Maria Mozzoli

Acetone Metabolism During Diabetic Ketoacidosis

The presence and the importance of acetone and its metabolism in diabetic ketoacidosis has largely been ignored. Therefore, we studied acetone metabolism in nine diabetic patients in moderate to severe ketoacidosis. The concentration of acetone in plasma, urine, and breath, and the rates of acetone production and elimination in breath and urine were determined and the rates of vivo metabolism were calculated. Plasma acetone concentrations (1.55–8.91 mM) were directly related and were generally > acetoacetate concentrations (1.16–6.08 mM). The rates of acetone production ranged from 68 to 581 μmol/min/1.73 m2, indicating the heterogeneous nature of the patients studied. The average acetone production rate was 265 μmol/min/1.73 m2 and accounted for about 52% of the estimated acetoacetate production rate. Urinary excretion of acetone remained constant and accounted for about 7% of the acetone production rate in all patients. There was a positive linear relationship between the percentage of the acetone production rate accounted for by excretion in breath and the plasma acetone concentration. At low plasma acetone concentrations, ∼ 20%, and at high plasma acetone concentrations, ∼ 80% of the production rate was accounted for by breath acetone. In contrast, there was a negative linear relationship between the percentage of acetone production rate undergoing in vivo metabolism and plasma acetone concentration. At low plasma acetone concentrations, ∼ 75%, and at high concentrations, ∼ 20% of acetone production rate was accounted for by in vivo metabolism. Radioactivity from 2-[14C]-acetone was variably present in plasma acetone, glucose, lipids and proteins. No radioactivity was found in plasma acetoacetate, beta-hydroxy butyrate or free fatty acids or other anionic compounds. Exchange rates of acetone into other metabolites could not be estimated because of non-steady-state precursor product relationships in these patients.

6Ketosis of starvation: A revisit and new perspectives

During starvation ketone bodies, acetoacetate (AcAc), 3-hydroxybntyrate (fl-OHB) and acetone accumulate in the body fluids. AcAc and /3-OHB are synthesized in the liver primarily from the partial oxidation of long-chain fatty acids. They are released into the blood as short-chain fatty acids, dissociate to become water-soluble anions and are distributed at different concentrations in the water components of the body (Owen et al, 1973). Acetone is probably formed by spontaneous decarboxylation of AcAc. Acetone is a neutral compound, and, unlike AcAc and/3-OHB, it does not affect blood bicarbonate concentration, arterial blood gases or pH (Sulway and Malins, 1970). Acetone is soluble in both water and lipids, and therefore it is distributed throughout the body (Reichard et al, 1979). During food deprivation, starvation ketosis is arbitrarily defined as being present when the minimum blood/plasma concentration of AcAc is about 1.0 mmol/1. Concurrent concentrations of/3-OHB and acetone are usually about 2.0 and 0.5 mmol/1, respectively. Such values are usually present after 2- 3 days of total starvation. Maximal blood/plasma concentrations of AcAc (2- 4 mmol/1),/3-OHB (5 - 12 retool/l) and acetone (3 -5 mmol/l) develop after several weeks of total fasting. The arbitrary definition of starvation ketosis is centred on the plasma/serum AcAc concentration, because the only semiquantitative test preparations, widely available to detect the presence of ketone bodies in biological fluids, depend upon the reagent nitroprusside to react with AcAc. Contrary to popula~ opinion, the presence of acetone or/3-OHB does not augment nitroprusside reactivity with AcAc. Plasma/serum/urine concentrations of AcAc below 0.5 mmol/1 are nonreactive with commercially available diagnostic tests. However, these semiquantitative tests develop reactions that are roughly concentration dependent. Nitroprusside tests show 1 + (trace to small) reactivity with 1 -2 mmol AcAc, 2 + (small to moderate) reactivity with 3 -4 mmol AcAc and 3 + (moderate to large) reactivity with 5 - 10 mmol AcAc. Thus, high

Effects of Therapy on the Nature and Quantity of Fuels Oxidized During Diabetic Ketoacidosis

