Novel therapeutic approaches based on the pathological role of gut dysbiosis on the link between nonalcoholic fatty liver disease and insulin resistance.

The growing global epidemic of obesity and type 2 diabetes mellitus has determined an increased prevalence of NAFLD (non-alcoholic fatty liver disease), making it the most common chronic liver disease in the Western world and a leading cause of liver transplantation. In the last few years, a rising number of studies conducted both on animal and human models have shown the existence of a close association between insulin resistance (IR), dysbiosis, and steatosis. However, all the mechanisms that lead to impaired permeability, inflammation, and fibrosis have not been fully clarified. Recently, new possible treatment modalities have received much attention. To reach the review purpose, a broad-ranging literature search on multidisciplinary research databases was performed using the following terms alone or in combination: "NAFLD", "gut dysbiosis", "insulin resistance", "inflammation", "probiotics", "Chinese herbs". The use of probiotics, prebiotics, symbiotics, postbiotics, fecal microbiota transplant (FMT), Chinese herbal medicine, antibiotics, diet (polyphenols and fasting diets), and minor therapies such as carbon nanoparticles, the MCJ protein, water rich in molecular hydrogen, seems to be able to improve the phenotypic pattern in NAFLD patients. In this review, we provide an overview of how IR and dysbiosis contribute to the development and progression of NAFLD, as well as the therapeutic strategies currently in use.

Risk factors for diabetic foot ulcers: an Albanian retrospective study of inpatients with type 2 diabetes.

OBJECTIVE: The aim of this study was to assess the impact of glucose control, diabetes-related complications and cardiometabolic risk factors on the risk of diabetic foot ulcers (DFUs) and DFU complications in Albanian adult inpatients with T2D. PATIENTS AND METHODS: We conducted a retrospective case-control study on 482 Albanian adult inpatients with T2D. DFU was defined as a full-thickness skin lesion requiring ≥14 days for healing and was classified at the time of hospital admission. Demographic and biochemical parameters of the study participants, the presence of comorbidities and diabetes-related complications at the time of hospital admission were evaluated through a retrospective chart review. RESULTS: Mean age of study participants was 54.8±10.7 years. Participants (284 males and 198 females) were divided into two groups: DFU (cases; n=104) and non-DFU (controls; n=378). Multivariate analysis (performed by a logistic regression model) revealed that the most relevant independent variables associated with DFU were BMI [OR=0.62; p=0.007], HDL-cholesterol [OR=0.00; p<0.0001], triglycerides [OR=7.48; p=0.0004], cigarette smoking [OR=26.46; p=0.005], duration of diabetes [OR=1.53; p<0.0001], fasting plasma glucose (FPG) [OR=1.06; p<0.0001], systolic blood pressure (SBP) [OR=1.13; p=0.0004] and insulin therapy alone [OR=0.11; p=0.02]. ROC curve analysis showed that FPG (AUC=0.83), glycated hemoglobin (HbA1c) (AUC=0.75), triglycerides (AUC=0.78) and HDL-cholesterol (AUC=0.82) were the most reliable biomarkers able to detect DFU. In the DFU group, the most relevant independent variables associated with previous minor lower-extremity amputations (LEAs) were represented by HbA1c [OR=1.47; p=0.03], age <55 years [OR=0.12; p=0.05] and female sex [OR=4.18; p=0.03]; whereas the most relevant independent variables associated with diabetic peripheral neuropathy (DPN) were HbA1c [OR=1.70; p=0.006], SBP [OR=1.08; p=0.05], BMI [OR=1.20; p=0.03] and lack of cigarette smoking [OR=0.07; p=0.01]. Correlation analysis (performed through the nonparametric Spearman's rank correlation test or through the parametric Pearson test, as appropriate) revealed a significant positive relationship between HbA1c and FPG (r=0.58; p<0.0001), ulcer surface area (r=0.50; p<0.0001), ulcer grade (r=0.23; p=0.02), minor LEAs (r=0.20; p=0.04), DPN (r=0.41; p<0.0001), and metformin therapy alone (r=0.72; p<0.0001). There was a significant inverse correlation between HbA1c and insulin therapy alone (r=-0.31; p=0.01) and combined metformin and insulin therapy (r=-0.60; p<0.0001). Both DFU and non-DFU groups exhibited suboptimal mean LDL-cholesterol levels (>100 mg/dl) and mean HbA1c values >7.5%. Moreover, in DFU group HbA1c values were markedly elevated (≥10%) particularly in patients with a grade 3 ulcer and an ulcer surface area ≥4 cm2, as well as in patients with history of minor LEAs and in patients affected by DPN. CONCLUSIONS: The present study suggested that longer duration of diabetes, cigarette smoking, lower HDL-cholesterol levels, poor glucose control, and elevated triglyceride and SBP values may all represent major risk factors for the development of DFU in Albanian patients with T2D. Thus, community interventions and health policies aimed to improve the management of diabetes and related cardiometabolic risk factors should be urgently implemented in Albania, in order to prevent DFUs and other diabetes complications in patients with T2D.

