Ileana Terruzzi

Regulation of glucose homeostasis in humans with denervated livers.

The liver plays a major role in regulating glucose metabolism, and since its function is influenced by sympathetic/ parasympathetic innervation, we used liver graft as a model of denervation to study the role of CNS in modulating hepatic glucose metabolism in humans. 22 liver transplant subjects were randomly studied by means of the hyperglycemic/ hyperinsulinemic (study 1), hyperglycemic/isoinsulinemic (study 2), euglycemic/hyperinsulinemic (study 3) as well as insulin-induced hypoglycemic (study 4) clamp, combined with bolus-continuous infusion of [3-3H]glucose and indirect calorimetry to determine the effect of different glycemic/insulinemic levels on endogenous glucose production and on peripheral glucose uptake. In addition, postabsorptive glucose homeostasis was cross-sectionally related to the transplant age (range = 40 d-35 mo) in 4 subgroups of patients 2, 6, 15, and 28 mo after transplantation. 22 subjects with chronic uveitis (CU) undergoing a similar immunosuppressive therapy and 35 normal healthy subjects served as controls. The results showed that successful transplantation was associated with fasting glucose concentration and endogenous glucose production in the lower physiological range within a few weeks after transplantation, and this pattern was maintained throughout the 28-mo follow-up period. Fasting glucose (4. 55+/-0.06 vs. 4.75+/-0.06 mM; P = 0.038) and endogenous glucose production (11.3+/-0.4 vs. 12.9+/-0.5 micromol/[kg.min]; P = 0.029) were lower when compared to CU and normal patients. At different combinations of glycemic/insulinemic levels, liver transplant (LTx) patients showed a comparable inhibition of endogenous glucose production. In contrast, in hypoglycemia, after a temporary fall endogenous glucose production rose to values comparable to those of the basal condition in CU and normal subjects (83+/-5 and 92+/-5% of basal), but it did not in LTx subjects (66+/-7%; P < 0.05 vs. CU and normal subjects). Fasting insulin and C-peptide levels were increased up to 6 mo after transplantation, indicating insulin resistance partially induced by prednisone. In addition, greater C-peptide but similar insulin levels during the hyperglycemic clamp (study 1) suggested an increased hepatic insulin clearance in LTx as compared to normal subjects. Fasting glucagon concentration was higher 6 mo after transplantation and thereafter. During euglycemia/hyperinsulinemia (study 3), the insulin-induced glucagon suppression detectable in CU and normal subjects was lacking in LTx subjects; furthermore, the counterregulatory response during hypoglycemia was blunted. In summary, liver transplant subjects have normal postabsorptive glucose metabolism, and glucose and insulin challenge elicit normal response at both hepatic and peripheral sites. Nevertheless, (a) minimal alteration of endogenous glucose production, (b) increased concentration of insulin and glucagon, and (c) defective counterregulation during hypoglycemia may reflect an alteration of the liver-CNS-islet circuit which is due to denervation of the transplanted graft.

Metabolic effects of successful intraportal islet transplantation in insulin-dependent diabetes mellitus

The intraportal injection of human pancreatic islets has been indicated as a possible alternative to the pancreas transplant in insulin-dependent diabetic patients. Aim of the present work was to study the effect of intraportal injection of purified human islets on: (a) the basal hepatic glucose production; (b) the whole body glucose homeostasis and insulin action; and (c) the regulation of insulin secretion in insulin-dependent diabetes mellitus patients bearing a kidney transplant. 15 recipients of purified islets from cadaver donors (intraportal injection) were studied by means of the infusion of labeled glucose to quantify the hepatic glucose production. Islet transplanted patients were subdivided in two groups based on graft function and underwent: (a) a 120-min euglycemic insulin infusion (1 mU/kg/min) to assess insulin action; (b) a 120-min glucose infusion (+75 mg/di) to study the pattern of insulin secretion. Seven patients with chronic uveitis on the same immunosuppressive therapy as grafted patients, twelve healthy volunteers, and seven insulin-dependent diabetic patients with combined pancreas and kidney transplantation were also studied as control groups. Islet transplanted patients have: (a) a higher basal hepatic glucose production (HGP: 5.1 +/- 1.4 mg/kg/ min; P < 0.05 with respect to all other groups) if without graft function, and a normal HGP (2.4 +/- 0.2 mg/kg/min) with a functioning graft; (b) a defective tissue glucose disposal (3.9 +/- 0.5 mg/kg/min in patients without islet function and 5.3 +/- 0.4 mg/kg/min in patients with islet function) with respect to normals (P < 0.01 for both comparisons); (c) a blunted first phase insulin peak and a similar second phase secretion with respect to controls. In conclusion, in spite of the persistence of an abnormal pattern of insulin secretion, successful intraportal islet graft normalizes the basal HGP and improves total tissue glucose disposal in insulin-dependent diabetes mellitus.

