Metabol ism II: In vitro

POS-002-038 MECHANISMS LIMITING GLYCOGEN STORAGE IN MUSCLE DURING PROLONGED INSULIN STIMULATION.

S A Hansen, B F Hansen, E A Richter August Krogh Institute, University of Copenhagen, Denmark

Skeletal muscle is the most important organ for disposal of a glucose load. Since glucose is stored as glycogen, it is of importance to deter- mine the maximum glycogen storage capacity of skeletal muscle as well as the mechanisms ultimately limiting further storage. Fed male rats had their isolated hindquarters perfused for up to 7 h with insulin at 20.000 ~U/ml and glucose at 11-13 mM. Glucose uptake decreased (mean~SE) from 34.9±1.2 ~mol/g/h at 0 h to "7.5±0.7 after 7 h of perfusion. Muscle glyco- gen concentrations increased linearly for the first 3 h and then plateaued at approximately 2, 3 and 3.5 times initial values in muscles representing fast-twitch white, slow-twitch red and fast-twitch red fibers, respective- ly. Intramuscular free glucose and glucose-6-phosphate increased after 3 h of perfusion, and glycogen synthase activity decreased during the 7 h perfusion whether expressed as activity ratio or as fractional velocity. These latter changes with perfusion time suggest that disposal of glucose was becoming increasingly difficult for the muscle cells. However, glu- cose transport as reflected by uptake of 14-C-3-O-methylglucose was mar- kedly decreased in all fiber types after 5 and 7 h of perfusion compared to initial values. It is concluded that in muscle, stimulation with maxi- mum insulin concentrations and glucose at 11-13 mM can increase glycogen as much as 2-3.5 times depending upon muscle fiber type. The mechanism ultimately limiting further net glycogen synthesis despite ongoing maximal insulin stimulation and abundant glucose seems to involve decreases in membran transport of glucose as well as in glucose disposal.

POS-002-039

This abstract is now being presented in

Oral Session No 18 under the abstract

number ORA-018-002

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POS-002-040 REGULATION OF GLYCOGEN SYNTHASE AND PHOSPHORYLASE ACTIVITIES IN HUMAN POLYMORPHONUCLEARLEUKOCYTES.

Y OHTSUKA, M SHIMADA, K MURAKAMI, T KONDO, Y KAWAKAMI First Department of Medicine, Hokkaido University School of Medicine, Sapporo, Japan.

Hormonal regulation of glycogen synthase and phosphorylase activities were studied in human polymorphonuclear leukocytes (PML). PML from normal subjects were incubated with glucose, insulin, isoproterenol and thyroxine, either independently or in different combinations, and changes of the enzyme activity ratios of glycogen synthase (active form (1) / total activity (T)) and glycogen phosphorylase (active form (a) / total activity (T)) were assessed.

Neither glucose nor insulin changed the I/T ratio. An increase of the I/T ratio was observed when PML were incubated with glucose and insulin simultaneously, but, on the contrary, isoproterenol or thyroxine diminished this effect and the I/T ratio decreased.

Insulin had no effect on the a/T ratio. Glucose decreased the a/T ratio. Being incubated with glucose and insulin at the same time~ no further changes were observed. However, in the presence of isoproterenol or thyroxine, this decreasing effect of glucose disappeared and the a/T ratio increased.

These results show that both thyroid hormone and a ~-adrener~ic agonist alter glycogen metabolism in human PML, and this may suggest the relatlon to glucose intolerance observed in hyperthyroidism.