We studied seven patients, in moderate to severe diabetic ketoacidosis (DKA), measuring respiratory exchanges of O2, GO2, and acetone and urinary excretion of nitrogen, ketone bodies, and glucose to calculate the respiratory quotient (RQ), nonprotein respiratory quotient (npRQ), metabolic requirements, and calories derived from fat, carbohydrate, and protein oxidation. Results from indirect calorimetry were related to circulating concentrations of glucose, free fatty acids, ketone bodies, and amino acids over a 14-h study consisting of a 2-h period I of rehydration with saline, a 4-h period II of rehydration and insulin therapy, and an 8-h period III of rehydration, insulin, and glucose administration. During period I, of about 2 h of saline rehydration, the RQ (0.55–0.80) and npRQ (0.58–0.88) varied among the patients but in general was low. The caloric requirements were 1.24 kcal/min/1.73 m2. Initially,fat contributed 78 ± 11%, glucose 17 ± 10%, and protein 5 ± 2% of the metabolic requirements. The circulating concentrations of fuels remained constant. During period II, after about 4 h of saline and insulin therapy, the RQ (0.62–0.88) and npRQ (0.55–0.91) remained rather stable, rising in only two of seven patients. Nevertheless, in all patients, saline and insulin therapy was associated with precipitous decreases in circulating concentrations of glucose, free fatty acids, acetoacetate, and beta-hydroxybutyrate and gradual decreases in plasma amino acids. During period III, after 8–12 h of insulin therapy, the RQ (0.68–0.92) and npRQ (0.48–1.01) increased, rising in five of six patients. Heightened RQ and npRQ values were observed only after plasma free fatty acid concentrations decreased to 0.44 ± 0.12 mM and plasma acetoacetate plus beta-hydroxybutyrate concentrations decreased to 5.27 ± 1.86 mM, while plasma glucose concentration remained elevated at 13.49 ± 3.67 mM because of intravenous glucose infusion. Caloric requirements diminished progressively throughout the study, and after about 4 h of saline and insulin therapy a reciprocal relationship between the contributions of fat and glucose to metabolic requirements was evident. At the end of period III the caloric requirements were 0.77 kcal/min/1.73 m2. Fat contributed 44 ± 16%, glucose 42 ± 22%, and protein 14 ± 8% of the metabolic requirements. We have observed a dissociation between the decrease in plasma glucose, free fatty acids, ketone bodies, and amino acids and the nature of fuels oxidized. This suggests that, during the initial hours of therapy for DKA, the predominant effect of insulin is to promote fuel storage rather than to promote glucose and ketone body oxidation. It was 8–12 h in the course of therapy before the npRQ rose, reflecting heightened glucose oxidation and diminished fat oxidation. Metabolic requirements progressively decreased with therapy.

Substrate, hormone, and temperature responses in males and females to a common breakfast

To evaluate the response to a mixed meal we studied oral temperature, metabolite, and hormonal responses to a common American breakfast containing 11 kcal/kg body weight (carbohydrate 43%, fat 42%, and protein 15%) in 12 normal volunteers (6 males and 6 females). There was a significant rise in oral temperature during the postcibal period. This change in oral temperature did not depend upon food consumption in males but was meal-dependent in females. Food ingestion caused increases in the peripheral circulating concentrations of glucose, lactate, pyruvate, and amino acids and reciprocal decreases in the concentrations of free fatty acids, glycerol, and urea nitrogen. Acetoacetate and beta-hydroxybutyrate decreased during the postcibal period but the changes were not statistically significant. Although peripheral venous serum insulin and plasma glucagon concentrations were indistinguishable between the sexes, males had higher concentrations of plasma triglycerides, plasma amino acids, and serum urea nitrogen. Peripheral venous plasma somatostatin and secretin remained unchanged, but pancreatic polypeptide hormone showed a large biphasic response to the meal. After breakfast the blood glucose concentration tended to be greater in males than in females and this difference was significant at 60 and 120 min postcibal. Furthermore, every female had a 120 min postcibal glucose concentration that was lower than her basal fasting glucose concentration. This suggests that postcibal glucose concentrations should be related to gender in making the diagnosis of carbohydrate intolerance or reactive hypoglycemia.

