Impaired glucose tolerance in the R6/1 transgenic mouse model of Huntington's disease.

Previous reports have highlighted a possible link between Huntington's disease (HD) and diabetes mellitus (DM), but the association has not been characterised in detail. A transgenic mouse model for HD, the R6/2 mouse, also develops diabetes. In the present study, we examined the R6/1 mouse, which carries a shorter CAG repeat than the R6/2 mouse, and found that, although not diabetic, the mice showed several signs of impaired glucose tolerance. First, following i.p. glucose injection, the blood glucose concentration was approximately 30% higher in young R6/1 mice (10 weeks) compared to wild-type mice (P = 0.004). In older mice (38 weeks), glucose tolerance was further impaired in both R6/1 and wild-type animals. Second, during glucose challenge, the R6/1 mice reached higher plasma insulin levels than wild-type mice, but the peripheral insulin sensitivity was normal as measured by injection of human or mouse insulin or when evaluated by the quantitative insulin sensitivity check index (QUICKI). Third, the beta cell volume was 17% and 39% smaller at 10 and 38 weeks of age, respectively, compared to age-matched wild-type littermates and the reduction was not caused by apoptosis at either age. Finally, we demonstrated the presence of the HD gene product, huntingtin (htt), in both alpha- and beta-cells in R6/1 islets of Langerhans. Since pancreatic beta cells and neurons share several common traits, clarification of the mechanism associating neurodegenerative diseases with diabetes might improve our understanding of the pathogenic events leading to both groups of diseases.

The (ProB27, ThrB28) human insulin analogue is more potent and more rapidly absorbed from subcutis than human insulin.

OBJECTIVE: To test the physiological properties of human insulin in which the amino acids Thr (B27) and Pro (B28) are interchanged (PT insulin). This was hypothesised to prevent dimerisation and accelerate the absorption from s.c. tissue without altering the affinity for the insulin receptor. DESIGN: PT insulin was expressed in Pichia pastoris and processed in vitro. The purified compound was used for physiological investigations. METHODS: Receptor binding activity to insulin and IGF receptors was evaluated in a competition assay using iodinated PT insulin and recombinant receptors while growth induction properties were evaluated by thymidine incorporation. Absorption kinetics from pig subcutis was investigated by measuring the disappearance of iodinated PT insulin. The potency was evaluated by measuring the blood glucose lowering activity in mice. RESULTS: The absorption of PT insulin was accelerated compared with human insulin, although still slower than Asp (B28) insulin. Human and PT insulin had similar affinities for the human insulin receptor (K(d)=3.6 x 10(-12) vs 5.2 x 10(-12) mol/l) while the affinity for the IGF receptor was four times higher for PT insulin than for human insulin (K(d)=3.4 x 10(-8) vs 1.3 x 10(-7) mol/l). This resulted in a slightly higher DNA synthesis when assayed in intermediary insulin concentrations. The blood glucose lowering effect in mice exceeded the effect of human insulin (integral 0-60 min: 61.4+/-7 vs 30+/-4, n=6, P=0.046). CONCLUSIONS: PT insulin is absorbed faster and is more potent than human insulin. Although PT insulin stimulates growth more than human insulin, this will not prevent its use in the clinic, but the main interest will probably focus on investigations to clarify the paradox of full biological activity in connection with the recently described lack of structure in the B-chain.

Sulfatide promotes the folding of proinsulin, preserves insulin crystals, and mediates its monomerization.

Sulfatide is a glycolipid that has been associated with insulin-dependent diabetes mellitus. It is present in the islets of Langerhans and follows the same intracellular route as insulin. However, the role of sulfatide in the beta cell has been unclear. Here we present evidence suggesting that sulfatide promotes the folding of reduced proinsulin, indicating that sulfatide possesses molecular chaperone activity. Sulfatide associates with insulin by binding to the insulin domain A8--A10 and most likely by interacting with the hydrophobic side chains of the dimer-forming part of the insulin B-chain. Sulfatide has a dual effect on insulin. It substantially reduces deterioration of insulin hexamer crystals at pH 5.5, conferring stability comparable to those in beta cell granules. Sulfatide also mediates the conversion of insulin hexamers to the biological active monomers at neutral pH, the pH at the beta-cell surface. Finally, we report that inhibition of sulfatide synthesis with chloroquine and fumonisine B1 leads to inhibition of insulin granule formation in vivo. Our observations suggest that sulfatide plays a key role in the folding of proinsulin, in the maintenance of insulin structure, and in the monomerization process.