POS-002-041 INFLUENCE OF A SELECTIVE THRCIgBOXANE SYNTHETASE INHIBITOR OKY-046 ON GLUCOSE METABOLISM

K.HORA, Y.AOKI, K.NISHIZAWA,*H.OGUCHI,*S.FURUTA Division of Internal Medicine, Nagano Red Cross Hospital, Nagano,Japan

*Dept. Int. Med., Shinshu Univ. Sch. Med., Matsumoto, Japan

To examine the influence of thromboxane(TX) A2 on glucose metabolism, we administered OKY-046, an inhibitor of TXA2 synthesis, to streptozotocin(STZ)- induced diabetic rats. Thirty rats were previously induced diabetes by an injection of STZ. Fifteen of these rats were given OKY-046 by garage at i00 mg/kg body weight daily for 8 weeks and 15 were given vehicle as controls. Rats were sacrificed 8 weeks after STZ administration. Blood sugar(BS) levels were determined every 2 weeks. Urinary excretion of TXB2, serum insulin(IRI) and plasma glucagon(IRG) levels and histochemical staining of islets were evaluated at the time of sacrifice. BS levels were significantly lower in the OKY-046 treated rats than in

controls(314.6±42.6 vs 433.8±55.4 mg/dl at the 8th week, P<0.05). Urinary TXB2 levels were also remarkably lower in the treated rats than in controls. IRI levels were similar in treated and control rats, but IRG levels were markedly lower in treated rats than in controls(145.9±46.7 vs 265.7±127.6 pg/ml, P<0.05) Area ratio(%) of insulin-positive cells and that of somatostatin-positive cells in the whole area of islet cells by staining PAP method were similar in the 2 groups, but that of glucagon-positive cells was significantly lesser in the treated rats compared with controls(31.1±10.7 vs 56.8~14.3 %, P<0.01). As a possible mechanism of reducing BS level of OKY-046 in diabetic rats, it

was suggested that OKY-046 might decrease glucagon secretion in islet. Therefore TXA2 might regulate glucose metabolism.

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POS-002-042 BIOLOGICAL EFFECTS AND METABOLISM OF PROINSULIN IN PERFUSION LIVER.

S.NISHIMURA, H.BESSHO, K.MACHIDA, T.OHOSHI, K.OKAI, M.KONDO, T.SANKE, K.NANJO, K.IWO*, K.MIYAMURA. The First Department of Medicine, Wakayama University of Medical Science and Health Care Center, University of Wakayama*, Wakayama, Japan.

It is known that the effect of proinsulin action is relatively more potent in the liver than in the periphery. To examine the presence or absence of the specific activity of proinsulin as well as the mechanism involved, we compared the inhibitory ability of proinsulin against glucagon~induced glycogenolysis in the liver with the insulin receptor binding ability in liver cells and fat cells. Perfusate was separated by HPLC to investigate proinsulin metabolism in the liver. Wistar rat liver in situ was perfused according to the flow-through method. The inhibitory ability of porcine insulin and porcine proinsulin against glucagon-induced glucose release was examined. Using cultured rat liver cells and isolated fat cells, the competitive binding ability of porcine insulin and porcine proinsulin with 125I- insulin was determined. Immunoreactive insulin in outflowing perfusate was extracted by Sep-pak and then separated by HPLC. The inhibitory ability of porcine proinsulin against glucagon-induced glucose release in the liver was 5~ of that of porcine insulin. The receptor binding ability of porcine proinsulin in both of liver cells and fat cells was 5 to 7~ of that of porcine insulin. Only porcine proinsulin was observed, and none of insulin and intermediate proinsulin were detectable in outflowing perfusate of porcine proinsulin by HPLC study. In conclusion, the inhibitory ability of porcine proinsulin against glucagon-induced glucose release in the liver was almost same as the binding ability in the liver and the periphery. The existence of any specific action of porcine proinsulin in the liver was denied. No conversion of porcine proinsulin into insulin or intermediate proinsulin in the liver was also observed in the perfusate.

POS-002-043

ABSTRACT WITHDRAWN

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POS-002-044 REDUCED GLUCOSE OXIDATION AND PYRUVATDEHYDROGENASE ACTIVITY (PDH) IN TYPE 1 DIABETICS (INSULIN-DEPENDENT) IN POOR CONTROL.

H BECK-NIELSEN, K WRIGHT, L VERITY, JM BELL, O KOLTERMAN, L MANDARINO Hvidore Hospital, Copenhagen, Denmark and University of California, San Diego, USA.

