A. Buongiorno

Acute hyperglycemia and acute hyperinsulinemia decrease plasma fibrinolytic activity and increase plasminogen activator inhibitor type 1 in the rat

Abstract Decreased plasma fibrinolysis may contribute to accelerated atherothrombosis in diabetes. To observe whether hyperglycemia and hyperinsulinemia, common findings in type 2 diabetes, acutely affect plasma fibrinolysis in vivo, we evaluated plasma fibrinolysis (lysis of fibrin plates, free PAI-1 activity and t-PA activity) in the rat after a hyperglycemic euinsulinemic clamp (n=8), an euglycemic hyperinsulinemic clamp (n=7) or a saline infusion (n=15). Plasma fibrinolytic activity was sharply reduced after both the hyperglycemic and hyperinsulinemic clamps as compared to the respective controls (mean lysis areas on the fibrin plate, 139±21 vs. 323±30 mm2, p<0.001; 78±27 vs. 312±27 mm2p<0.001, respectively). Plasma PAI-1 activity was greater after both hyperglycemic and hyperinsulinemic clamps as compared to saline infusion (6.6±2.6 vs. 1.6±0.6 IU/ml, p<0.001; 26±4 vs. 1.3±0.7 IU/ml, p<0.0001, respectively). Plasma t-PA activity was significantly reduced both after the hyperglycemic (0.36±0.15 vs. 2.17±0.18 IU/ml in controls, p<0.001) and the hyperinsulinemic (0.3±0.1 vs. 2.3±0.3 IU/ml in control, p<0.001) clamps. These data show that in vivo both acute hyperglycemia and acute hyperinsulinemia can decrease plasma fibrinolytic potential and that this is due to increased plasma PAI-1 and decreased free t-PA activities.

Increased adipose tissue PC-1 protein content, but not tumour necrosis factor-a gene expression, is associated with a reduction of both whole body insulin sensitivity and insulin receptor tyrosine-kinase activity

Summary In the present study we measured PC-1 content, tumour necrosis factor (TNF)-α gene expression, and insulin stimulation of insulin receptor tyrosine-kinase activity in adipose tissue from non-obese, non-diabetic subjects. These parameters were correlated with in vivo insulin action as measured by the intravenous insulin tolerance test (Kitt values). PC-1 content was negatively correlated with Kitt values (r = –0.5, p = 0.04) and positively with plasma insulin levels both fasting (r = 0.58, p = 0.009) and after 120 min during oral glucose tolerance test (OGTT) (r = 0.67, p = 0.002). Moreover, adipose tissue PC-1 content was higher in relatively insulin-resistant subjects (Kitt values lower than 6) than in relatively insulin-sensitive subjects (Kitt values higher than 6) (525 ± 49 ng/mg protein vs 336 ± 45, respectively, p = 0.012). Adipose tissue insulin receptor tyrosine-kinase activity in response to insulin was significantly lower at all insulin concentrations tested (p = 0.017, by two-way analysis of variance test) in insulin-resistant than in insulin-sensitive subjects (Kitt values lower or higher than 6, respectively). In contrast to PC-1, no significant correlation was observed between adipose tissue TNF-α mRNA content and Kitt values, and plasma insulin levels, both fasting and at after 120 min during OGTT. Also, no difference was observed in TNF-α mRNA content between subjects with Kitt values higher or lower than 6. These studies in adipose tissue, together with our previous studies in skeletal muscle raise the possibility that PC-1, by regulating insulin receptor function, may play a role in the degree of insulin sensitivity in non-obese, non-diabetic subjects. [Diabetologia (1997) 40: 282–289]

In vivo effects of glucosamine on insulin secretion and insulin sensitivity in the rat: possible relevance to the maladaptive responses to chronic hyperglycaemia.

SummaryWe tested the hypothesis that glucosamine, a putative activator of glucose toxicity in vitro through acceleration of the hexosamine pathway, may determine in vivo the two key features of glucose toxicity in diabetes, namely, peripheral insulin resistance and decreased insulin secretion. Two groups of awake rats were studied either with intraarterial administration of glucosamine (5 Μmol·kg−1· min−1) or saline. Insulin secretion was determined after arginine, glucose (hyperglycaemic clamp), and arginine/glucose infusions, while insulin-mediated glucose metabolism was assessed by the euglycaemic hyperinsulinaemic clamp in combination with [3-3H]-glucose infusion. Glucosamine had no effects on arginine-induced insulin secretion both at euglycaemia and hyperglycaemia, but significantly (40–50%) impaired glucose-induced insulin secretion (both first and second phases). During euglycaemic hyperinsulinaemic clamp studies, glucosamine decreased glucose uptake by ∼30%, affecting glycolysis (estimated from 3H2O rate of appearance) and muscle glycogen synthesis (calculated from accumulation of [3H]-glucosyl units in muscle glycogen) to a similar extent. Muscle glucose 6-phosphate concentration was markedly reduced in the glucosamine-infused rats, suggesting an impairment in glucose transport/phosphorylation. Therefore, an increase in hexosamine metabolism in vivo: 1) inhibits glucose-induced insulin secretion, and 2) reduces insulin stimulation of both glycolysis and glycogen synthesis, thereby mimicking in normal rats the major alterations due to glucose toxicity in diabetes.

