In Vivo Adenoviral Delivery of Recombinant Human Protein Kinase C-ζ Stimulates Glucose Transport Activity in Rat Skeletal Muscle*

An in vivo adenoviral gene delivery system was utilized to assess the effect of overexpressing protein kinase C (PKC)-ζ on rat skeletal muscle glucose transport activity. Female lean Zucker rats were injected with adenoviral/human PKC-ζ (hPKC-ζ) and adenoviral/LacZ in opposing tibialis anterior muscles. One week subsequent to adenoviral/gene delivery rats were subjected to hind limb perfusion. The hPKC-ζ protein was expressed at the same level (fast-twitch white) or at ∼80% of the level (fast-twitch red) of endogenous PKC-ζ, thus approximately doubling the amount of PKC-ζ in tibialis anterior. Basal glucose transport activity was elevated ∼3.4- and 2-fold, respectively, in fast-twitch white and red hPKC-ζ muscle relative to control. Submaximal insulin-stimulated glucose transport activity, corrected for basal transport, was ∼90 and 40% over control values, respectively, in fast-twitch white and red hPKC-ζ muscle. The enhancement of glucose transport activity in muscle expressing hPKC-ζ occurred in the absence of any change in GLUT1 or GLUT4 protein levels, suggesting a redistribution of existing transporters to the cell surface. These results demonstrate that an adenoviral vector can be used to deliver expressible hPKC-ζ to adult rat skeletal muscle in vivo and also affirm a role for PKC-ζ in the regulation of glucose transport activity.

Elucidation of the specific intermediates involved in the metabolic arm of the insulin signaling cascade initiated by insulin binding to its receptor and culminating in GLUT4 translocation to the cell surface has been an area of intense investigation. To date, several key regulatory proteins and their indispensability in propagating the insulin signal, such as the insulin receptor, insulin receptor substrate proteins (IRS), and phosphatidylinositol 3-kinase (PI-3-K), 1 have been identified and described (1)(2)(3)(4)(5)(6). Downstream of PI-3-K, however, the path is less clear. The majority of evidence currently compiled implicates the protein kinase Akt/PKB as a primary target of inositol 3-phosphates, and associated regulatory proteins such as PDK-1, supporting its role as an insulin signaling intermediate (7)(8)(9)(10)(11)(12). The existence of parallel or branched paths, however, at this point in the insulin signaling cascade have been suggested by the findings that inositol 3-phosphates and PDK-1 also activate a member of the atypical protein kinase C (PKC) family, PKC- (11,13).
PKC involvement in the stimulation of metabolism by insulin has been speculated on and investigated for many years, yet overall the evidence for or against a role for PKC in insulin signaling can best be described as equivocal (14). This ambiguity may stem, in part, from the diversity within the extensive PKC family that has been identified thus far. Normal insulinstimulated glucose transport in 3T3-L1 cells despite downregulation of conventional and novel PKC isoforms using phorbol esters suggests that these isoforms are not necessary for insulin-induced activation of glucose transport (15)(16)(17). Alternatively, atypical PKC isoforms, such as PKC-, are not diacylglycerol-sensitive and, as mentioned above, are activated by a well established arm of the insulin signaling pathway, products of PI-3-K (11,13). Interestingly, it has been demonstrated that PKC-is activated by insulin and that overexpression of wildtype PKC-increased, whereas overexpression of a dominantnegative PKC-decreased, basal and insulin-stimulated glucose transport in 3T3/L1 cells (18). Likewise, stable and transient expression of a kinase-inactive PKC-was shown to inhibit both basal and insulin-stimulated glucose transport in L6 myotubes (19). Collectively, these results suggest a potential role for PKCdownstream of PI-3-K in the insulin signaling pathway for the stimulation of glucose transport.
To examine the potential ability of exogenously administered PKC-to stimulate glucose transport activity in the primary insulin-responsive tissue, skeletal muscle, we expressed recombinant human PKC-(hPKC-) in rat tibialis anterior muscle using a novel in vivo adenoviral gene transfer system. Unlike classic transgenic studies, this approach affords several advantages including the following. Gene delivery and subsequent protein expression occurs in adult animals, and thus developmental compensatory responses are less likely to dictate the observed physiological outcome. Also, localization of gene delivery and protein expression within a specific muscle allows experimental and control genes to be delivered to the same animal, resulting in a highly controlled experimental paradigm. Finally, the ability to administer the adenoviral/gene construct to animals of various backgrounds or disease states eliminates the need for costly and time consuming breeding and backcrossing.