Combined Use of Rosiglitazone and Fenofibrate in Patients With Type 2 Diabetes Prevention of Fluid Retention

Elevated plasma free fatty acid (FFA) levels are responsible for much of the insulin resistance in obese patients with type 2 diabetes. To lower plasma FFA levels effectively and long term, we have treated eight obese patients with type 2 diabetes for 2 months with placebo followed by 2 months of treatment with a combination of rosiglitazone (RGZ) (8 mg/day) and fenofibrate (FFB) (160 mg/day) in a single-blind placebo-controlled study design. Compared with placebo, RGZ/FFB lowered mean 24-h plasma FFA levels 30% (P < 0.03) and mean 24-h glucose levels 23% (P < 0.03) and increased insulin-stimulated glucose uptake (glucose rate of disappearance [GRd], determined using euglycemic-hyperinsulinemic clamp) 442% (P < 0.01), oral glucose tolerance (area under the curve for 3-h oral glucose tolerance test) 28% (P < 0.05), and plasma adiponectin levels 218% (P < 0.01). These RGZ/FFB results were compared with results obtained in five patients treated with RGZ alone. RGZ/FFB prevented the fluid retention usually associated with RGZ (−1.6 vs. 5.6%, P < 0.05), lowered fasting plasma FFA more effectively than RGZ alone (−22 vs. 5%, P < 0.05), and tended to be more effective than RGZ alone in lowering A1C (−0.9 vs. −0.4%) and triglyceride levels (−38 vs. −5%) and increasing GRd (442 vs. 330%). We conclude that RGZ/FFB is a promising new therapy for type 2 diabetes that lowers plasma FFA more than RGZ alone and in contrast to RGZ does not cause water retention and weight gain.

Effects of Ethanol on Carbohydrate Metabolism in the Elderly

We have previously reported that in young men, ethanol caused acute insulin resistance, but compensatory insulin secretion prevented deterioration of glucose tolerance (1). In this study, we tested the hypothesis that elderly men, because of their pre-existing insulin resistance and compromised insulin secretory capacity, may experience worsening of their glucose tolerance after ethanol. Nine elderly men (65.7 ± 0.8 yr, BMI 25.8 ± 1.4 kg/m2) received ethanol (13 mmol/kg for 30 min i.v.) or saline followed 30 min later by i.v. glucose (2.8 mmol/kg for 5 min). To determine the mechanism of the ethanol effect, six of the men underwent euglycemic-hyperinsulinemic (∼350 pM) clamping with simultaneous infusion of ethanol or saline. Muscle biopsies were obtained before and 1 and 4 h after insulin infusion. In all nine men, glucose concentrations after i.v. glucose were higher after ethanol than after saline, whereas insulin was the same and glucose tolerance decreased by 23% (Kg 2.41 ± 0.2 vs. 1.86 ± 0.1%/min, P < 0.01). Ethanol reduced insulin-stimulated glucose uptake from 40.6 ± 3.1 to 25.6 ±1.9 μmo; · kg−1 · min−1 (−37%, P < 0.05), glucose oxidation from 11.7 ± 1.1 to 7.0 ±0.7 μmol · kg−1 · min−1 (−33%, P < 0.01), and glucose storage from 28.7 ± 2.4 to 18.6 ±1.7 μmol · kg−1 · min−1(−35%, P <0.01). Ethanol increased muscle lactate concentration from 0.49 ± 0.14 to 1.99 ± 0.99 μmol/mg protein (P < 0.05), but had no effects on muscle concentration of freeglucose, G-6-P, and citrate concentrations, nor did it affect muscle GS activity. We concluded that modest amounts of ethanol in elderly men impaired glucose oxidation and caused insulin resistance, which because of lack of compensatory insulin secretion, resulted in deterioration of glucose tolerance.

