Use of 3D Human Liver Organoids to Predict Drug-Induced Phospholipidosis.

Drug-induced phospholipidosis (PL) is a storage disorder caused by the formation of phospholipid-drug complexes in lysosomes. Because of the diversity of PL between species, human cell-based assays have been used to predict drug-induced PL in humans. We established three-dimensional (3D) human liver organoids as described previously and investigated their liver characteristics through multiple analyses. Drug-induced PL was initiated in these organoids and in monolayer HepG2 cultures, and cellular changes were systemically examined. Organoids that underwent differentiation showed characteristics of hepatocytes rather than HepG2 cells. The organoids also survived under PL-inducing drug conditions for 48 h and maintained a more stable albumin secretion level than the HepG2 cells. More cytoplasmic vacuoles were observed in organoids and HepG2 cells treated with more potent PL-induced drugs, but to a greater extent in organoids than in HepG2 cells. Lysosome-associated membrane protein 2, a marker of lysosome membranes, showed a stronger immunohistochemical signal in the organoids. PL-distinctive lamellar bodies were observed only in amiodarone-treated organoids by transmission electron microscopy. Human liver organoids are thus more sensitive to drug-induced PL and less affected by cytotoxicity than HepG2 cells. Since PL is a chronic condition, these results indicate that organoids better reflect metabolite-mediated hepatotoxicity in vivo and could be a valuable system for evaluating the phospholipidogenic effects of different compounds during drug development.

Exploiting ensemble learning to improve prediction of phospholipidosis inducing potential.

Phospholipidosis is characterized by the presence of excessive accumulation of phospholipids in different tissue types (lungs, liver, eyes, kidneys etc.) caused by cationic amphiphilic drugs. Electron microscopy analysis has revealed the presence of lamellar inclusion bodies as the hallmark of phospholipidosis. Some phospholipidosis causing compounds can cause tissue specific inflammatory/retrogressive changes. Reliable and accurate in silico methods could facilitate early screening of phospholipidosis inducing compounds which can subsequently speed up the pharmaceutical drug discovery pipelines. In the present work, stacking ensembles are implemented for combining a number of different base learners to develop predictive models (a total of 256 trained machine learning models were tested) for phospholipidosis inducing compounds using a wide range of molecular descriptors (ChemMine, JOELib, Open babel and RDK descriptors) and structural alerts as input features. The best model consisting of stacked ensemble of machine learning algorithms with random forest as the second level learner outperformed other base and ensemble learners. JOELib descriptors along with structural alerts performed better than the other types of descriptor sets. The best ensemble model achieved an overall accuracy of 88.23%, sensitivity of 86.27%, specificity of 90.20%, mcc of 0.765, auc of 0.896 with 88.21 g-means. To assess the robustness and stability of the best ensemble model, it is further evaluated using stratified 10×10 fold cross validation and holdout testing sets (repeated 10 times) achieving 84.83% mean accuracy with 0.708 mean mcc and 88.46% mean accuracy with 0.771 mean mcc respectively. A comparison of different meta classifiers (Generalized linear regression, Gradient boosting machines, Random forest and Deep learning neural networks) in stacking ensemble revealed that random forest is the better choice for combining multiple classification models.

Pathological changes associated with white striping in broiler breast muscles.

White striping is a condition in broiler chickens characterized grossly by the occurrence of white striations, seen parallel to the direction of muscle fibers, on broiler breast fillets and thighs. Based on visual evaluation of the intensity of white striping, breast fillets can be categorized into normal (NORM), moderate (MOD), and severe (SEV) categories. This study was undertaken to evaluate the details of changes in histology as well as proximate composition occurring in the fillets with respect to the 3 degrees of white striping. In experiment 1, representative breast fillets for each degree of white striping (n = 20) were collected from 45-d-old broilers, approximately 2 h postmortem. From each fillet, 2 skeletal muscle samples were obtained and fixed in 10% neutral buffered formalin. To identify and differentiate the histological changes, slides were prepared and stained using hematoxylin and eosin, Masson's Trichrome, and Oil Red O stains. In experiment 2, samples with 3 degrees of white striping were collected from 57-d-old birds for conducting proximate analysis. Major histopathological changes observed in the MOD and SEV samples consisted of loss of cross striations, variability in fiber size, floccular/vacuolar degeneration and lysis of fibers, mild mineralization, occasional regeneration (nuclear rowing and multinucleated cells), mononuclear cell infiltration, lipidosis, and interstitial inflammation and fibrosis. Microscopic lesions were visually scored for degeneration and necrosis, fibrosis, and lipidosis. The scale used to score the samples ranged from 0 (normal) to 3 (severe). There was an increase (P < 0.05) in mean scores for degenerative or necrotic lesions, fibrosis, and lipidosis as the degree of white striping increased from NORM to SEV. The results from the histopathological study were supported by the findings from proximate analysis confirming that the fat and protein contents of muscle increased (P < 0.05) and decreased (P < 0.05), respectively, as the degree of white striping increased. In conclusion, the histopathological changes occurring in white striping indicate a degenerative myopathy that could be associated with increased growth rate in birds.