Metabolic effects of liver transplantation in cirrhotic patients.

To assess whether liver transplantation (LTx) can correct the metabolic alterations of chronic liver disease, 14 patients (LTx-5) were studied 5+/-1 mo after LTx, 9 patients (LTx-13) 13+/-1 mo after LTx, and 10 patients (LTx-26) 26+/-2 months after LTx. Subjects with chronic uveitis (CU) and healthy volunteers (CON) were also studied. Basal plasma leucine and branched-chain amino acids were reduced in LTx-5, LTx-13, and LTx-26 when compared with CU and CON (P < 0.01). The basal free fatty acids (FFA) were reduced in LTx-26 with respect to CON (P < 0.01). To assess protein metabolism, LTx-5, LTx-13, and LTx-26 were studied with the [1-14C]leucine turnover combined with a 40-mU/m2 per min insulin clamp. To relate changes in FFA metabolism to glucose metabolism, eight LTx-26 were studied with the [1-14C]palmitate and [3-3H]glucose turnovers combined with a two-step (8 and 40 mU/m2 per min) euglycemic insulin clamp. In the postabsorptive state, LTx-5 had lower endogenous leucine flux (ELF) (P < 0.005), lower leucine oxidation (LO) (P < 0.004), and lower non-oxidative leucine disposal (NOLD) (P < 0.03) with respect to CON (primary pool model). At 2 yr (LTx-26) both ELF (P < 0.001 vs. LTx-5) and NOLD (P < 0.01 vs. LTx-5) were normalized, but not LO (P < 0.001 vs. CON) (primary and reciprocal pool models). Suppression of ELF by insulin (delta-reduction) was impaired in LTx-5 and LTx-13 when compared with CU and CON (P < 0.01), but normalized in LTx-26 (P < 0.004 vs. LTx-5 and P = 0.3 vs. CON). The basal FFA turnover rate was decreased in LTx-26 (P < 0.01) and CU (P < 0.02) vs. CON. LTx-26 showed a lower FFA oxidation rate than CON (P < 0.02). Tissue glucose disposal was impaired in LTx-5 (P < 0.005) and LTx-13 (P < 0.03), but not in LTx-26 when compared to CON. LTx-26 had normal basal and insulin-modulated endogenous glucose production. In conclusion, LTx have impaired insulin-stimulated glucose, FFA, and protein metabolism 5 mo after surgery. Follow-up at 26 mo results in (a) normalization of insulin-dependent glucose metabolism, most likely related to the reduction of prednisone dose, and, (b) maintenance of some alterations in leucine and FFA metabolism, probably related to the functional denervation of the graft and to the immunosuppressive treatment.

Insulin-mimetic action of conglutin-γ, a lupin seed protein, in mouse myoblasts

BACKGROUND AND AIMS Lupin seed is referred to as an antidiabetic product in traditional medicine. Conglutin-γ, a lupin seed glycoprotein, was found to cause a significant plasma glucose reduction when orally administered to rats in glucose overload trials. Conglutin-γ was identified as being responsible for the claimed biological activity, and the aim of this work was to envisage its hypothetical insulin-mimetic cellular mechanism of action. Insulin is responsible for proteosynthesis control through IRS/AKT/P70S6k/PHAS1 pathways modulation, glucose homeostasis through PKC/Flotillin-2/caveolin-3/Cbl activation and muscle differentiation/hypertrophy via muscle-specific MHC gene transcription control. METHODS AND RESULTS To assess whether conglutin-γ modulates the same insulin-activated kinases, myoblastic C2C12 cells were incubated after 72 h of differentiation with 100 nM insulin or 0.5 mg/mL (∼10 μM) conglutin-γ. Metformin-stimulated cells were used as a positive control. The effect on the above mentioned pathways was evaluated after 5, 10, 20 and 30 min. In the control cells medium insulin, conglutin-γ and metformin were not added. We demonstrated that insulin or conglutin-γ cell stimulation resulted in the persistent activation of protein synthetic pathway kinases and increased glucose transport, glut4 translocation and muscle-specific gene transcription regulation. CONCLUSIONS Our results indicate that conglutin-γ may regulate muscle energy metabolism, protein synthesis and MHC gene transcription through the modulation of the same insulin signalling pathway, suggesting the potential therapeutic use of this natural legume protein in the treatment of diabetes and other insulin-resistant conditions, as well as the potential conglutin-γ influence on muscle cells differentiation and regulation of muscle growth.