Application of this in vivo adenoviral/gene transfer system and subsequent assessment of physiological responses using the hind limb perfusion technique revealed that expression of hPKC-in skeletal muscle of normal lean rats results in enhanced rates of both basal and submaximal insulin-stimulated glucose transport activity.

MATERIALS AND METHODS
Generation of Recombinant Adenovirus-Recombinant adenovirus expressing ␤-gal was generated by homologous recombination between * The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. pBHG10 (20) and pCA17 (21) following techniques previously described (20). A recombinant adenovirus expressing hPKC-was constructed as follows. The hPKC-cDNA was isolated from hPKC-Bluescript (22) on an XbaI to EcoRI fragment and first inserted between the XbaI and EcoRI sites of the cloning vector pOK12 (23). The hPKC-cDNA was then removed on a 1.9-kilobase BclI fragment and ligated into the BamHI site of pCA13 (Microbix Biosystems Inc. Ontario, Canada), thus generating the transfer plasmid pCA13/. Using materials and protocols supplied by Microbix Biosystems Inc., recombination of PCA13/ and pBHG10 yielded the adenovirus Ad-hPKC-.
Amplification and Purification/Concentration of Recombinant Adenoviruses-Amplification of recombinant virus was performed in adherent 293 cells. Purification and concentration of the virus was achieved using cesium chloride ultracentrifugation. The resulting virus was dialyzed into phosphate-buffered saline (with calcium and magnesium) plus 10% glycerol. Viral titers were determined using 100-mm dishes of adherent 293 cells overlaid with 0.6% agarose.
Animals and Immunosuppressive Therapy-Female lean Zucker rats (Fa/?) were obtained from Charles River Laboratories (Raleigh, NC) at 12 weeks of age. All animal procedures were reviewed by and conformed with the Institutional Animal Review Board (Eli Lilly and Company, Indianapolis, IN). Rats were maintained in an environmentally controlled animal facility (22 C, 12 h light/dark cycle) and provided free access to food and water. Two days prior to adenoviral/gene injection, and on all days subsequent, rats received a 5 mg/kg subcutaneous injection of the immunosuppressive agent FK506 (Bergen Brunswig Drug Co., Louisville, KY) to curb host immune responses to the foreign gene and its delivery system. In preliminary investigations with the adenoviral/LacZ vector, this immunosuppressive strategy resulted in sustained high level ␤-gal protein expression relative to nonimmunosuppressed controls through a 2-week investigation. 2 Surgery and Adenoviral/Gene Intramuscular Injection-Surgical preparation and intramuscular injections were performed with the animals under anesthesia via the inhalant isoflurane in 2-3% O 2 . Initially, fur was shaved from the anterior portion of both hind limbs, and a 5-mm incision was made in the skin overlying the tibialis anterior. One hundred microliters of the adenoviral vector (2 ϫ 10 10 plaqueforming units/ml) containing either hPKC-or the control gene LacZ was then injected at six separate sites in the tibialis anterior. Preliminary studies revealed that this injection protocol resulted in the highest efficiency of gene transfer to the largest population of muscle fibers in the tibialis anterior. 2 Each animal received both the hPKC-test gene (right or left leg) and the Lac-Z control gene (opposite leg). In this manner, each animal served as its own control.
Hind Limb Perfusion Studies-Six days after adenoviral/gene delivery, rats were fasted overnight in preparation for hind limb perfusion. Prior to perfusion, rats were anesthetized with pentobarbital sodium (6 mg/100 g body weight), and the hind limb vasculature was surgically isolated for perfusion of both hind limbs as described previously (24). Prior to being placed in the nonrecirculating perfusion chamber, rats were euthanized via an intracardiac injection of pentobarbital sodium. Hind limbs were perfused for 30 min with well gassed (95% O 2 , 5% CO 2 ) Krebs-Henseleit bicarbonate buffer containing 2.5% bovine serum albumin, 8 mM glucose, 2 mM mannitol, and either the presence or absence of 50 units/ml insulin. Following the initial 30 min perfusion, the hind limbs were perfused for an additional 10 min in the presence of Krebs-Henseleit bicarbonate/bovine serum albumin containing 8 mM glucose (90 Ci/mmol 3 H-2-deoxy-D-glucose), 2 mM mannitol (120 Ci/ mmol 14 C-mannitol), and either the presence or absence of 50 microunits/ml insulin. At the end of the perfusion period, both tibialis anterior muscles were removed from the hind limbs and dissected into fast-twitch red, fast-twitch white, and mixed portions. These muscle pieces were frozen in tongs cooled to the temperature of liquid nitrogen and stored at Ϫ80 C for subsequent analysis of muscle glucose transport activity, Western blotting, and ␤-gal expression/activity in the presence of the substrate X-gal.
