A limit for liver lipid overload Hepatocytes respond to insulin by accumulating triglycerides and cholesterol. Excessive lipid accumulation in the liver can result in nonalcoholic fatty liver disease (NAFLD), the more severe forms of which are risk factors for the development of liver cirrhosis and cancer. Mayer et al. found that activation of PKD3 by insulin signaling served as a negative feedback mechanism to prevent hepatic lipid accumulation. Mice lacking PKD3 in the liver showed increased insulin signaling, triglyceride and cholesterol synthesis, and steatosis in response to a high-fat diet. In contrast, overexpression of a constitutively active form of PKD3 attenuated insulin signaling in the liver and resulted in insulin resistance. Thus, PKD3 activity curtails insulin signaling and, therefore, lipid synthesis and accumulation in the liver.

Abstract Hepatic activation of protein kinase C (PKC) isoforms by diacylglycerol (DAG) promotes insulin resistance and contributes to the development of type 2 diabetes (T2D). The closely related protein kinase D (PKD) isoforms act as effectors for DAG and PKC. Here, we showed that PKD3 was the predominant PKD isoform expressed in hepatocytes and was activated by lipid overload. PKD3 suppressed the activity of downstream insulin effectors including the kinase AKT and mechanistic target of rapamycin complex 1 and 2 (mTORC1 and mTORC2). Hepatic deletion of PKD3 in mice improved insulin-induced glucose tolerance. However, increased insulin signaling in the absence of PKD3 promoted lipogenesis mediated by SREBP (sterol regulatory element-binding protein) and consequently increased triglyceride and cholesterol content in the livers of PKD3-deficient mice fed a high-fat diet. Conversely, hepatic-specific overexpression of a constitutively active PKD3 mutant suppressed insulin-induced signaling and caused insulin resistance. Our results indicate that PKD3 provides feedback on hepatic lipid production and suppresses insulin signaling. Therefore, manipulation of PKD3 activity could be used to decrease hepatic lipid content or improve hepatic insulin sensitivity.

INTRODUCTION Hepatocytes are a major target for insulin. On the one hand, insulin stimulates hepatic glucose uptake, suppresses de novo glucose production, and, therefore, lowers systemic glycemia (1). On the other hand, excessive insulin signaling promotes de novo lipid synthesis and, consequently, the accumulation of triglycerides (TGs) and cholesterol in hepatocytes. This can lead to the development of nonalcoholic fatty liver disease (NAFLD), hepatic insulin resistance, and eventually to the development of nonalcoholic steatohepatitis (NASH) and, consequently, to liver cirrhosis (2). On the molecular level, insulin stimulates activity and expression of major transcription factors such as sterol regulatory binding proteins (SREBPs) that promote hepatic lipid production (3). Activation of SREBP-dependent transcription requires inputs from various insulin-evoked signaling cascades, which include AKT and mechanistic target of rapamycin complex 1 and 2 (mTORC1 and mTORC2) (3, 4). Obesity-related metabolic overload results in the accumulation of diacylglycerol (DAG) in the liver (5). Protein kinase C (PKC) isoforms mediate DAG-evoked insulin resistance (2, 5). The major PKC isoform expressed in the liver, PKCε, promotes insulin resistance by phosphorylating the insulin receptor to inhibit downstream signaling (6, 7). Protein kinase D (PKD) isoforms (PKD1, PKD2, and PKD3) are DAG and PKC effectors that integrate multiple nutritional and hormonal inputs (8). However, the impact of PKDs on hepatic metabolism has not been investigated so far. Different PKDs have been implicated in the regulation of muscle differentiation, function of adipose tissue, pathophysiological heart remodeling, immune response, carcinogenesis, blood coagulation, insulin secretion, actin remodeling, trans-Golgi network dynamics, cell proliferation, and migration (8–20). Here, we showed that PKD3 was the predominant PKD isoform in the liver and was activated in lipid-loaded hepatocytes. Furthermore, we demonstrated that PKD3 suppressed insulin signaling, resulting in impaired AKT phosphorylation and activation of mTORC1 and mTORC2. Mice lacking PKD3 in hepatocytes presented improved glucose and insulin tolerance and elevated SREBP-dependent lipogenesis, which resulted in increased hepatic TG and cholesterol content. These results indicate that PKD3 attenuates insulin signaling, thereby preventing the development of fatty liver.