Modulation of cell cycle progression by 5-azacytidine is associated with early myogenesis induction in murine myoblasts.

Myogenesis is a multistep process, in which myoblasts withdraw from the cell cycle, cease to divide, elongate and fuse to form multinucleated myotubes. Cell cycle transition is controlled by a family of cyclin-dependent protein kinases (CDKs) regulated by association with cyclins, negative regulatory subunits and phosphorylation. Muscle differentiation is orchestrated by myogenic regulatory factors (MRFs), such as MyoD and Myf-5. DNA methylation is crucial in transcriptional control of genes involved in myogenesis. Previous work has indicated that treatment of fibroblasts with the DNA-demethylating agent 5-azacytidine (AZA) promotes MyoD expression. We studied the effects of AZA on cell cycle regulation and MRFs synthesis during myoblast proliferation and early myogenesis phases in C2C12 cells. During the proliferation phase, cells were incubated in growth medium with 5µM AZA (GMAZA) or without AZA (GM) for 24 hours. At 70% confluence, cells were kept in growth medium in order to spontaneously achieve differentiation or transferred to differentiation medium with 5μM AZA (DMAZA) or without AZA (DM) for 12 and 24 hours. Cells used as control were unstimulated. In the proliferation phase, AZA-treated cells seemed to lose their characteristic circular shape and become elongated. The presence of AZA resulted in significant increases in the protein contents of Cyclin-D (FC:1.23 GMAZA vs GM p≤0.05), p21 (FC: 1.23 GMAZA vs GM p≤0.05), Myf-5 (FC: 1.21 GMAZA vs GM p≤0.05) and MyoD (FC: 1.20 GMAZA vs GM p≤0.05). These results propose that AZA could inhibit cell proliferation. During 12 hours of differentiation, AZA decreased the downregulation of genes involved in cell cycle arrest and in restriction point (G1 and G1/S phase) and the expression of several cyclins, E2F Transcription Factors, cyclin-dependent kinase inhibitors, specific genes responsible of cell cycle negative regulation. During 24 hours of differentiation, AZA induced an increment in the protein expression of Myf-5 (FC: 1.57 GMAZA vs GM p≤0.05), MyoD (FC: 1.14 DM vs GM p≤0.05; FC: 1.47 DMAZA vs GM p≤0.05), p21 (FC: 1.36 GMAZA vs GM p≤0.01; FC: 1.49 DM vs GM p≤0.05; FC: 1.82 DMAZA vs GM p≤0.01) and MyHC (FC: 1.40 GMAZA vs GM p≤0.01; FC: 2.39 DM vs GM p≤0.05; FC: 3.51 DMAZA vs GM p≤0.01). Our results suggest that AZA-induced DNA demethylation can modulate cell cycle progression and enhance myogenesis. The effects of AZA may open novel clinical uses in the field of muscle injury research and treatment.

Combined pancreas and kidney transplantation normalizes protein metabolism in insulin-dependent diabetic-uremic patients

