Insulin-degrading Activity Research Articles

Investigation into the presence of insulin-degrading enzyme in cultured type II alveolar cells and the effects of enzyme inhibitors on pulmonary bioavailability of insulin in rats.

The purpose of this study was to investigate the role of insulin-degrading enzyme (IDE, EC 3.4.22.11) in insulin degradation in alveolar epithelium. The primary culture of isolated rat type-II pneumocytes was used for the in-vitro characterization of IDE. Insulin was then administered intratracheally with various inhibitors to assess the improvement in its pulmonary bioavailability. In cultured type-II pneumocytes, the cytosolic insulin-degrading activity contributed 81% of total insulin degradation, reached a maximum at pH 7.5 and had an apparent Michaelis-Menten constant (Km) of 135 nM. N-Ethylmaleimide, p-chloromercuribenzoic acid and 1,10-phenanthroline inhibited insulin-degrading activity almost completely in both crude homogenate and cytosol. An immunoprecipitation study showed that IDE contributed 74% of cytosolic insulin-degrading activity. Western blot analysis showing a single band of 110 kDa on reduced SDS (sodium dodecylsulphate) gels confirmed the presence of IDE in cultured type-II cells. When given intratracheally with insulin, inhibitors including N-ethylmaleimide, p-chloromercuribenzoic acid, and 1,10-phenanthroline significantly enhanced the absolute bioavailability of insulin and the compound's hypoglycaemic effects. These results suggest that IDE is present in alveolar epithelium and might be involved in limiting insulin absorption in the lung.

The Presence of Insulin-degrading Enzyme in Human Ileal and Colonic Mucosal Cells

The aim of this research is to characterize the presence of insulin-degrading enzyme in human colon and ileal mucosal cells. Biochemical studies, including the activity-pH profiles, the effects of enzyme inhibitors, immunoprecipitation and western blots, were conducted. The majority of insulin-degrading activity in colon mucosal cells was localized in the cytosol. In both colon and ileum, cytosolic insulin-degrading activities had a pH optimum at pH 7.5, and were extensively inhibited by each of N-ethylmaleimide, p-chloromercuribenzoate, and 1,10-phenanthroline, but were very weakly affected by each of leupeptin, chymostatin, diisopropyl phosphofluoridate and soybean trypsin inhibitor. In the colon and ileum, more than 93% and 96%, respectively, of cytosolic insulin-degrading activities were removed by the mouse monoclonal antibody to human RBC insulin-degrading enzyme, as compared with less than 20% by the normal mouse IgG for both tissues. Further, a western blot analysis revealed that a cytosolic protein of 110 kD, in both human colon and ileum, reacted with the monoclonal antibody to insulin-degrading enzyme. It is concluded that insulin-degrading enzyme is present in the cytosol of human colon and ileal mucosal cells.

Nitric oxide inhibits insulin-degrading enzyme activity and function through S-nitrosylation

Insulin-degrading enzyme (IDE) is responsible for the degradation of a number of hormones and peptides, including insulin and amyloid β (Aβ). Genetic studies have linked IDE to both type 2 diabetes and Alzheimer's disease. Despite its potential importance in these diseases, relatively little is known about the factors that regulate the activity and function of IDE. Protein S-nitrosylation is now recognized as a redox-dependent, cGMP-independent signaling component that mediates a variety of actions of nitric oxide (NO). Here we describe a mechanism of inactivation of IDE by NO. NO donors decreased both insulin and Aβ degrading activities of IDE. Insulin-degrading activity appeared more sensitive to NO inhibition than Aβ degrading activity. IDE-mediated regulation of proteasome activity was affected similarly to insulin-degrading activity. We found IDE to be nitrosylated in the presence of NO donors compared to that of untreated enzyme and the control compound. S-nitrosylation of IDE enzyme did not affect the insulin degradation products produced by the enzyme, nor did NO affect insulin binding to IDE as determined by cross-linking studies. Kinetic analysis of NO inhibition of IDE confirmed that the inhibition was noncompetitive. These data suggest a possible reversible mechanism by which inhibition of IDE under conditions of nitrosative stress could contribute to pathological disease conditions such as Alzheimer's disease and type 2 diabetes.