Five fold increase of insulin concentration delays the absorption of subcutaneously injected human insulin suspensions in pigs.

Human NPH U100 (100 IU/ml), given once daily, is often absorbed too fast to cover the basal insulin demand throughout 24 h. The aim of the present study was to examine whether the absorption of human insulin suspensions would be delayed by increasing the insulin concentration from U100 to U500. In each experiment 10 IU of corresponding human Ul00 and U500 preparations, labelled with 125I-insulin, were injected subcutaneously and contralaterally in the neck of a pig followed by monitoring residual radioactivity over the injection sites. The time until 75, 50 and 25% residual radioactivity (T(75%), T(50%) and T(25%)) using NPH U500 was compared with NPH U100 in 14 experiments: T(75%): 5.0+/-0.5 (mean+/-SEM) vs 3.8+/-0.3 h (P=0.007, paired t-test), T(50%): 12. 2+/-0.9 vs 9.0+/-0.6 h (P=0.003) and T(25%). 24.2+/-1.2 vs 17.9+/-1. 2 h (P=0.001). The corresponding values for semilente U500 compared with semilente U100 in eight experiments were: T(75%): 2.8+/-0.4 vs 1.6+/-0.2 h (P=0.02), T(50%): 5.6+/-0.6 vs 3.4+/-0.3 h (P=0.01) and T(25%): 10.9+/-1.1 vs 7.2+/-0.7 h (P=0.009). Thus, the absorption of a given dose of human NPH or human semilente insulin in pigs is substantially delayed by changing the insulin concentration from Ul00 to U500. Human NPH U500 appears to be more appropriate than human NPH U100 for injection once daily in basal insulin therapy.

Failure of high-dose insulin treatment to increase beta-cell insulin content in diabetic non obese diabetic (NOD) mice.

High-dose insulin treatment in the first period after clinical onset of insulin-dependent diabetes mellitus (IDDM) has been found to reduce diabetic manifestations in humans. The aim of the present study was to examine whether high-dose insulin treatment of newly diagnosed diabetic non obese diabetic (NOD) mice would increase beta-cell insulin content after termination of treatment in this experimental IDDM animal model. Newly diagnosed diabetic female NOD mice were randomized into three groups composed of a low-dose insulin treated group (n = 10) injected subcutaneously with 15 IU/kg per day of NPH for 14 days followed by 5 days without insulin, a high-dose insulin treated group (n = 8) injected subcutaneously with 150 IU/kg per day of Actrapid for 14 days followed by 5 days without insulin and an untreated group sacrificed 3 days after diagnosis (n = 11). A reference group of age matched non-diabetic untreated female NOD mice (n = 11) was included in the study and sacrificed at the same time as the untreated diabetic mice. No significant difference in the amount of insulin extracted from the total pancreas was found by comparison of the three diabetic groups, consisting of the newly diagnosed untreated mice, the low-dose insulin treated mice and the high-dose insulin treated mice, respectively. The level was about 100-fold less than in the non-diabetic group. Blood glucose values in the two treated diabetic groups were at a high level (median > 18 mM) throughout the study. We conclude that no increase in beta-cell insulin content could be demonstrated in newly diagnosed diabetic NOD mice after early high-dose insulin treatment, at least not in the presence of high blood glucose values.

Nicotinamide prevents interleukin-1 effects on accumulated insulin release and nitric oxide production in rat islets of Langerhans.