DISCUSSION DAG-evoked activation of PKC-dependent signaling is critical for the development of hepatic insulin resistance (2, 5–7). Our study revealed PKD3 as a mediator of DAG-evoked insulin resistance in the liver. DAG is an intermediate product of TG synthesis, but it is also used by a subclass of G protein (heterotrimeric GTP-binding protein)–coupled receptors as a second messenger (5). Our data also demonstrated that PKD3 was activated in the livers of obese (HFD fed) mice. Moreover, lipid loading of primary hepatocytes and stimulation of cells with insulin resulted in activation of PKD3. However, the precise upstream mechanism promoting PKD3 activation and the relationship of this kinase to the classical PKC-dependent signaling pathway require further investigation. Although we showed that PKD3 is one of the signaling molecules aberrantly activated during metabolic overload evoked by high levels of lipids supplementation and its deletion partially ameliorated insulin resistance caused by lipid overload, PKD3 seems to also have a physiological function in the liver. We revealed that PKD3 expression was important to provide a negative feedback loop on lipogenesis evoked by refeeding after fasting/starvation, which would be in line with the increase in PKD3 abundance in response to prolonged refeeding and the increase in PKD activity in response to insulin stimulation. PKCε suppresses insulin action by directly phosphorylating the insulin receptor (6, 7). Our data suggest that PKD3 also suppressed insulin action, resulting in impaired AKT phosphorylation and reduced mTORC1 and mTORC2 activation in vitro and in vivo. Hepatic deletion of PKD3 not only improved glucose tolerance in mice but also resulted in an increased SREBP-dependent lipogenesis and, consequently, hepatic accumulation of TG and cholesterol. SREBP1c and SREBP2 are critical for the transcriptional activation of lipogenic machinery in hepatocytes (3). However, the phosphorylation of the mTORC2 downstream effector NDRG1 promotes lipogenesis in a PPARγ (peroxisome proliferator–activated receptor γ)–dependent manner in adipocytes (30). We found that phosphorylation of NDRG1 was enhanced in the absence of PKD3, suggesting that PKD3 might suppress lipogenesis by using mechanisms complementary to the suppression of SREBP-dependent transcription of lipogenic genes. Similarly, PKD3-dependent suppression of AKT and mTORC1/2 actions might influence lipogenesis by inhibiting factors such as DNA-dependent protein kinase (DNA-PK) or upstream stimulatory factors (USF), which also promote the expression of lipogenic enzymes (31). However, we showed that silencing of SREBP isoforms was sufficient to normalize the elevated rates of lipogenesis in PKD3-deficient hepatocytes, which indicates that PKD3 regulates lipogenesis primarily by suppressing SREBP-dependent transcription of lipogenic genes. PKD3 did not affect TG secretion in the form of VLDLs from hepatocytes. Moreover, deletion of PKD3 seemed to increase FA oxidation in hepatocytes. Increased rates of FA oxidation in hepatocytes would be expected to lead to reduced TG content in the liver. However, lipid accumulation in the livers of PKD3-deficient mice, despite increased FA oxidation and unaffected rates of VLDL secretion, indicates that elevated lipogenesis is the primary process responsible for the observed phenotype. Although the closely related PKD1 promotes lipid accumulation in adipocytes in an AMPK-dependent manner (19), PKD3 did not affect the activity of AMPK or its downstream target ACC1/2 in the liver. In addition, PKD3 seemed to be the predominant PKD isoform that regulates liver metabolism because PKD inhibition did not further stimulate expression of lipogenic genes in the absence of PKD3 and other PKD isoforms were only marginally expressed in hepatocytes. Therefore, our results suggest that different PKD isoforms have a specific set of substrates in different tissues. Our results also indicate that the expression of the constitutively active form of PKD3 was sufficient to attenuate insulin signaling (AKT and TORC1/2 activity) even in the absence of hyperlipidemia. Mice expressing the constitutively active form of PKD3 in the liver showed reduced insulin sensitivity and reduced AKT phosphorylation even when fed an ND. Cholesterol and TG content was not altered in these livers, and the expression of some SREBP target genes was only marginally reduced (fig. S9, C to E). These results might imply that increased insulin signaling is sufficient to enhance lipogenesis but that reducing insulin signaling does not necessarily block the lipogenic program. Alternatively, the ability of PKD3 to attenuate insulin signaling on lipogenesis might be present only in the livers of animals challenged with HFD. Multiple studies report that hepatic TG and cholesterol levels correlate with the development of insulin resistance [reviewed in (2)]. However, we found that deletion of PKD3 promoted glucose use and insulin sensitivity while, at the same time, enhancing lipid accumulation in the liver. Improved glucose use and increased lipid deposition are observed in several mouse models in which AKT activity is increased (23, 32). Nevertheless, we did not observe increases in apoptosis, immune cell infiltration, or fibrosis in PKD3-deficient livers, which are often associated with the progression of NAFLD into NASH (2). Therefore, PKD3 deletion results in the development of a metabolically healthy fatty liver. Last, our data indicate that the expression of the constitutively active form of PKD3, which preserves its activity in hepatocytes of animals independently of hormonal or metabolic challenges, suppressed insulin signaling and induced insulin resistance and glucose intolerance. The expression of PKD3ca in hepatocytes not only suppressed AKT- and mTORC-dependent signaling but also led to phosphorylation of the insulin receptor substrate at Ser612, which indicates that other signaling pathways [for example, mitogen-activated protein kinases (33)] might be activated by the expression of PKD3. Together, these data indicate that not only is PKD3 expression required to provide negative feedback on insulin signaling under conditions of lipid overload, but also its activation is sufficient to partially switch off insulin-dependent signaling.