Type 1 diabetics in poor control are insulin resistant in the periphery. Cellular mechanisms responsible for that are not known. Therefore, we have studied 8 normal weight Type 1 diabetics in poor control, (HbA. 10.0±1.5%) and a control group of 8 normal subjects. During euglycaemic glucos~Cclamp, glucose utilization and glucose oxidation (indirect calorimetry) were measured. Furthermore PDH activity (total and percent active) was measured in biopsies from skeletal muscles. We found that the glucose utilization at an insulin concentration of 50 NU/ml was significantly lower in diabetics than in control subjects (5.9±0.8 vs. 6.8±0.4 mg/kg/min). It was due to a reduction of the glucose oxidation (3.5±0.8 vs. 4.4±0.3 mg/kg/min, p<0.05). In parallel the total activity of PDH was significantly reduced in diabetics compared to controls (1326±360 vs. 3383±885, p<0.05). The active form of PDH (percent active) increased significantly in both groups during hyperinsulinaemic clamp. We conclude, that the peripheral insulin resistance in Type 1 diabetics in poor control may be due to a reduction in the total activity of PDH.

POS-002-045 MARKED ABNORMALITIES IN METAL METABOLISM IN DIABETES MELLITUS

H FUSHIMI, H HORIE, T INOUE, Y MATSUYAMA, F UDAKA*, H SAWADA*, M KAMEYAMA*, T MINAMI**, Y OKAZAKI** Department of Medicine and *Neurology, Sumitomo Hospital, **School of Pharmacology, Kinki University, Osaka, Japan

Urinary zinc excretion had been known to be increased in diabetic patients, but the metal metabolism and pathogenesis have not been clear. Therefore, we studied changes in Zn, Ca, Mg and Cu concentrations (by Hitachi 70- 7 atomic absorption spectrophotometer) and at the same time metal binding protein, metallothionein, concentrations (by Cd sedimentation and RIA method) in plasma and various tissues of streptozotocin diabetic rats (7 weeks of diabetes) to investigate abnormalities of metal metabolism in diabetes mellitus. Marked increases in urinary Zn, Ca and Mg excretion were found compared with those of controls. Urinary Zn was found increased markedly even with Zn defi- cient diet (Control: 21.7±2.8, DM: 45.3±8.4~g/day) . Zn, Mg and Cu concentrations were increased in diabetic liver, Zn and Cu concen- trations increased and Ca concentration decreased in diabetic kidney signifi- cantly but marked increase in Ca concentration in diabetic kidney with Zn deficient diet. Plasma, heart and brain showed no significant changes in metal concentrations. Then, metal concentrations were changed in diabetic rats of which roles were not understood yet. Metallothionein, regulator of metal concentrations in cells, were markedly increased in diabetic plasma (C: 102±19, DM: 188±54ng/ml), also liver (C: 5.4±1.5, DM: 106.8±26.5ng/g) and kidney (C: 31.5±2.9, DM: 66.4±12.8) and no significant changes in brain. These data suggest there exist marked changes in metal metabolism in diabetic rats and possibly in human diabetics.

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POS-002-046 DEHYDROEPIANDROSTERONE ALTERS INSULIN RESISTANCE IN PATIENTS WITH POLYCYSTIC OVARIAN DISEASE, ACANTHOSlS NIGRICANS, AND HYPERTESTOSTERONEMIA.

AE KITABCHI, CK BUFFINGTON, JR GIVENS. Dept of Medicine, and CLinical Research Center, Univers i ty of TN, Memphis, TN, USA