Nicotinamide (NA) prevents macrophage- and interleukin-1 (IL-1)-mediated beta-cell damage in vitro as well as diabetes development in animal models of insulin-dependent diabetes mellitus (IDDM). IL-1 beta-mediated inhibition of insulin release and damage to beta-cells are associated with intracellular production of nitric oxide (NO) radicals. Therefore, we studied whether NA prevented IL-1 beta-induced islet NO production, measured as nitrite release from isolated rat islets, and, if so, whether this action was associated with prevention of IL-1 beta-mediated inhibition of insulin release. NA dose- and time-dependently inhibited and delayed IL-1 beta-induced islet NO production. Light microscopy detected that 25 mM of NA protected against IL-1 beta-induced islet damage. Five to 50 mM of NA dose-dependently reduced inhibition of accumulated islet insulin release induced by 150 pg/ml of IL-1 beta. NA was not able to reverse the reduced ability of IL-1 beta-treated islets to respond to an acute glucose challenge. NO or nitrite did not interact directly with NA, because NA did not reduce sodium nitroprusside-generated nitrite. No-synthase inhibition with L-arginine depletion abolished NO production but only partially reduced IL-1 beta-induced inhibition of accumulated insulin release. Complete inhibition of IL-1 beta effects could not be obtained by adding L-arginine analogues to L-arginine-depleted medium, indicating that an NO-independent action of IL-1 beta on islet insulin release may exist.(ABSTRACT TRUNCATED AT 250 WORDS)

Pancreatic spasmolytic polypeptide: crystallization, circular dichroism analysis, and preliminary X-ray diffraction studies.

Pancreatic spasmolytic polypeptide (PSP) isolated from porcine pancreas has been crystallized by the hanging drop vapor diffusion method. Crystals suitable for X-ray diffraction analysis were grown at pH 4.7 from a solution of 6% saturated ammonium sulfate. The space group is orthorhombic I222 or I2(1)2(1)2(1) with unit cell parameters a = 54.38 A, b = 72.29 A, and c = 180.85 A. There are three molecules of PSP per asymmetric unit and a water content of 46.9%. The crystals diffracts to an estimated resolution of 2.7 A. The far-UV CD spectrum of PSP shows some exceptional features which cannot be accounted for thoroughly in terms of standard secondary structures commonly seen in protein CD spectroscopy. With this limitation, the secondary structure analysis predicts 15% alpha-helix, between 10 and 20% antiparallel beta-strand, 10% parallel beta-strand, 15% turn, and 25 to 40% of other structures.

Conventional and sprinkler needle injection of magnesium insulin.

Currently available short-acting insulin preparations fail to mimic the postprandial insulin profile of non-diabetic individuals. The activity of a novel insulin designed for faster absorption has been tested after subcutaneous injection. Magnesium insulin (50 U ml-1) given by sprinkler needle was compared with unmodified human insulin (100 U ml-1) given by conventional needle and unmodified human insulin (50 U ml-1) given by sprinkler needle in normal volunteers using a euglycaemic clamp. Magnesium insulin had a significantly faster onset of action resulting in a higher exogenous insulin level by 15 min, peak level was reached after 60 min compared with 75 min for the unmodified insulins, and duration of action was significantly shorter than both unmodified insulins. No significant differences were observed between the unmodified insulins for the first 5 h after injection, indicating that the observed differences to magnesium insulin could not be attributed to the insulin concentration or the type of needle used for insulin administration. Injection of magnesium insulin prior to a test breakfast in people with Type 2 diabetes resulted in significantly lower total and 0 to 120 min areas under the glucose curve, an earlier rise in exogenous insulin levels and a higher area under the insulin curve from 0 to 120 min compared with unmodified 100 U ml-1 human insulin.

Revised amino acid sequence of pancreatic spasmolytic polypeptide exhibits greater similarity with an inducible pS2 peptide found in a human breast cancer cell line.

The published amino acid sequence of pancreatic spasmolytic polypeptide (Thim, L., Thomsen, J., Christensen, M. and Jørgensen, K.H. et al. (1985) Biochim. Biophys. Acta 827, 410-418) has been checked by a combination of mass spectroscopy and Edman degradation. The pyroglutamyl blocking group was positively identified, and residue assignments at four positions were corrected: Lys48 (not Ser), Ser63 (not Lys), Cys68 (not Ser) and Ser74 (not Cys). The revised sequence exhibits greater similarity with pS2 peptide, a 60 residue polypeptide which is induced by oestrogen in the human breast cancer cell line MCF-7 and found in malignant but not in non-malignant breast tissue.

Growth stimulatory effect of pancreatic spasmolytic polypeptide on cultured colon and breast tumor cells.