[Pathogenesis of lipid storage diseases]. / Patogeneza lipidowych chorób spichrzeniowych.

Lipidoses are rare genetic disorders characterized by defects of the digestive system that impair the way the body uses dietary fat. When the body is unable to properly digest fats, lipids such as cholesterol, sphingolipids or glycolipids may accumulate in body tissues in abnormal amounts. It has been also suggested that molecular mechanisms leading to development of human diseases, including obesity, diabetes type II and atherosclerosis, consist of impaired transport and storage of lipids, as well as disturbed structure and function of lipid membrane microdomains. In this review we discuss probable mechanisms, including role of lipid membrane microdomains, which may participate in pathogenesis of lipid storage diseases such as Niemann-Pick type A/B and type C, as well as Gaucher type I diseases.

Glomerular basement membrane lipidosis in Alagille syndrome.

Alagille syndrome is characterized by a paucity of interlobular bile ducts with chronic cholestasis, cardiac, skeletal, and eye abnormalities and is associated predominantly with JAG1 mutations. Various renal abnormalities have been sporadically described. The classic renal histopathology described in Alagille syndrome is mesangiolipidosis, with lipid deposits predominately confined to the mesangium and minimal deposition within the glomerular basement membrane (GBM). We report a 5-year-old girl with Alagille syndrome who presented with persistent subnephrotic proteinuria and renal tubular acidosis. A renal biopsy showed GBM irregularities (mimicking membranous glomerulonephritis), mesangial sclerosis, and focal segmental glomerulosclerosis (FSGS) on light microscopy. Electron microscopy revealed few lipid inclusions within the mesangium but extensive inclusions along the GBM. These findings are mostly consistent with those reported previously in Alagille syndrome. However, the histologic distribution of lipid vacuoles is seemingly reversed in this patient and is uniquely accompanied by FSGS, emphasizing the spectrum of renal histopathology seen in Alagille syndrome. The proteinuria observed in this patient is likely attributed to significant GBM lipid deposition, which over time may contribute to the development of FSGS.

Amino acid pattern in the liver and blood of fattening turkeys suffering from hepatic lipidosis.

Hepatic lipidosis (HL) is a well-known disease in fattening and in parent turkey flocks. Among others, dietary effects like (a lack of) essential amino acids (AA) as lipotrophic factors (e.g., methionine) have been considered as potentially predispositing for HL. Several studies have reported abnormal AA profiles in hepatic diseases of humans and other livestock. The ratio of branched-chain amino acids (BCAA) to aromatic amino acids (AAA) in plasma is used to predict hepatic cirrhosis. In this study, the state of supply of AA was investigated by comparing non-affected (NA) animals and those affected by HL. The AA pattern in the liver and blood can provide potential indications of pathogenesis of HL. In cooperation with German poultry veterinarians, 3 cases of HL on 3 different fattening turkey farms were visited (13/14 wk old, "B.U.T. Big 6" and "TP7"). Overall, 73 birds were examined, of which 42 birds suffered from HL and 31 were not affected. Feeding samples of the respective actual feed were taken and analyzed. The selection of animals was carried out (NA randomly) by clinical signs such as apathy and dyspnea and the diagnosis was made at necropsy, which could be confirmed by crude fat content in liver tissue (HL: 309, NA: 155). In liver tissue, the CP and AA contents were lower among animals with HL than among NA (P < 0.05). In blood samples, the sum of AA, ammonia, and urea was more than 3 times higher among animals with HL (431 mg/dL serum) than among NA (114 mg/dL serum; P < 0.01). The ratio of BCAA to AAA was also significantly different between the groups (HL: 0.85, NA: 1.42; P < 0.05). In the case of HL, entire herds were not affected and the "non-affected" ones were comparable with healthy slaughtered animals. There seems to be a clear change in protein and AA metabolism of HL animals, which could lead to an optimization in feeding practice in repeated cases of HL.

