Distinct effects of saturated and monounsaturated fatty acids on beta-cell turnover and function

Glucotoxicity and lipotoxicity contribute to the impaired beta-cell function observed in type 2 diabetes. Here we examine the effect of saturated and unsaturated fatty acids at different glucose concentrations on beta-cell proliferation and apoptosis. Adult rat pancreatic islets were cultured onto plates coated with extracellular matrix derived from bovine corneal endothelial cells. Exposure of islets to saturated fatty acid (0.5 mmol/l palmitic acid) in medium containing 5.5, 11.1, or 33.3 mmol/l glucose for 4 days resulted in a five- to ninefold increase of beta-cell DNA fragmentation. In contrast, monounsaturated palmitoleic acid alone (0.5 mmol/l) or in combination with palmitic acid (0.25 or 0.5 mmol/l each) did not affect DNA fragmentation. Increasing concentrations of glucose promoted beta-cell proliferation that was dramatically reduced by palmitic acid. Palmitoleic acid enhanced the proliferation activity in medium containing 5.5 mmol/l glucose but had no additional effect at higher glucose concentrations (11.1 and 33.3 mmol/l). The cell-permeable ceramide analog C2-ceramide mimicked both the palmitic acid-induced beta-cell apoptosis and decrease in proliferation. Moreover, the ceramide synthetase inhibitor fumonisin B1 blocked the deleterious effects of palmitic acid on beta-cell viability. Additionally, palmitic acid but not palmitoleic acid decreased the expression of the mitochondrial adenine nucleotide translocator and induced release of cytochrome c from the mitochondria into the cytosol. Finally, palmitoleic acid improved beta-cell-secretory function that was reduced by palmitic acid. Taken together, these results suggest that the lipotoxic effect of the saturated palmitic acid involves an increased apoptosis rate coupled with reduced proliferation capacity of beta-cells and impaired insulin secretion. The deleterious effect of palmitate on beta-cell turnover is mediated via formation of ceramide and activation of the apoptotic mitochondrial pathway. In contrast, the monounsaturated palmitoleic acid does not affect beta-cell apoptosis, yet it promotes beta-cell proliferation at low glucose concentrations, counteracting the negative effects of palmitic acid as well as improving beta-cell function.     

Monounsaturated fatty acids prevent the deleterious effects of palmitate and high glucose on human pancreatic beta-cell turnover and function

Glucotoxicity and lipotoxicity contribute to the impaired beta-cell function observed in type 2 diabetes. Here we examine the effect of saturated and monounsaturated fatty acids at different glucose concentrations on human beta-cell turnover and secretory function. Exposure of cultured human islets to saturated fatty acid and/or to an elevated glucose concentration for 4 days increased beta-cell DNA fragmentation and decreased beta-cell proliferation. In contrast, the monounsaturated palmitoleic acid or oleic acid did not affect DNA fragmentation and induced beta-cell proliferation. Moreover, each monounsaturated fatty acid prevented the deleterious effects of both palmitic acid and high glucose concentration. The cell-permeable ceramide analogue C(2)-ceramide mimicked both the palmitic acid-induced beta-cell apoptosis and decrease in proliferation. Furthermore, the ceramide synthetase inhibitor fumonisin B1 blocked the deleterious effects of palmitic acid on beta-cell turnover. In addition, palmitic acid decreased Bcl-2 expression and induced release of cytochrome c from the mitochondria into the cytosol, which was prevented by fumonisin B1 and by oleic acid. Finally, each monounsaturated fatty acid improved beta-cell secretory function that was reduced by palmitic acid and by high glucose. Thus, in human islets, the saturated palmitic acid and elevated glucose concentration induce beta-cell apoptosis, decrease beta-cell proliferation, and impair beta-cell function, which can be prevented by monounsaturated fatty acids. The deleterious effect of palmitic acid is mediated via formation of ceramide and activation of the apoptotic mitochondrial pathway, whereas Bcl-2 may contribute to the protective effect of monounsaturated fatty acids.     