The effects of a novel polypeptide, pancreatic spasmolytic polypeptide (PSP) on a colon carcinoma cell line (HCT 116) were examined. PSP stimulated the incorporation of [3H]thymidine into HCT 116 cells as well as cell proliferation in a dose-dependent manner. Maximal increase in [3H]thymidine incorporation of 50-60% occurred at 3-300 microM PSP. The VIP-mediated-increase in cAMP levels was reduced by PSP at greater than 1 microM concentrations. PSP is highly homologous to the estrogen-induced pS2 protein in MCF-7 breast cancer cells. We find that PSP also enhanced [3H]thymidine incorporation in MCF-7 cells. These findings indicate for the first time that PSP has growth stimulatory properties.

The preparation of porcine (Tyr-A14-2H) insulin and its characterization by mass spectrometry.

The title compound was prepared by catalytic deiodination of (Tyr-A14-3-I) insulin with deuterium gas. Under special conditions (large excess of PdO in CH3OD/D2O at pH 9.2) developed to minimize problems due to poisoning of the catalyst, deuterated insulin was produced in yields of about 35% after purification by reversed phase HPLC. For analysis, the deuterated insulin was digested with V8 protease and was shown by mass spectrometry to have incorporated deuterium to an extent of 96.6 atom %, exclusively in a pentapeptide containing Tyr A14. The title compound should prove useful to those workers studying insulin by mass spectrometry, and use of the method with tritium gas in place of deuterium should permit the preparation of a specifically labelled radioactive insulin analogue which behaves identically to the natural hormone.

Receptor binding of pancreatic spasmolytic polypeptide (PSP) in rat intestinal mucosal cell membranes inhibits the adenylate cyclase activity.

The recently isolated pancreatic spasmolytic polypeptide, PSP, interacted with specific binding sites in the gastrointestinal tract and inhibited the adenylate cyclase activity in rat intestinal mucosal cell membranes. The binding sites appeared to be heterogeneous and Scatchard analysis of the binding data indicated the presence of at least two classes of sites. The high-affinity low-capacity binding sites and the low-affinity high-capacity binding sites had apparent dissociation constants of 1.3 X 10(-7) mol/l and 4.2 X 10(-6) mol/l, respectively. The PSP induced inhibition of the adenylate cyclase activity was independent of the stimulatory state of the enzyme. The basal activity as well as that stimulated by VIP and secretin was half maximally inhibited at approximately 3 X 10(-5) mol/l of PSP. The inhibitory effect of PSP was independent of the agonist concentration employed. PSP did not affect the receptor binding of VIP nor did VIP affect the receptor binding of PSP.

Single chain des-(B30) insulin. Intramolecular crosslinking of insulin by trypsin catalyzed transpeptidation.

Single chain des-(B30) insulin (SCI) has been synthesized from porcine insulin by trypsin in a medium with a low content of water. Trypsin catalyzes an intramolecular transpeptidation reaction in which the glycineA1 residue substitutes the alanineB30 residue, rendering a LysB29 -GlyA1 peptide link between the A- and B-chains of insulin. The insulin derivative has been purified by column chromatography and appears to be homogeneous in HPLC and disc electrophoresis. The structure was proven to be B(1-29)-A(1-21) insulin by proteolysis with Armilliaria mellea protease followed by a few steps of Edman degradation. The electrophoretic mobility indicates that SCI has a more condensed structure than that of insulin. Perfect rhombohedral crystals were obtained under conditions resembling those under which insulin crystallizes in the same form. SCI was devoid of effect in the blood sugar lowering assay in mice, the estimated potency being less than 0.1% of that of insulin.

The amino acid sequence of pancreatic spasmolytic polypeptide.

The sequence of porcine pancreatic spasmolytic polypeptide has been established by a variety of techniques including manual as well as automatic sequencing of fragments resulting from the cleavage of reduced and S-carboxymethylated pancreatic spasmolytic polypeptide with trypsin, chymotrypsin, clostripain, cyanogen bromide and formic acid. The N- and C-terminal sequences were established using pyroglutamate amino-peptidase and carboxypeptidase A, respectively. Pancreatic spasmolytic polypeptide contains 106 amino acid residues in a single chain with seven S-S bridges and a pyroglutamyl blocked N-terminal. The alignment of the sequences representing amino acids 14-49 and 63-98 shows pair-wise identical amino acid residues in 18 out of 36 positions, indicating that these two "domains" have been derived from a common gene.

Human proinsulin standards.