MATERIAL AND METHODS DAG quantification DAG analysis was performed as described in detail before (19). Briefly, lipids were isolated from liver homogenates by the butanol-methanol extraction method. 1,2-Dioctanoyl-sn-glycerol (Enzo Life Sciences) was used as internal standard. Subsequently, lipid extracts were fractionated into lipid classes on a silica matrix column (Phenomenex), and the DAG fraction was analyzed by liquid chromatography–mass spectrometry (LC-MS). Animals PKD3 floxed mice (PKD3f/f) (20) and loxP-STOP-loxP-(3xFlag)PKD3ca mice, in which the two serines in the PKD3 kinase domain are mutated to glutamic acid (S731E/S735E) (generated by Cyagen with the PiggyBac transgenic method), were mated with mice expressing transgenic Cre recombinase under the control of the albumin promoter/enhancer (the Jackson laboratory) (21) to generate homozygous liver-specific PKD3-deficient mice (PKD3liverΔ/Δ) and heterozygous liver-specific transgenic PKD3ca mice (TgPKD3caliver), respectively. All animal studies were approved by the local animal welfare authorities (Regierung von Unterfranken) with the animal protocol nos. AK55.2-2531.01-124/13 and AK55.2.2-2532-2-741-13. The mice were housed in cages from Tecniplast in a green line IVC rack system with ad libitum supply of water and normal chow diet (ssniff Spezialdiaeten). In that case of HFD (Research Diets, D12331i), mice received the high-calorie diet (58% kcal from fat) from the age of 3 weeks for a duration of 24 weeks, and BW measurements were performed weekly. For dissection, mice were sacrificed by cervical dislocation, and organs [liver, gonadal white adipose tissue (gWAT), subcutaneous white adipose tissue (sWAT), brown adipose tissue (BAT), and skeletal muscle (SKM)] were weighted and snap frozen in liquid nitrogen. For fasting/refeeding experiments, mice were fasted overnight for 16 hours with free access to water, and food was restored for 4 hours before livers were removed as described before. For insulin injections, mice received an intraperitoneal dose of 8 U/kg BW of insulin (Sigma), and livers were collected after 15 min according to standard procedures. For in vivo experiments using CRT0066101, C57BL/6JRj mice (Janvier Labs) were fed an HFD for 8 weeks and were randomly assigned to two groups that either received an intraperitoneal injection 10 mg/kg BW of CRT0066101 in 5% dimethyl sulfoxide (DMSO) or vehicle for five consecutive days. Glucose and insulin tolerance tests For glucose tolerance tests, mice were fasted for 4 hours before the experiment (starting at 8:00 a.m.) and received an intraperitoneal injection of a defined dose of glucose (Carl Roth) as indicated in the figure legends. Glucose concentrations in the blood were measured with Accu-Chek glucometer (Roche) at 0-, 15-, 30-, 60-, 90-, and 120-min time points. Insulin tolerance tests were performed similarly, except that the mice received a defined dose of insulin (Sigma) as indicated in the figure legends. Serum metabolite and HOMA-IR analysis Blood samples were collected after 20 weeks of ND or HFD feeding or after 6, 24, and 48 hours of fasting and 24 hours of refeeding for the fasting/refeeding experiment. Cholesterol (LabAssay Cholesterol, Wako) and TG (Triglycerides Kit, Sigma) concentrations in serum were determined according to the manufacturers’ protocols. Serum insulin levels were quantified either by using a magnetic bead based immunoassay kit (Bio-Plex Pro mouse insulin, Bio-Rad) and MAGPIX multiplex reader (Bio-Rad) or by the mouse insulin ELISA Kit (Crystal Chem) and a SPARK plate reader (Tecan) according to the manufacturers’ guidelines. HOMA-IR was calculated with the following formula: fasting insulin (mU/ml) × fasting glucose (mg/dl) divided by 405 using blood from mice that were fasted overnight. VLDL secretion Mice were fasted overnight before they received a retro-orbital injection of 0.5 mg/g BW tyloxapol. Blood was collected at the 0-, 1-, 2-, 4-, and 6-hour time points and used for serum TG analysis as described above. Metabolic measurements Metabolic parameters such as energy expenditure, food