Transgenic mice with dominant negative PKC‐theta in skeletal muscle: A new model of insulin resistance and obesity

Protein kinase C θ (PKC‐θ) is the PKC isoform predominantly expressed in skeletal muscle, and it is supposed to mediate many signals necessary for muscle histogenesis and homeostasis, such as TGFβ, nerve‐dependent signals and insulin. To study the role of PKC‐θ in these mechanisms we generated transgenic mice expressing a “kinase dead” mutant form of PKC‐θ (PKC‐θK/R), working as “dominant negative,” specifically in skeletal muscle. These mice are viable and fertile, however, by the 6–7 months of age, they gain weight, mainly due to visceral fat deposition. Before the onset of obesity (4 months of age), they already show increased fasting and fed insulin levels and reduced insulin‐sensitivity, as measured by ipITT, but normal glucose tolerance, as measured by ipGTT. After the 6–7 months of age, transgenic mice develop hyperinsulinemia in the fasting and fed state. The ipGTT revealed in the transgenic mice both hyperglycemia and hyperinsulinemia. At the molecular level, impaired activation of the IR/IRS/PI3K pathway and a significant decrease both in the levels and in insulin‐stimulated activation of the serine/threonine kinase Akt were observed. Taken together these data demonstrate that over‐expression of dominant negative PKC‐θ in skeletal muscle causes obesity associated to insulin resistance, as demonstrated by defective receptor and post‐receptorial activation of signaling cascade.

Relative contribution of glycogenolysis and gluconeogenesis to hepatic glucose production in control and diabetic rats. A re-examination in the presence of euglycaemia

Summary Several studies have suggested that, in non-insulin-dependent diabetes mellitus, augmented gluconeogenesis is responsible for increased endogenous glucose production (EGP) and in the end determines fasting hyperglycaemia. However, human and animal studies have been conducted by comparing euglycaemic control subjects to hyperglycaemic diabetic probands. We measured EGP and hepatic gluconeogenesis comparing control and diabetic rats in the fasting state (with diabetic animals in hyperglycaemia), re-examining them in the presence of identical euglycaemia (with diabetic rats made acutely euglycaemic through i. v. phloridzin) or during a hyperinsulinaemic clamp. All rats were infused with [3-3H]-glucose and [U-14C]-lactate; the ratio between 14C-uridine-diphosphoglucose (reflecting 14C-glucose 6-phosphate) and 2 ·14C-phosphoenolpyruvate specific activities (both purified by high performance liquid chromatography from liver) measured hepatic gluconeogenesis. In diabetic animals, although overall EGP ( ∼ 19.5 mg · kg–1· min–1) remained unaffected by experimental euglycaemia, the contribution of glycogenolysis largely increased (from 5.4 to 11.7 mg · kg–1· min–1, hyper- vs euglycaemia) while gluconeogenesis decreased (from 14.0 to 8.1 mg · kg–1æ min–1); both were responsible for the augmented EGP (control rats, EGP: 12.7 mg · kg–1· min–1; gluconeogenesis: 5.9 mg · kg–1· min–1; glycogenolysis: 6.7 mg · kg–1· min–1). Finally, during insulin clamp, gluconeogenesis and glycogenolysis were similarly decreased, and both contributed to the hepatic insulin-resistance of diabetic animals. We conclude that, in this model of non-insulin-dependent diabetes, augmented gluconeogenesis is not primarily responsible for fasting hyperglycaemia and hepatic insulin resistance. Finally, failure to accurately match the experimental conditions in which diabetic and control humans or animals are compared affects gluconeogenesis, overestimating its role in determining hyperglycaemia. [Diabetologia (1998) 41: 307–314]

Absence of clinically overt atherosclerotic vascular disease and adverse changes in cardiovascular risk factors in 70 patients with insulinoma.