PEPCK-C reexpression in the liver counters neonatal hypoglycemia in Pck1 del/del mice, unmasking role in non-gluconeogenic tissues.

Whole body cytosolic phosphoenolpyruvate carboxykinase knockout (PEPCK-C KO) mice die early after birth with profound hypoglycemia therefore masking the role of PEPCK-C in adult, non-gluconeogenic tissues where it is expressed. To investigate whether PEPCK-C deletion in the liver was critically responsible for the hypoglycemic phenotype, we reexpress this enzyme in the liver of PEPCK-C KO pups by early postnatal administration of PEPCK-C-expressing adenovirus. This maneuver was sufficient to partially rescue hypoglycemia and allow the pups to survive and identifies the liver as a critical organ, and hypoglycemia as the critical pathomechanism, leading to early postnatal death in the whole-body PEPCK-C knockout mice. Pathology assessment of survivors also suggest a possible role for PEPCK-C in lung maturation and muscle metabolism.

Hepatic lipidosis in a black-headed python (Aspidites melanocephalus).

The number of people keeping reptiles in captivity has markedly increased, and widespread inappropriate husbandry will result in an increasing incidence of the diagnosis of hepatic lipidosis(HL). It may become apparent that some species of snake have predisposition to develop HL. In the specific instance of the genus Aspidites, alterations to the usual husbandry that limit feeding and thereby body weight should markedly lessen the chance of the animal's developing HL. It is important for clinicians to develop a more aggressive approach to these cases and to build a bank of data with which to promote understanding of this common malady.

Hepatocarcinogenic and promoting action of a choline-devoid diet in the rat.

Male Fisher 344 rats were solely fed a choline-supplemented diet for 65 to 105 weeks or a choline-devoid diet for 24 to 102 weeks. Hepatocellular carcinomas developed in the latter animals, beginning at 24 weeks. Other groups of rats were given a single dose of 20 mg diethylnitrosamine/kg, 18 h after a partial hepatectomy and were fed, 4 weeks thereafter, either a choline-supplemented, or a choline-devoid diet for up to 48 weeks. In rats fed the choline-supplemented diet, the only relevant lesion observed was a small transect number of foci of enzyme-altered hepatocytes. On the other hand, a significant number of foci, of preneoplastic nodules, and of hepatocellular tumors developed in rats fed the choline-devoid diet. The results obtained are consistent with those previously reported by others, indicating that diets devoid of choline, or of choline and methionine, are carcinogenic. The diets appear to act as complete carcinogens, since they are also efficient promoters of chemical hepatocarcinogenesis, as shown again, in the present study, by the results obtained in the diethylnitrosamine-pretreated rats.

Hepatic Lipidosis in a Research Colony of Big Brown Bats (Eptesicus fuscus).

During a nearby construction project, a sudden decrease in food intake and guano production occurred in an outdoor colony of big brown bats (Eptesicus fuscus), and one animal was found dead. Investigation revealed that the project was generating a large amount of noise and vibration, which disturbed the bats' feeding. Consequently the bats were moved into an indoor enclosure away from the construction noises, and the colony resumed eating. Over the next 3 wk, additional animals presented with clinical signs of lethargy, weight loss, ecchymoses, and icterus and were necropsied. Gross necropsy of the affected bats revealed large, pale yellow to tan, friable livers with rounded edges that floated when placed in 10% neutral-buffered formalin. Some bats had ecchymoses on the webbing and skin and gross perirenal hemorrhage. Histologic examination showed hepatic and renal tubular lipidosis. The clinical and pathologic signs of hemorrhage and icterus were suggestive of hepatic failure. Hepatic lipidosis was attributed to stress and inappetence associated with environmental perturbations. Once the environmental stressor was removed, the colony morbidity and mortality decreased. However, 2 y later, a series of new environmental stressors triggered additional deaths associated with hepatic lipidosis. Over a 9-y period, 21 cases of hepatic lipidosis were diagnosed in this bat colony.

Apolipoprotein E deficiency and high-fat diet cooperate to trigger lipidosis and inflammation in the lung via the toll-like receptor 4 pathway.