Leptin effects on pancreatic beta-cell gene expression and function

The hormone leptin is secreted from white adipocytes, and serum levels of leptin correlate with adipose tissue mass. Leptin was first described to act on the satiety center in the hypothalamus through specific receptors (leptin receptor [ObR]) to restrict food intake and enhance energy expenditure. Important peripheral actions of leptin involve inhibition of insulin biosynthesis and secretion in pancreatic beta-cells. In turn, insulin stimulates leptin secretion from adipose tissue, establishing a hormonal regulatory feedback loop-the so-called "adipo-insular axis." Multiple signal transduction pathways are involved in leptin signaling in pancreatic beta-cells. We have identified the proinsulin gene and protein phosphatase 1 gene as leptin repressed genes and the gene for the suppressor of cytokine signaling 3 protein as a leptin-induced gene in pancreatic beta-cells. The molecular effects of leptin culminate to restrict insulin secretion and biosynthesis to adapt glucose homeostasis to the amount of body fat. In most overweight individuals, however, physiological regulation of body weight by leptin seems to be disturbed, representing "leptin resistance." This leptin resistance at the level of the pancreatic beta-cell may contribute to dysregulation of the adipo-insular axis and promote the development of hyperinsulinemia and manifest type 2 diabetes in overweight patients.     

Lipotoxicity of the pancreatic beta-cell is associated with glucose-dependent esterification of fatty acids into neutral lipids

Prolonged exposure of isolated islets to supraphysiologic concentrations of palmitate decreases insulin gene expression in the presence of elevated glucose levels. This study was designed to determine whether or not this phenomenon is associated with a glucose-dependent increase in esterification of fatty acids into neutral lipids. Gene expression of sn-glycerol-3-phosphate acyltransferase (GPAT), diacylglycerol acyltransferase (DGAT), and hormone-sensitive lipase (HSL), three key enzymes of lipid metabolism, was detected in isolated rat islets. Their levels of expression were not affected after a 72-h exposure to elevated glucose and palmitate. To determine the effects of glucose on palmitate-induced neutral lipid synthesis, isolated rat islets were cultured for 72 h with trace amounts of [14C]palmitate with or without 0.5 mmol/l unlabeled palmitate, at 2.8 or 16.7 mmol/l glucose. Glucose increased incorporation of [14C]palmitate into complex lipids. Addition of exogenous palmitate directed lipid metabolism toward neutral lipid synthesis. As a result, neutral lipid mass was increased upon prolonged incubation with elevated palmitate only in the presence of high glucose. The ability of palmitate to increase neutral lipid synthesis in the presence of high glucose was concentration-dependent in HIT cells and was inversely correlated to insulin mRNA levels. 2-Bromopalmitate, an inhibitor of fatty acid mitochondrial beta-oxidation, reproduced the inhibitory effect of palmitate on insulin mRNA levels. In contrast, palmitate methyl ester, which is not metabolized, and the medium-chain fatty acid octanoate, which is readily oxidized, did not affect insulin gene expression, suggesting that fatty-acid inhibition of insulin gene expression requires activation of the esterification pathway. These results demonstrate that inhibition of insulin gene expression upon prolonged exposure of islets to palmitate is associated with a glucose-dependent increase in esterification of fatty acids into neutral lipids.     

