Effects of small interfering RNA-mediated hepatic glucagon receptor inhibition on lipid metabolism in db/db mice.

Hepatic glucose overproduction is a major characteristic of type 2 diabetes. Because glucagon is a key regulator for glucose homeostasis, antagonizing the glucagon receptor (GCGR) is a possible therapeutic strategy for the treatment of diabetes mellitus. To study the effect of hepatic GCGR inhibition on the regulation of lipid metabolism, we generated siRNA-mediated GCGR knockdown (si-GCGR) in the db/db mouse. The hepatic knockdown of GCGR markedly reduced plasma glucose levels; however, total plasma cholesterol was increased. The detailed lipid analysis showed an increase in the LDL fraction, and no change in VLDL HDL fractions. Further studies showed that the increase in LDL was the result of over-expression of hepatic lipogenic genes and elevated de novo lipid synthesis. Inhibition of hepatic glucagon signaling via siRNA-mediated GCGR knockdown had an effect on both glucose and lipid metabolism in db/db mice.

used in mice to measure TG production rate in plasma. Mice were fasted overnight and injected with P407 (10 ml/kg body weight). Blood samples were collected at 0, 1, 2, 4, and 6 h after injection, and TG levels were quantifi ed, as described above.

Fast protein liquid chromatography
Lipoproteins were fractionated by fast protein liquid chromatography (FPLC) gel fi ltration using a Superose-6 size exclusion column (GE Healthcare LifeSciences, Inc.; Piscataway, NJ) attached to a Dionex HPLC system (Thermo Fisher Scientifi c, Inc.). The column effl uent was mixed with a commercially available enzymatic reagent for cholesterol (Wako Chemicals), and levels were continuously measured using a photodiode array detector at 600 nm absorbance. The fi rst, second, and third peaks were attributed to VLDL, LDL-C, and HDL-C, respectively, and the area under each peak was calculated using Chromeleon software, as previously described ( 17 ).

Real-time quantitative PCR analysis
Liver samples were homogenized, and total RNA was isolated using RNA Easy (Qiagen, Inc., Valencia, CA), according to the manufacturer's instructions. Two micrograms total RNA from each sample was reverse transcribed with a cDNA kit (Life Technologies Corp., Carlsbad, CA), and mRNA expression for the genes of interest was measured by RT-PCR, with SYBR Green PCR core reagents and primers (Qiagen, Inc.), as previously described ( 18 ). The relative amounts of specifi c target amplicons for each primer set were estimated by a cycle threshold (Ct) value and were normalized to the copy number of housekeeping genes ( 18 ). The P values were determined by two-tailed equal variance Student's t -test, comparing the ⌬ Ct values of the si-Control and si-GCGR groups.

GC-quadrupole-MS analyses of palmitate and cholesterol synthesis
At day 11 after siRNA injection, 2 H 2 O (Sigma-Aldrich) was injected at 20 ml/kg body weight into animals that had been fasted 4 h. Plasma samples were collected after 4 h of tracer incorporation, and stored at Ϫ 80°C until analysis. Total plasma (35 µl) was spiked with a known amount of heptadecanoic acid (C17:0, internal standard) and then saponifi ed in 1 N potassium hydroxide (KOH) in 80% ethanol (heated at 65°C for 60 minutes). Samples were acidifi ed (25 µl 6 N hydrochloric acid (HCl), and the lipids were then extracted with chloroform. Following evaporation of the solvent, the residue was fi rst reacted with trimethylsilyldiazomethane to form FA methyl esters, evaporated to dryness, and then reacted with pyridine-acetic anhydride (1:1, v/v) at 65°C for 30 min to generate cholesterol acetate. The reagents were evaporated to dryness. The 2 H labeling of the derivatized lipids was determined using an Agilent 5973N-MSD equipped with an Agilent 6890 GC system (Agilent Technologies; Santa Clara, CA), a DB5-MS capillary column (30 m × 0.25 mm × 0.25 µm). The mass spectrometer was operated in the electron impact mode, using selective ion monitoring of m/z 270 and 271 for palmitate, 284 and 285 for C17:0 (internal standard), and 368 and 369 for cholesterol; data were collected using selected ion monitoring at a dwell time of 10 ms per ion. To quantify the contribution of lipid synthesis, a precursor product labeling ratio was assumed and the general equation was used : newly made material product labeling (precursor labeling ) 100 for which product labeling = palmitate or cholesterol, precursor labeling = body water, and n = the number of exchangeable