SGK-1 protects kidney cells against apoptosis induced by ceramide and TNF-α.

Ceramide regulates several different cellular responses including mechanisms leading to apoptosis. Serum- and glucocorticoid-inducible protein kinase (SGK)-1 is a serine threonine kinase, which activates survival pathways in response to stress stimuli. Recently, we demonstrated an anti-apoptotic role of SGK-1 in human umbilical endothelial cells treated with high glucose. In the present study, since ceramide induces apoptosis by multiple mechanisms in diabetes and its complication such as nephropathy, we aimed to investigate whether SGK-1 may protect even against apoptosis induced by ceramide in kidney cells. Human embryonic kidney (HEK)-293 cells stable transfected with SGK-1 wild type (SGK-1wt) and its dominant negative gene (SGK-1dn) have been used in this study. Apoptotic stimuli were induced by C2-ceramide and TNF-α to increase endogenous synthesis of ceramide. Upon activation with these stimuli, SGK-1wt transfected cells have a statistically significant reduction of apoptosis compared with SGK-1dn cells (P<0.001). This protection was dependent on activation of caspase-3 and Poly-ADP-ribose-polymerase-1 (PARP-1) cleavage. SGK-1 and AKT-1 two highly homologous kinases differently reacted to ceramide treatment, since SGK-1 increases in response to apoptotic stimulus while AKT-1 decreases. This enhancement of SGK-1 was dependent on p38-mitogen-activated-protein kinases (p38MAPK), cyclic-adenosine-monophosphate/protein kinase A (cAMP/PKA) and phosphoinositide-3-kinase (PI3K) pathways. Especially, by using selective LY294002 inhibitor, we demonstrated that the most involved pathway in the SGK-1 mediated process of protection was PI3K. Treatment with inhibitor of SGK-1 (GSK650394) significantly enhanced TNF-α-dependent apoptosis in HEK-293 cells overexpressing SGK-1wt. Caspase-3, -8 and -9 selective inhibitors confirmed that SGK-1 reduced the activation of caspase-dependent apoptosis, probably by both intrinsic and extrinsic pathways. In conclusion, we demonstrated that in kidney cells, overexpression of SGK-1 is protective against ceramide-induced apoptosis and the role of SGK-1 can be potentially explored as a therapeutic target in conditions like diabetes, where ceramide levels are increased.

Diabetes-linked zinc transporter ZnT8 is a homodimeric protein expressed by distinct rodent endocrine cell types in the pancreas and other glands.

BACKGROUND AND AIMS: Zinc is abundant in pancreas, being required by endocrine islet cells for hormone secretion and by exocrine acinar cells as pancreatic juice component. ZnT8 is a member of the SLC30A family of zinc transporters whose overexpression in cultured pancreatic beta cells leads to increased insulin secretion in response to glucose, suggesting a possible role in regulating glycemia. ZnT8 was therefore proposed as a therapeutic target for diabetes, and recent genome-wide association studies identified polymorphisms in the ZNT8 gene conferring increased type 2 diabetes risk. METHODS AND RESULTS: As limited information was available on the biochemical properties of ZnT8 and on its endogenous expression, we have raised a specific polyclonal antibody and immunostained protein extracts, cell lines and tissue sections. We show that ZnT8 forms a very stable dimer that requires biological membranes to properly assemble. We demonstrate localization of murine ZnT8 to the secretory granules in pancreatic beta and alpha islet cells. Moreover, we show that ZnT8 is also expressed in other secretory cell types, namely the cubical epithelium that lines thyroid follicles and the cortex of the adrenal gland, suggesting a more widespread role in endocrine secretion. CONCLUSION: We provide novel insights into the features of the ZnT8 transporter, of special relevance in light of its proposed role as therapeutical target for diabetes treatment.