Apolipoprotein E deficiency (ApoE(-/-)) combined with a high-fat Western-type diet (WD) is known to activate the toll-like receptor (TLR4) pathway and promote atherosclerosis. However, to date, the pathogenic effects of these conditions on the lung have not been extensively studied. Therefore, the present study examined the effects of ApoE(-/-) and a WD on lung injury and investigated the underlying mechanisms. ApoE(-/-) and wild-type mice were fed a WD or normal chow diet for 4, 12 and 24 weeks. Lung inflammation, lung cholesterol content and cytokines profiles in broncho-alveolar lavage fluid (BALF) were determined. TLR4 and its main downstream molecules were analyzed with western blot analysis. In addition, the role of the TLR4 pathway was further validated using TLR4-targeted gene silencing. The results showed that ApoE(-/-) mice developed lung lipidosis following 12 weeks of receiving a WD, as evidenced by an increased lung cholesterol content. Moreover, dependent on the time period of receiving the diet, those mice exhibited pulmonary inflammation, which was manifested by initial leukocyte recruitment (at 4 weeks), by increased alveolar septal thickness and mean linear intercept as well as elevated production of inflammation mediators (at 12 weeks), and by granuloma formation (at 24 weeks). The expression levels of TLR4, myeloid differentiation primary response 88 (MyD88) and nuclear factor kappa B were markedly upregulated in ApoE(-/-) WD mice at week 12. However, these effects were ameliorated by shRNA-mediated knockdown of TLR4. By contrast, ApoE(-/-) ND or wild-type WD mice exhibited low-grade or no inflammation and mild lipidosis. The levels of TLR4 and MyD88 in those mice showed only minor changes. In conclusion, ApoE deficiency acts synergistically with a WD to trigger lung lipidosis and inflammation at least in part via TLR4 signaling.

Latrogenic lipidosis following prolonged intravenous hyperalimentation.

Intralipid was used as the main source of calories in the long-term therapy of a patient with severe nutritional failure and cachexia. The treatment was tolerated well for 64 days. The patient died of sepsis after a second therapeutic course which lasted 16 days adn was preceded by an impairment in liver function apparently related to starvation. At autopsy, free fat droplets and extreme foamy swelling of the cytoplasm of the reticuloendothelial cells were found in all examined organs. These findings constitute an unusual example of iatrogenic lipidosis. It is suggested that caution be exerted in the administration of Intralipid to patients with impaired liver function and that serum lipids be maintained regularly during therapy.

Dyslipoproteinemia in murine systemic lupus erythematosus.

Dyslipoproteinemia, a feature of systemic lupus erythematosus may contribute to premature atherosclerosis. In order to develop an experimental model for this dyslipoproteinemia we measured plasma concentrations of lipoproteins in juvenile NZB/W (lupus) and NZB/B (control) mice. Additionally to evaluate the effects of a diet rich in n - 3 fatty acids we measured lipoprotein concentrations in mice on normal or menhaden oil-enriched diets. The lupus mice had elevated triglycerides compared to the controls, similar to that seen in human SLE patients (161 +/- 31 vs 113 +/- 13 mg/dl, P less than 0.003). In contrast, the menhaden oil diet fed NZB/W mice had triglycerides similar to the NZB/B control fed group. In the NZB/W murine SLE model, dyslipoproteinemia is an early sign of disease as has been shown in man, therefore this model will be useful in elucidating the mechanism of dyslipoproteinemia in SLE.

Cusp disruption by massive lipid infiltration. A rare cause of porcine valve dysfunction.

A 68-year-old man underwent reoperation because of severe incompetence of a mitral valve xenograft, 96 months following implantation. Gross examination of the device showed yellow spots on the cusps, suggesting lipid infiltration, and a torn commissure. Plasmatic cholesterol and lipiprotein levels were normal. X-ray examination of the explants showed no calcific deposits. Histologic and electron microscopic studies disclosed massive accumulation of lipid clefts and droplets, predominantly at the level of the tear; focal loss and detachment of the endothelial lining and scanty porcine fibroblasts and collagen bundles with preserved periodicity were also noted. Primary disruption of porcine valvular bioprostheses without significant calcifications or collagen breakdown is uncommon. In the present case, lipid accumulation was the main determinant of tissue failure.

Pathological changes in organs of rats chronically exposed to hypoxia. Development of pulmonary lipidosis.