Glucose Transporter Proteins in Human Insulinoma

A major pathophysiologic abnormality in patients with insulinoma is their uncontrolled insulin secretion, particularly their inability to decrease and eventually completely shut off insulin release when plasma glucose concentrations decrease to lower than 2.0 to 3.0 mmol/L. This frequently causes hypoglycemia, the clinical hallmark of the disorder [1]. The cause for this abnormal insulin release during hypoglycemia is unknown; it is also not completely understood how under normal conditions hypoglycemia inhibits insulin release. It has been generally accepted, however, that glucose needs to be transported into -cells and metabolized to generate the biochemical signal (the nature of which remains elusive) responsible for the initiation of insulin secretion [2]. Glucose is transported into cells by several different transport proteins (Glut 1 through Glut 5), which are tissue-specific. Glut 2, a 522 amino acid peptide with a calculated molecular mass of 57 kd [3, 4], is the only glucose transporter protein reported to be present in normal -cells. Its low affinity for glucose (Km, 15 to 20 mmol/L) enables Glut 2 to increase glucose transport into -cells when the extracellular glucose concentration increases to greater than 5.0 mmol/L and to decrease the transport when the glucose concentration decreases. Glut 2 has therefore been proposed as a -cell glucose sensor [5, 6]. Oncogenic transformation of animal -cells has been associated with increased expression of Glut 1, a glucose transporter with high affinity for glucose (Km, 1.5 to 2 mmol/L) [7] and with abnormal insulin secretory responses to glucose. For instance, RINm5F insulinoma cells [8], which contain both Glut 1 and Glut 2 [3], released insulin maximally at a glucose concentration of 2.8 mmol/L [9]. Another insulinoma cell line (MIN7) obtained from transgenic mice also contains Glut 1 and Glut 2 and released similar amounts of insulin at glucose concentrations of 0.7 and 2.5 mmol/L [10]. These observations suggest that abnormal expression of glucose transport proteins (that is, an increased amount of Glut 1 or a decreased amount of Glut 2 or both) in islet cell tumors may be a reason for the abnormal insulin release from these tumors. The nature of the glucose transporter proteins present in human insulinoma is unknown and was, therefore, the objective of our study. Methods Patients and Tumors Clinical characteristics of five patients with the clinical insulinoma syndrome and their tumors are shown in Table 1. In four patients in the General Clinical Research Center of Temple University Hospital (patients 1, 2, 3, and 5) inappropriate hyperinsulinemia (insulin concentration, 30 pmol/L [5.0 U/mL]) occurred during hypoglycemia (glucose concentration, 2.2 mmol/L [40 mg/dL]) that was induced by fasting. In patient 4, who was on a surgical ward of the hospital, hypoglycemia was documented during an episode of hypoglycemia that occurred after an overnight fast before breakfast. As determined in our laboratory, none of the five patients had insulin-binding antibodies, and none had detectable levels of blood sulfonylurea (determined by Smith-Kline Bioscience Laboratories, King of Prussia, Pennsylvania). Pancreatic masses were identified in all patients by ultrasound or computed tomographic scans or during exploratory laparotomy. The surgically removed tumors were diagnosed as insulinomas by light and electron microscopy. After resection, the tumors were immediately rinsed with ice-cold saline, weighed, measured, and frozen at 80C. Table 1. Patients and Tumors Acid Ethanol Extraction Pieces of the frozen tumors were finely minced with scissors and extracted overnight at 4 C with acid ethanol [11] for measurement of immunoreactive insulin and glucagon content. Preparation of Solubilized Membranes Aliquots of frozen tumor or normal human liver were homogenized with a polytron in a medium containing 25 mmol/L of HEPES, 4 mmol/L of ethylenediaminetetraacetic acid, 25 mmol/L of benzamidine, and 1 M each of leupeptine, pepstatin, and aprotinin and 200 M of phenazine methosulfate. Triton X-100 (final concentration of 1%) (Union Carbide, Danbury, Connecticut) was then added to make the homogenate soluble. The suspension was shaken for 60 minutes at 4 C, and the solubilized membranes were centrifuged at 150 000 g for 35 minutes. The supernatant fluid was filtered through a 0.45-micrometer filter. Its protein concentration was determined [12], and samples were applied directly to nitrocellulose membranes for immune blotting (2 to 20 g of protein per assay) [13]. Immune Blotting of Glucose Transporter Proteins The nitrocellulose membranes into which test samples had been blotted were incubated for 2 hours with TRIS buffer containing 5% bovine serum albumin and specific antihuman Glut antiserum. The Glut 1 antiserum RAB 379 was directed against the amino acid sequence 480-492 of rat Glut 1, which is identical to the same sequence in human Glut 1 [14]. The Glut 2 antiserum was directed against the C-terminal 30 amino acids of human Glut 2. A mouse antirabbit IgG antibody (provided by Sigma Chemical Co., St. Louis, Missouri) and a Iodine-125-labelled sheep antimouse antibody were added as second and third antibodies. Membranes were then subjected to autoradiography and were counted in a counter [13]. Fasting Patients were admitted to the General Clinical Research Center of Temple University Hospital. A flexible intravenous catheter with a heparin lock was inserted into the antecubital vein of one arm. Patients were permitted to move around freely and had access to water as desired, but they did not receive any calories. Routine chemistries, complete blood count plus differential, and serum insulin and sulfonylurea concentrations were measured in blood samples at the beginning of the fast. Thereafter, blood samples were drawn every 2 hours for glucose determinations. Aliquots of these samples were kept for later determination of insulin. When the fasting blood sugar concentration decreased to less than 2.8 mmol/L (50 mg/dL), samples were taken every 30 minutes; when it decreased to less than 2.2 mmol/L (40 mg/dL), three blood samples were taken (one every 5 minutes), and the fast was terminated by intravenous infusion of glucose. Analytical Procedures Plasma glucose was measured with a Beckman Instruments (Palo Alto, California) glucose analyzer. Serum insulin [15] and plasma glucagon [16] concentrations were measured by radioimmunoassay and the C-peptide concentration was measured with a double-antibody radioimmunoassay kit (INCSTAR, Stillwater, Minnesota). Results Insulin and C-peptide Concentrations during Hypoglycemia Figure 1 and Table 2 show the insulin and C-peptide concentrations during hypoglycemia. Patients 1, 2, 3, and 5 underwent a fast in the General Clinical Research Center until hypoglycemia developed after 20, 10, 25, and 9 hours of fasting, respectively. In patient 4, hypoglycemia developed on a surgical ward after an overnight fast that lasted approximately 12 hours. Table 2. Insulin and C-peptide Concentrations and Symptoms during Hypoglycemia in Five Patients with Insulinoma Figure 1. Serum insulin concentrations at various plasma glucose concentrations observed during fasting in four patients with insulinoma. In patient 1, serum insulin concentrations increased paradoxically when blood sugar levels decreased during the fast. This patient was found to have a multinodular, malignant insulinoma and metastatic tumors in the liver. In patients 2 to 5, who had mononodular, nonmetastatic insulinomas, serum insulin and C-peptide concentrations decreased during the fast. However, in none of the five patients did serum insulin concentrations decrease to lower than 30 pmol/L, nor did C-peptide concentrations decrease to lower than 0.08 nmol/L when plasma glucose concentrations had decreased to 2.2 mmol/L or lower. Moreover, all five patients showed central nervous system signs of hypoglycemia, which disappeared promptly after intravenous administration of glucose. Glut 1 and Glut 2 Figure 2 shows the Glut 1 and Glut 2 concentrations in tumor membranes of four patients. Total insulinoma membranes of patient 1 contained exclusively Glut 1 protein. Tumors of the other four patients contained what appeared to be small amounts of Glut 2 and substantial amounts of Glut 1. Only Glut 2 protein was detected in a sample of a normal human liver. Thus, patterns of distribution of Glut 1 and Glut 2 in insulinoma were completely different than those in tissues from a normal liver. Figure 2. Glucose transporter protein concentrations in tumor membranes from four patients with insulinoma and in liver membranes from one healthy person. Discussion We studied five patients with the clinical insulinoma syndrome. During hypoglycemia induced by fasting (blood glucose concentration, <2.2 mmol/L), endogenous insulin release in all five patients was not appropriately suppressed. The islet cell tumors removed from these patients contained what appeared to be small amounts of Glut 2 protein (four patients) or no Glut 2 (one patient) and an abundance of Glut 1 protein as determined with specific anti-human Glut 1 and Glut 2 antisera. It should be emphasized, however, that this technique does not permit quantitative comparison between Glut 1 and Glut 2. Because normal human pancreatic islets were unavailable, we used a tissue sample from a normal human liver as a control. Both islets and the liver have been reported to almost exclusively contain Glut 2 and little or no Glut 1 protein [3, 4, 6, 17]. In concordance with these reports, only Glut 2 was detected in the liver membranes. To our knowledge, no reports have been published on glucose transporter protein content in human insulinoma. However, our findings are in accordance with a recent report by Seino and colleagues [18], who found Glut 1 messenger RNA but no Glut 2 messenger RNA in two cases of human insulinomas [18]. Their failure to dete