Molecular cloning and characterization of a highly basic protein, IA-4, expressed in pancreatic islets and brain.

A substraction library was constructed from mouse insulinoma (betaTC-1) and glucagonoma (alphaTC-1) cell lines. Differential screening and sequencing revealed a novel cDNA clone, IA-4, which was expressed in the islets of Langerhans and the brain. IA-4 cDNA is 1,007 bp in length and predicts a protein of 187 amino acids with a molecular mass of 19,940 D. Examination of the amino acid sequence showed a high content of arginine (18.7%), proline (14.4%), alanine (16.0%), leucine (13.4%) and glycine (9.6%). The deduced pI value is 12.5 indicating a highly basic protein. Northern blot analysis revealed a 1-kb mRNA highly expressed in brain, trigeminal ganglia and cell lines of neuroendocrine origin. Rabbit polyclonal antiserum raised against a synthetic IA-4 peptide, designated Pep-1, not only reacted with IA-4 recombinant protein, but also immunostained the islets of Langerhans and large neurons of the hippocampus, cerebral cortex, spinal cord, dorsal ganglia and Purkinje cells of the cerebellum. The high expression of IA-4 protein in neuroendocrine cells and its unique amino acid sequence suggest that IA-4 may have an important, but still undetermined, function in these special cell types.

Profile and differential expression of protein tyrosine phosphatases in mouse pancreatic islet tumor cell lines.

Protein tyrosine phosphatases (PTPs) play important roles in cell growth and differentiation of normal and tumor cells. In this study, we analyzed the PTP profile in two pancreatic islet tumor cell lines. Transcripts were isolated from alphaTC-1 (glucagon-secreting) and betaTC-1 (insulin-secreting) cell lines for templates. A pair of degenerative primers, based on the conserved regions of known PTPs, was used to amplify the transcripts by polymerase chain reaction. A total of 1,620 clones was examined by restriction enzyme analysis and cDNA sequencing. Twenty-one PTPs were identified, including nine cytosolic PTPs (TcPTP, P19PTP, PTP1B, PTPMEG, PTP1C, SYP, PTPH1, PTPL1, and PTPD1), nine transmembrane PTPs (PTPdelta, PTPgamma, PTPkappa, DEP-1, IA-2, LAR, PTPalpha, PTPNE3, and PTPepsilon), and three new PTPs--PTPmu-like PTPkappa-like, and IA-2beta. An RNase protection assay demonstrated that some of these PTPs were expressed predominantly in glucagonoma (i.e., PTPdelta and IA-2) and others in insulinoma (i.e., PTP1C, PTPkappa, and PTPNE3) cells. In this report, we present the first profile of PTPs in alpha and beta tumor cell lines.

Characterization and chromosomal localization of a new protein disulfide isomerase, PDIp, highly expressed in human pancreas.

Protein disulfide isomerase (PDI) catalyzes protein folding and thiol-disulfide interchange reactions. The enzyme is localized in the lumen of endoplasmic reticulum (ER) and is abundant in secretory cells of various tissues. In this study we describe the isolation and characterization from human pancreas of a new protein, PDIp, that is structurally and functionally related to PDIs. PDIp cDNA is 1,659 bp in length and predicts a protein with an open reading frame of 511 amino acids. PDIp amino acid sequence shows 46% identity and 66% similarity to that of human PDI. PDIp possesses two thioredoxin-like active sites (WCGHCQ and WCTHCK) and an endoplasmic reticulum retention signal sequence, KEEL, at the carboxyl terminus. Northern analysis of normal human tissues and various human tumor cell lines revealed PDIp mRNA (2.0 kb) expression only in the normal pancreas. Recombinant PDIp protein catalyzed reductive cleavage of insulin and renaturation of reduced RNaseA. Somatic cell genetics and fluorescence in situ hybridization localized the PDIp gene to the short arm of human chromosome 16. It is concluded that PDIp is a new member of the PDI family and is highly expressed in human pancreas.