Rats were exposed to chronic normobaric hypoxia of progressively increasing severity; down to 8% or 7% oxygen concentrations. In addition to loss of weight, pathology revealed congestion, haemorrhages, hypertrophy of the heart involving mainly the right ventricle, thickening of arteries, ischaemic changes in the myocardium and extramedullary haematopoiesis in the spleen. Changes not described up until now were: 1) sheets of foam cells in the pulmonary alveoli; 2) foamy and solid storing cells in the spleen; 3) mucoid changes in the atrioventricular valve leaflets; 4) hyperplasia of the juxtaglomerular apparatus; 5) atrophy of the adrenal glomerulosa and hyperplasia of medulla; 6) atrophy of the perifollicular B-cell zone in the spleen; and 7) lipid pigment deposition in various organs. The findings indicate that severe chronic hypoxia induces a significant pulmonary lipidosis similar to that caused by amphiphilic cationic drugs, presumably by inhibiting hydrolytic enzyme activities. The observations are of importance in human hypoxic conditions and open the possibility of their rational treatment.

Diet-induced lipofuscin and ceroid formation in growing pigs.

Seven piglets, aged 2 weeks at the beginning of the experiment, were fed a diet lacking antioxidants (vitamin E and selenium) and enriched with thermally oxidated cod liver oil (the peroxide value of which was 310 meqv/kg). From one month before to 4 weeks after farrowing (weaning) the sow also received the experimental diet. One pig died at 9 days of age, due to degeneration of the parenchymal tissues. The oxidative stress produced was aggravated in four of the seven remaining pigs by giving them an injection of iron-dextran (300 mg) one day before the animals were killed for necropsy at the age of 2 months. The three other piglets were given an injection of a control saline solution. The nutritional myodegeneration produced ('white muscle disease') was characterized by a pale, yellowish colour and translucence of skeletal muscle, with degeneration, including swelling of the muscle fibres and slackening of the fibrillar pattern. The arrangement of nuclei in chains indicated repair. Ultrastructurally, skeletal muscle showed vacuolization of the sarcoplasmic reticulum, irregularities in the mitochondria and focal dissolution of myofibrils. The sarcoplasmic reticulum displayed condensation and lamellar formations. Hyaline degeneration of the heart muscle occurred both in iron-treated and control pigs, and in six animals a yellowish pigment resembling lipofuscin and ceroid was observed. Ultrastructurally, the ceroid deposits appeared as myelin-like figures. The livers were pale and brittle, four with centrilobular degeneration. By light microscopy, lipidosis was distinguished in four and ceroid accumulations in three pigs. Thiobarbituric-acid-reactive substances, creatine kinase activity and iron increased significantly in the serum as a result of the injection of iron-dextran.

The occurrence of cardiac lipidosis and necrotic lesions in the hearts of rats following long-term feeding of different lipid supplemented diets.

The effects of "long-term" feeding of different lipid supplemented diets (12% added fat, w/w) on the incidence of lipidosis or the severity of necrosis was examined in the cardiac muscle of male Hooded Wistar rats, after at least 12 months on the diets. The effects of supplementation with either n-6 polyunsaturated fatty acids (PUFA's) added as sunflower seed oil (the SSO diet), or one enriched with n-3 PUFA's added as a low cholesterol, low vitamin (A & D) fish oil preparation obtained from eviscerated Southern Bluefin Tuna (the TFO diet) were compared to those found in the hearts of rats fed either a relatively low fat commercially available stock diet (REF) which contained 4% (w/w) of mixed fats of animal, vegetable and marine origin, or after this stock diet had been supplemented to the same extent by the addition of 12% (i.e. 12:88 g) sheep kidney (perirenal) fat, the SF diet. Extensive cardiac lipidosis was seen after feeding either the TFO or the SF diets, but was not observed in the hearts of experimental rats from either the SSO or REF fed groups. Conversely, in these mature animals, grade 1 necrotic lesions were uniformally found in the cardiac muscles of all rats examined, but neither their incidence nor severity could be attributed to any dietary effect. These necrotic lesions are therefore more probably a reflection of the age of the animals at the time of sacrifice, rather than to any of the dietary supplements employed. Some evidence of "Yellow Fat" disease was found by the presence of lipofuscin pigmentation in the storage fat of rats receiving n-3 PUFA's (the TFO diet) but was not observed in any other dietary group nor in the livers or kidneys of any animals. This extent of storage fat pigmentation was not associated with any retardation of growth in this dietary group.