Prolonged elevation of plasma free fatty acids impairs pancreatic beta-cell function in obese nondiabetic humans but not in individuals with type 2 diabetes

Our recent in vivo observations in healthy nonobese humans have demonstrated that prolonged elevation of plasma free fatty acids (FFAs) results in diminished glucose-stimulated insulin secretion (GSIS) when the FFA-mediated decrease in insulin sensitivity is taken into account. In the present study, we investigated whether obese individuals and patients with type 2 diabetes are more sensitive than healthy control subjects to the inhibitory effect of prolonged elevation of plasma FFAs on GSIS. In seven patients with type 2 diabetes and seven healthy nondiabetic obese individuals, we assessed GSIS with a programmed graded intravenous glucose infusion on two occasions, 6-8 weeks apart, with and without a prior 48-h infusion of heparin and Intralipid, which was designed to raise plasma FFA concentration approximately twofold over basal. The nondiabetic obese subjects had a significant 21% decrease in GSIS (P = 0.0008) with the heparin and Intralipid infusion, associated with a decrease in whole body insulin clearance. The impairment in GSIS was evident at low (<11 mmol/l) but not at higher plasma glucose concentrations. In contrast, the patients with type 2 diabetes had a slight increase in GSIS (P = 0.027) and no change in insulin clearance, although there was marked interindividual variability in response. Plasma proinsulin concentrations measured in a subset of subjects were not altered in either group by the infusion of heparin and Intralipid. In summary, 1) obese nondiabetic individuals are susceptible to a desensitization of GSIS with heparin and Intralipid infusion, and 2) patients with type 2 diabetes do not demonstrate such susceptibility when FFAs are elevated approximately twofold above basal with heparin and Intralipid. Our results suggest that FFAs could play an important role in the development of beta-cell failure in obese individuals who are at risk for developing type 2 diabetes. They do not, however, seem to further deteriorate the beta-cell function of patients who already have established type 2 diabetes and may even result in a slight increase in GSIS in this latter group.     

Tissue-specific deletion of the retinoblastoma protein in the pancreatic beta-cell has limited effects on beta-cell replication, mass, and function

Animal studies show that G(1/S) regulatory molecules (D-cyclins, cdk-4, p18, p21, p27) are critical for normal regulation of beta-cell proliferation, mass, and function. The retinoblastoma protein, pRb, is positioned at the very end of a cascade of these regulatory proteins and is considered the final checkpoint molecule that maintains beta-cell cycle arrest. Logically, removal of pRb from the beta-cell should result in unrestrained beta-cell replication, increased beta-cell mass, and insulin-mediated hypoglycemia. Because global loss of both pRb alleles is embryonic lethal, this hypothesis has not been tested in beta-cells. We developed two types of conditional knockout (CKO) mice in which both alleles of the pRb gene were inactivated specifically in beta-cells. Surprisingly, although the pRb gene was efficiently recombined in beta-cells of both CKO models, changes in beta-cell mass, beta-cell replication rates, insulin concentrations, and blood glucose levels were limited or absent. Other pRb family members, p107 and p130, were not substantially upregulated. In contrast to dogma, the pRb protein is not essential to maintain cell cycle arrest in the pancreatic beta-cell. This may reflect fundamental inaccuracies in models of beta-cell cycle control or complementation for pRb by undefined proteins.     

beta-cell function in obesity: effects of weight loss

In nondiabetic subjects, obesity is associated with a modest expansion of beta-cell mass, possibly amounting-according to the best available estimates-to 10-30% for each 10 kg of weight excess. Whether age of onset and duration of obesity, recent changes in body weight, and body fat distribution have any effect on beta-cell mass in humans is unknown. Both fasting insulin secretion and the total insulin response to oral glucose have the following characteristics: 1) they increase with BMI in an approximately linear fashion, 2) both fat-free and fat mass are significant positive correlates, and 3) BMI exerts a positive effect separate from that of insulin resistance (i.e., obesity may be a state of primary insulin hypersecretion). The mechanisms are currently unknown, though chronic small increments in plasma glucose may play a role. In contrast, dynamic properties of beta-cell function, such as glucose sensitivity (i.e., dose-response function), rate sensitivity, and potentiation, do not appear to be substantially altered by the presence of obesity, body fat distribution, or insulin resistance as long as glucose tolerance is maintained. Weight loss, by diet or restrictive bariatric surgery, is associated with consensual decrements in insulin resistance and insulin hypersecretion. The latter, however, seems to be more persistent, suggesting that the postobese state may reproduce the primary insulin hypersecretion of the obese state. Malabsorptive bariatric surgery, in contrast, normalizes insulin sensitivity and abolishes insulin hypersecretion even before achievement of ideal body weight. Lipid-triggered messages from the gastrointestinal tract to the insulin target tissues and endocrine pancreas are the subject of intense investigation.     