In order to assess the combined and separate effects of pancreas and kidney transplant on whole-body protein metabolism, 9 insulin-dependent diabetic-uremic patients (IDDUP), 14 patients after combined kidney-pancreas transplantation (KP-Tx), and 6 insulin-dependent diabetic patients with isolated kidney transplant (K-Tx), were studied in the basal postabsorptive state and during euglycemic hyperinsulinemia (study 1). [1-14C]Leucine infusion and indirect calorimetry were utilized to assess leucine metabolism. The subjects were studied again with a combined infusion of insulin and amino acids, given to mimic postprandial amino acid levels (study 2). In the basal state, IDDUP demonstrated with respect to normal subjects (CON): (a) higher free-insulin concentration (17.8 +/- 2.8 vs. 6.8 +/- 1.1 microU/ml, P < 0.01) (107 +/- 17 vs. 41 +/- 7 pM); (b) reduced plasma leucine (92 +/- 9 vs. 124 +/- 2 microM, P < 0.05), branched chain amino acids (BCAA) (297 +/- 34 vs. 416 +/- 10 microM, P < 0.05), endogenous leucine flux (ELF) (28.7 +/- 0.8 vs. 39.5 +/- 0.7 mumol.m-2.min-1, P < 0.01) and nonoxidative leucine disposal (NOLD) (20.7 +/- 0.2 vs. 32.0 +/- 0.7 mumol.m-2. min-1, P < 0.01); (c) similar leucine oxidation (LO) (8.0 +/- 0.1 vs. 7.5 +/- 0.1 mumol.m-2.min-1; P = NS). Both KP-Tx and K-Tx patients showed a complete normalization of plasma leucine (116 +/- 5 and 107 +/- 9 microM), ELF (38.1 +/- 0.1 and 38.5 +/- 0.9 mumol.m-2.min-1), and NOLD (28.3 +/- 0.6 and 31.0 +/- 1.3 mumol.m-2.min-1) (P = NS vs, CON). During hyperinsulinemia (study 1), IDDUP showed a defective decrease of leucine (42% vs. 53%; P < 0.05), BCAA (38% vs. 47%, P < 0.05), ELF (28% vs. 33%, P < 0.05), and LO (0% vs. 32%, P < 0.05) with respect to CON. Isolated kidney transplant reverted the defective inhibition of ELF (34%, P = NS vs. CON) of IDDUP, but not the inhibition of LO (18%, P < 0.05 vs. CON) by insulin. Combined kidney and pancreas transplanation normalized all kinetic parameters of insulin-mediated protein turnover. During combined hyperinsulinemia and hyperaminoacidemia (study 2), IDDUP showed a defective stimulation of NOLD (27.9 +/- 0.7 vs. 36.1 +/- 0.8 mumol.m-2.min-1, P < 0.01 compared to CON), which was normalized by transplantation (44.3 +/- 0.8 mumol.m-2.min-1).

Amino acid- and lipid-induced insulin resistance in rat heart: molecular mechanisms

Lipids compete with glucose for utilization by the myocardium. Amino acids are an important energetic substrate in the heart but it is unknown whether they reduce glucose disposal. The molecular mechanisms by which lipids and amino acids impair insulin-mediated glucose disposal in the myocardium are unknown. We evaluated the effect of lipids and amino acids on the insulin stimulated glucose uptake in the isolated rat heart and explored the involved target proteins. The hearts were perfused with 16 mM glucose alone or with 6% lipid or 10% amino acid solutions at the rate of 15 ml/min. After 1 h of perfusion (basal period), insulin (240 nmol/l) was added and maintained for an additional hour. Both lipids and amino acids blocked the insulin effect on glucose uptake (P<0.01) and reduced the activity of the IRSs/PI 3-kinase/Akt/GSK3 axis leading to the activation of glucose transport and glycogen synthesis. Amino acids, but not lipids, increased the activity of the p70 S6 kinase leading to the stimulation of protein synthesis. Amino acids induce myocardial insulin resistance recruiting the same molecular mechanisms as lipids. Amino acids retain an insulin-like stimulatory effect on p70 S6 kinase, which is independent from the PI 3-Kinase downstream effectors.

Effect of Pancreas Transplantation on Free Fatty Acid Metabolism in Uremic IDDM Patients