Dramatic Improvement of Subcutaneous Insulin Resistance with Nafamostat Ointment Treatment

Subcutaneous insulin resistance is characterized by a lack of biological efficacy of subcutaneously injected insulin, with retained sensitivity to intravenously injected insulin. The existence of increased insulin-degrading activity has been suggested as a possible underlying mechanism (1). It has been reported that protease inhibitors exert beneficial effects on the absorption of subcutaneously injected insulin (1). Although the effect of an ointment containing a protease inhibitor has been shown in normal volunteers (2), there has been no report on the ointment's effect in patients with subcutaneous insulin resistance. This is …

Subcutaneous insulin resistance successfully circumvented on long term by peritoneal insulin delivery from an implantable pump in four diabetic patients

Extreme subcutaneous insulin resistance is a rare syndrome characterized by a severe resistance to subcutaneous (S/C) insulin together with persistence of normal or near normal intravenous (IV) insulin sensitivity. Its pathophysiology is unknown, although increased insulin degrading activity has been reported in the S/C adipose tissue fraction in some cases. Until now, proposed treatments have been disappointing. We report 4 cases who were successfully treated by intraperitoneal (IP) route. The diagnosis of subcutaneous insulin resistance was based upon following combined conditions: resistance to hypoglycaemic action of subcutaneous insulin but normal or near normal sensitivity to IV or IP insulin. 4 patients among those followed by EVADIAC group met these criteria: 3 with type 1 diabetes (C peptide=0), the last one with unexplained non insulin-deficient diabetes (no anti-GAD antibodies, C peptide=5 ng/ml). All of them had been treated with subcutaneous insulin therapy without success despite huge doses (up to 4000 IU/day in two patients). The 3 type 1 diabetic patients presented with a history of repeated ketoacidosis episodes. A treatment of insulin mixed with aprotinin had been proposed to 2 patients without success. The IV insulin sensitivity was proved to be normal in two patients by euglycaemic clamp data. A skin biopsy was performed in 1 patient. An accumulation of insulin in the derma was revealed with no increase of degradation products of insulin. In these 4 patients, a dramatic improvement of diabetes control was obtained by IP insulin delivery from an implantable pump (HbA1c decrease by at least 3%). Although pathophysiology of the subcutaneous insulin resistance syndrome remains unexplained, our data show that intra-peritoneal insulin therapy from an implantable pump allows diabetes control in patients affected by this uncommon but severely disabling condition.

Insulin-degrading activity in wound fluid.

Patients with diabetes are at great risk of developing lower extremity ulcers. The management of diabetic foot ulcers typically includes early recognition and appropriate clinical care. Recent advances in wound treatment include topical growth factor therapy, which has been successful in diabetic wounds. Growth factors are decreased in wound fluid; this may be due to decreased supply, increased binding, or increased degradation of the naturally occurring growth factors. This study investigates the activity of the insulin-degrading enzyme in wound fluid. Wound fluid was obtained from patients with (n = 17) and without (n = 4) diabetes. Insulin degradation was assayed by incubating [(125)I]insulin with wound fluid and precipitation in trichloroacetic acid. Fluid from nondiabetics degraded 2.22 +/- 0.73%, whereas diabetic fluid degraded significantly more (6.13 +/- 1.48%; P < 0.05). In patients with diabetes, the degradation of insulin by wound fluid correlated with glucose control (hemoglobin A(1c); r(2) = 0.5353; P < 0.001), and patients with worse outcomes (i.e. amputation) had higher wound fluid insulin degradation. The biochemical characteristics of insulin degradation in the wound fluid were consistent with the characteristics of insulin-degrading enzyme. These data suggest that glucose control is a critical factor in wound healing, but a reduction in the insulin-degrading activity in the wound fluid is also a potential therapeutic target.

De novo emergence of growth factor receptors in activated human CD4 + and CD8 + T lymphocytes

Using phytohemagglutinin (PHA)-activated human T lymphocytes, we have demonstrated de novo emergence of growth factor receptors (insulin, insulin-like growth factor-1 [IGF-1], and interleukin-2 [IL-2]) in the CD4 + and CD8 + subsets determined by flow cytometry. This activation was also associated with development of insulin-degrading activity (IDA) in a time-dependent fashion. These events, which are actinomycin- and cycloheximide-sensitive, occur only in activated, but not nonactivated, CD4 + and CD8 + lymphocytes. The emergence of these receptors, as well as IDA, which is preceded by CD69 emergence, reaches a plateau by 72 hours and is comparable in both subsets. The IDA is localized in the cytosol, and insulin binding is limited to the cell membrane. T-lymphocyte activation also initiates expression of the IL-2 gene with the transcription of IL-2 mRNA, the level of which is further enhanced by 38% with the addition of insulin. In these activated lymphocytes, insulin binding to its receptor also caused an 83% upregulation of phosphorylated insulin receptor substrate-1 (IRS-1). In situ development of these growth factor receptors and signal transduction mechanisms in T lymphocytes upon activation, such as by proinflammatory cytokines or oxidative stress, could be an important defense mechanism in various disease states in man.