Regulation of pancreatic beta-cell glucokinase: from basics to therapeutics

Glucokinase (GK) serves as glucose sensor in pancreatic beta-cells and in other glucose sensor cells in the body. Biochemical genetic studies have characterized many activating and inactivating GK mutants that have been discovered in patients with hyperinsulinemic hypoglycemia or diabetes, all inherited as autosomal dominant traits. Mathematical modeling of the kinetic data of recombinant human wild-type and mutant GK accurately predicts the effects of GK mutations on the threshold of glucose-stimulated insulin release and glucose homeostasis. Structure/function studies of the enzyme suggest the existence of a hitherto unknown allosteric activator site of the enzyme that has significant implications for the physiological chemistry of GK-containing cells, particularly the pancreatic beta-cells. Glucose is the preeminent positive regulator of beta-cell GK expression and involves molecular mechanisms that are still to be elucidated in detail, but seem to have a specific requirement for increased glucose metabolism. Pharmaceutical chemists, motivated by the clear tenets of the GK glucose-sensor paradigm, have searched for and have discovered a novel class of GK activator molecules. The therapeutic application of this basic discovery offers a new principle for drug therapy of diabetes.     

Effect of epinephrine on pancreatic beta-cell and alpha-cell function in patients with NIDDM

The purposes of this study were to determine whether patients with non-insulin-dependent diabetes mellitus (NIDDM) have an enhanced glycemic response to epinephrine (EPI) and to quantitate the effect of physiological elevations of EPI on pancreatic islet function in these patients. The increment of plasma glucose (PG) in response to 45 min of EPI infusion (mean plasma EPI 2490 pM) was more than twofold greater in nine NIDDM patients than in 20 nondiabetic control subjects (mean +/- SE delta PG 3.9 +/- 0.3 vs. 1.7 +/- 0.1 mM, P less than 0.0001). The effects of EPI on beta-cell and alpha-cell function were compared in nine NIDDM patients and 9 age- and weight-matched control subjects during infusions of saline or two doses of EPI on separate days (mean plasma EPI 270, 1120, and 2490 pM). On each day, the acute insulin response (AIR) and acute glucagon response (AGR) to 5 g i.v. arginine were measured at three matched steady-state PG levels (means of 9, 14, and 29 mM). Beta-Cell sensitivity to glucose (slope of glucose potentiation) and beta-cell secretory capacity, or AIRmax (AIR at the highest clamped PG level), were calculated. In control subjects, EPI inhibited the AIR at PG concentrations of 9 and 14 mM (both P less than 0.05) but had no effect on the AIRmax, resulting in a rightward shift of the curve relating the AIR and PG and a decrease in the slope of glucose potentiation (P less than 0.01). In contrast in NIDDM patients, EPI inhibited the AIR at all PG levels, including the AIRmax (all P less than 0.05).(ABSTRACT TRUNCATED AT 250 WORDS)     

Molecular-genetic analysis and assessment of insulin action and pancreatic beta-cell function