A nuclear protein, synthesized in growth-arrested human hepatoblastoma cells, is a novel member of the short-chain alcohol dehydrogenase family.

We have described a protein (Hep27) [Donadel, G., Garzelli, C., Frank, R. & Gabrielli, F. (1991) Eur. J. Biochem. 195, 723-729] which is synthesized and accumulated in the nucleus of human hepatoblastoma (HepG2) cells, following growth arrest induced by butyrate treatment. The synthesis of Hep27 is inhibited in cells that, released from the butyrate block, have resumed DNA synthesis. This report describes the cloning and the characterization of the cDNA coding for the Hep27 protein. The translation of the Hep27 cDNA predicts an amino acid sequence that can be aligned with those of the known short-chain alcohol dehydrogenase enzymes (SCAD) family. Both the recognition of enzymic functional domains and the similarity with the SCAD family of proteins of several amino acid blocks throughout the molecule, strongly suggest that this protein is a new member of the SCAD family. In agreement with its nuclear localization Hep27 has a region similar to the bipartite nuclear-targeting sequence. The study of Hep27 mRNA expression and protein synthesis suggests the existence of a regulation at the post-transcriptional level. The possible nuclear role of the Hep27 protein is discussed.

Human polyreactive and monoreactive antibodies: effect of glycosylation on antigen binding.

The present experiments were initiated to determine whether the carbohydrate portions of antibody molecules contribute to polyreactivity. Cell lines making human monoclonal polyreactive or monoreactive antibodies of the immunoglobulin (Ig) M, IgG and IgA isotypes were treated with tunicamycin to block N-linked glycosylation of the proteins. Analysis of the secreted native and non-glycosylated proteins revealed a > 95% inhibition of [3H]mannose incorporation. Electrophoresis on sodium dodecyl sulphate-polyacrylamide gels of the proteins from tunicamycin-treated cells showed increased mobility and the absence of [3H]mannose incorporation of the immunoglobulin heavy chains, consistent with the lack of glycosylation. The native and non-glycosylated antibodies were then tested for their ability to bind different antigens. Despite the lack of glycosylation, both polyreactive and monoreactive antibodies bound to antigens with little if any loss of reactivity or specificity. It is concluded that the carbohydrate moieties do not contribute significantly to polyreactivity.

cDNA sequence and genomic organization of mouse secretin.

A murine insulinoma library was constructed by subtracting glucagonoma cDNAs from insulinoma cDNAs. Comparison of the nucleotide sequence of one of the clones with sequences from the GenBank database showed that it was a member of the secretin family. The clone, 490 bp long, encodes a protein of 133 amino acids consisting of a signal peptide, a N-terminal peptide, secretin, and a C-terminal peptide, which showed 88% and 80% homology with rat and porcine secretin precursor proteins, respectively. The translated secretin peptide showed a unique Met to Thr substitution at position 5 as compared to the secretins from other species. That the Met to Thr substitution was not tumor-related was demonstrated by the fact that an identical sequence was found in cDNA from normal mouse intestine. Studies on the mouse secretin gene revealed that it contains four exons separated by 81 bp, 110 bp, and 96 bp introns.

Half-life of polyreactive antibodies.

Monoclonal polyreactive antibodies bind to a variety of self and foreign antigens. In contrast, monoclonal monoreactive antibodies bind to a single or restricted number of known antigens. The rate at which polyreactive antibodies are removed from the circulation compared to monoreactive antibodies has not been determined. In the present experiments, human monoclonal polyreactive and monoreactive antibodies of different isotypes were injected intravenously into mice and the clearance from the circulation was determined. The half-life of polyreactive IgM, IgA, and IgG antibodies was 8.0, 8.2, and 9.8 hr, respectively, compared to 35.4, 26.6, and 280 hr for monoreactive IgM, IgA, and IgG antibodies, respectively. Examination of tissue sections from animals given intravenous antibody showed substantial deposition of polyreactive, but not monoreactive, antibodies in several organs, the liver being the principal site of deposition. It is concluded that polyreactive antibodies are cleared from the circulation substantially faster than monoreactive antibodies.

Identification of a novel nuclear protein synthesized in growth-arrested human hepatoblastoma HepG2 cells.