Extreme subcutaneous insulin resistance: a misunderstood syndrome

Extreme subcutaneous insulin resistance (SIR) is a rare syndrome characterized by severe resistance to subcutaneous insulin with normal intravenous insulin sensitivity. Its pathophysiology is unknown, though an increased insulin degrading activity has been suggested. We report the case of a 35 year-old female patient with type I diabetes since the age of 3. Despite five shots of insulin/day, the patient progressively developed permanent ketosis related to severe acquired SIR with insulin doses as high as 500 U/day. Subcutaneous infusion of insulin and lispro insulin through an external pump did not improve resistance: HbA(1c) levels remained between 14 and 18% (N<6.5%). After numerous ketoacidotic episodes, continuous ambulatory intravenous insulin infusion was attempted through a central port due to a lack of peripheral venous access. HbAlc improved (8.5%) and daily insulin needs decreased to below 40U. However, the treatment had to be discontinued because of thrombosis and infection at different times. Intraperitoneal insulin infusion with an external pump was then proposed. HbAlc improved to 8% during 18 months but several episodes of catheter infection and encapsulation led to its removal. An intraperitoneal pump was surgically implanted, leading to the stabilization of HbA(1c) to around 8%. An insulin degradation assay did not demonstrate any excess of insulin degrading activity in the patient's or controls' subcutaneous tissue; nevertheless, excessive amounts of insulin were found in the patient's derm compared to controls. This case report of acquired SIR raises the question of its treatment and mechanisms. Regarding treatment, intraperitoneal delivery of insulin appears to be the best solution, but the mechanisms underlying SIR still remain unclear.

Non-covalent interaction of ubiquitin with insulin-degrading enzyme

Insulin-degrading enzyme (IDE) is a metalloprotease implicated in insulin degradation and suggested to have a variety of additional functions, including the clearance of amyloid β peptides of Alzheimer's disease. Little is known about endogenous proteins that may interact with and modulate IDE's activity in the cell. We purified and characterized two proteins from mouse leukemic splenocytes that interact with IDE and inhibit its insulin-degrading activity. A protein of 14 kDa was similar to a competitive IDE inhibitor reported previously. The major inhibitor was identified by amino acid sequencing as ubiquitin, a protein that is post-translationally covalently attached to other intracellular proteins and regulates diverse cellular processes. Ubiquitin inhibited insulin-degrading activity of IDE and diminished crosslinking of 125I-insulin to IDE in a specific, concentration-dependent, reversible, and ATP-independent manner. Ubiquitin did not affect the crosslinking of 125I-insulin to insulin receptors or of 125I-atrial natriuretic peptide (ANP) to its receptor guanylate cyclase-A. These findings suggest a novel role for ubiquitin or perhaps proteins with ubiquitin-like domains in regulating the function of IDE.

Endosomal Proteolysis of Internalized Insulin at the C-terminal Region of the B Chain by Cathepsin D