Although the hereditary nature of non-insulin-dependent diabetes mellitus (NIDDM) is well recognized, the nature of the predisposing defect remains elusive. Individuals with a history of gestational diabetes had shown a reduced insulin-sensitivity index (S1) in the absence of fasting hyperglycemia. To determine whether this finding could result from an inherited defect of the insulin receptor, an NIDDM pedigree was ascertained through a former gestational-diabetic proband. The proband, her siblings, and her first cousins were clinically characterized for insulin sensitivity with the minimal-model-based S1 from a modified glucose tolerance test. Islet function was characterized by the incremental insulin response to 5 g i.v. arginine at baseline and at a plasma glucose level of 500-600 mg/dl. Genetic studies included linkage analyses for the insulin gene and the insulin-receptor gene with DNA polymorphisms (restriction-fragment-length polymorphisms, RFLPs) previously described. The pattern of inheritance in this large pedigree appeared to follow autosomal-dominant transmission. No defect in islet function was found, but as a group, third-generation family members had an S1 that was significantly lower than that of weight-matched control individuals, suggesting an inherited defect in insulin action. Genetic studies showed no sharing of insulin gene, insulin-receptor-gene alleles among the diabetic individuals, or insulin-receptor alleles among third-generation individuals with insulin insensitivity. The genetic analyses thus suggest that this pedigree has an inherited defect that is not linked to the insulin gene or the insulin-receptor gene. The diminished S1 may nonetheless suggest an inherited defect in insulin action.     

Correlations of in vivo beta-cell function tests with beta-cell mass and pancreatic insulin content in streptozocin-administered baboons

In vivo beta-cell function tests are used increasingly in humans during the preclinical phase of insulin-dependent diabetes mellitus (IDDM), but the severity of the beta-cell loss responsible for the abnormalities seen in these tests is unknown. We have measured several physiological beta-cell function tests--fasting plasma glucose, glucose disappearance constant, fasting insulin, acute insulin responses to arginine (AIRarginine) and glucose (AIRglucose), and glucose potentiation of AIRarginine (delta AIRarginine/delta G) and two direct objective measurements (pancreatic insulin content [PIC] and quantitative beta-cell mass)--in adolescent male baboons (Papio anubis/cyanocephalus). We have correlated in vivo measurements obtained within 3 days after the animals were killed with in vitro estimates of PIC and beta-cell mass in 15 animals, (2 nondiabetic requiring insulin treatment and 13 after varying doses of streptozocin to induce degrees of beta-cell damage ranging from normoglycemia to severe hyperglycemia). There was a strong linear correlation between beta-cell mass and PIC (r = 0.79, P less than 0.001). Physiological measures of beta-cell function were significantly correlated with both PIC and beta-cell mass. The correlations between physiological measures and beta-cell mass were linear and intercepted the beta-cell mass axis at 0.15-0.2 g, suggesting that in vivo measures of beta-cell function approach 0 when there is still approximately 40-50% of the beta-cell mass detectable histologically. With PIC, the linear correlations intercepted the axes close to 0. These findings provide considerable validity to the measurements of beta-cell function used in preclinical IDDM in humans.(ABSTRACT TRUNCATED AT 250 WORDS)     

Prolonged exposure to free fatty acids has cytostatic and pro-apoptotic effects on human pancreatic islets: evidence that beta-cell death is caspase mediated, partially dependent on ceramide pathway, and Bcl-2 regulated

In an effort to better understand the phenomenon of lipotoxicity in human beta-cells, we evaluated the effects of 48-h preculture with 1.0 or 2.0 mmol/l free fatty acid (FFA) (2:1 oleate to palmitate) on the function and survival of isolated human islets and investigated some of the possible mechanisms. Compared with control islets, triglyceride content was significantly increased and insulin content and glucose-stimulated insulin release were significantly reduced in islets precultured with increased FFA concentrations. These changes were accompanied by a significant reduction of glucose utilization and oxidation. By cell death detection techniques, it was observed that exposure to FFAs induced a significant increase of the amount of dead cells. Electron microscopy showed the involvement of beta-cells, with morphological appearance compatible with the presence of apoptotic phenomena. FFA-induced islet cell death was blocked by inhibition of upstream caspases and partially prevented by inhibiton of ceramide synthesis or serine protease activity, whereas inhibition of nitric oxide synthesis had no effect. RT-PCR studies revealed no major change of iNOS and Bax mRNA expression and a marked decrease of Bcl-2 mRNA expression in the islets cultured with FFA. Thus, prolonged exposure to FFAs has cytostatic and pro-apoptotic effects on human pancreatic beta-cells. The cytostatic action is likely to be due to the FFA-induced reduction of intraislet glucose metabolism, and the proapoptotic effects are mostly caspase mediated, partially dependent on ceramide pathway, and possibly Bcl-2 regulated.     