Animal study
Male, 8 week-old db/db mice were purchased from The Jackson Laboratory (Bar Harbor, ME) and fed a regular chow diet (Diet 7012; Harlan Teklad) throughout the study. Following a 2 week stabilizing period, 10 week-old mice were used for small interfering RNA (siRNA) injection. siRNA was packaged in lipid nanoparticles, as previously described ( 14 ). Chemically modifi ed siRNAs were synthesized and characterized, as previously described ( 14 ).
5 ′ -UGGUCAAGUGUCUGUUUGA-3 ′ and 5 ′ -GGACTTCTCTC-AATTTTCT-3 ′ were siRNA target sequences for Gcgr and a nontargeting control siRNA, respectively. The encapsulated siRNAs were injected via tail vein in a 3 mg/kg dose on days 0 and 6. Animals were euthanized at day 11, and blood and liver samples were collected immediately following euthanasia. Animal procedures were practiced in conformity with Public Health Service policy and the guidelines of the Institutional Animal Care and Use Committee of Merck.

Plasma characterization of metabolic phenotypes, and hepatic lipid determination
Ambient (nonfasted) plasma glucose levels were measured at 9:00 AM from tail bleeds with a glucometer (One Touch Ultra; LifeScan, Inc., Milpitas, CA) at day 4 and day 11 after siRNA injection. At day 11, mice were fasted for 5 h, and blood samples were collected. Plasma was separated by centrifugation and stored at Ϫ 80°C until analysis. Total plasma cholesterol and HDL-C were measured using commercial kits (Wako Chemicals; Richmond, VA) according to the manufacturers' protocol. Plasma insulin and TG levels were measured using an ALPCO ELISA kit (ALPCO Diagnostics; Salem, NH) and the Infi nity Triglycerides kit (Thermo Fisher Scientifi c; Waltham, MA), respectively, according to the manufacturers' instructions. Glucagon was measured using commercial ELISA kits (Linco Research Immunoassay; St. Charles, MO). Nonesterifi ed fatty acid (NEFA) was measured using commercially available enzyme-coupled spectrophotometric assays (Wako Chemicals, Inc.), and ␤ hydroxybutylate (bHB) was measured using the Liquid Enzymatic Reagent Kit (Stanbio Laboratory; Boerne, TX). Plasma apoB and apoA-I levels were measured by LC/MS assay, and protein convertase subtilisin/kexin type 9 (PCSK9) was measured using murinespecifi c ELISA, as previously described ( 14,15 ). All assays were performed following the recommended procedures for instrument operation, calibration, quality control, and assay guidelines. Hepatic TG and cholesterol content were measured as previously described ( 16 ).

Western blot analysis
To examine expression of proteins, liver samples were collected and cell lysates were prepared as described previously ( 16 ). Western blot analysis was carried out as described earlier ( 16 ) using anti-Ldlr antibody (Abcam, PLC; Waltham, MA) and anti-␣ tubulin (Abcam, PLC).

The effect of hepatic GCGR knockdown on plasma lipid levels
Lipoprotein analysis showed a 2-fold increase of LDL-C levels in si-GCGR-treated mice compared with those in the si-Control group, with no signifi cant difference in VLDL and HDL-C levels ( Fig. 2A ). In addition, there were no differences in hepatic LDL receptor expression, plasma PCSK9 levels, or lipoprotein lipase activity between these two groups ( Fig. 2B, C ; see supplementary Fig. IA). The use of ultracentrifugation to isolate lipoprotein fractions revealed a slight increase in apoB-100 content in LDL fractions, with a tendency toward increased total plasma apoB levels in si-GCGR mice ( Fig. 2D, E ). FPLC separation confi rmed an increase of LDL-C fraction in si-GCGR mice ( Fig. 2F ).

Statistics
Values are expressed as means ± SEM. The statistical significance of the differences was determined by Student's t -test, and a P value <0.05 was considered signifi cant.