Effects of hyperinsulinemia on hepatic metalloproteinases and their tissue inhibitors

To gain insight into the pathogenesis of hepatic fibrosis related to insulin resistance, we have examined the effects of euglycemic hyperinsulinemia on three matrix metalloproteinases (MMP-2, MMP-9, and MT1-MMP) and on two major tissue inhibitors of MMPs (TIMP-1 and TIMP-2) in liver of insulin-sensitive and insulin-resistant rats. Four hours of insulin infusion (4.8 mU.kg(-1).min(-1)) without or with lipid-heparin infusion (to produce insulin resistance) decreased hepatic MMP-2 mRNA (by RT-PCR), pro-MMP-2, MMP-2, MMP-9, and MT1-MMP (all by Western blots) and the gelatinolytic activity of MMP-2 (by gelatin zymography) by approximately 60-80%. Hyperinsulinemia ( approximately 1.6 mmol/l) increased TIMP-1 and TIMP-2 concentrations (by ELISA) in insulin-sensitive and insulin-resistant rats. Phosphoinositide 3-kinase was activated by insulin in insulin-sensitive rats and inhibited in insulin-resistant rats. Extracellular signal-regulated kinases 1/2 (ERK1/2) were activated by insulin in insulin-sensitive rats and partially inhibited in insulin-resistant rats; c-jun NH(2)-terminal kinase-1 (JNK1), JNK2/3, or p38 MAPK were only activated by lipid but not by insulin. We conclude that hyperinsulinemia, whether or not associated with insulin resistance, shifts the MMP/TIMP balance toward reduction of extracellular matrix degradation and thus may promote the development of hepatic fibrosis.

Improvement of metabolic control in diabetic patients during mebendazole administration: Preliminary studies

SummaryAfter the observation of decreasing insulin resistance in a diabetic patient during treatment with mebendazole for nematosis, we investigated the effect of mebendazole on metabolic control in six Type 1 (insulin-dependent) and six Type 2 (non-insulin-dependent) diabetic patients, eight of whom were chronically resistant to conventional treatment. Before and after mebendazole treatment for 1 month, plasma glucose and serum C-peptide concentrations were determined both fasting and 4 h after a mixed breakfast. Improvements in fasting blood glucose concentrations occurred in Typel (12.83±1.11 versus 6.56±0.56 mmol/l; p<0.05) and Type2 (10.22±0.56 versus 7.56±0.67 mmol/l; p < 0.05) diabetic patients and were associated with increases in post-cibal C-peptide responses in Type 1 and Type 2 diabetic patients. Following discontinuation of mebendazole, metabolic control deteriorated in five out of the six Type 1 diabetic patients and in all the Type 2 diabetic patients. We conclude that mebendazole increases insulin secretion, and decreases plasma glucose concentration in Type 1 and Type 2 diabetic patients. However, these beneficial effects may be transient. Keywords: Mebendazole (Vermox), insulin secretion, C-peptide, Type 1 and Type 2 diabetes.

Mebendazole and Insulin Secretion From Isolated Rat Islets

In a preliminary communication we reported that mebendazole, a vermicide, decreased plasma glucose and free fatty acid concentrations and increased plasma C peptide concentrations in both type II diabetic patients. Therefore, we suggested that mebendazole was an insulin secretagogue. However, these were uncontrolled studies, and improved metabolic control in these patients due to spontaneous remission rather than drug-induced insulin secretion was a possibility. To investigate the direct effect of mebendazole on insulin secretion we used intact islets isolated from normal rat pancreata. Mebendazole in concentrations as low as 10 to 20 mumol/L caused a twofold to threefold increase in acute-phase insulin release from isolated perifused rat islets. This heightened insulin release occurred in the presence of glucose-stimulated insulin secretion.