Enhancement of endocrine pancreatic secretions by essential fatty acids

Recent studies have suggested the beneficial effects of essential fatty acids in postoperative patients receiving total parenteral nutrition. While there is abundant information on the role of glucose and amino acids on insulin release, the effect of essential fatty acids on endocrine pancreatic secretions is not clear. Since linoleic and linolenic acids are constituents of TPN solutions as well as dietary fat, our aim was to examine their effect on the endocrine pancreatic function, using isolated islets. In each experiment, six islets microdissected from three mice were preperifused at the rate of 1 ml/min with Krebs-Ringer bicarbonate (KRB) buffer pH 7.4 containing 2% bovine albumin and 5.5 mM glucose (basal) with continuous supply of 95%/5%, O2/CO2 for 1 hr, after which basal samples were collected on ice every minute. The perifusion was continued for 20 min after the addition of a mixture of 10 mM linoleic acid and 5 mM linolenic acid to the KRB. During each perifusion phase, effluent samples were also collected for insulin and glucagon assay. The mean integrated area under the curve/20 min showed an increase in both insulin and glucagon secretions with the addition of fatty acids. Hence insulin increased from a basal 3154.8 +/- 953.7 to 8393.0 +/- 2073.1 pg (P less than 0.025, n = 6) and glucagon increased from 193.7 +/- 46.9 to 1566.1 +/- 411.2 pg (P less than 0.0025, n = 5). The fatty-acid-induced insulin but not glucagon secretion was blocked by the addition of 2 mM palmoxirate an inhibitor of fatty acid oxidation.(ABSTRACT TRUNCATED AT 250 WORDS)     

Fructose-1,6-bisphosphatase overexpression in pancreatic beta-cells results in reduced insulin secretion: a new mechanism for fat-induced impairment of beta-cell function

