ADP/O ratio is depressed only in the early stages of chemically induced DM, returning to normal ranges later on, indicating that mitochondria from DM hearts have normal ability to transfer electrons from NADH to O2 or to couple oxidation to phosphorylation. This suggests that the deficiency of energy production is related to a decrease in substrate oxidation through the Krebs cycle. Two important cellular functions which may contribute to abnormal ox-phos are alterations in mi-tochondrial transporter systems (Kazmi et al., 1985; Paulson et al., 1984) and decrease in substrate supply. One possible mechanism of impaired substrate oxidation in the DM heart is abnormal mitochondrial Ca2+ uptake (Baba and Kako, 1991). It is well known that Ca2+ plays an important role in regulation of several matrix dehydrogenases (including PDH

Substrate-level phosphorylation was observed under the conditions optimal for this process and opposite to those for oxidative phosphorylation. Polarographic registration of Ca2+ stimulated alpha-ketoglutarate oxidation and self-inhibition of uncoupled alpha-ketoglutarate (KG) oxidation was used. Acetylcholine (ACh) administration stimulated KG oxidation and substrate-level phosphorylation in isolated mitochondria. These effects are stronger in tissues with a higher level of endogenous acetylcholine, such as guinea pig liver vs rat liver and pancreas vs liver. The specific stimulation of KG oxidation by ACh is related to a decrease of succinate oxidation and is contrary to the specific stimulating effect of adrenaline on succinate oxidation. Therefore the existence of reciprocal hormone-substrate-nucleotide systems is suggested. The described set of conditions optimal for substrate-level phosphorylation observation by polarographic registration of respiration is as convenient as the ADP test for the investigation of oxidative phosphorylation.

Purpose: To compare the clinical outcomes and the rate of complications of 27-gauge transconjunctival nonvitrectomizing vitreous surgery (NVS) and of 25-gauge transconjunctival sutureless vitrectomy surgery for idiopathic epiretinal membrane removal. Methods: In this prospective randomized study, 83 phakic eyes of 83 consecutive patients with an idiopathic epiretinal membrane were randomized to receive 27-gauge NVS (NVS-group) or 25-gauge vitrectomy (Standard-group). Main outcome measures were best-corrected visual acuity, central retinal thickness, nuclear density units' changes, and rate of complications. Results: Thirty-nine eyes of the Standard-group and 40 of the NVS-group were considered in final analysis. Mean best-corrected visual acuity improved significantly in both groups, with a significant better result at 12 months in NVS-group (P 0.039; t-test). Central retinal thickness decreased significantly in both groups (P < 0.001, Tukey test), without significant difference between the two groups at any time point. At 12 months, nuclear density increased significantly in the Standard-group (analysis of variance, P < 0.001), and it did not change in the NVS-group (analysis of variance, P 0.537). Epiretinal membrane recurred in 5.1% of eyes in the Standard-group and in 7.5% of eyes in the NVS-group (Fisher's exact test, P 1.000). Conclusion: The 27-gauge NVS is an effective surgical procedure in eyes with epiretinal membrane and it induces less progression of nuclear sclerosis than 25-gauge vitrectomy.

Background/objectives: To compare the number of eye surgical procedures performed in Italy in the 2 months following the beginning of lockdown (study period) because of COVID-19 epidemic with those performed in the two earlier months of the same year (intra-year control) and in the period of 2019 corresponding to the lockdown (inter-year control). Methods: Retrospective analysis of surgical procedures carried out at 39 Academic hospitals. A distinction was made between elective and urgent procedures. Intravitreal injections were also considered. Percentages for all surgical procedures and incidence rate ratios (IRR) for rhegmatogenous retinal detachment (RRD) events were calculated. A p value Results: A total of 20,886 versus 55,259 and 56,640 patients underwent surgery during the lockdown versus intra-and inter-year control periods, respectively. During the lockdown, only 70% of patients for whom an operation/intravitreal injection was recommended, finally underwent surgery; the remaining patients did not attend because afraid of getting infected at the hospital (23%), taking public transportation (6.5%), or unavailable swabs (0.5%). Elective surgeries were reduced by 96.2% and 96.4%, urgent surgeries by 49.7% and 50.2%, and intravitreal injections by 48.5% and 48.6% in the lockdown period in comparison to intra-year and inter-year control periods, respectively. IRRs for RRDs during lockdown dropped significantly in comparison with intra- and inter-year control periods (CI: 0.65–0.80 and 0.61–0.75, respectively, p Conclusion: This study provides a quantitative analysis of the reduction of eye surgical procedures performed in Italy because of the COVID-19 epidemic.

Background: The Aim of This Study Was to Evaluate Intra- and Perioperative Outcomes of a Single High Volume Open Radical Prostatectomy (Orp) Surgeon, During His Learning Curve Period for Robot-Assisted Radical Prostatectomy (Rarp) and Extended Pelvic Lymph Node Dissection (Eplnd). Methods: The Study Included 264 Intermediate-High Risk Prostate Cancer Patients, Treated By Orp + Eplnd or Rarp + Eplnd, Prospectively Collected. Descriptive Statistics Compared Clinical and Pathological Variables Between Groups. Bivariate (Pearson) Correlation Analysis Assessed The Relationship Between The Number of Lymph Node (Ln) Removed, Positive Surgical Margins (Psm), Surgical Time and The Number of Procedures Performed Per Group. Results: Pt Stage and Gleason Score (Gs) Were Lower In Rarp Than In Orp Group (Both P=0.04), While Psm Were More Frequent In The Rarp Group (40% Vs. 25%; P=0.02). However, Psm Decreased With The Increase of Rarp ProCedures. The Number of Lns Removed Was 25 and 22, In Rarp and Orp Group (P=0.03). However, Ln+ Rate Did Not Differ Between Groups (11% Vs. 16%; P=0.216). In The Rarp Group, Overall Surgical Time and Eplnd Time Decreased With The Increase of Surgical Procedures (All P