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Ruminants are often fed a high-concentrate (HC) diet to meet lactating demands, yet long-term concentrate feeding induces subacute ruminal acidosis (SARA) and leads to a decrease in milk fat. Buffering agent could enhance the acid base buffer capacity and has been used to prevent ruminant rumen SARA and improve the content of milk fat. Therefore, we tested whether a buffering agent increases lipid anabolism in the livers of goats and influences of milk fat synthesis. Twelve Saanen-lactating goats were randomly assigned to two groups: one group received a HC diet (Concentrate: Forage=60:40, Control) and the other group received the same diet with a buffering agent added (10 g sodium butyrate, C(4)H(7)NaO(2); 10 g sodium bicarbonate, NaHCO(3); BG) over a 20-week experimental period. Overall, milk fat increase (4.25+/-0.08 vs. 3.24+/-0.10; P<0.05), and lipopolysaccharide levels in the jugular (1.82+/-0.14 vs. 3.76+/-0.33) and rumen fluid (23,340+/-134 vs. 42,550+/-136) decreased in the buffering agent group (P<0.05). Liver consumption and release of nonesterified fatty acid (NEFA) into the bloodstream increased (P<0.05). Phosphatidylinositol 3-kinase (PI3K), protein kinase B (AKT) and ribosomal protein S6 kinase (p70S6K) up-regulated significantly in the livers of the buffering agent group (P<0.05). It also up-regulated expression of the transcription factor sterol regulatory element binding protein-1c (SREBP-1c) and its downstream targets involved in fatty acid synthetic, including fatty acid synthetase (FAS), stearoyl-CoA desaturase (SCD-1) and acetyl-CoA carboxylase 1 (ACC1) (P<0.05). The BG diet increased insulin levels in blood (19.43+/-0.18 vs. 13.81+/-0.10, P<0.05), and insulin receptor was likewise elevated in the liver (P<0.05). Cumulatively, the BG diet increased plasma concentrations of NEFA by INS-PI3K/AKTSREBP-1c signaling pathway promoting their synthesis in the liver. The increased NEFA concentration in the blood during BG feeding may explain the up-regulated in the milk fat of lactating goats.

The methylotrophic yeast Pichia pastoris, which proved successful in producing many heterologous proteins, was used to express an insulin precursor. A transformant with a high copy number of the gene integrated into the chromosome was obtained by the dot-blotting method. In high-density fermentation using a simple culture medium composed mainly of salt and methanol, the expression level reached 1.5 g/L. A simple two-step method was established to purify the expression product from the culture medium with an overall recovery of about 80%. After tryptic transpeptidation, human insulin with full receptor binding capacity and biological activity was obtained. In the presence of zinc, the recombinant human insulin could be crystallized in the rhombohedral form., (Copyright 2001 John Wiley & Sons, Inc.)

Destetrapeptide insulin (DTI, human insulin with B27-30 removed) was obtained from a monomeric precursor (MIP) expressed in Saccharomyces cerevisiae through tryptic transpeptidation in the presence of synthetic tetrapeptide Gly-Phe-Phe-Tyr. The in vivo biological activity of DTI, determined by mouse convulsion assay, is 22 IU/mg. Its binding activity with insulin receptor on human placental membrane is 80% and its in vitro biological activity, determined by free fat cell assay, is 77%. Compared with native insulin, DTI molecules do not associate in solution but exist in the monomeric form, thus leading to its rapid utilization in vivo.

Single-chain insulin/IGF-1 hybrid-[Ins/IGF-1(C)], single-chain porcine insulin precursor-(PIP), and B10Asp PIP were prepared by protein engineering. Their growth-promoting activities in mouse breast cancer cell line GR2H6 are 10, 0.2, and 2 times that of insulin, respectively, and 29%, 0.6%, and 6% of that of IGF-1, indicating that the C domain and 9Glu of IGF-1 are important for its growth-promoting activity. Given these results and previous reports, we propose that the C domain, 9Glu, and 23Phe-26Asn beta bend are involved in the growth-promoting functional region of IGF-1.

Recombinant A17 Lys human insulin precursor expressed in yeast cells was isolated from the culture medium. After purification, the precursor was converted into A17 Lys human insulin. A17 Lys human insulin can be crystallized but has much lower receptor-binding and biological activities than porcine or human insulin where A17 is Glu. It is proposed that the A17 Glu residue may be involved in the receptor-binding or the active site of human insulin.

