In the liver of adult diabetics, the activity of two enzymes of the citrate-cleavage pathway was increased, the change being statistically significant for NADP-malate dehydrogenase (+ 46%, p less than 0.05) but not significant for ATP citrate-lyase (+ 55%, p greater than 0.10). The increased activity of NADP-malate dehydrogenase, together with the previously described elevation of pentose cycle dehydrogenases, suggests enhanced NADPH generation. Considering the recently proposed possibility of extramitochondrial acetyl-CoA formation by routes other than the citrate-cleavage (i.e., via cytoplasmic acetyl-CoA synthetase), our data is consistent with the occurrence of increased lipogenetic capacity.

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.

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.

With respect to the enzymes of NADPH-forming metabolic pathways in human leukocytes: (a) Glucose-6-phosphate dehydrogenase and phosphogluconate dehydrogenase (decarboxylating) were less active in leukocytes (mostly myeloblasts) from eight patients with acute myeloblastic leukemia (I) than in leukocytes (mostly granulocytes) from 16 normal subjects (II). (b) Of the enzymes of the citrate cleavage pathway, ATP citrate lyase and malate dehydrogenase (decarboxylating) (NADP+) were virtually absent in the cells studied. (c) Isocitrate dehydrogenase (NADP+), aspartate aminotransferase, and alanine aminotransferase, which, together with the much more active malate dehydrogenase, constitute a newly proposed NADPH-forming metabolic cycle, showed a higher activity in I than in II or III, and therefore could compensate, as concerns NADPH-generation, for the low activity of pentose cycle dehydrogenases. We are not sure whether the enzymatic characteristic of I cells is attributable to their immaturity or to their leukemic nature.

Abstract The average value for 24-h excretion of uropepsinogen increased by 64% (P < 0.05) in 44 uncomplicated diabetics as compared to 33 normal subjects. Uropepsinogen excretion was correlated with the daily output of urine (r = 0.43, P < 0.05) in the normals, but not in the diabetics (r = 0.24, P > 0.10). This shows that the enhanced excretion in diabetics is not merely a result of increased urine output, and suggests that diabetes is an interfering factor that affects uropepsinogen excretion. No correlation was found with age (in normal persons or diabetics), duration of disease, fasting glycemic level, or daily insulin requirement. Because gastric secretion is depressed in persons with diabetes mellitus, the increased uropepsinogen excretion is tentatively attributed to alterations of gastric mucosa, known to occur in this disease, which might result in a change of the "exocrine-endocrine partition" of pepsinogen in favor of the "endocrine" fraction, i.e., the fraction that enters the blood.

In 45 diabetics the 24-h urinary excretion of β- N -acetylglucosaminidase (E.C. 3.2.1.30) was increased by 40% ( P < 0.05) compared to 35 control subjects. The enzyme excretion was correlated with glycemia ( r = 0.58, P < 0.001), being little changed in diabetics with blood glucose concentrations of less than 200 mg/dl, and markedly elevated (+ 123%, P < 0.001) in those whose blood glucose was greater than 200 mg/dl. The rate of diuresis seemed to have no effect. These data indicate that the enhanced activity previously described in sera of diabetics for β- N -acetyl-glucosaminidase (as well as for other lysosomal enzymes) cannot be attributed to impairment of renal excretion, and support the hypothesis that in diabetes there is an activation of lysosomal enzymes in tissues that causes an increase in their activity in serum and, consequently, in urine.

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.

In 33 male patients with clinical manifestations of atherosclerosis (angina pectoris, myocardial infarction, or cerebral vascular accidents having occurred no less than six months before study) the serum activity of the lysosomal enzyme beta-N-acetyl-glucosaminidase (EC 3.2.1.30) was found increased by 30 per cent (p<0.001) compared to 76 control male subjects. The activity of enzymes located mainly or exclusively in the cytosol—aspartate aminotransferase (EC 2.6.1.1) and lactate dehydrogenase (EC 1.1.1.27)—was normal. This shows that there was no cell damage that could cause enzyme elevation. Based on the role of lysosomes in the catabolism of macromolecules of ground substance of connective tissue, it is speculated that the enhanced serum beta-N-acetyl-glucosaminidase activity in atherosclerosis might be a manifestation of extracellular secretion of lysosomal enzymes (exocytosis) by the cells of the arterial walls, occurring in response to the accumulation of mucopolysaccharides that takes place in the early stage of disease.

Serum enzymes that show changed activities in diabetes mellitus can be divided into four groups: Group I includes some lysosomal enzymes—β-glucuronidase N-acetyl-β-glucosaminidase, acid phosphatase, and amylase—that show increased activity correlated with blood sugar concentration. Because lysosomal enzymes as well as liver amylase show latency and may be "activated" by several agents, their increased activity in the serum of diabetics might be a manifestation of an activation occurring in tissues. Group II includes alkaline phosphatase and trehalase, which are increased but not correlated with blood sugar concentration. Their enhanced activity may reflect tissue metabolic disorders. Group III includes enzymes that increase in the postketotic period almost regularly—phosphohexose isomerase —or in only the most severe cases—aminotransferases and several dehydrogenases—because of tissue damage caused by metabolic and circulatory alterations. Cholinesterase, on the other hand, is decreased. Group IV includes any of the above-mentioned enzymes, and still others, that may be more active in diabetics with complications such as hepatic and renal involvement and obesity.