Hyperinsulinemia has been assumed to contribute to the pathogenesis of atherosclerosis. To assess the reliability of such claim we planned a retrospective study on a cohort of patients with pancreatic insulin producing neoplasm. A correlation was sought between fasting insulin plasma levels and the metabolic profile emerging from those parameters known to be cardiovascular risk factors, i.e. plasma triglycerides and cholesterol, insulin resistance, hypertension. Special attention was paid to the duration of disease, because the time exposure to hyperinsulinemia could play an important role in developing cardiovascular disease. Seventy patients, 41 females and 29 males, aged 44.9±1.96 yr (range 15–80), with surgically proved insulinoma were included in the study. Chronic exposure to hyperinsulinemia was documented through the measurement of insulin plasma levels either in the fasting state or post-prandially, resulting in 44.7±3.28 and 149.9±12.22 μU/ml, respectively. Fasting glycemia in average was 45.3±1.34 mg/dl. Plasma triglycerides and cholesterol concentrations were 136.3±7.93 and 195.8±5.18 mg/dl, respectively, their distribution overlapping that anticipated for the general population. No correlation arose between the degree of hyperinsulinemia and the lipidic profile. Preoperative blood pressure was 136.9±2.87 mmHg, systolic and 81.9±1.32 mmHg, diastolic. Hypertension was present in 5 (7.1%) out of 70 patients and persisted after tumor removal. A condition of insulin resistance (M=4.06±0.4 mg/kg min vs 7.41±0.21) was documented through the euglycemic hyperinsulinemic clamp technique in 20 patients and showed a positive and significant correlation with fasting insulinemia. In conclusion, these data suggest that insulinomas do not develop an increased risk of cardiovascular disease and we believe that insulin needs the interplay of other factors to manifest its atherogenic potential effect.

Chronic primary hyperinsulinaemia is associated with altered insulin receptor mRNA splicing in muscle of patients with insulinoma

SummaryAlternative splicing of the 36-base pair exon 11 of the human insulin receptor gene results in the synthesis of two insulin receptor isoforms with distinct functional characteristics (the isoform containing exon 11 has lower insulin binding affinity and lower internalization rate). Altered expression of these insulin receptor isoforms has been previously demonstrated in skeletal muscle of patients with non-insulin-dependent diabetes mellitus (NIDDM). However, this observation was not confirmed by other studies and is still a matter of controversy; furthermore, it is not known whether it represents a primary event or is secondary to hyperinsulinaemia and insulin resistance. In order to address this issue in patients with pure non-genetically determined hyperinsulinaemia, we examined the alternative splicing of insulin receptor mRNAs in skeletal muscle of eight patients with surgically confirmed insulinoma and insulin resistance and in eight healthy subjects, using the reverse transcriptase-polymerase chain reaction technique. The insulinoma patients displayed a significant increase in the expression of the insulin receptor isoform containing exon 11 (75.7±2.3%) when compared with normal subjects (57.9±1.5%); furthermore, this increase was positively correlated with plasma insulin concentration and negatively correlated with in vivo insulin sensitivity (glucose clamp). In conclusion, the increased expression of the insulin receptor isoform with lower insulin binding affinity in patients with primary non-genetically determined hyperinsulinaemia supports a role for insulin in the regulation of alternative splicing of insulin receptor pre-mRNA and suggests that in NIDDM an altered receptor isoform distribution might be secondary to the ambient hyperinsulinaemia rather than representing a primary defect.

Relationship between plasma free fatty acids and uncoupling protein‐3 gene expression in skeletal muscle of obese subjects: in vitro evidence of a causal link

objective To investigate whether skeletal muscle uncoupling protein‐2 (UCP2) and uncoupling protein‐3 (UCP3) gene expression is altered in massive obesity and whether it correlates with in vivo insulin sensitivity and with metabolic and hormonal status.

Rats that are made insulin resistant by glucosamine treatment have impaired skeletal muscle insulin receptor phosphorylation

The current study sought to verify whether glucosamine (GlcN)-induced insulin resistance is associated with impaired insulin receptor (IR) autophosphorylation. Rats were given either saline or primed continuous GlcN infusion (5 micromol x kg(-1) x min(-1)) 10 minutes prior to and during euglycemic hyperinsulinemic clamp (primed continuous infusion of 20 mU x kg(-1) x min(-1) insulin for 2 hours). IR autophosphorylation was measured in skeletal muscle after in vivo insulin stimulation (ie, during clamp) by Western blot and then retested after subsequent in vitro 0.1 to 100 nmol/L insulin stimulation (by enzyme-linked immunosorbent assay [ELISA]). Tissue PC-1 enzymatic activity was also measured. In vivo, insulin/GlcN rats had decreased (P <.01) whole body glucose uptake (37.7 +/- 2.1 v 49.7 +/- 2.7 mg x kg(-1) x min(-1) in respect to insulin/saline), receptor autophosphorylation (37 +/- 5 v 82 +/-.0 arbitrary units/mg protein), and insulin receptor substrate-1 (IRS-1) phosphorylation (112% +/- 15% v 198% +/- 23% of saline infusion rats). Receptor autophosphorylation was correlated with whole body glucose uptake (r = 0.62, P <.05). Skeletal muscle PC-1 activity (58.8 +/- 10.7 v 55.7 +/- 5.8 nmol x mg(-1) x min(-1)) was not different in the 2 groups. Our data show that GlcN-induced insulin resistance is mediated, at least in part, by impaired skeletal muscle IR autophosphorylation.

Vasoactive intestinal peptide (VIP): a new neuroendocrine marker of clinical progression in chronic heart failure?

objective  Vasoactive intestinal peptide (VIP) is a powerful vasodilatory neuropeptide with positive inotropic and chronotropic properties. The aim of the study was to investigate the pathophysiological role of VIP in heart failure.