Hepatic GCGR knockdown mice showed increased hepatic lipid contents and lipogenic gene expression
There were increased levels of TGs and cholesterol in the liver samples from si-GCGR mice ( Fig. 3A ). This increase may have potentially resulted from increased expression of hepatic lipogenic genes. There was no differential expression of sterol-regulatory element-binding protein isoform-1c (Srebf1) or SREBP-2 (Srebf2) mRNA in fed liver samples from si-GCGR mice compared with cholesterol (>2-fold) and palmitate (>5-fold) was observed in si-GCGR-treated groups relative to controls ( Fig. 4A, B ). Presumably, changes in the production of these plasma lipids refl ect hepatic metabolism, because the liver is a major source of newly made circulating lipids. To determine whether these changes lead to altered hepatic lipoprotein secretion, the lipase inhibitor P407 was administered by injection to both groups of mice. P407 blocks the clearance of apoB-containing lipoproteins from the circulation; thus, the rate of accumulation of lipoprotein refl ects the secretion rate in plasma. The results showed an increase in hepatic secretion of TGs in si-GCGR mice as well as increased basal plasma TG levels ( Fig. 4C, D ). Compared with control groups, there was no difference in plasma levels of NEFA, bHB, alanine transaminase, and aspartate aminotransferase in the si-GCGR group ( Fig. 4E, F ;

DISCUSSION
Targeting glucagon action to treat type 2 diabetes has been of great interest. This study demonstrated a strong and sustained effect on glucose lowering in siRNA-mediated hepatic Gcgr knockdown in db/db mice. Previous studies have shown that inhibiting glucagon action improves glycemic control in animals and humans ( 9,11,12,21 ). Consistent with those observations, the present results synthetase (Fdps) ( Fig. 3B ). A tendency of increased expression was shown by 3-hydroxy-3-methylglutaryl-CoA reductase (Hmgcr), whereas MLX interactor (Chrebp) mRNA expression was unchanged ( Fig. 3B ). Microsomal triglyceride transfer protein (Mttp) showed a moderate increase in expression, whereas other potential candidate molecules involved in the regulation of apoB-containing lipoprotein levels, including apoB, Lpl, PCSK9, or Ldlr mRNA expression, were unchanged ( Fig. 3B ). As expected with inhibition of glucagon signaling, the expression of glucokinase (Gck) and pyruvate kinase (Pklr) was increased, and the expression of phosphoenolpy ruvate carboxykinase (Pepck) was decreased in the hepatic Gcgr knockdown mice group ( Fig. 3B ). Upregulation of lipogenic and glycogen biosynthetic genes and reduction of gluconeogenic gene expression were observed with hepatic Gcgr knockdown. These fi ndings suggest that reduced levels of hepatic glucagon receptor in db/db mice produce an upregulation of lipogenic genes, which may eventually lead to increased hepatic lipid contents.