Muscle Homogenate Analysis-Muscle portions were homogenized (1:20 wt:vol) on ice in Hepes, EDTA buffer (25 mM Hepes, 1 mM EDTA, pH 7.4) in the presence of aprotinin, pepstatin, and leupeptin (0.1 g of each/25 ml of buffer). Protein content of the homogenates was determined with a Bio-Rad protein assay (Bio-Rad, Hercules, CA). For Western blotting/immunodetection experiments, 50 or 75 g of protein (as indicated) was subjected to SDS-PAGE and transferred to nitrocellulose membranes for subsequent detection with specific polyclonal antisera (PKC-, GLUT1, and GLUT4). Immunoreactive protein levels were quantified via detection of enhanced chemiluminescence (ECL-Plus; Amersham Pharmacia Biotech) using a STORM phosphoimager and ImageQuant (Molecular Dynamics, Inc., Sunnyvale, CA).
Expression/activity of ␤-gal in muscle homogenates was determined by incubating 50 l of each muscle homogenate with 100 l of the chromogenic substrate X-gal (1 mg/ml) for 18 h.
Statistical Analysis-All data are expressed as means Ϯ S.E. The data were subjected to analysis of variance with significant differences between means determined using Fisher's protected least significant difference (post hoc analysis). Significance was set at the p Ͻ 0.05 level.

RESULTS AND DISCUSSION
Previous work with transgenic animals demonstrated that single gene manipulations can significantly alter normal and/or disease (diabetic) phenotypes (25)(26)(27). The application of these genetic manipulations was of limited practical implication, however, as a result of the technical aspects of gene insertion at the pro-nuclear stage of development. Such early manipulation also raises the question of developmental compensatory alterations leading to the observed adult transgenic phenotype. To circumvent such confounding factors and to investigate the practicality of acutely altering genetic expression in adult animals, we developed/refined a system for delivering genes of interest to a primary target of insulin action and a key regulator of glucose homeostasis, skeletal muscle. By combining the intramuscular adenoviral/gene injections with subsequent hind limb perfusion experiments, we were able to determine whether transcription of our gene of interest (hPKC-) resulted in expression of a functional protein and, furthermore, determine whether the expression of that protein is physiologically relevant.
As shown in Fig. 1, lanes 2 and 4, intramuscular injection of the adenoviral/hPKC-construct into tibialis anterior muscle resulted in the appearance of a second immunoreactive PKCband of slightly greater molecular weight than the endogenous band. We have observed that this migratory difference, between endogenous and exogenous PKC-, on SDS-PAGE is species-related. Quantitative analysis revealed that hPKCexpression equaled endogenous PKC-expression in fasttwitch white muscle, whereas in fast-twitch red muscle hPKCwas expressed at ϳ80% of endogenous levels (p ϭ 0.01). There were no apparent systemic effects of the local injections as expression of hPKC- (Fig. 1) and expression/activity of ␤-gal (data not shown) were noted only in those muscles directly injected.
Upon establishment of hPKC-expression, attention was turned toward the metabolic consequences. Fig. 2 reveals glucose transport activity assessed during hind limb perfusion. In fast-twitch white muscle, hPKC-expression enhanced basal glucose transport 3.4-fold above the ␤-gal control. Similarly, basal glucose transport was elevated approximately 2-fold above control in fast-twitch red muscle expressing hPKC-. It may be speculated that the difference between fiber types, with regard to magnitude of increase in transport activity, reflects the subtle disparity noted in the level of hPKC-expression. The expression of ␤-gal in tibialis anterior muscle (fast- twitch  FIG. 1. Representative autoradiographic samples of fasttwitch white (lanes 1 and 2) and red (lanes 3 and 4) tibialis anterior muscle homogenates immunoblotted for PKC-protein. Lane 1, adenoviral/LacZ-injected white tibialis anterior (50 g of protein); lane 2, adenoviral/hPKC--injected white tibialis anterior (50 g of protein); lane 3, adenoviral/LacZ-injected red tibialis anterior (50 g of protein); lane 4, adenoviral/hPKC--injected red tibialis anterior (50 g of protein). red and white) did not alter basal glucose transport rates relative to control noninjected muscle (data not shown).