The endosomal compartment of hepatic parenchymal cells contains an acidic endopeptidase, endosomal acidic insulinase, which hydrolyzes internalized insulin and generates the major primary end product A(1--21)-B(1--24) insulin resulting from a major cleavage at residues Phe(B24)-Phe(B25). This study addresses the nature of the relevant endopeptidase activity in rat liver that is responsible for most receptor-mediated insulin degradation in vivo. The endosomal activity was shown to be aspartic acid protease cathepsin D (CD), based on biochemical similarities to purified CD in 1) the rate and site of substrate cleavage, 2) pH optimum, 3) sensitivity to pepstatin A, and 4) binding to pepstatin A-agarose. The identity of the protease was immunologically confirmed by removal of greater than 90% of the insulin-degrading activity associated with an endosomal lysate using polyclonal antibodies to CD. Moreover, the elution profile of the endosomal acidic insulinase activity on a gel-filtration TSK-GEL G3000 SW(XL) high performance liquid chromatography column corresponded exactly with the elution profile of the immunoreactive 45-kDa mature form of endosomal CD. Using nondenaturating immunoprecipitation and immunoblotting procedures, other endosomal aspartic acid proteases such as cathepsin E and beta-site amyloid precursor protein-cleaving enzyme (BACE) were ruled out as candidate enzymes for the endosomal degradation of internalized insulin. Immunofluorescence studies showed a largely vesicular staining pattern for internalized insulin in rat hepatocytes that colocalized partially with CD. In vivo pepstatin A treatment was without any observable effect on the insulin receptor content of endosomes but augmented the phosphotyrosine content of the endosomal insulin receptor after insulin injection. These results suggest that CD is the endosomal acidic insulinase activity which catalyzes the rate-limiting step of the in vivo cleavage at the Phe(B24)-Phe(B25) bond, generating the inactive A(1--21)-B(1--24) insulin intermediate.

Insulin-degrading enzyme identified as a candidate diabetes susceptibility gene in GK rats.

Genetic analysis of the diabetic GK rat has revealed several diabetes susceptibility loci. Congenic strains have been established for the major diabetes locus, Niddm1, by transfer of GK alleles onto the genome of the normoglycemic F344 rat. Niddm1 was dissected into two subloci, physically separated in the congenic strains Niddm1b and Niddm1i, each with at least one disease susceptibility gene. Here we have mapped Niddm1b to 1 cM by genetic and pathophysiological characterization of new congenic substrains for the locus. The gene encoding insulin-degrading enzyme (IDE:) was located to this 1 cM region, and the two amino acid substitutions (H18R and A890V) identified in the GK allele reduced insulin-degrading activity by 31% in transfected cells. However, when the H18R and A890V variants were studied separately, no effects were observed, demonstrating a synergistic effect of the two variants on insulin degradation. No effect on insulin degradation was observed in cell lysates, indicating that the effect is coupled to receptor-mediated internalization of insulin. Congenic rats with the IDE: GK allele displayed post-prandial hyperglycemia, reduced lipogenesis in fat cells, blunted insulin-stimulated glucose transmembrane uptake and reduced insulin degradation in isolated muscle. Analysis of additional rat strains demonstrated that the dysfunctional IDE: allele was unique to GK. These data point to an important role for IDE: in the diabetic phenotype in GK.

Insulin is degraded extracellularly in wounds by insulin-degrading enzyme (EC 3.4.24.56).

The exact mechanism by which insulin reverses impaired wound healing is unknown. Previous investigators have shown that insulin is degraded in experimental wounds, suggesting that the action of insulin may be locally modified. The following study corroborates these findings and identifies the major proteinase responsible for insulin degradation in wound fluid (WF). Adult male Fisher rats were wounded by subcutaneous implantation of polyvinyl alcohol sponges while under pentobarbital sodium anesthesia. WF and serum were collected on 1, 5, 10, and 14 days postinjury. Decreased insulin concentration in late WF correlated with an increased insulin-degrading activity. Multiple proteinases appear to participate in the overall degradation of insulin in WF. However, the primary enzyme responsible for insulin degradation in WF was characterized by immunoprecipitation and immunoblotting and identified as the neutral thiol-dependent metalloproteinase, insulin-degrading enzyme (EC 3.4.24.56). Exogenous steroid administration caused a decrease in WF insulin-degrading activity. Glucagon and adrenocorticotrophin degradation was also observed, whereas minimal degradation of insulin-like growth factors I and II and epidermal growth factor was detected in WF. The ability to extracellularly degrade insulin may represent a unique mechanism for the regulation of this hormone's role in healing wounds.

Two pathways for insulin metabolism in adipocytes

Using selected conditions, the appropriate collagenase, albumin and cell treatment, a preparation of isolated adipocytes was developed with no extracellular insulin degrading activity. Cell mediated insulin degradation rates were 0.68%±0.05%/100 000 cell/h using trichloracetic acid precipitability as a measure. Chloroquine (CQ) increased cell-associated radioactivity and decreased degradation while dansylcadaverine (DC), PCMBS and bacitracin (BAC) decreased degradation with no effect on binding. Extraction and chromatography of the cell-associated radioactivity showed 3 peaks, a large molecular weight peak, a small molecular weight peak and an insulin-sized peak. CQ, DC and BAC all decreased the small molecular weight peak while CQ and DC also increased the peak of large molecular weight radioactivity. Cell mediated insulin degradation in the presence of combinations of inhibitors suggested two pathways in adipocytes, one affected by inhibitors of the insulin degrading enzyme (IDE) (bacitracin and PCMBS) and the other altered by cell processing inhibitors (DC, CQ and phenylarsenoxide). Chloroquine altered the pattern of the insulin-sized cell-associated HPLC assayed degradation products, further supporting two pathways of degradation; one a chloroquine-sensitive and one a chloroquine-insensitive pathway.

