Francesco Belfiore

Animal and human tissue Na,K-ATPase in obesity and diabetes: A new proposed enzyme regulation.

Background:Na,K-ATPase is a membrane enzyme that energizes the Na-pump, hydrolyzing ATP and wasting energy as heat. It may play a role in thermogenesis, energy balance, and obesity development. Regulation of the enzyme by insulin is controversial. Methods:In animal and human obesity, tissue Na,K-ATPase was assayed by colorimetric measurement of released Pi. Results:Na,K-ATPase of hyperglycemic-hyperinsulinemic ob/ob mice (compared with lean control animals) was reduced in liver (−63%) and in kidney (−47%) (P < 0.001 in both instances). In contrast, in streptozotocin-treated hypoinsulinemic-diabetic Swiss mice, versus untreated animals, we found an increase of liver (+54%, P < 0.01) and kidney (+94%, P < 0.001) Na,K-ATPase. The enzyme was also increased (+99%, P < 0.05) in kidney from ob/ob mice made diabetic-hypoinsulinemic with streptozotocin (versus untreated obese animals). This is contrary to the occurrence of a genetic enzymatic defect and suggests regulation by hyperinsulinemia, present in ob/ob mice. A positive correlation between tissue enzyme activity and glycemia existed in both ob/ob and Swiss mice. In adipose tissue from obese patients (compared with lean subjects), Na,K-ATPase was reduced (−65%, P < 0.001) and negatively correlated with body mass index, oral glucose tolerance test—insulinemic area, and mean blood pressure. In vitro, in human liver tissue, 3 &mgr;g/mL glucagon exerted a statistically inhibitory effect on Na,K-ATPase (−44%). Conclusion:We hypothesize that animal and human obesity is associated with reduction of tissue Na,K-ATPase, linked to hyperinsulinemia, which may repress or inactivate the enzyme, influencing thermogenesis and energy balance.

Rheumatoid Syndrome Associated with Lung Interstitial Disorder in a Dental Technician Exposed to Ceramic Silica Dust. A Case Report and Critical Literature Review

Abstract Exposure to silica minerals is associated with silicosis and autoimmune disorders, especially systemic scleroderma. Evidence of this association has been increasingly reported in the last decade. The aim of this paper is to discuss, on the basis of a literature review, the case of a 28-year-old female dental technician who suffered from episodes of weakness, arthralgia, pain, swelling and stiffness of the fingers, dyspnoea with cough, a positive Waaler–Rose reaction, increased rheumatoid factor and normal ESR. She was a non-smoker. A rheumatoid syndrome with lung interstitial disorder, associated with silica exposure from dental ceramic products, was diagnosed. The patient had the HLA-A2-A31, HLA-B51-B18 and HLA-DR3-DR11 haplotypes, some of which are associated with autoimmune disease susceptibility. A 6-month follow-up, with adequate protection and without treatment, showed disappearance of the symptomatology and negative tests for Waaler–Rose reaction and rheumatoid factor. Exposure to silica should, therefore, be sought in the history of any patient with autoimmune or lupus-like syndrome and pulmonary changes. Symptoms associated with silica dust exposure from dental ceramic products should be recognised as being due potentially to an occupational disease, and dental technicians should be protected as workers at risk.

Animal and human tissue Na,K-ATPase in normal and insulin-resistant states: regulation, behaviour and interpretative hypothesis on NEFA effects.

The sodium(Na)‐ and potassium(K)‐activated adenosine‐triphosphatase (Na,K‐ATPase) is a membrane enzyme that energizes the Na‐pump by hydrolysing adenosine triphosphate and wasting energy as heat, so playing a role in thermogenesis and energy balance. Na,K‐ATPase regulation by insulin is controversial; in tissue of hyperglycemic‐hyperinsulinemic ob/ob mice, we reported a reduction, whereas in streptozotocin‐treated hypoinsulinemic‐diabetic Swiss and ob/ob mice we found an increased activity, which is against a genetic defect and suggests a regulation by hyperinsulinemia. In human adipose tissue from obese patients, Na,K‐ATPase activity was reduced and negatively correlated with body mass index, oral glucose tolerance test‐insulinemic area and blood pressure. We hypothesized that obesity is associated with tissue Na,K‐ATPase reduction, apparently linked to hyperinsulinemia, which may repress or inactivate the enzyme, thus opposing thyroid hormones and influencing thermogenesis and obesity development. Insulin action on Na,K‐ATPase, in vivo, might be mediated by the high level of non‐esterified fatty acids, which are circulating enzyme inhibitors and increase in obesity, diabetes and hypertension. In this paper, we analyse animal and human tissue Na,K‐ATPase, its level, and its regulation and behaviour in some hyperinsulinemic and insulin‐resistant states; moreover, we discuss the link of the enzyme with non‐esterified fatty acids and attempt to interpret and organize in a coherent view the whole body of the exhaustive literature on this complicated topic.