Two new batches of pancreatic human proinsulin have been compared with biosynthetic human proinsulin. Standards of these three proinsulin preparations were made on the basis of quantitative amino-acid analyses and compared in two proinsulin radioimmunoassays with a proinsulin standard prepared 14 years ago. The curves of the new standards were superimposable. However, they differed considerably from the curve of the old standard which proved to be only one-third of the strength of the new standards, thereby leading to a threefold over-estimation of proinsulin concentrations when the old standard is used. We conclude that the new standards should replace previously used standards.

Human monocomponent insulin. Chemistry and characteristics.

The primary structure of different insulins is reviewed and the properties, identification tests, purity, potency and immunogenicity of human insulin are summerized. Novo Research Institute has developed a method, simply using an enzymatic conversion reaction to substitute the B30 alanine of porcine insulin with threonine to manufacture human insulin. This process is basically an extension of the process currently used to manufacture the Novo purified insulins which are commercially available.

Pancreatic spasmolytic polypeptide (PSP): III. Pharmacology of a new porcine pancreatic polypeptide with spasmolytic and gastric acid secretion inhibitory effects.

Pancreatic spasmolytic Polypeptide (PSP) is a new porcine pancreatic polypeptide, which inhibits gastrointestinal motility and gastric acid secretion in laboratory animals after parenteral as well as oral administration. (1) PSP inhibits the amplitude of electrically stimulated contractions of the isolated guinea pig ileum. PSP's inhibitory effect is antagonized by phentolamine, but not by yohimbine. (2) PSP inhibits the motility of isolated guinea pig intestinal segments after intraluminal dosing. (3) PSP reduces intestinal motility in rabbits in vivo after intravenous and intraluminal administration, and in mice in vivo after subcutaneous injection. (4) PSP delays absorption of protein hydrolysate when it is administered orally in capsules to pigs and to pancreatectomized dogs. (5) PSP inhibits pentagastrin induced gastric acid secretion in rats after oral administration and in cats after subcutaneous and oral administration. The mechanism of action of PSP has so far not been finally elucidated. It seems likely that PSP interferes with endogenous acetylcholine release. Furthermore it might act by release of somatostatin from somatostatin cells in the gastrointestinal tract. It may have a direct or an indirect stimulant effect on alpha 2-receptors.

Pancreatic spasmolytic polypeptide (PSP): I. Preparation and initial chemical characterization of a new polypeptide from porcine pancreas.

A novel polypeptide, named Pancreatic Spasmolytic Polypeptide (PSP), was discovered in a side-fraction from the purification of porcine insulin. PSP was prepared by two different purification methods based on combinations of precipitations, anion-exchange and cation-exchange chromatography. The highest yield obtained, 52 mg PSP/kg pancreas, indicates that the content of PSP in porcine pancreas is about half the content of insulin. Both preparations appeared to be very pure as judged by basic disc electrophoresis, isoelectric focusing, analytical gel filtration and radioimmunoassays for various polypeptides known to be present in pancreas. The PSP molecule contains 106 amino acids (MW about 11 700). PSP is an acidic (pI 4.4), non-glycosylated protein without free N-terminal amino groups, and with high contents of proline and cystine. The high content of S-S bridges (7 per molecule), an unexpected low apparent MW determined by gel filtration, and a remarkable resistance towards treatment with trypsin and chymotrypsin, point to a compact structure of the PSP molecule.

Pancreatic spasmolytic polypeptide (PSP): II. Radioimmunological determination of PSP in porcine tissues, plasma and pancreatic juice.

A radioimmunoassay (RIA) for the determination of porcine Pancreatic Spasmolytic Polypeptide (PSP) has been developed. The antisera raised in rabbits were sensitive to 5 pgequiv. of PSP in a volume of 100 microliter. Immunoreactive PSP (IR-PSP) has been determined in extracts from 22 porcine organs. Pancreas was found to be the only organ containing substantial amounts of IR-PSP (0.1 mgequiv. IR-PSP/g wet weight). The fasting porcine plasma level of IR-PSP was about 10 ngequiv./ml, corresponding to 850 pM. The concentration of IR-PSP in porcine pancreatic juice varied from 0.2 mugequiv./ml in the fasting state to 46-116 mugequiv./ml after stimulation with pancreozymin or secretin. A linear correlation was found between the exocrine secretion of IR-PSP and total protein.