OBJECTIVE—Fructose-1,6-bisphosphatase (FBPase) is a gluconeogenic enzyme that is upregulated in islets or pancreatic β-cell lines exposed to high fat. However, whether specific β-cell upregulation of FBPase can impair insulin secretory function is not known. The objective of this study therefore is to determine whether a specific increase in islet β-cell FBPase can result in reduced glucose-mediated insulin secretion.RESEARCH DESIGN AND METHODS—To test this hypothesis, we have generated three transgenic mouse lines overexpressing the human FBPase (huFBPase) gene specifically in pancreatic islet β-cells. In addition, to investigate the biochemical mechanism by which elevated FBPase affects insulin secretion, we made two pancreatic β-cell lines (MIN6) stably overexpressing huFBPase.RESULTS—FBPase transgenic mice showed reduced insulin secretion in response to an intravenous glucose bolus. Compared with the untransfected parental MIN6, FBPase-overexpressing cells showed a decreased cell proliferation rate and significantly depressed glucose-induced insulin secretion. These defects were associated with a decrease in the rate of glucose utilization, resulting in reduced cellular ATP levels.CONCLUSIONS—Taken together, these results suggest that upregulation of FBPase in pancreatic islet β-cells, as occurs in states of lipid oversupply and type 2 diabetes, contributes to insulin secretory dysfunction.Type 2 diabetes is characterized by a chronic elevation of plasma glucose concentration, causing complications such as retinopathy, neuropathy, and nephropathy and increasing the risk of cardiovascular disease and stroke. Although insulin resistance may be the initiating defect, hyperglycemia in type 2 diabetes results from a relative deficiency of circulating insulin (1). Progressive deterioration in β-cell function is likely to result from exposure to the diabetic milieu (i.e., hyperglycemia and hyperlipidemia), thus setting up a positive feedback loop in which hyperglycemia and/or hyperlipidemia impairs β-cell function, leading to further hyperglycemia (2–7).Chronic high fatty acid exposure results in increased basal and blunted glucose-mediated insulin secretion and reduced β-cell mass (2,5,8,9). This is associated with the pancreatic β-cells undergoing adaptive changes such that genes that are highly expressed under normal conditions, for example, insulin, PDX-1, and GLUT2, are underexpressed and genes that are poorly expressed in the pancreatic β-cells, such as hexokinase I, glucose-6-phosphatase, c-Myc, and acetate dehydrogenase, are shown to be upregulated (10,11).One of the genes that is upregulated in β-cell lines under conditions of high fatty acid exposure is fructose-1,6-bisphosphatase (FBPase) (10–12), a regulated enzyme in the gluconeogenic pathway that catalyzes the dephosphorylation of fructose-1,6-bisphosphate to fructose-6-phosphate. FBPase is abundant in the liver and the kidneys but is poorly expressed in the pancreatic β-cells under normal conditions. In addition, FBPase was upregulated fivefold in islets from the diabetes-susceptible obese BTBR mouse compared with the diabetes-resistant C57BL/6 mouse (13). We have previously shown that FBPase is upregulated in the liver of mice or rats fed a high-fat diet (14,15) and in the New Zealand Obese (NZO) mouse, an obese model of type 2 diabetes (14,16). We have recently demonstrated that transgenic mice with specific overexpression of FBPase in the liver displayed increased glycerol gluconeogenesis (17).From the abovementioned studies, it is evident that upregulation of FBPase is induced by fatty acids. However, whether an increase in FBPase alone can be detrimental to cellular function and in particular to β-cell insulin secretory rates has not been investigated.To determine whether an increase in FBPase impairs insulin secretion, we generated both transgenic mice and stably transfected pancreatic β-cell lines (MIN6) overexpressing the human FBPase (huFBPase) gene. We demonstrated that overexpression of FBPase in β-cells results in impaired glucose-stimulated insulin secretion, which is associated with decreased glucose metabolism, resulting in reduced cellular ATP levels and cell proliferation.     

Quantitation of human pancreatic beta-cell function by immunoassay of C-peptide in urine.

Human proinsulin connecting peptide (C-peptide) was measured by immunoassay in urine from 25 normal subjects, 18 patients with diabetes mellitus, and 34 patients with various degrees of renal insufficiency. Assay validation studies showed that pancreatic C-peptide was quantitatively recovered when added to urine. Fractionation of urine by gel filtration indicated that most endogenous C-peptide eluted in fractions that corresponded to the C-peptide standard. In 34 nondiabetic subjects with normal kidney function or various renal diseases, C-peptide clearance was independent of creatinine clearance over a range of 6 to 190 ml./min. Urine C-peptide clearance (5.1 +/- 0.6 ml./min.) is greater than that of insulin (1.1 +/- 0.2 ml./min.), and the total quantity of C-peptide excreted in the urine per day represents 5 per cent of pancreatic secretion, as against only 0.1 per cent of secreted insulin. Healthy subjects excreted 36 +/- 4 mug. C-peptide per 24 hours, while this value in juvenile-onset diabetics was only 1.1 +/- 0.5 mug. Adult-onset diabetics excreted 24 +/- 7 mug./24 hr., the range overlapping the excretory rates of both normal subjects and juvenile-onset diabetics. Two insulin-requiring adult-onset diabetics showed significant beta-cell reserve during the course of acute infections. These results suggest that urine C-peptide provides a useful means of assessing beta-cell secretory capacity over a period of time and is especially advantageous when frequent blood sampling is not feasible.