Bilateral ocular lipidosis in a cottontail rabbit fed an all-milk diet.

Lipid infiltration of the corneal stroma, sclera, and uvea occurred in a 2-year-old cottontail rabbit that had been fed an all-milk diet for 20 months. Superficial keratectomy was performed on the left eye but the rabbit died. Histologically, lipid deposits were most extensive in the anterior corneal stroma and posterior iridal epithelium.

Hepatic lipidosis in llamas and alpacas: 31 cases (1991-1997).

OBJECTIVE: To identify factors associated with hepatic lipidosis (HL) in llamas and alpacas. DESIGN: Retrospective case series. ANIMALS: 30 llamas and 1 alpaca. PROCEDURES: Medical records were searched to identify llamas or alpacas in which a histologic diagnosis of HL was made. Information was retrieved on signalment, history, clinical and laboratory findings, and results of necropsy or examination of biopsy specimens. Data were analyzed using descriptive statistics and chi 2 analyses. RESULTS: Females were affected more often than males; however, the sex distribution was not different from that of the camelid population in the diagnostic laboratory's database. Fifty-four percent of the females were pregnant, and 46% were lactating. Most affected camelids were 6 to 10 years old. Anorexia and recent weight loss were common (51.6% of camelids). An infective agent was found in only one ilama, and toxins and mineral deficiencies were not identified. The most common abnormalities on serum biochemical analysis were a high concentration of bile acids, high activities of gamma-glutamyltransferase (GGT) and aspartate aminotransferase (AST), and hypoproteinemia. Concentrations of nonesterified fatty acids (NEFA) and beta-hydroxybutyrate (beta-HB) were high in those camelids in which these compounds were assayed. Twenty-nine camelids did not survive. CLINICAL IMPLICATIONS: Sick camelids should be considered at risk for developing HL, especially those with anorexia or the metabolic demands of pregnancy and lactation. Other stresses also appear to contribute. High concentrations of NEFA, beta-HB, and bile acids; high activities of GGT and AST; and hypoproteinemia may indicate that HL has developed.

Diabetic ketosis and ketoacidosis in cats: 42 cases (1980-1995).

OBJECTIVE: To determine clinical signs, clinicopathologic abnormalities, prevalence of concurrent disease, treatment, complications of treatment, and outcome in cats with diabetic ketosis (DK) or diabetic ketoacidosis (DKA). DESIGN: Retrospective study. ANIMALS: 42 cats with DK or DKA. PROCEDURE: Medical records of diabetic cats with ketonuria were reviewed. RESULTS: In 26 cats, diabetes was newly diagnosed; in 16, diabetes had been diagnosed previously and cats had been treated with insulin (n = 14) or sulfonylurea drugs (2). Common clinical findings were lethargy, anorexia, polyuria, polydipsia, and weight loss. Common laboratory findings were hyperglycemia, hyponatremia, hypochloremia, hypokalemia, hypocalcemia, hypophosphatemia, low total CO2 content, hyperosmolality, high serum alanine transaminase activity, azotemia, glycosuria, and ketonuria. Concurrent disorders were identified in 39 cats and included hepatic lipidosis, cholangiohepatitis, pancreatitis, chronic renal failure, urinary tract infection, and neoplasia. Treatment of DK and DKA included administration of regular crystalline (34 cats), NPH (6), or ultralente (2) insulin, intravenous (38) or subcutaneous (4) administration of fluids, and enterall parenteral or administration of antibiotics (42). Complications during treatment included abnormalities in serum electrolyte concentrations (27 cats), hemolytic anemia (4), hypoglycemia (3), and neurologic abnormalities unrelated to hypoglycemia (2). Eleven cats died or were euthanatized during the initial hospitalization period for treatment of DK or DKA. Azotemia, metabolic acidosis, and hyperosmolality were more severe in cats that died than in cats that survived. Differences in regard to treatment or complications were not apparent between cats that died and cats that survived. The 31 cats that survived were discharged 1 to 16 days (median, 5 days) after initiation of insulin treatment. Diabetic ketosis or ketoacidosis recurred in 13 (42%) of these cats. CLINICAL IMPLICATIONS: A thorough diagnostic evaluation should be performed on cats with DK or DKA to identify concurrent disorders, formulate an appropriate treatment plan, and provide prognostic information to the owner.