intake, activity, and respiratory exchange ratio were determined in the PhenoMaster (TSE Systems) system as described before (34) after 23 weeks on specific diets. Histological, immunohistochemistry, and immunofluorescence analyses For histology, liver tissues were fixed in 10% formalin and embedded in paraffin. H&E or Sirius red staining of 2- to 4-μm liver sections was performed according to standard laboratory procedures. For immunohistochemistry, Ki-67 (H2, 95°C), cleaved caspase-3 (H2), B220 (H2), CD3 (H2, 95°C), F4/80 (E1), and collagen type IV (E1) antibodies were used with EDTA pretreatment (H2) or an enzyme pretreatment kit (E1) (Leica), respectively. For TUNEL staining, a fluorescence in situ cell death detection kit (Roche) was used according to the manufacturer’s instructions. Lipid extraction and TLC Lipids were extracted according to the protocol of Bligh and Dyer with modifications (35). Briefly, 0.5 volumes of liver homogenate [1:100 in phosphate-buffered saline (PBS)] were acidified with 0.3 volumes of 0.2 N HCl. Subsequently, 3 volumes of MeOH/CHCl 3 (2:1, v/v), 1 volume of CHCl 3 , and 1 volume of H 2 O were added and mixed vigorously stepwise. The phases were separated by centrifugation, and the lower phase was transferred to a new tube and evaporated under a stream of nitrogen. The lipids were either resuspended in DMSO/H 2 O for enzymatic quantification of TG and cholesterol (as described before) or in MeOH/CHCl 3 (1:1, v/v) for separation by TLC on a silica gel 60 plate (Merck) in the solvent mixtures of CHCl 3 /MeOH/20% acetic acid (65:25:5, v/v/v), hexane/ethyl acetate/acetic acid (59:10:1, v/v/v), and pure hexane. Cholesteryl palmitate (for CE), triolein (for TG), oleic acid (for FFA), cholesterol (for FC), phosphatidylethanolamine (for PE), and phosphatidylcholine (for PL) were used as standards for the TLC. Lipid spots were visualized by dipping the plate in a hydrous solution of 2.5% 12-molybdophosphoric acid (Alfa Aesar), cerium (IV) sulfate (Sigma), and 6% H 2 SO 4 and heating at 200°C until the bands appear. Densitometric analysis was performed using ImageJ and known concentrations of the standards. Primary hepatocyte isolation and culture Primary mouse hepatocytes were prepared by the collagen perfusion method. Eight- to 12-week-old male mice were anesthetized with ketamine/xylazine, and the vena cava was cannulated. The portal vein was cut immediately, and the liver was perfused with Earle’s balanced salt solution (without Ca2+/Mg2+ ; Thermo Fisher Scientific) supplemented with 0.5 mM EGTA. Subsequently, the buffer was replaced to Hanks’ balanced salt solution with Ca2+/Mg2+ (Biochrom) containing type I collagenase (100 U/ml) (Worthington). After sufficient digestion, the liver was excised and the gall bladder was removed. Cells were liberated into Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum and 1% penicillin/streptomycin (P/S), filtered through a 100-μm cell strainer, and centrifuged (50g, 3 min, 4°C). Afterward, Percoll (GE Healthcare) mixed with respective amounts of 10× PBS and culture medium was used to form a gradient that allowed enrichment of the hepatocyte fraction (50g, 10 min, 4°C), which was washed three times with culture medium (50g, 3 min, 4°C). Hepatocytes were then plated on collagen type I–coated 6- or 12-well plates (BD). After 4 to 6 hours, to allow attachment, the medium was replaced by fasting medium [DMEM supplemented with 0.2% FFA-free BSA (Sigma) and 1× P/S], and the cells were used the following day unless otherwise noted. For PKD3 phosphorylation studies, primary hepatocytes were incubated either with 1,2-dioctanoyl-sn-glycerol (Enzo Life Sciences) for 4, 6, and 8 hours or with oleic acid bound to albumin (Sigma) for 4 and 20 hours. Adenovirus infection Primary hepatocytes were infected 4 to 6 hours after plating with adenoviruses expressing either enhanced green fluorescent protein (EGFP) (Ad-EGFP) or a constitutively active form of PKD3 (Ad-mycPKD3ca) at an multiplicity of infection (MOI) of 10. Medium was replaced the following morning, and cells were used for experiments 36 to 48 hours after