Glucose transport was also assessed in the presence of a physiologically relevant submaximal insulin concentration (50 microunits/ml). The absolute rates of glucose transport in fasttwitch white and red muscle portions are displayed in Fig. 2, however, as a result of the elevated basal glucose transport activity in muscle expressing hPKC-, the "true" insulin effect was obtained by subtracting basal rates of transport from the absolute insulin-stimulated values in the respective cases. These conversions revealed that increasing PKC-in fasttwitch white muscle enhanced the effect of a submaximal insulin concentration by approximately 90% over control (4.56 versus 2.39 mol/g/h). In fast-twitch red muscle, the true insulin effect was approximately 40% over control levels (10.33 versus 7.55 mol/g/h).
Previously, cell based assay systems have been utilized to examine the effects of expressing atypical PKC isoforms ( and ) on glucose transport activity (18,19,28). In 3T3-L1 cells, an approximate 2-fold overexpression of PKC-resulted in a significant enhancement of both basal and insulin-stimulated glucose transport (18), yet surprisingly similar overexpression of PKC-in L6 myotubes had no effect (19). Expression of a constitutively active mutant of PKC-in 3T3-L1 cells using an adenoviral vector system increased glucose transport in accord with the multiplicity of infection such that the stimulation paralleled the level of expression of the mutant protein (28). Additional evidence for the specificity of the effects of the atypical PKC isoforms was provided by overexpression of kinase-inactive mutants of PKC-and -, which resulted in inhibition of both basal and insulin-stimulated glucose transport in L6 myotubes and 3T3-L1 adipocytes, respectively (19,28). In the current study, we utilized the information from the cellbased experiments with implicit assumptions regarding the specificity of atypical PKC action and extend these findings to a higher level of organizational structure and physiological relevance by expressing hPKC-in vivo in arguably the most important insulin-responsive tissue, skeletal muscle. The highly controlled nature of these experiments, in which adenoviral/Lac-Z injections and subsequent ␤-gal expression served as the control for infection/gene transfer in the same animal as the hPKC-expression, revealed that increased expression of this kinase produces a physiologically relevant outcome, acceleration of glucose transport. Taken together with previous reports, these results provide strong evidence that PKC-is involved in stimulation of glucose transport activity in mammalian cells. In the current study, the mechanism through which these results were achieved, however, remained to be explored.
Insulin acutely accelerates glucose transport in skeletal muscle through the translocation of existing glucose transporters from an intracellular pool to the cell surface (29). Thus, it may be speculated that if PKC-is a downstream insulin signaling component, increased expression of the protein could result in enhanced glucose transporter translocation. Alternatively, PKC-has also been implicated in the ability of insulin to stimulate general protein synthesis (30). Therefore, it was equally as tempting to speculate that increased expression of PKC-would be capable of driving the synthesis of additional glucose transporters, which could also translate into increased glucose transport activity. To rule out the latter possibility, we quantified GLUT1 and GLUT4 protein levels in the injected muscle samples. We detected no difference in either GLUT1 or GLUT4 protein levels in fast-twitch red or white muscle expressing hPKC-relative to control (representative samples displayed in Fig. 3, A and B for GLUT1 and GLUT4, respectively). These findings, combined with previous in vitro observations of alterations in the subcellular distribution of glucose transporters in 3T3-L1 adipocytes overexpressing atypical PKC isoforms (18,28), suggest that the presently reported acceleration of glucose transport activity results from an enhancement of glucose transporters at the cell surface.
In summary, the results of the present study demonstrate that hPKC-can be expressed in adult rat skeletal muscle in vivo using an adenoviral/gene delivery system. Furthermore, expression of this kinase enhanced both basal and insulinstimulated muscle glucose transport, affirming in this unique setting a role for PKC-in the regulation of glucose transport. Establishment of these techniques and the resultant positive physiological outcome provides new insight into potential gene therapy approaches for the treatment of insulin resistance and diabetes mellitus.