Although insul in resistance (IR) has been commonly found in hyperinsul inemic women with elevated testosterone, l i t t l e is known about the associat ion of IR with hyperandrogenemia of adrenal o r i g i n . Recently, we demonstrated in vivo negative cor re la t ions between the adrenal androgen, dehydroepiandrosterone su l fa te (DHEAS) and insul in response to oral glucose chal lenge. Based on these observations we hypothesized that DHEA and/or DHEAS may improve t issue insul in s e n s i t i v i t y . To test th is hypothesis we have studied insul in binding and pyruvate dehydrogenase (PDH) responsiveness to insul in in phytohemagglutinin (PHA) act ivated T-lymphocytes of 5 pat ients with elevated DHEA; 7 hyperinsul inemic subjects wi th po lycyst ic ovarian disease (PCOD), acanthosis n igr icans, and hypertestosteronemia, and 6 normal cont ro ls . 1251-1nsulin binding to ce l ls of the DHEA group was 30% (p<O.01) greater than, and in the PCOD group 52% (P<0.001%) lower than control values. Concentrations of insu l in , which in control ce l ls produced half-maximal ac t i va t ion of PDH, maximally enhanced enzyme a c t i v i t i e s in DHEA ce l l s but produced only a minimal enzyme response (<10%) in ce l ls of the PCOD group. Impairments in insul in binding and PDH s e n s i t i v i t y to insul in of the PCOD pat ients were reversed in vivo by DHEA perfusions and in v i t r o by DHEA incubations. In T-lymphocytes of the perfusion studies, insu l in binding wi th DHEA was 40-60% greater than binding values observed for ce l ls from sal ine infusions. In v i t r o , addi t ion of DHEA (500-2,000 ng/d l ) to ce l ls of PCOD pat ients improved PDH s e n s i t i v i t y , reversed the i nh ib i t i on of insul in b inding, and in fac t , st imulated binding a c t i v i t i e s 10-20% above control values. In summary, we have demonstrated improvement of insul in s e n s i t i v i t y in act ivated T-lymphocytes of hypertestosteronemic pat ients with DHEA in vivo and in v i t r o .

POS-002-047 THE ELEVATIONS IN INSULIN LEVELS AND INSULIN/C-PEPTIDE MOLAR RATIOS SEEN IN THE MUTANT INSULIN SYNDROME RESULT FROM DECREASED CLEARANCE.

K NANJO, S NISHIMURA, T SANKE, M KONDO, M MIYANO, K MIYAMURA, K INOUYE #, B GIVEN*, K POLONSKY*, D S~EINER*, A RUBENSTEIN*. Dept of Med, Wakayama Univ of Med Sci, Wakayama, Japan; Shionogi Res Lab ~, Osaka, Japan; Depts of Med and Biochem & Mol Biol, Univ of Chicago*, Chicago, IL., U.S.A.

We have identified 4 unrelated families, some of whose members synthesize and secrete abnormal insu

~ i . . . . . B24 Insulzn [Leu ] B The molecular abnormalities have been determined in these families and include

insulin (Insulin Chicago), L ~ ] " " (Insulin LosAngeles) and [Leu A3] insulin ( 2

families, Insulin Wakayama). All affected family members have demonstrated fasting hyperinsulinemia and an increased insulin/C-peptide ~ar ratio- In an attemp~ to identify the

UP<, " factor(s) responsible for those observations, [Le ], [Ser B24] and [Leu aJ] insulins were synthesized for study in vitro and in vivo. Studies included rat adipocyte receptor binding, biological activity (stimulation of 2 deoxy-glucose uptake and glucose oxidation in rat adipocytes) and also determination of in vivo half-life (tl/2) in the circulation of somatostatin infused dogs. Results of these studies are shown below referenced to normal human insulin.

Normal Human [Leu B25 ] [Ser B24 ] [Leu A3 ]

Insulin/C-peptide ratio 0.12±0.01(n=22) 0.41(n=1) Receptor Binding (%) 100 1.9-2.8 Biological Activity (%) 100 1.9-2.8 ti/2 in Dogs (minutes) 3.6 15.0

0.80+0.04(n=5) 0.96+0.10(n=9) 0.5-1.8 0.3-0.8 0.5-1.8 0.3-0.8 24.5 35.0

There is a clear inverse correlation between in vitro measures of insulin binding and biological activity and in vivo fasting insulin/C-peptide molar ratios and circulating half-lives. We conclude that the hyperinsulinemia and increased insulin/C-peptide molar ratios seen in the mutant insulin syndrome are due to decreased clearance of the mutant insulins.

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