infection. Transduction efficiency, which was 100%, was assessed by analyzing the expression of the EGFP reporter (which was present in all adenoviruses). Small interfering RNA transfections Primary hepatocytes were transfected 4 to 6 hours after plating with siNonTargeting (60 nM), siSrebp1 (30 nM), or siSrebp2 (30 nM) (Dharmacon) using the Lipofectamine RNAiMAX transfection reagent (Invitrogen) according to the manufacturers’ recommendations. Medium was replaced the following morning, and cells were used for experiments 36 to 48 hours after infection. Lipogenesis assay De novo lipogenesis assay was performed 1 day after the isolation of primary hepatocytes. Cells were incubated with a lipogenic medium (DMEM with 25 mM glucose, 0.2% FFA-free BSA, and 1× P/S) supplemented with 0.5 mM sodium acetate and 3H-acetate (4 μCi/ml) (PerkinElmer) at basal or insulin (100 nM)–stimulated conditions for 4 to 6 hours. Cells were then washed twice with PBS and scraped into 0.1 N HCl. Subsequently, the lipids were extracted similarly as described before. Homogenate (0.8 volume) was mixed stepwise with 3 volumes of MeOH/CHCl 3 (2:1, v/v), 1 volume of CHCl 3 , and 1 volume of H 2 O. The phases were separated by centrifugation (3000g, 10 min), and the lower phase was transferred to a new tube, which was washed once with upper-phase MeOH/CHCl 3 /H 2 O (15:175:180, v/v/v) (3000g, 10 min). Last, the lower phase was transferred to a scintillation tube and evaporated under a stream of nitrogen. The dried lipids were resuspended in 4 ml of scintillation liquid before lipid scintillation counting. The lipogenesis rate was calculated as the amount of tritium incorporated into the newly formed lipids (measured in dpm) per total protein (Bradford, Bio-Rad) per hour. Alternatively, the extracted lipids were separated on a TLC plate as described before. The stained lipid spots were scraped and analyzed by lipid scintillation counting. To measure de novo lipogenesis in vivo, mice were fed an HFD for 8 weeks. After overnight fasting, mice were refed with an HFD for 4 hours before they received an intraperitoneal dose of 8 mCi [3H]-water/100 g BW. After 2 hours, the livers were excised, and liver pieces (three per mouse) were homogenized 1:5 in 0.1 N HCl, followed by lipid extraction as described above. For inhibitor treatments, 10 μM Akti-1/2, 0.7 μM KU0063794, and 0.7 μM rapamycin were added 4 to 6 hours after plating and 16 hours before the experiment, whereas 1 μM CRT0066101 was added 1 hour before the experiment. FA oxidation Hepatocytes were serum fasted overnight and incubated with DMEM containing 3H-oleic acid (2 μCi/ml) (PerkinElmer) and 0.2 mM oleic acid bound to BSA (Sigma) at basal or insulin (100 nM)–stimulated conditions for 3 hours. Subsequently, the supernatant was collected, and the cells were washed twice with PBS and scraped in 0.1 N HCl to determine the protein concentration. Then, 1 volume of MeOH/CHCl 3 (2:1, v/v) and 1 volume of 2 M KCl/2 M HCl (1:1, v/v) were added to 0.5 volume of the supernatant and mixed stepwise. The phases were separated by centrifugation (3000g, 10 min), and the upper phase was transferred to a new tube and the procedure was repeated once more. Last, the upper phase was transferred to a scintillation tube, and 4 ml of scintillation liquid was used for scintillation counting. The FA oxidation rate was calculated as the amount of tritium incorporated into 3H 2 O (measured in dpm) per total protein (Bradford, Bio-Rad) per hour. Immunoblotting Western blot was performed according to standard procedures. Briefly, 20 μg of protein lysates was separated by a 10% SDS–polyacrylamide gel electrophoresis and transferred on a polyvinylidene difluoride (PVDF) membrane and then probed overnight with corresponding primary antibodies (table S1). After incubation with corresponding mouse or rabbit horseradish peroxidase–conjugated secondary antibody, proteins were visualized using ECL Reagent in combination with x-ray films (Fuji) or with the Amersham Imager 600 (GE Healthcare) for densitometric analysis. Real-time quantitative polymerase chain reaction