Enzymes of Glucose Metabolism in Liver of Subjects with Adult-onset Diabetes

An enzyme study was made on needle biopsy specimens of liver from thirty-two subjects with adult-onset diabetes and normal body weight and thirty-two controls. The enzyme pattern in the patients with diabetes was different from that seen with alloxan diabetes. The activities of the two glucose phosphorylating enzymes tested were changed in opposite directions, hexokinase being enhanced and glucokinase moderately decreased. Total glucose phosphotransferase activity remained unchanged. Phosphofructokinase had a reduced activity, which suggested depressed glycolysis, especially if considered together with the enhanced activity of the opposing enzyme, fructose-1, 6-diphosphatase. Normal activity was found for most other glycolytic enzymes, as well as for key gluconeogenic enzymes, including glutamic oxalacetic and glutamic pyruvic transaminases, phosphoenolpyruvate carboxykinase and glucose-6-phosphatase. The finding suggests normal glucose release. Glucose-6-phosphate- and 6-phosphogluconate dehydrogenase activity was elevated. This would indicate an increased metabolism of glucose through the oxidative pathway and, therefore, increased formation of NADPH. This metabolic condition, which is known to favor fatty acid synthesis, might contribute to fatty liver changes. On the other hand, NADP-isocitrate dehydrogenase, which does not provide NADPH for fatty acid synthesis, was little changed.

Enzymes of Glucose Metabolism and of the Citrate Cleavage Pathway in Adipose Tissue of Normal and Diabetic Subjects

Enzyme activities operative in glucose degradation and citrate cleavage pathway were studied in the adipose tissue of twenty-four patients with adult-onset diabetes and normal body weight, aged 59±9 years, and twenty-four matched controls. In normal tissue, type II (heat-inactivated) hexokinase moderately predominated over type I (heat-resistant). 6-Phosphofructokinase had an extremely low activity, which was by far the lowest among the ten glycolytic enzyme activities investigated, and which therefore might greatly limit the glycolytic rate. The level of glucose-6-phosphate dehydrogenase and phosphogluconate dehydrogenase (decarboxylating) was elevated above that occurring in other tissues. This, especially if considered together with the low 6-phosphofructokinase activity, would suggest a major role of pentose cycle in glucose degradation. Of the citrate cleavage pathway enzymes, ATP citrate-lyase, although having a lower activity than malate dehydrogenase and malate dehydrogenase (decarboxylating) (NADP), was readily measurable, which contrasts with previous data by others. This finding is consistent with the occurrence of lipogenetic capacity in human adipose tissue. In diabetic tissue, there was a decreased activity, both on a protein and on a wet-weight basis, of enzymes concerned with the glucose entry into metabolic pathways, namely hexokinase (both type I and, especially, type II) and pentose cycle dehydrogenases, as well as of pyruvate kinase. This could be connected with the defective glucose utilization by adipose tissue in diabetes. Beside the above-mentioned dehydrogenases, malate dehydrogenase (decarboxylating) (NADP) was also diminished. The reduction of these NADPH-forming enzymes, which supply reducing equivalents for fatty acid synthesis, would suggest a depressed lipogenesis.

The glucose-6-phosphatase/glucokinase ratio in the liver of obese-diabetic subjects.

The study of G6Pase and GK activities in human liver (needle biopsies) in overnight fasted obese NIDDM patients has shown that, while G6Pase was unchanged, GK was higher (+ 55%, P less than 0.05) than in control subjects. Consequently, the G6Pase/GK ratio (which roughly reflects hepatic glucose production) was significantly reduced (-36%) in the obese diabetic group, due to more GK activity (glucose uptake). This contrasts with the activity in IDDM and nonobese NIDDM patients (where the G6Pase/GK ratio is elevated and normal, respectively) and would suggest that in the obese diabetic subjects, hepatic glucose production is not a major factor contributing to the maintenance of hyperglycemia in the overnight fasting state (leaving peripheral insulin resistance as the major cause of hyperglycemia).