DNA synthesis of human hepatoblastoma HepG2 cells is reversibly inhibited by butyrate. When butyrate is removed from the culture medium, cells re-enter the cell cycle, synthesizing DNA with a time lag of about 12 h. HepG2 cells, growth-inhibited for 30 h with butyrate, synthesize and accumulate a nuclear protein, called D. Protein D synthesis is inhibited in cells which, released from the butyrate block, have resumed DNA synthesis as well as in growing cells never exposed to butyrate. Protein D has been purified from growth-arrested cells and partially sequenced. The amino acid sequences of five internal trypsin peptides indicate that protein D is a novel nuclear protein.

The differentiation-inducing agent sodium butyrate produces divergent effects on albumin and thyroxine-binding globulin synthesis by human hepatoblastoma-derived (Hep G2) cells.

The addition of sodium butyrate, a differentiation-inducing agent, to the culture medium of human hepatoblastoma-derived (Hep G2) cells, produced a dose-dependent and time-dependent increase in albumin (ALB) and decrease in T4-binding globulin (TBG) synthesis and secretion. In the presence of 0.01 to 2.0 mM sodium butyrate, newly synthesized [35S]ALB progressively increased up to 139% of control cultures grown in the absence of sodium butyrate, whereas TBG synthesis was already slightly inhibited using the lowest concentrations of this agent and further diminished thereafter. The use of 5 mM and 10 mM sodium butyrate inhibited the synthesis of both proteins, probably as a consequence of toxic effects on cell cultures. The addition of 1 mM sodium butyrate for variable time intervals caused an increase in the amount of ALB recovered in the medium up to 146% after 72 h, and a decrease of TBG up to 44% of controls. These different effects on ALB and TBG occurred concomitantly with an inhibition of cell growth, as shown by the reduction in the cell number/flask compared to control cultures. At the highest sodium butyrate concentrations, a relevant impairment in the secretion of newly synthesized TBG, but not of ALB, also occurred. These divergent effects on ALB and TBG synthesis by Hep G2 cells might be related to biochemical differentiation induced by sodium butyrate in this tumoral cell system, suggesting that TBG synthesis is increased in Hep G2 cells because of their neoplastic nature.

[Removal of an endocavitary electrode with cardiopulmonary bypass for septicemia associated with right atrial thrombosis. Report of a case (author's transl)]. / Rimozione di elettrodo endocavitario mediante bypass cardiopolmonare totale per setticemia e trombosi atriale destra.

A case of staphylococcus epidermidis endocarditis associated with a right atrial thrombosis around a pacemaker electrode is presented. Stuce the antibiotic therapy had proved uneffective, the electrode had to be removed with cardiopulmonary bypass; the infection was subsequently eliminated. When a foreign body cannot be removed by closed techniques, open heart surgery with cardiopulmonary bypass may be necessary.

[The experience with permanent cardiac pacing at the Surgical Clinic of the University of Padua Medical School (author's transl)]. / Esperienza della Clinica Chirurgica dell'Università di Padova nella elettrostimolazione cardiaca permanente.

A computer analysis is reported of the most significant data concerning 1624 pacemaker implantations and 1386 battery replacements. The patients were treated by endocardial stimulation with transvenous electrodes in 91% of the cases; the remainder: 9% received epicardial electrodes. The mode of stimulation was 2189 times "demand", 138 "synchronized" and 683 "asyncronous". Pacers of 13 different brands were used. At the beginning of our experience, the catheter was introduced through the superficial or profunda giugular vein: since 1969 the cephalic vein has been preferred. Epicardial electrodes were implanted via a left thoracotomy but in the most recent years the extrapleural phrenico-pericardial approach has been adopted. In a number of cases external pacemakers have been employed for permanent stimulation. The most common post-operative complications were infection (3.9%), skin erosion (2.2%), catheter rupture (3.5%), electrode dislodgement (1.7%). Such complications are becoming less frequent since 1969, due to technical improvements.

[Infections in pacemaker carriers (author's transl)]. / Le infezioni nei portatori di pacemakers

Of 2243 cardiac pacemakers implanted at the cardio-surgical centre of the surgical clinic in the University of Padua, 150 cases of infection were observed, mostly caused by a delayed cutaneous decubitus from the electrode or the battery. The main causes for infection in pacemaker carriers and the most suitable means for their prevention are discussed.