Hepatic GCGR knockdown mice showed increased de novo lipid synthesis and TG secretion
Stable isotope tracer studies were performed to determine whether increased expression of hepatic lipogenic genes translated into increased lipid synthesis in si-GCGR mice. To quantify the rate of de novo lipogenesis, both groups of mice were injected with 2 H 2 O tracer, and the 2 H labeling of cholesterol and palmitate was quantifi ed using GC/MS. A signifi cant increase in the synthesis of The statistical signifi cance of the differences was determined by Student's t -test, and a P value <0.05 was considered signifi cant (* P < 0.05; **<0.01 vs. si-Control). B: Analysis of hepatic gene expression by real-time quantitative PCR in si-Control and si-GCGR mice (n = 8). Fold regulation indicates the ratio of expression levels in si-GCGR over si-Control. Livers were collected from mice in the fed condition. The statistical signifi cance of the differences was determined by Student's t -test, and a P value <0.05 was considered signifi cant.
contents, lipogenic gene expression, and TG production. All of these aggravated dyslipidemic characteristics are associated with an upregulation of de novo lipogenesis. We did not observe any increase in plasma NEFA concentrations to suggest increased FA fl ux to liver, nor a reduction in ␤ -hydroxybutyrate levels and/or reduction in the expression of FA oxidation genes in si-GCGR mice ( Fig.  4E, F ; data not shown). Hepatic LDLR protein and plasma PCSK9 levels were also unchanged; therefore, the increasing LDL-C is probably less due to the reduction in LDL fractional catabolism. On the basis of our results, we propose that the elevated rate of lipogenesis is a major driving force for the observed increases in LDL-C levels and increased TG production in si-GCGR db/db mice. Interestingly, siRNA-mediated knock down of fructose-1,6bisphosphatase 1, which converts fructose 1,6 biphosphate to fructose 6-phosphate in gluconeogenesis, also reduced plasma glucose levels, increased LDL-C levels, and upregulated expression of hepatic lipogenic gene expression in the db/db mouse model (see supplementary Fig. II; data not shown).
We also tested the effect of knockdown glucagon receptor in a mouse model without impairment of leptin signaling, CETP transgenic LDLR +/ Ϫ mouse, using siRNA tar geting Gcgr. The si-GCGR-treated mice showed ‫ف‬ 20% reduction in plasma glucose and ‫ف‬ 24% induction of LDL-C levels in the CETP-transgenic LDLR +/ Ϫ mouse model (see supplementary Fig. III).
The inhibition of hepatic gluconeogenesis and possible increase in glycolysis in si-GCGR mice could lead to indicate that blocking GCGR activation can improve glucose control in a diabetic mouse model.
Although such therapies have consistently demonstrated improvement of glucose in different mouse models and even human clinical trials, questions regarding the potential effects of blocking glucagon action on lipid metabolism have been raised. Interestingly, several other studies have also illustrated the potential hypolipidemic actions of glucagon, specifi cally, with respect to reduced plasma TG, VLDL secretion, hepatic TG contents, cholesterol levels, and increased FA oxidation (22)(23)(24)(25). These fi ndings underscore the importance of understanding the potential implications of downregulating glucagon signaling on lipid metabolism.
In the current study, inhibition of GCGR by si-RNA demonstrated effects on both glucose and lipid metabolism in the db/db mouse model. The si-GCGR db/db mouse showed robust glucose lowering and increased LDL-C levels. Si-GCGR mice showed increased hepatic lipid Fig. 4. Cholesterol and palmitate synthesis and TG production. Newly synthesized cholesterol (A) and palmitate (B) of si-Control and si-GCGR-injected mice. C: TG production in si-Control and si-GCGR mice. TG production was determined by measuring plasma TG concentrations at indicated times after P407 injection. Quantifi cations of plasma levels of TG (D), NEFA (E), and ␤ hydroxybutylate (bHB) (F) from si-Control and si-GCGR mice. All values are expressed as means ± SEM (n = 6-8). The statistical signifi cance of the differences was determined by Student's t -test, and a P value <0.05 was considered signifi cant (* P < 0.05; **<0.01 vs. si-Control).
build-up of acetyl-CoA, which can be used as carbon substrate for lipid synthesis. The newly synthesized hepatic lipid can be either stored or secreted as VLDL. Certainly, we observed increased hepatic lipid contents as well as increased TG production. Another explanation could be that knocking down GCGR sensitized the liver to insulinmediated lipogenesis. It is well documented that insulin upregulates FA biosynthesis and glycolysis genes. One study showed inhibitory effects of glucagon on hepatic lipogenesis by antagonizing the signaling pathway mediated by insulin ( 26 ). A recent study showed a global upregulation of lipid biosynthesis and glycolysis genes in Gcgr Ϫ / Ϫ mice liver at both mRNA and protein levels ( 27 ). Importantly, Gcgr knockout or Gcgr monoclonal antibodytreated preclinical animal models showed elevated plasma cholesterol and LDL cholesterol ( 8,28 ), which might be caused by increased hepatic cholesterol synthesis. Indeed, our stable isotope tracer studies demonstrated that cholesterol synthesis ( 2 H labeling of cholesterol) was signifi cantly increased in si-GCGR-treated mice. It is interesting to note that we did not observe robust change of gene expressions involved in cholesterol synthesis, which is somewhat inconsistent with the report that multiple genes of cholesterol biosynthesis were elevated in Gcgr Ϫ / Ϫ mice ( 27 ). This discrepancy might be due to differences in the genetic background, the dysmetabolic status of mice, and the degree of GCGR defi ciency. The implications of these fi ndings could be that glucagon/GCGR signaling not only plays a role in glucose metabolism but also protects against excessive hepatic lipid production.
Given that increased atherosclerosis is a major cause of morbidity and mortality in type 2 diabetes and metabolic syndrome ( 29 ), it is important to carefully eva luate the potential association of increased LDL cholesterol levels when GCGR is antagonized. The current study demonstrated the potential association between hepatic glucagon signaling and regulation of lipogenesis and plasma LDL in the db/db mouse model. Although there may be concerns with respect to directly applying results from a mouse model to human lipid metabolism, the hepatic Gcgr knockdown in db/db mice is a useful model by which to study the interaction of hepatic glucagon signaling and lipid metabolism. Whether the glucose and lipid effects can be decoupled at the glucagon receptor level will be of great interest to the fi eld. Understanding the glucagon/ GCGR mechanisms that control lipid metabolism will provide important insights to guide the design of the next generation of GCGR antagonists for the treatment of type 2 diabetes.