In a group of 90 diabetics, an increased serum activity (+137 per cent, p<0.001) of the lysosomal enzyme acid phosphatase was found. The increase was moderate (+55 per cent, p<0.01) in uncomplicated diabetics with slightly elevated glycemia (148 ± 24 mg glucose/100 ml), while it was more accentuated (about twice normal, p<0.001) in diabetics with either vasculopathies (micro- or macroangiopathy) or marked hyperglycemia (343 ± 108 mg glucose/100 ml). Serum aspartate and alanine aminotransferases were normal. Thus, acid phosphatase in diabetes behaves similarly to other lysosomal enzymes: beta-glucuronidase and N-acetyl-beta-glucosaminidase. This is a further evidence of lysosomal enzyme activation in diabetes, apparently linked to both the degree of metabolic decompensation and the presence of vasculopathies. Since acid phosphatase, unlike beta-glucuronidase and N-acetyl-beta-glucosaminidase, is not concerned with degradation of mucopolysaccharides and glycoproteins, the higher activity found in vasculopathic diabetics would indicate that in these patients the activation process is not restricted to the enzymes capable of degrading the aforementioned compounds, which accumulate in the walls of diseased vessels, but involves a variety of lysosomal hydrolases.

In sera of patients with diabetic coma, amylase activity was markedly elevated and closely correlated with the activity of some lysosomal enzymes, including β-glucuronidase, N-acetyl-β-glucosaminidase, and acid phosphatase. All these enzyme activities returned to normal with the normalization of glycemia. Activities of serum lipase, aspartate and alanine aminotransferases, aldolase, and lactate dehydrogenase were not changed. Since liver amylase, although primarily contained in microsomes, shows "latency" and is activated by several agents as are lysosomal enzymes, these findings might be regarded as a further evidence of a similarity between amylase and lysosomal enzymes, and make probable the hypothesis that a process of "activation" occurring in liver might be the cause of increased amylase activity in serum as well as of lysosomal enzymes.

In each of 10 highly hyperglycemic decompensated diabetics with ketoacidosis, we found a markedly increased serum activity of two lysosomal hydrolases (N-acetyl-β-glucosaminidase and β-glucuronidase). This was also true to a lesser degree of five diabetics with less severe decompensation and without ketoacidosis. The activity of both enzymes and the degree of hyperglycemia were highly correlated. We think these enzymatic changes result from a process of activation and release of tissue lysosomal enzymes, probably occurring in connection with the increased catabolism present in decompensated diabetes. Nonlysosomal (cytoplasmic or mitochondrial) enzymes were less changed (aspartate and alanine aminotransferases) or normal (aldolase, lactate- and malate dehydrogenase, and creatine kinase). This indicates that tissue damage alone could not account for the increased activity of the two lysosomal hydrolases; it therefore seems primarily to be due to involvement of lysosomes.

In sera of diabetics (115 patients) the activity of the lysosomal enzyme betaglucuronidase was found increased by 63 per cent (p<0.001) compared to normal individuals (75 subjects). The enzyme level seemed correlated to both the degree of glucose metabolic disorder and the vascular lesions. In fact, among the patients without vascular complications, beta-glucuronidase activity was roughly parallel to glycemia (r=0.647, p<0.001), reaching very high values (about four times the normal) in six decompensated ketoacidotic patients, while in diabetics with essentially similar glycemia, the enzyme was higher (p<0.005) in subjects with vascular complications, either microangiopathic or macroangiopathic in nature. Since lysosomal enzymes are capable of degrading various molecules, the increase of serum beta-glucuronidase in diabetics was regarded as an index of involvement of lysosomes in tissues, leading to activation of lysosomal hydrolases, apparently in response to the metabolic need of degrading either compounds which have accumulated in tissues, such as mucopolysaccharides and glycoproteins in diabetics with vasculopathies, or various constituents of cells themselves in a context of increased tissue catabolism, as occurs in diabetics with severe metabolic disorder.

In a group of seventy-three diabetics, a statistically significant (P < 0.001) increase (+ 41 per cent) of serum N-acetyl-beta-glucosaminidase activity was found. The enzyme level seemed to correlate with both the vascular complications (either microangiopathic or macroangiopathic in nature) and the blood sugar level measured simultaneously. In fact, in patients with moderately elevated glycemia (145 mg./100 ml. ± 41) and without vasculopathies, the enzymatic activity was slightly changed (P > 0.05), while the activity was significantly (P < 0.001) increased (+ 31 per cent), in diabetics with essentially similar gly-cemia (151 mg./100 ml. ± 30) but with vasculopathies, and even more elevated (+ 80 per cent, P < 0.001) in diabetics without vasculopathies but with marked hyper-glycemia (309 mg./100 ml. ± 160). Since lysosomal enzymes, to which N-acetyl-beta-glucosaminidase belongs, are capable of degrading various compounds, these enzymatic changes were regarded as due to an activation of lysosomal enzymes in tissues, occurring in diabetes in response to the metabolic need of degrading either mucopolysaccharides and glycoproteins (as in diabetics with vasculopathies), or various constituents of cells themselves in a context of increased tissue catabolism (as in decompensated diabetics). However, the possibility cannot be excluded that the enzyme is elevated merely because of decreased lysosome stability. Reduction in the destruction rate of the enzyme might also contribute to its elevation.