Effects of short-term metformin treatment on insulin sensitivity of blood glucose and free fatty acids

Aim:  Based on the known effect of metformin (MET) in improving insulin sensitivity in type 2 diabetes, with the scope to focus the effects on glycaemic and free fatty acids (FFA) levels, we studied the effects of a short‐term treatment with this drug in obese subjects and obese patients with diabetes or family history of diabetes (FHD). We used a method to allow us to evaluate the possible difference of insulin sensibility with regard to the insulin action on glycaemia and blood FFA, both in the basal state and during oral glucose tolerance test (OGTT).

A Mild Form of Alstrom Disease Associated with Metabolic Syndrome and Very High Fasting Serum Free Fatty Acids: Two Cases Diagnosed in Adult Age

&NA; Alstrom syndrome (ALMS) is a very rare genetic autosomal recessive disease, characterized by early‐onset severe abdominal obesity, impaired glucose tolerance or type 2 diabetes with insulin resistance, acanthosis nigricans, hyperlipidemia, childhood progressive retinal degeneration or retinitis pigmentosa and neurosensory hearing loss or deafness, cardiomyopathy, and other endocrine disorders. Genetic studies locate the ALMS gene on chromosome 2p12–13. The aim of this paper is to describe and discuss two unrelated cases of a mild ALMS form diagnosed after the age of 40 and 60, respectively, in adult fertile female patients. These cases showed several features of the disease plus other alterations characteristic of the classic “metabolic syndrome,” including hypertension, hyperfibrinogenemia, and thrombotic states. Moreover, the patients had very high fasting serum free fatty acid (FFA) levels (2150 and 1919 &mgr;mol/L, respectively), which proved to be sensitive to inhibition by oral glucose tolerance test (OGTT)–induced hyperinsulinemia as well as to caloric restriction. ALMS may have an adverse prognosis and is often underdiagnosed. Its mild form, which allows a long survival, may also be associated with the late complications of the metabolic syndrome, leading to increased vascular risk.

Enzymes related to lipogenesis in the adipose tissue of obese subjects

In a group of ten adult obese subjects, maintained for 15 days on a normal caloric intake and balanced diet, the activity of hexokinase (EC 2.7.1.1),6-phosphofructokinase (EC 2.7.1.11), and ATP citratelyase (EC 4.1.3.8) in the adipose tissue was significantly increased, both on a protein and on a fat cell number basis, compared to matched normal subjects. The activity of glucose-6-phosphate dehydrogenase (EC 1.1.1.49), malate dehydrogenase (EC 1.1.1.37), and malate dehydrogenase (decarboxylating) (NADP) (EC 1.1.1.40), on the other hand, was unchanged. Since both hexokinase and 6-phosphofructokinase are rate-limiting in glycolysis, their enhanced activity would indicate the occurrence of an increased capacity to metabolize glucose and therefore to generate alpha-glycerophosphate. The elevation of ATP citrate-lyase would suggest increased lipogenesis, owing to the regulatory role that this enzyme plays in fatty acid synthesis. The normal activity of glucose-6-phosphate dehydrogenase and malate dehydrogenase (decarboxylating) (NADP), which supply NADPH for the reduction of acetyl-CoA to fatty acids, would suggest that the change in lipogenesis is of moderate degree, thereb) affecting only the most rate-limiting enzyme, ATP citrate-lyase. These data, on the whole, are consistent with the occurrence of enhanced triglyceride formation. Whether the enzyme changes observed are adaptive or genetic in nature remains to be clarified.

A Large Spontaneous Splenorenal Shunt in a Patient with Liver Cirrhosis and Uncomplicated Portal Hypertension

The aim of this paper is to describe and discuss, on the basis of a thorough review of the literature, the case of a 70-year-old woman with probable cirrhosis secondary to chronic hepatitis B and C, uncomplicated portal hypertension (without ascites, encephalopathy or bleeding varices), splenomegaly and hypersplenism, and an unusual, spontaneous, large splenorenal shunt and recanalization of the umbilical vein. The tortuous and varicose splenorenal shunt was diagnosed by abdominal ultrasound and CT investigations. A duplex Doppler ultrasonography evaluation was performed to study shunt flow direction and velocity. No gastroesophageal varices were identified on endoscopic examination. The clinical relevance of spontaneous splenorenal shunt, often associated with fundic gastric varices, is discussed.