Super-CHO?A cell line capable of autocrine growth under fully defined protein-free conditions

Chinese Hamster Ovary (CHO) cells are widely used for the large scale production of recombinant biopharmaceuticals. Growth of the CHO-K1 cell line has been demonstrated in serum-free medium containing insulin, transferrin and selenium. In an attempt to get autocrine growth in protein-free medium, DNA coding for insulin and transferrin production was transfected into CHO-K1 cells. Transferrin was expressed well, with clones secreting approximately 1000 ng/10(6) cells/24h. Insulin was poorly expressed, with rates peaking at 5 ng/10(6) cells/24h. Characterisation of the secreted insulin indicated that the CHO cells were incompletely processing the insulin molecule. Site-directed mutagenesis was used to introduce a furin (prohormone converting enzyme) recognition sequence into the insulin molecule, allowing the production of active insulin. However, the levels were still too low to support autocrine growth. Further investigations revealed insulin degrading activity (presumably due to the presence of insulin degrading enzymes) in the cytoplasm of CHO cells. To overcome these problems insulin-like growth factor I (instead of insulin) was transfected into the cells. IGF-1 was completely processed and expressed at rates greater than 500 ng/10(6)cells/24h. In this paper we report autonomous growth of the transfected CHO-K1 cell line expressing transferrin and IGF-1 in protein-free medium without the addition of exogenous growth factors. Growth rates and final cell densities of these cells were identical to that of the parent cell line CHO-K1 growing in insulin, transferrin, and selenium supplemented serum-free media.

Transepithelial transport of insulin: I. Insulin degradation by insulin-degrading enzyme in small intestinal epithelium.

The purpose of this study was to determine the existence of insulin-degrading enzyme (EC 3.4.22.11) (IDE) in rat intestinal enterocytes. Subcellular fractionation, biochemical characterization, immunoprecipitation, and western blots were employed. Insulin-degrading activity was localized in the cytosol, constituting 92% of total insulin-degrading activity. Cytosolic insulin-degrading activity had a pH optimum of 7.5, was almost completely inhibited by IDE inhibitors (N-ethylmaleimide, 1,10-phenanthroline, EDTA, p-chloromercuribenzoate, bacitracin), but was not or only weakly inhibited by others (aprotinin, chymostatin, leupeptin, and diisopropyl phosphofluoridate.) Further, cytosolic insulin-degrading activity had a Km of 78 nM, sharing a similar Km value with insulin-degrading enzyme in non-purified forms. Approximately, 87 +/- 1.7% of cytosolic insulin-degrading activity was removed by the monoclonal antibody to IDE. On the SDS gel, the molecular weight of cytosolic IDE was 110 KD which is the same as that of human IDE. IDE is the major enzyme which degrades insulin in enterocytes.

Insulin-degrading enzyme in a human colon adenocarcinoma cell line (Caco-2).

The activity of insulin-degrading enzyme (IDE), a thiol metalloprotease degrading insulin in many insulin target cells, was determined in human colon adenocarcinoma (Caco-2) cells. Insulin-degrading activity was localized in the cytosol of Caco-2 cells, accounting for 88% of total activity. Western blots and immunoprecipitation showed that IDE was present in the cytosol of Caco-2 cells and contributed to more than 93% cytosolic insulin-degrading activity. Cytosolic insulin degradation was strongly inhibited by IDE inhibitors, including N-ethylmaleimide, 1,10-phenanthroline, p-chloromericuribenzoate, and EDTA, but was not significantly or not as extensively inhibited by strong inhibitors of proteasome, i.e., chymostatin, soybean trypsin inhibitor, leupeptin, and Dip-F. These results suggest that IDE is present in Caco-2 cells, that Caco-2 IDE has properties similar to those of its counterparts in insulin-target tissues, and that it significantly contributes to intracellular insulin degradation.