analysis Total RNA was extracted from tissue or cells using QIAzol Reagent (Qiagen) according to the manufacturer’s instructions. Reverse transcription of 1 μg of RNA was performed by using the First Strand cDNA Synthesis Kit (Thermo Fisher Scientific). Real-time quantitative polymerase chain reaction (QPCR) was performed using PowerUp SYBR Green Master Mix (Thermo Fisher Scientific) on a QuantStudio 5 Real-Time PCR System (Thermo Fisher Scientific). Relative amounts of all mRNAs were calculated using the comparative C T method normalized to the reference gene Rpl13a, 36B4, or Hprt1. The primer sequences (sense and antisense) for mouse are listed in table S2. Absolute quantification of PKD isoform copy numbers in the liver was performed according to the standard protocol of Applied Biosystems. Briefly, the primers were designed to be located within same exons (table S2), and genomic DNA of known concentration was used to create a standard curve reflecting copy numbers. Statistical analysis Data are presented as mean values ± SEM. Significances were determined by using two-tailed Student’s t test for independent groups or by using one-way analysis of variance (ANOVA), followed by the post hoc Tukey’s test for multiple comparisons. P values of 0.05 or lower were considered as statistically significant.

SUPPLEMENTARY MATERIALS stke.sciencemag.org/cgi/content/full/12/593/eaav9150/DC1 Fig. S1. Stimulation of hepatocytes with DAG suppresses expression of lipogenic genes. Fig. S2. PKD3 deletion is restricted to the liver. Fig. S3. Liver-specific PKD3 deletion does not affect metabolism of mice fed an ND. Fig. S4. PKD3 does not affect proliferation, immune cell infiltration, or apoptosis in the liver. Fig. S5. TG accumulation in the livers of PKD3liverΔ/Δ mice does not depend on FA oxidation or VLDL secretion. Fig. S6. The abundance and/or phosphorylation of mTORC1/2 components are not affected by deletion or overexpression of PKD3 in hepatocytes. Fig. S7. Quantifications of Western blots of control and PKD3-deficient primary hepatocytes. Fig. S8. Quantifications of Western blots of EGFP- and PKD3ca-transduced primary hepatocytes. Fig. S9. Liver-specific expression of PKD3ca improves glucose tolerance and insulin sensitivity. Table S1. List of antibodies used for Western blotting and immunohistochemistry. Table S2. Sequence of primers used for QPCR and genotyping.

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Acknowledgments: We thank O. Sumara for the critical comments to our manuscript, A. Schürmann and C. Baumeister for introducing us to primary hepatocyte isolation, J. Hetzer and D. Heide for performing the immunohistochemical stainings, and K. Aurbach, I. Becker, and D. Cherpokova for experimental assistance. Funding: This project was supported by the EFSD/Janssen Rising Star Fellowship Programme (2013) from the European Foundation for the Study of Diabetes and by the Starting Grant (SicMetabol) from the European Research Council (ERC) and internal funds of the Rudolf Virchow Center for Experimental Biomedicine. R.E.-M. and G.S. were also funded by the Emmy Noether grant from the German Research Foundation (no. Su 820/1-1). M.H. was supported by an ERC Consolidator grant (HepatoMetaboPath). A.S. was supported by a German Research Foundation grant (FOR2314, SCHU2670/1-1). Author contributions: A.E.M. and G.S. conceived the study, designed the experimental procedures, and wrote the manuscript. A.E.M. performed the majority of the experimental work and analyzed the data. M.C.L., A.E.L.V., R.E.-M., J.T.V., and M.E. performed parts of the experiments. W.S. participated in the experimental design and carried out the mass spectrometry. M.L., T.Z., and U.B. provided the PKD3f/f mice. M.H. and A.S. contributed to the experimental design. Competing interests: The authors declare that they have no competing interests. Data and materials availability: The PKD3f/f mice require a material transfer agreement by the PKC Research Consult (Cologne, Germany). All data needed to evaluate the conclusions in the paper are present in the paper or the Supplementary Materials.