Satisfactory single crystals of deshexapeptide (B25-B30) insulin for X-ray crystal structure analysis have been grown in citrate buffer by the method of hanging-drop gas phase diffusion. The crystal belongs to the monoclinic system with space group C2. The unit cell constants are a = 42.6 A, b = 37.9 A, c = 27.2 A, beta = 125.4 degrees and there is only one molecule of deshexapeptide insulin in an asymmetric unit.

Des-(B25-B30)-hexapeptide-insulin with B23-glycine replaced by D-alanine was prepared by a combination of enzymic and non-enzymic syntheses. The purified product was homogeneous in polyacrylamide-gel electrophoresis and could be crystallized. The biological activity in vivo of crystalline [B23-D-Ala]des-(B25-B30)-hexapeptide-insulin was determined as 58% of that of standard pig insulin (27 i.u./mg).

We have worked out a new adsorption method for the preparation of insulins from different species. At present, we are mainly involved in chicken and snake insulins. In all insulins so far studied, only chicken insulin has been reported to have much higher activity. However, results from different laboratories are quite different from one another. We have obtained chicken insulin in typical rhombohedral crystalline form and determined its activity in vivo which is not appreciably different from that of porcine insulin. From the pancreas of a non-venomous snake, Zaocys dhumnades dhumnades (Cantor), we have prepared snake insulin which is different from rattle snake insulin reported earlier. The dodecahedral crystals of the snake insulin are similar to those of zinc-free porcine insulin.

[B29-Arginine]porcine insulin was prepared from des-(B23-30)-insulin and synthetic octapeptide with the aid of trypsin. Comparison of reaction rates in trypsin-catalysed transamidation of this compound and porcine insulin with threonine ether ester showed that this reaction is determined only by conformational effects and structural features of amino acids leaving from and entering into B30, not by the structure and the pKa value of the basic amino acid in B29.

Snake insulin is isolated and crystallized from the pancreas of non-venomous snake (Zaocys dhumnades dhumnades, Cantor). The crystalline form is not rhombohedral but dodecahedral. The primary structure has been determined with the aid of an LKB solid-phase sequencer. In the primary structure of snake insulin, the presence of B5 Arg, B29 Arg, B16 Phe, B25 Tyr and B18 Ile is unusual in comparison with insulins from mammals, birds and fishes. However, the snake insulin we studied is very similar to rattle snake insulin, with the exception that A15 is glutamic acid instead of glutamine and B30 is threonine instead of serine. These differences are consistent with the higher acidity of the snake insulin we studied and with the threonine and serine content in amino acid analysis. The B10 residue of the snake insulin we studied is still histidine. The formation of dodecahedral crystals of the snake insulin has been discussed in connection with the presence of this histidine.

Despentapeptide (B26-30)-insulinamide (B25) prepared by a semisynthetic procedure was found to have about 65% of the hypoglycaemic activity of natural insulin. In contrast, the binding of the modified insulin analogue to insulin specific receptors was markedly increased. The discrepancy between the loss of biological potency and the apparent increase in binding affinity for membrane receptors reveals that not all of the biological activity of insulin is regulated by the receptor-binding system. The tetrapeptidamide of the B-chain of insulin (Arg-Gly-Phe-Phe-NH2) was clearly shown to have both insulin-like and insulin-potentiating actions in vivo although it had no effect on insulin receptor function in vitro. Evidence suggests that the small peptide fragment of insulin may be internalized and acts at the post-binding site(s) of the glucose metabolic pathway in target tissues. The present data support the general concept that insulin may exert its complex molecular actions through internalized hormonal fragment as well as the transmembrane mediators generated from receptor binding.

The semisynthesis of a series of B23-D-Ala deshexapeptide and despentapeptide analogues with B22-arginine replaced are described. The following semisynthetic scheme is used: desnonapeptide insulin pentamethyl ester is prepared from insulin hexamethyl ester through the action of trypsin and carboxypeptidase B, the amino groups are protected with Boc-groups, the protected product is condensed with synthetic peptides, and the protecting groups are removed with trifluoroacetic acid treatment and by saponification. The biological activities of these analogues indicate that the B22-Arg is non-essential and can be replaced by Lys or even Asp without any influence on the biological activity. The activity is reduced to one half when it is replaced by valine or to a very low level when replaced by Gly or D-Arg. The reason for the low activity of guinea pig insulin is discussed.