To assess the effect of pancreas transplantation on free fatty acid (FFA) and glucose metabolism, we studied seven uremic IDDM patients (HbA1c 9.1%), nine IDDM patients after combined kidney-pancreas transplantation (HbA1c 5.8%), seven patients with chronic uveitis (HbA1c 5.6%), and nine normal control subjects (HbA1c 5.5%) by means of the [3(- 3)H]glucose and [1(-14)C]palmitate infusion techniques combined with indirect calorimetry and euglycemic insulin clamp. In the postabsorptive state, pancreas-transplant patients had similar plasma glucose and FFA concentrations and non-statistically different rates of hepatic glucose production (HGP) and FFA turnover, while demonstrating a reduced rate of FFA oxidation (42 +/- 5 vs. 73 +/- 10 micromol x m-2 x min-1; P < 0.05) compared with control subjects. After 180 min of tracer equilibration, all subjects underwent a low-dose (100 min, 8 mU x m-2 x min-1) followed by a high-dose (100 min, 40 mU x m-2 x min-1) euglycemic insulin infusion. During insulin infusion, pancreas-transplant patients showed a greater inhibition of FFA concentration (609 +/- 76 to 58 +/- 15 micromol/l) compared with healthy subjects (681 +/- 90 to 187 +/- 25 micromol/l; P < 0.01 vs. pancreas-transplant patients). FFA turnover and oxidation rates during both low-dose and high-dose insulin infusions were lower in pancreas-transplant patients compared with healthy subjects (P < 0.03 and P < 0.01, for turnover and oxidation, respectively). Uremic IDDM patients demonstration altered basal and insulin-mediated glucose metabolism. Pancreas transplantation normalized only insulin-mediated glucose oxidation, leaving the stimulation of non-oxidative glucose disposal still markedly defective. In conclusion, patients after pancreas transplantation have normal basal FFA turnover and reduced basal FFA oxidation rates. During hyperinsulinemia, pancreas-transplant patients show a normal inhibition of FFA turnover and FFA oxidation. Insulin-mediated glucose metabolism remained abnormal after pancreas transplantation. Our findings may be related to the effect of chronic immunosuppressive therapy on glucose and FFA metabolism.

Recombinant human growth hormone: rationale for use in the treatment of HIV-associated lipodystrophy.

The role of hormonal and metabolic alterations in HIV-associated lipodystrophy syndrome is not yet clear. In patients with HIV-1 undergoing antiretroviral treatment, lipodystrophy is associated with peripheral fat wasting and central adiposity, dyslipidemia, insulin resistance, and increased intramuscular fat accumulation.In HIV lipodystrophy, changes in fat distribution are heterogeneous and can include reduced subcutaneous fat as well as increased visceral fat. In the literature, there is evidence showing that overnight growth hormone (GH) secretion and pulse amplitude decrease in patients with HIV lipodystrophy, with rates of response to standardized GH stimulation being abnormal in at least 20% of these patients.Excess accumulation of visceral fat, central obesity, and increased intra-abdominal adiposity are also typical features of patients with GH deficiency. Recombinant human GH (rhGH) is a potential treatment to diminish excess visceral fat. Our group recently demonstrated that GH therapy in HIV-infected patients with syndromes of fat accumulation produced a significant decrease in body fat and a gain in lean tissue. In this article, we discuss the origin of lipodystrophy in HIV patients, and the use of rhGH treatment (benefits and adverse effects) in HIV-related lipodystrophy.

Effect of L-acetylcarnitine on body composition in HIV-related lipodystrophy.

This study examined the impact of L-acetylcarnitine treatment on metabolic parameters and body composition in patients with lipodystrophy syndrome secondary to antiretroviral treatment in human immunodeficiency virus (HIV) infection. A total of 9 HIV-1 infected patients with lipodystrophy syndrome (4F/5M, age 41+/-5 years, HIV duration 8+/-2 years, BMI 23.7+/-3.4 kg/m(2), on protease inhibitors and nucleoside analogue Reverse Transcriptase inhibitors) were evaluated before and after 8 months of therapy with L-acetylcarnitine (2 g/die) and 9 matched healthy subjects served as control subjects. In all patients fasting plasma glucose, insulin concentrations (for evaluation of surrogate indexes of insulin sensitivity), lipid profile, lipid oxidation (by indirect calorimetry), body composition (by DEXA), and intramyocellular triglyceride (IMCL) content of the calf muscles (by (1)H NMR spectroscopy) were assessed. After this therapy, in HIV-1 patients, the IMCL content of the soleus had significantly decreased (p=0.03). Plasma FFAs (0.79+/-0.31 to 0.64+/-0.25; p<0.05) and Respiratory Quotient (0.83+/-0.18 to 0.72+/-0.16; p<0.03) also decreased. Insulin sensitivity was significantly lower prior (HOMA-IS 0.56+/-0.30) and nonstatistically different than controls after therapy (0.72+/-0.49 vs. 0.78+/-0.42) whilst the percentage of fat in the legs increased (p=0.05). Eight months of L-acetylcarnitine treatment increased lipid oxidation, decreased intramyocellular triglyceride content, and induced a more physiological distribution of fat deposits.