Detection of the substance immunologically cross-reactive with insulin in insulin ria is an artifact caused by insulin tracer degradation: involvement of the insulin-degrading enzyme

In previous literature, the existence of a new insulin-like substance found in tumor tissues, termed substance immunologically crossreactive with insulin (SICRI), has been proposed. In these studies, insulin-specific radioimmunoassay (RIA) was the only detection method for SICRI. The mouse melanoma B16BL6 cell line was found to be a rich source of SICRI. In this paper, we show that SICRI is not expressed in B16BL6 cells. Previous RIA measurements were wrongly ascribed to SICRI. What was really measured was a positive artifact caused by insulin tracer degradation in RIA. Several lines of evidence indicate that protease responsible for insulin degradation in B16BL6 cells is insulin-degrading enzyme (IDE; EC 3.4.22.11). First, SICRI activity of B16BL6 cytosol measured by insulin RIA was inhibited by thiol protease inhibitor N-ethylmaleimide (NEM). Thiol active agents as well as metal chelators, both potent IDE blockers, inhibited also the insulin-degrading activity of the same sample. Second, cross-linking to 125I-labeled insulin of partially purified sample with highest insulin RIA activity specifically labeled only a single protein with molecular mass similar to IDE (110 kDa). Labeling was blocked by ‘cold’ insulin in excess. Third, kinetic studies of insulin degradation by RIA active Chromatographic fractions revealed an apparent K d of 90 nM which is very similar to the reported affinity of insulin for IDE ( K d = 100 nM). Additionally, in B16BL6 as well as in mouse myeloid leukemia cells, IDE gene is actively transcribed and this expression was found to be much stronger than in normal mouse tissues. In conclusion, our results strongly question the real existence of SICRI. They showed that IDE is responsible for false positive measurements of SICRI in RIA. These artifacts were circumvented by IDE inhibitor NEM. Finally, increased expression of IDE gene in mouse tumor tissues suggests a possible important role for IDE in the process of malignant transformation.

Effects of bile salts on brush-border and cytosolic proteolytic activities of intestinal enterocytes

The effects of bile salts, i.e., sodium deoxycholate, sodium glycocholate, sodium glycodeoxycholate, sodium taurocholate, and sodium taurodeoxycholate, on the activities of individual rat intestinal brush-border membrane peptidases and cytosolic insulin-degrading activities were examined. At 2 mM, bile salts had no effects on aminopeptidases P and W, but did inhibit other brush-border peptidases though no single bile salt was able to inhibit every peptidase. Even at concentrations higher than the CMC, inhibition of brush-border insulin degradation is incomplete while that of cytosolic insulin degradation is complete at low concentrations. The results suggest that bile salts, inhibiting brush-border membrane and cytosolic proteolytic hydrolysis, may be useful for reducing intestinal degradation of peptide drugs; and that bile salts have stronger inhibitory effects on soluble than on membrane-bound insulin-degrading activity.

Androgen and glucocorticoid receptors interact with insulin degrading enzyme.

We recently identified a protein, designated receptor accessory factor (RAF), that interacts directly with and enhances the DNA binding of androgen (AR) and glucocorticoid (GR) receptor peptide fragments. To determine its identity, RAF was purified from HeLa cell extracts by anion-exchange and DNA affinity chromatography. RAF activity co-purified with a 110-kDa protein, the partial amino acid sequence of which shares 97.5% identity with insulin degrading enzyme (IDE), a metalloendoprotease implicated in the intracellular degradation of insulin. The identity of RAF was confirmed in gel shift assays by demonstrating that AR.RAF and GR.RAF DNA complexes shifted to a slower mobility in the presence of an IDE antibody. Purified preparations of RAF had insulin degrading activity, and this activity was inhibited by AR. In addition, the interaction of AR or GR with RAF was competed by insulin and bacitracin, a competitive inhibitor of IDE. The interactions of AR and GR with IDE may have important implications for both insulin- and steroid-mediated signaling.

Extracellular insulin degrading activity creates instability in a CHO-based batch-refeed continuous process.

In a batch-refeed continuous process involving a recombinant Chinese hamster ovary cell line, a brief upset was occasionally observed during which cell growth halted and cell viability dropped. This was found to be associated with depletion of insulin from the medium early during the affected passage. Insulin depletion was found to be primarily the result of insulin degrading activity released by the cells during the preceding passage.